Control of Postharvest Diseases of Rambutan Using Controlled

D. Sivakumar et a/.(2002) Phytoparasitica 30(4):xxx-xxx
Control of Postharvest Diseases of Rambutan Using
Controlled Atmosphere Storage and Potassium
Metabisulphite o r Trichoderma harzianum
1
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D. Sivakumar, R.S. Wilson Wijeratnam, R.L.C. Wijesundera
and M. Abeyesekere
1
Botryodiplodia theobromae, Colletotrichum gloeosporioides and Gliocephalotrichum microchlamydosporum are the causal fungi of three rambutan postharvest diseases, stemend rot, anthracnose and brown spot, respectively. Two different treatments of rambutan
fruits to control the diseases were investigated: application of potassium metabisulphite
(250 ppm) or culturefiltrateof Trichoderma harzianum (TrH 40) followed.by controlled
atmosphere storage (CA) at I3.5°C and 95% r.h. Potassium metabisulphite at 250 ppm under
CA effectively controlled the incidence and severity of the three postharvest diseases and
maintained the eating quality and color of the fruit for 21 days. The greatest effect of this
treatment was on brown spot disease, caused by G. microchlamydosporum. Application of
TrH 40 was less effective than potassium metabisuphite.
KEY WORDS: Gliocephalotrichum microchlamydosporum; Colletotrichum gloeospori­
oides; Botryodiplodia theobromae; Nephelium lappaceum; rambutan; potassium metabisul­
phite; Trichoderma harzianum; controlled atmospheric storage.
INTRODUCTION
Rambutan {Nephelium lappaceum L.) is a popular tropical fruit with high export
potential. However, during storage and transportation, postharvest diseases lower the
quality and value of this commodity (9). Anthracnose {Colletotrichum gloeosporioides
(Penz.) Sacc), brown spot {Gliocephalotrichum microchlamydosporum J.A. Mey, B.J.
Wiley & E.G. Simmons) and stem-end rot {Botryodiplodia theobromae Pat.) are the major
postharvest diseases of this fruit (2,5). These three postharvest diseases can be controlled
by the application of Bavistin (BASF, Colombo, Sri Lanka), but this leaves unacceptable
levels of residues on the fruit (1). A safe alternative method was developed to control these
three postharvest diseases which involved the combined application of biocontrol agent
Trichoderma harzianum (M. A. Rifai) (TrH 40) and a Generally Regarded As Safe (GRAS)
compound, potassium metabisulphite (at 250 ppm), in low temperature storage at 13.5°C
and 95% r.h for 18 days (7). However, sea shipment to European market destinations
takes about 21 days. Therefore, the objective of this study was to examine the effect of
potassium metabisulphite at 250 ppm or TrH 40 on naturally infected fruits under controlled
atmosphere (CA) storage in low temperature for 21 days.
Received Dec. 5,2001; received in final form April 4,2002; hrtp:/Avww.phytoparasitica.org posting July 8, 2002.
'Posharvest Technology Group, Industrial Technology Institute, Colombo 7, Sri Lanka [Fax: +94-1-683128;
e-mail: [email protected]].
DepL of Botany, University of Colombo, Colombo 3, Sri Lanka.
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Phytoparasitica 30:4,2002
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MATERIALS AND METHODS
Preparation of pathogen inoculum The fungi B. theobromae, C. gloeosporioides and
G. microchlamydosporum were isolatedfrominfected rambutan fruits. Pure cultures were
maintained on potato dextrose agar (PDA) at 28°C.
A mycelial disc of 1.2 cm diam obtained from a 4-5-day old culture of the fungus
on PDA was transferred to 100 ml of fresh, solidified PDA in a 250 ml conicalflaskand
incubated at 28°C. G. microchlamydosporum and C. gloeosporioides were incubated at
28°C for 4-5 days and B. theobromae for 14-15 days. (B. theobromae requires 14-15
days to sporulate.) At the end of the incubation period 30 ml of sterile distilled water was
added to each culture and theflaskswere shaken at 50 rpm for 30 min in an orbital shaker.
The content of each conical flask was thenfilteredthrough two layers of sterile muslin
cloth and the concentration of the spores was adjusted to 10 spores m l by diluting with
sterile water (7). This spore suspension was used for the inoculation of rambutan fruits in
the experiments described below.
