a comparative polyphasic study of 10 pratylenchus coffeae

Transcription

Doctoraatsproefschrift nr. 894 aan de faculteit Bio-ingenieurswetenschappen
van de K.U.Leuven
A COMPARATIVE POLYPHASIC STUDY
OF 10 PRATYLENCHUS COFFEAE
POPULATIONS FROM VIETNAM
NGUYEN Thi Tuyet
Promotor: Prof. Dirk De Waele, K.U.Leuven
Co-promotor: Assoc. Prof. Ho Huu Nhi, Vietnam
Academy of Agricultural Sciences.
Leden van de
examencommissie:
Prof. E. Decuypere, voorzitter, K.U. Leuven
Prof. J. Coosemans, K.U. Leuven
Proefschrift voorgedragen tot
Prof. B. Cammue, K.U. Leuven
het behalen van de graad van
Prof. M. Moens, ILVO & UGent
Doctor in de
Prof..A. Elsen, BDB & UGent
Bio-ingenieurswetenschappen
Dr. I. Van den Bergh, Bioversity International &
K.U. Leuven
April 2010
© 2009 Katholieke Universiteit Leuven, Groep Wetenschap & Technologie, Arenberg
Doctoraatsschool, W. de Croylaan 6, 3001 Heverlee, België
Alle rechten voorbehouden. Niets uit deze uitgave mag worden vermenigvuldigd
en/of openbaar gemaakt worden door middel van druk, fotokopie, microfilm,
elektronisch of op welke andere wijze ook zonder voorafgaandelijke schriftelijke
toestemming van de uitgever.
All rights reserved. No part of the publication may be reproduced in any form by print,
photoprint, microfilm, electronic or any other means without written permission from
the publisher.
ISBN 978-90-8826-137-4
Wettelijk depot D/2010/11.109/15
i
Acknowledgements
First of all, I would like to express my sincere gratitude and appreciation
to my promoter, professor Dirk De Waele, for giving me the opportunity to start
and finish my PhD adventure, for his enduring guidance, encouragement, patience
and support throughout my study.
I am truly grateful to the members of the examination commission,
professors Jozef Cooseman, Maurice Moens, Bruno Cammue, Annemie Elsen and
Inge Van den Bergh, for their time and efforts in reading this work. The invaluable
remarks and advice truly improved the quality of this thesis.
I am grateful to the Belgian Government, The Belgian Technical
Cooperation (BTC), for funding my research and studies in Vietnam and Belgium.
I would also like to express my gratitude to Inge Van den Bergh, who
suggested the topic of this study and provided me with the opportunity to apply for
a sandwich PhD scholarship from BTC-Hanoi.
My very heartfelt thanks go to professor Rony Swennen, head of the
Laboratory of Tropical Crop Improvement, Catholic University of Leuven, for
providing all necessary laboratory facilities. I wish to express my sincere thanks to
all the staff members of the laboratory for their kind assistance. Especially I would
like to thank Marleen Stockmans for her help in arranging accommodation for me
and other administrative matters, and to Wim Dillemans for providing microscopy
facilities.
Associate professor Ho Huu Nhi, my co-promotor in Vietnam, is very much
acknowledged for his support and continuous interest. I would like to thank my
former colleagues from the Agro-biotechnology Department, (VASI): Yen, Phuong,
Thanh, Ngan, Sen, Ninh, Ngoc, Tung, Linh, Thuy, Nguyet, Hanh, without whose
help I could not have achieved this work.
I am greatly indebted to Lieven Waeyenberge from the Instituut voor
Landbouw en Visserijonderzoek (ILVO) in Merelbeke, Belgium, for his assistance
and help with molecular work. I would also like to give my thanks to his wife,
Nancy de Sutter, for her friendship deeply.
I wish to thank professors Nguyen Vu Thanh and Nguyen Ngoc Chau from
the Institute of Ecology and Biological Resources in Hanoi, for providing facilities
that enabled me to conduct the morphological and morphometrical studies. Nguyen
Dinh Tu is acknowledged for help with photography.
I would like to thank Rita Van Deriessche and Marjolein Couvreur from
Department of Biology, Faculty of Sciences, Ghent University, for the scanning
electronmicrographs.
ii
I would like to express my gratitude to the board of Directors of the Plant
Resources Center (PRC), Vietnam Academy of Agricultural Science (VAAS), in Hanoi,
for allowing me to stay longer in Belgium to finish my work.
My special thanks are dedicated to the nematology team at the Laboratory
of Tropical Crop Improvement. I highly appreciate Annemie Elsen for her scientific
assistance, hospitality, encouragement and all the things that she has helped me
with since my first day working at the laboratory, and especially for giving me a lot
of care when I had an operation in 2004. And I will never forget the other members
of the team, Lieselot, Sugantha, Christine and Syarifah, for their great support
whenever I came to work at the laboratory. I would like to thank Lorna, Nguyet,
Nordalyn, Maung, Thuy, Papa and Teodora for the short but sweet time together. It
has been a pleasure to talk with you about our work as well as to have nice little
dinners full of chatting.
I wish to thank Tram and Trang from the Coffee Research and
Development Center, Northern Mountainous Agriculture and Forestry Science
Institute, for their assistance in the survey studies and for providing the coffee
seedlings.
I am grateful to Long, Phap, Syarifah, Hongmei and Sveta for their
guidance and help in the analysis of morphometrical and morlecular data.
I wish to extend my thanks to my colleagues from the Agro-biodiversity
Division, Plant Resources Center (PRC), Vietnam Academy of Agricultural Sciencces
(VAAS) in Hanoi, for your understanding and encouragement.
During the time of my study in Belgium, I met a lot of Vietnamese students
(Lien, Tuan, Thuy, Thanh, Mai+Lu, Ha Pham, Phuong, Hasa, Xa, Thoan, …), to
whom I am grateful for their supportive friendship.
Xin cảm ơn cha mẹ, anh chị em và các cháu đã luôn ủng hộ và động viên
tôi hoàn thành khoá học này.
Finally, I feel unlimited gratitude to my husband, Pham Van Luong, who
did his best to support me to go to the end of this study. To my daughters, Chi-Nay
and Minh-Bi, who had to overcome being separated from their mother by thousands
of kilometers for a long time. It is your unconditional love and patience that has
given me motivation and energy to complete this work.
There are so many people to thank. If I forgot someone, please forgive
me, but it was not my intention.
Leuven, 23rd April 2010
Nguyen Thi Tuyet
iii
Table of contents
Acknowledgement ............................................................................ i
Table of contents ............................................................................iii
List of tables ................................................................................. vii
List of figures .................................................................................ix
Summary .................................................................................... xiii
Samenvatting ...............................................................................xvii
CHAPTER 1: INTRODUCTION ............................................................... 1
1.1 Objectives of the study and outline of the thesis ............................. 1
1.2 Background: Pratylenchus coffeae............................................... 4
1.2.1
Classification and differential diagnosis ......................................4
1.2.2
Biology and life history ..........................................................7
1.2.3
Histopathology and symptoms of damage ....................................8
1.2.4
Resistance ....................................................................... 11
1.2.5
Geographical distribution, host plants and importance in agriculture.. 12
1.2.6
Biological diversity ............................................................. 13
CHAPTER 2: OCCURRENCE OF PRATYLENCHUS COFFEAE ON AGRICULTURAL
CROPS IN VIETNAM ............................................................ 15
2.1 Introduction ......................................................................... 15
2.2 Agro-ecological regions of Vietnam ............................................. 16
2.2.1
Northeast ........................................................................ 16
2.2.2
Northwest ....................................................................... 16
2.2.3
Red River Delta ................................................................. 17
2.2.4
North Central Coast ............................................................ 17
2.2.5
South Central Coast ............................................................ 17
2.2.6
Central Highlands .............................................................. 18
2.2.7
Southeast ........................................................................ 18
2.2.8
Mekong River Delta............................................................. 18
2.3 Materials and methods............................................................. 18
2.4 Results ................................................................................ 19
2.5 Discussion ............................................................................ 22
2.6 Conclusions .......................................................................... 23
2.7 Establishment of the collected Pratylenchus coffeae populations on in
vitro carrot disc cultures.......................................................... 23
CHAPTER 3: COMPARATIVE STUDY OF THE MORPHOLOGY AND MORPHOMETRICS
3.1
3.2
OF PRATYLENCHUS COFFEAE POPULATIONS FROM VIETNAM ......... 25
Introduction ......................................................................... 25
Materials and methods............................................................. 27
iv
3.2.1
Nematode populations ........................................................ 27
3.2.2
Killing and fixing of nematodes .............................................. 28
3.2.3
Mounting of nematodes ....................................................... 28
3.2.4
Light microscope observations ............................................... 28
3.2.5
Scanning electron microscope observations................................ 29
3.2.6
Canonical discriminant analysis .............................................. 30
3.3 Results ................................................................................ 30
3.3.1
Morphological observations ................................................... 30
3.3.2
Morphometrical observations................................................. 46
3.4 Discussion ............................................................................ 55
3.5 Conclusions .......................................................................... 59
CHAPTER 4: MOLECULAR CHARACTERISATION OF PRATYLENCHUS COFFEAE
POPULATIONS FROM VIETNAM .............................................. 61
4.1 Introduction ......................................................................... 61
4.2.1
4.2.2
Random amplified polymorphism DNA (RAPD) ............................. 64
4.2.2.1 DNA extraction............................................................... 64
4.2.2.2 RAPD-PCR reaction .......................................................... 64
4.2.2.3 Genetic data analysis and phylogeny ..................................... 65
4.2.3
D2/D3 sequencing.............................................................. 66
4.2.3.1 DNA extraction............................................................... 66
4.2.3.2 D2/D3 rDNA PCR ............................................................. 66
4.2.3.3 Purification of the PCR products .......................................... 67
4.2.3.4 Quantification of the purified PCR products............................. 67
4.2.3.5 Cloning of the PCR products ............................................... 67
4.2.3.6 Sequencing ................................................................... 70
4.2.3.7 Phylogenetic analyses....................................................... 71
4.3 Results ................................................................................ 71
4.3.1
Random amplified polymorphism DNA (RAPD) ............................. 71
4.3.2
D2/D3 sequencing.............................................................. 75
