Universities Research Journal - myanmar

Transcription

The Government of
The Republic of the Union of Myanmar
Ministry of Education
Department of Higher Education (Lower Myanmar)
and
Department of Higher Education (Upper Myanmar)
Universities
Research Journal
Vol. 6, No. 1
March, 2014
Universities Research Journal 2014, Vol. 6, No. 1
Contents
Page
Investigation of Curcuma longa L. and Antidiarrhoeal Activity of
Its Rhizome
Khin Cho Cho Oo
Effects of Holding Solutions on Post-harvest Quality and Vase-Life
of Ornamental Cut Flower, Chrysanthemum sp.
Soe Soe Aung
Quantitative Analysis of Forest Structure in Pahtaw Hill, Kyun-su
Township, Taninthayi Region
Wah Wah Khaing, Lae Lae Khaing, Htay Htay Win, Mi Mi Aye and
Sanda Hlaing
Antifungal Metabolites of Endophytic Strain YY20 Isolated from
Neomarica longifolia (Link & Otto) Sprague
Yee Yee Thu
Comparative Study on Preparation of Botanical Permanent Slides
by Different Methods
Bay Dar, Moe Moe Lwin, Ohnmar Than and Aye Aye Myint
Effects of Root Nodules Rhizobia on Growth of Vigna unguiculata
subsp. sesquipedalis (L.)Verdc.
Soe Myint Aye, Phyu Phyu Oo and Mu Yar Min
Taxonomic Study on Some Bryophytes from Southern Shan State
Soe Myint Aye and Win Win Aye
Effect of Isolated Azospirillum Strains on Germination and Growth
of Triticum aestivum L. (Wheat)
Yi Shan, Thi Thi Htun and Hnin Ei Phyu
Production of ɑ-Amylase Enzyme by Bacillus subtilis from Some
Raw Starchy Materials in Mon State
Khin Kye Mon
Significances of Urban Forest on the Environmental Conditions of
Magway City
Myat Thu
1
15
31
47
63
79
91
105
119
133
Page
Preservation of Lycopersicon esculentum Mill. Fruits through Jam
Production
Zin Moe Moe
Phytochemical Analysis of Cnestis palala (Lour.) Merr.and Its
Antimicrobial Activity
Mi San Mar Lar
A Study on Morphological Characters of Tea Plant and Effect of
Fertilizers on Growth of Camellia sinensis (L.) Kuntze
Kyaw Kyaw Sann
Effect of Spirulina on Growth, Yield and Nutritive Value of
Vigna unguiculata (L.) Walp.
Win Mar
Morphological Variations of Spirulina under Different
Environmental Parameters
Hlaing Nwe Thynn
Isolation and Identification of Pathogenic Fungi from the Fruits of
Capsicum annuum L. Grown in Hinthada Area
Khin Min Min Phyo
Taxonomical Studies on Some Species of Trees Commonly Found
in Lashio Area
Yee Yee Win
Some Orchid Species Found in Kalay Area
Htar Lwin
Plant-Pollinator Interactions of Bago University Campus, Bago
Region
Aye Aye Mar and Kyaw Zay Moe
Drinking water Analysis of Artesian wells found in Yinmabin
Township, Monywa District
Theingi Htay
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161
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203
217
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259
275
Universities Research Journal 2014
Vol. 6, No. 1
Editorial Board
Editors in Chief
Prof. Dr Thet Thet May, Head of the Department of Botany, University
of Yangon
Prof. Dr Malar Aung, Head of the Department of Botany, Mawlamyine
University
Prof. Dr Nu Nu Yi, Head of the Department of Botany, University of
Mandalay
Prof. U Nay Win, Head of the Department of Botany, Yadanarbon
University
Prof. Dr Tun Chun, Head of the Department of Botany, Meiktila
University
Editors
Prof. Dr Thet Thet May, Head of the Department of Botany, University
of Yangon
Prof. Dr Nu Nu Yi, Head of the Department of Botany, University of
Mandalay
Prof. Dr Malar Aung, Head of the Department of Botany, Mawlamyine
University
Prof. Dr San Wai Aung, Head of the Department of Botany, Dawei
University
Prof. Dr Than Than Nu, Head of the Department of Botany, Taungoo
University
Prof. Dr Moe Moe Shwe, Head of the Department of Botany, Bago
University
Prof. Dr Moe Moe Khaing, Head of the Department of Botany,
Hinthada University
Prof. U Nay Win, Head of the Department of Botany, Yadanarbon
University
Prof. Dr Swe Mar Tin, Head of the Department of Botany, Lashio
University
Prof. Dr Htar Lwin, Head of the Department of Botany, Banmaw
University
Prof. Dr Theingi Htay, Head of the Department of Botany, Shwebo
University
Prof. Daw May Than Su, Head of the Department of Botany, Magway
University
Prof. Dr Tun Chun, Head of the Department of Botany, Meiktila
University
The Government of
The Republic of the Union of Myanmar
Ministry of Education
Department of Higher Education (Lower Myanmar)
and
Department of Higher Education (Upper Myanmar)
Universities
Research Journal
Vol. 6, No. 1
March, 2014
Investigation of Curcuma longa L. and Antidiarrhoeal Activity
of Its Rhizome
Khin Cho Cho Oo1 and Kyawt Kyawt Khaing2
Abstract
The plant Curcuma longa L. is a perennial herb, locally known as “Nanwin”
which belongs to the family Zingiberaceae. The plant was collected from
Tingokkyi village, Tharrawaddy District, Bago Region. The morphological
characters of this plant were identified and the antimicrobial activity of 70%
ethanol, ethyl acetate and aqueous extracts of rhizomes of C. longa L. was
studied. The ethyl acetate extracts proved to be the best antimicrobial
activity against Bacillus pumalis. Minimum inhibitory concentration (MIC)
of various extracts of rhizomes of C. longa L. was investigated on
Escherichia coli and Vibrio cholera. The 70% ethanol and ethyl acetate
extracts of rhizomes of C. longa L. observed higher MIC than the aquerous
extract and showed the antidiarrhoeal activity.
Key words: Curcuma longa L., Minimum Inhibitory Concentration (MIC),
Antidiarrhoeal Activity
Introduction
Medicinal plants are important source for the verification of
pharmacological effects and can be natural composite sources that act as new
anti-infectious agents. Due to the increased resistance of many microorganisms
towards the currently available commercial antibiotics, investigation of the
antimicrobial properties in medicinal plants has become desirable. Globally,
plant extracts are employed for their antibacterial, antifungal and antiviral
activities. These plants have medicinal properties and this has made traditional
medicine cheaper than modern medicine.
Zingiberaceae has pantropical distributions chiefly occurring in
Indomalaysia. The majority of species are found in the humid, tropical
lowlands. The Zingiberaceae are rich in aromatic, volatile oils and are widely
used as condiments, herbs, dynes and medicinal plants. The rhizomes of
Curcuma spp. are important the world market (Heywood, 2007). Curcuma
longa L., a perennial herb, is a member of the family Zingiberaceae. This plant
is locally known as “Nanwin” in Myanmar and it is also called “Turmeric” in
English. C. longa L. was collected from Tingokkyi Village, Tharrawaddy
2
District, Bago Region. The plants were collected and identified with the
literatures of Hooker (1894), Backer (1968), Dassanayake (1983) and Wu
Delin (2000). C. longa L. yields turmeric, one of the main coloring and
aromatic ingredients of curry powder and also used as a yellow dye.
The rhizome of C. longa L. is bitter, carminative, diuretic and good for
affections of the liver and jaundice, urinary dischanges, scabies, burises
(Kirtikar et al., 1935). A decoction of the rhizome is said to relieve the pain of
purulent ophthalmia (The Wealth of India, 1950). This plant shows antiinflammatory and hepatoprotective effects and it stands as blood-purifier and it
also has antioxidant, antiasthmatic, anti-tumor, anticutaneous, antiprotozoal
and stomachic properties (Khare, 2007).
The development of bacterial resistance to presently available
antibiotics has necessitated the search for new antimicrobial agents.
Antimicrobial activities are conducted by using different available organisms.
These microorganisms and the diseases that they caused are as follow cited by
Cruickshank (1975) (Table 1):
Table 1. Type of microorganisms and the diseases that they caused
No.
Type of microorganism
Diseases
1. Bacillus subtilis
Ropiness and spoilage of food
2. Staphylococcus aureus
Skin infections and food poisoning
3. Pseudomonas aeruginosa
Pneumonia, septic shock, urinary tract infection,
septic shock, gastrointestinal infection, skin and soft
tissue infections
4. Bacillus pumalis
Eye infection, soft tissue and cutaneous infections
5. Canadida albicans
Oral and vaginal infection, skin and cardiac infections
6. Escherichia coli
Urinary tract infections, neonatal
septicemia, diarrhoea and dysentery
7. Vibrio cholerae
Diarrhoea, vomiting and abdominal cramps
8. Klebsiella pneumoniae
Pneumonia, urinary tract infections, lower biliary tract
and surgical wound site infection
9. Proteus mirabilis
Wound infections, septicemia, urinary tract infections
and pneumonias
meningitis,
3
Minimum inhibitory concentration (MIC) is the lowest concentration of
a particular antimicrobial drug which is necessary to inhibit the growth of a
particular strain of microorganism (Atlas, 1986). In this study, the efficiency of
rhizome extracts with 70% ethanol, ethyl acetate and distilled water, were
evaluated for the minimum inhibitory concentration on E. coli and Vibrio
cholera.
This research was aimed to be wider application of plants in Myanma
traditional medicine and promoting the Myanma traditional medicine
scientifically. This research has been made with the objectives of identifying of
Curcuma longa L., experimenting of antimicrobial activity of ethyl acetate,
70% ethanol and aqueous extract of rhizomes and determining of the minimum
inhibitory concentration (MIC) of these rhizome extracts.
Materials and Methods
Plant Materials
Rhizomes of Curcuma longa L. were collected from Tingokkyi Village,
Tharrawadddy District, Bago Region, which is situated at latitudes 17°44'
38.72''N and Longitudes 95°51' 25.26''E, 22.1 m (73 ft) above the sea level
(Fig. 1). The fresh rhizomes were washed and chopped into small pieces and
dried under shady place with good ventilation.
Source: Google Earth 2012 Digital Globe, Sport Image
Fig.1. Sample site of Tingokkyi Village, Tharrawaddy District area
4
Extraction Procedure
Dried rhizomes powder (100 g) was successively extracted with water,
ethyl acetate and also separately extracted with 70% ethanol on water bath for
6 hours at 70°C. After 6 hours, the extract was filtered and the solvents were
evaporated to dryness using water bath at 100°C so as to obtain a paste
(Harbone, 1998).
Testing by Microorganisms
The solvent extracts were tested against nine pathogenic
microorganisms by using agar-well diffusion method. The extent of
antimicrobial activity was measured at the diameter zone of inhibition. The test
organisms included Bacillus subtilis, Staphylococcus aureus, Pseudomonas
aeruginosa, Bacillus pumalis, Candida albicans, Echerichia coli, Vibrio
cholerae, Klebsiella pneumoniae and Proteus mirabilis.
Screening for Antibacterial Activity
The study of antimicrobial activities was performed by agar-well
diffusion method. Nutrient agar was prepared according to method described
by Cruickshank (1975). Nutrient agar was boiled and 20 - 25 ml of the medium
was poured into each test tube and plugged with cotton wool and sterilized at
121°C for 15 minutes in an autoclave. Then the tubes were cooled down to 30 35°C and the contents were poured into sterilized pertidishes and 0.1 - 0.2 ml
of test organism was also added into the dishes. The agar was allowed to set
for 2 - 3 hours. And then, 10 mm plate agar-well was made with the help of
sterilized agar-well cutter. After that, about 0.2 ml of sample was introduced
into the agar-well and incubated at 37°C for 24 hours. The inhibition zone
appeared around the agar-well, indicating the presence of antimicrobial
activity. The extent of antimicrobial activity was measured with the help of
transparent ruler at the diameter zone of inhibition including the agar-well.
Determination of Minimum Inhibitory Concentration (MIC)
The MIC for each sample was determined using agar-well method. In
order to determine MIC, serial dilutions of the extracts were prepared with
various concentration ranges. The MIC values were interpreted as the highest
dilution (lowest concentration) of the samples, which showed clear zone. The
experiments were repeated at exactly three times. The same parameters and the
mean results were taken. The MIC of 70% ethanol, ethyl acetate and aqueous
solutions were determined by test tube double fold serial dilution method.
5
Results
Scientific name
- Curcuma longa L.
Myanmar name
- Nanwin
English name
- Tumeric
Family
- Zingiberaceae
Distribution
- widely distributed in Myanmar
Outstanding Morphological Characters
Perennial herbs, 1.0 - 1.5 m high. Stems herbaceous, terete, rhizomes
many branched, bright yellow, aromatic. Leaves opposite in tuft up to 1.2 m,
simple, the lamina oblong-lanceolate, 80.0 - 85.0 cm long and 23.0 - 23.5 cm
wide, the tips acuminate, the margins entire, the bases annulate; petiole is
about as long as the blade. Inflorescence terminal, spikes, the peduncles
cylindrical, fertile bracts pale green, ovate-oblong, apex obtuse, coma bracts
spreading, white and pale greenish white, apex acute; sessile; bracteolate,
transluent white. Flowers yellow, 5.5 cm long and 2.3 cm in diameter,
complete, bisporangiate, zygomorphic, trimerous, epigynous; sepals (3),
synsepalous, tubular, light yellow; petals (3), synpetalous, funnel-shaped, light
yellow; lateral staminodes petaloid, obong, folded under the dorsal petal,
labellum with a thickened central portion and thinner side lobes which overlap
the lateral staminodes, fertile stamen 1, filament short and broad, constricted at
the apex, anther versatile, spurred at the base; ovary inferior, ovoid,
tricarpellary, trilocular, the placentation axile, the style long and filiform, the
stigma 2 lipped. Fruits and seeds unknown. Flowering and fruiting time;
September to December (Fig. 2).
(a)
(b)
(c)
(d)
Fig. 2. Habit of Curcuma longa L.: (a) Close up view of inflorescence, (b)
Inflorescence, (c) Close up view of flower, (d) L. S of flower
6
Screening for Antibacterial Activity
Yield percentage of dried rhizome powder of Curcuma longa L. in
different solvents were as shown in Table 2. Antimicrobial activities were
studied with 70% ethanol, ethyl acetate and aqueous extracts. Agar-well
diffusion method was used to determine the zone of inhibition of microbial
growth at particular concentration of various extracts as shown in Fig. 3, 4 and
Table 2.
Table 2.
Yield percentage in different solvent extracts of dried rhizome
powder of Curcuma longa L.
Yield Percentage in Solvent (%)
Name of Plant
Dried rhizome of
Curcuma longa L.
70% Ethanol
Ethyl Acetate
Aqueous Extract
16.5
13.2
8.5
Ethyl acetate, 70% ethanol and aqueous extracts did not show effective
antimicrobial activity on Candida albicans. Especially, ethyl acetate extracts
showed the most effective antimicrobial activity on eight different
microorganisms. Moderately effective antimicrobial activity of aqueous
extracts also found on eight different microorganisms (Table 3 and Fig. 3).
Table 3. Antimicrobial activity of different solvent extracts of dried rhizome of
Curcuma longa L.
Test Organisms
Extracts
Bacillus Staphylococcus Pseudomonas Bacillus Candida Escherichia Vibrio
Klebsiella Proteus
aeruginosa pumalis albicans
cholerae pneumoniae mirabilis
subtilis
aureus
coli
Ethyl
acetate
21 mm
25 mm
23 mm
30 mm
(+++)
(+++)
(+++)
(+++)
70%
ethanol
18 mm
20 mm
14 mm
15 mm
(++)
(+++)
(+)
(++)
15 mm
15 mm
14 mm
15 mm
(++)
(++)
(+)
(++)
Water
-
-
-
25 mm
25 mm
25 mm
25 mm
(+++)
(+++)
(+++)
(+++)
20 mm
25 mm
15 mm
15 mm
(+++)
(+++)
(++)
(++)
15 mm
14 mm
15 mm
15 mm
(++)
(+)
(++)
(++)
Agar well– 10 mm, 10 mm ~ 14 mm (+), 15 mm ~ 19 mm (++), 20 mm above (+++)
7
Fig. 3. Antimicrobial activity of different solvent extracts of dried rhizome of
Curcuma longa L.
EtOAc
EtOAc
H2O
70% EtOH
EtOAc
70% EtOH
H2O
70% EtOH
70% EtOH H O
2
Antimicrobial activity
Control
Bacillus subtilis
EtOAc
70% EtOH
Control
H2O
Staphylococcus aureus
EtOAc
EtOAc
70% EtOH
H2O
Pseudomonas aeruginosa
EtOAc
H2O
H 2O
70% EtOH
70% EtOH H O
2
Control
EtOAc
Control
Bacillus pumalis
8
EtOAc
EtOAc
EtOAc
EtOAc
70% EtOH
H2O
Control
70% EtOH H2O
70% EtOH H2O
EtOAc
H2O
Control
Candida albicans
70% EtOH
H2O
70% EtOH
Escherichia coli
EtOAc
EtOAc
70% EtOH
H2O
H2O
70% EtOH
Control
EtOAc
70% EtOH H2O
Vibrio cholerae
Control
Klebsiella pneumoniae
EtOAc
70% EtOH
EtOAc
H2O
H2O
70% EtOH
Control
Proteus mirabilis
Fig. 4. Antimicrobial activity of different solvent extracts against
microorganisms
Determination of Minimum Inhibitory Concentration (MIC)
The minimum inhibitory concentration (MIC) values of 70% ethanol,
ethyl acetate and aqueous extracts of dried rhizome of Curcuma longa L., were
investigated by using agar-well method as shown in Fig. 5, 6 and Table 4, 5).
9
Fig. 5. Minimum Inhibitory Concentration (MIC) values of ethyl acetate, 70%
Ethanolic and watery extracts from Cucurma longa L. against
Escherichia coli
Fig. 6. Minimum inhibitory concentration (MIC) values of ethyl acetate, 70%
ethanolic and watery extracts from Cucurma longa L. against Vibrio
cholerae
Table 4. Minimum Inhibitory Concentration (MIC) values against Escheriachia coli
Table 5. Minimum Inhibitory Concentration (MIC) values against Vibrio cholerae
11
Discussion and Conclusion
In the present study, Curcuma longa L., “Nanwin” grows
throughout of Myanmar. The perennial herb of C. longa L. was brightly
yellow rhizomes and strongly aromatic. The leaves were oblong-lanceolate
shaped which arrange opposite in tuft. The inflorescences were composed
of peduncled terminal spikes with pale green fertile bracts and another
white and pale greenish white coma spreading bracts. The flowers of
C. longa L. had petaloid lateral staminodes, yellowish labellum with central
yellow band and versatile anther. The ovary of C. longa L. had inferior and
axile placentation. These characters are in agreement with those of Hooker
(1894), Backer (1968), Dassanayake (1963) and Wu Delin (2000). The
plants in this experiment were collected from Tingokkyi Village,
Tharrawaddy District, Bago Region. The study was made for identification
of the morphological details of this plant. C. longa L. was chosen as the
subject of research because it is widely used in Myanmar herbal medicines.
In this experiment, antimicrobial activities of different solvents
showed that the ethyl acetate and 70% ethanolic extracts were more
effective than aqueous extracts. According to Singh et al. (2002), aqueous
extracts were evaluated for antimicrobial activity against Staphylococcus
aureus, Escherichia coli and Pseudomonas aeruginosa. It was evident from
the present results that aqueous extracts were more effective than those in
the experiment of Singh et al. (2002). In the investigation, it was found that
there was no antimicrobial activity of 70% ethanol, ethyl acetate and
aqueous extracts of C. longa L. on Candida albicans. Ethyl acetate extracts
proved to be the best antimicrobial activity against Bacillus pumalis which
causes eye infection, soft tissue infections and cutaneous infections.
Ong-ard Lawhavinit et al. (2010) reported that the antimicrobial activity of
ethanolic extract showed inhibitory effects for Vibrio cholera, Bacillus
subtilis, Staphylococcus aureus but it did not inhibit Escherichia coli,
Klebsiella pneumoniae and Proteus mirabilis. The results of the present
study with 70% ethanolic extracts, however, showed the inhibitory effects
on Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis and then
the best antimicrobial activity, among them, is against Vibrio cholerae
causing diarrhoea, vomiting and abdominal cramps. Based on the results of
antimicrobial activity, C. longa L. could be applied for the treatment on the
diseases resulting from Bacillus pumalis and Vibrio cholerae.
12
1
Universities Research Journal 2014, Vol. 6, No.
The MIC values of 70% ethanol and ethyl acetate extracts were
0.0012 × 102 μg ml-1 which showed as the best antimicrobial activity
against E. coli but the MIC value of aqueous extract was 5× 102 μg ml-1
which had antimicrobial activity against E. coli. In the current study, the
MIC values of 70% ethanolic extracts produced better results with Vibrio
cholerae than in the experiment of Ong-ard Lawhavinit et al. (2010). The
minimum inhibitory concentration (MIC) values of 70% ethanol and ethyl
acetate extracts were 0.0195 × 102 μg ml-1 and it proved to be the best
antimicrobial activity against Vibrio cholerae but the MIC value of aqueous
extract showing the least antimicrobial activity on Vibrio cholerae was
10 × 102 μg ml-1. The MIC values of 70% ethanol and ethyl acetate extracts
were higher than that of aqueous extract.
The 70% ethanol and ethyl acetate extracts had higher potential to
inhibit E. coli and Vibrio cholerae which cause diarrhoea and dysentery
than that of aqueous extract. It may be probably due to the presence of
relatively more polar constituents in former extracts. Most of secondary
metabolites are more soluble in polar solvents than non-polar. Therefore,
70% ethanol, ethyl acetate and aqueous extracts of dried rhizome of
C. longa L., could be useful as herbal medicine for the treatment of
diarrhoea and dysentery. For the future researches, the bioactivity of
C. longa L. should be investigated for antioxidant activity,
anti-inflammantory activity and anti-ulcer activity. Moreover, C. longa L.
possesses the medicinal value and then, the experiment on other bioactive
compounds should also be carried out.
Acknowledgements
We would like to express our gratitude to Professor Dr. Thet Thet May, Head of
Department of Botany, University of Yangon for kindly allowing us to undertake this
research in the Department and Professor Dr. Aye Pe, Department of Botany, University of
Yangon, for his kind permission. We would also like to greatly indebted to Professor
U Zenith Oo, Head of Botany Department (Retd.), West Yangon University, for his
numerous suggestions.
13
References
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New York.
Backer, C. A. and R. C., Bakhuizen Ven Den Brink (1968). Flora of Java. Vol. 3.
The Netherlands: Wolters-Noordhoff, N.V. Groningen.
Crucikshank, S. (1975). Handbook of Bacteriology. 10th Ed., E. and S. Churchill
Livingstone Ltd., Edinburgh, 121-125.
Dassanayake, M. D. (1983). A Revised Hand Book to the Flora of Ceylon. Vol. IV.
University of Peradeniya, Department of Agriculture, Peradeniya:
Sri
Lanka.
Harbone, J. B. (1998). Phytochemical Methods. A Guide to Modern Techniques of Plant
Analysis. 3rd ed., Edmundsbury Press, Great Britain.
Heywood, V. H., D. M., Moore, I.B.K., Richardson and W.T. Stearn (Eds.) (1978).
Flowering Plants of the World. London: Oxford University Press.
Hooker, J. D. (1894). The Flora of British India. Vol. VI. London: L. Reeve & Co., Ltd.
Hundley, H. G. and Chit Ko Ko (1986). List of Trees, Shrubs, Herbs and Principal
Climber, etc. Burma: Govt. Printing and Stationary.
Khare, C. P. (2007). Indian Medicinal Plants. Janak Puri. New Delhi, India.
KirtiKar, K. F. and B. D., Basu (1935). Indian Medicinal Plants. Vol. IV. 2nd ed. Latit
Mohan Basu, Alahabad, India.
Ong-ard Lawhavinit, Ngampong Kongkathip and Boonsong Kongkathip (2010).
Antimicrobial Activity of Curcuminoids from Curcuma longa L. on
Pathogenic Bacteria of Shrimp and Chicken. Kasetsart Journal. (Nat.
Sci.). Vol. 44. pp. 364-371, Kasetsart University, Bangkok, Thailand.
Singh, R., R. Chandra and P. M. Luthra (2002). Current Science. Vol. 83. No. 6, Delhi,
India.
The Wealth of India (1950). A Dictionary of Indian Raw Materials and Industrial Products.
Vol. II. Publication and Information Directorate, CSIR, New Delhi.
Wu Delin (2000). Flora of China. Vol. 24.
Effects of Holding Solutions on Post-harvest Quality and
Vase-Life of Ornamental Cut Flower, Chrysanthemum sp.
Soe Soe Aung
Abstract
The commercial ornamental cut flower, Chrysanthemum sp. (Gandama)
belongs to the family, Asteraceae. The post-harvest study was carried out on
Chrysanthemum sp. to investigate the effects of different holding solutions
using 0.1% sucrose, 0.3% sodium thiosulphate, 0.3% sodium nitrate, 0.05%
silver nitrate and tap water (control) to maintain their post-harvest quality
and to extend the longevity of their vase-life. Among different holding
solutions, the results showed that the longest longevity of vase life was
observed in the flower stems treated with 0.05% silver nitrate solution for 8
days room temperature and it gave the good post-harvest quality.
Key Word: cut flowers, vase life, sucrose, sodium thiosulphate, sodium
nitrate, silver nitrate.
Introduction
In Myanmar, the ornamental cut flowers and foliages were daily used
for the ceremonies of religious, funerals, birthday, wedding and other special
occasions, flowers decoration at home and business centres. However, the cut
flowers are very perishable horticultural crops which can easily decay or wilt
because of the occurrence of some decays, pests and diseases and fungal
infections, etc. after harvesting. In facts, keeping post-harvest quality and
longevity of their vase-life decreased immediately would be enhanced their
postharvest losses.
Storage of cut flowers is a new research and development area in
comparison to the storage of other horticultural products. Research on flower
storage has increased in conjunction with the expansion of the floriculture
industry. Storage methods are directed towards the increasing problems of
appropriate preservation of large volumes of flowers and their transport and
distribution to consumers. The greatest difficulty encountered in research
related to flower storage, derives from the great number of flower species and
Associate Professor, Department of Botany, University of Yangon
16
cultivars, and the short life of these particularly perishable products
(Goszczyѓiska and Rudnicki, n. d.). Ornamental plants or cut flower
production can be successful and economical only if the finished products are
excellent both in terms of quality and quantity (Manjula, 2005). Longevity of
vase-life is an important factor in consumer preference and considerable
research has been carried out on the causes of cut flowers senescence (Reid et
al., 1980 and Menguc and Usta, 1994).
However, in Myanmar, the ornamental postharvest researches and
technologies and its applications have been very limited for commercials. In
fact, this study needs to be implemented to produce the good quality of
ornamental cut flowers after harvesting for the commercial flower markets.
Therefore, this study was mainly aimed to maintain the post-harvest quality
and longevity of vase-life of ornamental cut flowers and foliages of
Chrysanthemum sp. (Gandama) in commercials. The specific objectives were
due to keep using the appropriate postharvest techniques vase solution
(holding solution) of ornamental cut flowers quality, longevity of its vast-life
and to extend the implements of postharvest technology to the flower growers,
producers, exporters and customers.
The post-harvest study of ornamental cut flower, Chrysanthemum sp.
(Gandama) was carried out at the laboratory of Department of Botany,
University of Yangon, from August 2011 to March 2012 (Fig. 1).
Fig. 1. Ornamental cut flower, Chrysanthemum sp. (Gandama)
The freshly harvested cut flower stems were obtained from the flower
markets of Yangon Region. During transporting, the cut flowers were covered
17
with fresh green banana leaves. Upon arrival to the laboratory, about 30 cm in
length of the flower stems with green foliages were maintained immediately
and the rest of the bottom parts were cut under the water using a sharp knife.
Then, sizing and grading of the cut stems were done immediately before
setting up the experiments under room temperature. Then, the prepared cut
stems were immediately immersed into the prepared holding solutions using
the transparent plastic containers as the flower vases. The plastic containers
(flower vases) were stored under room temperature until the flowers and
foliages become the poor marketable condition. During the study, storage room
temperature and room relative humidity (RH) were noted. The vase solutions
were removed and replaced with the new one at every 3 day intervals during
the holding period.
Experimental Layout
Total of 5 treatments of holding solutions such as T 1 (0.1% sucrose +
0.05% liquid detergent), T 2 (0.3% sodium thiosulphate + 0.05% liquid
detergent), T 3 (0.3% sodium nitrate + 0.05% liquid detergent), T 4 (0.05%
silver nitrate + 0.05% liquid detergent) and T 5 (tap water, control) were
assigned with 4 replications. Each replication consisted of 5 number of the cut
stems.
Data Collection and Statistical Analysis
The data were collected as follows: flower diameter, floret opening,
flowers and foliages quality, stem condition, marketable standard quality and
vase-life during the study. The treatments were arranged in CRD (Completely
Randomized Design). The treatment means were compared by Grand means
and SE (Standard Errors) which were calculated by Microsoft Excel.
Methods
Flower diameter
The measurement of flower diameters was determined using a calliper.
Floret opening
Florets opening of Chrysanthemum sp. (Gandama) were determined by
the following index of numerical rating. Numerical Rating: score 8 - 9 =
excellent, 25% of florets open; turgid; fresh appearance; free from damage;
score 6 - 7= 50% of florets open; turgid; fresh appearance; free from damage;
score 4 - 5 = 75% of florets open; turgid; fresh appearance; free from damage;
score 3 (limit of vase-life) = more than 75% of florets open, loss of turgidity;
18
leaves start to show signs of yellowing; minor defects on florets; score 1 - 2 =
mostly remaining buds fail to pen; inflorescence wilted; severe foliage
discoloration; more than 50% of florets deteriorated.
Flower quality
The flower quality was monitored as the following. Numerical Rating:
score 4 = excellent, fresh, colours bright; score 3 = good, saleable; score 2 =
good, but do not marketable; score 1 = poor, dull, faded colours; score 0 = very
poor, wilt.
Foliage quality
Foliage quality was determined using the index of numerical rating.
Numerical Rating: score 1 = foliage dark green; score 2 = foliage light green;
score 3 = yellowing of foliage including part of stem evident, dry; score 4 =
browning of discoloration evident; score 5 = browning of leaves, branches and
parts of the main stem.
Stem condition
Stem condition was monitored using the numerical scoring. Numerical
Rating: score 0 = none, no visible symptoms; score 1 = slight, limited
symptoms that are unlikely to impair saleability; score 2 (limit of vase-life) =
moderate, obvious symptoms that would impair saleability; score 3 = severe
widely distributed symptoms or intense localized symptoms; score 4 =
extreme, wide spread and intense symptoms.
Marketable standard quality
The marketable standard quality as determined as the follows.
Numerical Rating: score 3 = excellent, fresh, no wilting, strong colours; score
2 = good, marketable, very slight wilting, colours still bright; score 1 (limit of
vase-life) = good, not marketable but keep in a vase at home, noticeable
wilting, colours fading; score 0 = poor, unattractive, severely wilted,
discoloured or shrivelled.
Vase-life, storage room temperature and relative humidity
The vase-life was determined as the number of days to wilting of
flowers and foliages. During the storage of cut flowers with foliages, the room
temperature was ranged in 21.50 - 29.50°C and the relative humidity (RH) was
52 - 90%.
Results
19
Flower Diameter
In general, flower diameters of Chrysanthemum sp. were increased
overtime in all of the holding solutions during their vase-life (Table 1 and Fig.
2). At 4-day of vase-life, largest diameters of flowers among the holding
solutions were observed in silver nitrate holding solution (3.60 cm) followed
by sodium thiosulphate (3.45 cm). In contrast, smallest flower diameters were
observed in sodium nitrate holding solution (1.72 cm) followed by tap water
(1.85 cm). At 5-day, the largest flower diameters were found in sodium
thiosulphate holding solution (3.47 cm) followed by sucrose (2.75 cm) and tap
water (2.55 cm).
Table 1. Effect of different holding solutions on flower diameter of
Chrysanthemum sp. during the vase-life
Holding
Solutions
Flower Diameter (cm per stem)
Vase-Life (day)
0
1
2
3
4
5
T1
0.77
1.18
1.40
1.65
2.70
2.75
T2
0.65
0.73
1.13
1.57
3.45
3.47
T3
0.78
0.87
1.08
1.63
1.72
T4
0.80
0.87
0.93
1.55
3.60
2.15
T5
0.75
0.78
0.82
1.44
1.85
2.55
Means
± SE
0.75± 0.89± 1.07± 1.57± 2.66± 2.73±
0.03
0.08
0.10
0.04
0.39
0.28
6
7
8
2.90
3.27
3.95
-
-
-
SE = standard errors. T 1 = 0.1% sucrose + 0.05% liquid detergent, T 2 = 0.3% sodium
thiosulphate + 0.05% liquid detergent, T 3 = 0.3% sodium nitrate + 0.05% liquid detergent, T 4
= 0.05% silver nitrate + 0.05% liquid detergent, T 5 = tap water (control).
20
Fig. 2. Effect of different holding solutions on flower diameter of
Chrysanthemum sp. at 4- to 5-day of vase-life
Florets Opening
The bud stage of excellent, 25% of florets open; turgid; fresh
appearance; free from damage (score 8 - 9) was observed in sucrose and silver
nitrate holding solutions after 4 days of vase-life and in sodium thiosulphate,
sodium nitrate and tap water holding solutions after 3 days of vase-life (Table
2 and Fig. 3).
Table 2. Effect of different holding solutions on florets opening of
Holding
Solutions
Florets Opening*
Vase-Life (day)
0
1
2
3
4
5
T1
9.00
9.00
9.00
9.00
8.33
5.17
T2
9.00
9.00
9.00
9.00
7.33
4.17
T3
9.00
9.00
9.00
9.00
6.50
T4
9.00
9.00
9.00
9.00
8.00
7.33
T5
9.00
9.00
9.00
9.00
6.50
3.00
-
-
-
-
Means
± SE
7.33± 4.92±
0.38
0.92
6
7
8
6.50
5.67
4.67
-
-
-
21
* Numerical rating: 8-9 = excellent, 25% of florets open; turgid; fresh appearance; free from
damage; 6-7 = 50% of florets open; turgid; fresh appearance; free from damage; 4-5 = 75% of
florets open; turgid; fresh appearance; free from damage; 3 (limit of vase life) = less than 25%
of florets open, loss of turgidity; leaves start to show signs of yellowing; minor defects on
florets; 1-2 = mostly remaining buds fail to pen; inflorescence wilted; severe foliage
discoloration; more than 50% of florets deteriorated. SE = standard errors.
Fig. 3. Effect of different holding solutions on florets opening of
Among the holding solutions, 75% of florets open; turgid; fresh
appearance; free from damage (score 6 - 7) was observed in silver nitrate
holding solutions (score 4.67) after 8 days of vase life (Table 2). The limit of
vase life (score 3) was found in tap water at 5-day of vase-life.
Flower Quality
The excellent, fresh, colours bright of flowers (score 4) were observed
in silver nitrate holding solution after 8 days, in tap water after 5 days, in
sucrose and sodium thiosulphate holding solutions after 4 days and in sodium
nitrate holding solution after 3 days of vase-life (Table 3 and Fig. 4).
22
Table 3.
Holding
Solutions
Effect of different holding solutions on flower quality of
Flower Quality*
Vase-Life (day)
0
1
2
3
4
5
T1
4.00
4.00
4.00
4.00
4.00
1.83
T2
4.00
4.00
4.00
4.00
4.00
1.17
T3
4.00
4.00
4.00
4.00
2.33
T4
4.00
4.00
4.00
4.00
4.00
4.00 4.00
T5
4.00
4.00
4.00
4.00
4.00
3.50
-
-
-
-
-
Means
± SE
2.63±
0.67
6
-
7
8
4.00
3.67
-
-
* Numerical rating: 4 = excellent, fresh, colours bright; 3 = good, saleable; 2 = good but do not
marketable; 1 = poor, dull, faded colours; 0 = very poor, dead. SE = standard errors.
Fig. 4. Effect of different holding solutions on flower quality of
Chrysanthemum sp. at 5-day of vase-life
However, unmarketable flower quality (score 2) of Chrysanthemum
spp. was found in sucrose (score 1.83) at 5-day and in sodium nitrate holding
solutions at 4-day.
23
Foliage Quality
The foliage quality of Chrysanthemum sp. showed score 1 (dark green
of foliage) in silver nitrate holding solution after 7 days, in sucrose holding
solution after 4 days, in sodium thiosulphate and tap water after 2 days and in
sodium nitrate after 1 day of vase life (Table 4 and Fig. 5). Among the holding
solutions, silver nitrate holding solution showed score 2 (light green colour of
foliage) after 8 days of vase-life.
Table 4. Effect of different holding solutions on foliage quality of
Holding
Solutions
T1
T2
T3
T4
T5
Means
± SE
0
1.00
1.00
1.00
1.00
1.00
1
1.00
1.00
1.00
1.00
1.00
-
-
2
1.00
1.00
3.50
1.00
1.00
1.50±
0.50
Foliage Quality*
Vase-Life (day)
3
4
5
6
1.00 1.00 3.17
1.00 2.50 4.33
3.50 5.00
1.00 1.00 1.00 1.00
1.00 1.50 2.50
1.50± 2.20± 2.75±
0.50 0.75 0.70
7
8
1.00
2.17
-
-
* Numerical rating: 1= foliage dark green; 2 = foliage light green; 3 = yellowing of foliage
including part of stem evident; 4 = browning of discoloration evident; 5 = browning of leaves,
branches and parts of the main stem. SE = standard errors.
Fig. 5.
Effect of different holding solutions on foliage quality of
24
Stem Condition
No visible symptoms of stem condition was observed in the cut stems
of Chrysanthemum sp. treated with silver nitrate holding solutions after 7 days,
in sucrose after 4 days, in sodium thiosulphate, sodium nitrate and tap water
holding solutions after 3 days of vase-life (Table 5 and Fig. 6). Limit of vase
life (score 2) was found in sucrose holding solution at 5-day of vase life (score
2.33) and in sodium thiosulphate holding solution (score 2.40) at 3-day of
vase-life (Table 5).
Table 5. Effect of different holding solutions on stem condition of
Chrysanthemum spp. during the vase-life
Holding
Solutions
T1
T2
T3
T4
T5
Means
± SE
0
0.00
0.00
0.00
0.00
0.00
1
0.00
0.00
0.00
0.00
0.00
2
0.00
0.00
0.00
0.00
0.00
-
-
-
Stem Condition*
Vase-Life (day)
3
4
5
6
0.00 0.00 2.33
0.00 2.40 3.60
0.00 3.00
0.00 0.00 0.00 0.00
0.00 1.00 2.50
1.28± 2.11±
0.62 0.76
7
8
0.50
1.33
-
-
* Numerical rating: 0 = none, no visible symptoms; 1 = slight, limited symptoms that are
unlikely to impair saleability; 2 (limit of vase life) = moderate, obvious symptoms that
would impair saleability; 3 = severe widely distributed symptoms or intense localized
symptoms; 4 = extreme, wide spread and intense symptoms. SE = standard errors.
Fig. 6. Effect of different holding solutions on stem condition of
Chrysanthemum spp. at 4- to 5-day of vase-life
25
Marketable Standard Quality
The marketable standard quality attributes of Chrysanthemum sp.
showed score 3 (excellent, fresh, no wilting, strong colours) in silver nitrate
holding solution after 6 days, in sucrose and tap water holding solutions after 4
days, and in sodium thiosulphate and sodium nitrate after 3 days of vase life
(Table 6 and Fig. 7).
Table 6. Effect of different holding solutions on marketable standard quality
of Chrysanthemum sp. during the vase-life
Holding
Solutions
T1
T2
T3
T4
T5
Means
± SE
0
3.00
3.00
3.00
3.00
3.00
1
3.00
3.00
3.00
3.00
3.00
-
-
Marketable Standard Quality*
Vase-Life (day)
2
3
4
5
6
3.00 3.00 2.50 1.33
3.00 3.00 0.67 0.33
3.00 3.00 0.00
3.00 3.00 3.00 3.00 3.00
3.00 3.00 2.50 1.50
1.73± 1.54±
0.59 0.55
7
8
2.33
1.33
-
-
* Numerical rating: 3 = excellent, fresh, no wilting, strong colours; 2 = good, marketable, very
slight wilting, colours still bright; 1 = good, not marketable but keep in a vase at home,
noticeable wilting, colours fading; 0 = poor, unattractive, severely wilted, discoloured or
shrivelled. SE = standard errors.
Fig. 7. Effect of different holding solutions on marketable standard quality of
Chrysanthemum spp. at 4- to 5-day of vase-life
26
The post-harvest study of the commercial cut flower, Chrysanthemum
sp. (Gandama) was carried out for the maintenance of postharvest quality and
longevity of vase-life using different holding solutions such as 0.1% sucrose,
0.3% sodium thiosulphate, 0.3% sodium nitrate, 0.05% sliver nitrate and tap
water (control).
Among the treatments, the 0.05% sliver nitrate holding solution
showed the longest vase-life (8 days) under room temperature storage.
According to the present study, the postharvest quality and longevity of vaselife of Chrysanthemum sp. would be varied with different concentrations of its
holding solutions. Butt (2005) reported that influence of sucrose and silver
nitrate at different concentrations on the vase life of two roses (Rosa hybrida)
had significant effect on each cultivar.
In facts, longevity of the vase-life could extend about 8 days in the cut
stems treated with 0.05% silver nitrate holding solution among treatments
compared to tap water (control) which showed only 5 days of vase-life.
Moreover, using the 0.05% silver nitrate holding solution was benefited for the
longevity of vase-life of Chrysanthemum spp. because of the proper
maintenance on postharvest quality during the holding period in the flower
vases. Thus, the flower diameters, florets opening, flower and foliage quality,
stem condition and marketable standard quality showed high scores in cut
flower Chrysanthemum sp. treated with 0.05% silver nitrate holding solution
about 8 days of vase-life among the holding solutions (Appendix Table 1).
Butt (2005) also reported that in all the treatments containing sucrose
and silver nitrate (AgNO 3 ), the concentration of 150 ppm of AgNO 3 prolonged
the maximum number of days in both the rose cultivars, which were 4.3 and
3.2 days more in Whisky Mac and Trika as compared to control.
In the study, the vase-life of Chrysanthemum sp. was extended by silver
nitrate chemical. Regarding it, Paull and Goo (1985), silver nitrate was found
to decline the water uptake in Anthurium. Kofranek and Paul (1974) also
described that the silver ions could be acting as a biocide thus eliminating
microbial blockage of the xylem vessels. Paull and Goo (1985) and Sisler
(1982) who reported that silver ions also interfere with binding sites of wound
ethylene, thus preventing physiological blockage of cut stems.
In conclusion, using the chemicals as the vase solutions for cut flowers
showed benefits for flower growers, retailers and customers because of the
27
extension of their vase-life and flower quality in commercials. This study also
gave advantages for flower exporters by increased their incomes and foreign
currency for the country. Therefore, the further postharvest cut flowers studies
should be extended for the local and export markets using the proper
postharvest techniques and methods.
Acknowledgements
The author would like to express the sincere thanks to Department of Higher
Education (Lower Myanmar), Ministry of Education for the financial supports and permission
to do this research work; Professor Dr. Tin Tun, Rector, University of Yangon and Professor
Dr. Thet Thet May, Head of Botany Department, University of Yangon for allowing of this
paper establishment.
References
Butt, S. J., (2005). Extending the vase life of roses (Rosa hybrida) with different preservatives.
Int. J. Agri. Biol., Vol. 7, No. 1.
Goszczyѓiska, D. M. and R. M. Rudnicki (n. d.). Storage of cut flowers. Research Institute of
Pomology and Floriculture. Skierniewice, Poland.
Kende, H. B. and B. Baumgartner, (1974). Regulation of aging in flower of Ipomoea tricolor
by ethylene. Planta, 116:279-289.
Manjula, G., (2005). Performance of rose cultivars under naturally ventilated polyhouose.
M.Sc. (Agriculture) in Horticulture, Thesis. Department of Horticulture.
College of Agriculture, Dharwad. University of Agricultural Sciences,
Dharwad – 580 005. November.
Menguc, A. and E. Usta, (1994). Research on the effects of silver thiosulphate plus sucrose
pretreatment on the cold storage period and post storage vase life of cut
flowers of carnation cv. Astor harvested at different maturities. Acta
Horticult., 368: 802-807.
Paull, R. E. and T. T. C. Goo, (1985). Ethylene and water stress in the senescence of cut
Anthurium flowers. Journal of American Society of Horticultural Science,
10:84-88.
Reid, M. S., J. L. Paul, M. B. Farhoomand, A. M. Kofranek and G. L. Staby, (1980). Pulse
treatments with the silver thiosulphate complex extend the vase life of cut
carnations. J. Am. Soc. Horticult. Sci., 105: 25-27.
Sisler, E. C. (1982). Ethylene-binding properties of a triton X-100 extract of mung bean
sprouts. Journal of Plant Growth Regulators, I:211-218.
28
Appendix Table 1. Summary on effect of holding solutions on postharvest quality and longevity of vase-life of cut
flower Chrysanthemum sp.
Holding
Solutions
Vase-Life (day)
0
1
2
3
4
5
0.1% Sucrose
FD
0.77 1.18 1.40 1.65 2.70 2.75
FloO
9.00 9.00 9.00 9.00 8.33 5.17
FQ
4.00 4.00 4.00 4.00 4.00 1.83
FolQ
1.00 1.00 1.00 1.00 1.00 3.17
StC
0.00 0.00 0.00 0.00 0.00 2.33
MSQ
3.00 3.00 3.00 3.00 2.50 1.33
0.3% Sodium thiosulphate
FD
0.65 0.73 1.13 1.57 3.45 3.47
FloO
9.00 9.00 9.00 9.00 7.33 4.17
FQ
4.00 4.00 4.00 4.00 4.00 1.17
FolQ
1.00 1.00 1.00 1.00 2.50 4.33
StC
0.00 0.00 0.00 0.00 2.40 3.60
MSQ
3.00 3.00 3.00 3.00 0.67 0.33
0.3% Sodium nitrate
FD
0.78 0.87 1.08 1.63 1.72
FloO
9.00 9.00 9.00 9.00 6.50
FQ
4.00 4.00 4.00 4.00 2.33
FolQ
1.00 1.00 3.50 3.50 5.00
StC
0.00 0.00 0.00 0.00 3.00
MSQ
3.00 3.00 3.00 3.00 0.00
6
7
8
29
Appendix Table 1. (continue):
Holding
Solutions
Vase-Life (day)
0
1
2
3
4
5
6
7
8
0.05% Silver nitrate
2.15
0.77 1.18 1.40 1.65 2.70 2.75
7.33
9.00 9.00 9.00 9.00 8.33 5.17
4.00
4.00 4.00 4.00 4.00 4.00 1.83
1.00
1.00 1.00 1.00 1.00 1.00 3.17
0.00
0.00 0.00 0.00 0.00 0.00 2.33
3.00
3.00 3.00 3.00 3.00 2.50 1.33
Tap water (control)
2.55
0.65 0.73 1.13 1.57 3.45 3.47
3.00
9.00 9.00 9.00 9.00 7.33 4.17
3.50
4.00 4.00 4.00 4.00 4.00 1.17
2.50
1.00 1.00 1.00 1.00 2.50 4.33
2.50
0.00 0.00 0.00 0.00 2.40 3.60
1.50
3.00 3.00 3.00 3.00 0.67 0.33
FD = flower diameter, FloO = florets opening, FQ = flower quality, FolQ =
foliage quality, StC = stem condition, MSQ = marketable standard quality.
Quantitative Analysis of Forest Structure in Pahtaw Hill,
Kyun-su Township, Taninthayi Region
Wah Wah Khaing1, Lae Lae Khaing2, Htay Htay Win3, Mi Mi Aye4 and Sanda Hlaing5
Abstract
Pahtaw hill is located in Western part of Myeik, Kyun-su Township,
Taninthayi Region. The field data was conducted in January 2012. Diversity
indices are better measure of the species diversity of a forest and more
informative than species counts alone. In order to assess plant species
diversity and forest structure, seven quadrats (25mx25m each) for canopy
layer and 20 quadrats (5mx5m each) for ground layer were established. To
clarify the forest structure, all woody plants with ≥10cm GBH (girth at breast
height) were recorded, counted and measured GBH and height in each
quadrat. Floristic diversity index of canopy layer was (5.24, 0.96), of ground
layer was (4.34, 0.92) (i.e. Shannon-Wiener Index, Simpson Index).
Ecological successful species with the highest important value were Gluta
renghas L., (Lay tha yet) (22.23%), Semecarpus pandurata Kurz., (Chee)
(21.43%) and Syzygium cymosum DC. (Thabye-htat ta ya) (17.64%). A total
relative basal area was 4.39 m2 ha-1. This research will show to recognize the
information and phytosociological data of the actual natural vegetation and
to develop practical technology for environmental management of forest
community of Pahtaw hill, Kyun-su Township, Taninthayi Region.
Keywords : species diversity, phytosociological data, natural vegetation.
Introduction
Vegetation ecology includes the investigation of species composition
and sociological interaction of species in communities (Mueller-Dombois and
Ellenberg, 1974). The structural property of a community is the quantitative
relationship in between the species growing around. The quantitative study of
vegetation is called phytosociology and its principal aim is to describe the
vegetation, explain or predict its pattern and classify it in a meaningful way
(Ilorka and Khatri, 2003). It indicates species diversity which determines the
distribution of individuals among the species in a particular habitat. A sound
understanding of species diversity is necessary for appropriate conservation
and restoration of the biological diversity.
1. Lecturer, Department of Botany, University of Yangon
2. Assistant Lecturer, Department of Botany, Myeik University
3,4. Lecturer, Department of Botany, Myeik University
5. Professor and Head, Department of Botany, Myeik University
32
Species diversity is a measure of both the species richness and evenness
of a community. Species richness refers to the number of species in a
community whereas species evenness refers to the relative abundance of
individuals within a species. A higher number equals a more diverse
community. Diversity indices are designed to combine both species richness
and the evenness or equitability of the distribution of individuals among those
species.
The most widely used indices for measurement of diversity are the
‘information theory indices’. Among the various such indices, the Shannon–
Wiener index is most commonly used. This index has been used for the present
study, since sampling was done randomly and also because it is the most
widely used measure of diversity and thus the findings of the present work
could be easily compared with other studies done in the surrounding areas.
Species richness is essentially a measure of the number of species in a defined
sampling unit. This is the basic component of diversity of any community and
is relatively simple to measure. Species richness measures also provide an
easily comprehensible expression of diversity.
Measure of diversity is regarded as indicators of the well-being of
ecological systems (Magurran, 1988). Species diversity can be measured and
calculated by recording the number of species, by describing their relative
abundances or by using a measure which combines the two components richness and abundance. Current species diversity reflects historical as well as
environmental factors since environmental change and human activities leads
to changes in species composition and competition (Babour et al., 1998).
Species of high Importance Value Index (IVI) value in a given area can
also be considered as representative species of a forest type studied and they
should be taken into account as ecologically important species in reforestation
operations. The ecological significance of a species can be compared by IVI in
a given forest types (Lamprecht, 1989).
The results of quantitative inventory have enormous significance for
the conservation and management of forest of study area. Quantitative
inventories help in identification of economically and ecologically useful
species as well as species of special concern, i.e. rare, uncommon and
vulnerable species.
The dominant species can be quantified by calculating a statistic known
as “importance value” (Smith and Smith, 2001). Once importance values are
33
determined, a specific community can be described in terms of its most
important species. Importance values can be calculated after the size and
number of individual trees of the various species is measured. The trees with
the highest importance values will be those that exist in the greatest number or
are of the greatest size, these are the trees that may have the greatest effect on
the community.
Every forest types has a different vertical structure, almost by
definition, due to the particular climate, soil, tree species and plant life forms
that produce a definable type (Malcolm and Hunter, 1999).Forest structure is
the physical and temporal distribution of trees in a stand and include within the
description; the distribution of the species, vertical and horizontal spatial
patterns, size of trees or tree parts, tree age, or combination (Oliver and Larson,
1990).
The basal area is used to analyze the cross-sectional area, which refers
to the area of ground actually penetrated by stem. The basal coverage or area
cover by a species is used to express its dominance. Basal area provides a
better measure of the relative importance of the species than simple stem count
(Bekele, 1994). Therefore, species with the largest contribution in basal area
can be considered as the most important woody species in the forest.