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'Preparation of spore suspension of T. harzianum A 100-ml aliquot of of potato
dextrose broth in a 250 ml conicalflaskwas inoculated with three mycelial discs (1.2 cm
diam) obtained from 5-7-day old cultures of T. harzianum grown on PDA. The inoculated
medium was incubated at 28°C in an orbital shaker (80 rpm) for 12 days, after which the
culture was harvested byfilteringthrough two layers of muslin cloth. The preparation
(culturefiltrateat 10 conidia m l concentration) was stored at 10°C until use (6).
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Effect of CA and potassium metabisulphite/TrH 40 on inoculated fruit A set of 30
rambutan fruits var. 'Malwana' special selection at color stage 6 (pericarp 75% red and 25%
orangish red) was surface sterilized with 75% ethanol and thefruitsurface was wounded
with a No. 2 cork borer (1.2 cm diam) to a depth of 3 mm. The wound was inoculated
with 0.1 ml of the spore suspension (10 spores m l ) of the test pathogen, and incubated
for 3 h at 28°C. The inoculated wounds of the fruits were then treated immediately with
0.1 ml potassium metabisulphite (250 ppm) or 0.1 ml of the culturefiltrateof TrH 40 (7).
The inoculated and TrH 40- or potassium metabisulphite-treatedfruitswere placed in tray
packs and packed in corrugated cardboard boxes lined with pin-pricked polystyrene film
and kraft paper (8). The packed fruits were stored under CA at 13.5°C and 95% r.h. for 10
days. A set of fruits inoculated and treated with sterile distilled water or autoclaved culture
filtrate of TrH 40 served as control.
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Recovery of the pathogens and TrH 40 was conducted by removing the pericarp of the
fruit and homogenizing the tissue in 0.1 M acetate buffer, pH 4.5 (w/v 2:1). The preparation
was serially diluted and 0.1 ml of each dilution was plated on PDA by the spread plate
method. The plates were incubated at 28°C for 5-7 days and observed for growth of the
postharvest pathogens and TrH 40 (7).
A complete randomized design (CDR) was adopted and the experiment was repeated
twice. Data were processed for analysis of variance. The treatment means were compared
by Duncan's Multiple Range Test (DMRT) at P=0.05.
Controlled atmosphere storage The CA consisted of an impermeable storage chamber
made of stainless steel with a perspex lid and a gas inlet and outlet. The atmosphere in the
chamber consisted of 3% O2 and 7% CO2 (3), applied at a continuous flow rate of 30 ml
m i n at 95% r.h. over the fruits. O2 and CO2 concentrations were measured using a gas
chromatograph GC-9A. Specifications were: column, Carboxen 1000; detector, Thermal
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D. Sivakumar et al.
Conductivity Detector (TCD); carrier gas (He) at 30 ml min
225° C, at increments of 20°C per min.
oven temperature, 40-
Naturally infected fruit A set of 45 naturally infected fruits was harvested and dipped
in 3 / of potassium metabisulphite (250 ppm) or 3 / of TrH 40 culture filtrate for 20 min
and then left to air-dry for 5 min (7). Then, the fruits were packed and stored as described
above under CA storage at 13.5°C and 95% r.h. for 21 days. Another set of 45 naturally
infected fruits dipped in 3 / sterile distilled water or 3 / autoclaved TrH 40 and packed as
described above, served as control. The observations were recorded as described below
after 21 days in storage. The experiments were repeated twice.
Assessment of disease incidence and disease severity Disease incidence was expressed
as the ratio of number of fruits with disease symptoms (brown spot, anthracnose or stemend rot) to total number of fruits in a commercial pack. The severity was evaluated by
measuring the lesion diameter of the disease symptom in cm (7). Statistical analysis was
conducted using a Statgrafic program for CDR design analysis of variance. The treatment
means were compared by DMRT at P=0.05.
Assessment of quality Eating quality was assessed hedonically by a panel of six people.
Six fruits were randomly selected from the package containing fruits from different
treatments under CA and rated for eating quality (1 = poor to 9 = excellent) (4). The skin
color of the fruit was scored according to the hedonic scalefrom1 = poor to 5 = excellent
skin color quality (6). At the same time the skin color of the mid-side of each fruit was
measured using a Minolta chomameter (9 mm aperture) and Commission International
de l'Eclairage (CIE) color space (1* a* b*). The results were analyzed according to the
Freidman two-way test.