4.4 Discussion ............................................................................ 85
4.5 Conclusions .......................................................................... 87
CHAPTER 5: EFFECT OF TEMPERATURE ON THE IN VITRO REPRODUCTIVE
FITNESS OF PRATYLENCHUS COFFEAE POPULATIONS FROM VIETNAM
................................................................................... 89
5.1 Introduction ......................................................................... 89
5.2.1
5.2.2
Experimental set-up ........................................................... 91
5.2.3
Assessment of the nematode reproduction................................. 91
v
5.2.4
Data analysis .................................................................... 92
5.3 Results ................................................................................ 92
5.3.1
In vitro reproductive fitness of 10 Pratylenchus coffeae populations
from Vietnam and a P. coffeae population from Ghana .................. 92
5.3.2
Effect of temperature on the reproductive fitness of three Pratylenchus
coffeae populations from Vietnam........................................... 95
5.4 Discussion .......................................................................... 100
5.5 Conclusions ........................................................................ 102
CHAPTER 6: HOST RANGE CHARACTERISATION, IN VIVO REPRODUCTION AND
PATHOGENICITY OF PRATYLENCHUS COFFEAE POPULATIONS FROM
VIETNAM....................................................................... 103
6.1 Introduction ....................................................................... 103
6.2 Materials and methods........................................................... 104
6.2.1
Host range experiment set-up...............................................105
6.2.2
Pathogenicity experiment set-up ...........................................106
6.2.3
Preparation of nematode inoculum and inoculations ....................106
6.2.4
Assessment of the nematode reproduction ................................107
6.2.5
Assessment of the host plant range.........................................107
6.2.6
Assessment of the pathogenicity............................................107
6.2.7
Data analysis ...................................................................108
6.3 Results .............................................................................. 108
6.3.1
Host range of Pratylenchus coffeae populations from Vietnam ........108
6.3.2
In vivo reproduction and pathogenicity of Pratylenchus coffeae from
Vietnam.........................................................................110
6.3.2.1 In vivo reproduction and pathogenicity of Pratylenchus coffeae on
banana .......................................................................110
6.3.2.2 In vivo reproduction and pathogenicity of Pratylenchus coffeae from
Vietnam on coffee ..........................................................112
Vietnam on sugarcane......................................................114
Vietnam on maize ..........................................................116
6.4 Discussion .......................................................................... 119
6.5 Conclusions ........................................................................ 122
CHAPTER 7: GENERAL CONCLUSIONS AND PERSPECTIVES ......................... 123
REFERENCES ............................................................................... 127
LIST OF ACCEPTED PUBLICATIONS IN INTERNATIONAL REFEREED JOURNALS . 141
vi
vii
List of tables
Table 2.1: Sampling period, localities (districts), provinces and agro-ecological
regions sampled...................................................................... 19
Table 2.2: Occurrence of Pratylenchus coffeae in the Northern Uplands and Red
River Delta............................................................................ 20
Table 2.3: Occurrence of Pratylenchus coffeae in the North Central Coast. ....... 21
Table 2.4: Occurrence of Pratylenchus coffeae in the Central Highlands. .......... 21
Table 2.5: Occurrence of Pratylenchus coffeae in the Southeast and Mekong River
Delta................................................................................... 22
Table 2.6: Origin and population codes of the 10 Pratylenchus coffeae populations
collected in Vietnam and of the P. coffeae population from Ghana used in
our study. ............................................................................. 24
Table 3.1: List of the 41 Pratylenchus coffeae populations for which the
morphometrics were reported. .................................................... 27
Table 3.2: Measurements taken for the comparative morphometrical study of the
10 Pratylenchus coffeae populations from Vietnam and their abbreviations.
......................................................................................... 29
Table 3.3: Frequency of occurrence of the tail tip shapes of Pratylenchus coffeae
females from Vietnam. ............................................................. 36
Table 3.4: Morphometrics of the females of the Pratylenchus coffeae populations
collected in Vietnam. ............................................................... 49
Table 3.5: Morphometrics of the males of the Pratylenchus coffeae populations
collected in Vietnam. ............................................................... 51
Table 3.6: Standardised coefficients for canonical variates of females and males of
the Pratylenchus coffeae populations from Vietnam. .......................... 53
Table 4.1: Oligonucleotides used for the RAPD-PCR study............................. 65
Table 4.2: Origin, accession numbers, codes and number of base pairs of
Pratylenchus accessions from Genbank (deposited by Duncan et al., 1999)
used for comparison with the Pratylenchus coffeae populations from
Vietnam. .............................................................................. 71
Table 4.3: Pairwise distances between 10 Pratylenchus coffeae populations from
Vietnam and a P. coffeae population from Ghana based on RAPD bands
generated by 10 10-oligonucleotide-long primers............................... 74
Table 4.4: Pairwise distances between 10 Pratylenchus coffeae populations from
Vietnam, 10 P. coffeae and two Pratylenchus jaehni populations obtained
from GenBank based on D2/D3 28S rDNA expansion segments sequences... 84
Table 5.1: Comparative in vitro reproduction on carrot discs of 10 Pratylenchus
coffeae populations from Vietnam and one P. coffeae population from
viii
Ghana, 10 weeks after inoculation with 25 females, incubated at 25oC in the
dark.................................................................................... 94
Table 5.2: Effect of time and temperature on the in vitro reproduction factor (RF)
on carrot discs of three Pratylenchus coffeae populations from Vietnam after
inoculation with 25 females, incubated at 15, 20, 25 and 30oC. ............. 96
Table 5.3:
Regression
analysis
of
the population dynamics
at
different
temperatures of three Pratylenchus coffeae populations from Vietnam. ... 98
Table 6.1: Experiments carried out to study the host range, in vivo reproduction
and pathogenicity of 10 Pratylenchus coffeae populations from Vietnam on
13 agricultural crops commonly grown in Vietnam. ........................... 105
Table 6.2: Reproduction factors (RF) for 10 Pratylenchus coffeae populations from
Vietnam on 13 selected agricultural crops, 14 weeks after inoculation with
1,000 vermiforms/plant. .......................................................... 109
Table 6.3:
In vivo reproduction and percentage root lesions caused by 10
Pratylenchus coffeae populations from Vietnam on banana cv. Ngop Dui Duc,
14 weeks after inoculation with 1,000 vermiforms/plant. ................... 111
Table 6.4: Plant height, shoot and root fresh weights of banana cv. Ngop Dui Duc
inoculated with 10 Pratylenchus coffeae populations from Vietnam, 14
weeks after inoculation with 1,000 vermiforms/plant. ....................... 112
Table 6.5: In vivo reproduction of 10 Pratylenchus coffeae populations from
Vietnam on coffee cv. Catimor, 14 weeks after inoculation with 1,000
vermiforms/plant................................................................... 113
Table 6.6: Plant height, shoot and root fresh weights of coffee cv. Catimor
Table 6.7: In vivo reproduction and percentage root lesions caused by 10
Pratylenchus coffeae populations from Vietnam on sugarcane var. ROC20, 14
Table 6.8: Plant height, shoot and root fresh weights of sugarcane var. ROC20
Table 6.9: Reproduction of 10 Pratylenchus coffeae populations from Vietnam on
maize var. LVN10, 14 weeks after inoculation with 1,000 vermiforms/plant
........................................................................................ 117
Table 6.10: Plant height, shoot and root fresh weights of maize var. LVN10
ix
List of figures
Figure 1.1: Research outline of the present study. .......................................3
Figure 1.2: Female (A) and male (B) of Pratylenchus coffeae as observed under the
light microscope.. .....................................................................5
Figure 1.3: Pratylenchus coffeae. A: female stylet; B: entire female; C: female
head region; D: female lateral field; E: female pharyngeal region; F: female
vulval region; G: male pharyngeal region; H: female en face view; I-L:
female tail shapes; M: male tail. ....................................................6
Figure 1.4: Life and disease cycle of lesion nematodes (Pratylenchus spp.)..........7
Figure 1.5: Pratylenchus coffeae in the tip of coffee roots.. ...........................9
Figure 1.6: Symptoms caused by Pratylenchus coffeae on coffee. A: wilting; B-C:
lesions on roots. .......................................................................9
Figure 1.7: Symptoms caused by root-lesion nematodes on banana roots (A and B)
and corm (C). ........................................................................ 10
Figure 1.8: Symptoms caused by Pratylenchus coffeae on tuber of yam (A) and taro
(B)...................................................................................... 11
Figure 1.9: Geographical distribution of Pratylenchus coffeae. ...................... 12
Figure 2.1: Agro-ecological regions of Vietnam. ........................................ 17
Figure 2.2: Provinces in Vietnam surveyed for the occurrence of Pratylenchus
coffeae. ............................................................................... 20
Figure 3.1: Light microscope photographs of Pratylenchus coffeae populations from
Vietnam. A-C, F & I: anterior region females; D: anterior region male; E:
pharyngo-intestinal junction female; H: hemizonid and excretory pore
female; G & J: tail region males................................................... 33
Figure 3.2: Light microscope photographs of Pratylenchus coffeae populations from
Vietnam. A-B: anterior genital branch females; C: post-vulval uterine sac
female; D: lateral field at level of the median pharyngeal bulb female; E, H-I
& M: lateral field at mid-body females; F-G, J-L: spermatheca females. ... 34
Figure 3.3: Light microscope photographs of the tail and tail tip shapes of
Pratylenchus coffeae females from Vietnam.. .................................. 35
Figure 3.4: Light microscope photographs of the tail tip shapes of Pratylenchus
coffeae females from Vietnam: truncated (a); obliquely truncated (b);
broadly rounded (c); hemispherical (d); sub-hemispherical (e); bluntly
pointed (f); pointed (g); convex (h); digitated (i); cleft (j); crenated (k);
bilobed (l)............................................................................. 36
Figure 3.5: En face and lateral head scanning electron microscope photographs of
Pratylenchus coffeae specimens from Vietnam. ................................ 37
x
Figure 3.6: Lateral field at mid-body and at vulva level scanning electron
microscope photographs of Pratylenchus coffeae specimens from Vietnam..