One of the measures of the well-being and stability of any ecosystem is
the estimation of its species diversity and richness. These values also indicate
the nature of the forests in a region. Along with the composition of the forest,
information on the diversity of the communities and of the landscape as a
whole provides better insight into the state of the forests of an area.
The objective of the present study is to clarify floristic diversity and to
analyze the community structure for species richness, evenness, stand
population structure, density, frequency, abundance, and species girth class
relationship.
Ecological Description of Study Site
Study area
Pahtaw hill is situated in Western part of Myeik, Kyun-su Township,
Taninthayi Region of the Southern part of Myanmar. This area is located
between Latitude: 12º 26' N latitude and Longitude: 94º 35' E longitude. The
total area is 8.57 sq km. Location map of study areas are shown in Fig. (1).
34
Fig.1. Location map of study area
Climate
Taninthayi Region has a tropical monsoon climate. The land which is
located nearest to the Equator in Myanmar is warm year round with only slight
changes in temperatures. The average highest temperature of Myeik is 35.83°
C and the lowest average temperature is 18.33° C. Annual average rainfall in
Myeik is 411.48cm.
Soil
Soil pH and texture of study sites and the nutrient contents are shown
in Table 1.
Table.1 Physical and chemical properties of soil in Pahtaw hill
K
(meq/100g)
K2O
(mg/100)
10.40
2
T10
11.35 55.50 30.10 96.95 Silty clay loam 15.51 3.80 1.43
0.31
2.48 0.064 4.04
0.24
11.43
Total(%)
P (ppm)
0.22
Na (ppm)
2.81 0.012 3.87
Soil pH
0.26
Loam
Moisture(%)
1.71 4.55 1.63
Soil Class
30.65 44.60 23.50 98.75
Clay(%)
T1
Silt(%)
1
Sample
Sr.No
No.
Sand(%)
Humus (%)
Available Nutrients
Total N2 (%)
Texture
35
Organic carbon
(%)
3
T20
26.85 50.50 20.30 97.65
Silt loam
1.94 4.72 1.75
0.26
3.01 0.097 2.24
0.20
9.79
4
TT1
6.00 41.70 50.20 97.90
Silty clay
4.63 4.22 3.81
0.29
6.58 0.075 2.73
0.18
8.84
5
TT10 22.40 60.20 16.30 98.90
Silt loam
13.52 3.33 4.17
0.40
7.19 0.114 3.48
0.20
9.07
6
TT20
Silt loam
14.33 3.29 3.07
0.32
5.29 0.088 3.52
0.13
6.28
6.00 71.70 20.10 97.80
Source: Land Use Section, Myanmar Agriculture Service, Yangon
The structure and nutrient content of the soil is important, particular for
plants. The soil is affected by the vegetation that grows on it, and, in turn, can
affect the nature of vegetation. Soil pH in Pahtaw hill was nearly slightly acid.
The soil texture was generally loam, silt loam to silty clay loam. The nutrient
content of soils was also nearly the same.
Methodology
Data Collection
To clarify the tree species diversity and to calculate the important value
index (IVI), seven quadrats (25m x 25m each) were set up and observed. In
order to analyze flora of ground cover, 5x100m belt transect were laid down
and observed. In each sub-plot along the belt transect every plant species were
listed and counted.
5m
5m
Fig. 2. Belt transect method
100m
Plant specimens were collected, pressed, dried and identified by
matching Herbarium specimens of Botany Department, Y.U and by checking
with Backer et al., 1963 and Kress et al., 2003. The spatial location (latitude,
longitude and altitude) of each quadrat was collected using a Global
Positioning System (GPS). Care has been taken to cover different elevation,
slope, aspects, rainfall and temperature gradients to study overall spectrum of
tree species diversity. To know climatic variation of Pahtaw hill, temperature,
36
rainfall and relative humidity of Myeik District were taken from Department of
Meteorology and Hydrology, Myeik station.
Fig. 3 Monthly mean rainfall, temperature and
relative humidity of Myeik (2010)
Fig. 4 Monthly mean rainfall, temperature and
relative humidity of Myeik (2011)
Data Analysis
The field data collected were analysed for diversity (Shannon-Wiener,
1963 and Simpson, 1949), Jackknife estimate of species richness (Heltshe &
Foerster, 1983), evenness (Shannon-Wiener function, 1963), coefficient of
similarity (Sorenson, 1948) and stand density (trees) per hectare, basal area per
hectare. To study the quantitative analysis, the importance value index (IVI)
for the tree species was determined as the sum of the relative values of
frequency, density and dominance (Curtis, 1959).
Measurement of plant species diversity
Shannon-Wiener Index (1963)
s
H = −∑ ( pi )( log 2 pi )
i =1
H = index of species diversity
S = number of species
p i = proportion of total sample belonging to the ith species
Simpson Index (1949)
s
D = 1 − ∑ ( pi ) 2
i =1
D = Simpson's index of species diversity
p i = proportion of individual of species i in the community
37
Evenness (Shannon-Wiener function, 1963)
E=
H
H max
H max = Log 2 S
E = evenness (range 0-1)
H = index of species diversity
H ma x = species diversity under conditions of maximal equitability
Jackknife estimate of species richness (Heltshe & Foerster, 1983)
Ŝ=S
Ŝ = Jackknife estimate of species richness
S = observed total number of species in “n” sample plots
n = Total number of plots sample
k = number of unique species
Results
Plant species diversity, Species richness, Evenness
In this study, diversity index of canopy layer was (5.24, 0.96) and
diversity index of ground layer was (4.34, 0.92) by the method of ShannonWiener’s Index (H) and Simpson’s Index (D) respectively. According to the
result of Jackknife estimate, species richness at canopy layer was 69.09 and
ground layer was 60.49 respectively. The results of diversity indices and
evenness are shown in Table (2).
Table 2. Consolidated detail of species inventory in Pahtaw hill
Description
Canopy Layer
Ground layer
No of Sample Plots
7 (25mx25m)
20 (5mx5m)
No of Tree Species
69
60
Individual Tree Species
528
1169
Jackknife Estimate of Species Richness
69.09
60.49
Simpson Evenness Index (E)
0.86
0.73
Shannon-Winner Diversity Index (H)
5.24
4.34
Simpson Diversity Index (D)
0.96
0.92
38
Importance Value Index (IVI)
The highest IVI of major tree species possess high value of relative
dominance and relative frequency. These species could be considered as
ecological indicator species of Pahtaw hill.
Among the tree species Gluta renghas L., (Lay tha yet),
Semecarpus pandurata Kurz., (Chee), Schima wallichii (DC.) Korth.
(Thityah), Lophopetalum fimbriatum Wight. (Taung-yemane) and Diospyros
crumentata Thwaites. (Taung-bok) have same highest relative frequency of
3.55%. Table (3) and Fig. (5) So they can occur everywhere and contain in all
sample plots.
Among the tree species, Lophopetalum fimbriatum Wight. (Taungyemane) and Vatica maingayi Dyer. (Kanyin-kyaung-che) have same highest
relative density of 6.64%, followed by Gluta renghas L. (Lay tha yet)
6.26%.Table (3) and Fig. (6). So they have high species richness in study area.
Highest relative dominance tree species were Semecarpus pandurata
Kurz., (Chee) 13.32%, Gluta renghas L. (Lay tha yet) 12.41%,
Syzygium cymosum DC. (Thabye-htat ta ya) 11.17% respectively. Table (3)
and Fig. (7). So they possessed larger basal diameter and occupy the area more
than other tree species.
As a combination result of relative density, relative frequency and
relative dominance of all recorded tree species, the highest IVI of major
dominant tree species were Gluta renghas L., (Lay tha yet) (22.23%),
Semecarpus pandurata Kurz., (Chee) (21.43%) and Syzygium cymosum DC.
(Thabye-htat ta ya) (17.64%). Table (3) and Fig. (8). So these species are
ecologically successful in the study area than the others.
39
Table 3. Importance Value Index of Tree Species in Pahtaw hill
Sr. no.
1
Botanical Name
Gluta renghas
Family
Anacardiaceae
Vanicular Name
Lay tha yet
RF
(%)
RD
(%)
RDm
(%)
IVI
(%)
3.55
6.26
12.41
22.23
2
Semecarpus pandurata
Anacadiaceae
Chee
3.55
4.55
13.32
21.43
3
Syzygium cymosum
Myrtaceae
Thabye-htat ta ya
3.05
3.42
11.17
17.64
4
Schima wallichii
Theaceae
Thityah
3.55
4.74
8.88
17.17
5
Vatica maingay i
Dipterocarpaceae Kanyin-kyaung-che
3.05
6.64
3.53
13.21
6
Litsea laurifolia
Lauraceae
Ondon
3.05
5.88
4.03
12.96
7
Lophopetalum fimbriatum Celastraceae
Taung-yemane
3.55
6.64
1.33
11.53
8
Eugenia oblata
Myrtaceae
Thabye-ni
3.05
1.90
5.50
10.44
9
Myristica malabarica
Myristicaceae
Kywe thwe
2.03
3.80
3.21
9.04
Ebenaceae
Taung-bok
3.55
4.74
0.71
9.00
Others
68.02
51.42
35.90
155.34
Total
100
100
100
300
Relative Frequency (%)
10 Diospyros crumentata
3.60
3.50
3.40
3.30
3.20
3.10
3.00
2.90
2.80
2.70
Relative Density (%)
Fig. 5.Relative Frequency of Tree Species in Pahtaw hill
7.00
6.00
5.00
4.00
3.00
2.00
1.00
0.00
Fig. 6. Relative Density of Tree Species in Pahtaw hill
Relative Dominance (%)
40
12.00
10.00
8.00
6.00
4.00
2.00
0.00
Impotance Value Index (%)
Fig. 7.Relative Dominance of Tree Species in Pahtaw hill
25.00
20.00
15.00
10.00
5.00
0.00
Fig. 8. Importance Value Index of Tree Species in Pahtaw hill
Forest structure
Population Structure and size class distribution
The distribution of the basal area across GBH interval classes reveals
the dominance of small stemmed individuals in the study area. Out of total
number of stems inventoried, 54 species and 60.11% of total species were
accumulated in the ≤30cm GBH class, 36 species and 22.24% of total species
in the 31-60cm, 22 species and 6.62% of total species in the
61-90cm, 15
species and 4.78% of total species in the 91-120cm, 8 species and 2.21% of
total species in the 121-150cm, 7 species and 0.55%of total species in the151180cm,4 species and 0.37% of total species in the 181-210cm, and only one
species and 0.18% of total species in the 211-240cm. Table(4), Fig. (9)
41
Table 4. Population Density of tree species across GBH class interval
GBH Class
(cm)
No of Species
Total number
of individual
% of Total
species
≤30
54
327
60.11
31-60
36
121
22.24
61-90
22
36
6.62
91-120
15
26
4.78
121-150
8
12
2.21
151-180
7
3
0.55
181-210
4
2
0.37
211-240
1
1
0.18
Total
68
528
100
60
50
No. of Species
40
30
20
10
0
Population Density of Tree Species Across GBH Classes
Fig. 9. Population Density of tree species across GBH class interval
Horizontal structure of tree species across GBH class interval
Horizontal structure of tree species across GBH class interval shows
that (0.39 m2/ha) belong to GBH class interval ≤30cm, (0.81 m2/ha) belong to
GBH class interval 31-60cm,(0.64 m2/ha) belong to GBH class interval 6190cm, (1.03 m2/ha) belong to GBH class interval 91-120cm, (0.76 m2/ha)
belong to GBH class interval 121-150cm, (0.29 m2/ha) belong to GBH class
interval 151-180cm,(0.27 m2/ha) belong to GBH class interval 181-210cmand
(0.20 m2/ha) belong GBH class interval 211-240cm.Total relative basal areas
per hectare were found 4.39 m2 ha-1.Table (5), Fig. (10)
In the stand portion of Pahtaw hill, tree species distribution across the
GBH class interval shows that 143.06 tree/ha were found in GBH class interval
≤30cm, 52.94 tree/ha in 31-60cm, 15.75 tree/ha in the 61-90, 11.38 tree/ha in
the 91-120cm, 5.25 tree/ha in the 121-150cm, 1.31 tree/ha in the 151-180cm,
0.88 tree/ha in the 181-210cm, 0.44 tree/ha in the 211-240cm. Table (5), Fig.
(11)
42
Table 5.Horizontal structure of tree species across GBH class interval
GBH Class
(cm)
Total BA
BA/ha
(m2ha-1)
Total
Individual
Tree/ha
≤30
0.89
0.39
327
143.06
31-60
1.84
0.81
121
52.94
61-90
1.47
0.64
36
15.75
91-120
2.36
1.03
26
11.38
121-150
1.73
0.76
12
5.25
151-180
0.66
0.29
3
1.31
181-210
0.62
0.27
2
0.88
211-240
0.46
0.20
1
0.44
Total
10.03
4.39
528
231
Basal Area/ha (m2 ha-1)
1.20
1.00
0.80
0.60
0.40
0.20
0.00
Horizontal Structure of Tree Species Across GBH Classes
Fig.10. Horizontal structure of tree species across GBH class interval
160
140
120
Tree / ha
100
80
60
40
20
0
Stand Structure of Tree Species Across GBH Classes
Fig.11. Stand Structure of Tree Species across GBH Classes Interval
Population density of tree species across height class intervals
Population density of tree species in height class intervals shows that
among the 528 total number of individual, 219 individuals (41.48%) were
belong to 6-10m category, followed by 128 individuals (24.24%) in ≤5m
category, 103 individuals (19.51%) in 11-15m, 65 individuals (12.31%) in 1620m, 10 individual (1.89%) in 21-25m and 3 individuals (0.57%) in above
25m category. Table (6), Fig. (12)
43
Table 6. Population density of tree species across height class intervals
Height Class
(m)
≤5
% of Total
species
24.24
42
Total number of
individual
128
6-10
46
219
41.48
11-15
16
103
19.51
16-20
31
65
12.31
21-25
6
10
1.89
No of Species
25<
3
3
0.57
Total
68
528
100
Total Number of Individual
Species
250
200
150
100
50
0
Population Density of Tree Species Across Height Classes
Fig. 12. Population density of tree species across height class intervals
Floristic diversity index of canopy layer was (5.24, 0.96) and diversity
index of ground layer was (4.34, 0.92) by the method of Shannon-Wiener’s
Index (H) and Simpson’s Index (D) respectively.
As a combination result of relative density, relative frequency and
relative dominance of all recorded tree species, the highest IVI of major
dominant tree species were Gluta renghas L., (Lay tha yet) (22.23%),
Semecarpus pandurata Kurz., (Chee) (21.43%). Syzygium cymosum DC.
(Thabye-htat ta ya) (17.64%). They possess all of the high value of relative
dominance, relative density and relative frequency so they occupied the area
more than other tree species.
The population structure and size class distribution have generally been
used by many researchers for understanding regeneration and magnitude of
disturbances and future stability of tree species population in forest
communities (Upreti, 1982). From the present study, the overall pattern of
distribution of the trees in different GBH classes reveals dominance of small
trees were more in young diameter classes.
44
Horizontal structure of tree species across GBH class interval shows
that (0.39 m2/ha) belong to GBH class interval ≤30cm, (0.81 m2/ha) belong to
GBH class interval 31-60cm, (0.64 m2/ha) belong to GBH class interval 6190cm, (1.03 m2/ha) belong to GBH class interval 91-120cm, (0.76 m2/ha)
belong to GBH class interval 121-150cm, (0.29 m2/ha) belong to GBH class
interval 151-180cm, (0.27 m2/ha) belong to GBH class interval 181-210cm and
(0.20 m2/ha) belong GBH class interval 211-240cm.
Population density of tree species in height class intervals shows that
among the 528 total number of individual, 219 individuals (41.48%) were
belong to 6-10m category, followed by 128 individuals (24.24%) in ≤5m
category, 103 individuals (19.51%) in 11-15m, 65 individuals (12.31%) in 1620m, 10 individual (1.89%) in 21-25m and 3 individuals (0.57%) in above
25m category.
Rarely found species, Payena paralleloneura Kurz. (Kan-zaw) possess
high medicinal value of Taninthayi Region. Economically important tree
species of Shorea obtusa Wall (Thitya) and Dipterocarpus costatus Gaertn.
(Kanyin-ni) that recorded in study area were assessing in IUCN Red List of
Globally Threatened and Endangered Species (Ashton, 2009). So these species
should be paid special attention for conservation.
According to this study, various tree species were found and they are
not only considered for their economic status but also stand as essential
ecological value for rural people of the study area. All recorded data about
floristic diversity assessment and quantitative inventories in the forest of
Pahtaw hill will be continue to provide valuable information for management
and biodiversity conservation.
Acknowledgements
I would like to express my thanks to Acting Rector Dr Thet Htun Aung and Pro Rector
Dr. Than Htut Oo, Myeik University for their kind help to carry out this research. Especially
thanks to Botany students, Department of Botany, Myeik University for their participation and
support throughout field works.
References
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N.V.P. Noordhoff Groningen. Nether Lands.
Barbour, M.G., Burk, J.H., Pitts, W.d., Gilliam, F.S & Schwartz, M.W., (1998). Terrestrial
Plant Ecology, 3rd Ed. Addison Wesley Longman, Menlo Park, California,
649.
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Bekele, T. (1994). Studies on remenantafromontane forests on the Central Plateau of Shewa,
Ethiopia. Ph D dissertation at Uppsala University.
Curtis, J.T. (1959). The vegetation of Wisconsin, An Ordination of Plant Communities.
University Wisconsin Press, Madison, Wisconsin.
Heltshe, J. F. & Foerster, R. E. (1983). Estimating species richness using Jackknife procedure.
Biometric 39, 1; 1-11.
Ilorkar, V.M. and P.K. Khatri, (2003). Phytosociological Study of Navegaon National Park
(Maharashtra).Indian Forester, 129: 377 387.
Kress J. W., Robert A. De Filipps, Ellen Far and Yin Yin Kyi (2003). A Checklist of the Trees,
Shrubs, Herbs, and Climbers of Myanmar.
Lamprecht, H. (1989). Silviculture in the tropics: Tropical forest ecosystems and their longterm utilization. (GTZ) GmbH, Eschborn, Germany.
Magurran, A. E. (1988). Ecological diversity and its measurement. New Jersey, USA:
Princeton University Press.
Malcolm, L. & Hunter, M. L. (1999). Maintaining biodiversity in forest ecosystems.
Cambridge, UK: The Edinburgh Building.
Mueller-Dombois, D. and H. Ellenberg, (1974). Aims and Methods of Vegetation Ecology.
John Wiley and Sons, New York, USA.237 pp.
Oliver, C. D. & Larson, B. C. (1990). Forest stands dynamics. New York: McGraw-Hill Inc.
Shannon, C. E. & Wiener, W. (1963). The mathematical theory of communication. Urbana,
USA: University of Illinois Press.
Simpson, E. H. (1949). Measurement of diversity. Nature, 163, 688.
Smith, R.L and T. M. Smith, (2001). Ecology and Field Biology, 6th edition. Addison Wesley
Longman, San Francisco, 771 pages.
Sorenson, T. A. (1948). Method of establishing groups of equal amplitude in a plant society
based on similarity of species content. K. Dan Vidensk. Selsk.,5, 1–34.
Upreti, N. (1982): A Study on Phytosociology & State of Regeneration of Oak-Forest at
Nainital.– Ph.D. Thesis, Kumaum University, Nainital.
Ashton, P. (1998). Shorea siamensis, Shorea obtusa, Dipterocarpus tuberculatus. In: IUCN
2009, 2009 IUCN Red List of Threatened Species. Version 2009.1.
Retrieved October 21, 2009, from Web site: <www.iucnredlist.org>.
46
Appendix
Importance Value Index of Tree Species in Pahtaw hill
Sr. no.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
Gluta renghas L.
Anacardiaceae
Lay tha yet
RF
(%)
3.55
RD
(%)
6.26
RDm
(%)
12.41
IVI
(%)
22.23
Semecarpus pandurata Kurz.
Syzygium cymosum DC.
Schima wallichii (DC.) Korth.
Diospyros crumentata Thwaites.
Anacadiaceae
Myrtaceae
Theaceae
Dipterocarpaceae
Lauraceae
Celastraceae
Myrtaceae
Myristicaceae
Ebenaceae
Chee
Thabye-htat ta ya
Thityah
Kanyin-kyaung-che
Ondon
Taung-yemane
Thabye-ni
Kywe thwe
Taung-bok
3.55
3.05
3.55
3.05
3.05
3.55
3.05
2.03
3.55
4.55
3.42
4.74
6.64
5.88
6.64
1.90
3.80
4.74
13.32
11.17
8.88
3.53
4.03
1.33
5.50
3.21
0.71
21.43
17.64
17.17
13.21
12.96
11.53
10.44
9.04
9.00
Garcinia merguensis Wight
Hypericaceae
Khet Mya
3.05
2.66
3.25
8.95
Gmelina arborea Roxb.
Verbenaceae
Yemane
1.02
5.88
1.31
8.21
Schleichera trijuga Willd.
Sapindaceae
Gyo nyin
2.54
3.61
1.86
Botanical Name
Vatica maingayi Dyer.
Litsea laurifolia (Jacq.) Kurz
Lophopetalum fimbriatum Wight
Eugenia oblata Roxb.
Myristica malabarica Lam.
Family
Local Name
8.00
Unknown 3
Dipterocarpaceae Mi chaung chee
2.03
1.71
4.09
7.83
Litsea salicifolia (Nees) Hook. f.
Lauraceae
Ta gu shwe wah
2.03
2.28
2.84
7.14
Shorea gratissima Dyer.
Dipterocarpaceae
U ban
2.03
0.95
3.12
6.10
Elaeocarpus robustus Roxb.
Elaeocarpaceae
Tawmagyi
2.03
1.14
2.64
5.81
Unknown 4
Min gyo
2.03
2.66
0.47
5.15
Unknown 1
Hin cho
2.54
1.71
0.42
4.67
Sageraea elliptica (A. DC.) Hook. f. &Annonaceae
Thabut
2.03
1.52
0.61
4.16
Vitex pubescens Vahl.
Verbenaceae
Kyet yo
1.52
1.52
1.03
4.08
Anisoptera scaphula (Roxb.) Pierre
Dipterocarpaceae
Kaung mhu
1.52
0.57
1.83
3.92
Myrtaceae
Syzygium malaccense (L.) Merr. & L.M
Thabye-phyu
2.03
1.33
0.52
3.88
Litsea elongata (Nees) Benth.
Ta gu
1.52
1.71
0.61
3.84
Lauraceae
Syzygium attenuatum (Miq.) Merr. & L
Myrtaceae
Thabye
1.52
1.90
0.27
3.69
Unknown 5
Myauk gamone
2.03
0.95
0.59
3.57
Nephelium laurinum Blume.
Sapindaceae
Taw kyet myauk
2.03
1.33
0.17
3.53
Getonia floribunda Roxb.
Combretaceae
Gyut new
1.52
1.71
0.22
3.45
Cinnamomum inunctum Meissn.
Lauraceae
Kayaway
1.02
0.57
1.40
2.98
Palaquium obovatum (Griff.) Engl.
Sapotaceae
Artocarpus lakoocha Roxb.
Moraceae
Pinle-byin
1.52
0.95
0.39
2.86
Myauk pa lote
1.52
0.76
0.46
2.74
Cinnamomum pachyphyllum Kosterm.Lauraceae
Mhan thin
1.52
0.95
0.19
2.66
Unknown 13
2.66
Tha mook
1.02
0.38
1.26
Rhodamnia trinervia Blume.
Myrtaceae
Taung-kamyaing
1.52
0.95
0.13
2.60
Samadera indica Gaertn.
Simaroubaceae
Ka thae
1.52
0.95
0.11
2.58
Shorea obtusa Wall.
Dipterocarpaceae
Thit-ya
0.51
0.38
1.47
2.36
Se phore
1.52
0.57
0.22
2.31
Unknown 11
Hopea oblongifolia Dyer.
Dipterocarpaceae
Eugenia spp.
Myrtaceae
Tanyin-byan
1.52
0.57
0.22
2.31
Thabye-wah
1.02
0.76
0.38
2.15
Sapium insigne (Muell. Arg.) Trimen Euphorbiaceae
Taung-kala
1.02
0.38
0.49
1.88
Phoebe tavoyana (Meissner) Hook. f. Lauraceae
Kyè sae
1.02
0.57
0.15
1.73
Ficus altissima Blume
Moraceae
Nyaung-peinne
1.02
0.38
0.34
Dipterocarpus kerrii King.
Dipterocarpaceae
Sibin
1.02
0.57
0.14
1.73
Eugenia operculata Roxb.
Myrtaceae
Thabye-ywet chaw
0.51
0.19
0.91
1.61
Garcinia microstigma Kurz.
Hypericaceae
Taung thale
1.02
0.38
0.06
1.45
Calophyllum amoenum Wall.
Hypericaceae
Tharapi
1.02
0.38
0.05
1.44
Myay o kyal
1.02
0.38
0.05
1.44
Unknown 10
Sa nwin new
1.02
0.38
0.03
1.42
Unknown 7
Nalakhat
0.51
0.57
0.12
1.20
Ywe-gyi
0.51
0.57
0.10
1.18
Taung ta yaw
0.51
0.19
0.40
1.10
Unknown 6
Adenanthera pavonina L.
Mimosaceae
Unknown 12
1.73
Cinnamomum tavoyanum Meissner.
Lauraceae
Tauktu-ywe
0.51
0.19
0.33
1.02
Barringtonia Spp.
Lecythidaceae
Kyè
0.51
0.38
0.11
1.00
Syzygium kurzii (Duthie) N.P. Balakr. Myrtaceae
Thabye-nyo
0.51
0.38
0.10
0.99
Derris spp.
Fabaceae
Kyar ma naing
0.51
0.38
0.04
0.93
Syzygium Spp. 2
Myrtaceae
Thapye-nge
0.51
0.19
0.11
0.81
57
Madhuca longifolia (Koen.) MacbrideSapotaceae
Kan zaw
0.51
0.19
0.10
0.79
58
59
60
61
62
63
64
65
66
67
68
Dipterocarpus costatus Gaertn. f.
Dipterocarpaceae
Kanyin ni
0.51
0.19
0.09
0.78
Gyrocarpus jacquinii Gaertn.
Hernandiaceae
Pinle-thit-kauk
0.51
0.19
0.03
0.73
Kan ba lai new
0.51
0.19
0.02
0.72
Unknown 2
Cedrela toona Roxb.
Meliaceae
Thit ka toe
0.51
0.19
0.01
0.71
Embelia sessiliflora Kurz.
Lythraceae
Ate mwe new
0.51
0.19
0.01
0.71
Ficus spp.
Moraceae
Nyaung pa ong
0.51
0.19
0.01
0.71
Syzygium cerasoides (Roxb.) Raiz.
Myrtaceae
Thabye-gyin
Syzygium spp. 1
Myrtaceae
Thapye-khaung long
0.51
0.19
0.01
0.71
Nwe pha pyoke
0.51
0.19
0.01
0.71
Unknown 8
Unknown 9
Bouea burmanica Griff.
Anacardiaceae
0.51
0.19
0.01
0.71
Pa yel chin
0.51
0.19
0.00
0.70
Ma yan
0.51
0.00
0.02
0.53
100
100
100
300
Total
Antifungal Metabolites of Endophytic Strain YY20 Isolated
from Neomarica longifolia (Link & Otto) Sprague
Yee Yee Thu
Abstract
In the course of screening of bioactive secondary metabolites producing
microorganisms, an endophytic bacterial strain was isolated from the leaves
of Neomarica longifolia (Link & Otto) Sprague. It was temporarily
designated as strain YY20. The six metabolites including a novel crystal
compound “Cyclo-D-Prolyl-D-leucyl” from the fermented broth (7 L) of
strain YY20 were isolated and purified by using column chromatographic
and preparative thin layer chromatographic techniques. The pure isolated
compounds were identified and characterized by spectroscopic techniques
such as UV, IR, EI-MS or ESI-MS, 1H-NMR and 13C-NMR spectra.
Biological activity of the pure compounds was determined by paper disc
diffusion assay on fungal test organisms. The three compounds (YY20 A, C,
and D) indicated high antifungal activity on Malassezia furfur whereas the
compound YY20 E showed bioactivity against Candida albicans in 20 μL of
1.0 mg/mL (MIC) in vitro in this research.
Keywords: Antifungal activity, Bioactive metabolites, Endophytic
strain, Neomarica longifolia (Link & Otto) Sprague.
Introduction
The use of microorganisms to produce natural products and in
production processes has been a part of human history since the days of early
civilization. Since the discovery of penicillin in 1929, intensive studies of
bacteria and fungi have shown that microorganisms are a rich source of
pharmaceutically important bioactive substances (Fenical, 1993 and Fleming,
1929).
Endophytic microorganisms such as bacteria, fungi and Actinomycetes
have had a profound effect on the development of medical science (Berdy,
1989). Endophytic microorganisms have been defined as those that reside at
some phase of their life cycle within living plant tissues (Carroll, 1986; Petrini,
1991), or which can be isolated from surface disinfected plant tissues or
extracted from inner plant parts without causing apparent damage to them (ElShanshoury et al., 1996 and Hallmann et al. 1997).
Lecturer, Department of Botany, University of Yangon
48
The use of microorganisms to produce natural products and in
production processes has been a part of human history since the days of early
civilization. Since the discovery of penicillin in 1929, intensive studies of
bacteria and fungi have shown that microorganisms are a rich source of
pharmaceutically important bioactive substances (Fenical, 1993 and Fleming,
1929).
Endophytic microorganisms such as bacteria, fungi and Actinomycetes
have had a profound effect on the development of medical science (Berdy,
1989). Endophytic microorganisms have been defined as those that reside at
some phase of their life cycle within living plant tissues (Carroll, 1986; Petrini,
1991), or which can be isolated from surface disinfected plant tissues or
extracted from inner plant parts without causing apparent damage to them (ElShanshoury et al., 1996 and Hallmann et al. 1997).
Endophytes include both commensal microorganisms, which have no
direct effect on the host plant, and mutualistic symbionts that could be used in
the biological control of pathogens or for plant growth promotion (Petrini,
1991 and Murray et al., 1995). Antibiotics such as vancomycin, daptomycin,
cephalosporin, streptomycin, the antifungal amphotericin B, griseofulvin, the
antiviral aciclovin, doxorubicin and many others were isolated from
endophytic microorganisms. Over the past 60 years, about 28000 natural
products have been isolated from microorganisms. More than 10000 of these
compounds are biologically active and more than 8000 are antibiotic and
antitumor agents (Demain, 1992).
The needs of novel and effective compounds that can fight serious
deadly diseases are essential since life-threatening fungal and bacterial
infections are increasing. Nowadays, microbial agents (bacteria and fungi)
continue to play a major role in drug discovery and development in the
pharmaceutical industry.
The main aim and objectives of the present investigation are screening
of bioactive strain from the leaves of Neomarica longifolia (Link & Otto)
Sprague possessing selective toxicity against human diseases, isolation of the
antifungal metabolites from bioactive strain, and evaluation of antifungal
activity (MIC) of the isolated compounds.
49
Screening of Endophytic Strain from Plant Parts
In the course of screening of the antifungal compounds producing
microorganisms, an endophytic bacterial strain was isolated from the leaves of
Neomarica longifolia (Link & Otto) Sprague in the family Iridaceae. The plant
sample was collected from the campus of Yangon University in Myanmar.
Isolation of endophytic strain can be carried out by the following scheme.
1. The leaves were washed in running tap water for 10 min. They were cut into
about 1 cm pieces. 2. The surfaces of cut pieces were sterilized by soaking
in 75% ethanol for 2 min.
3. Next, sterile surfaces were socked in 5.3% sodium hypocloride for 3 min.
4. Cut pieces were socked in 75% ethanol for 0.5 min to wash out sodium
hypocloride.
5. They were dried and cut into smaller pieces, and placed on nutrient broth
agar plates and then incubated for 3 days to 1 week (Lee et al., 1996 and
Phay, 1997).
sterilized
cut
small pieces
-------
socked
by soaking in 75%
ethanol for 2 min
washed in running
water for 10 min
in 5.3% sodium
hypocloride for 3 min
socked
pure culture
in 75% ethanol
for 0.5 min
dried
transferred
to the test tube
transferred
placed
to new plate
on agar plate
incubated for 3 days
to 1 week
Figure 1. Isolation procedure of endophytic fungal strain
50
Antifungal Activity of Isolated Endophytic Strain
As the preliminary study on antifungal activity of isolated bacterial
strain, its bioactivity was determined by paper disc diffusion assay with fungal
test organisms such as Candida albicans and Malassezia furfur (Cruickshank
et al., 1975; Phay, 1997).
Test organisms
Candida albicans: It occurs both in the form of oval yeast-like bodies and as
thick septate pseudo-hyphae. It occurs as a normal inhabitant of the mouth,
skin, nail, alimentary tract, vagina or many parts of the body. It also occurs
naturally as a commensal of mucus membranes and in the digestive tract of
humans and animals. A similar condition may occur on the vaginal and vulval
mucosa giving rise to vaginal irritation and discharge. This type of infection is
particularly likely to occur in pregnancy and in diabetic (Stewart, 1968).
Malassezia furfur: Malassezia yeasts are a type of fungus. Malassezia species
inhabit the skin of about 90% of adults without causing harm. The yeasts
produce chemicals that reduce the pigment in the skin, causing whitish patches
(Frey, et al., 1979; Isenberg, 1992). Malassezia furfur is a lipophilic yeast
living on the skin. It is the causative agent of Pityriasis versicolor, Pityriasis
folliculitis, seborrhoeic dermatitis and dandruff (Elewski, 1992; Ajello and
Hay, 1997).
Seed culture and fermentation of isolated strain
A bacterial strain grown on nutrient agar plate was transferred into a 50
mL falcom tube containing 10 mL of nutrient medium. Then, this tube was
incubated for two days as seed culture. After two days, seed culture (1%) of
the strain was transferred into 250 mL conical flask containing 100 mL of
fermentation medium. Fermentation was carried out for 5 days at 180 rpm on
glass shaker. At the end of fermentation, the fermentation broth was used to
check antifungal activity by paper disc diffusion assay (Monaghan, et al.,
1999).
Paper Disc Diffusion Assay
Assay medium (malt extract agar medium 25g/L, pH 7.0) was utilized
for two fungal test organisms. Test organisms were inoculated in 10 mL assay
broth in 50 ml falcon tubes at 37ºC and incubated overnight. After an
51
overnight incubation, 100 μL of each test organism was added in each assay
plate and spread on the agar plate. Then, the paper discs impregnated with the
fermented broth were applied on the test plates and the plates were incubated at
37°C for 24 hrs.
After 24 hrs, inhibitory zone surrounding the test disc indicates the
presence of the bioactive compounds which inhibits growth of test organism.
The disc size is 6 mm (width) and 0.5 mm (thickness) while volume of
fermented broth is 10 μL/disc. Inhibitory zones are measured as weak activity:
10-12 mm, high activity: 13-17 mm and highest activity: above 18 mm (David
et al., 1949; Gavin, 1956; David and Stout, 1971).
Paper disc
diffusion assay
Plate culture
Seed culture
Fermentation
Inhibitory zone
Figure 2. Procedure of paper disc diffusion assay
Fermentation Studies of Bioactive Strain
Screening for the best medium
The most bioactive strain YY20 grown on nutrient broth agar plate was
transferred into 50 mL falcom tube containing 10 mL of nutrient broth medium
and the falcom tube was incubated for two days as seed culture. After two days
incubation, seed culture (1%) was transferred into 50 mL conical flask
containing 10 mL of various fermentation media such as M1: nutrient broth
medium, M2: glucose yeast extract medium and M3: LB medium. Then,
fermentation was carried out for five days (Zeeck, et al., 2001).
Kinetic growth of inoculums
In order to produce the bioactive compounds from the most active
strain YY20, fermentation studies were investigated by optimizing the proper
growth kinetic (cultivation and transfer) of inoculum.
Strain YY20 grown on nutrient agar plate was transferred into a 50 mL
falcom tube containing 10 mL of nutrient broth medium and incubated for two
52
days as seed culture. After two days, seed cultures (size of inoculum; 0.5%,
1.0%, 1.5%, 2.0%, 2.5%) were transferred into the five tubes of 50 mL falcom
tube containing 10 mL fermentation medium in each tube. Fermentation was
carried out for five days. The fermented broths were used to check bioactivity
by paper disc diffusion assay (Monaghan, et al., 1999).
Cultivation and Fermentation of Bioactive Strain
Two days old seed culture (140 mL) was transferred into the seven
flasks of 2.5 L conical flask containing 1 L of fermentation medium in each
flask. Then, the flasks were incubated at 30°C at 180 rpm on shaker.
Fermentation was carried out for three days (Strobel & Sullivan, 1999).
Extraction of Bioactive Metabolites from Stain YY20
On the third day of fermentation, the fermented broth (7 L) was
centrifuged at 4500 rpm for 20 minutes. Then, the mycelium cake was
extracted with acetone and the cultural filtrate (the supernatant) was extracted
with ethyl acetate (pH 4.5) for three times. The extracted samples were
concentrated by using rotary evaporator, applied on TLC plates and allowed to
dry. The TLC plates were developed in the solvent of chloroform-methanol,
9:1 and 8:2 (Grabley et al., 1999).
.
Isolation and Purification of Bioactive Metabolites from Strain YY20
Bioactive secondary metabolites were isolated and purified by using
silica gel columns and preparative thin layer chromatography (PTLC) with
various solvent systems (chloroform or dichloromethane : methanol, 95:5, 9:1,
8:2, 6:4, 2:1 and 1:1 (Grabley et al., 1999; Zeeck et al., 2001).
Structural Elucidation of Bioactive Compounds
The molecular weight of the isolated compounds was determined by
EI-MS or ESI-MS spectra. Their structural elucidation was identified and
characterised by IR, UV, 1H-NMR and 13C-NMR spectra at The University of
New South Wales, Sydney in Australia.
53
Bioactivity of Isolated Compounds
Minimum inhibitory concentration (MIC) of the isolated compounds
from strain YY20 was determined by paper disc diffusion assay. The paper
discs (6 mm) were soaked in 20 μL of a solution of the pure compound in a
suitable solvent (concentration: 1 mg/mL), the discs were dried under sterile
flow box and put on agar plates inoculated with test organisms C. albicans and
M. furfur.
Results
In the course of screening of the antifungal compounds producing
microorganisms, an endophytic bacterial strain was isolated from the leaves of
Neomarica longifolia (Link & Otto) Sprague in the campus of Yangon
University, Myanmar (Figure 3).
Figure 3. Endophytic bacterial strain YY20 growing on nutrient agar plate
Antifungal Activity of Isolated Strain
According to the result of inhibitory test, isolated strain YY20 showed
antifungal activity against both Candida albicans (18 mm) and Malassezia
furfur (20 mm). Therefore, strain YY20 indicated highly antifungal activity.
54
Fermentation Studies of Strain YY20
Screening the best medium
According to the result, the fermented broth of strain YY20 in medium
M1 showed high activity (20 mm) against C. albicans whereas the broth in
medium M2 indicated high activity (22 mm) against M. furfur on the three day
fermentation as shown in Table 1. The nutrient broth medium or glucose yeast
extract medium was used for large scale fermentation of strain YY20.
Table 1. Inhibitory zones (mm) of strain YY20 on various media
Candida albicans
Strain
Malassezia furfur
1 day 2 days 3 days 4 days
1 day 2 days
3 days
4 days
YY 20: M1
11
15
20
16
12
15
18
14
M2
10
12
18
15
13
17
22
16
M3
10
11
17
14
10
14
17
13
M1: Nutrient broth medium, M2: Glucose yeast extract medium, M3: LB
medium
Size of inoculum
As regard the sizes of inoculum (0.5%, 1.0%, 1.5%, 2.0%, 2.5%), 2.0%
size of inoculum showed the highest inhibitory zone against Candida albicans
and Malassezia furfur on the third day fermentation for large scale
fermentation strain YY20 as can be seen in Table 2 and 3.
Table 2. Inhibitory zones (mm) of strain YY20 against Candida albicans
Fermentation
0.5%
1.0%
1.5%
2.0%
2.5%
Fer. 2nd day
11
15
15
16
16
Fer. 3rd day
12
19
20
20
18
Fer. 4th day
14
16
17
17
16
Fer. 5th day
10
12
13
13
12
55
Table 3. Inhibitory zones (mm) of YY20 against Malassezia furfur
Fermentation
0.5%
1.0%
1.5%
2.0%
2.5%
Fer. 2nd day
10
15
12
13
13
Fer. 3rd day
11
18
20
24
19
Fer. 4th day
13
15
15
16
15
Fer. 5th day
10
11
11
12
11
Extraction of Bioactive Compounds from Strain YY20
The mycelia extract (0.3 g) and the ethyl acetate extract (5.5 g) were
obtained from the seven liters fermented broth of strain YY20. The ethyl
acetate extract showed more bioactive activity than the mycelia extract. Thus,
ethyl acetate residue was extracted for isolation and purification of the
bioactive compounds from strain YY20 in this research.
Isolation of Bioactive Metabolites from Strain YY20
There were seven fractions which were separated from the ethyl acetate
crude extract by silica gel column (5 cm × 20 cm) with various solvent systems
(dichloromethane: methanol, 95:5, 9:1, 4:1, 2:1 and 1:1). Among these
fractions, fractions F2, F3, F4 and F5 showed antifungal activity on Candida
albicans and Malassezia furfur, whereas fractions F1 showed no bioactivity.
Therefore, active fractions F2, F3, F4 and F7 were purified by rechromatography to produce their bioactive compounds as shown in Figure 4.
56
Ethyl acetate extract (5.5 g)
Silica gel, CH 2 Cl 2 :MeOH (95:5, 9:1, 8:2, 6:4, 2:1, 1:1)
Fractions: F1
F2
F3
F4
1.8 g 405 mg
140 mg
115 mg
PTLC
PTLC
CHCl 3
CH 2 Cl 2
F5
1.0 g
PTLC
Silica gel
CH 2 Cl 2 :MeOH (95:5) CH 2 Cl 2 :MeOH (9:1,4:1,2:1)
Oily substances
PTLC;CH 2 Cl 2 :MeOH(8:2)
YY20A YY20B
20.2 mg
7.1 mg
YY20C
YY20D
YY20E
YY20F
6.2 mg
4.0 mg
15.2 mg
18.5 mg
Figure 4. Isolation procedure of the compounds YY20 A, B, C, D, E and F
Characterization of Isolated Metabolites of Strain YY20
The compound "YY20A" was isolated from fraction F2 by silica gel
column and preparative thin layer chromatography (PTLC). It has an UV
absorbing band at 254 nm and its R f is 0.98 (chloroform/methanol, 9:1). It
showed an intensive brown colour with anisaldehyde/sulphuric acid reagent.
This substance dissolves well in chloroform and dichloromethane. It has high
antifungal activity on Malassezia furfur.
During the isolation of strain YY20, the second compound "YY20B"
was isolated from fraction F3 and has R f 0.95 (chloroform/methanol, 9:1). It
showed light brown colour with anisaldehyde/sulphuric acid reagent. This
57
substance dissolves well in chloroform, dichloromethane and acetone. It has no
biological activity against Candida albicans and Malassezia furfur.
The compound "YY20C" was isolated from fraction F4 as an UV
absorbing band at 254 nm and has R f 0.24 (chloroform/methanol, 9:1). It was
colourless with anisaldehyde/sulphuric acid reagent. This substance dissolves
well in acetone and methanol. It showed high antifungal activity on M. furfur.
The crystal compound "YY20D" was isolated from fraction F4 by
silica gel column and PTLC method as an UV absorbing band at 254 nm. It has
R f 0.35 (chloroform/methanol, 9:1) and 0.72 (chloroform/methanol, 8:2). It
was colourless with anisaldehyde/sulphuric acid reagent. This substance
dissolves well in acetone and methanol. It is identified as “cyclo-D-Prolyl-Dleucyl” according to crystallographic X-ray (Figure 5). This compound was
previously isolated from marine bacteria by Salvatore De Rosa et al., 2003.
But, Salvatore De Rosa et al. did not isolate it in crystal form so that its crystal
form in this research was the first report. It has high antifungal activity on M.
furfur.
Working up of fraction F5 led to the compound "YY20E" as an UV
absorbing band at 254 nm and has R f 0.60 (chloroform/methanol, 8:2). It did
not show any colour with anisaldehyde/sulphuric acid reagent. This substance
dissolves well in methanol. It has high antifungal activity on
C. albicans.
An additional bioactive compound "YY20F" was isolated from fraction
F5 by silica gel column and PTLC method. It has R f 0.77
(chloroform/methanol, 8:2). It showed an UV absorbance under 254 nm and
gave no colour with anisaldehyde/sulphuric acid reagent. This substance
dissolves well in methanol.
O
HO
N
NH
O
X-ray crystal structure
Figure 5. New crystal compound "Cyclo-D-Prolyl-D-leucyl"
58
Antifungal Activity of Isolated Metabolites
The isolated compounds YY20A, YY20C and YY20D indicated high
antifungal activity on Malassezia furfur whereas the compound YY20E also
showed high bioactivity against Candida albicans (Table 4 and Figures 6 & 7).
Table 4. Inhibitory zones (mm) of the isolated compounds
No.
Compound
Candida albicans
Malassezia furfur
1
YY20A
-
18
2
YY20C
-
24
3
YY20D
-
17
4
YY20E
14
-
Figure. 6. Inhibitory zone of YY20 E
against Candida albicans
Figure. 7. Inhibitory zone of YY20
A, C & D against Malassezia furfur
In this strategic research an endophytic bacterial strain was isolated
from the leaves of Neomarica longifolia (Link & Otto) Sprague in the campus
of Yangon Uiversity, Myanmar. According to the result of antifungal activity,
strain YY20 showed highly biological activity against Candida albicans and
Malassezia furfur.
59
Demain has studied different kinds of microorganisms and their
secondary metabolism since 1971 while many researchers have also
investigated microbial metabolism continuously. Carroll (1986) studied
different kinds of endophytes in plants, especially woody perennials whereas
Stroble and Sullivan (1999) reported that improvement of fermentations is very
important to food and pharmaceutical industries.
Kinetic growth of inoculum of bioactive strain YY20 was investigated
in order to produce the bioactive compounds. As regard the size of inoculum
(0.5%, 1.0%, 1.5%, 2.0%, 2.5%), 2.0% showed the highest inhibitory zone
against two test organisms on the third day fermentation for strain YY20.
In the course of isolation and purification of the bioactive compounds
from strain YY20, the six compounds including a novel crystal compound
“Cyclo-D-Prolyl-D-leucyl” from 7 L fermentation were isolated by using silica
gel columns, preparative thin layer chromatography with various solvent
systems. The isolated compounds (YY20 A, C, D and E) selectively showed
high antifungal activity against Candida albicans and Malassezia furfur in 20
μL of 1.0 mg/mL (MIC) in vitro.
Lee et al (1996) has isolated carboxymethyl-cellulase and other
metabolites from Bacillus sp. KD1014. Berdy (1989) has also isolated
bioactive metabolites from microorganisms from different sources in nature.
Petrini (1991) also reported that the bioactive compounds have been isolated
from fungal endophytes of tree leaves. Moreove, Zeeck et al (2001) stated the
chemistry screening approach of microorganisms from nature in drug
discovery, and then they isolated various types of secondary metabolites from
microbial sources.
In conclusion, it is essential to search for newly antifungal drugs since
life-threatening fungal diseases are increasing nowadays. In this research, the
four isolated compounds have shown high antifungal activity on two
pathogenic microorganisms: C. albicans and M. furfur. Among bioactive
metabolites isolated, the other three including the new crystal compound
indicated high antifungal activity on M. furfur which is the causative agent of
Pityriasis versicolor, Pityriasis folliculitis, seborrhoeic dermatitis and
dandruff. Therefore, these bioactive metabolites should be used as the active
compounds in the field of medicine and can render beneficial effects for health
of human beings.
60
Acknowledgements
I would like to express my sincere thanks to Professor Dr. Thet Thet May, Head of
Botany Department, University of Yangon, Myanmar for her encouragement to conduct this
research paper. I wish to mention my deep gratitude that comes from my heart to Professor Dr
David St. C Black, Prof. Dr. Naresh Kumar and Dr. Carola, Schools of Chemistry and
Biological Sciences, The University of New South Wales (UNSW), Sydney, Australia for their
valuable instructions and constructive criticisms. My warmest thanks are due to all staff from
NMR and MS Departments at UNSW, Sydney, Australia for their generous assistance. My
deepest indebtedness to United Nations Educational, Scientific and Cultural Organization
(UNESCO) is acknowledged for its financial support to conduct some of research work in
Australia.
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Comparative Study on Preparation of Botanical Permanent
Slides by Different Methods
Bay Dar1, Moe Moe Lwin2, Ohnmar Than3and Aye Aye Myint4
Abstract
In the study of biology and medicine, historical permanent slides are widely
demanded for effective teaching and learning. Thus the main aim of this
paper is to prepare botanical permanent slides by different methods and to
study their quality. Sandoricum koetjape Merr. (Thit-to) and Eupatorium
odoratum L. (Bi-zat) growing in University of Yangon campus were
selected. The selected samples can mainly support the biological study (in
Basic Education High School (BEHS) level and undergraduate level. It may
be useful for school and college students to know the technique of preparing
histological slides as a part of their study of biology. In this paper,
permanent slides for histological study in plants were prepared by tertiary
butyl alcohol (TBA), xylene-alcohol method and free hand technique. The
quality of 537 permanent slides out of 1100 slides were classified as class A.
TBA method was found to be the best and cost effective in this work.
Key words:
Sandoricum koetjape Merr., Eupatorium odoratum L., TBA,
xylene-alcohol, free hand
Introduction
In the biology of high schools and undergraduate courses, the
permanent tissue slides are used for learning the histology of plant parts, tissue
and microorganisms. Normally, they are imported and they are very expensive,
so the main purpose of this research is to make cost effective teaching aid
materials (Tissue slides) in the country instead of importing them. Medicinal
plants still play an important role in Myanmar people for curing many diseases.
This study will provide histological information on two plant species in
University of Yangon campus.
The aim of the present study is to find out various medicinal plants
which could be classified and identified using their histological characters
based on the prepared permanent slides.
The objective of this research was to evaluate botanical permanent
slides using three methods: Tertiary Butyl alcohol (TBA), Xylene-alcohol and
Free hand method.
1 .Associate Professor (Botany), Universities' Research Centre (URC), University of Yangon
2,3. Lecturer, Department of Botany, University of Yangon
4. Professor (chemistry), Universities' Research Centre, University of Yangon.
64
The plant samples were collected from University of Yangon campus
and verified at the Department of Botany, University of Yangon. Preparation
of permanent slides was conducted at Universities’ Research Centre,
University of Yangon.
(A)
(B)
Figure 1. Dehydration and cleaning the tissue in the tissue processor
(A) Tissue processor (Citadel TM Shandon, USA) set up at URC
(B) Placing the tissue cassette into the cassette hanger
The samples of Dicot: the lamina, midrib, petiole and stem of
Sandoricum koetjape Merr. (Thit-to), Family-Meliaceae and lamina, midrib,
petiole, stem and root, of Eupatorium odoratum L. (Bi-zat), FamilyAsteraceae were cut in transverse section (15 - 25 µm). Plant tissues were
divided into soft tissues and hard tissues. CRAF III solution was used for soft
tissues and Formalin Aceto-Alcohol (FAA) solution was used for hard tissues
to carry out fixation of plant tissue. There are five steps in the histological
process including: 1. Fixation, 2. Dehydration and Clearing, 3. Embedding, 4.
Slicing by Microtome, 5. Staining and Mounting. In this study, the tissue
processors were programmed for fixation, dehydration, cleaning (Figure. 1),
and infiltration into paraffin (Figure. 2).
65
Figure 2. Tissue embedding by paraffin dispenser
The embedded paraffin was then poured into a mold and cooled
on the Shandon Histocentre TM 3 cold plate. When paraffin block was frozen,
they were kept in the refrigerator (Figure. 3).
Figure 3. Chilling the mould on the Shandon Histocentre TM 3 cold plate
The cooled wax block with the tissue inside was sliced into very thin
ribbons that have the thickness of 5 μm, using a microtome (Leica, RM 2155)
(Figure. 4). The tissue ribbon was transferred to a tissue floating bath, not
exceeding 40°C. The section was then quickly picked up on the slide and dried
on the slider warmer for 24 hours.
66
Figure 4. Microtome for tissue slicing
For the examination of histological tissues, the staining reagent for the
specific tissue was systematically conducted. After 3 days or 5 days fixing,
tissue samples were processed using with TBA or Xylene-alcohol method and
then blocked with paraffin. Then lamina and midrib sections of 10-15 µm
thickness and stem, petiole and root sections of 20-30 µm thickness were cut
by microtome. Then the sliced tissue sections were placed on glass slides using
warm water (35-40 °C) and they were dried in incubator (37-40 °C) overnight.