RESULTS
Fruits inoculated and treated with potassium metabisulphite at 250 ppm or culture
filtrate of TrH40 and stored under CA at low temperature had significantly smaller
(P<0.05) lesion diameters for all three postharvest diseases in comparison with the control
fruits (Fig. 1). Potassium metabisulphite treatment resulted in smaller lesion diameter than
the TrH 40 culture filtrate treatment. The smallest lesions were seen in brown spot caused
by G. microchlamydosporum, but the difference was not significant.
TABLE 1. Combined effect of potassium metabisulphite (PM) or Trichoderma harzianum (TrH
40) under CA at low temperature storage on color retention of rambutanfruits(Color values were
determined with a Minolta chromameter using CIE 1*, a*, b*)
a*
b*
1*
21.94
+1.6
PM, 250 ppm
+57.0
+18.5
Sterile distilled water
27.93
+20.45
+4.6
Culturefiltrateof TrH 40
+40.0
27.95
+22.17
+16.2
Autoclaved culturefiltrateof TrH 40
27.95
*1, lightness; a and b, chromatic coordinates; +a value, red color; +b value, yellow color; the numerical value
expresses the intensity of these colors.
Fruits dipped in:
Phyloparasitica 30:4,2002
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b
PM + CA
TrH 4 0 +
SDW+CA
A. TrH 4 0
+
CA
CA
I Brown spot S Anthracnose • Stem-end rot
Fig.
1.
Mean lesion diameter of inoculated fruits (n=30) treated with 2 5 0 ppm potassium
metabisulphite (PM) or Trichoderma harzianum (TrH 40) and held under controlled atmosphere ( C A )
at low temperature. SDW, sterile distilled water; A . TrH 4 0 , autoclaved TrH 4 0 . Statistical analysis
was done separately for each of the tested organisms (P=0.05).
PM + CA
Fig.
2.
C.
SDW + CA A.TrH 40 +
CA
63 a.
• TrH 40
•
TrH 40 +
CA
gloeosporioides
Q B.
microchlamydosporum
theobromae
Percentage recovery of the postharvest pathogens from inoculated fruits treated with
potassium metabisulphite (PM) or Trichoderma harzianum (TrH 4 0 ) and held under controlled
atmosphere ( C A ) at low temperature. S D W , sterile distilled water; A . TrH 40, autoclaved TrH 4 0 .
Statistical analysis was done separately for each of the tested organisms (P=0.05).
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D . Sivakumar et al.
PM + CA
TrH 4 0 + SDW + CA A. TrH 4 0
CA
+CA
Fig. 3. Disease incidence of naturally infected fruits treated with potassium metabisulphite (PM) or
Trichoderma harzianum (TrH 40) and held under controlled atmosphere (CA) at low temperature.
SDW, sterile distilled water; A. TrH 40, autoclaved TrH 40. Disease incidence is expressed as the
ratio of number offruitswith disease symptoms to total number of fruits in a commercial pack.
CA
+
C
A
Fig. 4. Disease severity (as measured by lesion diameter) of naturally infected fruits treated with
potassium metabisulphite (PM) or Trichoderma harzianum (TrH 40) and held under controlled
atmosphere (CA) at low temperature. SDW, sterile distilled water; A. TrH 40, autoclaved TrH 40.
Phytoparasitica 30:4, 2002
5
5
2
PM + CA
TRH 40 + SDW + CA A TRH 40
CA
+CA
Fig. 5. Color and eating quality of naturally infected fruits treated with potassium metabisulphite
(PM) or Trichoderma harzianum (TrH 40) and held under controlled atmosphere ( C A ) at low
temperature. SDW, sterile distilled water; A . TrH 4 0 , autoclaved TrH 40. Statistical analysis was
done separately for each of the tested organisms (P=0.05).
The recovery of the postharvest pathogens was significantly low (P<0.05) in all the
treatments in comparison with the control fruits. In inoculated fruits treated with potassium
metabisulphite, the recovery was 2-5%. Fruits inoculated and treated with TrH 40 revealed
13% recovery of the antagonist and 20-24% recovery of the three postharvest pathogens
(Fig. 2). In naturally infected fruits, potassium metabisulphite or TrH 40 under CA
significantly reduced (P<0.05) both disease incidence and mean lesion diameter (Figs.
3, 4). However, of the two treatments, potassium metabisulphite under CA at low
temperature was the more effective one. The naturally infected fruits dipped in potassium
metabisulphite and held under CA showed significantly higher (P<0.05) scores for color
(Table 1) and eating quality (Fig. 5) than the other treatments.