......................................................................................... 40
Figure 3.7: Female tail scanning electron microscope photographs of Pratylenchus
coffeae specimens from Vietnam. ................................................ 43
Figure 3.8: Male tail scanning electron microscope photographs of Pratylenchus
coffeae specimens from Vietnam. Northwest (A), Northeast 1 (B), Northeast
2 (C), Northeast 3 (D), Northeast 4 (E), Red River Delta 1 (F), Red River Delta
2 (G), North Central Coast 1 (H), North Central Coast 2 (I) and Central
Highlands (K-L) populations. ....................................................... 45
Figure 3.9: Canonical discriminant analysis of 10 Pratylenchus coffeae populations
from Vietnam for females (A) and males (B) performed with 12 female and
five male morphometrical characters. ........................................... 54
Figure 4.1: Structure of the ribosomal DNA gene family in nematodes. Coding
regions of the 18S small subunit (SSU), 5.8S and 28S large subunit (LSU); noncoding regions of the internal transcribed spacer (ITS) and external
transcribed spacer (ETS) regions; external nontranscribed spacer (NTS)
region. ................................................................................ 61
Figure 4.2: RAPD bands generated by 10 10-oligonucleotide-long primers for 11
Pratylenchus coffeae populations. L: 100 bp DNA ladder; M: 1 Kb DNA
ladder; 1: Northwest; 2: Northeast 1; 3: Northeast 2; 4: Northeast 3; 5:
Northeast 4; 6: Red River Delta 1; 7: Red River Delta 2; 8: North Central
Coast 1; 9: North Central Coast 2; 10: Central Highlands population; 11:
population from Ghana. ............................................................ 73
Figure 4.3: Neighbour-Joining tree of 10 Pratylenchus coffeae populations from
Vietnam and a P. coffeae population from Ghana based on RAPD bands
generated by 10 10-ologonucleotide-long primers.............................. 75
Figure 4.4: Alignment of the D2/D3 28S rDNA expansion segments sequences of 10
Pratylenchus coffeae populations from Vietnam and 10 P. coffeae
populations as well as two Pratylenchus jaehni populations obtained from
GenBank. ............................................................................. 77
Figure 4.5: Single maximum parsimony tree of 10 Pratylenchus coffeae populations
from Vietnam, 10 P. coffeae, two Pratylenchus jaehni, one Pratylenchus
loosi and one Radopholus similis population obtained from GenBank based
on the sequence alignment of the D2/D3 28S rDNA expansion segments.... 85
Figure 5.1: Population dynamics of three Pratylenchus coffeae populations from
Vietnam at different temperatures (15, 20, 25 and 30oC). Pf: final nematode
population density................................................................... 97
xi
Figure 5.2: Effect of time and temperature on the in vitro population composition
on carrot discs of three Pratylenchus coffeae populations from Vietnam after
inoculation with with 25 females, incubated in the dark. ..................... 99
Figure 6.1: Average monthly soil temperature in the soil in the pots from March
2004 to November 2006............................................................104
Figure 6.2: Nematode population density per 10 g fresh roots and percentage root
necrosis caused by 10 Pratylenchus coffeae populations from Vietnam on
banana cv. Ngop Dui Duc, 14 weeks after inoculation with 1,000
vermiforms/plant...................................................................111
Figure 6.3: Nematode population density per 10 g fresh roots and percentage root
necrosis caused by 10 Pratylenchus coffeae populations from Vietnam on
sugarcane
var.
ROC20,
14
weeks
after
inoculation
with
1,000
vermiforms/plant...................................................................116
Figure 6.4: Damage caused by Pratylenchus coffeae on the roots of banana (A),
coffee (B), sugarcane (C) and maize (D), 14 weeks after inoculation with
1,000 vermiforms/plant. ..........................................................118
xii
xiii
Summary
Root-lesion nematodes rank second only to root-knot and cyst nematodes
in terms of their wordwide impact on agricultural crops. Many of these species are
of little or no economic importance but some are responsible for substantial
damage and high yield losses in a variety of agricultural crops. Pratylenchus
coffeae (Zimmermann, 1898) Filipjev and Schuurmans Stekhoven, 1941 is one of
these root-lesion nematodes that are considered important plant pathogens. It has
a worldwide distribution and a wide host plant range.
In Vietnam, the presence of P. coffeae on agricultural crops has been
reported for the first time in the 1970s. However, its impact on agricultural crops
in Vietnam is largely unknown. The general objective of our study was to study the
biodiversity of P. coffeae populations from Vietnam and to contribute to the
polyphasic taxonomy of the species.
In the first part of our study (Chapter 2), the occurrence of P. coffeae on
the most commonly cultivated agricultural crops in Vietnam was established. With
the exception of the South Central Coast, samples were taken in all agro-ecological
regions of Vietnam. In total 95 soil samples and 95 root samples were collected
from 21 agricultural crops in 20 provinces. About 25% of the root samples were
infected with this nematode species. In Vietnam, P. coffeae has a remarkable
geographical distribution: its occurrence decreases from north to south. These data
confirm earlier observations that the occurrence of P. coffeae is apparently
restricted to North and Central Vietnam. Pratylenchus coffeae was found in 59 and
36% of the banana and coffee fields, respectively, sampled. In contrast, P. coffeae
was only found in one out of 16 pineapple fields sampled. In our study, P. coffeae
was found in nine of the 21 agricultural crops examined which reconfirms that this
nematode species has a wide host range.
In the second part of our study (Chapter 3), the morphological and
morphometrical characters of 10 P. coffeae populations from Vietnam in vitro
established on carrot discs were compared in detail. Variability in morphology and
morphometry within and between these populations was observed. However, these
differences fall within the range of the morphological and morphometrical
variability described previously in P. coffeae populations from other parts of the
world. Scanning electron microscopy observations further confirm that in P.
coffeae there is a complete fusion of the 1st (lip) annule with the oral disc resulting
in an undivided en face view with no division between the lateral and median (subdorsal and sub-ventral) segments of the 1st (lip) annule. This is an important
xiv
morphological character typical for this nematode species and a small number of
related Pratylenchus species. Of the 19 morphometrical characters studied, the
position of the vulva (V-value) in the females and the stylet length in both females
and males had the lowest coefficient of variation while the length of the ovary in
the females and the length of the testis in the males had the highest coefficient of
variation. The 10 P. coffeae populations from Vietnam examined were divided in
three groups based on a combination of five morphological characters for the males
by canonical discriminant analysis. However, there was no relationship nor
between these groups and their geographic origin or between these groups and the
host plants from which they were originally isolated.
In the third part of our study (Chapter 4), RAPD bands analysis of the
complete genome and sequencing of the D2/D3 expansion segments of the 28S
rDNA gene were done to compare the intraspecific genomic variability of the 10 P.
coffeae populations collected from different agricultural crops in different agroecological regions in Vietnam and to compare these with the sequences available in
the GenBank database. As determined by RAPD bands analysis of the complete
genome, genomic similarity did not correspond nor with geographic or original host
plant origin. As determined by sequence analysis of the D2/D3 28S rDNA expansion
fragments, all 10 P. coffeae populations from Vietnam examined were closely
related among each other and with the P. coffeae populations of which the D2/D3
28S rDNA expansion fragments sequences were obtained from GenBank. Both the
RAPD bands analysis of the complete genome and the sequence analysis of the
D2/D3 28S rDNA expansion fragments indicate genetic divergence among the 10 P.
coffeae populations from Vietnam examined on the one hand and the P. coffeae
population from Ghana on the other hand, supporting previous suggestions made in
the nematological literature that taxonomic clarification of the population from
Ghana is required.
In the fourth part of our study (Chapter 5), the in vitro reproductive
fitness on carrot discs of the 10 P. coffeae populations collected from different
agricultural crops in different agro-ecological regions in Vietnam was studied and
compared to those of a P. coffeae population originally isolated from banana in
Ghana. Few major differences in in vitro reproductive fitness on carrot discs were
observed among the 10 P. coffeae populations from Vietnam. The in vitro
reproduction fitness on carrot discs of five out of the 10 P. coffeae populations
from Vietnam examined was not statistically different from the reproduction
fitness of the P. coffeae population from Ghana. Also, no differences were
observed in the in vitro reproductive fitness on carrot discs between P. coffeae
populations originally isolated from either banana or coffee. The effect of
xv
temperature on the in vitro reproductive fitness on carrot discs of three selected
P. coffeae populations from Vietnam (collected in three different agro-ecological
regions: (Northwest, North Central Coast and Central Highlands) was compared
over time. Temperature influenced the three populations in a similar way. The
optimum temperature for reproduction of this nematode species is 25 to, at least,
30oC. The three P. coffeae populations examined are tolerant to low temperatures
(15 to 20oC) and this enables the P. coffeae populations in Vietnam to survive the
low temperatures which occur during the winter in the northern and central parts
of the country.
In the fifth and last part of our study (Chapter 6), in general, the in vivo
reproduction of all the 10 P. coffeae populations collected in Vietnam on the 13
agricultural crops included in our experiments was very similar. Of the 13 crops
studied, banana, sugarcane, maize and upland rice were observed to be good hosts
of P. coffeae. Soybean is considered a poor host and groundnut, tomato, sweet
potato, ginger, sesame, pineapple and citrus can be considered as very poor hosts
or non hosts of P. coffeae. The in vivo pathogenicity on banana, coffee, sugarcane
and maize of all the 10 P. coffeae populations from Vietnam examined was very
similar. These populations were able to cause considerable damage to the
vegetative growth of banana and coffee but not to sugarcane and maize. In view of
the low reproduction on coffee, the extensive damage the P. coffeae populations
from Vietnam examined caused on this agricultural crop is surprising and illustrates
the high damage potential of P. coffeae on coffee. Based on the in vitro
reproduction on carrot discs as well as the in vivo reproduction on 13 agricultural
crops and the in vivo pathogenicity on banana, coffee, sugarcane and maize, there
was no indication of the existence of different biotypes or pathotypes among the
10 P. coffeae populations from Vietnam.
xvi
xvii
Samenvatting
Wortellesienematoden (Pratylenchus spp.) zijn na de wortelknop- en
cystnematoden, de belangrijkste plantenparasitaire nematoden die voorkomen op
landbouwgewassen. Veel Pratylenchus soorten zijn van weinig of geen economisch
belang maar sommige soorten kunnen substantiële schade en oogstverliezen
toebrengen aan landbouwgewassen. Pratylenchus coffeae (Zimmermann, 1898)
Filipjev & Schuurmans Stekhoven, 1941 is één van de wortellesie-nematoden die als
belangrijke plantenpathogenen worden beschouwd. Deze soort heeft een
wereldwijde verspreiding en talrijke waardplanten.