And then tissue samples were stained stepwise by the procedure of staining
method using Saffranin (Avilla, 2000). Dehydration and clearing (TBA) series
for plant tissues were carried out as listed in Table (1). Xylene-alcohol series
for plant tissues were prepared as shown in Table (2) (Donald, 1940 and Mya
Mya, 2003). The staining procedure for plant tissue was summarized in Table
(3) (Avilla, 2000). Finally, the stained tissue slides were mounted with Canada
balsam and dried overnight. The permanent slides were labeled and kept in
slide boxes for microscopic studies.
Table 1. Dehydration and clearing series for plant tissue by TBA method
95 %
Step
No.
Alcohol (mL)
1
100 %
Absolute
alcohol (mL)
Tertiary ButylAlcohol (mL)
Distilled
water (mL)
Time (hr)
5
95
2-4
2
10
90
2-4
3
20
80
2-4
4
30
70
2-4
5
40
60
2-4
95 %
100 %
Absolute
alcohol (mL)
67
Distilled
water (mL)
Time (hr)
50
2-4
10
40
2-4
50
20
30
2-4
9
50
35
15
2-4
10
50
50
2-4
75
2-4
12
100 + erythrosin
2-4
13
100
12
14
100
12
Step
No.
Alcohol (mL)
6
50
7
50
8
11
25
Tertiary ButylAlcohol (mL)
15
Soft Paraffin
2
16
Hard Paraffin
2
Table 2. Dehydration and clearing series for plant tissue by Xylene-alcohol
method
Distilled
water (mL)
Time
5
95
2-4
2
10
90
2-4
3
20
80
2-4
4
30
70
2-4
5
40
60
2-4
6
50
50
2-4
7
60
40
2-4
8
70
30
2-4
9
85
15
2-4
10
95
5
2-4
11
100
-
4-12
Step
No.
98% Alcohol
(mL)
1
Xylene (mL)
(hr)
68
Distilled
water (mL)
Time
-
4-12
-
2-3
Step
No.
98% Alcohol
(mL)
12
100
13
95
5
14
90
10
2-3
15
85
15
2-3
16
75
25
2-3
17
50
50
2-3
18
25
75
2-3
19
100
12
20
100
12
Xylene (mL)
21
Soft Paraffin
2
22
Hard Paraffin
2
(hr)
Table 3. Staining procedure for sliceable plant tissue
Step
No.
Chemical Reagents
Time (min)
1
Xylene I (pure xylene)
10
2
Xylene II (pure xylene)
10
3
3:1 (xylene: aniline)
10
4
10
5
1:1:1 (xylene: aniline: 95 % ethanol)
10
6
97% ethanol
10
7
85% ethanol
10
8
70% ethanol
10
9
50 % ethanol
10
10
Distilled water
10
11
1% aqueous water Saffranin (staining)
12
50% ethanol
3
13
70% ethanol
3
6-24 hrs
Step
No.
69
Chemical Reagents
Time (min)
14
85% ethanol
3
15
95% ethanol
3
16
0.5% Fast green in 95% ethanol (counterstains)
3
17
1:1:1 (xylene: aniline: 95 % alcohol)
3
18
3
19
3
20
Xylene III (pure xylene )
3
21
Xylene IV (pure xylene)
3
Results
In this process, a total of 1100 permanent tissue slides were obtained by
TBA and Xylene-alcohol methods. The high quality permanent slides (537)
were recorded as class A. Some samples (Class A slides) were shown in
Figures 5-27. The taxonomy and structure of laminar, midrib, root, various
types of stems, trichomes and calcium oxalate crystals were clearly observed.
About 300 slides were damaged due to the thinness or thickness of cell or
imperfection including loss of tissue orientation, teared section and round holes
while sectioning and they were classified as B. During staining, 263 out of 800
slides were damaged and classified as class C.
Some High Quality Tissue Slides Samples (Class A) by Different Methods
Upper epidermis
Palisade parenchyma cell
Intercellular space
Spongy mesophyll cell
Lower epidermis
Figure 5. Classs A - T.S* of Lamina of Sandoricum koetjape ( × 10) by TBA method
70
Intercellular space
Parenchyma cell
Vascular bundle
Lower collenchyma cell
Lower epidermis
Figure 6. Class A - T.S* of Midrib of Sandoricum koetjape ( × 10) by TBA method
T.S*= Transverse Section
Epiblema
Xylem
Phloem
Figure 7. Class A - T.S of Root of Eupatorium odoratum L. ( × 4) by TBA method
Xylem
Phloem
Cortex
Epiblema
Figure 8. Class A - T.S of Root of Eupatorium odoratum L. ( × 20) by TBA method
71
Class A - Quality tissue slides by Xylene-alcohol method
Upper epidermis
Intercellular space
Spongy mesophyll cell
Lower epidermis
Figure 9. Class A-T.S of Lamina of Sandoricum koetjape Merr. ( × 10) by Xylenealcohol method
Upper epidermis
Vascular bundle
Lower parenchyma cell
Lower epidermis
Figure 10. Class A-T.S of Midrib of Sandoricum koetjape Merr. ( × 4) by Xylene–
alcohol method
Xylem rays
Phloem
Pericyclic fibres
Epidermis
Figure 11. Class A - T.S of Stem of Sandoricum koetjape Merr. ( × 10) by Xylene –
alcohol method
72
Pith
Xylem rays
Figure 12. Class A - T.S of Stem of Sandoricum koetjape Merr. ( × 20) by Xylene-alcohol
method
Epiblema
Xylem
Phloem
Figure 13. Class A - T.S of Root of Eupatorium odoratum L. ( × 4) by Xylene-alcohol
method
Xylem
Cortex
Figure 14. Class A - T.S of Root of Eupatorium odoratum L. ( × 20) by Xylenealcohol method
73
Unicellular, uniseriate
Trichomes
Figure 15. Class A - Unicellular, uniseriate Trichomes of Sandoricum koetjape Merr.
(× 20) by Xylene-alcohol method
Unicellular, uniseriate Trichomes
Figure 16. Class A - Unicellular, uniseriate trichomes, epidermal cells and the cortex
of Sandoricum koetjape Merr. ( × 20) by Xylene-alcohol method
Class A Tissue slides by Free hand
Figure 17. Surface view of upper epidermis showing straight anticlinal wall of
Sandoricum koetjape Merr. ( × 20)
74
Figure 18. Surface view of lower epidermis paracytic stomata ( × 20)
Figure 19. Calcium oxalate (Raphides) ( × 40)
Figure 20. Watery trichomes ( × 20)
75
Pith
Xylem rays
Phloem
Figure 21. T.S of Stem of Sandoricum koetjape Merr. ( × 20) by Free hand
Xylem
Pericycle
Pith
Epidermis
Figure 22. T.S of Stem of Eupatorium odoratum L. ( × 4) by Free hand
Epidermis
Collenchyma cell
Pericycle
Phloem
Xylem
Pith
Figure 23. T.S of Stem of Eupatorium odoratum L. ( × 10) by Free hand
76
Epidermis
Xylem
Intercellular space
Figure 24. T.S of Root of Eupatorium odoratum L. (× 4) by Free hand
Epidermis
Phloem
Intercellular spaces
Xylem rays
Figure 25. T.S of Root of Eupatorium odoratum L. (× 10) by Free hand
Xylem rays
Figure 26. Vascular bundles of Root of Eupatorium odoratum L. (× 10) by Free hand
77
Unicellular, Uniseriate trichome
Glandular trichome
Figure 27. Unicellular, Uniseriate trichome and glandular trichome of Eupatorium
odoratum L. ( × 20) by Free hand
The sliced tissues prepared by TBA method were the first experience
for the researchers in making permanent slides. While making permanent
sliced sample tissue with no previous experience and while not very skillful in
microtome techniques, many slides were damaged. When Xylene-Alcohol
method was used for making slides for the second time, there was less damage.
For the process of cutting microtome section, it is better to have three persons
at the same time instead of two only. When conducting free hand section, air
bubbles were found in tissues during staining although the section was thin and
good. In the process of permanent slides by free hand techniques, the cells
inside the tissues were found to be damaged. Thus more has to be attempted
for better quality. This research was first attempted to prepare permanent slides
applying microtome technique in the Botany department.
After studying the three methods used systematically TBA method was
found to be the best and the most cost effective. Thus, it can be used as
teaching aid in biological syllabus of high schools and undergraduate courses.
This project will be of help in providing information on the histological
characteristics of plant applying different techniques. Microtome techniques
were found to be more suitable for Dicot (hard tissue) than Monocot (soft
tissue). Based on these findings and experience, quality permanent slides of
spore formation of bacteria and fungi that can be used as teaching aids should
be prepared for further studies.
78
Acknowledgements
We would like to express our deepest thanks to Professor Dr. Tin Tun, Rector,
University of Yangon, for his permission and providing us with essential references. We would
also like to express our gratitude to Professor Dr Pho Kaung, Pro-Rector and Head of
Universities' Research Centre (URC), University of Yangon for providing us with research
facilities. We would like to convey our sincere thanks to all the URC staff who helped in this
project. Finally, we are most indebted to Dr Thet Thet May, Professor, Head of the Department
of Botany for her kindness and unstinting support in every way.
References
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Dassanayake, M. D., (1983). "A Revised Hand Book to the Flora of Ceylon". Vol. IV,
Amerind Publishing Co. Pvt. Ltd., New Delhi.
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Company, Inc, New York & London.
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Ltd., New Delhi.
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Rangoon University Press, Rangoon, Burma.
Mya Mya, (2003). "Plant Micro Technique". Department of Botany, Dagon University.
Metcalfe, C. R. and L. Chalk, (1960). "Anatomy of the Dicotyledons". Vol II. The Clarendon
Press, Oxford.
Pandey, S. N. and A. C. Chadha, (1996). "Plant Anatomy and Embryology". Vikas Publishing
House Pvt. Ltd.
Sundara Rajan, S., (2000). "Plant Anatomy and Embryology". Anmol Publications (Pvt) Ltd.,
India.
Trease, G. E. and W. C. Evans, (1978). "Pharmacognosy". 11th Ed. Baillere Tindoll London.
Trease, G. E. and W. C. Evans, (2002). "Pharmacognosy". 15th Ed. Baillere Tindoll London.
Effects of Root Nodules Rhizobia on Growth of Vigna
unguiculata subsp. sesquipedalis (L.) Verdc.
Soe Myint Aye1, Phyu Phyu Oo2 and Mu Yar Min3
Abstract
The effect of rhizobial strains on Vigna ungiuculata subsp. sesequipedalis (L.)
Verdc., have been performed. Four rhizobia strains, MYM-1 from
V.
trilobata (L.) Verdc., MYM-2 from V. unguiculata subsp. sesquipedalis (L.)
Verdc., MYM-3 from Crotalaria pallida Ait. and MYM-4 from Clitoria
ternatea L., were isolated respectively. The host specificity test on V.
unguiculata subsp. sesquipedalis (L.) Verdc., was carried out in the growth
chamber for one month. Among the four strains, MYM-2 was found to be
most effective in the nodule formation. In pot culture experiment, the
germinated seeds of V. unguiculata subsp. sesquipedalis (L.) Verdc.
inoculated with four isolated rhizobial strains were grown from the months of
August to October in 2012. In this experiment also, Bradyrhizobium strain,
MYM-2 showed the most highly significant effects on the fresh and dry
weight of plant, nodules and pods and the pod length.
Key words: Vigna unguiqulata subsp. sesquipedalis, Rhizobial strains,
growth
Introduction
Biological nitrogen fixation by the legume-Rhizobium system is the
main natural source of nitrogen in agricultural systems. Biological nitrogen
fixation (BNF) is an effective alternative natural source of nitrogen made
available to the soil (Predeepa and Ravindran, 2012).
Rhizobium is the most well known species of a group of bacteria that
acts as the primary symbiotic fixer of nitrogen. These bacteria can infect the
roots of leguminous plants, leading to the formation of lumps or nodules where
the nitrogen fixation takes place. The bacterium's enzyme system supplies a
constant source of reduced nitrogen to the host plant and the plant furnishes
nutrients and energy for the activities of the bacterium. About 90% of legumes
can become nodulated (Anonymous, 2002).
Most of the Rhizobium species nodulate only one or a few genera of
legumes. On the other hand, Bradyrhizobium species tend to nodulate diverse
legumes including a number of the agriculturally most important genera
Arachis, Glycine, Vigna and so forth (Lewin et al., 1990).
1. Associate Professor, Department of Botany, University of Mandalay
2. Assistant Lecturer, Department of Botany, Yadanapon University
3. M.Res.-student, Botany, University of Mandalay
80
Legume nitrogen fixation starts with the formation of nodule. A
common soil bacterium, Rhizobium, invades the root and multiplies within in
the cortex cells. The plant supplies all the necessary nutrients and energy for
the bacteria. In the field, small nodules are visible 2-3 weeks after planting,
depending on legume species and germination conditions (Lindemann and
Glover, 2003).
Black (1968) suggested that higher grain yield in food legumes
inoculated with Rhizobium was due to an increase in nodulation.
Biological Nitrogen Fixation (BNF) has been used in farming systems
to cut down on fertilizer expenses (Mwangi et al. 1994 as cited in Otieno et al.,
2009). Inoculation with an effective and persistent rhizobium strain has
numerous advantages, which include non-repeated application of nitrogen
fertilizers and higher pod yield due to increased nodulation (Sanginga et al.,
1994 cited in Otieno et al,. 2009). It has been reported that rates of N 2 fixation
of 1 to 2 kg N ha–1 growing season per day is possible in most legumes in
tropical cropping systems (Giller, 2001 cited in Otieno et al,. 2009).
Vigna unguiculata subsp. sesquipedalis (L.) Verdc. is a cultivated
legume which can be eaten as green pods. It is known as the yardlong bean,
bora, long-podded cowpea, asparagus bean, snake bean, or chinese long bean.
The crisp, tender pods are eaten both fresh and cooked. They are at their best
when young and slender (Anomynous, 2012).
The yardlong bean pods are widely cultivated in most of the region of
Myanmar, especially in the rainy season. The farmers get some income by
cultivation of this crop and the young pods are very common vegetables in
Myanmar traditional foods. The nutritionally and healthy young pods can be
produced, that is commercially demanded and free of undesirable chemicals by
using biofertilizer of rhizobia, it will be very practically applicable research
for the country.
Therefore, the aim and objectives of the present research is forecasting
to study the nature of root nodules and its rhizobial strains that infected in
some leguminous species, to investigate the cultural characteristics of infected
rhizobial strains on the culture media, to specify the host specificity of studied
strains on the Vigna unguiculata subsp. sesquipedalis (L.) Verdc., to know the
effect of selected strains on cultivation of commercially important crop,
yardlong bean and to share the knowledge of the root nodule bacteria in
81
legume reduces the costly plant chemical fertilizers by their nitrogen fixation
process and that plant can use it for growth.
The nodule samples were collected from Vigna trilobata (L.)Verdc.,
Vigna unguiculata subsp. sesquipedalis (L.) Verdc., Crotalaria pallida Ait.
and Clitoria ternatea L. In the isolation of rhizobial strains, Yeast Mannitol
Broth (YMB) and Yeast Mannitol Agar (YMA) were used as the basal culture
media according to Vincent (1970). N- free nutrient solution for plant
experiment of Leonard Jar methods were used sterilized N-free nutrient
solution (Broughton and Dilworth 1970 as cited in Somasegaram and Hoben
1994). Isolation of rhizobial strains and bacterial culture followed Vincent
(1970). Isolated bacteria strains were labeled as MYM 1, MYM 2, MYM 3 and
MYM 4 depending on sourced plants of Vigna trilobata (L.)Verdc., Vigna
unguiculata subsp. sesquipedalis (L.) Verdc., Crotalaria pallida Ait. and
Clitoria ternatea L. respectively (Fig. 1, 2, 3 and 4).
The germinated seeds were inoculated with rhizobial strains and then
inoculated germinated seeds were used for testing host specificity by growing
them in pots. Germinating seeds were used to record the rhizobial effect on
crop for host specificity and pot culture experiment. Leonard Jar Method of
Somasegaram and Hoben (1994) used for test of host specificity.
Soil samples were collected from the cultivated field of leguminous
crop from Shangalay Kyun, Mandalay Region. Seeds of Vigna unguiculata
subsp. sesquipedalis (L.) Verdc. for all experiments were received from
Myanmar Agriculture Service, Mandalay Region. The nodulation assays and
plant growth promoting were prepared in pots, which is 30 cm in diameter and
35 cm high, filled with 9 kg of soil. After sowing, the pots were placed in the
green house in order to receive sufficient light and water. No fertilizers were
added to the plants. At 70 days (the completion of growth phase), the plants
were harvested and yields characteristics like number and length of pods, fresh
weight and dry weight of plant, and root nodules were recorded.
82
Fig. 1. Host plant of Vigna trilobata and its infected rhizobial strains (MYM 1)
Fig. 2. Host plant of Vigna unguiculata and its infected rhizobial strain (MYM 2)
Fig. 3. Host plant of Crotalaria pallida and its infected rhizobial strain (MYM
3)
83
Fig. 4. Host plant of Clitoria ternatea and its infected rhizobial strain (MYM 4)
Results
Host Specificity
To specify the host specificity of the rhizobial strains the nodule
number, fresh weight and dry weight of the seedlings were recorded (Fig. 5).
In the experiment the nodule number in MYM-2 was higher than the
other strains. The average number was found as 4.17 in MYM-1, 2.9 in MYM3, and 2.8 in MYM-4. In control there was no formation of nodule.
The fresh weight of the plant was heavier by using the MYM-2 strain
with 3.36 g than that grown by other strains. The control as 1.84 g, MYM-1 as
3.28 g, MYM-3 as 3.25 g, and MYM-4 as 3.20 g respectively.
The dry weight of the plant was also showed by using the MYM-2 with
0.35 g. They were found as 0.23 g in control, 0.34 g in MYM-1, 0.32 g in
MYM-3, and 0.29 g in MYM-4.
All the resulting data for host specificity was shown in Table 1.
Table 1.Effect of Rhizobial strains on V. unguiculata subsp.sesquipedalis (L.)
Verdc.
Strain No
Control
MYM-1
MYM-2
MYM-3
MYM-4
Nodule number
0
4.17 ± 1.50
5.17 ± 1.91
2.9 ± 0.96
2.8 ± 1.89
Fresh weight
1.84 ± 0.30
3.28 ± 0.25
3.36 ± 0.29
3.25 ± 0.42
3.20 ± 0.17
Dry weight
0.23 ± 0.15
0.34 ± 0.15
0.35 ± 0.10
0.32 ± 0.10
0.29 ± 0.17
84
A
B
C
D
Fig. 5. Experiment of host specificity in light control growth chamber
A. Seedling at 7 DAS
B. Seedling at 14 DAS
C. Seedling at 21 DAS
D. Seedling at 28 DAS
Effect of Rhizobia on Crop Growth
The rhizobia strains MYM-1, MYM-2, MYM-3 and MYM-4 were used
as biofertilizer in yardlong bean cultivation (Fig. 7).
The results revealed that MYM-2 showed the highest average fresh
weight with 15.66 g of plant while the other strain MYM-1 with 15.4 g, MYM3 with 15.39 g, MYM-4 with 15.22 g, and 15.15 g in control without strains.
A
B
C
D
E
F
Fig. 7. Cultivation of Vigna unguiculata subsp. sequipedalis (L.)
A. Young plants at 7 DAS
B. Plants at 21 DAS
C. Plants at 35 DAS
D. Plants at 49 DAS
E. Plants at 63 DAS
F. Plants at 70 DAS
85
86
In the average dry weight of cultivated plant, MYM-1 and MYM-2
strains showed the significant effect with 2.62 g. MYM-3 strains showed 2.12
g and MYM-4 strain showed 2.09 g, and control showed 1.99 g.
Although the fresh weight of the pods were heavier than the others by
using MYM-2 with 8.76 g, 8.18 g was found in control, 8.73 g in MYM-1,
8.72 g in MYM-3, and 8.39 g in MYM-4.
The dry weight of the pod was found to be 1.50 g by using the rhizobia
strain MYM-1 and MYM-2 while 1.11 g in control, 1.32 g in MYM-3 and 1.29
g in MYM-4 .
The fresh weight of the nodule showed the significant effect with the
strain of MYM-2 with 4.22 g while they were 4.19 g in the strain of MYM-1,
4.12 g in MYM-3, 4.12 g in MYM-4 and 4.09 g in control.
The average dry weight of the nodule was 1.89 g in control, 2.01 g in
MYM-1, 2.05 g in MYM-2, 1.99 g in MYM-3 and MYM-4 (Table 4.8).
The pod length showed the significant effect with the strain of MYM-2
with 14.55 cm while they were 14.16 cm in MYM-1, 13.88 cm in MYM-3,
13.72 cm in MYM-4 and control showed 12.13 cm in pod length.
The resulting data on plants, pods, nodules were shown in Table 2.
Table 2. Effect of Rhizobia strains on V. unguiculata subsp. sesquipedalis (L.)
Verdc.
Item
Plant fresh
weight
Plant dry
weight
Pod fresh
weight
Pod dry
weight
Nodule
fresh
weight
Nodule dry
weight
Pod length
Control
MYM-1
MYM-2
MYM-3
MYM-4
15.15±0.17
15.40±0.18
15.66±0.19
15.39±0.16
15.22±0.16
1.99±0.13
2.62±0.12
2.62±0.13
2.12±0.12
2.09±0.10
8.18±0.14
8.73±0.18
8.76±0.13
8.72±0.15
8.39±0.13
1.11±0.10
1.50±0.10
1.50±0.13
1.32±0.10
1.29±0.13
4.09±0.08
4.19±0.08
4.22±0.12
4.12±0.12
4.12±0.09
1.89±0.14
2.01±0.15
2.05±0.14
1.99±0.14
1.99±0.11
12.13 ±2.18
14.16±2.29
14.55±3.05
13.88±0.64
13.72±2.77
87
The present study deals with the effect of rhizobial biofertilizer on
cultivated crop Vigna unguiculata subsp. sesquipedalis (L.) Verdc. (yardlong
bean). The various rhizobial species, MYM-1, MYM-2, MYM-3 and MYM-4,
were isolated from Vigna trilobata (L.) Verdc., Vigna unguiculata subsp.
sesquipedalis (L.) Verdc., Crotalaria pallida Ait and Clitoria ternatea L.
respectively.
Colonies of rhizobia were obtained on YMA agar medium after
incubation at 30°C for 3-5 days. The colonies seemed to be sticky in
appearance showing the production of mucous. The morphology of colony
indicated rounded colonies, white colored until 2-4 days of growth and turning
yellow after 4 days.
Somasegaran and Hoben (1994) stated that rhizobia have specified time
for growth, 3 to 5 days for fast growers Rhizobium and 7 to 10 days for slow
growers Bradyrhizobium. In the test of Bradyrhizobium strains, the resulting
strains of MYM-1, MYM-2 and MYM-3. growed in yeast mannitol agar after
5 days. Lewin et al. (1990) also reported that the strains isolated from the
nodule of genus Vigna are under Bradyrhizobium. Therefore the three strains
are the strains under the genus Bradyrhizobium. MYM-4 strains are fastgrowing root nodule bacteria, medium-sized, rod shaped cells, gram-negative.
Therefore this strain of bacteria is under the genus Rhizobium.
In the experiment of host specificity, sterilized soil and nitrogen free
nutrient solution were used to test the nodule formation of the leguminous
plants. It was found that the number of nodules, the fresh weight and dry
weight of the plants are the highest in plants inoculated with MYM-2 strains.
The other strains (MYM-1, MYM-3 and MYM-4) also showed better effect
compared to control. It was found that there was no nodule formation in the
control plants. Therefore, it can be concluded that the inoculation of Rhizobium
strain to the plants induce the nodulation of the leguminous plant in the
nitrogen free nutrient solution. It was observed that the MYM-2 strain
(Bradyrhizobium strain) from Vigna unguiculata subsp. sesquipedalis (L.)
Verdc., possessed higher host specificity than the other strains.
Although Weaver (1974) postulated that the presence of nodules on
roots did not mean for fixation of nitrogen for good growth of host plant, in the
present study, it was found that the rhizobia strains can promote nodules
formation and also in promoting the plant growth; these characters were in
88
agreement with Ravikumar (2012) on ground nut, Zahran (2001) in several
legumes, Warge (1989) in Acacia, peanut and cowpea, Mahmood and Athar
(2007) in Leucena leucocephala.
Khachani (1981) and Million (1989) as cited in Otieno et al. (2007)
reported that an increase pod yield was due to inoculation of French bean.
Mahmood (1992) also noticed that the isolated rhizobial strains of Albizia
lebbeck, Pithecellobium dulce and Vigna unguiculata were most effective in
nitrogen fixation. A significant increase of dry weight and nitrogen content of
the host plant were found. In the present study, the inoculation of rhizobial
strains was also found to be better effect than the control that had not been
used by any strain.
In the study on pot culture, all the isolated strains were inoculated into
Vigna unguiculata subsp. sesquipedalis (L.) Verdc. The Bradyrhizobium strain
MYM-2 showed the more amount of fresh and dry weight of whole plants and
pods. MYM-2 is also found to be most significant in fresh weight and dry
weight of total nodules per plant. Otieno et al. (2007) suggested that higher
grain yield in food legumes inoculated with Rhizobium was due to increase in
nodulation. Therefore the present research work is in agreement with those
findings by Black (1968).
Nitrogen is one of the major components in seed development or seed
yield. Nitrogen fertilizer is quite expensive due to the high prize of petroleum.
Under these circumstances, the ability of legumes in symbiosis with rhizobia to
obtain the atmospheric nitrogen is important in crop production. Biological
nitrogen fixation reduces the cost of production and helps to reduce pollution.
Therefore, the use of Bradyrhizobium strain (MYM-2) in the cultivation of
yardlong bean cannot only be very applicable for the production of crops but
also it may be beneficial to the agroecosystems.
Acknowledgements
We would like to deeply express our profound gratitude to Dr. Khin Swe Myint,
Rector of Mandalay University, for her kind encouragement during our research work and
providing the necessary facilities.
89
References
Anonymous (2012). Vigna unguiculata subsp. sesquipedalis from Wikipedia. The free
encyclopedia.
Lindemann, W.C. and C.R. Glover (2003). Nitrogen Fixation by legumes. New Mexico State
University is and United State Department of Agriculture cooperating.
Lewin, A., E. Cervantes, W. Chee-Hoong and J.B. William (1990). Nodsu, Two New Nod
Genes of the Broad Host Range Rhizobium Strain NGR 234 Encode hostSpecific Nodulation of The Tropical Tree Leucaena leucocephala. University
of Sains Malaysia.
Mahmood, A. and M. Athar (2007). Cross Inoculation Studies: Respone of Vigna Mungo To
Inoculation With Rhizobia From Tree Legumes Growing Under Arid
Environment. Department of Botany. University of Karachi. Pakistan.
Otieno, P.E., J.W. Muthomi, G.N. Chemining’wa and J.H. Nderitu (2007). Effect of Rhizobia
Inoculation, Farmyard Manure and Nitrogen Fertilizer On Nodulation and
Yield of Food Grain Legumes. Department of Plant Science and Crop
production. University of Nairobi.
Otieno, P.E., J.W. Muthomi, G.N. Chemining’wa and J.H. Nderitu. 2009. Effect of Rhizobia
Inoculation Farmyard Manure and Nitrogen Fertilizer On Growth,
Nodulation and Yield of Selected Food Grain Legumes. Vol. VIII. p. 805312. African Crop science Conference Proceedings.
Predeepa, R.J. and D.A. Ravindran (2010). Nodule Formation Distribution and Symbiotic
Efficacy Or Vigna Unguiculata L. Under Differend Soil Salinity Regimes.
Department of Biology. The Unviersity of Western Australia Australia.
Ravikumar, R. (2012). Growth Effects of Rhizobium Inoculation In Some Legume Plants.
Somasegaran, P. and Hoben (1994). Hand Book of Rhizobia. Methods In Legume Rhizobium
Technology. Springer-Verleg, New Youk, Inc.
Vincent, J.M. (1970). A Manual For The Practical Study of The Root Nodule Bacteria IBP 15.
Oxford: Black well Scientific Publications.
Taxonomic Study on Some Bryophytes from Southern Shan
State
Soe Myint Aye and Win Win Aye
Abstract
Bryophytes from Southern Shan State were collected and studied in 2012.
Ten species belong to 9 genera and 8 families of mosses from Bryophytes
were found as tufts on damp soil, rocks, tree trunks, old walls, hardly
calcareous soil. They are Philonotis rigida Brid., Bryum argenteum
var.argenteum Hedw., B. caespiticium var. caespiticium Hedw.,
Octoblepharum albidum Hedwig, Funaria fascicularis (Hedw.) Lindb.,
Taxiphyllum wissgrillii (Garov.) Wijk & Marg., Pterogonium gracile
(Hedw.) Sm., Pogonatum urnigerum (Hedw.) P. Beauv., Desmatodon
cernuus (Hiib.) Br. and Weissia controversa var. controversa Hedw. The
artificial key to the species were constructed and their diagnostic characters
of study species were described.
Key words: Bryophytes, Taxonomy, Southern Shan State
Introduction
Bryophytes are small “leafy” or flat plants that most often grow in moist
locations in temperate and tropical forests or along the edges of wetlands and
streams. Bryophytes include liverworts, hornworts and mosses. They are a very
ancient group of land plants that first migrated and colonized bare land around
450 million years ago. They are non-vascular plants that have neither flowers
nor fruits, and they disperse by spores, instead of seeds. Today, the bryophytes
are estimated to be more than 18,000 species worldwide. Estimated to consist
of well over 10,000 species, mosses are the second largest plant group of land
plants today after the flowering plants. About 2000 species of mosses occur in
Southern Asia (Tan & Boon-Chuan 2008).
Bryophytes are amphibians of the kingdom plantae. Plants grow in two
well defined habitats called the amphibious zone (Vashishta, 1963). Mosses,
liverworts, and hornworts are superficially very similar. However, there are
several ways to describe the three groups apart. Mosses have leaves that are
spirally arranged along the stem and a vein (or costa) that runs at least part way
along the middle of each leaf (Bjorkman, 2008).
1. Associate Professor, Department of Botany, University of Mandalay
2. Demonstrator, Department of Botany, Taunggyi University
92
Bryophytes contribute significantly to plant biodiversity and are also
important in some parts of the world for the large amounts of carbon they
store, thereby playing a significant role in the global carbon cycle (Raven et al.
2005).
Southern Shan State is situated in the eastern part of Myanmar.
Southern Shan State lies between the latitudes of 19° 23' and 22° 15' north and
between the longitudes 96° 13' and 98° 36' east. The present study areas are
Taunggyi Township, Pindaya Township, and Panglong Township of Southern
Shan State.
In 1981, Hla Hla Ko studied 53 species from 27 genera of mosses of
Yangon and 20 species belonging to 14 genera from 8 orders of mosses of
Mandalay Region have been described by Kin Maung Win in 1994.
Only a few researchers studied the bryophytes of Myanmar. Because of
the small size of plants and the identification characters are mainly basing on
very thin small leaves and sporophytes, the study on mosses faced with much
difficulties. However, no bodies studied on that lower plant group in Southern
Shan State and the valuable information will be partially fulfilled for the Flora
of Shan State. .
The aims and objectives of the present research works were to identify
and classify the bryophytes from Southern Shan State, to record the
identification characters, and to partially fulfill the accomplished information
of bryophytes from Shan State.
Specimens were collected during the field exploration months of June
to October in 2012. The 10 species of bryophytes from Southern Shan State
have been collected, identified, classified and described in the present study.
Photographic record and data collection were taken to know precise localities,
and external features of the collected species. The literature that have been
used for identification are followed to Smith (1978), Meinunger and Schroder
(2007), Zander (2007), Tan and Boon-Chuan (2008), Gudino et al. (2011).
93
Results
The bryophytes growing in Southern Shan State were collected, studied,
classified, and described their taxonomic characteristics. Totally 10 species
belonging to 8 families of 7 orders were recorded in study area. List of the
collected mosses species were stated in Table 1. The artificial key to all
collected mossess species were constructed and the diagnostic characteristics
were also described as follows:
Table 1. List of the collected species of Mosses
Order
1.Polytrichales
Family
1.Polytrichaceae
Species
1. Pogonatum urnigerum
(Hedw.) P. Beauv.
Location
Taunggyi,
Shwe Phone
Pwint Pagoda
2.Pottiales
2.Pottiaceae
2. Desmatodon cernuus (Hueb.)
Br. & Schimp.
3. Weissia controversa var.
controversa Hedw.
3.Funariales
4.Bryales
3.Funariaceae
4.Bryaceae
4. Funaria fascicularis (Hedw.)
Lindb.
5. Bryum argenteum var.
argenteum Hedw.
6. Bryum caespiticium var.
caespiticium Hedw.
5.Bartramiaceae
7. Philonotis rigida Brid.
Taunggyi,
Sularmuni Pagoda
Pindaya
Taunggyi,
Taungchun
Panglong
Taunggyi,
Sularmuni Pagoda
Taunggyi
Taungchun
5.Dicranales
6.Calymperaceae
8. Octoblepharum albidum
Hedwig
6.Isobryales
7.Leucodontaceae
9. Pterogonium gracile (Hedw.)
Sm.
7.Hypnobryales
8.Plagiotheciaceae
10. Taxiphyllum wissgrillii
(Garov.) Wijk &Marg.
Taunggyi,
Shwe Phone Pwint
Pagoda
Taunggyi University
Campus
Taunggyi,
Taungchun
94
An artificial key to the studied species
1. Plants erect …………………………………………………..………....3.
1. Plants prostrate ………………………………………………………... 2.
2. Leaves broadly ovate ; nerve absent ………..9. Pterogonium gracile
2. Leaves ovate to oblong-lanceolate ; nerve about quarter way up
leaf…..……………………………………10. Taxiphyllum wissgrillii
3. Nerve present; calyptra absent or not short……………………..….......4.
3. Nerve absent; calyptra short………………. 8. Octoblepharum albidum
4. Calyptra absent or cucullate………………………………………..…5.
4. Calyptra long or hairy……………………………………………...…8.
5. Operculum convex…………………………………..…………….….…6.
5. Operculum mamillate…………………………………………….…......7.
6. Leaves lanceolate-spathulate to oblanceolate-spathulate; nerve below the
apex; capsule obovoid symmetrical or shortly pyriform……………
………………………………………….….……4. Funaria fascicularis
6. Leaves lanceolate; nerve strongly excurrent; capsule globose…………
……………………………………….………...….7. Philonotis rigida
7. Leaves ovate to broadly ovate; nerve below the apex…………...............
……………………………………5.Bryum argenteum var. argenteum
7. Leaves ovate to ovate-oblong; nerve excurrent ……...…..……………
………………………………..6. Bryum caespiticium var. caespiticium
8. Nerve excurrent; operculum rostrate or mamillate……………………
…………………………………………………………………..…… 9.
8. Nerve percurrent; operculum longly rostrate…………..……………
……………………………….3. Weissia controversa var. controversa
9. Leaves lanceolate to narrowly lanceolate; calyptra hairy…………………
………………………………………………. 1. Pogonatum urnigerum
9. Leaves oblanceolate-spathulate; calyptra long ………………………….
…………………………………………………. 2. Desmatodon cernuus
95
1. Pogonatum urnigerum (Hedw.) P. Beauv., Prodr., 1805
Plants dioecious, erect. Leaves lanceolate to narrowly lanceolate, acute
at the apex, margin plane with coarse spinose teeth; nerve excurrent; basal cells
rectangular, and above cells rounded, papillose, rectangular, sinuose. Seta red;
capsule erect, shortly cylindrical; lid rostellate, yellowish green; peristome
recurved, pale red; calyptra as a bird, ending at the base of capsule with hairs,
yellow (Figure 1 A - D).
This species was found scattered plants on banks, crevices of walls.
Specimens investigated: Taunggyi, Shwe Phone Pwint Pagoda;
20.10.12; Win Win Aye # 21.
2. Desmatodon cernuus (Hueb.) Br. & Schimp., Bryol. Eur.2: 58.1843
Plants autoecious, erect. Leaves oblanceolate-spathulate, acuminate at
the apex, margin bordered, denticulate; nerves excurrent; basal cells
rectangular - hexagonal, some cells hyaline, narrower at margin, above cells
variable in shape and size, marginal rows longer and narrower. Seta reddish at
the base; capsule erect to horizontal, some inclined, ovoid; lid mamillate,
reddish; peristome teeth more or less straight; calyptra ending at the middle of
capsule, pale green (Figure 1. E-H).
This species was found on tree trunks.
Specimens investigated: Taunggyi, Sularmuni Pagoda; 2.8.12; Win
Win Aye # 8.
3. Weissia controversa var. controversa Hedw., Sp. Musc., 1801
Plants autoecious, erect. Leaves oblong-lanceolate; basal part abruptly
narrowed to linear-lanceolate upper part, apex acute, margin plane, entire;
nerve ending at the apex; basal cells rectangular, hyaline, above cells quadrate.
Seta yellowish; capsule erect or slightly inclined, ovoid to narrowly ellipsoid,
yellow, reddish brown when mature; lid longly rostrate; peristome present or
poorly developed; calyptra long, ending at the almost middle of capsule, pale
green (Figure 1.I-L).
This species was found on roadsides and cliffs.
Specimens investigated: Pindaya; 21.8.12; Win Win Aye # 13.
96
4. Funaria fascicularis (Hedw.) Lindb., Ofv. K.V.A. Forh. 1865
Plants autoecious, erect. Leaves lanceolate-spathulate to oblanceolatespathulate, acuminate at the apex, toothed towards the middle; nerve ending
below the apex; cells rectangular, but above cells slightly hexagonal. Seta
straight, brown; capsule erect, obovoid symmetrical or shortly pyriform; lid
convex, without an apiculous, brown; peristome absent; calyptra cucullate,
ending at the apex of capsule, brown (Figure 2.A-C).
This species was found on moist soil.
Specimens investigated: Taunggyi, Taungchun; 20.10.12; Win Win
Aye # 27.
5. Bryum argenteum var.argenteum Hedw., Sp. Musc. 1801
Plants dioecious, erect. Leaves ovate to broadly ovate, acuminate at the
apex, base decurrent, margin entire; basal cells rhomboid-hexagonal, above
cells rhomboid, cells in upper part of leaf pellucid with colourless walls. Seta
reddish brown; capsule small, pendulous, ellipsoid, green; lid mamillate;
peristome double, long, outer teeth yellow and inner white; calyptra cucullate,
ending at the apex of capsule, reddish (Figure 2.D-G).
This species was found on damp soil.
Specimens investigated: Southern Shan State, Panglong; 20.9.12; Win
Win Aye # 20.
6. Bryum caespiticium var. caespiticium Hedw., Sp. Musc., 1801
Plants dioecious, erect. Upper leaves ovate to ovate-oblong, accuminate
at the apex, widest below middle, margin more or less entire; nerve excurrent,
yellowish to reddish- brown; basal cells shortly rectangular, above cells
narrowly hexagonal. Seta reddish brown basally; capsule pendulous, narrowly
pyriform, striate, green, wide- mouthed, reddish brown; lid mamillate, reddish;
peristome teeth long; calyptra cucullate, ending at the apex of capsule, reddish
brown (Figure 2. I-K).
This species was found on tree trunks.
Specimens investigated: Taunggyi, Sularmuni Pagoda; 2.8.12; Win
Win Aye # 9.
97
7. Philonotis rigida Brid., Br Univ. 1827.
Plants autoecious. erect. Leaves lanceolate, acuminate to subulate at the
apex, margin plane, toothed; nerve strongly excurrent; basal cells rectangular,
above cells narrowly rectangular to linear, mamillose. Seta red; capsule
globose, striate, brownish; lid convex, brown; peristome double, teeth long;
calyptra absent (Figure 3 A - D).
This species was found on moist soil.
Aye # 22.
8. Octoblepharum albidum Hedwig, Sp. Musc. Frond. 50. 1801
Plants autoecious, erect, glossy. Leaves ligulate to lanceolate, apiculate
at the apex, margin entire; nerve absent; chlorophyllose cells in a single layer,
above cells hexagonal except margin, basal cells longly rectangular; capsule
erect, ovoid - cylindrical, reddish brown; lid obliquely rostrate, reddish;
peristome teeth triangular; calyptra short, ending at the apex of capsule,
reddish (Figure 3.E - H)
.
This species was found spreading branches on tree trunks.
Specimens investigated: Southern Shan State, Shwe Phone Pwint
Pagoda; 10.6.12; Win Win Aye # 3.
9. Pterogonium gracile (Hedw.) Sm., Eng. Bot., 1802
Plants doecious, prostrate. Leaves broadly ovate; nerve absent; cells
linear - rhomboidal, alar cells strongly differentiated, extending almost halfway
of leaf. Seta reddish; capsule exserted, erect, cylindrical, brown when mature;
lid conical to rostrate, reddish basally; peristome double; calyptra long, ending
at the middle of capsule, pale yellow (Figure 3. I - L).
This species was found at the base of big trees and branches.
Specimens investigated: Southern Shan State, Taunggyi University
Campus; 2.8.12; Win Win Aye # 11.
10. Taxiphyllum wissgrillii (Garov.) Wijk & Marg., Taxon, 1960
Plants dioecious, prostrate. Leaves ovate to oblong-lanceolate, acute at
the apex, margin denticulate above to middle; nerve double, ending about
quarter way up leaf; cells more or less linear, angular cells shortly rectangular.
Seta reddish; capsule erect, inclined, cylindrical, green; lid rostrate, yellow,
98
reddish when mature; peristome long, double; calyptra cucullate, ending at the
almost base of capsule, yellow (Figure 4. A - D).
This species was found on bark of tree trunks.
Aye # 25.
A
B
C
E
F
G
I
J
D
H
K
L
Figure 1. A. Habit. B. Leaf, C. Capasule & D. Operculum of Pogonatum urnigerum
(Hedw.) P. Beauv. E. Habit. F. Leaf, G. Capasule & H. Operculum of Pogonatum
urnigerum (Hedw.) P. Beauv. of Desmatodon cernuus (Hueb.) Br. & Schimp.I. Habit.
J. Leaf, K. Capasule & L. Operculum of Weissia controversa var. controversa Hedw.
A
99
B
C
A
D
E
G
F
A
H
I
J
K
Figure 2. A. Habit, B. Leaf & C. Capsule of Funaria fascicularis (Hedw.)
Lindb. D. Habit, E. Laf, F. Capsule & G. Operculum of Bryum argenteum
var. argenteum Hedw. H. Habit, I. Laf, J. Capsule & K. Operculum of
Bryum caespiticium var. caespiticium Hedw.
100
A
B
C
D
A
A
E
F
I
J
A
G
K
H
L
Figure 3. A. Habit, B. Leaf , C. Capsule & D. Operculum of Philonotis rigida Brid.
E. Habit, F. Leaf , G. Capsule & H. Operculum of Octoblepharum albidum Hedwig
I. Habit, J. Leaf , K. Capsule & L. Operculum of Pterogonium gracile (Hedw.) Sm.
A
101
B
C
D
Figure 4. A. Habit, B. Leaf , C. Capsule & D.Operculum of Taxiphyllum
wissgrillii (Garov.) Wijk & Marg.
The present research work deals with taxonomic study on bryophytes
growing in Taunggyi Township, Pindaya Township, and Panglong Township
of Southern Shan State. In the present work, as a first attempt, by extending the
field collection to 2012, 10 species belong to 9 genera of 8 familes and 7
order under class Bryopsida were identified and described.
In the study area mosses are abundantly occur as epiphytes are
Taxiphyllum wissgrillii (Garov.) Wijk & Marg., Desmatodon cernuus (Hiib.)
Br. and Bryum caespiticium var. caespiticium Hedw. Funaria fascicularis
(Hedw.) Lindb., Bryum argenteum var. argenteum Hedw., Philonotis rigida
Brid., are grown on moist soil. Pogonatum urnigerum (Hedw.) P. Beauv. and
Weissia controversa var. controversa Hedw., and were grown on banks, on
crevices of wall. Most of the plants were erect and Pterogonium gracile
(Hedw.) Sm. and Taxiphyllum wissgrillii (Garov.) Wijk & Marg. were
prostrate.
The nature and shapes of the leaves were variously occured as oblonglanceolate, oblong-spathulate, linear-lanceolate, ovate, ligulate, etc. The ending
of the nerves of leaves are also found as a very useful character. It was ending
below the apex in Funaria fascicularis (Hedw.) Lindb. and Bryum argenteum
var. argenteum Hedw., absent in Octoblepharum albidum Hedwig and
Pterogonium gracile (Hedw.) Sm.; percurrent in Weissia controversa var.
controversa Hedw.; excurrent in Pogonatum urnigerum (Hedw.) P. Beauv.,
102
Desmatodon cernuus (Hiib.) Br., Bryum caespiticium var. caespiticium Hedw.
and Philonotis rigida Brid.
The shapes of the capsules are found as cylindrical in Pogonatum
urnigerum (Hedw.) P. Beauv., Octoblepharum albidum Hedwig, Taxiphyllum
wissgrillii (Garov.) Wijk & Marg.; ovoid in Desmatodon cernuus (Hueb.) Br.&
Schum.; narrowly ellipsoid on Weissia controversa var. controversa Hedw.
and Bryum argenteum var. argenteum Hedw., obovoid in Funaria fascicularis
(Hedw.) Lindb., pyriform in Funaria fascicularis (Hedw.) Lindb. and Bryum
caespiticium var. caespiticium Hedw., ellipsoid in Bryum argenteum var.
argenteum Hedw., globose in Philonotis rigida Brid.
The shapes of the opercula are rostrate in Octoblepharum albidum
Hedwig, Taxiphyllum wissgrillii (Garov.) Wijk & Marg.; rostellate Pogonatum
urnigerum (Hedw.) P. Beauv.; mamillate in Desmatodon cernuus (Hueb.) Br.&
Schum, Bryum argenteum var. argenteum Hedw., B. caespiticium var.
caespiticium Hedw.; convex in Funaria fascicularis (Hedw.) Lindb. and
Philonotis rigida Brid.
The calyptras are large in Desmatodon cernuus (Hueb.) Br.& Schum,
Weissia controversa var. controversa Hedw., Pterogonium gracile (Hedw.)
Sm.; hairy in Pogonatum urnigerum (Hedw.) P. Beauv.; cucullate in Funaria
fascicularis (Hedw.) Lindb., Bryum argenteum var. argenteum Hedw., B.
caespiticium var. caespiticium Hedw., Philonotis rigida Brid. and Taxiphyllum
wissgrillii (Garov.) Wijk & Marg., short in Octoblepharum albidum Hedwig,
and absent in Philonotis rigida Brid.
According to the previous studies, Pogonatum urnigerum was recorded
in Mandalay Region (Khin Maung Win, 1994). This species was also found in
Southern Shan State area.
The morphological chacteristics of the species are variable among the
studied species. The most distinguished characters are habit of the plants,
shapes of the leaves with their margin, nerve, cell-shape, the characters of
capsule, operculum, calyptra, peristome and spore. The identification of the
species were also based on characters of nerve, operculum, leaves shape and
cells shape.
Although the higher vascular plants were taxonomically studied by
many researchers in Myanmar, the taxonomic characterization on mosses is
still rare. Therefore, the present study partially fulfilled the valuable
information of bryophytes for Myanmar.
103
Acknowledgements
Our thanks are due to Professor Dr Nu Nu Yee, Head of the Department of Botany,
University of Mandalay, for her kind suggestion and encouragement. We are also very
thankful to Professor Dr Aye Aye Win Kyi, Head of the Department of Botany, University of
Taunggyi, for her encouragement, for providing the departmental facilities during our research
work.
References
Bjorkman, A. (2008). About Bryophytes, Bryophytes in BC / Ecology and Evolution /
terms, University of British Columbia.
Key
Gudino, J., S. A. Noris and V. M. Konrat. (2011). A key to bryophytes distinguishing between
liverworts, hornworts and mosses, Chicago.
Hla Hla Ko. (1981). Mosses of Yangon. MSc Thesis, Department of Botany, University of
Yangon.
Khin Maung Win. (1994). Mosses of Mandalay Region. MSc Thesis, Department of Botany,
University of Mandalay.
Meinunger, L. & W. Schroder. (2007). Bildatlas der Moose Deutschlands , Germany.
Raven, P. H., R. F. Evert and S. E. Eichhorn. (2005). Biology of Plants, seventh edition, W.
H. Freeman and Company, New York 345 -367.
Smith, A. J. E. (1978). The Moss Flora of Britain and Ireland, W & J Mackay Ltd., London.
Tan, B. C. & H. Boon-Chuan. (2008). A guide to the Mosses of Singapore, Science Center,
Singapore 4 - 29.
Vashishta, B. R. (1963). Bryophyta. Part III. Botany for Degree Students, S. Chand Co. Ltd.,
New Delhi.
Zander, H. R. and M. P. Eckel. (2007). Bryophyte Flora of North America, Flora of North
America Association, Inc., North America.
Effect of Isolated Azospirillum Strains on Germination and
Growth of Triticum aestivum L. (Wheat)
Yi Shan1, Thi Thi Htun2and Hnin Ei Phyu3
Abstract
The isolation of Azospirillum strains from three grasses namely Imperata
cylindrica (L.) Raeuschel. (Thetke), Eleusine indica (L.) Gaertn. (Sin ngo
myet) and Panicum paludosum Roxb. (Unknown) has been undertaken.
This experiment was carried out at the Microbiology Laboratory,
Department of Botany, University of Mandalay, from June 2012 to
February 2013. Two specific media such as N-Free semisolid malate
medium (NFb) and Congo Red Agar (CRA) medium were used to isolate
the Azospirillum strains. Azospirillum strain HEP-1 was isolated from the
root of Imperata cylindrica (L.) Raeuschel., HEP-2 from Eleusine indica
(L.) Gaertn. and HEP-3 from Panicum paludosum Roxb. In germination
test, these strains were treated to Triticum aestivum L. (Wheat) seeds and
tested for germination and growth. It was found that Azospirillum strain
HEP-2 showed higher germination percentage than other treated plants and
control. This strain HEP-2 also possessed higher length of plumule and
radicle than the others. In pot culture experiment, these 3 strains were
treated to wheat plants to study of growth. It was observed that Azospirillum
strain HEP-2 had significance in plant height and flag leaf area, which were
better than the other treated plants and control.
Key words: Isolation, grasses, Azospirillum strains, Triticum aestivum
L.,
Introduction
In recent years, concepts of Integrated Plant Nutrient Management
(IPNM) have been developed, which emphasize maintaining and increasing
soil fertility by optimizing all possible sources (organic and inorganic) of
plant nutrients required for crop growth and quality. This is done in an
integrated manner appropriate to each cropping system and farming
situation. Improvement in agricultural sustainability requires optimal use
and management of soil fertility and soil physical properties, both of which
rely on soil biological processes and soil biodiversity (Boddey and
Döbereiner, 1994).
Biological nitrogen fixation is important in non-leguminous crops
e.g., rice and wheat, because it is an inexpensive source of nitrogen for
1. Professor, Department of Botany, University of Mandalay
2. Assistant Lecturer, Department of Botany, University of Mandalay
3. Demonstrator, Department of Botany, Yadanapon University
106
higher yields. This process diminishes the need for expensive chemical
fertilizers, which have been associated with numerous health and
environmental problems. The knowledge of nitrogen biological fixation in
non-leguminous plants, mainly in Graminae, among them several cereals,
became one of the largest challenges, since these represent the more
important alimentary base of the population, mainly in developing countries
(Boddey and Döbereiner, 1994).
In view of a positive influence of bacteria from the genus
Azospirillum on plants, attempts have been made to inoculate crops with
these bacteria. The effect of inoculation to a large degree was showed
dependent on the ability of microorganisms to survive in the soil. The
genera Azospirillum, Herbaspirillum, Azotobacter, and Acetobacter show
the generalized occurrence in economically important cultures such as corn,
wheat, rice, sorghum and sugar cane, like this being with frequency, in
experiments seeking the agronomic utilization as biofertilizers (Bashan and
Levanony, 1990).
Puente et al. (2005) observed greater tiller numbers, root dry matter
and number of spikelets per plant when wheat seeds were inoulated with
several Azospirillum strains. Azospirillum species are commonly found in
soils and in association with roots of plants namely rice, maize, wheat and
legumes. Rhizosphere colonization by Azospirillum species has been shown
to stimulate the growth of a variety of plant species. Chemotaxis is one of
the several properties which may contribute to survival, rhizosphere
colonization and the initiation of mutualistic interactions by Azospirillum
species (Lopez-de-victoria, 1989).