DISCUSSION
Carbon dioxide is known to have a fungistatic effect at concentrations of 15% or above,
but high CO2 concentrations have detrimental effects on the quality of tropical fruits (3).
A CO2 concentration between 7% and 12% is reported to retardflavorand color loss when
the O2 concentration is 3% (M. Abeyesekere, unpublished results). However, the optimal
CO2 concentration depends on the cultivar of the fruit (3).
Potassium metabisulphite acts as an antifungal agent, and an effective antioxidant
(7), and its stability may have been retained in the I0W-O2 environment. Therefore, the
application of potassium metabisulphite at 250 ppm to rambutan fruit, and storage under
CA (CO2 7% and 0 3%) at low temperature (13.5°C), effectively controlled the diseases
caused by B. theobromae, C. gloeosporioides and G. microchlamydosporum; color and
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D. Sivakumar et al.
eating quality of the fruits were maintained for up to 21 days. Application of TrH 40 was
less effective than potassium metabisulphite under CA,. Probably, the CA storage affected
the activity and survival of T. harzianum due to the high C 0 concentration (7%). This
was demonstrated by the low level of recovery of T. harzianum (Fig. 2). However, the
low temperature (13.5 °C) did not affect the recovery of T. harzianum (8). A similar effect
of CA has been observed on many other fungi (3). Therefore, potassium metabisulphite
application and CA storage, in combination, can be used commercially to extend the
storage life of rambutan fruits up to 21 days.
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ACKNOWLEDGMENTS
The financial assistance by the National Research Council in Sri Lanka for research grant No. 99-52 is
acknowledged. The authors also wish to thank Ms. D. Shiranthi Pererh and Ms. L.N.P. Nimali Abeyeratna for
their valuable technical assistance
REFERENCES
1. Abeyesekere.M., Nettikumara, N.A.L.A., Wilson Wijeratnam, R.S., Sivakumar, D. and Kurukutadithya, P.
(1997) Studies on preharvest treatments for control of postharvest pathogens of rambutans. Proc. Australian
Postharvest Horticultural Conf. (Sydney, Australia), pp. 152-154.
2. Farungsang, U., Farungsang, N. and Sagchote, S. (1991) Postharvest diseases of rambutan during storage.
Proc. 8th Australian Plant Pathological Society Conf. (Canberra, Australia), p. 114.
3. Kader, A. (1993) Modified and controlled atmosphere storage of tropical fruits, pp. 239-249. in: Champ, B.R.,
Highley, E. and Johnson, G.I. [Eds.] Postharvest Handling of Tropical Fruits. ACAIR, Canberra, Australia.
4. O'Hare, T.J., Prasad, A. and Cooke, A.W. (1994) Low temperature and controlled atmosphere of rambutan.
Postharvest Biol. Technol. 4:147-157.
5. Sivakumar, D., Wilson Wijeratnam, R.S., Wijesundera, R.L.C. and Abeyesekere, M. (1997) Postharvest
diseases of rambutan (Nephelium lappaceum) in the Western province. J. Natl. Sci. Counc. Sri Lanka 25:225229.
6. Sivakumar, D., Wilson Wijeratnam, R.S., Wijesundera, R.L.C. and Abeyesekere, M . (2000) Effect of
potassium metabisulphite on low-temperature storage of rambutan. Trap Sci. 40:29-31.
7. Sivakumar, D., Wilson Wijeratnam, R.S., Wijesundera, R.L.C. and Abeyesekere, M. (2002) Combined
effect of Generally Regarded As Safe (GRAS) compounds and Trichoderma harzianum on the control of
postharvest diseases oframbutan.Phytoparasitica 30:43-51.
8. Sivakumar, D., Wilson Wijeratnam, R.S., Wijesundera, R.L.C., Marikar, F.M.T. and Abeyesekere, M.
(2000) Antagonistic effect of Trichoderma harzianum on postharvest pathogens of rambutan (Nephelium
lappaceum). Phytoparasitica 28:240-247.
9. Visarananoth, N. and Hag, L.L. (1987) Postharvest diseases of rambutan. pp. 51-57. in: Lam, P.F. and
Kosiyachinda, S. [Eds.] Rambutan: Fruit Development, Postharvest Physiology and Marketing in ASEAN.
Food Handling Bureau, Kuala Lumpur, Malaysia.
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