In Vietnam werd de aanwezigheid van P. coffeae op landbouwgewassen
voor het eerst gerapporteerd in de jaren 70. De impact van deze nematodensoort
op landbouwgewassen in Vietnam is evenwel grotendeels onbekend. De algemene
doelstellingen van onze studie waren om een bijdrage te leveren aan de
biodiversiteit
van
Vietnamese
P.
coffeae
populaties
en
de
zogenaamde
“polyphasic” taxonomie van deze nematodensoort.
In het eerste deel van onze studie (Hoofdstuk 2) werd de aanwezigheid
van P. coffeae op de belangrijkste landbouwgewassen in Vietnam onderzocht.
Hiervoor werden stalen verzameld in, op één na, alle agro-ecologische regio’s van
Vietnam. In totaal werden 95 grondstalen en 95 wortelstalen onderzocht afkomstig
van 21 landbouwgewassen in 20 Vietnamese provincies. Ongeveer 25% van deze
stalen waren geïnfecteerd met P. coffeae. Pratylenchus coffeae heeft een
opmerkelijke geografische verspreiding in Vietnam: zijn aanwezigheid neemt af van
noord naar zuid. Onze studie bevestigt vorige studies dat het voorkomen van P.
coffeae blijkbaar beperkt is tot Noord en Centraal Vietnam. Pratylenchus coffeae
werd aangetroffen in 59% en 36%, respectievelijk, van de onderzochte bananen- en
koffievelden. In tegenstelling hiermee werd P. coffeae maar aangetroffen in één
van de 16 onderzochte ananasvelden. Aangezien P. coffeae aanwezig was in negen
van de 21 landbouwgewassen bevestigen onze resultaten dat deze nematodensoort
talrijke waardplanten heeft.
In het tweede deel van onze studie (Hoofdstuk 3) werd de morfologie en
morfometrie van 10 P. coffeae populaties verzameld in Vietnam en geteeld in vitro
op wortelschijfjes in detail vergeleken. Binnen en tussen deze populaties werd een
hoge variabiliteit in morfologie en morfometrie vastgesteld. Deze variabiliteit is,
evenwel, vergelijkbaar met de variabiliteit die vastgesteld werd in P. coffeae
populaties in andere werelddelen. Scanning electronenmicroscopie bevestigde dat
de 1ste lippenring volledig gefusioneerd is zodat het zogenaamde “en face” beeld
xviii
van deze nematodensoort een onverdeelde mondschijf toont zonder een scheiding
tussen de laterale en mediane (sub-dorsale en sub-ventrale) segmenten van de 1ste
lippenschijf. Dit is een zeer belangrijk morfologische kenmerk van P. coffeae en
een beperkt aantal andere Pratylenchus soorten. Negentien morfologische
kenmerken werden bestudeerd. Van deze kenmerken vertoonde de positie van de
vulva (V-waarde) in de vrouwtjes en de lengte van de mondstekel in de vrouwtjes
en mannetjes de laagste variatiecoefficiënt. De lengte van het ovarium in de
vrouwtjes en de lengte van de testis in de mannetjes vertoonden daarentegen de
hoogste variatiecoefficiënt. Op basis van vijf morfologische kenmerken in de
mannetjes konden de Vietnamese populaties d.m.v. zogenaamde “canonic
discriminant” analyse ingedeeld worden in drie groepen. Maar er werd geen
verband vastgesteld tussen deze groepen en hun geografische oorsprong en tussen
deze groepen en de waardplanten waarop zij oorspronkelijk werden aangetroffen.
In het derde deel van onze studie (Hoofdstuk 4) werd RAPD analyse van
het volledige genoom en sekwentiebepaling van de D2/D3 expansiesegmenten van
het 28S rDNA gen uitgevoerd met de doelstelling de intraspecifieke genomische
variabiliteit van de 10 Vietnamese P. coffeae populaties te onderzoeken en te
vergelijken met informatie die beschikbaar is in de Genbank database. Er werd
geen verband vastgesteld tussen genomische similariteit van de P. coffeae
populaties noch met hun geografische oorsprong
noch met de waardplanten
waarop zij oorspronkelijk werden aangetroffen. Op basis van de sekwentiebepaling
van de D2/D3 expansiesegmenten van het 28S rDNA gen kon vasgesteld worden dat
de 10 onderzochte Vietnamese P. coffeae populaties nauw verwant zijn met elkaar
en met de P. coffeae populaties waarvan genetische informatie beschikbaar is in de
Genbank database. Opvallend is dat zowel de RAPD analyse van het volledige
genoom als de sekwentiebepaling van de D2/D3 expansiesegmenten van het 28S
rDNA gen erop wijzen dat de onderzochte Vietnamse P. coffeae populaties
genetisch verschillen van een P. coffeae populatie van Ghana. Onze resultaten
bevestigen
vroegere
suggesties
in
de
nematologische
literatuur
dat
de
taxonomische positie van deze P. coffea populatie van Ghana nader onderzocht zou
moeten worden.
In het vierde deel van onze studie (Hoofdstuk 5) werd de in vitro
reproductie-capaciteit op wortelschijfjes van de 10 Vietnamese P. coffeae
populaties bepaald en vergeleken met de reproductie-capaciteit van de P. coffeae
populatie van Ghana. Tussen de Vietnamese P. coffeae populaties onderling
werden geen grote verschillen in in vitro reproductie-capaciteit vastgesteld. De
reproductie-capaciteit van de helft van de Vietnamese P. coffeae populaties was
statistisch niet verschillend van de reproductie-capaciteit van de P. coffeae
xix
populatie van Ghana. Er werd op de wortelschijfjes ook geen verschil vastgesteld in
reproductie-capaciteit van P. coffeae populaties die enerzijds van bananenplanten
en anderzijds van koffieplanten waren geïsoleerd. Verder werd de invloed van
temperatuur op de reproductie-capaciteit van drie Vietnamese P. coffeae
populaties, afkomstig uit drie verschillende agro-ecologische regio’s, nader
onderzocht. De invloed van temperatuur op deze drie P. coffeae populaties was
vergelijkbaar. De optimale temperatuur voor reproductie van P. coffeae variëerde
van 25oC tot (minstens) 30oC. De drie Vietnamese P. coffeae populaties waren wel
tolerant voor lage temperaturen (15 tot 20oC) en dit kan verklaren waarom P.
coffeae populaties in Vietnam in staat zijn om de relatief lage temperaturen die
voorkomen tijdens de winter in het noorden en het centrum van Vietnam te
overleven.
In het vijfde en laaste deel van onze studie (Hoofdstuk 6) werd de in vivo
reproductie van de 10 Vietnamese P. coffeae populaties op 13 landbouwgewassen
onderzocht. De reproductie was vergelijkbaar. Banaan, suikerriet, mais en rijst
waren goede waardplanten van P. coffeae; soyaboon een slechte waardplant;
aardnoot, tomaat, zoete aardappel, gember, sesamzaad, ananas en citrus zeer
slechte tot geen waardplanten. Eveneens werd de in vivo pathogeniciteit van de 10
Vietnamese P. coffeae populaties op banaan, koffie, suikerriet en mais onderzocht.
De pathogeniciteit was vergelijkbaar. Alle Vietnamese P. coffeae populaties waren
in staat grote schade toe te brengen aan de vegetatieve ontwikkeling van banaan
en koffie maar niet van suikerriet en mais. De reproductie van de Vietnamese P.
coffeae populaties op koffie was zeer laag maar toch slaagden deze populaties erin
om grote schade aan de koffieplanten toe te brengen wat de hoge pathogeniciteit
van P. coffeae voor koffie illustreert. Op basis van hun in vivo reproductie op 13
landbouwgewassen en in vivo pathogeniciteit op banaan, koffie, suikerriet en mais
menen wij te mogen concluderen dat de onderzochte Vietnamese P. coffeae
populaties behoren tot hetzelfde bio- en pathotype.
Chapter 1
1
Chapter 1: Introduction
1.1
Objectives of the study and outline of the thesis
Plant-parasitic nematodes (Phylum Nematoda Potts, 1932) are important
pathogens of many agricultural crops worldwide. Among the most common and
damaging plant-parasitic nematodes are the root-knot nematodes (Meloidogyne
spp.), cyst nematodes (Globodera and Heterodera spp.), burrowing (Radopholus
spp.), rice root (Hirschmanniella spp.) and root-lesion nematodes (Pratylenchus
spp.). This latter group of nematodes causes typical elongated necrotised lesions
on and in the roots of the plants they attack and colonise, hence their name. Rootlesion nematodes rank second only to root-knot and cyst nematodes in terms of
their wordwide impact on agricultural crops (Sasser & Freckman, 1987).
At the end of 2006, the genus Pratylenchus Filipjev, 1936 counted 76
nominal species (De Waele & Elsen, 2007). Many of these species are of little or no
economic importance but some are responsible for substantial damage and high
yield losses in a variety of agricultural crops. Pratylenchus coffeae (Zimmermann,
1898) Filipjev and Schuurmans Stekhoven, 1941 is one of these root-lesion
nematodes that are considered important plant pathogens. It has a worldwide
distribution and a wide host plant range (Siddiqi, 1972; Castillo & Vovlas, 2007).
In nematology, species identification is based primarily on morphological
and morphometrical characters mainly of females and males observed and
measured with a light microscope (Coomans et al., 1978). However, many
nematode
genera,
especially
plant-parasitic
nematode
genera
including
Pratylenchus, exhibit little morphological diversity. Intraspecific variability of the
morphological and morphometrical characters important for distinguishing species,
the possibility of observational and interpretative mistakes and several other
factors make the precise and reliable identification of nematode species a
formidable task, even for well-qualified taxonomists (Coomans, 2002).