The isolation of Azospirillum spp. is capable of high rates of N 2
fixation and suitable for use as biofertilizers and is also potentially of great
importance to modern agriculture. In the present study, the effects of
isolated Azospirillum on germination, growth and yield of Triticum
aestivum L. (Wheat) had been undertaken. The aims and objectives of this
research were to study of biofertilizer that can be substituted instead of
chemical fertilizer and to know the biofertilizer which can significantly
effect on the plant growth.
107
Materials and methods
Three grass plants such as Imperata cylindrica (L.) Raeuschel.
(Thetke), Eleusine indica (L.) Gaertn.(Sin ngo myet) and Panicum paludosum
Roxb. (Unknown) were collected from University of Mandalay and Yadanabon
University from June 2012 to February 2013. Wheat seeds (Gyon Phyu) were
collected from Zalote Research Farm, Monywa, Sagaing Region. The
experiments were carried out at the Microbiology Laboratory, Department of
Botany, University of Mandalay. Azospirillum strains were cultured in the
nitrogen free semi solid malate medium (NFb) and Congo Red Agar Medium
(CRA) according to Dobereiner (1980). Leaf area was measured by method of
Yoshida, 1981. Macroscopical and microscopical characters of Azospirillum
strains isolated from roots of three grasses were studied by using Bergey
(2006). The staining procedure was carried out according to the methods
described by Santra et al. (1998). These data analysis was carried out by using
student t-test Excel 2007.
Results
Isolated Azospirillum Strains
HEP-1, 2 and 3 strains were isolated from the roots of Imperata
cylindrica (L.) Raeuschel., Eleusine indica (L.) Gaertn. and Panicum
paludosum Roxb., respectively (Table 1).
Table 1. Isolated Azospirillum strains
No.
Source Plants
Scientific name
Myanmar name
Strain No.
Part
Used
1
Imperata cylindrica
(L.) Raeuschel.
Thetke
HEP-1
Root
2
Eleusine indica (L.)
Gaertn.
Sin ngo myet
HEP-2
Root
3
Panicum paludosum
Roxb.
Unknown
HEP-3
Root
108
Morphological Characters of Azospirillum strain of HEP-1
Azospirillum strain of HEP-1 was found pink colour on CRA
medium. Colonies were irregular. Cells were slightly curved, rod-shaped,
the cells 1.0-1.8 µm in diameter and 1.0-3.0 µm in length, gram-negative.
The optimal temperature was 33ºC and pH was 7.0 (Fig. 2C and 2D).
Morphological characters of Azospirillum strain of HEP-2
HEP-2 strain was found red colour on CRA medium. Colonies were
irregular. Cells were curved rod-shaped, the cells 1.2-2.0 µm in diameter
and 1.5-4.5 µm in length, gram-negative. The optimal temperature is 33ºC
and pH was 7.0 (Fig. 3C and 3D).
Morphological characters of Azospirillum strain of HEP-3
HEP-3 was found dark pink colour on CRA medium. Colonies were
irregular. Cells were straight rod-shaped, the cells 1.0-1.8 µm in diameter
and 1.0-2.5 µm in length, gram-negative. The optimal temperature is 33ºC
and pH was 7.0 (Fig. 4C and 4D).
Effect of Azospirillum on Plumule Length
In germination test, the mean plumule length was ranged from 1.31
to 4.06 cm. The plumule length was found to be highest in treated plant
with HEP-2. The lowest length was observed in control. All treated plants
possessed higher plumule length than the control ones. The treated plants
with HEP-2 have significantly higher plumule length than HEP-1, HEP-3
and control (Table 2).
Table 2. Effect of Azospirillum on plumule length
Mean ± Sd
value (cm)
Control
HEP-1
HEP-2
HEP-3
Control
1.31 ± 1.08
-
-
-
-
HEP-1
3.33 ± 2.05
3.03**
-
-
-
HEP-2
4.06 ± 1.86
6.66**
2.61**
-
-
HEP-3
3.57 ± 1.58
4.92**
–0.91ns
2.00**
-
Treatment
ns = non significant,
*
= significantly different at 0.05 %, ** = significantly different at 0.01 %
109
General characteristics of Azospirillum
Phylum
- Proteobacteria
Class
- β-proteobacteria
Family
- Rhodospirillaceae
Genus
- Azospirillum
Scientific Name
- Azospirillum sp.
The Azospirillum strains are excellent grown in NFb medium,
forming with pellicles on CRA medium, forming dark pink or red colour
(Fig. 1). The diameter of colonies is 1.0 to 2.0 µm, irregular form, undulated
edge along the margin. The genus Azospirillum is curved or straight rodshaped, Gram-negative, motile, cell length range from 1.0 µm to 4.5 µm.
The optimal temperature is 30-40ºC and optimal pH is 5.0 - 8.0 (Bergey,
2006).
C
HEP-
HEP-
HEP-
A
B
Fig. 1. NFb medium and CRA medium
A. NFb medium showing pellicle form
B. CRA medium forming dark pink or red colour colonies
110
A
B
C
D
Fig. 2.
10µm
Isolated Azospirillum strain HEP-1 from Imperata cylindrica (L.)
Raeushel.
A. Habit of Imperata cylindrica (L.) Raeushel.
B. Fibrous root
C. Colonies of Azospirillum on CRA medium
D. Azospirillum strain HEP-1
A
B
C
D
111
10µm
Fig. 3. Isolated Azospirillum strain HEP-2 from Eleusine indica (L.)
Gertn.
A. Habit of Eleusine indica (L.) Gertn.
B. Fibrous root
112
A
B
C
D
10µm
Fig. 4. Isolated Azospirillum strain HEP-3 from Panicum paludosum Roxb.
A. Habit of Panicum paludosum Roxb.
B. Fibrous root
113
Effect of Azospirillum on Radicle Length
In germination test, the mean radicle length was ranged from 2.614.66 cm. The highest length was found in treated plant with HEP-2 and the
lowest length was observed in control. All treated plants possessed higher
radicle length than the control ones. The treated plants with HEP-2 have
significantly higher radicle length than HEP-1, HEP-3 and control (Table 3,
Fig. 5). The germination percentage of HEP-1, HEP-2, HEP-3 and control
were 96%, 99%, 98% and 87%, respectively. It was observed that treated
plants were higher germination percent than the control (Fig. 6).
Fig. 5. Effect of plumule and radicle length, inoculated with HEP-1, 2, 3
and control
Table 3. Effect of Azospirillum on radicle length
Mean ± Sd
value (cm)
Control
HEP-1
HEP-2
HEP-3
Control
2.61 ± 1.05
-
-
-
-
HEP-1
4.05 ± 1.51
7.56**
-
-
-
HEP-2
4.66 ± 1.13
12.84**
3.18**
-
-
HEP-3
4.16 ± 1.41
8.61**
– 0.56ns
2.70**
-
Treatment
ns = non significant, ** = significantly different at 0.01 %.
114
Fig. 6. Effect of wheat germination, inoculated with HEP-1, 2, 3 and
control
Effect of Azospirillum on Plant Height
The mean of plant height was measured from 52.38 to 60.94 cm.
The highest length was found in treated plant with HEP-2 (60.94 cm)
whereas the lowest length was observed in control (52.38 cm). All treated
plants possessed higher plant height than the control ones. The plant height
of treated plants with HEP-2 had significantly higher than HEP-1, HEP-3
and control (Table 4).
Table 4. Effect of Azospirillum on plant height
Treatment
Mean ± Sd
value (cm)
Control
52.38 ± 5.04
HEP-1
58.38 ± 3.06
1.16ns
HEP-2
60.94 ± 1.47
5.64**
– 2.12*
HEP-3
59.88 ± 4.59
3.74**
– 0.77ns
Control
HEP-1
HEP-2
HEP-3
0.62ns
ns = non significant, * = significantly different at 0.05 %, ** = significantly different at 0.01 %.
Effect of Azospirillum on Flag Leaf Area
The mean of flag leaf area was ranged from 5.48 to 7.61 cm2. The
highest area was recorded in treated plant with HEP-2 (7.61cm2) while the
lowest area was observed in control (5.48 cm2). All treated plants possessed
higher flag-leaf area than the control ones. The flag leaf area of treated plant
115
with HEP-2 showed significantly larger than HEP-1, HEP-3 and control
(Table 5).
Table 5. Effect of Azospirillum on flag leaf area
Control
Mean ± Sd
value (cm2)
5.48 ± 1.48
HΕP-1
6.69 ± 0.96
1.95*
HΕP-2
7.61 ± 1.17
3.20*
– 1.72ns
HΕP-3
7.14 ± 0.72
2.87**
– 1.07ns
Treatment
ns = non significant,
0.01%
*
Control
HEP-1
= significantly different at 0.05 %,
HEP-2
HEP-3
0.96ns
**
= significantly different at
Elmerich et al. (1992) stated that several microorganisms can
colonize plant roots and establish useful plant-bacteria associations.
Azospirillum is a plant-growth-promoting-rhizobacterium (PGPR) which
can able to produce hormone-like substances and fix atmospheric nitrogen
in association with grasses. In this research work, Azospirillum strains were
isolated from three grasses. Azospirillum strains HEP- 1, 2 and 3 were
isolated from the roots of Imperata cylindrica (L.) Raeuschel., Eleusine
indica (L.) Gaertn. and Panicum paludosum Roxb. respectively. Therefore,
roots of grasses are the rhizosphere of genus Azospirillum strains. This
observation was similar to those given by Elmerich et al. (1992).
Oh et al. (1999) mentioned that members of the genus Azospirillum
are Gram-negative to Gram-variable, have a curved rod-shaped and are
motile by a single polar flagellum in liquid media and by polar and lateral
flagella on solid media. They also produce a rising pellicle in semi-solid
nitrogen free (NFb) media. In this research work for isolation of
Azospirillum from three grasses were used as NFb and CRA media. The
morphological characters of isolated Azospirillum were similar to the
findings of Oh et al. (1999).
According to Patriquin et al. (1983), Triticum aestivum L. (wheat)
roots was inoculated with Azospirillum brasilense revealed that the massive
adsorption of bacterial cells to the root surface and less adsorption to root
hairs. Root colonization is the key factor in the successful interaction of
116
plants with Azospirillum, which is known to positively affect plant growth.
Azospirillum species are known to colonize root surfaces of several plant
species as well as the interior cortex of cereal roots. In the present study, the
plant treated with Azospirillum showed the greater germination percentage
than the non-treated plant (control). Plumule and radicle lengths of treated
plants were also found higher than control plant. Therefore, it was observed
significantly that the plant treated with Azospirillum affected to plant
growth, these characters were in agreement with Patriquin et al. (1983).
In the present work, the germination percentage of wheat seeds
treated with the isolated Azospirillum strains HEP 1, 2 and 3 were obtained
96%, 99% and 98%, respectively but the control plant was 87%. Therefore,
germination percentages of the treated plants were higher than the control
ones.
Okon (1985) and Wani (1990) mentioned that inoculation of plants
with Azospirillum can result in a significant change in various growth
parameters, viz. increase in plant biomass, nutrient uptake, tissue N content,
plant height, leaf size, tiller numbers, root length and volume in different
cereals.
In the present study, the plant inoculated with Azospirillum showed
the significant change in various growth, such as plant height, flag leaf area.
These changes of growth was similar to those reported by Okon 1985 and
Wani 1990, Okon and Labandera-Gonzalez, 1994.
It was concluded that among the three Azospirillum strains HEP-1, 2
and 3, Azospirillum strain HEP-2 isolated from the root of Eleusine indica
(L.) Gertn. showed that the best growth of wheat. Therefore, according to
Plant-Growth-Promoting-Bacteria (Azospirillum) strain HEP-2 which may
be used as suitable biofertilizer for wheat and also it can be beneficial to the
environmental surroundings of soil. This experiment concerning stimulation
of wheat by effective Azospirillum strains must be followed by investigation
under field condition.
Acknowledgements
We wish to extend our sincere thanks to Dr. Nu Nu Yee, Professor and Head,
Department of Botany, University of Mandalay, for her kind permission, encouragement
and suggestions to carry out the research work under the present topic and for providing the
required facilities for the research.
117
References
Bashan, Y. and H. Levanony (1990). Curent Status of Azospirillum Inoculation
Technology: Azospirillum as a challenge for agriculture. Can. J.
Microbiol, 36, 591.
Bergey’s Mannual for Bacteriolog (2006). Biofertilizer Development, Proceeding of The
Third Asia Pacific Conference On Agricultural Biotechnology. p. 199206 Bowen, G.D. and Reddel, P. 1986. Nitrogen fixation in
Casuarinaceae. In proceeding of 18th IURO world congress Ljublijana,
Yugoslavia.
Boddey, R. M. and J. Döbereiner (1994). Biological Nitrogen Fixation Associated With
Graminaceous Plants. In: Okon, Y.(Ed.). Azospirillum Plant
Associations. USA: CRC-Press. pp: 119-130.
Döbereiner, J. and V. L. D. Baldani (1980). Host-Plant Specificity In The Infection of
Cereals With Azospirillum spp. Soil Biol. Bioche., 12:433-439.
Elmerich, C., W. Zimmer and C. Vieille (1992). Associative Nitrogen Fixing Bacteria. In
Biological Nitrogen Fixation. p. 212-258. Edited by G. Stacey, R.H.
Burris, and H.J. Evans, Chapman and Hall, New York.
Lopez-de-Victoria, G. (1989). Chemotactic Behaviour of Deep Subsurface Bacteria
Toward Carbohydrates, Amino Acids and A Chlorinated Alkene. M.Sc.
Thesis (unpublished), University of Pureto Rica. Rio Piedras.
Oh, K. H., C. S. Seong, S.W. Lee, O. S. Kwon and Y. S. Park (1999). Isolation of
Psychrotrophic Azospirillum sp., and Characterization of Its Extracellular
Protease. FEMS Microbiol. Lett. 174: 173-178.
Okon, Y. (1985). Azospirillum As Pontential Inoculants For Agriculture. Trends in
Biotechnol., 3:223-228.
Okon Y. and CA. Labandera-Gonzalez (1994). Agronomic Application of Azospirillum. An
evaluation of 20 years worldwide field inoculation. Soil Biol. Biochem.
26: 1591-1601.
Patriquin, D. G., J. Döbereiner and D. K. Jain (1983). Sites and Processes of Association
Between Diazotrophs and Grasses. Can. J. Microbiol. 29: 900-915.
Puente, M., M. S. Montecchia and A. Perticari (2005). Evluation of Azospirillum inoculant
Strains In Wheat. in: SAGPy A-INTA (Ed.), 7th International Wheat
Congress, Mar del Plata, BA. Argentina, SAGPyA-INTA, Argentina,
Pen, C.D.
Santra S. C., T. P. Chantterjee and A.P. Das (1998). College Botany Practical. Vol. II. New
Central Book Agency (P) Ltd.
Wani, SP. (1990). Inoculation With Associative Nitrogen Fixing Bacteria: Role In Cereal
Grain Production Improvement. Indian J. Microbiol., 30, 363-393.
Yoshida, S. (1981). Fundamentals of Rice Crop Science. The International Rice Research
Institute (IRRI). Los Baños, Laguna, Philippines.
Production of ɑ-Amylase Enzyme by Bacillus subtilis from
Some Raw Starchy Materials in Mon State
Khin Kye Mon
Abstract
The ɑ-amylase enzyme was fermenting by Bacillus subtilis. Bacillus subtilis
was isolated from soil by soil dilution method for the preliminary
investigation of ɑ-Amylase. The secretion of ɑ-amylase enzyme showed the
hydrolyzing activity of starch, preliminarily tested on the starch agar
medium. The end products appeared from the hydrolysis reaction of soluble
starch by B. subtilis ɑ-amylase had identified by TLC technique. The effect
of pH on the production of ɑ-amylase was experimented in pH 5, 6, 7, 8 and
9 and the effect of various temperatures on the ɑ-amylase production were
investigated at 30ºC, 40ºC, 50ºC, 60ºC, 70ºC, 80ºC and 90ºC in soluble
starch medium. The hydrolyzing activity of crude ɑ-amylase on the some
raw starchy materials was comparatively studied using corn starch, rice
starch, and tapioca starch. The microscopic examinations of the hydrolysis
reaction of ɑ-amylase on the starch grains of some raw materials were also
investigated at reaction time and comparatively recorded in
microphotographs.
Key words: amylase, enzyme, hydrolysis, investigated, isolated, materials,
medium, starch
Introduction
Enzymes are proteins with catalytic properties due to their power of
specific activation. The enzymes are always present in all living cells. They
perform a vital function by controlling the metabolic process where nutrients
converted into energy.
The studies of enzymes started in early nineteenth century but the great
development had come during the last 40 years. In 1833, Payen and Persoz
made the clear recognition of amylase. They found that an alcohol
precipitation of malt extract contained a thermostable substance named
“diatase” which converted starch into sugar. Now this substance is calling as
“amylase” (Horikoshi, 1974).
The amylase or starch digesting enzyme of wheat was probably one of
the first enzymes discovered by Kirchoff in 1811. The amylase can divide into
three groups: (1) ɑ-amylase, (2) β-amylase (3) Debranching enzyme. The ɑ-
Lecturer, Department of Botany, Mawlamyine University
120
amylase hydrolyzes the internal ɑ-1, 4 linkages in amylose and amylopectin.
β-amylase splits off the β-1,4 linkages of the amylose and amylopectin.
Debranching enzymes cleaves the ɑ-1, 6 linked branch points of starch
(Mitsugi, 1977).
Structure of amylose
Structure of Amylopectin
Enzyme technology has recently been finding several directions for its
development. These may classified as industrial catalysts, tools for food
production and processing, pharmaceutical uses and analytical and measuring
tools.
Researcher had discovered the amylase enzyme from different sources
such as plants, animals and microorganisms and these enzymes were been
extensively studied. Among them, most of the thermostable amylase can found
only in bacteria. Bacteria are essential participants of important elemental
cycles in nature such as of nitrogen, sulfer, carbon and phosphorous
(Andersson, 1985).
Amylase enzyme can produce from the species of Bacillus, bacteria. In
1917, Boidin and Effornt introduced the production of amylase by B. subtilis.
Later, many researchers around the world extensively studied the enzyme in
different ways, such as production, purification, characterizing and application
in various industries (Andersson, 1985).
121
In Myanmar, being Mon State is an agricultural land, it is enormously
rich in many different kinds of starchy raw materials such as rice, tapioca,
corn, sugarcane, etc., and natural flora of microorganisms. In Mon State,
according to the list of cultivated land of 2012-2013, rice produced from
813,099 acres, corn produced from 3,329 acres, tapioca produced from 851
acres and sugarcane produced from 1,864 acres. It is very applicable to use
these raw materials as a source of substrates and source of microorganism in
the production of enzymes.
In the present research, the preliminary investigation of enzyme
secretion in agar plate culture of B. subtilis, production of amylase in some
raw starchy medium, estimation of enzyme activity and other optimal
condition are throughly studied. The objectives of present study were to
introduce the microbiology, to know the isolation of bacterial colonies from
soil by serial soil dilutions method and to understand the knowledge of
biotechnology.
Apparatus and Glassware
Pyrex and local made glasswares were used in the experiment. Firstly,
these glasswares were cleared with chromosulfuric acid, and rinsed several times
with tap water and then sterilized with autoclave under pressure of 15 lb per
square inch for 15 minutes.
Organism Isolation
Bacillus subtilis was isolated from the soil using by soil dilution
method for the present investigation. It was maintained in the enzyme activity
preliminary test medium (Young, 1976) and then transferred into Starch Agar
Medium and used throughout the present research work.
Preparation of Soil Serial Dilutions
One gram of soil sample was introduced in the conical flask containing
99 ml of sterile distilled water to make a dilution of 1:100. The mixture was
then shaken for about 20 times to separate soil particles. The soil solution was
further diluted to 1:1000, 1:10,000, 1:100,000 and 1:1,000,000 and each of
these dilution was separately poured into sterile test tubes, each time using a
separate sterile pipette under aceptic condition (Fig. 1) .
122
(b)
(a)
Fig. 1 (a-b). Preparation of Serial Soil Dilutions Method
Preparation of Nutrient Agar Medium
Peptone
0.9%
Yeast extract
0.1%
Malt extract
0.5%
MgSO 4
0.1%
KH 2 P0 4
0.1%
Agar
1.5%
pH
6.5
Plating
The sterile medium cooled to 45ºC and poured into each of the
petridishes containing the respective serial dilutions. The plates then incubated
at 30ºC for 3-5 days.
Enzyme Activity Preliminary Test Medium (Young, 1976)
Soluble starch
1.00%
Peptone
0.10%
Yeast extract
0.05%
Malt extract
0.05%
KH 2 PO 4
0.10%
KNO 3
0.01%
MgSO 4
0.01%
ZnS0 4
0.01%
FeSO 4
0.01%
Agar
1.50%
pH
5.50
123
Preliminary Test of Starch Hydrolysis Activity
A two days-old culture of B. subtilis was inoculated onto the center of
presolidified starch agar medium and incubated at room temperature. After 2
days, iodine reagent poured on to the culture agar surface.
Thin Layer Chromatography (TLC) for Identification of Action Patterm
of ɑ-Amylase
The initial hydrolysis products from the substrates by the action of
crude ɑ-amylase enzyme was identified by TIC as follow: as soon as the
hydrolysis reaction started 1 - 2 ml amount of sample taken out at various time
intervals and the reaction terminated by placing the sample tubes in boiling
water for 5 minutes. They centrifuged to remove the higher polysaccharides.
An aliquote of 1 to 2 µg of each sample was subjected to precoated silica gel
TLC plate (20 × 20 cm, E. Merk, Germany) by three ascents of the solvent
system of Isopropyl, Butanol, water (2 : 5 : 3 by volume). After developing the
three ascents in the solvent system, sugar detected by spraying with 20%
sulfuric acid in methanol and charring for 5 minutes at 110ºC.
Enzyme Assay
The activity of ɑ-amylase enzyme was determined by detecting the
amount of reducing sugar liberated by the action of enzyme described by
Madsen (1973), Norman and Slott (1973).
The reaction mixture containing 0.5 ml of 1% soluble starch in 0.1 M
acetate buffer, pH 4.5 and 0.5 ml of crude enzyme in final volume of 1 ml was
incubated at 40ºC for 30 minutes. The amount of reducing sugar liberated by
the reaction which measured by DNS method. One unit of ɑ-amylase activity
was defined as the amount of enzyme catalyze the liberation of 1µ mol of
reducing sugar as maltose per minute from the soluble starch substrate under
the above experimental conditions. The unit of enzyme activity was calculated
by using the formula below.
Unit =
μ mol of Maltose × Total reaction volume × Dilution
Reaction time × Enzyme ml or mg Protein
124
Determination of Reducing Sugar by Dinitrosalicylic Acid Method.
The reducing sugar appeared by the hydrolytic action of ɑ-amylase
produced by the bacteria was determined by the method of Dinitrosalicylic
Acid (DNS Method) (Miller, 1959).
Results and Discussion
Characteristic Features of Bacillus subtilis
The morphology of B. subtilis was rod shaped 2.0 - 4.0 µm to 0.6 - 0.9
µm. It was a kind of gram positive, aerobic spores-forming bacteria. They
possessed peritrichous flagella and hence show the motility. The spores are
1.0-3.0 by 0.5-0.8 µm and oval or spherical in shape (Fig. 2).
(a)
(b)
(c)
5µm
Fig. 2 (a-c). (a) Culture of agar plate, (b) Subculture of organisms made
on agar slant, (c) Characteristic feature of B. subtilis
Preliminary Test of Starch Hydrolyzing Activity of B. subtilis ɑ-Amylase
The secretion of amylase enzyme showed the hydrolyzing activity of
starch by preliminarily tested on the starch agar medium. The B. subtilis spores
inoculated. After 24 hours incubation at 30ºC, a small amount of iodine
reagent waspoured onto the surface of the medium. The results were shown in
Fig. 3. Formation of clear zone around the bacterial streak indicated that the
enzyme hydrolyzed the starch molecules into smaller units such as maltose,
maltodextrin, dextrin, etc., which did not show blue-black colour reaction with
iodine molecule. This showed the rapid lost of viscosity and of the iodine
staining of the amylose. Other places away from secreting enzyme was stained
blue black indicated that there was no hydrolyzing activity with starch and
enzyme. Therefore, it confirmed that the isolated bacteria, which used in this
experiment undoubtedly secreted enzyme that could be hydrolyzed the starch.
125
Fig. 3. Formation of clear zone around the colony of B. subtilis indicates
the secretion of starch hydrolyzing enzyme
Analysis of End Products in the Reaction Mixture Containing Soluble
Starch and Crude B. subtilis ɑ-Amylase by TLC
The reaction mixture contained 0.2 ml of B. subtilis ɑ-amylase and 1%
soluble starch suspended in 10 ml of 0.1 M acetate buffer, pH 4.5. The other
optimal conditions in this experiment were same as though formerly described.
The aliquote amount of sample (5 ml) was taken out at 0 minute, 30 minutes, 1
hour, 2 hours, 3 hours and 4 hours. The samples subjected to TLC analysis.
The results of TLC were shown in Fig. 4 and it found that the maltose is the
dominant sugar appeared as the reaction time lenghthen. Some spots of
glucose could found in 3 hours and 4 hours samplings.
G = Standard Glucose
M= Standard Maltose
S = 1% Soluble Starch
E = Crude ɑ-Amylase
O = 0 hours
1 = 30 minutes
2 = 1 hour
3 = 2 hours
4 = 3 hours
Fig. 4. TLC of crude ɑ-Amylase which produced by B. subtilis on 1%
soluble starch.
In 0 hour sample only minute amount of maltose 6.7 µg was observed
and its concentration gradually increased in 1 and 2 hours samples. However,
it decreased in 3 hours sampling. The higher oligomer was observed in TLC.
126
By analyzing the data obtained from TLC, the B. subtilis ɑ-amylase was a kind
of liquefying amylase and no glucoamylase activity found.
Effect of Different pH on the Activity of Bacillus subtilis ɑ-Amylase
The effect of different pH on the activity of B. subtilis ɑ-amylases was
experimented in a series of various pH. The reaction mixture contained 2%
soluble starch as substrate, 0.754 unit ɑ-amylase dissolved in total 10 ml of 0.1
N acetate buffer. The pH of the reaction mixture in different flasks adjusted to
4, 5, 6, 7, 8, 9 and 10 and they incubated at 80ºC for 2 hours in the shaking
water bath. The enzyme activity in different flask were determined and shown
in Table 1 and Fig. 5.
Table 1. Effect of pH on ɑ-amylase produced by B. subtilis
pH Amount of Reducing sugar (mg ml-1)
Enzyme Activity (µ ml-1)
4
1.83
0.33
5
3.68
0.68
6
3.84
0.79
7
4.85
0.89
8
4.49
0.83
9
2.49
0.46
10
2.49
0.46
1
0.9
0.8
Relative Acitivity
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
4
5
6
7
8
9
10
pH
Enzyme Activity (µ/ml)
Fig. 5. Effect of pH on the ɑ-amylase activity produced by B. subtilis
127
The highest amount of reducing sugar 4.85 mg ml-1 and the maximum
amount of enzyme 0.89 µg ml-1 were observed in the flask adjusted to pH 7.0.
The minimum amount of reducing sugar and enzyme were detected in the
flask of pH 4 and 9.
In 1972, Boyer and Ingle reported that an alkaline ɑ-amylase extracted
from the Bacillus species showed the maximum activity at pH 9 to 9.2.
According to Horikoshi et al. (1974), a liquefying ɑ-amylase from Bacillus
species No. 38.2 showed the broad optimal pH values of 4.5 to 9. A
theriaophillic ɑ-amylase, which isolated from B. licheniformis was broad pH
activity between 5.0 and 8.0 (Saito, 1973).
The present results showed the optimal activity at pH 7 closed to the
results of Saito (1973). The two other bacterial ɑ-amylases with optimal pH7
was found in Thermoactinomyces vulgaris and thermophile V-2. In general,
the fungãl amylase and some bacterial acidic ɑ-amylase always showed the
low optimal pH value 3.5 to 5.5. On the other hand, the alkaline amylase
possessed the high optimal pH value. The present result of B. subtilis ɑamylase indicated that the enzyme was a the mostable enzyme (80ºC
optimum) and neutral pH 7.0. It was in accordance with the findings of Ingle
and Erickson (1978). A heat stable type of ɑ-amylase from Bacillus
amyloliguefaciens which showing optimal pH 7 by Madsen (197). It was
generally known that most of the thermophillic ɑ-amylase lost their activity if
pH fell below 6.
Effect of Various Temperatures on the Activity of B. subtilis ɑ-Amylase
The effect of various temperature on the activity of B. subtilis was
experimented at 30ºC, 40ºC, 50ºC, 60ºC, 70°C, 80°C and 90ºC in a reaction
mixture containing 2% soluble starch as substrate. About 0.754 unit of crude ɑamylase dissolved in 10 ml of 0.1 M acetate buffer pH 4.5 used in each conical
flask. The enzyme activity in each flask incubated at different temperatures
was determined after the incubation periods of 15 minutes,
30 minutes, 1
hour, 2 hours, 3 hours and 4 hours and shown in Table 2.
The highest activity, 2.01 units per ml observed in the flask incubated
at 80ºC after 2 hours. At 30ºC only 0.027 units ml-1 was detected. These results
indicated that the B. subtilis ɑ-amylase showed a thermostable amylase. At
90ºC, the lesser amount (1.52 units ml-1) was measured at 1 hour incubation
period but it gradually declined to 0.91 unit ml-1 at 4 hours reaction time. The
temperature curve of B. subtilis ɑ-amylase shown in Fig. 6 and it noticed that
128
the enzyme activity increased directing with the increased temperature up to
80ºC. Above that temperature, the enzyme activity decreased gradually. This
indicated that 80ºC was the maximum temperature for this enzyme activity.
Table 2. Effect of temperature on the ɑ-amylase activity produced by B. subtilis
TºC
40
50
60
70
80
90
30 mins
1 hr
2 hrs
3 hrs
4 hrs
R-Sugar
0.150
0.260
0.100
0.150
-
-
µg/ml
0.027
0.048
0.018
0.027
-
-
R-Sugar
0.540
0.480
0.420
0.480
-
-
µg/ml
0.090
0.080
0.077
0.080
-
-
R-Sugar
0.100
0.990
0.990
1.190
-
-
µg/ml
0.018
0.180
0.180
0.190
-
-
R-Sugar
1.980
1.980
1.980
1.990
-
-
µg/ml
0.360
0.360
0.360
0.367
-
-
R-Sugar
2.550
3.860
5.430
4.880
3.860
4.090
µg/ml
0.460
7.710
1.000
0.900
0.710
0.750
R-Sugar
3.490
6.010
7.750
10.910
8.540
5.380
µg/ml
0.640
1.110
1.460
2.010
1.580
0.990
R-Sugar
4.700
5.090
8.250
5.090
5.880
4.930
µg/ml
0.860
0.940
1.520
0.940
1.080
0.910
2.5
Amount of enzyme activity (µg/ml)
30
15 mins
2.0
1.5
1.0
0.5
0.0
15
30
60
120
180
240
Tim e (m in)
30º
40º
50º
60º
70º
80º
90º
Fig. 6. Effect of temperature on the ɑ-amylase activity produced by
B. subtilis
129
In 1973, Saito reported that a thermophillic extra-cellular ɑ-amylase from
B. licheniformis showed the optimal temperature of 76ºC at pH 9.0. Ingle and
Erickson (1978) reported their review that the thermostable amylase could be
isolated from Thermomonosora curvata and Thermoactinomyces vulgaris where
optimum activities were shown at 65ºC and 60ºC, respectively. Moreover, the
thermosabie ɑ-amylase from B. stearotbermophilus showed the optimum activity
at 70ºC and an acidic ɑ-amylase from B. acidocaldarius possessed the temperature
optimum of 75ºC.
According to Madsen (1973), the optimal temperature for the
liquefaction reaction of starch by ɑ-amylase from B. amyloliquefaciens was
90ºC. In the present investigation, it found the B. subtilis ɑ-amylase gave the
maximum activity at 80ºC. Therefore, it can be assumed that enzyme secreted
by B. subtilis was thermostable ɑ-amylase.
Hydrolysis Activity of Crude Emzyme on the Indigenous Raw Starch
The hydrolyzing activity of crude B. subtilis ɑ-amylase on different
indigenous raw materials comparatively investigated and the results shown in
Table 3 and plotted in Figure 7. Therefore, at the beginning of hydrolysis up to
15 minutes, the ɑ-amylase showed more hydrolysis capacity on rice than on
soluble starch and others. However, the more reaction time lengthen the higher
hydrolyzing activity appeared on the soluble starch. It was reasonable to infer
that the less complexity of soluble starch gave the higher percentage than more
complex structure of natural raw starch. It noted that at the time of one-hour
reaction period only 56.9% observed in rice relative to soluble starch.
Table 3. Effect of the hydrolysis activity of enzyme on the indigenous raw
starch
S. starch
R-sugar
(mg/ml)
Rice
R-sugar
(mg/ml)
Corn
R-sugar
(mg/ml)
Tapioca
R-sugar
(mg/ml)
5
2.80
4.41
2.75
2.81
15
3.49
4.80
3.94
2.97
30
6.01
4.90
3.94
3.37
60
7.75
4.41
3.97
2.81
120
10.91
3.62
3.15
2.81
180
8.54
3.62
3.15
2.81
12
10
Amount of reducing sugar
Time
(min)
8
6
4
2
0
5
15
30
60
120
180
Tim e (m in)
S. starch R-sugar (mg/ml)
Rice R-sugar (mg/ml)
Corn R-sugar (mg/ml)
Tapioca R-sugar (mg/ml)
Fig. 7. Hydrolyzing activity
of Crude B. subtili ɑamylase on different
indigenous starchy raw
materials
130
Udaka et al. (1984) discovered a Bacillus strain, which could secrete
endolytic ɑ-amylase. That enzyme showed the 50-70% digestibility on corn
and tapioca relative to rice starch. The conditions of Hydrolysis at one-hour
period of ɑ-amylase on the different starchy materials, were comparatively
recorded in photomicrographs and shown in Fig. 8. The conditions of
degradation in each starch granules supported the results of digestibility in
present study.
(a)
(c)
Soluble starch
Rice starch
(b)
Corn starch
Fig. 8 (a-d).
(d)
Tapioca starch
Microphotographys showing the conditions of enzyme
hydrolysis on various starch grains of indigenous raw
materials by crude B. subtilis ɑ-amylase. They are taking after
1-hour enzyme hydrolysis at 80ºC.
Conclusion
Starch is an important raw material for food, beverage and
pharmaceutical industries in Myanmar. The use of starch in industrial sector is
demanding more and more as our country is going through open marketing
policy with foreign countries.
Out of many enzymes that hydrolyze the starch-molecules the ɑamylases catalyzed a random hydrolysis of ɑ-1, 4 bond of starch. One of the
advantages in the use of bacterial ɑ-amylase is that the operation is quite heat
stable and it can be used in starch hydrolysis up to 90ºC.
131
According to Madsen et al. (1973), the B. licheniformis ɑ-amylase is
capable of starch hydrolysis in 105º to 110ºC. Another favor of bacterial
ɑ-amylase is the relatively low cost.
In the present investigation, the production and characterization of B.
subtilis ɑ-amylase were investigated. The aim of the present investigation is to
initiate the enzyme technology. It is sure to get achievement in the
manufacture of various materials by using ɑ-amylase because it is not only
thermophilic but also shows a wide optimal pH and temperatures. Although
there were very limited facilities and insufficient chemicals, the concrete data,
fundamentally important in the production of such as industrially useful
enzymes were thoughly evaluated.
This paper attempts to give the knowledge of one of the industrial
materials, ɑ-amylase enzyme, which can be produced from the raw starchy
materials by microorganisms, Bacillus subtilis and to transfer this enzyme
technology to the entrepreneurs in Mon State who are interested in the enzyme
industry, which is potentially, prosperous in the near future.
Acknowledgements
I wish to express my sincere thanks to Rector, Dr. Htay Aung and Pro-rector
Dr. Aung Myat Kyaw Sein of Mawlamyine University for giving the opportunity to carry out
this present paper. I would also like to express my special thanks to Dr. Marlar Aung,
Professor and Head of Botany Department, Mawlamyine University for her permission and
kind help to write this research paper. I also want to show my gratitude to Dr. Khin Lat Lat
Mon, Professor, Botany Department, Mawlamyine University, for her encouragement. Special
thanks extended to Dr. U Win, Rector (Retd), Hinthada University for his guidance and
valuable suggestions. I also extend my thanks to Dr. Khin Lay Nwe, Lecturer, Department of
Botany, Mawlamyine University for her help in aspects during the preparation of this work.
References
Andersson, E., A. C. Johansson and B. Hahn-Hagerdal (1985). ɑ-Amylase Production in
Aqueous Two-Phase System with B. subtilis. Enzyme and Micro. Technol.
7:333.
Boyer, E. W. and M. E. Ingle (1972). An Alkaline Amylase from Pseudomonas stutzeri.
J. Bacterial. 110:992.
Horikoshi, K.; Y. Ikeda and Y. Tanaka (1974). Amylolytic Enzymes from Bacteria. Industrial
Enzymes from Microbial Sources. Halpern. M.G.H., 1981. Data
Corporation, New Jersey, U.S.A P. 31.
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Ingle, M. B. and R. J. Erison (1978). Bacteria ɑ-Amylase, Advances in Applied Microbiology.
p. 24, 259.
Madsen, G. B., B. E. Norman and S. Slott (1973). Bacterial ɑ-Amylase, Applied Biochemistry
and Bioengineering. Vol. 2. p. 59. Enzyme Technology. Lemucl B. Wingard,
Jr., 1979. Academic Press. New York.
Mitsugi, K. (1977). US Patent 4, 022, 666. Industrial Enzymes from Microbial Sources. G.H.
Halpem. 1981. p. 29.
Miller, G. L. (1959). Use of Dinitrosalicylic Acid Reagent for Determination of Reducing
Sugar. Anal. Chem. 31 (3): 426.
Saito, N. (1973). A Thermophilic Extracellular ɑ-Amylase from Bacillus licheniformis.
Archieves of Biochemistry and Biophysics, Achdemic Press, New York and
London. 155(2):290.
Udaka, et. al. (1984). Isoglucose or fructose syrups. p. 166-170. Feeding Tomorrow’s World.
Sextant. Sasson, A. 1990.
Young, M. M. (1976). Patent U.S. 3:944, p. 323.
Significances of Urban Forest on the Environmental Conditions
of Magway City
Myat Thu
Abstract
This paper deals with the factors of urban forest that affect some
environmental conditions like temperature variation and how monitoring of
urban forest can reduce air pollution. The role of urban vegetation on the soil
water holding capacity is tested to compare the temperature differences
within under urban forest and out of urban forest. It can be seen differences
between that urban forest can reduce the temperature significantly than out
of urban forest area. From the results, urban forest can greatly mitigate from
air pollutants compared with out of urban forest. Results from test models,
show that urban vegetation area is the best and beneficial to soil water purity.
For each study area, inventory of to the plants that include in each area was
presented and also calculate the percentage of canopy was calculated and
relevant photographs are described.
Key words : urban forest, air pollution, temperature, canopy
Introduction
Magway city is located in the eastern bank of Ayeyarwaddy river and
also the capital of Magway region. The area of Magway City is about (18.1)
∙
′
∙
′
square mile and situated between East longitude (94 55.31 ) and (94 .55.68 )
∙
′
∙
′
and North latitude (22 07.47 ) and (22 09.27 ). It is included in the central
dry zone of Myanmar. So the weather condition is almost semidescent.
Therefore in summer, the temperature is so high, in rainy season, the
precipitation is fewer than other regions but in winter, it is moderately cool. By
this reason, in Magway City, the role of urban forest is so significant. If there
is no dense urban forest, all the people who live in this city might suffer from
many impacts like very high temperature in summer, very few precipitations in
rainy season and very cool condition in the winter. By looking overall point of
view, the situation of urban forest in Magway City is satisfactory for now but
most of the trees and other plants are growing wild and there is no systematic
maintenance. So on the landscape point of view, there are many things to do
though overall canopy is acceptable.
Assistant Lecturer, Department of Botany, Magway University
134
The role of urban forest is very significant not only in this city but also
all the cities that include in all countries. It gives us many benefits in our daily
life. They can reduce percentage of carbon present in the atmosphere by using
it in the process of photosynthesis. So they play a vital role to solve the
problem of green house effect. This is a global concerning problem. Another
important factor is landscape decoration. If a city is decorated with different
kinds of plants that means systematically plotted urban forest, this city would
be is so graceful and everybody who come this place would get immeasurable
pleasure. Urban forest gives not only pleasure but also protection against air
pollutions, soil erosion and some other bad environmental conditions. In this
paper, four sample selected areas in Magway City are used to do research
about urban forest. For each area, firstly inventory the plants that include in
each area was made and then measuring circumferences of these plants and
calculating their Diameter at Breast Height (DBH). Secondly, percentage of
canopy in each study area by using densitometer is measured. And then, the
temperature differences between the places where almost completely canopy
and very few canopy for each study area are measured. At the same time, how
the urban forest plants can reduce from air pollution by retaining the pollutants
on their plant parts was measured. Finally, experiments concerning this urban
vegetation can improve the purity of soil water and reduce the soil erosions
were conducted.
The aims of this paper are (1) to share the knowledge on the impact of
urban forest in our daily life (2) to estimate the benefits of urban forest by
looking at the different data of inner urban forest and outer urban forest and
(3) to know the value of urban forest and its sustainable maintenance.
Experimental sites
To study some significances of urban forest on environmental
conditions, four sample areas in Magway City are (1) in front of Botany
Department, University of Magway, (2) beside the Strand Road (3) in the
Independence Park and (4) in the campus of State High School No.(3) were
selected. Materials to study for each area are ruler, densiometer, thermometer,
(9½) inches filter paper, hand lens, three artificial models with pollutants, steel
tray and 500 ml beaker.
135
To study the selected areas, firstly inventory of each area was made.
Every tree that include in it was measured. When measuring the trees, there are
two kinds of measurements, they are their circumference (C) and Diameter at
Breast Height (DBH). For measuring circumference, trees are measured (4½)
feet above the ground. To calculate the Diameter at Breast Height of each
plant, measurement of circumference is divided by 3.14. The result is the
Diameter of Breast Height (DBH) of the trees. (Bebette, et al., n.d.)
Construction of Densiometer
Secondly, the canopy of each area is measured by using densiometer.
This is a hand made instrument. For making densiometer;
(1) 3-inche PVC pipe is cut into 6-inches length pieces.
pieces.
(2) The nylon strings are cut off to a length of 6 inches into three
(3) Tape the two 6-inches strings to make (X) shape at the top of the
PVC pipe.
(4) A nut is inserted on the other 6-inches string and tape it the bottom
of the pipe.
Estimation of canopy by using densiometer
By this way, a densiometer measures the canopy of the urban forest. To
use it, densitometer is placed vertically and the alignment of densitometer is
checked by looking up through the nut (from the bottom) and (X) cross point
as a target. To calculate the canopy, two diagonal distances for every three feet
are used. If there is a canopy at a point, a tick () is marked and there is not a
canopy a cross (x) is marked. Then the percentage of canopy is calculated by
using these recorded data.
Estimation of temperature differences
In the third step, the temperature differences of each area are compared
between canopy and that at without shaded area. To measure this, two
thermometers are placed at two places at the same time. One is hanged on the
tree where there is almost complete shade and another one is hanged very few
or no shaded tree for three days and the temperatures were recorded at 10:00
a.m., 12:00 noon, 2:00 p.m. and 4:00 p.m. daily. And then the recorded data
between them are compared.
136
Estimation of the presence of air pollutant between canopy and non
canopy area
The fourth step is testing how the urban forest mitigates air pollution.
To test it, the two same size (9½-inches in diameter) filter papers are placed on
the different trees that were not far from one another. One is on the tree which
is covered by the leaves around it and the other one is on the tree which has no
leaf cover. Both papers are sprayed with water three times a day and placed for
(5) days. After that the sticking rate of particles and other air pollutants on the
papers are compared.
Estimating the condition of different soils by using artificial models
Finally, how urban vegetation is important to the purity of soil water
and reduction of erosion is tested. To perform this process, artificial conditions
of urban ground is used. There are mainly three conditions for urban area.
They are ground without vegetation, ground that is covered by concrete, tar or
pavement and ground with plants vegetation. So three, 2 liters bottles are used
for three conditions. The first bottle is filled with soil and pressed down with
hand to be more dense. This represents the urban area which has no vegetation
or any other cover. The second bottle is filled with the soil and covered with
three layers of plastic and tuck around edges inside the bottle. This represents
the urban area covered by pavement, tar or concrete. The third bottle is filled
with the soil and caused with a small section of grass and other plants. This
condition represents the urban area where there is vegetation. These three
models are represents urban compacted soil area, paved area and forest area,
respectively.
The steps to prepare for testing these models are;
(1) Fill the varieties of pollutants that can be present in the urban area.
(a) 1 spoon of dry leaves powder to represent the rubbish.
(b) 1 spoon of color chalk powder to represent some coloring wastes
(c) 1 spoon of engine oil to represent motor oil.
(2) Prop up the models 20 degree with a cork block.
(3) Place the empty tray at the open end of the model to catch runoff.
(4) Pour slowly 500 ml of water to represent rain on model for two minutes.
(5) Note the time in seconds when runoff starts to enter the empty tray.
137
(6) Pour water from the tray into a measure cup (beaker) and measure its
volume and check the color of water and then record it on the data sheet.
(b)
(a)
Fig. (1) (a) Measuring the circumference, measure 4½ feet above the ground
(b) Top view of densiometer
Fig. ( 2) Hand magnify glass to watch out the rate
of pollutant particles on the filter paper
Fig. (3) Add pollutants to the models.
(a) color powder
(b) rubbish
(c) engine oil
(a)
(b)
(c)
Fig. (4) Props up the model
20 degree with a cork block
Fig. (5) Measure 500 ml of water to pour
in the models
138
Results
The following data are resulted from the study area (1) that are located
in front of Botany Department, University of Magway. In this study area, the
length of East to West is (130) feet and North to South is (85) feet. So the area
is (11050) square feet.
Study area (2) – Beside the Strand Road
-
Canopy percentage of study area is 75.8%.
-
Temperature range between shaded and non-shaded areas is 3 C -
∙
∙
4 C.
-
Sticky pollutant particles on the filter papers between shaded and
non- shaded areas are much difference.
Study area (3) In the Independent Park
-
-
∙
∙
6 C.
-
non- shaded areas are not much difference.
Study area (4) In the Campus of State High School No. (3)
-
-
∙
∙
9 C.
-
non- shaded areas are much difference.
all the results are very significantly between urban forest area and other
area (Table 1-4 and Fig. 1-12).
139
Table 1. Inventorying the plants located in front of Botany Department
Circumference
Inches (C)
DBH
C/3.14
DBH(Inches)
Approximate
Butter tree
30.5
9.7
10
Egayit
Indian cork
tree
20.5
6.52
7
Example 3
Yetama
India mast
tree
9.5
3.02
3
Example 4
Zi
Plum
13
4.14
4
Example 5
Tama
Neem
21.5
6.84
7
Example 6
Egayit
Indian cork
tree
36.6
11.02
11
Example 7
Yetama
India mast
tree
16
5.09
5
Example 8
Seinpangyi
Peacock
flower
34.5
10.89
11
Example 9
Seinpangyi
Peacock
flower
25
7.96
8
Example
10
Yetama
India mast
tree
16
5.09
5
Example
11
Tama
Neem
30.5
9.75
10
Example
12
Egayit
Indian cork
tree
22.5
7.16
7
Example
13
Egayit
Indian cork
tree
44
14.01
14
Example
14
Khaye
Star flower
18
5.73
6
Example
15
Egayit
Indian cork
tree
23.5
7.5
8
Tree No.
Myanmar
Name
Example 1
Myintzutha
kanatpan
Example 2
English
Name
140
Circumference
Inches (C)
DBH
C/3.14
DBH(Inches)
Approximate
Butter tree
12.5
3.72
4
Seinpangyi
Peacock
flower
27
8.59
9
Example
18
Hthanaung
White barked
acacia
32.5
10.35
10
Example
19
Egayit
Indian cork
tree
32.5
10.35
10
Example
20
Egayit
Indian cork
tree
20.5
6.52
6
Example
21
Yetama
India mast
tree
13.5
4.2
4
Example
22
Tama
Neem
22
8.91
9
Example
23
Seinpangyi
Peacock
flower
34.5
10.98
11
Example
24
Egayit
Indian cork
tree
27.5
8.75
9
Example
25
Egayit
Indian cork
tree
28
8.91
9
Example
26
Egayit
Indian cork
tree
25
7.96
8
19
6.05
6
Tree No.
Myanmar
Name
Example
16
Myintzutha
kanatpan
Example
17
English
Name
Indian cork
tree
Example
27
Egayit
Example
28
Egayit
Indian cork
tree
20.5
6.5
7
Example
29
Egayit
Indian cork
tree
10.5
3.34
3
141
Circumference
Inches (C)
DBH
C/3.14
DBH(Inches)
Approximate
India mast
tree
14
4.5
5
Egayit
Indian cork
tree
37
11.84
12
Example
32
Yetama
India mast
tree
32
19.5
20
Example
33
Thabyut
Maseen
19.5
6.21
6
Example
34
Kokko
Rain tree
81
25.8
26
Example
35
Seinpangyi
Peacock
flower
35
8.59
9
Example
36
Egayit
Indian cork
tree
37.5
11.9
12
Example
37
Yetama
India mast
tree
12
3.82
4
Example
38
Tama
Neem
16
5.05
5
Example
39
Tama
Neem
17
5.5
6
Example
40
Tama
Neem
14
4.5
5
Sha
Acacia
10.5
3.34
3
Example
42
Egayit
Indian cork
tree
17.5
5.6
6
Example
43
Maezel
Butter tree
8.5
2.7
3
Tree No.
Myanmar
Name
Example
30
Yetama
Example
31
Example
41
English
Name
142
Circumference
Inches (C)
DBH
C/3.14
DBH(Inches)
Approximate
India mast
tree
7.5
2.4
2
Tama
Neem
18.5
5.9
6
Example
46
Seinpangyi
Peacock
flower
43.5
13.85
14
Example
47
Tama
Neem
26.5
8.5
9
Example
48
Egayit
Indian cork
tree
29.5
9.32
9
Tree No.
Myanmar
Name
Example
44
Yetama
Example
45
English
Name
Table 2. Measuring canopy by using Densioneted from study area that
located in front of Botany Department
No. of
Measuring
Point
Diagonal (1)
Diagonal (2)
(From North East to South West)
(From South East to North West)
Point 1
Point 2
Point 3
Point 4
X
Point 5
X
X
Point 6
X
X
Point 7
X
X
Point 8
X
Point 9
X
Point 10
X
No. of
Measuring
Point
Diagonal (1)
Diagonal (2)
Point 11
Point 12
Point 13
Point 14
Point 15
Point 16
Point 17
Point 18
Point 19
Point 20
Point 21
Point 22
Point 23
Point 24
Point 25
Point 26
X
Point 27
X
Point 28
Point 29
Point 30
Point 31
Point 32
143
144
No. of
Measuring
Point
Diagonal (1)
Diagonal (2)
Point 33
Point 34
X
Point 35
X
Point 36
Point 37
Point 38
Point 39
Point 40
Point 41
Point 42
Point 43
X
Point 44
X
Point 45
Point 46
Point 47
X
Point 48
X
Point 49
X
Point 50
Point 51
X
Point 52
X
Point 53
X
X
In the above study area, total canopy point is (83) and non canopy is
(23). So the canopy percentage of this area is 78.3%.