During the past two decades, taxonomists have increasingly used
molecular techniques, both protein- and DNA-based, to confirm the validity of
existing nematode species and to assist in the identification and description of new
species. Compared with protein-based diagnostics, DNA-based diagnostics have
several advantages, the most important being that the effects of environmental
and developmental variation can be excluded (Powers, 2004; Subbotin & Moens,
2006).
Chapter 1
2
Closer examination of the intraspecific variability in morphology and
morphometry (Román & Hirschmann, 1969; Tarjan & Frederick, 1978; Bajaj &
Bhatti, 1984; Inserra et al., 1998), isozyme phenotypes (Andrés et al., 2000) and of
polymorphism as detected with rDNA-RFLP (Waeyenberge et al., 2000) of many
populations of P. coffeae from around the world and of closely related species has
lead to the conclusion that there exist a P. coffeae species complex consisting of
several species, many of which remain undescribed (Duncan et al., 1999).
Correct identification up to the species level is of vital importance: the
prevention, locally and internationally, of the spread of pathogenic nematodes and
the success of effective nematode management strategies depend on it.
The general objective of our study was to study the biodiversity of P.
coffeae populations from Vietnam and to contribute to the polyphasic taxonomy of
the species. In Vietnam, this nematode species can be found in the roots of many
agricultural crops including sugarcane, coffee, ginger, pineapple and banana.
Surveys, mainly of banana, have shown that it is present in most areas in North and
Central Vietnam (Chau et al., 1997). In Chapter 2, some additional information on
the occurrence of P. coffeae on agricultural crops in Vietnam is presented. In the
next two chapters, 10 P. coffeae populations from Vietnam are characterised and
identified based on their morphology and morphometry (Chapter 3) and DNA
(Chapter 4). In Chapter 5, the in vitro reproductive fitness on carrot discs of the
Vietnamese populations is compared. In Chapter 6, their host range, in vivo
reproduction and pathogenicity on selected agricultural crops is examined. In the
final chapter (Chapter 7), conclusions and suggestions for further research are
formulated.
The term polyphasic taxonomy has been coined for integrated taxonomical
studies of nematode species based not only on the description of the phenotype
and genotype of species but also on other biological characteristics such as their
host range and ability to reproduce on and cause damage to agricultural crops
(pathogenicity; De Waele & Elsen, 2007).
Chapter 1
3
Introduction (Chapter 1)
Survey (Chapter 2)
Establishment of
in vitro cultures on carrot discs
Morphological study
(Chapter 3)
Molecular study
(Chapter 4)
RAPD analysis
Morphological
observations
Sequencing
In vitro study
(Chapter 5)
Reproductive
fitness
In vivo study
(Chapter 6)
Effect of temperature
on reproductive
Morphometrics
Host range
Conclusions and perspectives
(Chapter 7)
Figure 1.1: Research outline of the present study.
Pathogenicity
Chapter 1
1.2
4
Background: Pratylenchus coffeae
1.2.1 Classification and differential diagnosis
Pratylenchus coffeae was first described from coffee roots in Java,
Indonesia (Zimmerman, 1898) as Tylenchus coffeae. The genus Pratylenchus was
erected by Filipjev in 1936. In 1941, Filipjev and Schuurmans Stekhoven transferred
T. coffeae to the genus Pratylenchus.
The following classification of P. coffeae is based on Siddiqi (2000):
Phylum: Nematoda
Class: Secernentea
Subclass: Tylenchia
Order: Tylenchida
Suborder: Tylenchina
Superfamily: Hoplolaimoidea
Family: Pratylenchidae
Subfamily: Pratylenchinae
Genus: Pratylenchus
Species: Pratylenchus coffeae (Zimmermann, 1898) Filipjev and Schuurmans
Stekhoven, 1941.
Differential diagnosis of the genus Pratylenchus (Hunt et al., 2005;
Castillo & Vovlas, 2007): Small nematodes (< 1 mm long) dying slightly curved
ventrally on application of gentle heat (Fig. 1.2). No marked sexual dimorphism in
form of anterior region. Lateral fields with four to six lines at vulval region. Deirids
absent. Phasmids near middle of tail. Labial region strongly sclerotised, low,
rounded to flattened, usually appearing as a dark flat cap under the
stereomicroscope, divided in two, three or four annules and continuous with or
sometimes slightly set off from the body contour. Amphideal apertures pore-like,
near labial disc, indistinct. Stylet is approximately 20 µm or less in length (i.e. less
than three labial region diameters long), moderately sclerotised and with rounded
to oblong or anteriorly concave knobs. Pharynx equally developed in both sexes,
median bulb well developed, oval to round, very muscular. Pharyngeal gland lobes
overlapping intestine mostly ventrally, usually less than two body diameters long.
Female: Vulva well posterior at 70-80% of body length. Genital system with a
single, anteriorly directed branch (monoprodelphic) and a variable post-vulval
uterine sac which may show some differentiation, but which is never functional.
Spermatheca large, oval to rounded, usually filled with sperm when males are
present. Tail subcylindrical1 or more or less conoid2, usually about two to three
anal body diameters long with a broad to narrowly rounded or truncated3 tip,
which may be smooth, crenated4 or cleft. Male: Tail short, dorsally convex-conoid
with subterminal pore on dorsal side. Bursa extending to tail tip. Spicules tender,
arcuate.
___________________________
1
Subcylindrical: tail tapering but little, with broad tip.
Conoid: tail tapering strongly, with narrow tip.
3
Truncated: tail with a square tip.
4
Crenated: transverse cuticular striations extending around the tail tip (= annulated).
2
Chapter 1
5
A
B
Figure 1.2: Female (A) and male (B) of Pratylenchus coffeae as
observed under the light microscope. Scale bar: 53 µm. Source
of the picture: Timmer and Graham (2002).
The following morphological and morphometrical characters have been
used in tabular and dichotomous keys to distinguish Pratylenchus spp. (Castillo &
Vovlas, 2007): number of lip annules, presence or absence of males, stylet length,
shape of spermatheca, vulva position (V-value), length of post-vulval uterine sac,
female tail and tail tip shape, length of pharyngeal overlap, number of lateral field
lines at vulval region, presence or absence of areolated bands on the lateral field
at vulval region.
Identification keys for the species of the genus Pratylenchus have been
published by Sher and Allen (1953), Corbett (1969), Café Filho and Huang (1989),
Frederick and Tarjan (1989), Handoo and Golden (1989), Loof (1991), Siddiqi
(2000), Ryss (2002) and Castillo and Vovlas (2007).
Differential diagnosis of P. coffeae (based on Sher & Allen, 1953; Loof,
1978, 1991; Román & Hirschmann, 1969; Siddiqi, 1972; Bajaj & Bhatti, 1984; Inserra
et al., 1998, 2001; Mizukubo, 1992; Duncan et al., 1999; Ryss, 2002; Castillo &
Vovlas, 2007): Female (Fig.1.3): Body rather slender in young females, thicker in
old ones. Labial region slightly set off from the body contour, rounded, divided in
two annules. Occasionally three annules on one side of labial region. En face view
characterised by fusion of subdorsal, subventral and lateral lips with oral disc.
Outer margins of heavily sclerotised labial framework extend into body about one
body annule. Cuticular annulation fairly conspicuous. Lateral fields distinct,
normally with four to five, occasionally with six lines, outer ones crenate in the tail
region. Stylet well developed with broadly round to oblong basal knobs. Dorsal
pharyngeal gland orifice about 2 µm behind stylet base. Hemizonid just anterior to
excretory pore, about two body annules long. Ovary does not extend to pharyngeal
gland, consisting of a single row of oocytes, except for a double row near the
anterior end. Spermatheca large, broadly oval to nearly rounded, often with
sperm. Post-vulval uterine sac variable in length, 1 to 1.5 times body diameters
long, sometimes reaching 90 µm. Tail tapering slightly, its length in young
Chapter 1
6
specimens 2 to 2.5 times, in old specimens 1.5 to 2 times the anal body width. Tail
tip indented, sometimes appearing smoothly rounded or truncated, in some
specimens appearing weakly and irregularly crenated. Phasmids slightly posterior
to the middle of the tail. Four lateral lines extend past the phasmids. Male (Fig.
1.3): Similar to females. Spicules very slender, shaft ventrally concave. Gonad
extends over about one-half of the body length. Testis shorter than vas deferens.
Bursal edges faintly crenated.
Figure 1.3: Pratylenchus coffeae. A: female stylet; B: entire female; C: female head region;
D: female lateral field (note striae and crenate lines); E: female pharyngeal region; F: female
vulval region; G: male pharyngeal region; H: female en face view; I-L: female tail shapes; M:
male tail (note the faintly crenated bursal edges). Source of the figure: Inserra et al. (2001).
Chapter 1
7
1.2.2 Biology and life history
Pratylenchus coffeae, a bisexual species, is a migratory endoparasite of
the root cortex, corms or tubers, where it feeds and multiplies (Fig. 1.4). But P.
coffeae can also wander out of the plant, live for some time in the soil and migrate
to and penetrate new host plant roots. When decay sets in, for instance due to
secondary invasion by bacteria or fungi, the nematodes abandon the dying roots
and migrate through the soil to attack healthy roots.
Figure 1.4: Life and disease cycle of lesion nematodes (Pratylenchus spp.). Source of the
figure: Agrios (2005).
Eggs are laid in the root tissues and the whole life cycle of P. coffeae is
completed within the root cortex in about 4 weeks under optimal conditions. From
egg to adult, the nematode passes through four juvenile stages. The first moult
takes place in the egg, the following three moults occur outside the egg. Eggs
hatch in 6 to 8 days at 28-30oC in water. In potato tubers in Japan, adult P. coffeae
appeared in about 2 weeks after hatching and the average life span was about 27
days at 25-30oC (Gotoh, 1964, cited by Siddiqi, 1972). In yam, P. coffeae is also
assumed to have a life cycle of 3 to 4 weeks (Thompson et al., 1973). The life cycle
of P. coffeae was even completed in only 20 days in citrus callus and roots
produced from citrus leaves (Inserra & O´Bannon, 1975). In coffee roots, (Lordello,
1986 cited by Inomoto & Oliveira, 2008) observed the first-stage juveniles (J1) 8
days after egg laying, the second-, third-, fourth-stage juveniles (J2-J4) and the
Chapter 1
8
adults 14, 21, 28 and 29 to 32 days after egg laying, respectively. Eggs frequently
undergo cleavage and sometimes reach full development in the uterus (Wehunt &
Edwards, 1971).
Pratylenchus coffeae populations from Florida and Puerto Rico have seven
chromosomes (n = 7). The chromosome number is the same in males and females.
There are no sex chromosomes (Román & Triantaphyllou, 1969).
The optimum temperature for penetration, colonisation and development
of P. coffeae is 25-30oC (Yokoo & Kukoda, 1966, cited by Radewald et al., 1971b;
Acosta & Malek, 1979). In rought lemon (Citrus jambhiri) roots, the highest
reproduction rate of P. coffeae was observed at 29.5oC with the optimum
temperature for reproduction ranging from 26 to 32oC (Radewald et al., 1971b).
Pratylenchus coffeae survived in excised roots of rough lemon stored in soil with an
initial moisture content near field capacity at temperatures ranging from 10 to
32oC and was still infective after 4 months but did not survive extended storage in
either moist or dry soils at 38oC (Radewald et al., 1971b). However, in an apple
orchard in Australia (Colbran, 1954, cited by Siddiqi, 1972), P. coffeae survived in
moist soil for up to 7 months in the absence of host plants. All stages of P. coffeae
survived equally well in storage (Radewald et al., 1971a).
1.2.3 Histopathology and symptoms of damage
All mobile developmental stages of P. coffeae from the second-stage
juvenile (J2) onwards may enter the roots and invade all locations along the
seedling tap roots, including root cap, apical meristem, region of elongation,
region of maturation and mature tissues. The nematodes feed mainly on cortex
cells and form cavities. There is no evidence that P. coffeae feeds on vascular
tissue. However, the nematode is seen probing the endodermis and migrating
parallel to the vascular tissue in cells adjacent to the endodermis (Pinochet, 1978).
According to Inomoto and Oliveira (2008), P. coffeae penetrates the roots of coffee
1 day after inoculation mainly at the root tip (Fig. 1.5). Pratylenchus coffeae does
not cause hyperplasia or detectable cellular reactions and growth stimulus in the
pericycle and endodermis. When large numbers of the nematodes entered a root at
a single location, both the epidermal and cortical tissues were destroyed, resulting
in an exposed lesion extending to the stele tissues (Radewald et al., 1971a). Males
are necessary for reproduction. Males continually migrate in and out of the roots
destroying tissue in the same manner as migrating females and juveniles but not
forming cavities (Radewald et al., 1971a). Pratylenchus coffeae seems to directly
affect the root uptake capacity by damaging root cells during penetration and
feeding (Vaast et al., 1998).
Chapter 1
9
Figure 1.5: Pratylenchus coffeae in the tip of coffee roots. Source
of the picture: Inomoto and Oliveira (2008).
Coffee roots infected with P. coffeae turn yellow then brown. Infected
coffee seedlings with advanced root rot symptoms are stunted, have few and small
chlorotic leaves and most lateral roots are rotten. The earliest symptoms of P.
coffeae infection in newly transplanted trees are yellowing of leaves, loss of young
primary branches and stunting of the shoot (Figs 1.6A-C). A gradual wilt sets in,
followed by death of the whole tree (Whitehead, 1969).
A
B
C
Figure 1.6: Symptoms caused by Pratylenchus coffeae on coffee. A: wilting; B-C: lesions on
roots. Source of picture A: http://www.biologico.sp.gov.br/artigos_ok.php?id_artigo=106, B:
Souza (2008) and C: own pictures.
In banana and plantain, P. coffeae causes purple or black necrosic lesions.
The necrotic lesions enlarge and become more deeply colored over time. When
infected roots are cut longitudinally, typical brownish-red lesions can be seen along
the cortex (Figs 1.7A-C). Cross-sections show that the area most damaged is the
inner cortex which is destroyed producing a circular cavity, detached from the
Chapter 1
10
vascular tissues of the central stele (Pinochet, 1978). The above ground symptoms
of damage are similar to those observed when bananas and plantains are attacked
by Radopholus similis: stunting of plants, lengthening of the vegetative cycle,
reduction in size and number of leaves and in bunch weight, reduction of the
productive life of the plantation, and toppling (Gowen et al., 2005).
A
B
C
Figure 1.7: Symptoms caused by root-lesion nematodes on banana roots (A and B) and corm
(C). Source of picture A: http://plp3002.ifas.ufl.edu/pdfs/slides/nematodes.pdf, B and C:
Speijer and De Waele (1997).
In yam, a dry rot disease of the tubers is caused by P. coffeae (Fig. 1.8A).
The rot occurs as a dark brown band of necrotic tissues along the outer edges of
the tubers which can be clearly observed when the tubers are cut in half (Bridge et
al., 1996; Acosta & Ayala, 1975, 1976). A population of only 600 P. coffeae per
plant can produce significant damage (severe necrosis and deep cracks) to the
tubers, while 1,000 P. coffeae per plant can cause complete deterioration and
severe reduction in tuber quality (Acosta & Ayala, 1975). In taro, infection with P.
coffeae causes poor plant growth, root decay and reduced number of cormels. Two
months after planting, roots turn brown and then rot (Fig. 1.8B).
Chapter 1
11
A
B
Figure 1.8: Symptoms caused by Pratylenchus coffeae on tuber of yam (A) and taro (B).
Source of the picture A: Bridge et al. (2005), B: Anonymous (2008).
In citrus, P. coffeae causes citrus slump (Tarjan & O’Banon, 1969).
Destruction of the root cells results in a reduced tree vigor which in turn causes a
serious decline of the trees and small fruits. Growth reduction of young citrus trees
measured during 4 years in the field ranged from 49 to 80% depending on the
multiplication rate of P. coffeae on different rootstocks (O’Bannon & Tomerlin,
1973).
1.2.4 Resistance
Although efforts to screen agricultural crop germplasm for resistance to
plant-parasitic nematodes have mainly been aimed at identifying resistance to
sedentary endoparasitic nematodes such as root-knot (Meloidogyne spp.) and cyst
(Globodera and Heterodera spp.) nematodes, resistance to some migratory
endoparasitic Pratylenchus species was found in several agricultural crops, such as
cereals (i.e. maize, wheat, rice), root and tuber crops (potato, sweet potato),
banana and plantain, lucerne (alfafa), etc. (De Waele & Elsen, 2002).
Very few studies have been undertaken to study the mechanism(s)
responsible for host plant resistance to Pratylenchus spp. (Castillo & Vovlas, 2007).
Baldridge et al. (1998) found preliminary evidence for the involvement of several
known plant defence response genes in lucerne resistance to Pratylenchus
penetrans. Pratylenchus penetrans-resistant plants had higher constitutive levels
of transcripts for key enzymes involved in the biosynthesis of isoflavanoid
phytoalexins which are known to play a role in fungal resistance. Soriano et al.
(2004) reported that phytoecdysteroids (plant hormones inducible in response to
mechanical damage or herbivores) inhibit penetration and development of
Pratylenchus neglectus in spinach and may confer a mechanism for nematode
resistance.
Chapter 1
12
1.2.5 Geographical distribution, host plants and importance in
agriculture
Pratylenchus coffeae is probably a native of the Pacific and Pacific Rim
countries, from which it may have been spread through infected planting material.
Today it has a pantropic distribution (Fig. 1.9; Bridge et al., 1997; CABI/EPPO,
2007).
Figure 1.9: Geographical distribution of Pratylenchus coffeae. ●: widespread; ●: present, no
further details; ○: present, localised. Source of the map: CABI/EPPO (2007).
Bridge et al. (1997) suggested that P. coffeae may have the same
geographical origin as bananas and plaintains, i.e. the Pacific islands and
neighbouring Asian countries, from which it may have been spread through the
movement of infected planting material. In the Pacific, many food crops
(especially root and tuber crops) and cash crops (such as sugarcane and ginger)
that are propagated vegetatively are infected with P. coffeae (Bridge, 1988) and
this nematode species may also have spread with infected planting material of
these crops.
Pratylenchus coffeae is a polyphagous species with a large host range. A
host plant list of P. coffeae including 128 plant species and varieties was published
by Esser (1969). Approximately 30 of these host plants were ornamentals. Recently,
Jackson et al. (2003) reported that the host plant range of P. coffeae includes over
250 plant species covering almost all plant families.
On coffee, P. coffeae is the most widely reported root-lesion nematode
species worldwide (Campos & Villain, 2005). In Central and South America, it was
found in the Dominican Republic, El Salvador, Guatemala, Costa Rica, Colombia,
Venezuela and Brazil. In the Caribbean, P. coffeae was detected in Martinique,
Cuba and Puerto Rico. In Africa, it was reported in the Democratic Republic of
Congo and Tanzania. Pratylenchus coffeae also occurs on coffee in Madagascar. In
Chapter 1
13
Asia, besides Java (Indonesia), its typical site, it was reported in India and the
Indochina region. Pratylenchus coffeae also occurs on coffee in Hawai.
Also on bananas and plantains (Musa spp.), P. coffeae is the most widely
reported root-lesion nematode worldwide (Gowen et al., 2005). Although this
nematode species is found in banana roots, it is most frequently associated with
root injury in plantain in Central America, the Caribbean and West Africa (see for
instance Díaz-Silveira & Herrera, 1998; Brentu et al., 2004; Moens et al., 2006),
and diploid and triploid bananas in Asia and the Pacific (Bridge et al., 1997). In
Africa, althought it is widespread and important in some countries, such as Ghana
and Nigeria (Speijer et al., 2001; Brentu et al., 2004), in other African countries,
such as Ivory Coast and Cameroon (Adiko, 1988; Bridge et al., 1995), its frequency
of occurrence is very low indicating that in this continent its distribution is much
more localised.
On yam, P. coffeae is the cause of a tuber dry rot disease. On this crop it
has been recorded in Brazil, Belize, the Caribean (Barbados, Jamaica, Puerto Rico),
China, Taiwan and in several Pacific islands (Papua New Guinea, Fiji, Niue, Tonga,
Vanuatu and the Solomon Islands; Bridge et al., 2005). In Japan, P. coffeae is also
reported to cause serious losses to two other tuber crops: sweet potato (Scurrah et
al., 2005) and taro (Bridge et al., 2005).