Table 3. Temperature comparison between shaded area and non shaded area
145
Fig. (6) Study area located in front of Botany Department
Fig. (7) Thermometers are hanged
on the trees
(a) under the canopy
(b) out of the canopy
(a)
(b
Fig. (8)The filter papers placed
on the trees to monitor
the present of pollutant
(a) on the branch with leaves
(b) on the branch without leaves
146
(a)
(b)
Fig. (9) Sticky pollutant particles
seen under hand lens
(a) occurrence of few particles from
the branch with leaves
(b) occurrence of few particles from
the branch without leaves
(a)
(b)
(a)
(b)
(c)
Fig. (10) (a) Pour 500 ml of water like a rain onto the without vegetation
compact soil model (b) water flowing down into the tray (c) measuring
and examine the color of water
147
(b)
(c)
(a)
Fig. (11) (a) Pour 500 ml of water like a rain onto the pave area
model (b) water flowing down into the tray (c) measuring
(b)
(a)
(c)
Fig. (12) (a) Pour 500 ml of water like a rain onto the urban forest
area model (b) water flowing down into the tray (c) measuring
Table 4. Analysis of recorded data from the three model experiments
Observes
Time of the water
runoff start
flowing into the
tray (second)
Color of water in
the tray
Pollutants
Measurement of
water in the tray
Model change or
not
Fi (29) M
Compacted Soil
without
Vegetation
Paved Area
Urban Forest
10s
4s
45s
Dark brown
Red brown
Pale red brown
much amount of
much amount of
pollutants (rubbish,
pollutants
color particles, oil)
(rubbish, color
and also soil
particles, oil)
particles
very small
amount of
pollutants and
just a few soil
particles
300 ml
450 ml
200 ml
Much change by
water current
No change
Moderate change
i
i
h b k
d
i
h
148
Observes
Water holding
capacity
Compacted Soil
without
Vegetation
Paved Area
Urban Forest
Moderate
Bad
Good
In this paper, four study areas were selected and the influence of
environmental conditions on the urban forest was estimated and how urban
forest is significant for the environment were investigated. But just one study
area could not be accommodated because the results of all study areas were
investigated due to the length of the paper. All the results are very significantly
different between urban forest area and other areas.
Firstly by measuring circumference and Diameter at Breast Height
(DBH) of the plant, the relation between size of trees and percentage of canopy
is calculated. In the area with abundant of trees and large measurement of
DBH, the canopy percentage is high. Another beneficial factor that results
from this practical work is measuring temperature from different places in the
same area.
From this work temperature differences between canopy (shaded) area
and non canopy (non shaded) area can be seen. Looking the collected data,
temperature of non canopy area is more than canopy area but there is some
variation from day to day. One of the conclusions in this study is that the
presence of canopy can reduce the local temperature of this specific site.
The other important factor of urban forest is it reduced pollution for
urban area because tree can retain the pollutant by their leaves. So if there is
many trees in the urban area, people who live in this area will have fewer
chances from direct contact of pollutant. It was observed that the fall of
pollutants on the filter paper, under the canopy is less than that of the paper
with less branches. So urban forest can be reduced pollutants like dust, carbon
particles that are released from exhaust and other sources.
Urban forest can reduce not only pollution and temperature but also
maintain the water holding capacity of soil and can mitigate the rate of soil
erosion of the current water. Tray under the models can be assumed as a river,
lake or stream near the urban area. So if the results of water in the tray are
149
good, water in the river, lake or stream located near the urban area would be
good, but if the results are not good, the flower could not be good. By these
results, in the compacted soil area model, little water was retained in the soil
because there was no vegetation and the rate of water current is fast. So, out of
the 500 ml of input water poured, nearly 300 ml of water flowed down into the
tray and just 200 ml of water are retained in the soil. Beside, this water has
many pollutants, soil particles and oil. So after pouring the water that the
simulates the rain, some configuration of erosion is found in this model. The
color of water in the tray is dark brown. It is in the stage of polluted water. In
the paved area model, water cannot retain in the soil because nearly 450 ml of
water flowed down into the tray. The color of water in the tray is red brown
and there are many pollutants. In the urban forest model, water was highly
maintained because only about 200 ml of water is measured in the tray from
500 ml of input water. Moreover, water in the tray has very low pollutant, very
few soil particles (i.e. can reduce soil erosion) and can decrease the rate of
water current. The color of water in the tray is also pale red brown color. So by
comparing these three models for urban forest, compact soil or soil with no
vegetation can retain little amount of water. Water current can erode to the
surface ground and cannot reduce the rate of pollutants that would be included
in the ground water. In the paved area, soil erosion cannot occur but pollutants
are flowed out together with water current and water current is also so fast that
there becomes flood for urban area. In the urban forest area, the rate of water
flow, pollutant’s content and soil erosion is least among other areas. Moreover,
soil water could be retained moderately and could make less pollutant in it. So
by these results, it clearly inferred that urban forest can naturally benefit our
environments.The role of urban forest is partly important for our ecosystem.
Rural area and forest area are also important, but urban area is mostly contact
with men and suffers many impacts than any other regions because most of the
population of human lives in this area. Human always try to improve their life
and at the same time also destroy their environment. In urban area, many
factories, vehicles and workshops are located and their waste products are
released to the environment and that can become air, water and soil pollution.
If a city that has no or very few urban forest, this city will suffer from these
pollutants effect but if there is dense urban forest, it will mitigate these effects.
So every urban forest of the cities has its benefits and economic and
aesthetic values. The most obvious city in Myanmar is Pyin Oo Lwin. Also
urban forest of Magway City has these values more or less. Although the urban
forest of Magway City cannot give aesthetic beauty, it can reduce pollutions
150
and mitigate climate impacts. By looking the results, temperatures under
canopy and out of canopy are so different; pollutant rate of in the tree and out
of the tree are also very different and urban area with plants can maintain soil
erosion, reduce air and soil pollution and retain soil water for a long time. But
there is something to notice that if most of the plants in the urban area are
deciduous plants, canopy cannot be existed throughout the year. So like the
central dry zone, Magway City, should make plans for planting ever green
trees such as Magyi, Yetama, Kokko and Khayay to release summer heat
impact and is necessary in other places as well.
Thus all the plants in the urban forest are valuable for the environment.
Every tree can give shade and reduce temperature and also mitigate from
pollutants for all houses and all persons. Shrubs and herbs can reduce soil
erosion and also help to purify soil water and aid to clear water into the rivers
and streams. Therefore, all the people who live in city should acknowledge
about the value of urban forest and help their maintenance to existence and
make effort to improve urban forest in the future. In this paper, the important
role of urban forest on positively sustainable environment is experimented
from different aspects. It is hoped that this paper can help to taking notice the
benefits and values of urban forest in our city and try to contribute the long life
existence and improvement.
Acknowledgement
I would like to express my heartfelt thanks to Daw May Than Su, Professor and Head,
Department of Botany, Magway University for her permission to write this paper and for her
thorough guidance throughout this paper.
References
Website
Bebette de, Vera, Martha C.Moroe, and Jennifer A.Seitz. School of Urban Forest Resources
and Conservation, (pdf). University of Florida.
Liisa Tyrvainen, Stephan Pauleit, Klaus Seeland and Sjerpde Vries. Benefits and Uses of
Urban Foersts and Trees (pdf).
http://www.sustainablecitiesinstitute.org
http://en.wikipedia. org/wiki/Urban – forest.
Preservation of Lycopersicon esculentum Mill. Fruits through
Jam Production
Zin Moe Moe
Abstract
In Myanmar, tomato (Lycopersicon esculentum Mill.) is the important
vegetable crop of the country. A wide variety of tomato fruits were available,
and it constitutes part of the daily diet. In this experiment, the constituents
sound tomato fruits were chosen and processed into jams. The physicochemical properties of product were analyzed by pH, acidity, total soluble
solids and moisture content.
Key words: tomato, jams, physic-chemical properties
Introduction
Tomato, Lycopersicon esculentum Mill., is one of the world's most
popular vegetable, which are grown both in home garden and commercially.
Tomatoes are now grown worldwide for its edible fruits. Tomato is originated
from Western South America and it was introduced into European gardens in the
early sixteenth century (Wien, 1997).
Tomato (Lycopersicon esculentum Mill.) is a member of the
Solanacease family. Tomato (Lycopersicon esculentum Mill.) plant has a main
stem and a system of lateral branches. The main stem is erect for the first
alternate, compound, relatively large, well developed, with rather broad
leaflets, and has glandular hairs. The flowers are borne in clusters on the main
axis and on lateral branches. Individual flowers contain a green calyx, yellow
corolla, five or more stamens, and a single superior pistil. The ripe fruit is
large, juicy posses a fleshy ovary (Edmond, 1957; Lawerence, 1969).
The tomato fruits are eaten raw or cooked. Large quantities of tomatoes
are used to produce soup, juice, sauce, ketchup, puree, pasta and powder.
Tomatoes are an important component in salad along with onions, garlic,
peppers, cumin and lime juice (Decoteau, 2000). The seeds of tomato contain
24% oil and this is extracted from the pulp and residues of the canning
industry. Ripe tomatoes contain approximately water 94%, protein 1%, fat
Lecturer, Department of Botany, Taungoo University
152
0.1%, carbohydrate 4.3%, fiber 0.6%, vitamin A 250 IU, and ascorbic acid 25
mg/100 g (Purseglove, 1968).
The postharvest technology is essential to maximize the shelf-life of
fruit for markets and utilization. Shelf-life is determined by the intensity of
physiological activity or rate metabolism. The metabolic rate of the commodity
is essential for extending the shelf-life and maintaining consumer acceptance
of harvested produce (FAO, 1989).
Fruit jam manufacturing is one of the ways of preserving fruits. It can
be prepared by home-made or commercial scale. A jam is a preparation
consisting of whole fruit boiled with sugar, having consistency firm enough to
meet the demands of confectioners and to withstand the accidents of transport
without altering its position in the container or having its surface broken. In
other words, jam consists of fruit tissues embedded in reasonable firm pectinsugar-acid gel (Website 1).
One important feature of preserves like jam is the high acidity, which
prevents the growth of food poisoning bacteria and also helps to maintain the
color and flavor for most fruits. Some mould and yeasts are able to grow at the
high acidity and these can spoil the food. They are prevented by ensuring that
the sugar content of the preserve is at least 68% (Website 2). It should contain
not less than 68.6% soluble solid as determined by refractometer when jam is
cold (Alyward, 1999).
The aim of this paper is to increase the shelf life of foods, to prepare
new product, jams. Such products are enjoyed by everyone and of the year
round and to utilize the foodstuff when available excess.
Material and Methods
Experimental Site
Tomato (Lycopersicon esculentum Mill.) fruits were used in the
experiments. The experimental site was conducted at the Department of
Botany.
Equipments Required for Tomato Jam
Equipments required for tomato jam were stainless steel vessel, sharp
knife, sieve plate, ladle, wooden chopping board, glass bottle, thermometer,
refractometer, pH meter, oven and balance.
153
Ingredients Required for Tomato Jam
Ingredients for jam preparations are tomato, sugar, a little salt, lime
water, lemon juice, pectin and potassium sorbate.
Collection, Sorting and Cleaning the Fruits
The relative ripeness of fruits may be used due to the primary
importance in determining the ultimate quality of all products derived from
tomato. Sorting is the sound ripe deep red color fruits without damage for this
experiment. The selected tomato fruits were weighed, then washed with tap
water and rinsed about 10 minutes in dilute solution to depress the
development of microorganism (Fig. 1.).
Fig. 1. Collection, sorting and cleaning the fruit
Procedure for Tomato Jam Preparation
The cleaning tomato fruits (500 g) for sample 1 were cut with knife and
tomato seeds were removed. And then the tomato slices were dipped into the
lime water for two hours. These slices were rinsed with tap water and were
placed into the pan. Sugar (250 g), salt (1 g) and lemon juice (1 teaspoon) were
added and stirred to form syrupy sugar solution for about 30 minutes. Pectin (2
g) was then added and boiling was continued and stirred constantly in order to
prevent the pectin from clotting. The tomato jam was stirred until the setting
point was reached (Fig. 2).
Sample 2 was also prepared as sample 1 except the addition of
potassium sorbate (0.5 g) into the sample (Fig. 2).
154
Fig. 2. Tomato jam preparation
Procedure for Tomato Jam Preparation
For preparing sample 3, sugar (125 g), salt (1 g) and lemon juice (1
teaspoon) were added into tomato slices in the pan. Pectin (2 g) was then added
until the setting point was reached. Sample 4 was prepared as sample 3 except
the addition of potassium sorbate (0.5 g). Finally, the prepared jams were
cooled and stored into the glass bottle. (Fig. 2)
Data Collection
The following data were collected from these experiments: the total
soluble solids (TSS) (°Brix), the percentage of titratable acidity (TA), pH value
and mixture content.
Determination of Total Soluble Solids (TSS)
The total soluble solids content or sugar (sucrose) content of tomato
jam was measured by using Abbe refractometer at room temperature. The
refractive index was then read on a long scale which is clearly visible with
naked eye (Thanh, 2008).
Determination of Titratable Acidity (TA)
Titratable acidity (TA) of the product was obtained by titration of jam
and using the following formula (Thanh, 2008).
% TA =
155
(V × N ) NaOH × Predominant acid
× 100
Fresh weight sample in g
× V aliquot
VH 2O + Fresh weight sample
where, TA
= Titratable acidity
V
= titrated volume of NaOH (m)
N
= concentration of NaOH, normality (N)
VH 2 O
= volume in ml or weight in gm of sample
V aliquot = weight equivalent of aliquot
Determination of pH Value
The pH was determined by using pH meter. The glass electrode was
first standardized by placing in two known buffer solutions of pH 4 and 7 and
the pH meter was adjusted to those values. Then the pH value of jam was
measured.
Determination of Moisture Content
Tomato jam (10 g) was weighed in aluminum foil and dried in hot air
oven at 105°C for 3 hours, cooled and weighed. Drying was repeated until a
constant weight was obtained and placed in a desiccator for 30 minutes and the
moisture was calculated as follows (FSSAI, 2012):
Moisture Content, % =
where a
( b − c)
× 100
a
= sample weight in gram
b
= sample before drying
c
= sample after drying
(b–c) = loss in weight of sample after drying
Results
Prepared Jam Samples and Yields of Tomato Jam
In this experimental work, Tomato (Lycopersicon esculentum Mill.)
was used as major raw material and the different ratios of ingredients were
used as shown in Tables 1. The prepared jam samples are also shown in Fig. 3
and Fig. 4.
156
Table 1. Preparation of tomato jam (Procedure 1 and Procedure 2)
Ingredients
Quantity
Quantity
(Procedure 1)
(Procedure 2)
Sample 1
Sample 2
Sample 3
Sample 4
Tomato fruit (g)
500
500
500
500
Sugar (g)
250
250
1.25
1.25
Salt (g)
1
1
1
1
Pectin (g)
2
2
2
2
Potassium benzoate (g)
-
0.2
-
0.2
Lemon juice (teaspoon)
1
1
1
1
Sample 1
Sample 2
Sample 3
Fig. 3. Tomato jam for procedure 1
Sample 4
Fig. 4. Tomato jam for procedure 2
The yields of resultant tomato jams were shown in Table 2. The yield
percent for samples 1 and 2 were higher than samples 3 and 4 since the weight
of sugar used was quite different.
Table 2. Yield of tomato jam
Samples
Yield of Jam (g/500 g of Tomato)
Yield (%)
Sample 1
440
88.0
Sample 2
440
88.0
Sample 3
380
76.0
Sample 4
380
76.0
157
Chemical Analysis of Tomato Jam
The resultant data of analysis are tabulated in Tables 3 and 4. Titratable
Acidity (TA) percent was measured by using titration methods. This result
showed that 0.080% for sample 1, 0.083% for sample 2, 0.081% for sample 3
and 0.083% for sample 4.
Using pH meter, pH values of tomato jams were observed 5.7
(sample1), 6.1 (sample 2), 5.1 (sample 3) and 6.0 (sample 4), respectively.
Therefore, these jams were classified as acidic food (pH > 4.5).
The total soluble solids (TSS) (°Brix) were analyzed by measuring with
refractometer. The TSS (°Brix) of tomato jams were 68 for sample 1, 70 for
sample 2, 70 for sample 3 and 69 for sample 4, respectively. The result
revealed that the total soluble solids values of jams were ranged in 68 - 70°
Brix.
Next, the moisture content of jam was 19% (sample 1 and 3), 18%
(sample 2) and 14% (sample 4), respectively. This result showed that the
moisture contents of jams were 14 - 19%.
The shelf-life of jam was 14 days (sample 1) and 18 days (sample 3)
under room temperature. Tomato jam for sample 2 and 4 had no mould during
their shelf-life.
Table 3. Composition of tomato jam (Procedure 1)
Parameter
Sample 1
Sample 2
Remarks
0.080
0.083
pH
5.7
6.1
TSS (°Brix)
68
70
Moisture content (%)
19
18
Shelf-life (day)
14
To-date (14 days)
sample 1 as
found mould
after 14 days but
sample 2 has no
mould after 14
days
TA (%)
158
Table 4. Composition of tomato jam — Procedure II
Parameter
Sample 3
Sample 4
0.081
0.083
pH
5.1
6.0
TSS (°Brix)
70
69
19
14
Shelf-life (day)
18
To-date (18 days)
TA (%)
Remarks
sample 3 as
found mould
after 18 days but
sample 4 has no
mould after 18
days
Discussion
In the experiments, tomato jams were prepared using different sugar
contents and different amount of preservatives. In samples 1 and 2, 250 g of
sugar was used for 50 g of tomato fruits. But 125 g of sugar was used in
samples 3 and 4. The resultant yield percents were quite different with respect
to amount of sugar used. About 440 g of jam was obtained using 250 g of
sugar added whereas the yield of jam was changed to 380 g when 125 g of
sugar was used.
Su Lat Win (2010) used 262.47 g of sugar and the brix of the products,
the sauce, became 38.0. The difference might be due to the different
ingredients of the different products.
Some physico-chemical parameters of tomato fruit jam such as pH,
titratable acidity (TA), total soluble solids (TSS) and moisture contents were
then investigated.
The acidity prevented the growth of spoilage bacteria and yeast. In this
research, titratable acidity (TA) was attained 0.080% to 0.083%. Low acidity
of fruit was made by addition of acid. The amount used varies 0.1% and 0.2%
of total of weight of jam. The pH value of tomato jam in this experiment is
greater than 4.5. The pH value plays an important role in fruit jam which it
ensures the shelf stability of the jam during storage. But, Sethi (2007) reported
that the optimum pH for jam to set is 4 or less.
In this research, the total soluble solids (TSS) value of tomato jam 68 70°Brix. According to Aylward (1999), the total soluble solids of jam are 65 68°Brix and should not exceed 72°Brix. This value will influence the gel
159
strength of the jam and reduce the growth of microorganisms in jam (Aylward,
1999).
Next, the moisture content of jam was observed 14 - 19% in the present
study. The reduction in moisture content may be due to the evaporation in jam
during heating process.
FSSAI (2012) revealed that pectin acts as a gelling agent in fruit jam
and provides the desired gel strength and consistency. The pH value and the
amount of sugar added in jam also influence the intermolecular force
contributing to the gel strength and responsible for shelf stability of the jam
during storage. Tartness of the jam is important to prevent growth of
microorganisms that might cause spoilage in processed products. The
preservative must be non-toxic for food products, and using it to the products
could be stored under room temperature.
Conclusion
The pH, titratable acidity and total soluble solids of the produce is
depended on the ingredients applied in the respective prepared jams and also
by the different ways of their processing procedures. Adding the postharvest
shelf-life of the produce will be extended by the application of suitable
preservatives. The postharvest losses of crops can be maintained by using food
technology to the respective products. The preserved food products can be
used year round. To be economical, jam should be produced during growing
season of tomato, properly preserved and stored to extend its availability.
Acknowledgements
The author would like to appreciate Dr. Aung Thu, Rector and Dr. San San Mar, ProRector of Taungoo University, for their permission to submit this research paper. The author is
also grateful to Dr. Than Than Nu, Professor and Head, Botany Department, Taungoo
University for her kind permission and help in various ways.
References
Alyward, F. (1999). Food Technology Processing and Laboratory Controls. Allied Scientific
Publishers, India.
Decoteau, D. R. (2000). Vegetable Crops, Prentice Hal. Upper Saddle River, NJ 0745.
160
Edmond, J. B. (1957). Fundamentals of Horticulture. A Textbook. A.M. Musser and Designed
for Courses in General Horticulture, McGraw-Hill Book Company, Inc. New
York, Toronto, London.
FAO (Food and Agriculture Organization) (1989). Preservations of Postharvest Food Losses,
Fruits, Vegetables and Root Crops. A Training Manual. Food and
Agriculture Organization of the United Nation. Printed in Rome, Italy.
FSSAI (Food Safety and Standards Authority of India) (2012). Manual of Method of Analysis
of Foods: Fruit and Vegetable Products. Ministry of Health and Family
Welfare, New Delhi, India.
Kartikar, K. F. and B. D., Basu (1933). Indian Medicinal Plant, Vol. I, Second Edition.
Lawerence, G. H. M. (1969). Taxonomy of Vascular Plants. The Macmillan Co. New York.
Purseglove, J. W. (1968). Tropical Crops Dicotyledons (2). Longmans Green and Co., Ltd.
London and Harlow, Associated Companies, Branches and Representatives
throughout the World.
Su Latt Win (2010). Postharvest Technology on Shelf-Life and Food Processing of
Lycopersicon esculentum Mill., Botany Department. PhD (Dissertation)
(unpublished), University of Yangon.
Thanh, C. D. (2008). Training Manual on Postharvest Research and Technology Development
for Tomato and Chilli in RETA 6208.
Wien, H. C. (1997). The Physiology of Vegetable Crops Science. pp. 207. Cornell University,
Ithaca, NY, USA.
Websites
1.
http://www.World foodscience.org
2.
http://www.Food processing.com/ff/index.html
3.
www.botanical-online.com
Phytochemical Analysis of Cnestis palala (Lour.) Merr.and Its
Antimicrobial Activity
Mi San Mar Lar
Abstract
The medicinal plant Cnestis palala (Lour.) Merr. is belonging to the family
Connaraceae.. This wild medicinal plant is collected from Myeik and Dawei
Township, Tanintharyi Region, in Southern Myanmar. In this research,
preliminary phytochemical test, physicochemical properties and
antimicrobial activities have been carried out. In phytochemical test,
glycoside, triterpenoid, reducing sugar, saponin, phenolic compound, αamino acid, carbohydrate, tannin, flavonoid and coumarins were present but
alkaloids were found to be absent. In addition, fats, fibers, proteins and
carbohydrates were observed as nutritional content. According to the
physicochemical examination, the raw sample and ash roots were more
soluble in water and moderately soluble in pet-ether. In elemental analysis,
calcium was examined to be major element. In antimicrobial activities,
chloroform extract showed the most significant activity, while pet-ether and
watery extracts did not show any activity.
Key
words:
Cnestis palala (Lour.) Merr., phytochemical test,
physicochemical properties, lemental analysis, antimicrobial
activity
Introduction
The medicinal plant, Cnestis palala (Lour.) Merr., is commonly
known as Kyet-mauk-ni, Gwe-dauk, Taw-kyet-mauk, Kyet-babyin and
Mayan-sikkaw in Myanmar, Stinging-hair cnestis in English, Shi mao guo in
Chinese (Roy, 1922; Hundley and Chit Ko Ko, 1987; Kress, 2003).
In Malaysia, a decoction of roots of Cnestis palala (Lour.) Merr. is
used as a drink to treat stomach-ache and urinary troubles. It is also applied as
a tonic after childbirth, and as an anticoagulant as a remedy for stroke and
blood clotting. In Laos, an infusion of the roots is used internally to treat
dysentery, and a decoction of the leaves externally to treat scabies and
wounds (Lemmens and Bunyapraphatsara, 2003).
Most people are unaware of the medicinal values of the roots of this
wild plant except in Tanintharyi Region and some areas of Myanmar where
the infusion of fragrant powdered roots were used as folk medicine for the
Professor, Department of Botany, Dawei University
162
treatment of dysentery and diarrhoea. The aim of this study is to investigate
the phytochemical and physicochemical properties, nutrient content and
antimicrobial activities of the crude drug from this plant.
Preliminary Phytochemical Investigation of Cnestis palala (Lour.) Merr.
Phytochemical investigation on powdered leaves and roots of Cnestis
palala (Lour.) Merr. was carried out to examine the plant constituents. The
method of Tin Wa (1970), Central Council for Research in Unani Medicine
(1987) and Trease and Evans (2002) were applied for investigation of
phytochemical studies.
Physicochemical characterization of Cnestis palala (Lour.) Merr.
The physicochemical characters such as moisture content, content of
total ash, acid insoluble ash, water soluble ash and various organic solvents
soluble content were carried out according to the method of British
Pharmacopoeia (1965).
Elemental Analysis from Leaves and Roots of Cnestis palala (Lour.) Merr.
By Using EDXRF
In this research, the energy dispersive X-ray fluorescence spectrometer
(EDX 700, Shimadzu) was used to analyze the samples of interest. The
parameters of each part of the spectrometer are given below:
Detector Type
: Si (Li) detector
Liquid N 2 Supply
: Only during measurement
Liquit N 2 tank capacity
: 3 liters
Liquid N 2 consumption
: less than 1 liter per day
Detection area
: 10 mm2
Resolution
: less than 155eV (Mink, 1500H 2 )
The EDX 700 spectrometer can detect a wide range of the elements
from sodium (Na) to Uranium (U). The required data can be produced in a few
minutes and it has a high degree of resolution for the spectrum evaluation. Due
to its high sensitivity, the spectrometer can detect the relative concentration of
163
elements in the percentage range. The spectrometer produces the characteristic
X-ray spectrum of each sample, consisting of the respective elements.
Determination of Nutrient Contents In the Powdered Leaves and Roots
from Cnestis palala (Lour.) Merr.
The protein, fat, fibre and carbohydrate contents in the powdered leaves
and roots of Cnestis palala (Lour.) Merr. were determined according to Willam
(1980) and Myanmar Traditional Medicine Formulary (1969). The experiments
were conducted at the National Nutrition Centre, Department of Health and
Yangon.
Antimicrobial Activities of Different Solvent Extracts from Roots of
Cnestis palala (Lour.) Merr.
For the determination of antimicrobial activity of the roots extract from
the plant in vivo, agar-well diffusion method was used because of its
simplicity, speed of performance, economy and reproducibility (Cruickshank,
1970 and Finegold et al., 1978).
Results
Morphological Characters of Cnestis palala (Lour.) Merr.
Scientific name
- Cnestis palala (Lour.) Merr.
Family name
- Connaraceae
Myanmar name
- Kyet-mauk-ni, Gwe-dauk, Taw-kyet-mauk
Flowering period
- November to January
Shrub and liana; branchlets densely brown pubescent. Leaves alternate,
unipinnately compound, imparipinnate, stipules absent. Inflorescences an
axillary fascicled raceme or panicle. Flower white to creamy, bisexual, regular,
5 merous. Stamens 10, free, all fertile. Pistils 5 - 7, usually 6, apocarpous, the
ovary ovoid, hairy. Fruit obovoid to oblongoid follicle, 1-3 per flower, bright
orange red, slightly falcate, distinctly beaked, velvety hairy outside and long
silky or yellowish hairs inside, opening by a longitudinal ventral slit. Seed
solitary, ovoid, black, with a yellow fleshy aril at the basal part (Fig. 1).
164
Habit with Flowers
Young fruits
Habit with Fruits
Mature fruits
Inflorescence
Seeds
Fig. 1. Morphology of Cnestis palala (Lour.) Merr.
Preliminary Phytochemical Investigation of Cnestis palala (Lour.) Merr.
Preliminary phytochemical screening was done on the aqueous extract
and the ethanolic extract of the powdered leaves and roots of Cnestis palala
(Lour.) Merr., according to the standard methods. These tests are usually based
on the results of color changes or precipitation that showed the presence or
absence of organic constituents present in the study plant (Table 1).
165
Table 1. Preliminary phytochemical investigation of Cnestis palala (Lour.)
Merr.
No.
Tests
Extract
10
11
Cyanogenic
glycosides
Reducing
sugar
Flavonoids
Triterpenoids
H2O
Extract
H2O
Extract
H2O
Extract
H2O
Extract
H2O
Extract
H2O
Extract
H2O
Extract
H2O
Extract
H2O
Extract
EtOH
EtOH
12
Alkaloids
EtOH
1
Saponin
2
α-Amino acid
3
Carbohydrate
4
Phenolic
Compound
5
Glycosides
6
Tannins
7
Starch
8
9
13
Coumarins
+ = present, - = absent
EtOH
Test Reagent
Distilled water
Observation
Results
Leaves Roots
Frothing
+
+
Ninhydrin reagent
Pink
+
+
10% α-napthol +
Conc. H 2 SO 4
Red ring
+
+
K 3 Fe(CN) 6 and FeCl 3
Deep blue
+
+
10% Lead acetate
White ppt
+
+
3% FeCl 3 solution
White ppt
+
+
+
+
+
+
+
+
+
+
+
+
No ppt
No ppt
No ppt
_
_
_
_
_
_
Pink colour
+
+
I 2 solution
Conc.H 2 SO 4 +
Sodium picrate sol:
Fehling's A and B
HCl / Mg
Acetic anhydride +
Conc. H 2 SO 4
(1)Dragendroff's
reagent
(2)Mayer's reagent
(3)Wagner's reagent
KOH solution
Blue black
ppt
Brick- red
ppt
Brick- red
ppt
Pink colour
Deep blue
According to the results, that saponin, α- amino acid, carbohydrate,
phenolic compounds, glycosides, tannins, starch, cyanogenic glycosides,
reducing sugar, flavonoids, triterpenoids and coumarins were present and
alkaloids was absent in both plant parts.
Physicochemical Characterization of Cnestis palala (Lour.) Merr.
The physicochemical characterization was useful for the quality control
of medicinal plants or herbal drugs. According to the solubility matter results
the powdered samples were more soluble in polar solvents (Table 2).
166
Table 2. Physicochemical characterization of Cnestis palala (Lour.) Merr.
No.
Physicochemical Characters
Leaves
Roots
1
6.70
3.90
2
Total ash content (%)
4.50
4.00
3
Acid insoluble ash (%)
0.39
7.18
4
Water soluble ash (%)
24.42
10.21
5
Water soluble matter (%)
17.60
11.77
6
Ethyl alcohol soluble matter (%)
1.46
5.90
7
Petroleum ether soluble matter (%)
0.97
0.37
Determination of Elemental Analysis from Leaves and Roots of Cnestis
palala (Lour.) Merr. by Using EDXRF Spectrometer
The contents of the elements in the leaves and roots were measured by
EDXRF method (Table 3). The dendrogram of elemental analysis was shown
in Fig. 2 and 3.
Table 3. Elemental analysis on powdered leaves and roots of Cnestis palala
(Lour.) Merr. by using EDXRF spectrometer
No.
1
2
3
4
5
6
7
8
9
10
Elements
Calcium (Ca)
Potassium (K)
Sulphur (S)
Chlorine (Cl)
Manganese (Mn)
Iron (Fe)
Rubidium (Rb)
Strontium (Sr)
Zinc (Zn)
Copper (Cu)
Concentration Value (%)
Leaves
Roots
50.920
80.195
33.442
8.998
7.101
6.940
3.647
1.944
0.700
1.254
1.962
1.061
0.407
0.276
0.641
0.184
0.171
0.158
167
According to this result, Calcium (Ca), Potassium (K) and Sulphur (S) were
found as principal elements in both plant parts but Chlorine (Cl) and Zinc (Zn)
were absent in roots. Manganese (Mn), Iron (Fe), Rubidium (Rb) were
moderately present and Strontium (Sr) and Copper (Cu) were found to be a
trace elements in both samples. Calcium (Ca) is a major element in both
samples.
Fig. 2. Elemental analysis of
Fig. 3. Elemental analysis of Cnestis palala
(Lour.) Merr. roots by EDXRF
leaves by EDXRF
Analysis of Nutrient Content
The determination of percentage of the fats, proteins, fibers and
carbohydrates contents of dried powder samples, Cnestis palala (Lour.) Merr.
were carried out according to the procedures described by Willam (1980) and
Myanmar Traditional Medicine Formulary (1989) (Table 4).
Table 4. Analysis of nutrient content of Cnestis palala (Lour.) Merr.
No.
Constituents
Nutritional Value
Nutritional Value
in Leaves (%)
in Roots (%)
1.7
0.7
1
Fat content
2
Protein content
13.9
5.9
3
Fiber content
21.1
32.5
4
Carbohydrates content
49.3
52.8
168
Antimicrobial Activities of Seven Different Solvent Extracts from Roots of
Different solvent extracts from the roots of Cnestis palala (Lour.) Merr.
were used to perform the antimicrobial activities by agar well diffusion
method. The result was shown in Table 5.
Table 5. Antimicrobial activity of seven different solvent extracts from roots of
Test Organisms
Sample
Solvent
Pseudomonas
Bacillus
Candida
Escherichia
coli
subtilis
aureus
aeruglnosa
pumalis
albicans
-
-
-
-
-
-
CHCl 3
17 mm
18 mm
20 mm
18 mm
18 mm
18 mm
MeOH
-
-
16 mm
15 mm
15 mm
-
Acetone
-
-
17 mm
18 mm
16 mm
-
EtOAc
13mm
12 mm
15 mm
14 mm
14 mm
11 mm
EtOH
13 mm
16 mm
15 mm
17 mm
16 mm
13 mm
-
-
-
-
-
-
Petether
Roots
Bacillus Staphylococcus
H2O
Agar-well 10 mm
According to this experiment, chloroform, ethyl acetate and ethanol
extracts exhibited antimicrobial property against all test organisms with the
inhibition zone diameters ranged between 11~20 mm. Methanol and acetone
extracts were showed the antimicrobial activity against on Pseudomonas
aeruginosa, Bacillus pumalis and Candida albicans but non effected against
on Bacillus subtilis, Staphylococcus aureus and Escherichia coli. While the
petroleum ether (60 – 80˚C) and watery extracts did not show inhibition zone
of all tested microorganisms.
From these experimental results, it can be seen that chloroform extract
were the most significant antimicrobial agents especially against Pseudomonas
aeruginosa (20 mm). These observations show the presence of antimicrobial
active compounds in the extract (Fig. 4).
CHCl3
PE
MeOH
PE
EtOAc
Acetone
CHCl3
EtOH H2O
Bacillus subtilis
PE
CHCl3
MeOH
CHCl3
EtOH H2O
PE
EtOH
H2O
Acetone
PE
CHCl3
Candida albicans
EtOH
MeOH
EtOAc
EtOH
EtOAc
H 2O
Bacillus pumalis
Acetone
MeOH
Acetone
PE
EtOAc
Pseudomonas aeruginosa
CHCl3
EtOAc
Staphylococcus aureus
Acetone
MeOH
169
H2O
EtOAc
Acetone
MeOH
EtOH H2O
Escherichia coli
Fig. 4. Treatment of various extracts of Cnestis palala (Lour.) Merr. on test
organisms
The medicinal plant, Cnestis palala (Lour.) Merr., was observed shrub
at first and later becomes liana. Leaves were alternate, unipinnately compound,
imparipinnate; Inflorescence was an axillary raceme or panicle or cauliflorous
on older branch. Flowers were white or creamy, bisexual, regular, 5-merous
and fragrant. Stamens were 10; Pistils 5 to 7, free and superior. Fruit consisting
of 1 - 3, obovoid to oblongoid follicles; Seed was solitary and arillate. These
characters were in agreement with those given by literatures (Hooker, 1879;
170
Hutchinson, 1964; Bhattacharyya
Bunyapraphatsara, 2003).
and
Johri,
1998;
Lemmens
and
In this research, preliminary phytochemical and physicochemical tests
were investigated to determine the presence of various phytoconstituents and to
determine the moisture, fat, ash and soluble matter composition. The nutritional
values such as protein, fat, carbohydrates and fibres of leaves and roots from
Cnestis palala (Lour.) Merr., were also studied. For the determination of
antimicrobial activity of the roots extract from the plant in vivo, agar-well
diffusion method was used. According to the preliminary phytochemical tests,
saponin, α-amino acid, carbohydrate, phenolic compounds, glycosides, tannins,
starch, cyanogenic glycosides, reducing sugar, flavonoids, triterpenoids and
coumarins were present and alkaloids were absent. In the physicochemical
study, the powdered leaves and roots of this plant were more soluble in water.
Elemental analysis (EDXRF) revealed that calcium (Ca), potassium (K)
and sulphur (S) were found as principal elements and Manganese (Mn), Iron
(Fe), Rubidium (Rb), Strontium (Sr) and Copper (Cu) were found as trace
elements. Among them, Calcium was examined as a major element in both
plant parts. In addition, protein, carbohydrate, fiber and fats were found as
nutrient in this plant. From the investigation of the antimicrobial activity of
seven crude extracts were tested on six microbial strains. According to the
results, chloroform extract was the most significant antimicrobial agents
especially against Pseudomonas aeruginosa (20 mm). The larger the inhibition
zone diameter, the higher the antimicrobial activity. These observations
indicate the presence of anti-microbial active compounds in these extracts.
From this finding, it can be inferred that Cnestis palala (Lour.) Merr., can be
effective for the treatment of diseases, such as stomach-ache, urinary troubles,
diarrhoea, dysentery, scabies and wounds (Lemmens and Bunyapraphatsara,
2003).
Acknowledgements
Firstly, I would like to express my gratitude to Professor Dr Thet Thet May, Head of
the Botany Department, University of Yangon, for her help and invaluable suggestion. I wish
to express my deepest appreciation and special thanks to my supervisor, Professor Dr Aye Pe,
Department of Botany, University of Yangon, for his invaluable advice, and constant
encouragement.
171
References
Backer, C. A. and R. C. B. Van Den Brink (1965). Flora of Java. Vol. 2. The Netherland:
N.V.P. Noordhoff-Groningen.
Bhattacharyya, B. and B. M. Johri (1998). Flowering Plants (Taxonomy and Phylogeny).
Narosa Publishing House, New Delhi Madras Bombay Calcutta, London.
British Pharmacopeia. (1965). Department of Health Social Security Home and Health
Department Welsh Office, Ministry of Health and Social Services for
Northern Ireland, London. Her majesty; Stationary Office.
Central Council for Research in Unani Medicine. (1987). Physicochemical Standard of Unani
Formulation. Ministry of Health, Government of India, Delhi.
Cruickshank, R. J. P. (1970). Medical Microbiology. 11th ed., London: Churchill Livingstone
Ltd. London.
Finegold, S. M., W. J. Martin and E. G. Scott (1978). Diagnostic Microbiology. London: The
C.V. Mosby Co., Ltd.
Hooker, J. D. (1879). The Flora of British India. Vol. II. L. Reeve & Co. Ltd. The Oat House,
Brook, NR. Ashford, Kent. England.
Hundley, H. G. and Chit Ko Ko (1987). List of Trees, Shrubs, Herbs and Principle climbers of
Myanmar (Burma). 3rd ed., Government Printing Press, Yangon.
Hutchinson, J. (1964). The Genera of Flowering Plants. Vol. 1. Dicotyledon. Clarendon Press,
Oxford.
Kirtikar, K. R. and B. D. Basu (1975). Indian Medicinal Plants. (Vol. 1). Lalit Mohan Basu,
India.
Kress, J. W. and A. Robert (2003). A Checklist of the Trees, Shrubs, Herbs, and Climbers of
Myanmar. Vol. 1. Washington D.C, New York.
Lemmens, R. H. M. J. and N. Bunyapraphatsara (2003). Plant Resources of South-East Asia.
No.12 (3). Medicinal and Poisonous Plants Bogor, Indonesia.
Myanmar Traditional Medicine Formulary (1989). Pharmacology Research Division.
Department of Medical Research, Yangon. 302.
Roy, J. S. B. (1922). Flora of China. Vol. 1. p. 435. Cnestis palala in Flora of China@
efloras.org. Asiat. Soc. 85: 201. 1922.
Tin Wa (1970). Phytochemical Screening; Methods and Procedures. Phytochemical Bulletin of
Botanical Society of America. Inc.5. (3). p. 4-10.
Trease, G. E. and W. C. Evans (1978). A Texbook of Pharmacognosy. Bailliere, Tindall. 11th
ed., Spinger. Berlin.
Willam, H. (1980). Official Methods of Analysis of the Association of official analytical
chemists. 13th ed., Washinton, DC: Association of official analytical
chemists.
A Study on Morphological Characters of Tea Plant and Effect of
Fertilizers on Growth of Camellia sinensis (L.) Kuntze
Kyaw Kyaw Sann
Abstract
In Myanmar, Camellia spp., are very important for tea products. Among
these species, C. sinensis (L.) Kuntze, C. kissi Wallich, C. drupifera
Loureiro, and C. olifera Abel are cultivated for local production in
Myanmar. C. sinensis was cultivated for production of green tea. In this
paper, the study on morphological characters and effect of fertilizers on
growth of C. sinensis in polythene bag experiment. In the study of plants
growth rate of tea by using five fertilizers: Spirulina, cowdung, urea, Armo
and ash of paddy husk. The Spirulina suspension and cowdung
biofertilizers were more effective on shoot height, leaf number, leaf area of
tea plants than those of control and other fertilizer at 1% significant level.
The results showed that Myanmar Spirulina actually promotes the shoot
height, leaf number and leaf area.
Key words: Camellia spp., Spirulina, polythene bag, biofertilizers
Introduction
Tea is derived from the leaves of Camellia spp. and is considered by
about half of the world population. It has long been used in the orient as an
item of commerce and as a custom and that of China dates from the 5th century
AD (Sammbanurthy and Subrahmanyam, 1998).
The tea was considered to be a native of Assam and adjoining area of
Upper Burma, and some regarded that it is to be a native of Southern Yunan
and Upper Indochina (Pandey, 2000). But some of the authors stated that
Camellia sinensis L. is undoubtedly native to China. Asians have much legend
describing the first uses of tea.
Tea has long been used in the orient at first probably medicinally. Its
precise mode and date of origin are uncertain, but it was an item of commerce
with the Mongols for thousand of years ago. Tea was introduced to Europe in
1610 by the Dutch, London in 1664, and Boston in 1714 (Schery, 1972).
The beverage prepared from the dried leaves of Camellia spp.
(Theaceae) which is known in different parts of the world as tea, Chai, or tea is
the most popular nonalcoholic drink in Asia. Tea plant is native to India
Assistant Lecturer, Department of Botany, Yadanabon University
174
(Assam) or China. Tea contains 2-5% theine, 13-18% tannin, a small amount
of caffeine and volatile oil when an infusion if made with hot water, the
alkaloid and the oil dissolve resulting in a beverage with characteristic taste
and aroma of tea are provided by the essential oil theols, tannins, along with
pectins and dexitrins provide the colour and astringency (Sammbanurthy and
Subrahmanyam, 1998).
In Myanmar, over 120,000 acres of tea is grown in the Shan State
(North), at Lashio, Muse, Kyaukme, Kwanlon and Laukki District, over
50,000 acres in the Southern Shan State in Loilem and Linkhei District, about
7178 acres in the Homalin Township, Sagaing Region. Also in Eastern regions
of Thanlwin, in the Thandaung, Kayin State, in Southern-Northern Chin State,
tea is grown a lot Myanmar tea differs in quality according to the cultivation
and processing Myanmar products of tea plants are Laphet-so, green tea and
black tea. Fermented pickle tea, also known as "Laphet" in Myanmar is
directly consumed by Myanmar People. It is a well-known traditional
Myanmar food used as appetizer. Myanmar pickled tea was produced from
that of common tea plants.
Tea is cultivated on a large scale in areas with moist and warm climate.
It grows from almost the sea level to an altitude of 2,460 m. A good rainfall,
well distributed throughout the year, absence of strong dry wind and freezing
temperature are some of the essential requirements. The average annual
rainfall of 150 cm-370 cm is well suited for cultivation of tea. However, an
annual rainfall of 750 cm does not seem to produce any harmful effect on the
plant, provided the soil is well drained and there is no waterlogging (Sen,
1996).
Spirulina has autotrophic characters and nitrogenase activities.
Besides, Spirulina are abundantly found in Myanmar. Therefore Spirulina is
considered for the production of the most suitable potential biofertilizer in
Myanmar Agriculture. By studying the effect of fertilizer on plantation of tea
plants, the resulting data can be used as information for growth of tea plant
and commercial production.
175
Morphological Study
The specimens were collected from Mogok during the flowering and
fruiting period September to December, 2008; Kyaukme and Pyin-Oo-Lwin
during the flowering and fruiting period September to December, 2009;
Panglong and Pindaya during the flowering and fruiting period September to
December, 2010. According to the resulting morphological characteristics, the
scientific name was identified or key out using the floristic literatures or
references. The name of the genus was referred according to the book of
vascular plant families and general written by Benson (1965), Hutchinson
(1959) and Cronquist (1981). The valit name of the species has been based on
A Revised Hand book to the Flora of Ceylon by Dassanayake (1996), Flora of
Java by Backer (1965) and Flora of British India by Hooker (1894).
12 ft
10 ft
1 ft
C
T2
T5
T4
T1
T3
T1
T3
T4
C
T2
T5
T4
T5
T2
T1
C
T3
T2
T1
T3
C
T4
T5
T4
T5
C
T1
T2
T3
T1
C
T5
T3
T4
T2
2ft
Randomized Complete Blocks Design (RCBD)
176
Effect of Fertilizers on Growth of Camellia sinensis (L.) Kuntze
For the experiment of plant plots using growth in cultivation, about one
year old tea plants were transplanted into polythene bag. Each plot consisted
of 36 plants. There were 6 replications (6 × 6 = 36 plots). Randomized
completely blocks design (RCBD) was used. Total of 1296 one year old tea
plants with about 12.5 cm in height were treated with five fertilizers; the
Spirulina powder (lot. no 324) obtained from MPF (Myanmar Pharmaceutical
Factory, Yekharr, Sagaing Region), cowdung, urea (46% of nitrogen), Armo
(15% : 15% : 15%) and ash of paddy husk. The graph paper method was used
(Santra, Chatterjee and Das, 1999). Preparation of fertilizer suspension was as
shown in Table 1. The fertilizers were applied in suspension about 50 ml
plant–1 once a month. These transplanting plants were sprayed with water. The
watering was done every morning with 50 ml plant–1.
Table 1. The ratio of fertilizer suspension used
No.
Types of
fertilizer
Weight of fertilizer
to be used (g)
be used (ml)
Used
Suspension per
plant (ml)
Water to
1
Control
–
1000
50
2
Cowdung
20
1000
50
3
Urea
20
1000
50
4
Armo
20
1000
50
5
Ash of
20
1000
50
20
1000
50
Paddy Husk
6
Spirulina
First treatment started at 1st January 2009 with five kinds of fertilizers
and a control with no fertilizer. After one month, the growth rate of shoot
height (cm) and leaf number were measured. The procedure is repeated in the
2nd (1st February 2009) and 3rd (2nd March 2009) month. And then the resulting
growth of shoot height (cm) and leaf number were recorded at 30 Days after
treatment (DAT), 60 DAT, 90 DAT and 120 DAT. The measurement of leaf
area was performed only 120 DAT. For every experiment, the comparisons of
177
the results of the growth rate in shoot height (cm), leaf number and leaf area
(cm2) were analyzed by "t" test method (Fowler, 1992).
Results
Morphological Characters of Camellia sinensis (L.) Kuntze, Fl. Cochinch
2:411. 1790.
Shrubs or trees. Stem and branches cylindrical; internode 2.0 to 3.5 cm
long, 2 - 4 mm wide, grayish-brown; current year branchlets reddish brown,
glabrous. Leaves simple, alternate, exstipulate, petiolate; petiole5 - 7 mm,
glabrous. Blade elliptic to broadly elliptic; 5.0 - 12.0 × 3.5 cm, leathery,
abaxillary pale green and glabrous, adaxillary dark green, shiny and hirtellous
along midvein, margin serrulate, apex acute shortly acuminate but with an
obtuse tip, base rounded to obtuse. Lateral nerves or veins 7 - 9 on each side of
midvein. Flower, bisexual, actinomorphic, hypogynous, white, axillary,
solitary, fragrant, 4 - 6 cm in diam, subsessile, ebracteate, bracteolate,
pedicellate; bracteoles and sepal 10 - 12 or more, caducous, glabrous, margin
ciliolate; other bracteoles and sepals semiorbicular, 1 - 2 mm; inner bracteoles
and sepals sepaloid, ovate to suborbicular to 1 cm. Petals 5-8 white, nearly
distinct, obovate, 3 - 6 × 1.5-3.5 cm, apically 2-parted for 7 - 10 mm. Stamen
numerous, 1.2 - 1.7 cm, glabrous; outer filament whorl basally connate for 1-2
mm to nearly distinct. Ovary globose, tomentose, 3 – 5-loculed. Style 3 - 5, 1 1.5 cm, glabrous, distinct nearly to base. Capsule globose to ovoid, 3- to 5loculed with 1 - 4 seeds per locule; pericarp 6-8 mm thick. Seeds brown, (Fig.
1 and 2).
Flowering time
: September to October
Fruiting time
: December to January
Specimens examined : Ye-chan-oke village, Pyin-Oo-Lwin
Township; Kyaw Kyaw Sann Coll. no.
10, Wanmai village, near the University of
Panglong; Kyaw Kyaw Sann Coll. no. 15.
178
Fig. 1. Habit of Camellia sinensis
(L.) Kuntze
Fig. 2. Flowering branch of
Camellia sinensis (L.) Kuntze
Effect of Fertilizers on Growth of Camellia sinensis (L.) Kuntze
Comparison on Mean Shoot Height of Camellia sinensis (L.) Kuntze with
Different Fertilizers and Control
The result of comparison effect of different mean shoot height (cm) of
tea plant with different effect of fertilizers and control at 30 DAT, 60 DAT, 90
DAT and 120 DAT were as shown in Table 2. In this study, the height of tea
plants treated with Spirulina suspension fertilizer of tea plants were more than
other fertilizers and control at 30 DAT, 60 DAT, 90 DAT and 120 DAT (Fig.
A-E). According to the result of 30 DAT, the shoot height of tea plants were
40 cm with treatment of Spirulina suspension fertilizer, followed by Armo,
urea, cowdung, ash of paddy husk and control, at 60 DAT followed by
cowdung, Armo, urea, ash of paddy husk and control; but at 90 DAT and
followed by cowdung, urea, Armo, ash of paddy husk and control (Table 2-6
and Fig. 9).
179
Table 2. Mean shoot height (cm) of C. sinensis with different fertilizers
and control
Fertilizers
30 DAT
60 DAT
90 DAT
120 DAT
Mean ±
Mean ±
Mean ±
Mean ±
Sd value
Sd value
Sd value
Sd value
Control
13.083±0.204 14.000±0.524 14.833±0.540 15.083±0.283
Cowdung
13.625±0.378 14.791±0.178 16.583±0.228 17.000±0.088
Urea
13.791±0.226 14.375±0.159 15.541±0.316 16.083±0.221
Armo
13.875±0.293 14.541±0.176 15.250±0.186 15.708±0.233
Ash of
Paddy Husk
Spirulina
13.625±0.302 14.000±0.267 14.875±0.275 15.166±0.516
14.000±0.219 14.833±0.257 16.625±0.301 17.250±0.379
Sd = standard deviation. DAT = day after treatment.
At 30 DAT, the effect of Spirulina suspension, urea and Armo
fertilizers on the tea plants were most significant than the control at 1% level
by the 't' test method. The treatment of ash of paddy husk and cowdung
fertilizers on the tea plants were more significant than the control at 5 % level
by the 't' test method (Table 3).
At 60 DAT, the effect of Spirulina suspension on the tea plants was
most significant than the ash of paddy husk at 1% level by the 't' test method
and more significant than the cowdung, urea and control at 5% level. The
treatment of cowdung fertilizer on the tea plants was most significant than the
ash of paddy husk and urea fertilizer at 1% level and more significant than the
control at 5% level. The treatment of urea fertilizers on the tea plants was more
significant than the Ash of paddy husk at 5% level. The treatment of Armo on
the tea plants was most significant than the Ash of paddy husk at 1% level and
more significant than the control at 5% level by the 't' test method (Table 4).
At 90 DAT, the treatment of Spirulina suspension on the tea plants was
most significant than the control, urea, Armo and ash of paddy husk fertilizer
at 1% level. The treatment of cowdung fertilizer on the tea plants was most
significant than the control, Armo, urea and ash of paddy husk at 1% level. The
treatment of urea fertilizer on the tea plants was more significant than the
control and ash of paddy husk at 5% level by the 't' test method (Table 5).
180
At 120 DAT, the Spirulina suspension on the tea plants was most
significant than the control, urea, Armo and ash of paddy husk at 1% level.
The treatment of cowdung fertilizers on the tea plants was most significant
than the control, urea, Armo and ash of paddy husk at 1% level. The treatment
of urea on the tea plants was most significant than the control at 1% level and
more significant than the Armo and ash of paddy husk at 5% level. The
treatment of Armo on tea plants was most significant than the control at 1%
level by 't' test method (Table 6 and Fig. 9).
Table 3. Comparison on mean shoot height (cm) of C. sinensis (L.) Kuntze
with different fertilizers and control at 30 DAT
Fertilizers
Spirulina
APH
A
U
CD
– 3.085*
Control
– 7.495**
– 3.640*
– 5.431**
– 5.697**
Cowdung
– 2.100
0.000ns
– 1.279
– 0.926
Urea
– 1.620
+ 1.082
– 0.551
Armo
– 0.837
+ 1.456
Ash of
– 2.461
Paddy Husk
*, ** = Significantly different at 5 % and 1 % level respectively.