In addition to coffee, bananas and plantains, root and tuber crops, P.
coffeae is known to cause damage to several other crops such as citrus in the USA,
South Africa, Oman, India, Japan and Taiwan (Duncan, 2005).
1.2.6 Biological diversity
The worldwide distribution, wide host plant range and economic
importance of the genus Pratylenchus has resulted in the description of many
populations and species. Although the genus Pratylenchus as such is easy
recognisable from other plant-parasitic nematode genera, the gross morphology of
the species within the genus Pratylenchus is very similar while the variability of the
morphometrical characters usually used by taxonomists to distinguish nematode
species is very high. In fact, the genus Pratylenchus is notorious for the high intraand interspecific variability of the small number of morphological characters that
can be observed either with the light microscope or the scanning electron
microscope and the overlapping of morphometrical characters (Mizukubo, 1992;
Bajaj & Bhatti, 1984).
This is also the case for P. coffeae. In addition to a high variability in
morphology and morphometry (see Chapter 3), genotype (see Chapter 4), also
Chapter 1
14
differences in in vitro reproductive fitness (see Chapter 5), host range, in vivo
reproduction and pathogenicity on agricultural crops (see Chapter 6) have been
observed and reported. To prevent repetition, details of these observations and
reports will be given in Chapter 3 (morphology and morphometry), Chapter 4
(genotype), Chapter 5 (in vitro reproductive fitness) and Chapter 6 (host range, in
vivo reproduction and pathogenicity).
Chapter 2
15
Chapter 2: Occurrence of Pratylenchus coffeae on
agricultural crops in Vietnam1
2.1
Introduction
In Vietnam, the presence of Pratylenchus coffeae on agricultural crops has
been reported for the first time in the 1970s (Chau et al., 1997). In 1997 and 2002,
Chau et al. and Van den Bergh, respectively, documented the occurrence of this
root-lesion nematode species on bananas in the country. Since then, P. coffeae has
been reported as the most common and widespread Pratylenchus species in
Vietnam (Chau & Thanh, 2000).
Although it is known that P. coffeae can cause considerable damage to
several agricultural crops worldwide (Bridge et al., 1997), its impact on agricultural
crops in Vietnam is largely unknown. Van den Bergh et al. (2006) compared
bananas inoculated with P. coffeae with nematode-free control plants in terms of
plant growth, crop cycle duration and yield under field conditions in Hanoi, North
Vietnam. Infection with P. coffeae did not affect the crop cycle duration or the
plant height, the pseudostem girth or the number of standing leaves at harvest of
any of the cultivars, but did significantly reduce the bunch weight of cv. Ngu Tien
from 6.6 to 5.3 kg (20% reduction), the bunch weight of cv. Tay Tia from 7.3 to 5.9
kg (19% reduction) and the bunch weight of cv. Grand Naine from 6.9 to 6 kg (13%
reduction). The bunch weight of cvs Hot and Ben Tre was not significantly
affected. More recently, studies were initiated to examine the precise role of P.
coffeae in the occurrence of yellow-leaf disease in the main coffee-producing areas
in Vietnam after in certain provinces about 10 to 25% of the coffee plants were
observed showing yellow-leaf disease symptoms and damage caused by P. coffeae
to coffee roots had been reported (Nghi et al., 1996; Trung et al., 2000; Sung et
al., 2001).
The objectives of this part of our study were a) to establish the
occurrence of P. coffeae on agricultural crops in the agro-ecological regions of
Vietnam, b) to collect as many as possible P. coffeae populations from as many as
possible different agricultural crops and agro-ecological regions in the country, and
c) to establish the collected P. coffeae populations in vitro on carrot discs for
further studies.
__________________________________
1
The results presented in this chapter have been published in: Tuyet N.T., Nhi H.H., Van den
Bergh I., Elsen A. and De Waele D. 2008. Occurrence of Pratylenchus coffeae on agricultural
crops in Vietnam. International Journal of Nematology 18:174-180.
Chapter 2
2.2
16
Agro-ecological regions of Vietnam
Vietnam occupies the south-eastern part of the Indochinese peninsula in
Southeast Asia. The country has a long and narrow shape, and covers a land area of
approximately 330,000 km2 extending from latitude 9 to 23oN and longitude 102 to
110oE. It is bordered in the north by China, in the west by Laos and Cambodia, and
in the south and east by the South China Sea with a long coastal line.
Vietnam has a tropical monsoon climate with an air humidity averaging
84% throughout the year. In the north four seasons can be distinguished (spring,
summer, autumn and winter) while in the south only a dry and a rainy season can
be distinguished. The annual rainfall is substantial in all regions, ranging from
1,200 to 3,000 mm. Nearly 90% of the precipitation occurs during the summer or
rainy season. The average annual temperature is in general higher in the south
than in the north and in the lowlands than in the highlands. In the north, it ranges
from 5oC in December and January, the coolest months, to more than 37oC in July
and August, the hottest months. In the south, the difference in temperature
between months and between seasons is very small, except in the highlands. The
temperature in the south normally ranges from 21 to 28oC.
Based mainly on differences in climate and topography, eight agroecological regions can be distinguished in Vietnam (Fig. 2.1).
2.2.1 Northeast
This region is situated on average at 500 to 600 m altitude. During the
summer (March to November), the average temperature is over 20oC. Because of
the northern situation and the higher altitude winter starts early in this region with
average temperatures which can be up to 10oC lower than in the other agroecological regions in the north. The rainy season starts between May and
September and its duration can vary from 4 to 10 months with an annual rainfall of
more than 1,200 mm except for the coastal areas.
2.2.2 Northwest
The average annual temperature in this region is 23oC. Summer comes
earlier in this region compared with the other agro-ecological regions in the north.
June is the hottest month with temperatures which may reach 40oC due to hot
winds. During the winter minimum temperatures may be 0oC. The annual rainfall is
about 2,500 mm. As the valleys are sheltered from wind by mountain ranges, the
dry season may last longer (4 to 5 months) compared with the other agro-ecological
regions in the north.
Chapter 2
17
Figure 2.1: Agro-ecological regions of Vietnam.
2.2.3 Red River Delta
The Red River Delta is a flat (< 3 m altitude), triangular region of about
3,000 km2, enriched with alluvial soil from the numerous rivers of the Red River
water system. The average annual temperature in this region is also about 23oC. In
summer, the average temperature is 29.2oC, in winter 17.2oC. The annual rainfall
ranges from 1,600 to 2,200 mm.
2.2.4 North Central Coast
In contrast with the South Central Coast, this region has a cold winter
lasting from December to February with an average monthly temperature below
20oC (about 16 to 19oC). In July (summer), the average temperature is 28-29oC. The
rainfall is high and unevenly distributed, ranging from about 2,400 mm/year in the
northern part of the region to about 1,300 mm/year in the southern part of the
region.
2.2.5 South Central Coast
In contrast with the North Central Coast, this region has no winter. The
average annual temperature is higher than 25oC. The rainy season lasts from
September to December or January. The rainfall is also high and unevenly
distributed, ranging from about 1,600 to 4,000 mm/year in the northern part of the
region to about 1,300 to 1,400 mm/year in the southern part of the region.
Chapter 2
18
2.2.6 Central Highlands
The Central Highlands consist of an area of 51,800 km2 with rugged
mountain peaks, extensive forests and fertile soil. The average annual temperature
is about 22oC. The warmest months are March and April, the coldest month is
January. Rainfall is low during the dry season (December to March). In many places
it rains continuously during the 5-months-long rainy season with a rainfall on
average higher than 200 mm/month.
2.2.7 Southeast
This region has an average annual temperature of about 21oC. The dry
season occurs from November to April. The months with the highest rainfall are
July and August. The annual rainfall ranges from 1,100 to 1,200 mm in the
mountainous parts of the region and from 1,400 to 1,600 mm in the coastal parts of
the region.
2.2.8 Mekong River Delta
This region has a high average annual temperature (26-27oC). The daily
temperatures range from 17 to 34oC in January and from 22 to 33oC in July. Rainfall
is very variable: from the western parts of this region, with a rainy season of 8
months, the rainfall gradually decreases towards the eastern parts of this region
from more than 2,000 mm to 1,400-1,600 mm.
2.3
Materials and methods
The sampling of the P. coffeae populations was carried out during 2000-
2005. With the exception of the South Central Coast, samples were taken in all
agro-ecological regions of Vietnam (Table 2.1; Fig. 2.2). In five of the seven agroecological regions visited, samples were taken both in summer and in winter or
both in the rainy and dry season.
In each agro-ecological region sampled, the presence of P. coffeae was
examined on the most commonly cultivated agricultural crops. Agricultural crops
sampled included coffee, banana, pineapple, ginger, cinnamon, tea, black pepper,
citrus, durian, longan, mango, cashewnut, a grass, maize, custard apple, soybean,
papaya, sugarcane, groundnut and coconut.
In each province, one to three localities (districts) were visited. At each
locality, three to 10 plants of each agricultural crop examined were uprooted.
Samples from banana were collected according to the methodology described by
Speijer and De Waele (1997). For the other agricultural crops, primary and
Chapter 2
19
secondary roots were collected. Soil samples consisted of soil from the rhizosphere
of the plants. Samples were stored in plastic bags at low temperature until
nematode extraction was carried out.
Table 2.1: Sampling period,
sampled.