Fertilizers
Spirulina
*
APH
A
U
*
CD
Control
– 3.495
0.000ns
– 3.731
– 1.676
Cowdung
– 0.326*
+ 6.040**
+ 0.432
+ 4.271**
Urea
– 3.710*
+ 2.950
– 1.717
Armo
Ash of
– 2.292
+ 4.144
– 3.501*
**
**
– 5.504
Paddy Husk
*, ** = Significantly different at 5 % and 1 % level respectively. ns
= Non signifiant.
181
Fertilizers
Spirulina
APH
A
U
CD
– 7.311**
Control
– 7.100**
– 0.168
– 1.787
– 2.771*
Cowdung
– 0.270
+ 11.718**
+ 11.100**
+ 6.539**
Urea
– 6.079**
+ 3.897*
+1.947
Armo
– 9.531**
+ 2.771
Ash of
– 10.527**
Paddy Husk
Fertilizers
Spirulina
APH
A
– 4.172**
U
Control
– 11.208** – 0.347
– 6.817**
Cowdung
– 1.572
+ 8.571**
+ 12.687** + 9.432**
Urea
– 6.508**
+ 3.997*
+2.859*
Armo
– 8.479**
+ 2.341
Ash of
**
CD
– 15.821**
– 7.963
Paddy Husk
Comparison on Mean Leaf Number of C. sinensis with Different
Fertilizers and Control
The result of the comparison on leaf number of tea plants with different
effect of fertilizers and control at 30 DAT, 60 DAT, 90 DAT and 120 DAT are
as shown in Table 7. In this study, the leaf number of Spirulina suspension
fertilizer of tea plants, were better than other fertilizers and control at 30 DAT.
60 DAT, 90 DAT and 120 DAT. According to the result of 120 DAT, the leaf
number of tea plants was 7 with the treatment of Spirulina suspension fertilizer
followed by cowdung, urea, Armo, ash of paddy husk and control (Table 7-10
and Fig. 10).
182
Table 7. Mean leaf number of C. sinensis with different fertilizers and control
30 DAT
60 DAT
90 DAT
120 DAT
Mean ± Sd
value
Mean ± Sd
value
Mean ± Sd
value
Mean ± Sd
value
Control
3.500±0.547
3.833±0.752
4.166±0.408
4.500±0.547
Cowdung
4.666±0.577
5.000±0.707
5.833±0.752
6.333±0.516
Urea
4.166±0.841
4.667±0.577
4.833±0.753
5.333±0.943
Armo
4.167±0.456
4.500±1.046
4.666±0.577
5.166±0.752
Ash of
3.833±0.527
4.167±0.527
4.500±0.707
5.000±0.816
5.000±0.707
5.333±0.577
6.000±1.000
7.000±0.577
Fertilizers
Paddy Husk
Spirulina
In this experiment (30 DAT), the treatment of Spirulina suspension
fertilizer on the leaf number of tea plants was significantly superior than the
control at 1% level and more significant than the ash of paddy husk at 5% level
by the 't' test method. The treatment of cowdung fertilizer on the leaf number
of tea plants was more significant than the control and the ash of paddy husk at
5% level by the 't' test method (Table 8).
At 60 DAT, the treatment of Spirulina suspension fertilizer on the leaf
number of tea plants was most significant than the control at 1% level and
more significantly than the ash of paddy husk at 5% level by the 't' test method.
The treatment of cowdung fertilizer on the leaf number of tea plants was more
significant than the control at 5% level by the 't' test method (Table 9).
After 90 days of treatment, the treatment of Spirulina suspension
fertilizer on the leaf number of tea plants was most significantly than the
control at 1% level and more significantly than the Armo and ash of paddy
husk at 5% level. The treatment of cowdung fertilizer on the leaf number of
tea plants was significantly superior than the control at 1% level and more
significant than the Armo and ash of paddy husk at 5% level by the 't' test
method (Table 10).
At 120 DAT, the treatment of Spirulina suspension fertilizer on the leaf
number of tea plants was significantly superior than the control, Armo and ash
of paddy husk at 1% level and more significant than the urea at 5% level. The
treatment of cowdung fertilizer on the leaf number of tea plants was
183
significantly superior than the control at 1% level and more significant than
the Armo and ash of paddy husk at 5% level by the 't' test method (Table 11
and Fig. 10).
Table 8. Comparison on mean leaf number of C. sinensis (L.) Kuntze with
different fertilizers and control at 30 DAT
Fertilizers
Spirulina APH
A
U
CD
**
Control
– 4.429
– 1.257 – 2.290
– 1.626 – 3.590*
Cowdung
– 0.894
+2.611* + 1.664
+ 1.200
Urea
– 1.856
+ 0.822 0.000ns
Armo
– 2.425
+ 1.171
*
Ash of Paddy Husk – 3.240
*, **= Significantly different at 5 % and 1 % level respectively. ns = non signifiant.
Fertilizers
Spirulina
APH
A
U
CD
Control
– 4.543**
– 1.053
– 1.732
– 1.907
Cowdung
– 0.894
+3.162*
+ 3.012*
+ 2.301
Urea
– 2.000
+ 0.791
+ 0.430
Armo
– 1.704
+ 0.447
Ash of Paddy Husk
– 3.656*
– 4.767**
Fertilizers
Spirulina
APH
A
U
CD
Control
– 4.205**
– 1.053
– 1.732
– 1.907
– 4.767**
Cowdung
– 0.326
+3.162*
+ 3.012*
+ 2.301
Urea
– 2.283
+ 0.791
+ 0.430
Armo
– 2.828*
+ 0.447
Ash of Paddy Husk
*
– 3.000
184
Fertilizers
Spirulina
**
APH
A
U
Control
– 7.694
– 1.245
– 1.754
– 1.872
Cowdung
– 2.108
+3.380*
+ 3.130*
+ 2.278
*
+ 0.655
+ 0.338
**
+ 0.367
Urea
Armo
Ash of
– 3.692
– 4.734
CD
– 5.965**
**
– 4.898
Paddy Husk
Mean of leaf area (cm2) of C. sinensis with different fertilizers and control
The result of the comparison on leaf area (cm2) of the tea plants with
different effect fertilizers and control at 120 DAT are as shown in Table 12. In
this study, the effect of Spirulina suspension fertilizer on leaf area of tea plants
were 6.5 cm2 and followed by cowdung, urea, armo, ash of paddy husk and
control (Table 12 and Fig. 11).
Table 12. Mean Leaf Area (cm2) of C. sinensis with Different Fertilizers and
Control
30 DAT
Mean ± Sd value
Control
5.455 ± 0.121
Cowdung
6.455 ± 0.158
Urea
6.302 ± 0.219
Armo
6.273 ± 0.190
Ash of Paddy Husk
5.847 ± 0.506
Spirulina
6.568 ± 0.059
In this experiment, the treatments of Spirulina, cowdung, Armo and
urea fertilizers on the tea plants were most significant than the control at 1%
level by the "t" test method. The treatment of Spirulina fertilizer on the tea
plants was more significant than the Armo, ash of paddy husk and urea
fertilizer at 5% level by "t" test method. The cowdung fertilizers was
significant than the ash of paddy husk fertilizer at 5% level by "t" test method
(Table 13 and Fig. 11).
Fertilizers
185
Table 13. Comparison on mean leaf area (cm2) of C. sinensis (L.) Kuntze with
Spirulina
Fertilizers
**
APH
A
U
**
CD
**
Control
– 20.188
– 1.845 – 7.992
– 9.188
Cowdung
– 1.641
+2.811* + 1.644
+ 1.512
Urea
*
+ 2.062 + 0.239
*
– 1.895
– 3.272
Armo
– 3.174
*
– 3.471
Ash of
Paddy Husk
APH
A
U
CD
C
ns
Sd
't'
=
=
=
=
=
=
=
=
Ash of Paddy husk
Armo
Urea
Cowdung
Control
Non-significant
Standard Deviation
t-value
– 12.289**
186
Fig. 3. Comparison of tea plant and polythene bag with different fertilizers at
30 DAT
60 DAT
90 DAT
120 DAT
Fig. 7. Comparison on shoot height (cm) of C. sinensis control and Spirulina
fertilizer
Fig. 8. Comparison on leaf area of C. sinensis control and Spirulina fertilizer
20
TC
Cowdung
Urea
Armo
Ash of paddy husk
Spirulina
18
Mean shoot height (cm)
16
14
12
10
8
6
4
2
0
30 DAT
60 DAT
90 DAT
120 DAT
DAT = day after treatments
Fig. 9. Comparison on mean shoot height (cm) C. sinensis by using different
fertilizers and control
8
TC
Cowdung
Urea
Armo
187
Ash of paddy husk
Spirulina
7
Mean leaf number
6
5
4
3
2
1
0
30 DAT
60 DAT
90 DAT
120 DAT
DAT = day after treatments
Fig. 10. Comparison on mean leaf number C. sinensis by using different
7
2
Mean leaf area (cm )
6
5
4
3
2
1
0
Control
Cowdung
Urea
Armo
Treatment of fertilizers
Ash of paddy
husk
Spirulina
Fig. 11. Comparison on mean leaf area (cm2) C. sinensis by using different
In this study, the uniform sizes of one year old plants (about 12.5 cm
tall) were treated with Spirulina and cowdung fertilizers. After 120 DAT,
fertilizers enhanced shoot height (cm) significantly than the control and other
fertilizers at 1% level. In leaf number, the Spirulina fertilizer enhanced the
number significantly than the other fertilizers at 1% level. Also in leaf area,
the Spirulina fertilizer enhanced the area (cm2) significantly than the other
fertilizers at 1% level (Fig. 8). Urea and Armo are the chemical containing
fertilizers, cowdung and ash of paddy husk are natural and Spirulina is a
biofertilizer. Benefit obtained from using Spirulina in tea cultivation can
improve the soil fertility and no side-effect on soil and also to the tea plants.
According to these result, the treatments of Spirulina and cowdung fertilizer
188
were the best for nursery stage of tea plants. These fertilizers treatment can
establish the size of tea plant to be transplanted in the field within shorter
period than other fertilizers and control. Therefore, Spirulina (biofertilizer)
should be used for cultivation of tea plant and other crop plants.
At present, tea is widely cultivated throughout Myanmar, especially in
hilly region of Chin State, Shan State, Kachin State and Kayin State. By using
the Spirulina fertilizer, the young plants can be produced faster and in the
plantation also the plants can reach adult stage and the leaves plucked for
commercial tea production.
Acknowledgements
I would like to express my sincere thanks to Dr. Khin Maung Oo, Rector and
Dr. Si Si Hla Bu, Prorector of Yadanabon University for their permission to submit this
article. I also indebted to U Nay Win, Professor and Head for their invaluable suggestions and
guidance. I am grateful to Dr. Soe Myint Aye, Associate Professor, Department of Botany,
Mandalay University for providing all kindly necessary instruction.
References
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Cronquist, A. (1981). An Integrated System of Classification of Flowering Plants, p. 320-323.
Columbia University Press New York.
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Lane, Chichester, West Sussex. Po 19 IUD, England.
Hooker, J. D., C. B. K. C. S. I. (1894). Flora of British India. Vol. I. Reeve and Co. Ltd.
London.
Huchinson, J. (1959). The families of flowering plants. Vol. I (Dicotyledons). Second Edition.
p. 320-323. Oxford University Press.
Pandy, B. P. (2000). Economic Botany, S. Chand & Company Ltd. Ram nagar, New Delhi. p.
421-430.
Sammbanurthy, A. V. S. S and N.S. Subrahmanyam (1998). A Text book of Modern
Economic Botany, New Delhi.
Santra S. C., T. P. Chatterjee and A. P. Das (1999). College Botany Practical. Vol. 1. p 110.
8/1 Chintamoni Das Lane, Calcutta 700009.
Schery, W. R. (1972). Plants for Man. p. 589. PRENTICE-HALL, INC. Englewood (Lifts
New Jersery).
Sen, S. (1996). Economic Botan. New Central Book Agency. Allen Press, Inc. New York.
Effect of Spirulina on Growth, Yield and Nutritive Value of
Vigna unguiculata (L.) Walp.
Win Mar
Abstract
The effect of Spirulina suspension on growth, yield, nutritional value of
seeds, nitrogen content of nodule, chemical analysis of soil and colony
forming unit of soil microorganisms were studied in field experiment. The
cowpea, Vigna unguiculata (L.) Walp. (pe lun phyu) was grown with
Spirulina suspension (2 g L-1) at Kengtung University Campus. The
Spirulina suspension enhanced more effectively on root length, nodule
number, and nodule weight than those of control. The seed yield of
treatment was 59.29% greater than control. In this result, nitrogen content of
nodule in treatment was 13.08% greater than control (5.10% vs. 4.51) and
crude protein content of cowpea seeds in treatment was 15.45% which was
greater than in control (26.38% vs. 22.85%). The colony forming unit of soil
microorganisms in treatment was 45.65% greater than in control (67×106 vs.
46×106). The amount of nitrogen, phosphorous, potassium and moisture in
treatments were 24.49%, 9.52%, 32.52%, 32.70%, respectively greater than
in control. Spirulina biofertilizer helps to provide some nutrients required by
the plants and helps to increase the quality of the soil with soil
microorganisms. Thus, the Spirulina biofertilizer can be increased in yield
due to a prebiotic effect.
Key words: Spirulina, biofertilizer, Vigna unguiculata (L.) Walp., yield,
nutritional values, chemical analysis
Introduction
Myanmar is a developing country whose economy is mainly based on
agricultural product. At present, Myanmar is a leading country of pulses
production among ASEAN countries and exported 866 thousand metric tons in
2007. Cowpea is one of the major export legume crops in Myanmar and it is
used both for local consumption and export. It covered about 4.5℅ of the total
sown area of pulses (MOAI, 2010).
Moreover, the rapid growth population of global population, there is a
need to supply enough food for the growing population. Pulses are of major
importance as protein-rich foods. However, the growth of a plant depends on a
Assistant Lecturer, Department of Botany, Yadanabon University
190
sufficient supply of each nutrient and the yield is limited by the nutrients. To
obtain high yields, fertilizers are needed to supply the crops with the soil
lacking nutrients. With fertilizers, crop yields can often be doubled or even
tripled (FAO, 1965).
Use of such natural products like biofertilizers in crop cultivation will
help in safeguarding the soil health and also the quality of crop products
(Vince et al., 1998). Microalgae are used in agriculture as biofertilizers and
soil conditioners by adding innocula to the soil (Metting, 1998). Biofertilizer
improves soil fertility and enhance nutrient uptake and water uptake in
deficient soils, thereby aiding in better establishment of plants. Blue-green
algae, cyanobacteria, is the evolutionary bridge between bacteria and green
plants. Some blue green algae can fix atmospheric nitrogen into organic forms.
This is very important because organic nitrogen is essential for building
proteins and amino acid complexes in plants and animals. Blue green algae,
Spirulina can be regarded as a high quality complementary protein of
vegetable origin as it contains about 60% protein with 18 kinds of amino acids
including all essential amino acids. It is also a comparatively rich natural
source of beta carotene (pro-vitamin A), vitamin B12, calcium, potassium,
phosphorous, iron and gamma linolenic acid. Thus, Spirulina from Myanmar
can also be used as a nutritional supplement or health food as produced and
marketed in some countries (Min Thein, 1987).
Spirulina is one of the most important blue-green algae, cyanobacteria,
for our planet with several purposes. Spirulina are also important in reducing
soil erosion and raising the organic content of the soil and probably in
producing certain substance which enhance the growth of higher plants (Fox,
1996). The addition of Spirulina biomass and of a derived aqueous extract led
to an at least 10-fold increase in growth rate of the lactobacilli compared to the
control (Pulz, 2004). Application of plant hormones and synthetic plant growth
regulators on legume and grain crops might indirectly influence
microbiological processes in plants rhizosphere followed by the intense
formation of plant bacteria symbioses and associations resulting in activation
of biological nitrogen fixation and enhancement of mineral plants nutrition
(Volkogon et al., 2008).
Soil microorganisms play an important role of great economic and
environmental importance in the decomposition of organic matter and nutrient
cycling of agro-ecosystems. Several microorganisms influence nutrient uptake
in plants. Agriculturally important microorganisms that fix atmospheric
191
nitrogen and help maintain or restore soil fertility are the soil bacteria
Rhizobium, Bradyrhizobium, Azotobacter, Azospirillum, Acetobacter, and
several cyanobacteria.
The aim of this study was to analyze the effect of different
concentration of Spirulina suspension on growth and yield of cowpea cultivars;
to determine some nutritional values of cowpea seeds and nitrogen content of
nodule and to find out some chemical compositions and colony forming unit of
soil microorganisms of cowpea cultivated soil.
The cowpea used in this experiment was Yezin 1. The Spirulina
biomass used in this study were obtained from Myanmar Pharmaceutical
Factory (M.P.F). The land used for the experiment study was virgin land at
Kengtung University Campus during June 2011 to August 2011. The wild
grasses were cut and the land was plouged to clear the root-stocks and to clean
the land at the week before the experiment was started. The experiment was
conducted in a Randomized Complete Block Design (RCBD) with five
replications (Fig. 1). The plot size was 5.4 m × 6 m with 19 rows per plot. The
rows were spaced 0.3 m apart from each other with 41 plants per row. Plant to
plant distance was 0.15 m apart. Before planting, some seeds were soaked in
water as control and other seeds were presoaked in 2 g L-1 of Spirulina
suspension as treatment for 5 hours. Thinning was done at two weeks after
sowing to maintain one plant per hole and the crop was sprayed with Spirulina
biomass 0.2 kg ha-1 in 100 liters of water.
6m
5.4m
0.9m
5.4m
0.9m
CR1
6m
TR4
6m
0.9m
6m
CR3
0.9m
CR4
TR2
0.9m
6m
0.9m
6m
TR5
6m
0.9m
TR1
0.9m
6m
CR5
6m
CR2
0.9m
6m
TR3
Fig. 1. Randomized completely block design (RCBD)
192
Data Collection
For this investigation, seed yield per plant, harvest index, and total dry
matter were recorded on five randomly selected plants from each location
according to the International Board for Plant Genetic Resources Descriptior
(IBPGR, 1983).
Seed Yield Per Plant
The seed yield was measured in gram as in average from five randomly
collected plants.
Harvest Index (HI)
The proportion of biological yield represented by economic yield has
been called the harvest index (HI). All these terms characterize the movement
of dry matter to the harvest part of the plant (Gardner et al., 1985).
HI
=
Economical yield
Biological yield
Total Dry Matter
Five randomly selected plants from each replication were separated into
°
stems, leaves, roots and pods are dried to constant weight in an oven at 95 C
for dry matter determination. All of the data were analyzed by using the
Genstat, sixth edition (Lawes Agriculture Trust, 2002).
Determination of Nitrogen Content of Nodules
Five plants were taken each sample replication and number of nodules,
nodule dry weight and root length per plant were recorded at 42 DAS. Nodule
samples were spreading out at air drying. Then, nitrogen content of nodules
was analyzed in Quality Control Department, Myanmar Pharmaceutical
Factory (MPF), Ye khaar, Sagaing.
Determination of Some Nutritional Contents
Five replicated 100 seeds were weighed and nutritional contents of
seeds were analyzed in Quality Control Department, Myanmar Pharmaceutical
Factory (MPF), Ye khaar, Sagaing. Kjeldahl method was used for the
determination of crude protein percentage. Determination of carbohydrate was
done by phenol-sulphuric colorimetric method. Moisture content was
193
determined by moisture analyzer (XM-60). The content of ash was determined
by combustion in a Muffle furnace. The determination of lipid was carried out
by using Soxhlet Extraction method.
Determination of Soil Microorganisms Culture
Soil microorganisms culture were carried out by using Hi Media-M091
method in Quality Control Department, Myanmar Pharmaceutical Factory
(MPF),Ye khaar, Sagaing.
Soil Sampling and Soil Testing Method
Soil samples were taken before and after cultivation from four corners
and one center point of each plot according to FAO (1965). Soil analysis was
carried out in Quality Control Department, Myanmar Pharmaceutical Factory
(MPF), Ye khaar, Sagaing.The composition of nitrogen and phosphorous were
determined by Phosphovandomolydate procedure.The potassium content was
determined with Atomic Absorption Spectrophotometer. Moisture content was
determined by moisture analyzer.
Results
Total Dry Matter (TDM), Harvest Index (HI) and Seed Yield of
Vigna unguiculata (L.)Walp. at Harvest
The effect of Spirulina suspension on total dry matter, harvest index
and seed yield of cowpea, harvest index and seed yield showed highly
significant differences at p ≤ 0.01 probability level as in Table 1. In total dry
matter, it was 1258.2 gm-2 in treatment and 890 gm-2 in control (LSD = 52.24).
In harvest index, it was 0.788 in treatment and 0.699 in control (LSD =
0.0275). For seed yield, it was 994 gm-2 in treatment and 624 gm-2 in control
(LSD = 62.9) as in Table 1 and Figure 4.The relationship between seed yield
and total dry matter was described by a close positive exponential model (R2 =
0.995) at 1% significant level (Fig. 2). Similarly, seed yield and harvest index
demonstrated a close linear relationship (R2 = 0.954) at 1% significant level
(Fig. 3).
194
Table 1. Effect of Spirulina suspension on total dry matter, harvest index and
seed yield of Vigna unguiculata (L.)Walp. after harvesting
TDM (g m-2)
HI
Control
890.0
0.6994
624.0
Treatment (2 g L–1)
1258.2
0.7888
994.0
Fpr
<0.001
<0.001
<0.001
LSD 0.05
52.24
0.0275
62.9
4.4
2.9
6.9
Treatment
CV %
Yield (g m-2)
Fpr = F probability, LSD 0.05 = Least significant level of 5% level,
Total
drymatter
matter(gm
(g-2 )m–2)
Total dry
CV % = Coefficient of Variation
1600
1400
1200
1000
800
600
y = 0.982x + 279.9
400
R2 = 0.9958
200
0
0
200
400
600
800
1000
1200
Yield (gm-2 )
Seed yield (g m–2)
Fig. 2. Relationships between seed yield and total dry matter of
Vigna unguiculata (L.)Walp.
0.9
Harvest index
Harvest
index
0.8
0.7
0.6
0.5
0.4
0.3
y = 0.0003x + 0.5406
0.2
R2 = 0.9542
0.1
0
0
200
400
600
800
1000
1200
–2
-2
Seed
yield
Yield
(gm(g
)m )
Fig. 3. Relationships between seed yield and harvest index of Vigna
unguiculata (L.) Walp.
1200
T reatment
Yield (g m–2)
1000
800
195
Control
600
400
200
0
Spirulina suspension (g l–1)
Fig. 4. Effect of Spirulina suspension on seed yield of Vigna unguiculata
(L.)Walp. after harvesting
Root Length, Nodule Number and Nodule Weight of V. unguiculata after
42 DAS
The results of root length, nodule number and nodule weight of cowpea
after 42 DAS are presented in Table 2. The root length of treatment was
43.23% longer than control (26.64 vs. 18.60 cm). The nodule number per plant
in treatment was also 49.21% greater than control (54.64 vs. 36.62). Similarly,
the nodule weight per plant in treatment was 72.37% greater than control
(0.262 g vs. 0.152 g) as shown in Fig. 6.
Table 2. Effect of Spirulina suspension on root length, nodule number
and nodule weight of V. unguiculata after 42 DAS
Root length per
plant (cm)
No. of nodules per
plant (no.)
Dry weight of
nodules per plant (g)
Control
18.60
36.62
0.152
26.64
54.64
0.262
Fpr
<.001
<.001
0.049
LSD 0.05
0.257
0.816
0.109
6.6
6.2
5.9
Treatment
CV%
Effect of Spirulina Suspension on Nitrogen Content of Nodule of
Vigna unguiculata (L.)Walp. after 42 DAS
196
Effect of Spirulina suspension on nitrogen content of cowpea nodule
after 42DAS was shown in Table 3. In this result, nitrogen content of nodule in
treatment was 13.08% greater than control (5.10% vs. 4.51% )
Table 3. Effect of Spirulina suspension on nitrogen content of nodule of
Vigna unguiculata (L.)Walp. after 42DAS
Parameter
Control
Nitrogen (%)
4.51
5.10
Effect of Spirulina Suspension on Nutritional Compositions of Seeds of
Vigna unguiculata (L.)Walp. after Harvesting
The effect of Spirulina suspension on nutrional compositions of
cowpea seeds after harvesting was showed in Table 4. This study was done to
estimate protein, carbohydrate, moisture, lipid and ash. A wide range of crude
protein (22.85%), carbohydrate (31.63%), moisture (11.38%), lipid (1.92%)
and ash (3.21%) were found in control. The protein (26.38%), carbohydrate
(40.98%), moisture (10.96%), lipid (1.92%) and ash (3.33%) were found in
treatment.
Table 4. Effect of Spirulina suspension on nutrient compositions of
seeds of Vigna unguiculata (L.)Walp. after harvesting
Control
Crude protein (%)
22.85
26.38
Carbohydrate (%)
31.63
40.98
Moisture (%)
11.38
10.96
Lipid (%)
1.92
1.92
Ash (%)
3.21
3.33
Parameter
Effect of Spirulina Suspension on the Soil Microorganisms Culture after
42 DAS
Effect of Spirulina suspension on the soil microorganisms culture in the
cowpea cultivated soil as shown in Table 5. The colony forming unit of
treatment was 45.65 % greater than in control (67 × 106 vs. 46 × 106) as shown
in Fig. 6.
197
Table 5. Effect of Spirulina suspension on the soil microorganisms
culture in Vigna unguiculata(L.) Walp. cultivated soil
Parameter
Control
Standard Plate Count fu/g
46 x 106
67 x 106
Effect of Spirulina Suspension on Chemical Compositions of Soil
The effect of Spirulina suspension on chemical compositions of
cowpea cultivated soil after harvesting was showed in Table 6. Some
nutritional compositions of cowpea cultivated soil in control were nitrogen
(0.245%), phosphorous (569 ppm), potassium (981 ppm), moisture (13.70%)
and pH (6.90). The chemical compositions of treatment were nitrogen
(0.245%), phosphorous (569ppm), potassium (1300 ppm), moisture (18.18%)
and pH (6.95). In before cultivation, chemical compositions of soil are nitrogen
(0.210%), phosphorous (516.28 ppm), potassium (935.2 ppm), moisture (11.46
%) and pH (6.87).
Table 6. Effect of Spirulina suspension on chemical compositions in Vigna
unguiculata (L.) Walp. cultivated soil
Parameter
Before
cultivation
After cultivation
Control
Nitrogen (%)
0.210
0.25
0.30
Phosphorous (P) ppm
516.28
569.01
623.16
Potassium (K) ppm
935.2
981.00
1300.00
Moisture (%)
11.46
13.70
18.18
pH
6.87
6.90
6.95
A
B
198
Fig. 5. Field experiment of Vigna unguiculata (L.) Walp.
A. Replication no. 2 of control
B. Replication no. 2 of treatment (2 g L-1)
A
Control
Treatment (2 gl–1)
B
Fig. 6. Effect of Spirulina suspension on root nodule and colony forming
unit of soil microorganisms at 42 DAS
A. Root nodules of Control and Treatment (2 g l-1)
B. Colony forming unit of Control and Treatment (2 g l-1)
In this study, the effects of Spirulina suspension on growth, yield,
nutritional value of seeds, chemical analysis of soil, nitrogen content of nodule
and colony forming unit of cowpea cultivated soil were studied. In the present
study, the application of Spirulina were significantly promoted yield. The seed
yield of treatment was 59.29% greater than control. The highest yield of
cowpea (4124 kg acre-1) was obtained by using of Spirulina suspension (0.1 kg
acre-1). Thet Naing Htwe (2008) stated that the seed yield a 20% significant
increase with treatment over control in chickpea.
The result of this study showed that the root length of treatment was
43.23% longer than control. The nodule number and nodule weight per plant
of treatment was also 49.21% and 72.37% greater than control. Khin Lay
Nandar Aung (2011) reported that number of root nodules with using Spirulina
suspension in green gram was significantly higher than control. In this study,
the amount of nitrogen in treatment was 13.08% greater in nodule than control.
IRRI (1983) reported that soybean accumulated a large amount of nitrogen by
N 2 fixation. Legume N 2 fixation can therefore be enhanced by increasing total
N yield (Herridge et al., 2001). Therefore, Spirulina suspension does not only
increase seed yields and protein quality but also nodulation and the amount of
199
nitrogen in cowpea nodules. Spirulina biofertilizer also promoted the growth of
bacteria and concentration of nitrogen content in soil.
In this research, cowpea seed treated with Spirulina suspension had
15.45% and 29.56% increased in crude protein and carbohydrate content
respectively than control after harvesting. It agreed with Thet Naing Htwe
(2008). She reported that the protein content of Spirulina 6% treatment in
chick pea was 25.59% higher than control. In this result, the colony forming
unit of soil microorganisms with use of Spirulina was 45.65% greater than in
control.This finding is agreed with Moe Moe Kyaw (2001). She reported that
the presence of Spirulina extract or biomass in the fermentation media
produced higher bacterial growth. Spirulina increased lactobacillus by five
times over control group.
According to the soil analysis, the soil sample of cultivated cowpea
treated with Spirulina suspension was 24.49%, 9.52% and 32.52% increased in
nitrogen, phosphorous and potassium respectively than control after
cultivation. In addition, the soil sample treated with Spirulina suspension had
the highest moisture content after harvesting than in control. Mukerji (2005)
reported soil microorganisms may influence the availability of moisture and
nutrients. Metting (1996) reported that the utilization of macroalgae or their
extraction residues is the increase in water-binding capacity and mineral
composition of the soil.
These results recommended Spirulina platensis could be used as a
successful biofertilizer. There were an increase in cowpea, growth, yield and
protein content of seeds by using Spirulina. The quality crops could be
produced by biofertilizer Spirulina as well as it is more safe from the health
point of view. The poverty of essential nutrients from the soil especially
nitrogen, phosphorous and potassium are highly needed for vegetable crops
production. Therefore, the use of the bio-fertilizer Spirulina may cover
shortage in these nutrients. Biofertilizers are beneficial to the soil, as they
enrich the soil micro-organisms that help in recycling organic nutrients
(Nitrogen, Phosphorous and Potassium). It can be concluded that using
Spirulina suspension is an advantage for cowpea production on deficient soils.
200
Acknowledgements
I am grateful to Dr. Khin Maung Oo, Rector and Dr Si Si Hla Bu, Prorector of
Yadanabon University and U Nay Win, Professor and Head of Department of Botany,
Yadanabon University for their permission to submit this paper. I would like to express my
heartfelt thanks to Dr. Nu Nu Yee, Professor and Head, Department of Botany, University of
Mandalay, for her permission to carry out this research work and for providing me the
necessary facilities. My grateful to my supervisor, Dr. Min Thein (Part-Time Professor),
Myanmar Pharmaceutical Factory, Ministry of Industry No. (1), Ye Kharr, Sagaing Township,
for his valuable supervision, constructive suggestions and for providing his laboratory
facilities.
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Fox, R. (1996). Spirulina Production and Potential. EDISUD.
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State University Press.
Herridge, D. F. (2001). Inoculants and Nitrogen Fixation of Legumes in Vietnam. Proceedings
of a workshop held in Hanoi. Vietnam 17-18 February.
International Board for Genetics Resources (IBPGR) (1983). Descriptors for Cowpea.
International Board for Genetics Resources. Rome. Italy.
IRRI (International Rice Research Institute) (1983). Symposium on Potential Productivity of
Field Crops Under Different Environments. International Rice Research
Institute, Los Baños, Laguna, the Philippines.
Lawes Agricultural Trust (2002). GenStat, BSN International. 6th edition. Wilkisonson House.
Jordan Hill Road. Oxford. U.K.
Metting, D. (1988). Micro-algae in Agriculture. In Borowitzka. M.A. & L.J. Borowitzka.
[Eds.]. Micro-algal Biotechnology. Cambridge University Press. Cambridge.
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Ministry of Agricultural and Irrigation. Nay Pyi Taw. Myanmar.
Min Thein (1987). Laboratory Examination of Spirulina Samples from Burma and a Study of
Spirulina Production and Use. UNIDO/UNDP/BUR/ 85/85/018 in
Netherlands. USA and Mexico.
Moe Moe Kyaw (2001). Effect of Spirulina platensis and Its Extract on Lactobacillus sp. MSc.
(Thesis) (unpublished). Department of Zoology. University of Taunggyi.
Mukerji, K. G., C. Manoharachary, and J. Singh (2005). Microbial Activity in the Rhizosphere.
Department of Botany. University of Delhi.
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Pulz, O. and W. Gross (2004). Valuable Products from Biotechnology of Microalgae. Institute
for Biologic. Freie University. Berlin. Germany.
Thet Naing Htwe (2008). Effect of Spirulina on the Germination and Growth of Pea, Soybean
and Butter bean. PhD Dissertation (unpublished). Department of Botany.
University of Mandalay.
Vince, O. A. W. and V. S. Johannes (1998). Identification of the Cytokinin Isopenteny
Ladenice in a Strain of Arthronemia africanum (Cyanobacteria). J. Phycol.
Volkogon M. V., I. V. Dragovoz and V. K. Yavorska (2008). Using Spirulina platensis. In
book of abstract Synposium on Microalgae and Seaweed products on
Agriculture Mosonmagyarovar. Hungary.
Morphological Variations of Spirulina under Different
Environmental Parameters
Hlaing Nwe Thynn
Abstract
The trichome morphology of Spirulina Yekharr strain from Yekharr
Spirulina natural lake was studied under different environmental parameters
such as light intensity, temperature, pH and salinity. These were designed for
10 days laboratory experiment to study the morphology variations of
Spirulina. The tight spiral trichomes disappeared under low light intensities
(2.245Wm-2 and 3.115Wm-2) in laboratory culture comparing with the high
light intensity (solar radiation) in natural lakes. The light intensity 2.245Wm2
could response significantly to trichome pitch. Under three different
temperature (20°C, 25°C and 30°C), the response of natural trichomes was
distinct at 30°C. Most of the trichomes changed into barrel-shaped. At pH
10, pH 11 and pH 12, the colourless trichomes without lysis were found at
pH 12. Out of three salinity concentration; 20ppt, 40ppt and 60ppt, the
almost straight trichomes could be seen at 60 ppt salinity concentration. It
was therefore reported that the salinity concentration 60 ppt, light intensity
2.245 Wm-2, temperature 30°C and pH 12 had the significant responses to
the trichome morphology of Arthrospira platensis (Nordst.) Gom.
Morphological characters of Spirulina could change depending on the
environmental parameters. Therefore, it could be proposed that DNA
characterization is necessary to identify Spirulina species.
Key words : Spirulina, trichome, morphology, light, temperature, pH,
salinity, DNA
Introduction
Cyanobacteria (blue-green algae) are pioneers and have existed for over
3 billion years. Since then, blue-green algae created today's oxygen-filled blue
sky and helped regulate our planet's biosphere (Henrikson, 1997).
In the case of Cyanobacteria, most attention has been drawn to species
belonging to the genus Arthrospira, strains of which are marketed as a health
food under the name Spirulina (Belay, 2008). Among the various species
included in the genus Arthrospira, the most widely distributed, Arthrospira
platensis, is mainly in Africa (Chad, Kenya, Egyt, Ethiopia, Sudan, Libya,
Demonstrator, Department of Botany, Meiktila University
204
Algeria, Congo, Zaire, Zambia), Asia (Pakistan, India, Sri Lanka, Myanmar,
Thailand) and South America (Uruguay, Peru) (Vonshak and Tomaselli, 2000).
In present days one of the most promising blue-green algae is Spirulina
because of its contents. Many observations have pointed out the use of
Spirulina as a food supplement for undernourished people in many parts of the
world due to its high protein content (65%), high digestibility and specific
amino acid content (Henrikson, 1997).
Because of the marvellous quality of Spirulina, it is needed to
understand thoroughly the nature, morphology, physico-chemical parameters,
chemical compositions, phylogenetic relationships and genetic levels of this
beneficial Spirulina to fulfill the great demand of the world.
Regular helical coiling or spirals are the key characteristics of
Arthrospira spp. and have been used as a taxonomic criterion and in the
ranking of product quality (Belay, 1997).
Some researchers use the identification methods depending on the
measurement and the shape of the filament for identification while others
advise genetic tests. However, some other researchers claimed that the
biochemical composition of algae could be a criterion for taxonomic
identification (Dalay, 2002).
The species of this genus are difficult to distinguish. The origin of these
problems is the morphological variability shown in nature and in culture by
many taxa. This plasticity is especially striking in the degree of spiralization
and the arrangement of the spirals. In particular, the spontaneous appearance of
straight trichomes in a previously helical strain culture is a well-documented
phenomenon (Tomaselli, 1997).
Environmental factors, physical and chemical conditions, may affect
the helix geometry (Jeeji Bai and Seshadri, 1980). Although the helical shape
of the trichome is considered a stable and constant property maintained in
culture, there may be considerable variation in the degree of helicity between
different strains of the same species and within the same strain. Even in natural
monospecific populations, variations in trichome geometry may be observed
(Tomaselli, 1997).
As the statement of Fox (1996) which straight trichomes may be found
in the artificial commercial ponds and laboratory, when the strains from natural
lakes were isolated and cultured in laboratory, it was found that there was no
tight spiral in laboratory as time goes by.
205
Therefore, it became an interest to know why the tight trichomes
disappear, how helix pitch of Arthrospira platensis are regulated under
different conditions, which environmental conditions can make changes from
tight trichomes to loose trichomes and linear forms, how much chemical
composition and quality for commercial production differs between tight
trichomes and loose trichomes.
Because of the high responsibility of morphological features of
cyanobacteria to their different environments, Dadheech et al. (2010) informed
that the morphological features of a cyanobacterium in its natural environment
and in culture are very complicated and confusing and it is needed to include
the molecular characterization for identification of a cyanobacterial taxon.
The principal aims of the present study are to prove that the
morphological characters of Spirulina strains and to propose the reason why
the DNA characterization is needed to identify Spirulina species.
Experimental organism
Spirulina Yekharr strains were used to study the effect of
environmental parameters on helix morphology. The helix length, helix
diameter, pitch (the distance between neighboring spirals) and cell diameter of
each 100 trichomes were measured under the light microscope using a
calibrated ocular meter (Anagonostidis and Komarek, 1989).
Effects of environmental parameters on helix morphology variations
In this laboratory experiment, four essential environmental stress such
as light intensity, temperature, pH and salinity were chosen. Throughout the
experiment, the modified Zarrouk's media was used as in Myanmar
Pharmaceutical Factory (MPF media). The continuous illumination and six
hour aeration per day was done along the experimental period. All of the
experiment was carried out at room temperature (28°C) except temperature
stress experiment and illuminated with two 40W fluorescent light tubes except
light stress experiment. Daily investigation was done under the light
microscope with the help of calibrated ocular meter. From the first day to the
10th day of the experiment, the growth rate of the Spirulina strain was
recorded.
206
Light intensity effect on helix morphology variations
The natural tight-formed strains from Yekharr Lake were cultured in
200 ml MPF media at room temperature (28°C) in the laboratory and tested by
continuously illuminating with two 40 W fluorescent light tubes (2.245 Wm-2)
and three 40 W fluorescent light tubes (3.115 Wm-2).
Temperature effect on helix morphology variations
200 ml MPF media at different temperature (20°C, 25°C and 30°C) in the
laboratory by continuously illuminating with two 40 W fluorescent light tubes
(2.245 Wm-2).
pH effect on helix morphology variations
200 ml MPF media added Sodium hydroxide (NaOH) 0.2g/l for pH 10, 1g/l for
pH 11 and 4g/l for pH 12 at room temperature (28°C) in the laboratory by
continuously illuminating with two 40 W fluorescent light tubes ( 2.245 Wm2
).
Salinity effect on helix morphology variations
200 ml MPF media added Sodium chloride (NaCl) 18g/l for salinity 20 ppt,
37g/l for salinity 40 ppt and 59g/l for salinity 60 ppt at room temperature
(28°C) in the laboratory by continuously illuminating with two 40 W
fluorescent light tubes ( 2.245 Wm-2).
Results
Experimental organism
According to morphology, the collected Spirulina strains were
collected from Yekharr Spirulina Lake, belonging to the family
Phormidiaceae, Order Oscillatoriales, Class Cyanophyceae, Phylum
Cyanophyta.
207
Light intensity effect on helix morphology variations
The morphological variations of tight and loose natural trichomes were
tested under two different light intensities (2.245 Wm-2 and 3.115 Wm-2). One
day after experiment, there was any changes under both light intensities. On
the 5th day of the experiment, the trichomes under 3.115 Wm-2 became loose a
little, but the trichomes under 2.245 Wm-2 became looser than the ones under
3.115 Wm-2. On the 7th day of the experiment, the tight spirals were fewer
under both light intensities. On the 9th and 10th days of the experiment, there
was not very tight trichome under both light intensities (Figure 1). But the
trichome pitch was the greatest (nearly straight forms) under 2.245 Wm-2
(Table 1). Colour changes did not occurr along the experimental period. In this
experiment, 2.245 Wm-2 light intensity could response significantly to
trichome pitch.
Table 1. Effect of light intensity on helix's morphological variations on
day of the experiment
10th
Light intensity
Trichome
Diameter (µm)
Trichome Pitch
(µm)
Trichome Colour
2.245 Wm-2
18
46
Green
3.115 Wm-2
29
39
Green
µm
- micron meter
The values were the average of 100 trichomes.
A
B
C
Figure 1. Effect of light intensity on helix morphology variations on 10th day
(scale bar = 50 µm)
A. the morphology of natural strain (YK)
B. the morphology under 2.245 Wm-2
C. the morphology under 3.115 Wm-2
208
Temperature effect on helix's morphological variations
tested at three different temperatures (20°C, 25°C and 30°C). One day after
experiment, there was any changes under all temperature. On the 3rd day of the
experiment, the trichomes started loosening at 20°C and at 25°C, but at 30°C,
the tight spirals were a little large in the middle of the trichomes and the loose
spirals were a little short and tight. The tight trichomes became looser and
looser day by day at 20°C and at 25°C. On 10th day of the experiment, in the
comparison of loose trichomes at 20°C and at 25°C, the trichomes at 20°C are
longer than the ones at 25°C. On the other hands, the trichomes at 20°C
possessed over ten coils and the trichomes at 25°C possessed between 3 and 7
coils. No colour change was observed. The trichome pitch was the greatest at
20°C (Table 2). Most of the trichomes at 30°C were barrel-shaped and the
colour of trichomes changed yellowish green on the 10th day of the experiment
(Figure 2). In this experiment, the significant barrel-shaped morphology and
colour change were found at 30°C.
Table 2. Effect of temperature on helix morphology variations on the 10th
day of the experiment
µm
Temperature
Trichome
Diameter (µm)
Trichome Pitch
(µm)
Trichome Colour
20°C
26
37
Green
25°C
31
30
Green
30°C
52
9
Yellowish green
- micron meter
A
B
C
209
D
Figure 2. Effect of temperature on helix morphology variations on 10th day
B. the morphology at 20°C
C. the morphology at 25°C
D. the morphology at 30°C
pH effect on helix's morphology variations
tested at three different pH ranges (pH10, pH 11 and pH12). On first day of the
experiment, there was any changes in two media which possessed pH 10 and
pH 11. In the media which possessed pH 12, the trichomes were pale colour on
the first day of the experiment. On the second day of the experiment, the
trichomes were colourless without lysis. This condition was stable until the
10th day of the experiment. And then, the trichomes were lysis. In the media
which possessed pH 10 and pH 11, the trichome pitch increased day by day
(Table 3). In the media which possessed pH 11, unequal trichomes were found.
Most of the trichomes possessed decreased trichome pitch in the middle of the
trichome and increased trichome pitch at the edges of the trichomes (Figure 3).
In this experiment, the significant morphological variations and colourless
trichome were occured in the media which possessed pH 12.
Table 3. Effect of pH on helix's morphological variations on 10th day of the
Experiment
pH
µm
10
11
12
- micron meter
Trichome
Diameter (µm)
29
29
30
Trichome Pitch
(µm)
20
31
24
Trichome Colour
Green
Green
No colour
210
A
B
C
D
Figure 3. Effect of pH on helix morphology variations on 10th day
B. the morphology at pH 10
C. the morphology at pH 11
D. the morphology at pH 12
Salinity effect on helix's morphological variations
tested at three different salinity concentrations (20 ppt, 40 ppt and 60 ppt). On
the 2nd day of the experiment, the trichomes started to be loose at all of the
salinity concentration. On 10th day of the experiment, it was found that the
trichomes pitch increased more than usual. The trichomes which possessed
under 7 coils were not observed in all media (Figure 4). In the media which
possessed 60 ppt salinity concentration, the trichomes had gone nearly straight
trichomes in yellowish green and possessed the greatest trichome pitch (Table
4). This was the significant morphological variations in this experiment.
211
Table 4. Effect of salinity on helix morphology variations on 10th day of the
experiment
Salinity (ppt)
Trichome
Diameter (µm)
Trichome Pitch
(µm)
Trichome Colour
20
31
28
Green
40
32
30
Green
60
18
61
Yellowish green
µm
- micron meter
ppt
- parts per thousand
The value is the average of 100 trichomes.
A
B
C
D
Figure 4. Effect of salinity on helix morphology variations on 10th day
B. the morphology at 20 ppt salinity
C. the morphology at 40 ppt salinity
D. the morphology at 60 ppt salinity
212
Discussion
In the present study, it was found that the amounts of tight spirals are
more than the amounts of loose spirals in nature. Min Thein (1987) stated that
the tight spiral forms are considered to be mature forms and the loose helices
as intermediate forms. In the present study, laboratory experiment under
different environmental stress showed that there were no very tight spirals in
culture like the ones in nature. If the loose spirals were young spirals, they
should change into tight forms again in the laboratory like the ones in nature.
But, this condition did not happen in the present experiment.
Fox (1996) believed that Spirulina are spiraled as a protection against
photolysis. In natural lakes without agitation, the helix had to contact with high
solar radiation all day. Therefore, the helix became tighter due to self-shading.
In this present laboratory study under low light, the spirals became looser and
appeared as nearly straight filaments. Because of shaking and low light, the
spirals did not need to prevent photolysis like in natural lakes.
According to these statements, and the results of the present study, it
could be assumed that the trichomes under high light intensity as solar
radiation with no agitation in natural lake possessed the tighter spiral forms to
defend themselves against photolysis. The trichomes under low light intensity
in laboratory with well agitation in artificial ponds possessed the looser spiral
forms (Table 1 and Figure 1).
In this study, it could also be seen that temperature, one of the
environmental stresses, can change the morphology of the trichomes. Mühling
(2003) observed that 30% of the studied trichomes had reversed orientation
over 30°C. In the present study, the spiral shape in nature changed barrelshaped in laboratory culture (Table 2 and Figure 2). A fusiform or barrelshaped helical morphology may also be easier to reverse than a regular one
(Mühling 2003). The barrel-shaped trichomes in this result were tight coils.
Therefore, although this condition agreed with the statement of Van
Eykelenburg (1979) which mentioned tight coils form in high temperature, it
did not agree with the statement of Thammathorn (2001) which described
loose coils form in high temperature. If so, the helicity of trichomes may not be
affected by temperature only. It may also be affected by other environmental
stresses.
Not only pH is very important for Spirulina growth but also it can
affect on Spirulina morphology and colour of trichomes. In the present study,
213
the trichomes were colourless without lysis in the media which possessed pH
12 on second day of the experiment. It seemed that the pigments diminish in
the cells of trichomes. In Spirulina cells, carotenoids, chlorophyll and
phycocyanin are major pigments and they were used as colouring agents
(Belay 1997). In this experiment, the colour of the trichomes in the media
which possessed pH 10 and pH 11 did not change colour. Therefore, according
to these experiments, high alkalinity can damage the major pigments of
Spirulina. In the media which possessed pH 11, most of the trichomes are
unequal size in the same trichome (Table 3 and Figure 3). This condition may
lead to reproduction. Because the reproduction of Spirulina is fragmentation,
the place to fragment may need to coil tightly. Min Thein (1987) stated that the
tight spirals had necridia and fragmentation occurred at the place of the
formation of necridia.
In the present study, salt stress led to the morphology changes of
Spirulina. All of the tight spirals and the loose spirals in nature became nearly
straight trichomes in the media which possessed 60 ppt salinity concentration.
The morphological transition from helical to nearly straight may be influenced
by salt stress (Table 4 and Figure 4). Among the present environmental stress,
salt stress can change morphology of Spirulina into nearly straight trichomes
more than others.
In this designed experiment, there was no spiral breakage which caused
unsatisfactory conditions for harvesting. Breakage of filaments negatively
influences the quality and yield of the harvested biomass (Wu et al. 2005). The
looser spirals could make the cells receive more light for photosynthesis while
the tighter spirals could facilitate the cells self-shading from excessive solar
radiation (Ma and Gao 2009).
Therefore, without considering the chemical compositions of
trichomes, it could be said that not only tight spirals but also loose spirals are
suitable for application. According to the morphological point of view, both
two forms of trichomes did not result in unsatisfactory conditions for
harvesting. But it is noticed that the species identification among
cyanobacteria.
Consequently, it was proposed that when the organisms which have a
great sensitivity to the environmental parameters are also required to identify
avoiding misunderstanding and misidentification. Based on the above study, it
is therefore suggested to do not only to do morphological identification but
also DNA characterization for species identification.
214
Acknowledgements
I would like to express my heartfelt thanks to Dr Maung Thynn, Rector, Meiktila
University, Dr Khin Phyu Phyu Aye and Dr Khin Maung Htay, Pro-rectors, Meiktila
University and Dr Htun Chun, Professor and Head of Botany Department, Meiktila University
for their permission to write this paper and invaluable advice.
I am especially indebted to Dr Min Thein, part-time Professor and Dr Moat War Dine
Naw, Lecturer, Department of Botany, Mandalay University for their brilliant supervision and
invaluable guidance.
My special thanks are due to all of the members of Myanmar Pharmaceutical Factory,
Yekharr, Sagaing for their kind help and for providing me the necessary facilities of my study.
I willingly thank all of the members concerning with the publication of the
Universities Research Journal in Myanmar.
Last, but not the least, I would like to express my thanks to my parents for their moral
and financial support and encouragement throughout this work.
References
Anagnostidis, K. and J. Komarek. (1988). Modern Approach to the Classification System of
Cyanophtes 3. Oscillatoriales. Algological Studies 80: 327-472.
Belay, A. (1997). Mass Culture of Spirulina Outdoors- The Earthrise Farms Experience. In
Vonshak A. (eds.) Spirulina platensis (Arthrospira): Physiology, Cellbiology and Biotechnology. Taylor and Francis, London.
Belay, A. (2008). Spirulina (Arthrospira): Production and Quality Assurance. In Gershwin
M. E. and Belay A. (eds.) Spirulina in Human Nutrition and Health. Taylor
and Francis Group, Boca Raton.
Dalay, M. C. (2002). Arthrospira maxima (= Spirulina maxima (Stiz.) Geitl., 1930) Aci Lake
Strain. E.U. Journal of Fisheries and Aquatic Sciences. Cilt/Volume 19,
Sayi/Issue (1-2) : 241-245.
Dadheech, P. K., A. Ballot, P. Casper, K. Kotut, E. Novelo, B. Lemma, T. Proschold and L.
Krienttz. (2010). Phylogenetic Relationship and Divergence among
Planktonic Strains of Arthrospira (Oscillatoriales, Cyanobacteria) of
African, Asian and American Origin Deduced by 16S-23S ITS and
Phycocyanin Operon Sequences. Phycologia 49: 361-372.
Fox, R. D. (1996). Spirulina Production and Potential. Edisud, Aix en Provence, France.
Henrikson, R. (1997). Earth Food Spirulina (revised edition). Ronore Enterprise, Inc.,
Kenwood, California.
Jeeji Bai, N. and C. V. Seshadri. (1980). On Coiling and Uncoiling of trichomes in the genus
Spirulina, Arch. Hydrobiol., Suppl. 60, Algol. Stud., 26, 32.
215
Min Thein. (1987). Laboratory Examination of Spirulina Samples from Burma and a Study of
Spirulina Production and Use. UNIDO, Yangon.
Ma, Z. and K. Gao. (2009). Photosynthetically Active and UV Radiation act in an Antagonistic
Way in Regulating Buoyancy of Arthrospira (Spirulina) platensis
(Cyanobacterium). Elsevier. Environmental and Experimental Botany 66:
265-269.
Mühling, M., N. Harris, A. Belay and B. A. Whitton. (2003). Reversal of Helix Orientation in
the Cyanobacterium Arthrospira. J. Phycol 39:360-367.
Thammathorn, S. (2001). Factors Affecting Coiling and Uncoiling of Spirulina platensis C1.
M.Sc Thesis of Biotechnology Program, School of Bioresources and
Technology, King Mongkut's University of Technology Thonburi.
Tomaselli, L. (1997). Morphology, Ultrastructure and Taxonomy. In Vonshak,
A. (eds.)
Spirulina platensis (Arthrospira): Physiology, Cell-Biology and
Biotechnology. Taylor and Francis Ltd, London, pp. 1-15.
Van Eykelenburg, C. (1979). The Ultrastructure of Spirulina platensis in relation to
temperature and light intensity. Antonie Van Leeuwenhoek 45:369-390.
Vonshak, A. and L. Tomaselli. (2000). Arthrospira (Spirulina): Systematics and
Ecophysiology. In Whitton, B. A. and Potts, M. (eds.) The Ecology of
Cyanobacteria. Kluwer, Dordrecht.
Wu, H., K. Gao, V. E. Villafane, T. Watanabe and E. W. Helbling. (2005). Effects of Solar UV
Radiation on Morphology and Photosynthesis of Filamentous
Cyanobacterium Arthrospira platensis. Applied and Environmental
Microbiology, p. 5004-5013.
Isolation and Identification of Pathogenic Fungi from the Fruits
of Capsicum annuum L. Grown in Hinthada Area
Khin Min Min Phyo
Abstract
The dried ripe fruits of Capsicum annuum L. (Solanaceae) with infected
pathogenic fungi grown in Hinthada Area were collected. The media
preparation was selected for the isolation of pathogenic fungi from the dried
ripe fruits according to the literature. In the isolation procedure, the
pathogenic fungi were inoculated on the nutrient agar media by serial
dilution and direct inoculation methods. Two pathogenic fungi were isolated
and repeatedly inoculated to get the pure isolated pathogenic fungi.
According to the inoculation of the isolated pathogenic fungi on the various
media, the two pathogenic fungi can be keyed out as Aspergillus awamori
and Aspergillus niveus based on their macroscopical and microscopical
characters.
Key words: Capsicum annuum L., pathogenic fungi, Aspergillus awamori,
Aspergillus niveus
Introduction
The agricultural crops cultivated today are the result of selection of
plants from wild populations for several centuries with the aim of improving
the quantity and quality of the produce. Ever since man began to select plants
from among wild plant populations for domestication to meet the needs for
food, feed and fiber, the existence of several factors that might adversely affect
crop production was recognized. Among the limiting factors, diseases due to
different causes accounted for significant losses in various kinds of products
such as grains, fruits, vegetables, fodder and other products obtained from
different crops of economic importance (Espinosa-Garcia, 1991).
Diseases are caused by biotic agents such as microbial pathogens and
nematodes and abiotic factors such as unfavorable environmental conditions.
Crops may also suffer due to injury or damage caused by birds, animals and
man. Diseases due to biotic causes are more numerous than those due to
abiotic causes. Fungi, bacteria, phytoplasmas, viruses, viroids, phanerogamic
parasites and nematodes are able to infect various plant species causing
Lecturer, Department of Botany, Hinthada University
218
diseases of great historical and economic importance (Ferrman and Rodriguez,
1993).
Crop plants suffer due to several pathogenic and physiogenic diseases.
Microbial pathogens responsible for most plant diseases may be fungi, bacteria,
phytoplasmas, viruses and viroids. Most microbial pathogens induce
characteristic external symptoms on susceptible host plant species, whereas
some viruses cause no observable symptom of infection in certain plant species
known as carriers. This type of infection is termed latent infection. Symptoms
of diseases may be seen on certain plant parts such as leaves, stem, flowers of
fruits and pathogen may be confined to such tissues or organs showing
symptoms. This kind of infection is called local infection (Kohlmeyer and
Kohlmeyer, 1979).
Diseases such as leaf spots, blights, powdery mildews, and rusts are
due to local infection by fungal pathogens. Some pathogens may be able to
spread from the point of entry into the plant to different organs in which
symptoms are induced. Fungal pathogens inducing wilts and smuts, viruses
and phytoplasmas cause systemic infection in their respective host plants. The
pathogen causing the diseases in question may be isolated by various methods
and grown in appropriate culture media.
The fungal pathogens causing powdery mildews, downy mildews and
rusts and viruses require living hosts to complete their life cycle. They are
known as obligate parasites. Pathogens that can be cultured on cell-free media
are termed either facultative saprophytes or facultative parasites depending on
the extent of their ability to survive on dead organic matter in the absence of
living host plants (Petrini, 1985).
The microbial pathogens, fungi, bacteria, phytoplasmas, viruses and
viroids have some common distinguishing characteristics based on which they
are calssified into class, order, family, genus and species. The taxonomic
characters may be studied using various traditional methods involving light
microscopy or electron microscopy. Light microscopic techniques are
employed for the study of fungal. Fungal pathogens infect different plant parts
such as roots, stem, leaves, flowers and seeds. Fruits and vegetables may be
affected either in the field or during storage (Scott, 1996).
In the course of isolation for pathogenic fungi, two kinds of pathogenic
fungi were isolated from dried ripe fruits of Capsicum annuum L. (Solanaceae)
grown in Hinthada Area. Several other diseases caused by microbial pathogens
219
have been reported from time to time from various countries with differing
magnitude of losses.
Some microbes have the ability to interfere in the life process of plant
pathogens. The effectiveness of biocontrol depends on the choice of suitable
microbes and methods for introducing and maintaining them in a crop to be
protected. Different species and isolates of antagonistic microbes are screened
for their efficacy by employing various methods both under in vitro and in vivo
conditions. Microbes may produce different kinds of antibiotics capable of
inhibiting the growth of pathogens.
During the latter half of the 20th century many research programs with
the main objective of increasing the yield of cultivars without much concern
for disease resistance were implemented in different international, national and
provincial research institutes, leading to the release of crop cultivars which
were found to be highly susceptible to many diseases and insect pests
(Vanderplank, 1963).
Isolation of Pathogenic Fungi from Dried Ripe Fruit of Capsicum annuum
L.
Seeds may carry spores of some pathogenic fungi. In such cases, a
sedimentation or seed-washing test is useful for detecting spores. Seeds with
spores samples were placed in test tube containing 10 ml of 0.85% saline. The
aqueous suspension (1 ml) was transferred into other test tube containing 5 ml
of distilled water and then 0.5 ml suspension into 4.5 ml distilled water tube
after that 1 ml suspension into 4 ml distilled water. After serial dilution for
spore suspension, each of 1 ml were inoculated onto the nutrient agar plates at
three points, equidistant from the center and incubated for 7 days at room
temperature (Fig. 1). The pathogenic fungi were inoculated on the three kinds
of sterile growth media (MEA - sucrose 1%, malt extract 0.3%, agar 1.8% at
pH 7.0: CYA – sucrose 1%, yeast extract 0.3%, K 2 HPO 4 0.3%, czapek 0.5%,
agar 1.8% at pH 7.0: CY20S – sucrose 20%, yeast extract 0.3%, K 2 HPO 4
0.3%, czapek 0.5%, agar 1.8% at pH 7.0) for the macroscopical and
microscopical characters of identification (Omura, 1985).
When the fungi were sporulating on the surface of the agar media, the
pure culture can be obtained. In the direct inoculation, the fungal spores and
mycelia were placed on to the sterile growth medium ( MEA - sucrose 1%,
malt extract 0.3%, agar 1.8% at pH 7.0) in sterile petridishes at three points,
220
equidistant from the center and incubated for 7 days at room temperature (Fig.
2). After 7 days, the developing fungal colonies were examined by color of
colonies with the reverse phases and spore formations for identification.
1 ml
0.5 ml
1 ml
Dissolved for 10 min
Dried ripe fruit of
Capsicum annuum L.
10 ml 0.85% saline 5 ml DW 4.5 ml DW 4 ml DW
7 days
incubation
1 ml
Nutrient agar plate
Two types of pathogenic fungi
Fig. 1. Isolation Procedure of Pathogenic Fungi from the seeds of
Capsicum annum L.
direct
inoculation
Fig. 2. Direct isolation procedure of the pathogenic fungi from the seeds of
Capsicum annuum L.
221
Results
In the isolation of pathogenic fungi (Fig. 1 and 2), two types of pathogenic
fungi from the seeds of Capsicum annuum L. belonging to the family of
Solanaceae were isolated. According to the macroscopical characters of 1st
pathogenic fungus, the colony diameter at 7 days incubation in MEA medium is
60 mm (Fig. 3). Conidia colors are dark brown to black and mycelium color is
white and reverse colors is dull brown. The colony diameter at 7 days incubation
in CYA medium is 65 mm (Fig. 4) and the conidia colors are dark brown to black.
The mycelium color is white and reverse color is dull brown. The colony diameter
at 7 days incubation in CY20S medium is 55 mm (Fig. 5) and the conidia colors
are dark green to brown. The mycelium color is white and reverse color is dull
brown. According to the microscopical characters of this pathogenic fungus,
conidial heads are radiate, the stipe is smooth-walled, uncolored, the vesicle is
globose-shaped and biseriate, metulae cover at least the upper half of the vesicle
(Fig. 6). According to these macroscopical and microscopical characters, this
pathogenic fungus can be identified as Aspergillus awamori by the following key
steps.
According to the macroscopical characters of 2nd pathogenic fungus,
the colony diameter at 7 days incubation in MEA medium is 23mm (Fig. 7).
Conidia colors are dull orange white and mycelium color is white and reverse
colors is yellow (soluble pigment). The colony diameter at 7 days incubation in
CYA medium is 17 mm (Fig. 8) and the conidia colors are yellowish white.
The mycelium color is white and reverse color is pale yellow. The colony
diameter at 7 days incubation in CY20S medium is 13 mm (Fig. 9) and the
conidia colors are dull yellowish white. The mycelium color is white and
reverse color is yellow (soluble pigment).
Reverse phase
Fig. 3. Macroscopic characters of 1st pathogenic fungi in MEA
222
Reverse phase
Fig. 4. Macroscopic characters of 1st pathogenic fungi in CYA
Reverse phase
Fig. 5. Macroscopic characters of 1st pathogenic fungi in CY20S
X400
Fig. 6. Microscopic characters of 1st pathogenic fungi (Aspergillus awamori)
223
Key Steps for Identification of 1st Pathogenic Fungi (Klich, 2002)
1. Biseriate …………………………………………………………. 2
1. Uniseriate ………………………………………………………. 43
2. Colony diameter > 45 mm ………………………………….…… 3
2. Colony diameter < 45 mm ……………………………………… 22
3. Conidia are green on CYA ……………………………….……… 4
3. Conidia are not green on CYA …………………………….…… 11
11. Conidia in black or dark brown colors …………………….….. 12
11. Conidia in yellow colors ……………………………………… 16
12. Conidia rough walled ………………………………………….. Aspergillus
sp.
12. Conidia with smooth to finely roughened walls …………….... 14
14. Colony diameter on CYA 65-70 mm, mycelia uncolored … A. awamori
14. Colony diameter on CYA < 65 mm, mycelia yellow ……... A. sp.
Reverse phase
Fig. 7. Macroscopic characters of 2nd pathogenic fungi in MEA
224
Reverse phase
Fig. 8. Macroscopic characters of 2nd pathogenic fungi in CYA
Reverse phase
Fig. 9. Macroscopic characters of 2nd pathogenic fungi in CY20S
X 400
Fig. 10. Microscopic characters of 2nd pathogenic fungi (Aspergillus niveus)
225
According to the microscopical characters of this 2nd pathogenic fungus,
conidial heads are radiate to columnar, the stipe is smooth-walled, uncolored,
the vesicle is pyriform-shaped and biseriate, metulae crowed covering the
upper two-thirds of the vesicle (Fig. 10). According to these macroscopical and
microscopical characters, this pathogenic fungus can be identified as
Aspergillus niveus by the following key steps.
Key Steps for Identification of 2nd Pathogenic Fungi (Klich, 2002)
1. Biseriate …………………………………………………………….. 2
1. Uniseriate ……………………………………………………..……. 43
2. Colony diameter > 45 mm …………………………………………... 3
2. Colony diameter < 45 mm …………………………………….…… 22
22. Conidia > 4 µm in length …………………………………...…….. 23
22. Conidia < 4 µm in length …………………………………..…….. 26
26. Conidia in green to blue-green on CYA ………………….……… 27
26. Conidia in other colors on CYA ………………………….……… 34
34. Colony > 40 mm on CY20S & 40 mm on CYA ………..……….. 16
34. Colony diameter not as above …………………………….……… 35
35. Vesicles > 20 µm in diameter …………………………………….. 36
35. Vesicles < 20 µm in diameter …………………………………..… 37
37. Colony diameter 55-70 mm on CYA, CY20S ………… Aspergillus sp.
37. Colony diameter < 55 mm on CYA, CY20S ………………… ….. 38
38. Conidia on CYA, white to pale yellow ………………………… . 39
38. Conidia on CYA, pink color …………………………………..…...40
39. Vesicles > 15 µm, metulae covering the entire surface of the vesicle ..…
…………………………………………………………. Aspergillus sp.
39. Vesicles < 15 µm, metulae covering the upper one to two thirds of the
vesicle ………………………………………………….. Aspergillus niveus
226
Outstanding Characters of Capsicum annuum L. (Solanaceae)
Shrubs. The leaves simple, alternate, exstipulate, petiolate; the blade
oblong, the base oblique, the tip acute, the margin entire. Inflorescences
solitary and axillary cymes. Flowers white, bracteate, bracteolate,
actinomorphic, bisexual, 5 merous, hypogynous. Calyx (5), campanulate,
minute, nerved teeth, sepaloid. Corolla (5), rotate, the lobes deeply, valvate,
petaloid (white). Androecium 5, petalostemonous, filament short, anther linear,
dithecous, greenish yellow, basifixed, longitudinal dehiscence. Ovary with 2
fused carpel, axile placentation, style curved, stigma subcapitate. Fruit berry,
green, red when ripe, indehiscence and 5 -10 cm long. Seed many, compressed,
discoid, dark-brown. Flowering and fruiting periods July to September (Fig.
11).
.
As seen
Fig. 11, Habit and flower of Capsicum annuum L. (Solanaceae)
In the isolation of the pathogenic fungi, two types of pathogenic fungi
were isolated from the dried ripe fruits of Capsicum annuum L. (Solanaceae)
and it was grown in Hinthada Area. In such cases, a sedimentation or seedwashing test is useful for detecting spores. Seeds with spore samples from
Capsicum annuum L. with pathogenic fungi were placed onto the nutrient agar
plates at three points, equidistant from the center and incubated for 7 days at
room temperature.
In the direct inoculation of pathogenic fungi from the seeds of
Capsicum annuum L., the fungal spores and mycelia were placed on to the
sterile growth medium in sterile petridishes at three points, equidistant from
227
the center and incubated for 7 days at room temperature. After 7 days
incubation, the developing fungal colonies were examined by color of colonies
with the reverse phases and spore formations.
According to the macroscopical characters of pathogenic fungi from the
seeds of Capsicum annuum L., the colony diameters at 7 days incubation in
MEA media were 60 mm and 23 mm, conidia colors were dark brown to black
and dull orange, mycelium colors were white and reverse colors were dull
brown and yellow (soluble pigment), in CYA media were 65 mm and 17 mm,
the conidia colors are dark brown to black and yellowish white, reverse colors
were dull brown and pale yellow, in CY20S media were 55 mm and 13 mm,
the conidia colors were dark green to brown and dull yellowish white, reverse
colors were dull brown and yellow (soluble pigment) respectively.
According to the microscopical characters of these pathogenic fungi,
conidial heads were radiate, the stipe was smooth-walled, uncolored, the
vesicle was globose-shaped and biseriate, metulae cover at least the upper half
of the vesicle and conidial heads were radiate to columnar, the stipe was
smooth-walled, uncolored, the vesicle was pyriform-shaped and biseriate,
metulae crowed covering the upper two-thirds of the vesicle respectively.
Therefore, this would be concluded that two pathogenic fungi can be
identified as Aspergillus awamori and Aspergillus niveus based on their
macroscopical and microscopical characters of mycelia, pure colonies and
spore formations.
Acknowledgements
I wish to express my sincere thank to Dr. San Linn, Acting Rector and Dr. Aung Win,
Pro-Rector, Hinthada University, for their kind permissions to perform the present study. I
would like to acknowledge to Dr. Khin Khin Sann, Professor and Head, Department of Botany,
Hinthada University, for her permission and her constant encouragement to do this research. I
would also like to record my thank to Dr. Moe Moe Khine, Professor, Department of Botany,
Hinthada University, for her patient helps and valuable suggestions.
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89-97.
Ferrman, S. and R.J. Rodriguez (1993). Genetic conversion of a fungal plant pathogen to a
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Taxonomical Studies on Some Species of Trees Commonly
Found in Lashio Area
Yee Yee Win
Abstract
The collected twenty species of dicotyledonous trees which belong to 14
families of Angiosperms from Lashio area, Northern Shan State were
recorded, identified and presented with its scientific names, local names and
colour plates in this paper. The presented species are useful both
economically and environmentally for people. These interested species are
abundantly found in the studied area. Because of their usefulness, the plant
resources not only in this research area should be maintained as
systematically management and utilization but also must be protected greater
than before as the resources of our national profit. It is hoped that this paper
makes a payment to the taxonomic information for supporting the valuable
tree species which grow in Myanmar.
Key words: Lashio area, twenty tree species, taxonomy
Introduction
The Forests of Myanmar is one of the greatest natural resources
because they cover up large area and most of the trees have been used for
timber, firewood and many other products. The great variety of natural habitat
is shown as rich in biodiversity. Shan State is famous for its rich biodiversity
and its valuable natural resources. In Northern Shan State, Lashio area is
situated between 22° 39' 53'' and 23° 4' 27'' N. latitude and 97° 30' 10'' and 97°
50' 40'' E. longitude. The total area of Lashio is 4832 sq km and positioned on
the elevation 855 m above sea level.
Topographically, the study area is Shan plateau, characterized by the
plain areas and the mountainous areas. In this study area, average annual
rainfall is about 166 cm and average temperature is about 16.31°C. According
to latitudinal location, temperature, and rainfall, Lashio area set
down with humid subtropical climate. The soils of Lashio area is one of the
basic geographical features. The majority types of soils are found as
Mountainous Red Earth and Red Earth.
Assistant Lecturer, Department of Botany, Lashio University
230
Due to the climate, topography and kinds of soil, the natural vegetation
varies in Lashio area, i.e. evergreen forest, deciduous forest and moist mixed
deciduous forest are found. Due to this variation, many different tree species
from different families have been collected. These trees are valuable for people
both economically and environmentally. In addition, many tree species in this
area were studied and identified for taxonomic information. As a result, it was
found that not only deciduous trees, but also evergreen trees widely grow wild
and are also cultivated in the study area.
The aims and objectives of the study are; to identify the tree species of
Angiosperm in Lashio area, to give the knowledge for valuable information of
tree species that are beneficial the local people and also to contribute
taxonomic information for natural scientific researchers.
Taxonomic study of tree species in Lashio area was conducted from
2010 to 20123. All the collected specimens of woody plants were recorded by
colour photographs. Field notes were made of detailed plant description,
habitat types, and precise location by using the GPS. The specimens which
including leaves, inflorescences, flowers and fruits were collected, described,
identified and recorded as herbarium sheets. Herbaria sheets were stored in
Herbarium at Department of Botany, Lashio University.
Identification of woody specimens was carried out by using hand lens,
dissecting microscope, dissecting pin and blade and by referring the literatures
as Flora of Indian Trees (1907), Flora of Java (1968), Flora of Ceylon (1998)
and Flora of Hong Kong (2007). The final verification was made by probing
based on the earlier recorded of the herbarium specimens. Local names were
furnished and received from local inhabitants.
Results
Morphological Characters
1. Castanopsis indica DC. Prodr. 3: 186. 1830.
Local name - Thit-e
Family
- Fabaceae
Monoecious trees; stems glabrous, with brown barks. Leaves simple,
alternate; blades elliptic or oblong, obliquely obtuse at the base, sharply
serrate along the margins, acuminate at the apex, dark green above, pale
231
green beneath, glabrous on both surfaces. Inflorescences terminal
racemiform erect spike. Flowers unisexual, actinomorphic, apetalous,
epigynous, hexamerous, bracteate, ebracteolate, creamy colour. Sepals 6,
ovate, spreading, creamy colour, glabrous. Petals absent. Stamens 12, free,
exserted; filaments filiform; anthers dithecous. Ovary inferior, globoid,
unilocular, one ovule in the locule on the basal placentae; style 3, filiform,
curved; stigma capitates (Figure 1. A).
2. Chionanthus albidiflora Thw., Enum. Pl. Zeyl. 189. 1860.
Local name - Thit-hla
Family
- Oleaceae
Evergreen trees; stems glabrous, with grey barks. Leaves simple,
opposite and decussate; blades elliptic-lanceolate, cuneate at the base,
entire along the margins, acuminate at the apex, dark green above, pale
green beneath, glabrous on both surfaces. Inflorescences axillary, fasiculate
cymes. Flowers bisexual, actinomorphic, hypogynous, tetramerous,
bracteate, bracteolate, cream, fragrant. Calyx cup-shaped, 4-lobed, greenish
yellow, pubescent, persistent. Corolla tubular, 4-lobed, united in pairs for
half of their length, cream, glabrous. Stamens 2, free, included; filaments
filiform, glabrous; anthers dithecous. Ovary superior, ovoid, bilocular, two
ovules in each locule on the axile placentae; styles filiform, pilose; stigma
bifid (Figure 1. B).
3. Elaeocarpus stipularis Blume, Bijdr. Tot. de. Flora. 12. 121. 1825.
Local name - Unknown
Family
- Elaeocarpaceae
Evergreen trees; stems and branches glabrous. Leaves simple, spirally
arranged; blades narrowly ovate, attenuate and slightly asymmetrical at the
base, serrate-crenate along the margins, acuminate at the apex, with
domatia in axils of lateral veins beneath. Inflorescences unbranched
racemes, usually born on the leaves behind. Flowers bisexual,
actinomorphic, hypogynous, pentamerous, ebracteate, ebracteolate, white,
fragrant. Sepals 5, lanceolate, greenish brown, pubescent. Petals 5,
obovate, fimbriate, white, pubescent. Stamens many free, included;
filaments short; anthers dithecous, with tuft of hairs. Ovary superior, ovoid,
bilocular, with two ovules in each locule on the axile placentae; styles
short, silky hairy, with pointed stigma (Figure 1.C).
Formatted: Space After: 6 pt
232
.
4. Gluta renghas L. Sp. Pl. 671. 1753.
Local name - Thayet-thitsi
Family
- Anacardiaceae
Evergreen trees; stems terete; barks greyish-brown, often resinuous,
glabrous. Leaves simple, alternate; blades elliptic, attenuate at the base,
entire along the margins, acute at the apex, green and glabrous above, pale
green and tomentose beneath. Inflorescences terminal, paniculate cymes.
Flowers bisexual, actinomorphic, hypogynous, pentamerous, bracteate,
ebracteolate, white. Sepals 5, elliptic-ovate, pale green, tomentose. Petals 5
or 6, elliptic, white, spreading. Stamens 20, free, exserted; filaments
filiform; anthers dithecous. Ovary superior, oblongoid, unilocular, one
ovule in the locule on the basal placentae; styles single, terminal, filiform;
stigma capitates (Figure 1. D ).
5. Gmelina arborea Hort. Beng. 46, basonym. 1814.
Local name - Ye-me-ne
Family
- Verbenaceae
Large trees; stems creamy-brown barks, tomentose. Leaves simple,
opposite and decussate; blades broadly ovate, cordate at the base, entire
along the margins, long acuminate at the apex, tomentose above, tomentose
with stellate hairs beneath. Inflorescences terminal and axillary paniculate
cymes. Flowers bisexual, zygomorphic, hypogynous, pentamerous,
bracteate, ebracteolate, brownish yellow, slightly fragrant. Calyx broadly
campanulate, equally 5-toothed, with two glands, greenish brown,
yellowish tomentose without, persistent. Corolla bilabiate, unequally 5lobed; tubes ventricose upward; the middle lobe much larger than the other.
Stamens 4, didynamous, exserted; filaments cylinder, puberulous; anthres
dithecous. Ovary superior, ovoid, tetralocular, one ovule in each locule on
the axile placentae; styles terminal slender; stigma shortly bifid (Figure 1.
E).
6. Grevillea robusta A. Cunn. ex R. Br. Pl. Corom. 1:127.1795.
Local name - Khar-daw-hmi
Family
- Proteaceae
233
Evergreen, medium sized trees; stems and branches rusty-brown.
Leaves pinnately compound, unipinnate, imparipinnate, spirally arranged;
pinnate with mostly 5 to 11 pairs of opposite; pinnae deeply pinnatifid;
leaflets lanceolate, attenuate at the base, entire along the margins, acute at
the apex, brownish-tomentose beneath. Inflorescences axillary or behind
the leaves, second raceme, 1- to 5-nate in defoliolate leaf-axils, with
multiflorous; peduncles robust. Flowers bisexual, zygomorphic,
hypogyonus tetramerous, apetalous, bracteate, ebracteolate, orange-yellow.
Perianth tubular, 4-fid, orange-yellow or golden brown, near the base of the
inside dark red, pubescent without; lobes narrowly spathulate, revoluted at
the anthesis, acute at the apex. Stamens 4, free, included; filaments, short,
with 4 hypogynal glands; anthers dithecous. Ovary superior, ovoid,
unilocular, two ovules in the locule on the basal placentae; styles filiform,
persistent, curved at the anthesis, straight after the anthesis, with clubshaped stigma
(Figure 1. F).
.
7. Holarrhena pubescens (Buch-Ham.) Wall. ex G. Don, Gen. Syst. 4: 78.
1837.
Local name - Lethtok-gyi
Family
- Apocynaceae
Deciduous trees, milky latex; stems terete; barks pale brown,
glabrous. Leaves simple, opposite and decussate; blades oblong-ovate,
obtuse at the base, entire along the margins, acute at the apex, glabrescent
above, pubescent beneath. Inflorescences axillary and terminal, corymbose
cymes. Flowers bisexual, actinomorphic, hypogynous, pentamerous,
bracteate, ebracteolate, creamy-white, fragrant. Calyx campanulate, 5lobed, pubescent. Corolla tubular, 5-lobed; lobes elliptic-oblong,
overlapping to the left, creamy-white, pubescent. Stamens 5, included;
filaments slenders; anthers dithecous, saggitate. Carpel 2, free; ovaries
superior, oblongoid, unilocular, numerous ovules in the locule on the
parietal placentae; styles terminal, slender; stigma oblong-fusiform (Figure
21. A G)..
8. Lagerstroemia speciosa (L.) Pers., Syn. Pl. 2: 72. 1806.
Local name
: Pyinma
Family
: Lythraceae
234
Deciduous trees; barks creamy brown. Leaves simple, opposite and
decussate; blades elliptic-ovate, obtuse at the base, entire along the
margins, emarginate at the apex, green above, pale green and glabrous.
Inflorescences terminal, paniculate racemes. Flowers bisexual,
actinomorphic, hypogynous, hexamerous, bracteate, ebracteolate, rosepurple. Calyx campanulate, 6-lobed, woody, with 12-ribs, yellowish green,
tomentose, persistent. Petals 6, orbicular, with claws, corrugate along the
margins, purple. Stamens numerous, free, exserted; filaments slender;
anthers dithecous. Ovary superior, globoid, hexalocular, many ovules in
each locule on the axile placentae; styles slender; stigma capitates (Figure
22. AB ).
9. Ligustrum confusum Dcne in Nauv. Arch. Mus. 2.2: 24.1942.
Local name - Waso-pan
Family
- Oleaceae
Deciduous, small trees; stems and branches, pubescent to glabrous.
Leaves simple, opposite and decussate; blades elliptic-lanceolate, attenuate at
the base, entire along the margins, acute or acuminate at the apex, dark green
above, pale green beneath, glabrous. Inflorescences terminal, paniculate
racemes at the end of branchlets. Flowes bisexual, actinomorphic, hypogynous,
tetramerous, bracteate, bracteolate, yellowish white, fragrant. Calyx cupular, 4lobed, yellowish white, persistent. Corolla tubular, 4-lobed, yellowish white;
lobes reflexed at the anthesis. Stamens 2, exserted; filaments filiform, adnate
near the middle of corolla tubes; anthers dithecous. Ovary superior, oblongoid,
bilocular, with two ovules in each locule on the axile placentae; style filiform,
persistent, with bifid stigma (Figure 2. BC)..
10. Magnolia grandiflora L., Sp. Pl. 531. 1753.
Local name - Ta-daing-hmwe
Family
- Magnoliaceae
Evergreen trees; stems terete; barks pale brown. Leaves simple,
alternate; blades elliptic, attenuate at the base, entire along the margins,
acute or obtuse at the apex, dark green above, rustly beneath. Flowers
solitary and terminal, bisexual, actinomorphic, hypogynous, polymerous,
bracteate, ebracteolate, creamy colour, fragrant, large and showy. Tepals
16, supulate, concave, with long claws, caducous, creamy colour. Stamens
235
numerous free, included; filament flat, glabrous; anthers dithecous. Ovary
with numerous free carpels, spirally arranged on an elongated receptacles,
superior, ellipsoid, unilocular, two ovules in the locule on the basal
placentae; styles very short with curved stigma (Figure 2. CD).
11. Michelia champaca L., Sp. Pl. 536. 1753.
Local name - Sagawa
Family
- Magnoliaceae
Deciduous trees; stems terete; barks pale brown. Leaves simple,
alternate; blades elliptic, obtuse at the base, entire along the margins,
acuminate at the apex, dark green above, pale green beneath, glabrous.
Flowers solitary and axillary, bisexual, actinomorphic, hypogynous,
polymerous, bracteate, ebracteolate, bright orange-yellow, fragrant. Tepals
11 or 12, lanceolate, orange yellow, glabrous. Stamens numerous, free,
exserted; filaments flat, glabrous; anthers dithecous, with short appendage.
Ovary with numerous free carpels, spirally arranged on a elongated
receptacles, superior, ovoid, unilocular, two ovules in the locule on the
basal placentae; styles very short with simple stigma (Figure 2. DE).
12. Pavetta indica
L., Sp. Pl. 110. 1753.
Local name - Za-gwe-pan
Family
- Rubiaceae
Deciduous, small trees; stems and branches glabrous. Leaves simple,
opposite and decussate; blades broadly elliptic, acute at the base, entire
along the margins, obtuse or subacute at the apex, softly puberulous above,
densely tomentose beneath. Inflorescences terminal corymbose cymes.
Flowers bisexual, actinomorphic, epigynous, tetramerous, bracteate,
ebracteolate, white, fragrant. Clayx cupular, 4-lobed, pubescent. Corolla
hypocrateriform, 4-lobed, white. Stamens 4, subexserted; filaments short;
anthers dithecous. Ovary inferior, ovoid, bilocular, with single ovule in
each locule on the axile placentae; styles filiform, exserted, twice as long
as corolla tube, with bilobed stigma (Figure 2. EF).
13. Santalum album L. Sp. Pl. 349.1753.
Local name - Santagu
Family
- Santalaceae
236
Evergreen, small trees; stems and branches glabrous. Leaves
simple,opposite and decussate; blades elliptic- lanceolate, attenuate at the
base, entire along the margins, acute at the apex, green and glabrous on
both surfaces. Inflorescences terminal and axillary paniculate cymes.
Flowers bisexual, actinomorphic, perigynous, apetalous, tetramerous,
bracteate, ebracteolate, red. Perianth campanulate, petaloid, 4-lobed, red
with orange centre; lobes triangular-ovate, reflexed after the anthesis.
Stamens 4, subexserted; filaments short; anthers dithecous. Ovary semiinferior, ovoid, unilocular, three ovules in the locule on the free central
placentae; styles filiform, with inconspicuous stigma (Figure 23. FA).
14. Saurauia roxburghii Wall. Pl. Cat. 112. 1: 242.1832.
Family
- Saurauiaceae
Evergreen, small trees; stems and branches glabrous. Leaves simple,
alternate; blades narrowly obovate, obtuse at the base, double serrate along
the margins, acute at the apex, pubescent above, glabrous beneath.
Inflorescences faciculate cymes on old wood stem. Flowers bisexual,
actinomorphic, hypogynous, apetalous, pentamerous, ebracteate,
ebracteolate, white. Perianth biseriate, outer series 5-lobed, orbicular,
concave, petaloid or white; inner series cup-shaped. Stamens 35 - 40, free,
included; filaments filiform, short; anthers dithecous, versatile. Ovary
superior, globoid, pentalocular, numerous ovules in each locule on the axile
placentae; styles filiform, with five spreading stigmas (Plate 33.. A B).
15. Schefflera heptaphylla (L.) Frodin, Bot. J. Linn. Soc. 104(3): 312.1990.
Family
- Araliaceae
Evergreen, unarmed trees; stems and branches glabrous. Leaves
palmately compound, alternate; leaflets 5 - 7, unequal; blades elliptic,
obtuse to cuneate at the base, entire along the margins, acuminate at the
apex, dark green above, pale green beneath. Inflorescences terminal, large
paniculate racemes with branched umbels. Flowers bisexual,
actinomorphic, epigynous, pentamerous, bracteate, ebracteolate, greenish
yellow, fragrant. Calyx cupuliform, 5-minute teeth, inconspicuous, adnate
to the ovary, greenish yellow, caducous. Petals 5, ovate, broad at the base,
arising from the disc, pinkish yellow, spereading before falling, caducous.
237
Stamens 5, free, exserted; filaments filiform; anthers dithecous. Ovary
inferior, globoid, pentalocular, with one ovule in each locule on the axile
placentae; styles very short, with small boss stigma (Figure 3. BC).
16. Tectona hamiltoniana Wall. Pl.As. Rar.3:63. t.294.1832.
Local name - Dahat
Family
- Verbenaceae
Medium sized trees; stems and branches densely stellate hairy when
young. Leaves simple, opposite and decussate; blades rhombic-ovate,
cuneate at the base, entire along the margins, acute at the apex, densely
stellate-hairy and densely glandular pubescent beneath. Inflorescences
axillary or terminal, paniculate dichasial cymes. Flowers bisexual,
zygomorphic, hypogynous, pentamerous, bracteate, ebracteolate, pale
whitish blue, slightly fragrant. Calyx campanulate or urceolate, 5- or 6lobed, pale green, tomentose, accrescent; tubes short; lobes triangular.
Corolla rotate, funnel form, 5 or 6 lobed, whitish blue, long hairy at the
throat within. Stamens 5 or 6, subexserted; filaments filiform; anthers
dithecous, sagittate. Ovary superior, oblongoid, woolly, tetralocular, with
one ovule in each locule on the axile placentae; styles filiform, glabrous
with bifid stigma (Figure 3. CD).
17. Terminalia tomentosa
Wight & Arn. Prod.314.1856.
Local name - Htauk-kyant
Family
Formatted: Space After: 6 pt, Line spacing:
single
- Combretaceae
Deciduous trees; stems and branches tomentose when young. Leaves
simple, subopposite and distichous; blades oblong, oblique and cordate at
the base, entire along the margins, acute at the apex, sparsely tomentose
and with two stalked glands on the midribs near the leaf base beneath.
Inflorescences terminal and axillary, spike like racemes. Flowers bisexual,
actinomorphic, epigynous, pentamerous, apetalous, bracteate, ebracleolate,
creamy color. Calyx campanulate, 5-lobed, creamy color, densely
tomentose within, persistent. Stamens 10, in two series, free, exserted;
filaments filiform; anthers dithecous. Ovary inferior, globoid, densely
pubescent, unilocular, with three pendulous ovules in the locule; styles
filiform, pubescent, with simple stigma (Figure 3. DE).
238
18. Wendlandia bicuspidata Wight & Arn. Prod. 1:403. 1834.
Local name - Thit ni
Family
- Rubiaceae
Small tree; stems brown, glabrous. Leaves simple, opposite and
decussate; blades broadly elliptic-ovate, cuneate at the base, entire along
the margins, acute at the apex, pale green and pubescent beneath.
Inflorescences terminal, pyramindal, paniculate racemes. Flowers bisexual
actinomorphic, epigynous, pentamerous, bracteate, ebracteolate, creamy
colour, fragrant. Calyx cup-shaped, 5-toothed, persistent. Corolla tubular,
5-lobed; white, glabrous. Stamens 5, free, exserted; filaments filiform;
anthers dithecous. Ovary inferior, ovoid, bilocular, many ovules in each
locule on the axile placentae; styles filiform; (Figure 3. EF).
19. Wendlandia tinctoria Roxb. DC. Prodr. 4:411. 1830.
Local name
: Taung-sagyin
Family
: Rubiaceae
Small trees; stems reddish-brown or brick-red bark. Leaves simple,
opposite and decussate; blades elliptic, cuneate at the base, entire along the
margins, acute at the apex, dark green and shining above, pale green
beneath. Inflorescences terminal, pyramidal, paniculate racemes. Flowers
bisexual, actinomorphic, epigynous, pentamerous, bracteate, ebracteolate,
white, fragrant. Calyx cup-shaped, 5-toothed, puberulous, persistent.
Corolla tubular, 5-lobed, white, sparsely hairy within. Stamens 5, free,
exserted. Ovary inferior, ovoid, bilocular, many ovules in each locule on
the axile placentae; styles filiform; (Figure 3. G ).
20. Wrightia tomentosa Roem .&Schult. ,Syst.Veg. 4:414.1819.
Local name - Lettok-thein
Family
- Apocynaceae
Small trees, with milky juice; stems and branches tomentose when young.
Leaves simple, opposite; blades elliptic-lanceolate, cuneate at the base, entire
along the margins, retuse at the apex, pubescent on both surfaces.
Inflorescences terminal or axillary, few-flowered, dichasial cymes. Flowers
bisexual, actinomorphic, hypogynous, pentamerous, bracteate, ebracteolate,
turning greenish-yellow, with unpleasant odours. Calyx campanulate, 5-lobed,
pubescent without. Corolla salverform, 5-lobed, salmon pink within, bright
239
green without. Corona of two series, shorter than the anther, segment, truncate,
alternipetalous segments deeply bifid; filaments short; anthers dithecous,
sagittate. Ovary superior, 2 carpels, free, oblongoid, unilocular, many ovules in
the carpel on the parietal placentae; styles long; stigma yellow colour. (Figure
3. FH).
A
B
C
D
A
F
A
A
Figure 1. A. Castanopsis indica DC.
B. Chionanthus albidiflora Thw.
C. Elaeocarpus sitipularis Blume
D.Gluta renghas L.
E. Gmelina arborea Hort.
240
F. Grevillea robusta A. Cunn
G.
Holarrhena pubescens (Buch-Ham) Wall.
H. Lagerstroemia speciosa (L.) Pers.
241
A
C
BC
C
D
E
F
Figure 2. A. Holarrhena pubescens (Buch-Ham) Wall.
B. Lagerstroemia speciosa (L.) Pers.
Wall.
C. Ligustrum confusum DcneHolarrhena pubescens (Buch-Ham.)
Formatted: Indent: First line: 0.5"
BD. Liustrum confusum Dcne
C. Magnolia grandiflora L.
DE. Michelia champaca L.
FE. Pavetta indica L.
F.
Santalum album L.
Formatted: Indent: First line: 0"
242
A
B
C
D
E
F
G
H
Figure 3. A. Santalum album L.
D B. Saurauia roxburghii
E Wall.
A
F
C. Saurauia roxburghii Wall.
B. Schefflera heptaphylla (L.) Frodin
CD. Tectona hamiltoniana Wall.
DE. Terminalia tomentosa Wight & Arn.
E F. Wendlandia bicuspidata Wight & Arn.
G. Wendlandia tinctoria Roxb.
B
C
Formatted: Indent: First line: 0.5", Space After: 3
pt
243
HF. Wrightia tomentosa Roem. & Schult.
The present study deals with the tree species growing in Lashio area,
Northern Shan State of Myanmar. Lashio area is not very wide but containing
a large number of woody trees. Among them, selected eighteen twenty tree
species under 2314 dicotyledonous families has been studied.
In Lashio area the large woody trees such as Castanopsis indica,
Gmelina arborea, Lagerstroemia speciosa and Terminalia tomentosa are
found. The crown of each tree almost touches the other trees, forming a close
canopy.
The area of Lwe-Ngu forest band composed of multistorey of dense trees.
This forest including Grevillea robusta, Holarrhena pubescens, Pavetta
indica, Terminalia tomentosa, and Wendlandia bicuspidata. This forest is not
too large even if rich in plant resources. In addition, the study area, naturally
and artificially regenerated forest plantation of Tectona grandis are also
formed according to the reforestation programme of Forest Department. These
woody plants can also provide timber, firewood, building materials and
numerous useful plant products. In addition, Castanopsis sp., Gmelina
arborea, Michelia champaca, and Terminalia tomentosa are commercial
valuable timber species. Out of these species, Tectona grandis, and Terminalia
tomentosa have demand in overseas and domestic markets.
The cultivated species of Chionanthus albidiflora, Grevillea robusta,
Magnolia grandiflora, Michelia champaca and Santalum album can be
commonly found.
Although Lashio area fall humid subtropical climate, some tropical
woody species of Tectona hamiltoniana, Terminalia tomentosa, and Wrightia
tomentosa can be found.
In this research, important valuable woody species can not only be
recorded but also various tree species can be found. Because of their
usefulness, the plant resources in this research area are maintaining as
systematically management and utilization, it can be increased the resources of
the national profit. It is hoped that the present research gives the information of
tree species for sustaining the valuable tree species which are abundantly
grown in Myanmar.
Formatted: Centered
244
Acknowledgements
I am greatly indebted to Rector Dr Maung Maung, Lashio University for his kind
permission to conduct this paper. I am grateful to Prorector Dr Htun Hlaing, Lashio University
for providing opportunity throughout the research work. I am also thankful to Dr Swe Mar
Tin, Professor and Head, Department of Botany for providing all the necessary guidance and
encouragement.
References
Anonymous, (1980-2001). A Reviced Handbook to the flora of Ceylon, Vol. 1 to 14
University of Peradeniya, Department of Agriculture, Peradeniya, Sri Lanka.
Anonymous, (2009). Flora of Hong Kong. Hong Kong Herbarium, Agriculture, Fisheries
and Conservation Department, South China Botanical Garden, Chinese
Academy of Science.
Backer, C. A & R. C. (1963). Flora of Java, Vol. 1, 2. Rijksherbarium, Leyden, N. V. P.
Noordhoof.
Brandis, D. (1906). Indian Trees. Assisted by Indian Foresters, Archibald Constable & Co.Ltd.
16 James Street Haymarket S. W. London.
Davis, H. J. (1960). The Forests of Burma. Department of Botany, University of Florida,
U.S.A.
Heywood, V. H. (1978). Flowering Plants of The World. Oxford University press, London.
Hooker, J. D., (1879). The Flora of British India, Vol. 1 to 7, L. Reeve & Co. 5 Henrietta
Street, Covent Garden, London.
Hyndley, H. G. (1987). List of Trees, Shrubs, Herbs and Principal Climbers. etc. fourth
Revised Edition, Shwe Daw Oo Press, Mayangon, Rangoon, Burma.
Kurz, S. (1877). Forest Flora of British Burma. Office of the Superintendent of Government
Printing, Calcutta.
Lawrence, George H. M., (1964). Taxonomy of Vascular plants. The Macmillan Company,
New York.
Some Orchid Species Found in Kalay Area
Htar Lwin
Abstract
The studied area is situated on the South-Western part of Sagaing Region,
Kalay area. It lies between 22° 16' and 23° 41' N latitude and 93° 57' and 94°
46' E longitude. The studied area was characterized by mountain ranges,
pleasant climate and rich in natural plant resources. The wild species of
orchids were collected during the years of 2006 - 2007. The research work
consisted of 12 species belonging to 9 genera of family Orchidaceae. It
provided Taxonomic knowledge and distinctive characters of studied
species. Although the terrestrial orchids were widely distributed in
throughout the studied area, some epiphytic species like Pholidota articulata
Lindl., Rhynchostylis retusa Blume and Vanda thwaitesii Hook. were found
upon the giant trees in the unreserved dense-forest.
Key words: Sagaing Region, Kalay area, Orchidaceae, Taxonomic,
epiphytic species, unreserved dense-forest
Introduction
Myanmar forms the north-west corner of the Indo-Chinese or Further
Indian region. Although extending from latitude 11°- to -25°, still by far the
greatest portion of it is situated within the influence of the monsoons. The
greater part of the country is hilly or mountainous and thus favourable to the
existence of forests (Kurz, 1977). Kalay area is surrounded by Chin Hills and
Pontaung Ponnyar Ranges, and north latitude is passed through northern part
of this area, so it has advantages like pleasant surroundings and climate, and
grows a variety of natural plant resources. Kalay area due to its distinct
physiological background favoured with a rich diversity flowering species, it
includes some of the wild orchid species are growing.
Orchids are best known for their conspicuous and complex floral
structure (Kress et al., 2009). The flowers of orchids are exceptionally varied
in size and form, and the habitats of the plants are equally diverse. The flowers
of one Venezuela species have less than one millimeter in diameter, while
those of a species native to Madagascar may be more than fourty-five
centimeters long. One species of Dendrobium orchid from Java has flowers
that are so delicate they perish within five or six minutes of opening. Many
Professor and Head, Department of Botany, Banmaw University
246
orchids are epiphytic on bark of trees, others are aquatic or terrestrial, and a
saprophytic species, native to Western Australia grows and flowers entirely
underground (Nyo Maung, 2007). The floral structure are distinctive in
consisting of resupinate (resulting in a 180˚ shifted floral parts) flowers with a
showy labellum (the posterior inner median tepal, early in development), the
androecium and gynoecium adnate (termed a column, gynostegium, or
gynostemium), the pollen grains often fused into 1-several masses(pollinia),
bearing a sticky-tipped stalk (Simpson, 2009).
The aim and objectives of this research were to record the native
terrestrial orchids of Kalay area to be evaluated the valuable orchid species as
the part of orchid field studies in Myanmar.
The plant specimens were properly collected during the flowering and
fruiting periods from the year 2006 to the year 2007. Field notes were made of
precise locations and of habitat types. Firstly, identification of the collected
specimens were carried out by referring to Flora of British India (Hooker, 1954),
Flora of Java (Backer, 1963), and Flora of Ceylon (Dassanayake, 2001) to
know its generic name. The next steps were due to determine the specific
identity of the unknown plants. The index for nomenclatural data was referred
to in accordance of Index Kewensis (Hooker, 1895) by which the names and
synonyms of plants up to the rank of species had being confirmed. The
inflorescences of all the collected specimens were taken into the photographs.
The herbarium specimens were deposited at the herbarium of Mandalay
University for references and other scientific studies.
Results
Coelogyne nitida Lindl. in Wall., Cat. 1954.
Local name
: Unknown
Flowering period
: December to May
Sympodial epiphytes. Roots clinging. Pseudobulbs one-jointed, erect,
fusiform or ovate, yellowish-green to green. Leaves ovate-lanceolate, 2 leaves
per pseudobulb, deciduous, leafy at anthesis. Inflorescences basal racemes,
erect, 1- on each pseudobulb, 2- to 4-flowered; peduncular bracts 6 to 7, ovate
to ovate-oblong, creamy white; floral bracts ovate-lanceolate, creamy white to
pale yellow. Flowers 3.0 - 4.2 cm in diameter, creamy white to white; dorsal
sepals elliptic-lanceolate; lateral sepals oblong-lanceolate; petals elliptic-
247
lanceolate, labellum distinctly 3-lobed, creamy white to white with yellow
blotch; lateral lobes oblong to sub-orbicular; mid lobes oblong, creamy white
or white with brown striations and yellow blotch at the tip; spur not distinct,
column flat, white, with membranous wings; anthercaps ovoid to sub-globose,
2-locules, white; pollinia 4, oblanceolate to sub-clavate cohering in pairs by a
granular viscus, yellow, waxy; stigma sub-quadrangular, creamy white; ovary
oblongoid (Fig. 1A).
This species is growing in the wet places of the forest.
Specimen examined: Se Gyi Chaung area, N 23° 12´075" and E
93°12´147", Elevation 720.3m; January 20th, 2007; Htar Lwin.
Cymbidium sundaicum Schiltr., Flora of Java. 3: 395. 1965.
Local name
: Unknown
Flowering period
: February to May
Sympodial terrestrial. Roots fiberous and tuberous; fiberous root fleshy,
cylindrical, white; tubers ovoid, white; stems leafy, erect, hidden by the leafsheaths. Leaves lorate, deciduous, leafy at anthesis; leaf-sheaths white.
Inflorescences axillary, raceme, erect, 1- to 2 on each pseudobulb; penduncular
bracts 4 to 5, oblong-lanceolate, persistent, pale green to brownish-white; floral
bracts narrowly triangular, deciduous, pale green. Flowers 4.5 - 5.5 cm in
diameter, light yellowish green with reddish-brown stripes; dorsal sepals
lanceolate, yellowish-green with reddish-brown stripes; lateral sepals falcately
ovate, spreading or drooping, yellowish-green with reddish-brown stripes;
petals lanceolate, yellowish-green with reddish-brown stripe; labellum 3-lobed;
lateral lobes light yellowish green with reddish brown, interrupted
transverse streaklets; mid-lobe ovate, light yellowish green with irregularly
reddish-brown blotched; column yellowish white, with reddish brownspeckled, basally thickened; anther terminal, 2-loculed; pollnia 2, yellowish,
waxy; viscidium white; stigma light yellowish green, shinning; ovary
oblongoid, with 6-longitudinal ridges; fruit capsule, pyriform (Fig. 1B).
This species is growing in the wet places of the dense forest.
Specimen examined: Zi Chaung area, N 23°20´135" and E 94°08´
112", Elevation 689.2 m; February 28th, 2007; Htar Lwin.
Dendrobium transparens Wall., Cat. No. 2008. 1829.
248
Local name
: Unknown
Flowering period
: March to May
Sympodial epiphytes. Roots clinging. Pseudobulbs many jointed, erect,
or pendulous, terete, green to grayish green. Leaves alternate and distichous,
linear-lanceolate to oblong-lanceolate, deciduous, leafless at anthesis; sheaths
membranous, white. Inflorescences axillary racemes, 3 to many on each
pseudobulb, 1- to 3-flowered; peduncular bracts ovate to oblong, persistent,
membranous; floral bracts oblong, deciduous. Flowers 3.5 - 4.0 cm in
diameter, pale purplish white and pale purplish white with light or bright violet
central labellum; dorsal sepals lanceolate; lateral sepals falcately lanceolate;
petals elliptic to broader ovate; labellum elliptic-oblong from a convolute
cuneate base, attached to the base of the column-foot, not differentiated into
lateral lobes and midlobes; spur conical; column short, white with violet base;
anthercaps oblong; pollinia 4, dark yellow, waxy; caudicles and viscidium
absent; stigma oblong, white with violet margins, shining; ovary oblongoid;
fruits not seen (Fig. 1C).
This species is growing in the wet places of the forest.
Specimen examined: Khon Tha area, N 23°35´119" and E 94°25´ 013",
Elevation 348.04 m; February 28, 2007; Htar Lwin.
Geodorum purpureum R.Br. in Ait., Hort. Kew. ed. 2: 5. 207. 1810.
Local name
: Unknown
Flowering period
: April to May
Sympodial terrestrials. Roots fibrous, white. Rhizomes tuberous, sub-globose,
white. Leaves simple, alternate, elliptic-lanceolate, deciduous, green, leafy at
anthesis; sheath membranous, pale green. Inflorescences lateral racemes, erect,
flowering part drooping, 12- to 20-flowered; peduncular bracts about 4, basally
sheathing; floral bracts lanceolate, membranous, deciduous. Flowers about 9.0
mm in diameter, membranous to sub-coriaceous, white and white with violet
streaks and yellow labellum; dorsal sepals linear lanceolate; lateral sepals
linear lanceolate; petals ovate-oblong; labellum cymbiform, shallowly 3-lobed;
lateral lobes white with violet streaks and striations; mid lobe white with
yellow blotch; basal spur present, white; column short, stout purplish white,
column-foot absent; anther caps sub-globose, white with violet tips; pollinia 2,
sub-globose, yellow, waxy; stipes short and membranus; stigma sub-orbicular;
ovary oblongoid (Fig. 1D).
249
This species is growing in the moist places of the forest.
122", Elevation 689.2 m; May 21st, 2006; Htar Lwin.
Habenaria horsfieldiana Krzl., Flora of Java. 3: 253. 1965.
Local name
: Unknown
Flowering period
: November to January
Sympodial terrestrials. Roots fibrous and tuberous; fibers fleshy; tubers
ellipsoid, white. Stems leafy, erect, partially covered by the leaf-sheaths.
Leaves simple, alternate; oblong-lanceolate, persistent, green, leafy at anthesis;
sheaths pale green. Inflorescences terminal spikes, lax, solitary, 4- to 6flowered; peduncular bracts about 4, oblong-lanceolate, deciduous, pale green;
floral bracts lanceolate. Flowers white, about 2.5 cm in diameter; dorsal sepals
ovate, concave; lateral sepals ovate; petals ovate; the labellum sub-orbicular,
distinctly 3-lobed; lateral lobes ovate, margins entire; mid-lobes ovate, margin
entire, acute at the tips; spur narrow, straight or slightly incurved, white with
pale green tip; anther cells broad, divaricate, adnate to the column, white; the
pollinia 2, one in each cell, clavate, yellow, granular. Ovary oblongoid, with 6longitudinal grooved and ribbed; caudicles about 3.0 mm long, white, turned
towards the base of the anther, the viscidium membranous; stigma 2, distinct
(Fig. 1E).
This species is growing in the moist places under the shade of trees.
Specimen examined: Zi Chaung area, N 23°20´145" and E 94°08´ 100",
A
B
C
250
D
E
F
Fig. 1. A. Coelogyne nitida Lindl.
B. Cymbidium sundaicum Schiltr.
C. Dendrobium transparens Wall.
D. Geodorum purpureum R. Br.
E. Habenaria horsfieldiana Krzl.
F. Habenaria plantaginia Lindley.
Habenaria plantaginia Lindley, Gen. et Sp. Orch. 323. 1853.
Orchis platyphyllos Roxb., Fl. Ind. 3: 450. 1832.
Local name
: Unknown
Flowering period
oblong, white. Stems leafy, erect, partially covered by the leaf-sheaths. Leaves
simple, alternate; narrowly-oblong persistent, green, leafy at anthesis; sheaths
pale green. Inflorescences terminal spikes, lax, solitary, 3- to 5-flowered;
peduncular bracts many, lanceolate, deciduous, pale green; floral bracts
subulate-lanceolate. Flowers white, about 1.6 cm in diameter; dorsal sepals
ovate-oblong; lateral sepals falcately oblong; petals linear-lanceolate; labellum
more than twice as long as the sepals, distinctly 3-lobed; lateral lobes halfovate, margins entire; mid-lobes as long, narrowly linear; spur slender,
pendulous, slightly incurved, white with pale green tip; anther cells divergent
below, adnate to the column, white; the pollinia 2, one in each cell, pyriform,
granular. Ovary oblongoid, slender and beaked; caudicles broad, inserted on a
concave, lanceolate gland dividing longitudinally, the viscidium membranous;
stigma 2, distinct (Fig. 1F).
251
Specimen examined: Zi Chaung area, N 23°20´145" and E 94°08´ 100",
Habenaria dichopetala Thw., Enum. Pl. Zeyl. 309. 1861.
Local name
: Unknown
Flowering period
oblong, white. Stems leafy, erect, partially covered by the leaf-sheaths. Leaves
simple, alternate, narrowly-oblong persistent, green, leafy at anthesis; sheaths
pale green. Inflorescences terminal spikes, lax, 3- to 5-flowered; peduncular
bracts many, lanceolate, deciduous, pale green; floral bracts subulatelanceolate. Flowers white, about 1.6 cm in diameter; dorsal sepals ovateoblong; lateral sepals falcately oblong; petals linear-lanceolate; labellum more
than twice as long as the sepals, distinctly 3-lobed; lateral lobes half-ovate,
margins entire; mid-lobes as long, narrowly linear; spur slender, pendulous,
slightly incurved, white with pale green tip; anther cells divergent below,
adnate to the column, white; the pollinia 2, one in each cell, pyriform, granular.
Ovary oblongoid, slender and beaked; caudicles broad, inserted on a concave,
lanceolate gland dividing longitudinally, the viscidium membranous; stigma 2,
distinct (Fig. 2A).
100", Elevation 689.2 m; February 13, 2007; Htar Lwin.
Pecteilis sussannae (L.) Rafin., Flor. Tell. 2: 38.1836.
Orchis sussannae L., Sp. Pl. 939. 1753.
Local name
: Padein-ngo
Flowering period
: June to August.
Sympodial terrestrials. Roots fibrous and tuberous; fibers fleshy, white;
tubers ellipsoid, white. Stems leafy, erect, partially covered by the leafsheaths. Leaves simple, alternate, ovate-lanceolate, persistent, leafy at anthesis;
leaf-sheaths pale green. Inflorescences terminal, racemes, erect, 1- to 3flowered; floral bracts ovate, sub-coriaceous, persistent. Flowers 4.5-5.0 cm in
diameter, pure white; dorsal sepals obovate, concave; lateral sepals ovateoblong, reflexed; petals subulate; labellum suborbicular, attached to the base of
the column, distinctly 3-partite; lateral segments cuneate, divided into several
252
narrow segments; median segments broadly linear; spur narrow linear,
elongate, white with pale green apex, slightly curved; column white, columnfoot absent; anthers oblong, broad, divaricate, white; pollinia 2, one on each
cell, clavate, yellow, granular, caudicles white, turned towards the base of the
anther; viscidium membranous, the rostellum smooth and shining , between
the 2 cells of the anther; stigmas 2; ovary broadly oblongoid, slightly curved,
with 6-ridges, resupinate (Fig. 2B).
Specimen examined: Zi Chaung area, N 23° 12´85" and E 93°12´47",
Elevation 720.3 m; July 12th, 2006; Htar Lwin.
Peristylus plantagineus (Lindl.) Lindl., Gen. et. Sp. Orch. 300. 1835.
Herminium plantagineum Lindl., Bot. Reg. 18: 1832.
English name
: Unknown
Flowering period
: June to September
Sympodial terrestrials. Roots fibrous and tuberous; fibers fleshy, white;
tubers ellipsoid, white. Stems leafy, erect, covered by the leaf-sheaths. Leaves
simple, alternate, oblong-lanceolate, persistent, leafy at anthesis.
Infloerscences terminal, spike, erect, dense-flowered; floral bracts lanceolate,
persistent. Flowers 4.5-5.0 mm in diameter, white; dorsal sepals ovate, lateral
sepals ovate-oblong; petals orbicular; labellum ovate, concave base, attached to
the base of the column, shortly 3-lobed; spur globose, very small; column
continuous into the lip, white; anthers cells 2-locule, lateral, white; pollinia 2,
one on each cell, clavate, , yellowish, granular, the caudicles white; viscidium
membranous, small, the rostellum short, acute; stigmatic process short; ovary
oblongoid, erect, green (Fig. 2C).
Specimen examined: Zi Chaung area, N 23°20´515" and E 94°08´702",
Elevation 689.2 m; July 29th, 2007; Htar Lwin.
Pholidota articulata Lindl.in Wall., Cat. n. 1992.
Local name
: Unknown
Flowering period
: March to May
Sympodial epiphytes. Roots clinging, glabrous, greenish-white to
brownish-white. Pseudobulbs many-jointed, erect, oblong, terete to slightly
253
quadrangular. Leaves elliptic-lanceolate, mostly 2-leaves per pseudobulb, at
the top of the internodes, slightly pliated, deciduous. Inflorescences terminal
racemes, arising from the leaves axil, about 30-flowered; peduncular bracts not
found; peduncles zig-zag in flowering part; floral bracts rhombic-ovate,
caducous, creamy white to pale yellowish-green. Flowers about 1.2 cm in
diameter, sub-coriaceous, creamy white to yellowish-white; pedicels pale
green; dorsal sepals ovate, concave, creamy white to yellowish-white; the
lateral sepals ovate-lanceolate, slightly falcate, creamy white to yellowishwhite; petals elliptic - lanceolate, creamy white; labellum cymbiform with
didymous mid-lobe, with 5 basal lamellate nerves, creamy white with yellow
disk; lateral lobes saccate; the mid lobe bi-fid; spur indistinct; column short
and slender, white to yellowish-white, the column-foot not distinct; anthercaps
ovoid, glabrous, pale yellow to orange; pollinia 4, sub-globose to pyriform,
yellow, waxy, cohering in pairs by a viscus; stigma orbicular, about 1.0 mm
long and wide; ovary trigonous, glabrous, pale green. Fruits capsules, ovoid,
about 1.5 cm long, glabrous, pale green (Fig. 2D).
This species is growing upon the tree in the moist places of the forest.
Specimen examined: Se Gyi Chaung area, N 23°21´715" and E 94˚6´
151", Elevation 800.4 m; March 27th, 2007; Htar Lwin.
254
Fig. 2. A. Habenaria dichopetala Thw.
B. Pecteilis sussannae (L.) Rafin.
C. Peristylus plantagineus (Lindl.) Lindl.
D. Pholidota articulata Lindl.
E. Rhynchostylis retusa Blume
F. Vanda thwaitesii Hook.
Rhynchostylis retusa Blume, Bijdr. 286. Pl. 49. 1825.
Local names
: Foxtail orchid, Kyaung-mi-tu
Flowering period
: March to May
Sympodial stout epiphytes. Roots clinging. Pseudobulbs absent. Leaves
alternate and distichous, linear, recurved, unequally lobed at the tip, persistent,
leafy at anthesis; sheaths membranous, brown. Inflorescences axillary racemes,
many flowered; peduncular bracts oblong, persistent, membranous; floral
bracts cordate-acute, persistent. Flowers 1.5-2.0 cm in diameter, white with
light violet pink; dorsal sepals ovate; lateral sepals obliquely ovate; petals
oblong-ovate; labellum 3-lobed, clawed; lateral lobes obscure, mid-lobe
elongate; spur saccate, rounded; column short, white, rostellum shortly beaked;
anthercaps long; pollinia 2, pinkish white, waxy; caudicles short; stigma
oblong, white; ovary oblongoid; fruits obconicle, with ridge (Fig. 2E).
This species is growing upon the large trees of the forest.
Specimen examined: Zi chaung area, N 23°20´119" and E 94°8´ 613",
Vanda thwaitesii Hook. f. in Trimen, Handb. Fl. Ceylon 4: 193. 1898.
Local name
: Unknown
Flowering period
: January to March
255
Monopodial epiphytes. Roots long drooping and clinging, white. Stems leafy,
erect, lower internodes covered with brown coriaceous sheath. Leaves
alternate and distichous; blades linear oblong, rigid, falcately recurving,
coriaceous, persistent, glabrous on both surfaces, green, entire along the
margim, bifid at the tips, leafy at anthesis. Inflorescences axillary racemes,
more or less erect, 2- to 3-flowered; peduncle yellowish green; peduncular
bracts 4, sheathing, membranous, persistent, dark brown; floral bracts ovate,
whitish green. Flowers about 3.7 cm in diameter, fleshy, yellowish- green,
streaked and spotted with reddish-brown and pal yellow; pedicels angular,
glabrous, white, twisted (resupinate); dorsal sepals obovate-oblong, yellowishgreen with brownish-red speckles; lateral sepals orbicular-ovate, yellowishgreen with brownish-red speckles; petals obovate-oblong; labellum
infundibuliform, sessile on the base of the column, 3-lobed; lateral lobes
small, erect; mid-lobes broad,ovate, 2-lobulate; spur shorter than the lobes,
straight, acute; column short, stout; the anther caps broadly ovoid; pollinia 2,
obovoid, yellow, waxy, the caudicles short flat; viscidium very minute,
white; the stigma obovoid; ovary narrowly oblongoid with ridges (Fig. 2F).
This species is growing in the moist places of the forest.
Specimen examined: Se Gyi Chaung area, N 23°21´035" and E 94˚6´
791", Elevation 800.4 m; Apri16th, 2007; Htar, collected No.181.
In this research, there were 12 species belonging to 10 genera under the
family Orchidaceae. All the studied species were growing in wild. The species
of Coelogyne nitida Lindl., Dendrobium transparens Wall., Pholidota
articulata Lindl., Rhynchostylis retusa Blume, and Vanda thwaitesii Hook.
were found as epiphytic and the species of Cymbidium sundaicum Schiltr.,
Geodorum purpureum R. Br., Habenaria horsfieldiana Krzl., Habenaria
plantaginia Lindley, Habenaria dichopetala Thw., Pecteilis sussannae (L.)
Rafin., and Peristylus plantagineus (Lindl.) Lindl. are occurred as terrestrial.
Three species such as Coelogyne nitida Lindl., Dendrobium transparens Wall.
and Pholidota articulata Lindl. were showed the jointed pseudobulbs, two
species of Rhynchostylis retusa Blume and Vanda thwaitesii Hook. were found
as non-pseudobulb and the rest species are occurred as tuberous. Monopodial
256
type is found in Vanda thwaitesii Hook. and the others are showed the
spmpodial type. In the species of Coelogyne nitida Lindl., Dendrobium
transparens Wall. and Pholidota articulata Lindl., the number of pollinia was
four and the rest species have two pollinia. Among the studied species, Vanda
thwaitesii Hook. was probably extinct now as no other collecter has found and
there are no specimens in the Peradeniya Herbarium nor in Kew. Rhynchostylis
retusa Blume is rare species and occurred in 11 countries including Myanmar
mentioned by The Flora of Ceylon, Volume two.
The Orchidaceae is monophyletic with all orchids being derived from a
unique single orchid ancestor that lived perhaps over 80 million years ago. The
orchids are most closely related evolutionarily to all the other plants in the
Asparagales, such as asparagus, onions, irises, and amaryllis. Pollination of
orchid flowers is effected by various insects, birds, bats, or frogs. A single
fertilized orchid flower may produce over one million of tiny seeds, which lack
any type of nutritive cells. When the seeds reach an appropriate habitat, they
must form an antimate ecological relationship with a particular fungus. This
relationship between the orchids and their fungal partners had resulted in the
evolution of the large number of species in this family.
Hooker (1894) indicated 123 genera and 1104 species in the book of
“The Flora of British India”. Although Kress et al. (2003) recorded that 128
genera and 739 species in the book of Check List of Myanmar. However, the
species of Cymbidium sundaicum Schiltr., Habenaria horsfieldiana Krzl.,
Habenaria plantaginia Lindley, Habenaria dichopetala Thw., Peristylus
plantagineus (Lindl.) Lindl. and Vanda thwaitesii Hook. were not recorded in
this book.
The wild orchids were still naturally distributed in everywhere in
Myanmar and it would be finally hoped that this research work will be partially
fulfilled the orchid informations in Myanmar.
Acknowledgements
I wish to express my sincere gratitude to Dr. Kyi Shwin, Rector, Banmaw University,
for his permission. Further I wish to thank to Dr. Phone Myint Aung, Prorector, Banmaw
University, for his valuable suggestion.
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Plant-Pollinator Interactions of Bago University Campus, Bago
Region
Aye Aye Mar and Kyaw Zay Moe
Abstract
This observation was carried out in the Bago University Campus of Bago
Town, from June, 2011 to May, 2012. A total of 51 seasonal flowering plant
species based on numbers of 968 flowers and 676 total visits of visitors were
analyzed for the interactions of plants and their pollinators of Bago
University Campus. Insects and bird pollinated plant species were mainly
conducted, but wind and aquatic pollinated plant species were not
emphasized in this research. The results indicate that nectar is the most
common floral resources. The floral traits of the study area was mainly
composed by nectar providing plant species in floral rewards, dish-to-bowl
type in floral shapes, large in floral sizes, actinomorphic in floral symmetries
and white in floral colours. In insect pollinators, small bee and bee pollinated
plant species were mainly found.
Key words: floral traits, insects, pollinated plant species, Bago
University Campus
Introduction
Pollination is a critical stage in plant reproduction and thus in the
maintenance and evolution of species and communities. A community
perspective is important for comparing different ecosystems, for understanding
on sharing and competition for resources and their effects on community
structure, and for guiding conservation programs in threatened and fragmented
ecosystems (Machado and Lopes, 2004).
Pollination is a crucial part of the ecosystems in which they live and it
is an important ecological role, especially in the transfer of pollen from one
flower to another, which helps to reproduce for their next generation or
community (Kyaw Zay Moe, 2008).
The Convention on Biological Diversity (CBD) has recognized
pollination as a key driver in the maintenance of biodiversity and ecosystem
function. Pollination is vital for completing the life cycle of plants and is
1. Lecturer, Department of Botany, Bago University
2. Assistant Lecturer, Department of Botany, Bago University
260
essential for crop production and biodiversity conservation. Pollination is an
ecological process based on the principle of mutual interactions or interrelationships between the pollinated plant and the pollinator (Collette, 1999).
Floral traits are related to pollination vectors, and an analysis of these
attributes can help in the prediction of the pollinator of a species (Machado and
Lopes, 2004). Moreover, the interactions between floral traits and pollinator
behavior has been an important force in the coevolution of plants and their
animal pollinators (Gegear and Laverty, 2000).
Therefore, pollination studies were needed to provide awareness in the
role of floral traits and its pollinators for biodiversity conservation programs in
Myanmar.
This paper observed and compiled the data that the plant-pollinator
interactions between characterized floral traits of plant species and its various
pollinators for this campus from the aspects of botanical portion. These
relationships are very important to value and conserve for the seasonal
flowering plants of the Bago University Campus. From this concept, we should
accept that all of these relationships are a gift of nature and necessary to
conserve for the communities of plant and pollinator or forest ecosystem in
Myanmar.
The aim of this present paper was to investigate the pollinated plant
species from the interactions of plants and its pollinators based on floral traits
of the observed plant species, to seek out the information of high contribution
of floral traits for visitors as well as pollinators of the study area, to understand
the pollination processes in nature.
Field Methodology
Study area
The study area, Bago University Campus is situated to the east side of
Yangon-Mandalay Highway Road and located about 5 miles to the southern
part of Bago city (Figure 1). The global positional system point of the study
area campus is between North Latitude 17º 2′ to 18º 0′ N and between East
Longitude 96º 2′ to 96º 36′ E.
261
The study area,
Bago University
Campus
Map source: Geography Department, Bago University
Fig. (1) Wards of Bago Town and the study area Bago University Campus in
Bago Town
Observation, data collection and photo preparations
During field observations, family names, scientific names, habit, floral
traits, number of observed flowers, visitors for each plant species, their total
visits and estimated pollinators were carried out. In this research, observations
were made in daytime.
Taking photographs for floral morphologies were carried out after each
observation and prepared by photoshop CS-2 to get the clear focus and for
262
picture sizes. But, some photographs from internet were presented because a
previous recorded data storage device was inconvenient to use.
Data collection of floral traits
The floral traits such as floral symmetries, floral rewards, floral shapes,
floral sizes and floral colours were recorded according to Kearns and Inouye
(1993).
Floral symmetry
Floral symmetries were classified into actinomorphic and zygomorphic.
Floral rewards
Floral rewards such as nectar major source (N), both nectar and pollen
major source (N/P) and pollen major source (P) were mainly considered from
visitation rate observation.
Floral shapes
Floral shapes were classified according to floral types (“Structural
blossom classes”) from Faegri and Pijl, 1979. Six floral types were considered
as (1) bell-funnel; (2) tube; (3) dish-to-bowl; (4) gullet;
(5) brush; and
(6) flag.
Floral size
Floral sizes (flower diameter) in approximately 5-10 flowers per species
were measured. Flowers were classified by measuring floral diameter: (1)
small, ≤ 10mm; (2) medium, >10≤20mm; (3) large, >20≤30mm; and (4) very
large, >30mm.
263
Floral colours
Six categories of flower colour were considered with regard to the
conspicuous colour as: (1) white; (2) red; (3) yellow (including orange); (4)
purple (including blue and violet); and (5) rose (including light and pink).
Visitors
Visitors were classified into (1) small bees (< 10 mm); (2) bees (>10
mm); (3) wasp; (4) beetle; (5) butterfly; (6) moth; (7) fly; (8) small diverse
insects (SDIs); and (9) birds. Estimated pollinators are considered according to
their highest visitation rates and dichotomous key to floral syndromes table
(Parrish, 2004).
30-minute observation for estimated pollinators
Each flowering plant species were emphasized by 30-minutes visitation
rate observations to record the visitation rates of each visitor according to Judy
Parrish, 2004.
Calculation on percentages of visitation rates and floral traits
Visitation rate percentages were calculated based on total visits of each
visitor in the total visits of observed plant species (676) and floral trait
percentage was based on numbers of each floral trait per plant species in
numbers of observed plant species (51).
Plant and insect identifications
Plant specimens were verified by using literatures. Insect visitors were
recorded by taken photographs and identified by internet information using
field photographs.
264
Results and Discussions
Observed plant species
Numbers of 51 plant species together with their habits, floral traits and
numbers of observed flowers of plant species were recorded in the study area.
Observations made flower visitors on a total of 968 selected flowers of 51
plant species from 46 genera and 27 families (Fig. 2).
Floral traits for plant-pollinator interactions
In floral rewards of 51 plant species, rewards of nectar major source
plant species (N) were registered in 72.55 % followed by both nectar and
pollen major source (N/P) was 19.61 % and only pollen major source (P) was
7.84 %. Therefore, the majority of nectar providing plant species (N and N/P)
was 92.16 % (47 species) for insects in study area. This is related to the high
percentage of species pollinated by nectar-seeking insects together with
ornithophilous species.
Floral morphology can also result in placement of different species of
pollen on different parts of a common pollinator's body (Kerans and Inouye,
1993).
In floral shapes, a great variation of floral types or their morphologies
were observed with a predominance of dish-to-bowl flowers (D-B) in 14
species (27.45 %), followed by gullet (G) flowers in 10 species (19.61 %), bell
funnel (B-F) flowers in 8 species (15.69 %), tube (T) in 7 (13.73 %) and the
remaining floral types of brush (B) and flag flowers (F) were 6 species (11.76
%) in each, respectively.
In flag types, 3 plant species may pollinate by small diverse insects
(SDIs). These small diverse insects were found in the keel petals together with
the development of reproductive organs until not opened flowers. Therefore,
plant species which possessed flag types were assumed as pollinated by small
diverse insects (SDIs) although other visitors visit to these observed flowers.
In floral size, the size of the flower is generally associated with the
size of respective pollinator (Opler, 1980). In the study area, plant species
which possessed the very large sized flowers (VL) were 17 (33.33 %),
followed by large sized flowers (L) were 15 (27.45 %), the medium sized
flowers (M) were 11 (23.53 %) and finally small sized flowers were 8
(15.69 %) (Table 1).
265
In floral symmetry, actinomorphic flowers were found in majority of
species (64.71 %), mainly due to dish-to-bowl, bell-fnnel, brush and tube. The
remaining (35.29 %) was corresponding to zygomorphic species because of
flag and gullet floral types. According to Sargent (1985), zygomorphic flowers
need specific pollinators because of the reproductive isolation. In zygomorphic
flowers, gullet type plant species were mainly pollinated by small bees and
bees. Therefore, the specific pollinators of these plant species also need to
conserve for next generation of these plant species.
The behavior of potential pollinators is strongly influenced by the
colour of a flower (Scogin, 1980). In flower colours, a high contribution of
species with colourful flowers of the study area, Bago University Campus was
56.89 % in 29 plant species (including red, yellow, purple and rose) in which
rose (Rse) was 9 plant species (17.67 %), followed by red and yellow were
each 7 plant species (13.73 %), and finally purple colour was 6 plant species
(11.7 %). The remaining 22 plant species (43.14 %) were pale colour white
flowers (Table 1).
Visitor groups and their activities
In a total of 676 visits of the visitors, the highest visitors were small
bees with 225 times (33.28 %), followed by bees with 181 (26.78 %),
butterflies with 134 (19.82 %), beetles with 37 (5.47 %), flies with 26 (3.85
%), SDIs with 24 (3.55 %), moths with 19 (2.81 %), wasps with 16 (2.37 %)
and birds were 14 (2.07 %) respectiverly (Figure 2). From this point of view,
the highest visitation rate was the small bees, followed by bees and butterflies
were mainly found in the study area, Bago University Campus. Wasp from
insects and birds as animals were found in the lowest visitation rates. Wasp
and moth pollinated species were not found among insect pollinated plant
species and the two plant species of Spathodea campanulata Beauv. and Butea
frondosa Roxb. were recorded as bird pollinated plant species in animals.
Abundant visitor groups were found in Heliotropium indicum Linn. in
which beetles, small bees, bees, wasp, fly, butterfly, moth and small diverse
insects (SDIs) were included (Table. 1 and Figure 4). This plant species can be
recognized as the highest attractiveness plant species for visitor groups, and
then need to conserve for the generations of insect visitors and its values.
266
Observed pollinated plant species
In the study area, insect pollination was also the most frequent (96.08
%) and bird pollination was rarely found (3.92 %) in the study area.
In the pollinated plant species of the study area, Each 15 plant species
were pollinated by small bees and bees (29.41 %), followed by butterflies in
10 (19.61 %), small diverse insects (SDIs) in 4 plant species (7.84 %), flies
were in 3 (5.88 %) and finally beetle and bird were in each 2 plant species
(3.92 %) (Table 1 and Fig. 3).
In entomophilous plant species (combination of small bees and bees),
58.82 % were considered to be melittophilous with the remaining 41.18 % was
pollinated by other estimated pollinators in the study area. But, wasp and moth
pollinated plant species were not found although they visited to flowers.
Table (1) Numbers of pollinated plant species with respect to floral traits
Floral traits
Floral
rewards
N
N/P
P
Floral shapes
Dish-to-bowl
Bell-funnel
Gullet
Flag
Tube
Brush
Floral sizes
Small (≤10mm)
Medium
(>10≤10mm)
Large
(>20≤30mm)
Very large
(>30mm)
Floral
symmetry
Interactions between each floral traits of plants and its estimated pollinators
Beetl-e
Sma-ll
bee
Bee
Wasp
Fly
Butterfly
Moth
SDIs
Bird
2
-
8
4
3
11
4
-
-
2
1
9
1
-
-
3
1
-
2
-
1
1
-
3
1
5
1
1
4
5
4
3
1
2
-
-
1
2
4
1
1
4
-
-
1
3
-
1
1
-
-
2
1
-
2
3
-
-
-
-
3
6
4
4
-
1
-
1
2
-
2
2
1
2
4
6
-
-
4
-
-
1
Floral traits
Actinomorphic
Zygomorphic
Floral colour
White
Purple
Yellow
Rose
Red
Pollinated
plant species
267
Interactions between each floral traits of plants and its estimated pollinators
Beetl-e
Sma-ll
bee
Bee
Wasp
Fly
Butterfly
Moth
SDIs
Bird
2
-
9
6
11
4
-
3
-
7
3
-
4
2
1
1
2
5
4
3
2
1
15
7
3
2
2
1
15
-
2
1
3
6
1
3
10
-
1
1
1
1
4
2
2
N: Nectar major source; N/P: both nectar and pollen major source; P: pollen major source
1
2
3
4
5
6
7
8
9
10
12
13
14
15
11
268
16
17
21
22
26
27
19
20
23
24
25
28
29
30
18
31
32
33
34
35
36
37
38
39
40
41
46
42
47
269
44
43
48
49
50
45
51
Fig. (2) The flowers of observed plant species (51) in the Bago
University Campus
(1) Catharanthus alba (L.) G. Don; (2) Nerium indicum Mill.; (3) Plumeria
obtusa L.; (4) Thevetia peruviana (Pers.) Schum; (5) Allamanda cathartica L.;
(6) Eupatorium odoratum Linn.; (7) Enhydra fluctuans Lour.; (8) Thumbergia
laurifolia Lindl.; (9) Heliotropium indicum Linn.; (10) Spathodea campanulata
Beauv.; (11) Cassia alata Linn.; (12) Cassia fistula Linn.; (13) Caesalpinia
pulcherrima Linn.; (14) Cassia javanica L..; (15) Bouhinia acuminata L.; (16)
Amherstia nobilis Wall.; (17) Gynandropsis gynandra (L.)Pers; (18) Tridax
procumbens L.; (19) Terminalia catappa L.; (20) Impomoea bona-nox Linn.;
(21) Ipomaea pupurea (L.) Roth.; (22) Crotalaria striata Schrank.; (23)
Canavalia ensiformis DC.; (24) Desmodium triquetrum DC.; (25) Acacia
auriculiformis Benth.; (26) Meusa ferrea Linn.; (27) Hyptis suaveolens (L.)
Poit.; (28) Leea rubra Blume.; (29) Lagerstroemia macrocarpa Wall.; (30)
Sida rhombifolia Linn.; (31) Urena rigida Wall.; (32) Hibiscus rosasinensis
L.; (33) Callistemon lanceolatus DC.; (34) Melastoma malabathrium L.; (35)
Acacia pennata (L.) Willd.; (36) Albizzia lebbek Benth.; (37) Albizzia procera
Benth.; (38) Mimosa pudica Linn.; (39) Moringa oleifera Lamk.; (40) Butea
frondosa Roxb.; (41) Clitoria ternatia L.; (43) Ziziphus jujuba Mill & Lan.;
(44) Minusops clengi L.; (45) Torenua flurnieri Linden.; (46) Microcos
270
pollinated plant species (% )
paniculata L.; (47) Turnera ulmifolia L.; (48) Tectona grandis Linn.f.; (49)
Clerodendrum serratum Spreng.; (50) Costus speciosus (Koenig) Smith.; (51)
Globba sessiflora Sims.
30.00%
25.00%
20.00%
15.00%
10.00%
5.00%
0.00%
29.41% 29.41%
Small bee
19.16%
Bee
7.84% 5.88%
3.92% 3.92%
Butterfly
SDIs
Fly
Beetle
Bird
Estimated pollinators
Fig. (3) Pollinated plant species by estimated pollinators (%) in Bago
University Campus
numbers of observed plant species (51)
Globba sessiflora Sims.
Costus speciosus (Koenig) Smith.
Clerodendrum serratum Spreng.
Tectona grandis Linn.f.
Turnera ulmifolia L.
Microcos paniculata L.
Torenua flurnieri Linden.
Minusops clengi L.
Ixora rosea K. Schum.
Ziziphus jujuba Mill & Lan.
Clitoria ternatia L.
Butea frondosa Roxb.
Moringa oleifera Lamk.
Mimosa pudica Linn.
Albizzia procera Benth.
Albizzia lebbek Benth.
Acacia pennata (L.) Willd.
Melastoma malabathrium L.
Callistemon lanceolatus DC.
Hibiscus rosasinensis L.
Urena rigida Wall.
Sida rhombifolia Linn.
Lagerstroemia macrocarpa Wall.
Leea rubra Blume.
Hyptis suaveolens (L.) Poit.
Meusa ferrea Linn.
Acacia auriculiformis Benth.
Desmodium triquetrum DC.
Canavalia ensiformis DC.
Crotalaria striata Schrank.
Ipomaea pupurea (L.) Roth.
Impomoea bona-nox Linn.
Terminalia catappa L.
Tridax procumbens L.
Gynandropsis gynandra (L.)Pers
Amherstia nobilis Wall.
Bouhinia acuminata L.
Cassia javanica L.
Caesalpinia pulcherrima Linn.
Cassia fistula Linn.
Cassia siamea Linn.
Spathodea campanulata Beauv.
Heliotropium indicum Linn.
Thumbergia laurifolia Lindl.
Enhydra fluctuans Lour.
Eupatorium odoratum Linn.
Allamanda cathartica L.
Thevetia peruviana (Pers.) Schum
Plumeria obtusa L.
Nerium indicum Mill.
Catharanthus alba (L.) G. Don
271
Beetle
Small
bee
Bee
Wasp
Fly
Butterfly
Moth
0
5
10
15
20
25
Visitation rates (%) and various visitor groups per plant species
Fig. (4) Visitor groups and their visitation rates in plant species (51)
30
35
272
Conclusion
Most of plants species of the study area are also mainly pollinated by
insect visitors among observed plant species. Insect pollination was the most
frequent, occurring in approximately 96.08% and bird pollinated plant species
were rarely found (3.92 %) in 51 observed plant species.
The plant species of Heliotropium dindicum Linn. was found in the best
providing attractants for insect visitor groups. This plant species can attracts 7
visitor groups, followed by Eupatorium odoratum Linn., Ipomoea pupurea (L.)
Roth. and Crotalaria striata Schrank. (with 6 visitor groups), followed by
Thumbergia laurifolia Lindl., Enhydra fluctuans Lour., Urena rigida Wall.,
Moringa oleifera Lamk., Ziziphus jujuba Mill & Lan., Torenua flurnieri
Linden., and Amomum corynostachyum Wall (with 5 visitor groups). The plant
species of Spathodea campanulata Beauv. and Butea frondosa Roxb. were
found in only bird (animal) pollinated plant species (Fig. 4).
All of above these plant species possess the characterized attractants or
floral traits for various visitor groups including birds, and thus need to
conserve these plant species for the generations of both plant and its visitors of
the Bago University Campus.
In the study area, the majority floral traits were mainly composed by
nectar providing plant species in floral rewards, dish-to-bowl type in floral
shapes, large in floral sizes, actinomorphic in floral symmetries and white in
floral colours. These traits were mainly provided ecological services for the
plant-pollinator interactions of Bago University Campus.
The highest contribution of insect pollinated pollinators which
dependent on the floral traits in the study area were characterized by the
dominance of small bees and bees (melittophily) because small bees and bees
were the world known pollinators, followed by butterflies (psychophily), small
diverse insects (SDIs), and finally fly and beetle pollinated plant species. Wasp
and moth pollinated plant species were not found although they visit to
observed flowers (Table 1).
In suggestion, bee species are the best performance pollinators in
pollination processes. The conservation of small bees and bees pollinated plant
species should be conducted in order to conserve for the world known
pollinators in nature. Moreover, bee plants are critical to observe for a local
area in accordance with the bee community, medicines and ecological roles.
Nowadays, bee pollination systems have been urgently declined because of the
273
climate changes and anthropogenic actions. In this research paper, small bees
and bees visiting plant species can also be noticed as the bee plants of Bago
University Campus. Finally, other researches which are concerned in small bee
and bee visiting plant species or the plant species which provide nectar and or
pollen as food for small bees and bees should be carried out from the botanical
aspects or a combination of botany and zoology in order to conserve our own
ecosystems in Myanmar.
Acknowledgements
We would like to express my gratitude to Dr. Kyi Soe, Rector, Bago University for
allowing us to conduct this research. We are greatly indebted to Dr. Moe Moe Shwe, Professor
and Head, Department of Botany, Bago University for the constructive guidance to carry out
pollination study in Bago University campus. Moreover, we warmly like to express grateful
acknowledge to professor Dr. San San Aye and Associate Professors, Lecturers, Assistant
Lecturers and Demonstrators of Botany Department for their invaluable advices and
encouragement throughout the study period.
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Drinking water Analysis of Artesian wells found in Yinmabin
Township, Monywa District
Theingi Htay
Abstract
In this research, Analysis of drinking water from artesian well storage ponds
found in Yinmabin Township, Monywa District, Sagaing Region were
presented. Water samples were collected from all of the study area within the
year 2010. According to the water analysis results, 85 wells were recorded as
potable and 14 wells were unpotable, out of 99 artesian wells. Most of the
artesian wells storage ponds have been used for irrigation nearby the
villages. The parameters of drinking water samples were collected and
presented in six study area. The water used for drinking water to determine
Biochemical Oxygen Demand (BOD) and Chemical Oxygen Demand
(COD) found them to exceed the maximum permissible limit of WHO
Guideline Values (2000). Heavy metals lead, cadmium, mercury, and arsenic
were more than 1 ppb which was more than WHO guideline values. The
amount of coliform bacteria in the drinking water remains constant for all
stations during the dry and wet seasons.
Key words: BOD, COD, coliform bacteria
Introduction
Generally water is fulfilled by rain water which gets deposited in
surface and ground water resources (Goel 1997). As the rain water has to be
stored in water resources like lakes, reservoirs and underground aquifers.
Water wells tap into this underground water supply (Goel 1997). Wells drilled
into confine aquifer or artesian aquifers are called artesian wells and
commonly yield large quantities of high quality water (Spellman 2008). There
is a large artesian system under Queensland, Australia and in North America
and the Great Plains. "Artesian" comes from the name of the town of Artois,
France (Frank 2002).
According to Gray (1978), water from natural environment, especially
the rainwater and groundwater, is of high quality. Changes in water quality are
often reflected by changes in the algal community. Excessive algal growth can
cause a number of water quality problems, including: bad taste and odor,
deoxygenation of water.
Professor and Head, Department of Botany, Shwebo University
276
Very high values of alkalinity are harmful to aquatic organisms (Goel
1997). Aquatic organisms are also useful as indicators because the cope with
chemical, physical, and biological influences in their habitat over the course of
their entire aquatic life cycle. Phytoplankton long as been used as indicators of
water quality (APHA 1985). Algae are also an important component of the
ecosystem in river and streams, making them a valuable indicator of water
quality (Schimidt 1987).Water resources are useful or potentially useful to
humans, especially agriculture, industrial, household, recreational and
environmental activities.
It is estimated that 69% of worldwide water use is for irrigation. 8% of
worldwide water use is for household purposes. These include drinking water,
bathing, cooking, sanitation, and gardening. Most water is purified for human
consumption but water purification may also be designed for a variety of other
purpose, including meeting the requirements of medical, pharmacology,
chemical and industrial applications.
In this research, an attempt has been made to contribute to the
knowledge of drinking water of different artesian well storage ponds found in
Yinmabin Township, Monywa District. This township is situated at the west of
Monywa and Chindwin River. It is located at latitude 21°59'N and longitude
94°36'E. The total urban area of Yinmabin is 362.7 square miles. The elevation
of this area is 600 feet above sea level. The climate condition of this area is
tropical. The hottest months are April and May with a humidity of 52%.
January is coldest month and the annual average temperature is about 38°C.
The heaviest rainfall is in September with an average rainfall of 30.47 inches.
There are 99 artesian well storage ponds in this township since 1994
according to project of the Water Resource Utilization Department, Monywa
District. Measurements of these ponds are (61×61×3) metre. There are 42
village tracts in Yinmabin Township. Among them thirteen village tracts will
benefit from the groundwater irrigation project. Kyisintaung, Sabetaung are at
the south and Pontaung regions are at the west side. Groundwater from
Wazeintaung (north side), Alongdaw Kathapha region (northwest side) are
become an aquifer under Yinmabin Township, which can not flow to the
Chindwin river because of the barrier set up by Sabetaung, Kyisintaung and
Letpandaungtaung.
The aim of the present study is to record drinking water quality of
artesian wells. The objectives of the study are to asses the current status of
water quality in study areas whether it is beyond the quality thresholds value or
277
not, to evaluate the correlations between the characteristics of the physical,
chemical and biological parameters variation of water quality in dry and wet
season and to find out the environmental risk area due to human impact.
Study Area
Samples of water were collected six drinking water samples from
artesian wells storage ponds. These ponds are situated at the Yinmabin
Township, Monywa District, Sagaing Region. Head of Department of Water
Resource Utilization, Monywa District had given the names of these artesian
wells since 1994, such as N, R, L and A which were denoted according to the
location of their sides of the Monywa to Pale main road. N is indicated at the
north side of road and includes 36 wells. R is indicated at the south side of the
road and includes 39 wells. L is at the west side of road and includes 16 wells.
A is the accessory wells and includes 8 wells shown in Figure 1.
Figure 1. Location Map of Artesian Wells in Study Area
278
Six different ponds were selected for drinking water analysis. Station
L1, L 2 and R 1 are in the south of Yinmabin; stations N 18 and N 23 were
Yahtaung village in the north of Yinmabin and station N 32 is Lethloke village
in the west of Yinmabin shown in Figure 2.
A
B
C
D
E
F
Figure 2. Locations of drinking water samples
A. N-18
B. N-23
C. N-32
D. L-1
E. L-2
F. R-1
279
Sampling and Analysis of Water
Drinking water samples were collected from six sampling sites near
Yinmabin which were currently used as drinking and potable water for public
use. The drinking water samples were collected directly from the pumping
water pipe-line during the dry (May, 2010) and Wet seasons (August, 2010).
The names of drinking water sampling sites were L1, L2, R1, N 18, N 23 and
N 32. The water samples for physico-chemical and bacteriological properties
were analyzed at the Water Laboratory, Water and Sanitation Department,
Public Health Laboratory, Mandalay and Department of Fisheries, Freshwater
Fisheries Research Center, Chemical Laboratory (Thaketa, Yangon). Then,
concentrations of heavy metals were analyzed at the Universities Research
Center, Yangon.
Physical and Chemical Analysis of Drinking Water
Total Solids
Porcelair crucible (3 and 4 cm diameter), a desicator, a hot plate and an
electric oven were used.
Total Hardness
The 50 ml water sample was mixed with 1 ml ammonia buffer solution
at pH 7.5 and 5 drops of indicator solution and the mixture was titrated with
ethylene diamine tetra acetic (EDTA) solution, until a clear blue colour
appeared and it was calculated by the following equation
Total Hardness mg/L =
ml of titrant used × 1000
ml of sample
Biochemical Oxygen Demand
Water sample were filled into glass bottles and initially dissolved
oxygen content was determined by modified laboratory method. One ml
solution of 0.05% urea and phosphate buffer solution at pH 4.5 were added in
to the bottles. The bottles were incubated at 20 dC for 5 days. After incubation,
the oxygen concentration was measured. Finally, 5 days biochemical oxygen
demand was obtained from the difference between the initial DO content and
DO after 5 days incubation (i.e., B.O.D 5 in mg/l).
280
Chemical Oxygen Demand
The water sample 50 ml was placed in a conical flask. Five solution of
potassium permanganate was added to the water sample and the flask was
placed in a water bath at 100 dC for an hour. Then the sample was cooled for 10
minutes, 5 ml of potassium iodide solution was added, followed by 10ml of
sulphuric acid solution. The solution was titrated with standard sodium
thiosulphate solution until a pale yellow colour was obtained.
pH was measured by using a pH meter. The total alkalinity was
determined by titration of the sample with a standard solution of sulphuric acid
using phenolphthalein and methyl orange as indicators.
The amount of trace elements such as calcium, magnesium, chloride
and sulphate in the samples of drinking water were determined by using a
flame photometer and that of lead, cadmium, mercury and arsenic by using
atomic absorption spectrophotometer.
Six water samples for detection of pathogenic microorganisms were
collected from different stations. Glass petridishes, filter membranes,
autoclave, 10 ml graduated pipettes, 1000 ml conical flask and 1000 ml
volumetric flask were used.
Results
Drinking Water Samples
Parameters of drinking water quality typically fall under two
categories: chemical and physical parameters include heavy metals, trace
organic compounds, total solids and turbidity.
In this research, the temperature of water sample varies ranging from
29 °C to 30°C. Colour of the samples is ranging from 5 to 11 TCU (True
colour unit). Total solids content from sampling sites vary from 586 mg/L to
1234 mg/L. The range of total hardness varied from 150 mg/L to 400 mg/L in
dry season, from 134 mg/L to 300 mg/L in wet season (Table 1,2) (Figure 3,
4).
Biochemical Oxygen Demand and Chemical Oxygen Demand unit in
most samples exceeded the maximum permissible limit of guideline values.
In this research, concentration of calcium in sample ranges from 15
mg/L to 80 mg/L during the rainy season and 12 mg/L to 86 mg/L in the
summer time. The range of magnesium in the rainy season was higher than that
281
in the dry season. Chloride values were found to be in the range of 75 mg/L to
120 mg/l during the summertime and 79 mg/L to 132 mg/L in the wet season.
Sulphate levels of water samples ranges from 40 mg/L to 220 mg/L for all
seasons (Table 3, 4) (Figure 5, 6).
In the rainy season, the values of lead, cadmium, and mercury were
higher than that in dry season. Seasonally measured arsenic units in all samples
exceed the maximum permissible limits of WHO guideline values. The amount
of coliform bacteria in drinking water remains constant for all stations during
dry and wet seasons (Table 5, 6) (Figure 7, 8).
Table 1. Physical characteristics of drinking water samples during dry season
(May, 2010)
Parameters
Drink
ing
Stations
(mg/L)
(WHO)
Potable
(WHO)
L1
L2
R1
N18
N23
N32
Temperature
ºC
29
29
30
29
29
30
-
-
Colour
5
7
11
7
7
5
5
50
Total Solids
700
700
850
1100
600
1230
500
1500
Total Hardness
360
150
320
250
220
400
100
500
Dissolved
Oxygen
2.10 3.74
2.48
2.88
3.33
2.52
>10
4-6
Biochemical
Oxygen
Demand
2.07 1.13
1.81
1.45
1.35
1.86
1
5
Chemical
Oxygen
Demand
2.2
1.03 1.588
1.14
1.59 1.675
-
<10
282
Table 2. Physical characteristics of drinking water samples during wet season
(August, 2010)
Parameters
Stations
(mg/L)
Drinking
Potable
(WHO)
(WHO)
L1
L2
R1
N18
N23
N32
29.1
29
29
30
29
30
-
-
7
10
9
9
11
5
5
50
Total Solids
700
724
586
800
1100
1234
500
1500
Total
Hardness
294
134
300
214
210
412
100
500
Dissolved
Oxygen
2.12
4.04
3.70
2.84
3.6
2.41
> 10
4-6
Biochemical
Oxygen
Demand
1.93
1.05
1.62
1.25
1.21
1.14
1
5
Chemical
Oxygen
Demand
2.12
1.9
1.42
1.821
1.6
1.93
-
< 40
Temperatue
dC
Colour
(TCU)
Note: Values of Temperature and colour are not mg/L.
TCU = True Colour Unit
283
1300
1200
1100
Total Solids
Elements (mg/L)
1000
900
800
Total Hardness
700
600
Dissolved Oxygen
500
400
Biochemical Oxygen
Demand
300
200
100
0
L1
L2
R1
N 18
N 23
N 32
Chemical Oxygen
Demand
Stations
Figure 3. Physical characteristics of drinking water samples during dry season
(May, 2010)
1300
Total Solids
1200
1100
Total Hardness
Elements (mg/L)
1000
900
Dissolved Oxygen
800
700
600
Biochemical Oxygen
Demand
Chemical Oxygen Demand
500
400
300
200
100
0
L1
L2
R1
N 18
N 23
N 32
Stations
Figure 4. Physical characteristics of drinking water samples during wet season
(August, 2010)
284
Table 3. Chemical characteristics of drinking water sample during dry season
(May, 2010)
Parameters
Drinking Potable
Stations
(mg/L)
(WHO)
(WHO)
L1
L2
R1
N18 N23 N32
pH
7.1
7.1
7.3
7.1
7.6
7.4
7-8.5
6.5-9.2
Total
Alkalinity
650
400
250
520
390
780
600
950
Calcium
80
80
15
80
40
64
75
200
Magnesium
29
29
35
25
14.4
24
30
150
Chloride
75
110
100
75
75
120
200
600
Sulphate
85
120
40
40
58
220
200
400
Table 4. Chemical characteristics of drinking water samples during wet season
(August, 2010)
Parameters
Drinking Potable
Stations
(mg/L)
L1
L2
R1
pH
6.8
6.2
7
Total
Alkalinity
530
Calcium
82
(WHO)
(WHO)
N18 N23 N32
7.1
7.8
7.6
7-8.5
6.5-9.2
560 280
510
320
730
600
950
86
12
82
40
67
75
200
Magnesium 31.7
30
29
35
25
27
30
150
Chloride
79
121 110
80
82
132
200
600
Sulphate
86
110
48
63
211
200
400
Note: pH value is not mg/L.
59
285
800
700
Element (mg/L)
600
500
pH
400
Total Alkalinity
Calcium
300
Magnesium
200
Chloride
100
0
Sulphate
L1
L2
R1
N 18
N 23
N 32
Stations
Element (mg/L)
Figure 5. Chemical characteristics of drinking water sample during dry season
(May, 2010)
800
700
600
500
400
300
200
100
0
pH
Total Alkalinity
Calcium
Magnesium
Chloride
L1
L2
R1
N 18 N 23 N 32
Sulphate
Stations
Figure 6. Chemical characteristics of drinking water samples during wet season
(August, 2010)
286
Table 5. Concentration of heavy metals in drinking water samples during dry season
(May, 2010)
Elements
Drinking
Potable
(WHO)
(WHO)
0.197 0.221 0.190 0.227 0.258 0.257
0.01
-
Cadmium 0.006 0.004 0.031 0.029 0.027 0.053
0.003
-
Mercury
2.342 2.294 2.312 2.305 2.342 2.302
0.001
-
Arsenic
7.124 6.839 6.756 7.132 6.829 7.182
0.01
≤ 10
Stations
(mg/L)
L1
Lead
L2
R1
N18
N23
N32
Table 6. Concentration of heavy metals in drinking water samples during wet season
(August, 2010)
Elements
Drinking Potable
Stations
(mg/L)
L1
N18
N23
N32
0.490 0.495 0.526 0.529
0.522
0.557
0.01
-
Cadmium 0.106 0.119 0.026 0.031
0.056
0.075
0.003
-
Mercury
4.690 4.216 4.078 3.780 0.3823 3.840
0.001
-
Arsenic
5.672 4.908 5.493 5.766
0.01
< 10
Lead
L2
R1
(WHO) (WHO)
5.764
6.084
8
Element (mg/L)
7
6
5
Lead
4
Cadmium
3
Mercury
2
Arsenic
1
0
L1
L2
R1
N 18 N 23 N 32
Stations
Figure 7. Concentration of heavy metals in drinking water samples at dry season
(May, 2010)
287
Elemental (mg/L)
7
6
5
Lead
4
Cadmium
3
Mercury
2
Arsenic
1
0
L1
L2
R 1 N 18 N 23 N 32
Stations
Figure 8. Concentration of heavy metals in drinking water samples at wet season
(August, 2010)
According to WHO (2004), water is a tasteless, odorless liquid at
standard qualities. Parameters of drinking water quality typically fall under
three categories: chemical, physical and microbiological. Chemical and
physical parameters include heavy metals, trace organic compounds, total
solids and turbidity. In this research, the concentrations of total solid in six
water samples are found ranging from 586-1234 mg/L. Water is commonly
classified in terms of the degree of hardness as 0-75 mg/L consider to be soft,
75-150 mg/L is moderately hard, and 150-300 is hard and above 300 mg/L is
very hard water, (WHO 2004).
The DO content in samples were found to be 2.10 to 4.04 mg/L during
dry and wet seasons. The BOD ranges are more than maximum permissible
limits of WHO guidelines values of drinking water. In this data, COD content
was found in the range of 1.03 to 2.12 mg/L in dry and wet seasons. In this
study, the pH value in sample were found to be in the range of 6.2 to 7.8
during dry and wet seasons. The present pH values are in accordance with the
literature range of 7.0 to 8.5 for drinking water. Goel (1997) reported that for
human health, alkalinity has got little significance but highly alkaline waters
produce an alkaline taste and become unpalatable or unpotable.
288
The calcium contents in water samples were found to be in the range of
15 to 80 mg/L. This range of calcium was exceeded the literature value of 75
mg/L for drinking water The range of magnesium was ecxceeded the literature
value of 30 mg/L for drinking water. In this study, the chloride content of
sample were found to be in the range of 25.3 to 120 mg/L, respectively.The
heavy metals lead, cadmium, mercury and arsenic were not considerd as good
quality for drinking. Acute lead poisoning in humans causes severe
dysfunction in the kidneys, reproductive system, liver, the brain and central
nervous system. Lead poisoning from environmental exposure is thought to
have caused mental retardation in many children and anemia. Lead is probable
not a major problem in drinking water, except old lead pipe still in use.
The amount of coliform bacteria in drinking water remains constant for
all stations. An acceptable content of coliform organism in drinking water is 1
MPN (most probable number). Heavy metals like mercury and cadmium that
can be dangerous, to health of both human and aquatic lives, even in trace
amount, are present in all drinking water samples.
According to the results of the present study, it can be concluded that
water environment of study area is facing with water contamination problems
due to the environmental degradation with respect to lake sedimentation,
development of aquatic biota, organic waste contamination and rich in nutrient
concentration due to human induced activities.
Acknowledgements
I am especially grateful to Dr Nu Nu Yee Professor and Head, Department of Botany,
University of Mandalay for the permission to use the laboratory and library during this work. I
am greatly thankful to my supervisor Dr Min Thein, Part-time Professor, Department of
Botany, University of Mandalay and Co-supervisor Dr Moat War Dine Naw, Lecturer,
Department of Botany, University of Mandalay for their supervising this research with
invaluable suggestion. I wish to express my thanks to U Kyaw Kyaw Lwin, Assistant Lecturer,
Department of Botany, University of Monywa for his kind helps in collecting specimens
during in my field trips.
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