Sampling dates
May & December 2004
March 2005
December 2001
June & November 2001
February 2001
August 2005
March & November 2001
July 2003
April 2001
June & December 2003
June 2001; July, September
& December 2004
April 2002; July 2003
April 2002; July 2003
May & November 2001
May & November 2001
May & November 2001
May & November 2001
July 2003
July 2003
July 2003
September 2003
July 2003
October 2004
October 2004
July 2003; October 2004;
September 2005
October 2004
localities (districts), provinces and agro-ecological regions
Locality (district)
Dien Bien
Ky Son
Van Chan
Tran Yen
Phu Ho
Bach Thong
Ba Vi
Province
Dien Bien
Hoa Binh
Yen Bai
Yen Bai
Phu Tho
Bac Kan
Ha Tay
Hung Yen
Thanh Hoa
Thanh Hoa
Nghe An
Agro-ecological region
Northwest
Northwest
Northeast
Northeast
Northeast
Northeast
Red River Delta
Red River Delta
North Central Coast
North Central Coast
North Central Coast
Ben Luc
Cai Be
Tan Phuoc
Chau Thanh
Quang Tri
Thua Thien Hue
Kon Tum
Gia Lai
Dak Lak
Dak Lak
Tay Ninh
Tay Ninh
Dong Nai
Ba Ria Vung Tau
Long An
Tien Giang
Tien Giang
Tien Giang
North Central Coast
North Central Coast
Central Highlands
Central Highlands
Central Highlands
Central Highlands
Southeast
Southeast
Southeast
Southeast
Mekong River Delta
Mekong River Delta
Mekong River Delta
Mekong River Delta
Giong Trom
Ben Tre
Mekong River Delta
Nghi Son
Trieu Son
Phu Quy
Khe Sanh
A Luoi
Dak Ha; Sa Thay
Dak Doa
Buon Ma Thuot
Krong Ana
Tan Chau
Hoa Thanh
Thong Nhat
Nematodes were extracted from the soil using a tray method modified
from the Baermann funnel method (Hooper et al., 2005) and from the roots by
maceration and sieving (Speijer & De Waele, 1997). Soil was placed on a sieve in a
dish of water and left at room temperature for 48 hours. Roots were washed, cut
into pieces of 1 cm length, macerated in a blender and passed through a series of
sieves with 250, 160 and 40 µm apertures. The extracted samples were examined
using a stereomicroscope for the presence of P. coffeae.
2.4
Results
In total 95 soil samples and 95 root samples were collected from 21
agricultural crops in 20 provinces (Fig. 2.2).
Chapter 2
20
1. Dien Bien
2. Hoa Binh
3. Yen Bai
4. Phu Tho
5. Bac Kan
6. Ha Tay
7. Hung Yen
8. Thanh Hoa
9. Nghe An
10. Quang Binh
11. Thua Thien Hue
12. Kon Tum
13. Gia Lai
14. Dak Lak
15. Tay Ninh
16. Dong Nai
17. Ba Ria Vung Tau
18. Long An
19. Tien Giang
20. Ben Tre
Figure 2.2: Provinces in Vietnam surveyed for the occurrence of Pratylenchus coffeae.
Twenty soil and 20 roots samples were collected from seven provinces in
the Northern Uplands (Northeast and Northwest) and in the Red River Delta (Table
2.2). Pratylenchus coffeae was found in all banana fields sampled while it was
present in only one out of three coffee fields sampled. Pratylenchus coffeae was
not found in any of the six pineapple fields examined. Pratylenchus coffeae was
found as well in the winter as in the summer.
Table 2.2: Occurrence of Pratylenchus coffeae in the Northern Uplands and Red River Delta.
Crop
Northwest
Northeast
Red River Delta
Dien Bien
Hoa
Yen Bai
Phu
Bac
Binh
Tho
Can
Coffee
+
0
Banana
+
+
+
+
+
Pineapple
0
0
0
0
0
Ornamental tree
Ginger
0
Cinnamon
+
Tea
0
(+): P. coffeae present; (0): P. coffeae not found; (-): not sampled.
Ha Tay
Hung Yen
0
+
0
0
+
-
Chapter 2
21
Fifteen soil and 15 root samples were collected from four provinces in the
North Central Coast (Table 2.3). Again, P. coffeae was found in all banana fields
sampled while it was present in two out of four coffee fields sampled. Pratylenchus
coffeae was not found in any of the four pineapple fields examined. Pratylenchus
coffeae was also found as well in the winter as in the summer.
Table 2.3: Occurrence of Pratylenchus coffeae in the North Central Coast.
Crop
North Central Coast
Thanh Hoa
Nghe An
Quang tri
Thua Thien Hue
Coffee
+
+
0
0
Banana
+
+
Black pepper
0
0
0
Pineapple
0
0
0
0
Citrus
0
0
Thirty-two soil and 32 root samples were collected from three provinces in
the Central Highlands (Table 2.4). Again, P. coffeae was found in all banana fields
sampled while it was also present in both coffee fields sampled. Pratylenchus
coffeae was this time found in one out of three pineapple fields examined. In this
region, P. coffeae was also found on several other agricultural crops: durian,
citrus, longan and maize.
Table 2.4: Occurrence of Pratylenchus coffeae in the Central Highlands.
Crop
Central Highlands
Dak Lak
Gia Lai
Banana
+
+
Coffee
+
+
Black pepper
0
0
Durian
0
0
Citrus
0
0
Pineapple
0
+
Ginger
0
Longan
+
Mango
0
0
Cashewnut
0
0
Grass
0
Maize
+
Custard apple
Kon Tum
+
0
0
+
+
0
0
0
0
0
0
0
Twenty-eight soil and 28 root samples were collected from six provinces in
the Southeast and in the Mekong River Delta (Table 2.5). Pratylenchus coffeae was
found in none of the fields sampled.
Chapter 2
22
Table 2.5: Occurrence of Pratylenchus coffeae in the Southeast and Mekong River Delta.
South East
Mekong River Delta
Crops
Tay Ninh
Dong Nai
Ba Ria Vung Tau
Long An
Banana
0
0
0
0
Coffee
0
0
Pineapple
0
0
0
Durian
0
Pepper
0
0
Soybean
0
Papaya
Sugarcane
0
0
0
Citrus
Groundnut
Coconut
2.5
Ben Tre
Tien Giang
0
0
0
0
0
0
0
0
0
0
0
0
-
Discussion
In our study, in total 95 root samples were examined on the presence of P.
coffeae. About 25% of these root samples were infected with this nematode
species. In Vietnam, P. coffeae has a remarkable geographical distribution: its
occurrence decreases from north to south. In the Northern Uplands (Northwest,
Northeast and the Red River Delta), 45% of the root samples examined were
infected with this nematode species. In the North Central Coast and Central
Highlands, this figure was 27 and 31%, respectively. In the Southeast and the
Mekong River Delta, however, P. coffeae was not found. These data confirm earlier
observations (Chau et al., 1997; Van den Bergh, 2002) that the occurrence of P.
coffeae is apparently restricted to North and Central Vietnam. The reason for this
remarkable geographical distribution is unknown. According to Gowen (2000),
temperatures of 25 to 30oC are optimal for the development and reproduction of P.
coffeae. This is precisely the average annual temperature that prevails in the
Mekong River Delta in South Vietnam. Although the northern part of Vietnam has a
cold winter, summer temperatures are high and well within range of the minimum
temperature (15oC) required for the development and reproduction of P. coffeae
(Bridge et al., 1995; Pinochet et al., 1995).
In our study, P. coffeae was found in 59 and 36% of the banana and coffee
fields, respectively, sampled. These data confirm that this nematode species is the
most common plant-parasitic nematode associated with banana in Vietnam (Chau
et al., 1997; Van den Bergh, 2002) and that it is common on coffee in this country
(Sung, 1976; Cuc et al., 1990). In contrast, P. coffeae was only found in one out of
16 pineapple fields sampled. This observation confirms that pineapple is not a good
host of this nematode species (Sipes et al., 2005). The remarkable geographical
distribution of P. coffeae in Vietnam as mentioned above, can also not be caused
Chapter 2
23
by the absence of suitable host plants in the south as banana, coffee, etc. are also
cultivated in South Vietnam.
2.6
Conclusions
Our observations confirm that P. coffeae is the most common plant-
parasitic nematode species associated with banana in Vietnam, that it is common
on coffee in this country and that its geographical distribution is apparently
restricted to North and Central Vietnam. Our observations also reconfirm that P.
coffeae has a wide host range.
2.7
Establishment of the collected Pratylenchus coffeae
populations on in vitro carrot disc cultures
Ten populations of P. coffeae (Table 2.6) collected from coffee, banana
and the roots of an ornamentral tree were established on in vitro carrot discs
according to the method described by Speijer and De Waele (1997). The nematodes
were surface-sterilised with streptomycine sulphate 2,000 ppm. After one night,
the nematodes were rinsed twice with sterile water. Carrot discs were prepared
using fresh carrots with green foliage. The carrots were carefully washed in tap
water to remove soil debris and other adhering materials. They were surfacesterilised with 95% ethanol, flamed and peeled with a sterile potato peeler. The
peeled carrots were cut into 5 mm thick discs. The carrot discs were placed
individually into sterile 3.5 cm diameter Petri dishes, which were sealed with
parafilm and stored at room temperature in the dark for 1 week. Before the carrot
discs were used for inoculation they were checked for bacterial or fungal
contamination.
Females of P. coffeae were inoculated on each carrot disc by hand picking
gravid females using a stereomicroscope with a needle and placing the females
near to the edge of the carrot disc. After inoculation, the Petri dishes were sealed
with parafilm and incubated at 25oC in the dark.
The cultures were sub-cultured at intervals of 8 to 10 weeks when
nematodes started moving out of the carrot discs.
Chapter 2
24
Table 2.6: Origin and population codes of the 10 Pratylenchus coffeae populations collected
in Vietnam and of the P. coffeae population from Ghana used in our study.
No.
Host plant
Province
Agro-ecological region
Population code
1
Banana
Dien Bien
Northwest
NW
2
Coffee
Yen Bai
Northeast
NE1
3
Banana
Yen Bai
Northeast
NE2
4
Banana
Phu Tho
Northeast
NE3
5
Banana
Bac Kan
Northeast
NE4
6
Banana
Ha Tay
Red River Delta
RRD1
7
Ornamental tree
Hung Yen
Red River Delta
RRD2
8
Banana
Thanh Hoa
North Central Coast
NCC1
9
Coffee
Nghe An
North Central Coast
NCC2
10
Coffee
Dak Lak
Central Highlands
CH
11*
Banana
Kade
Ghana (Africa)
Gha
* Reference population previously studied by Stoffelen et al. (1999).
Chapter 3
25
Chapter 3: Comparative study of the morphology
and morphometrics of Pratylenchus coffeae
populations from Vietnam1
3.1
Introduction
In nematology in general, and thus also in the taxonomy of the genus
Pratylenchus, species identification has been based primarily on light microscope
observations
(morphology)
(morphometrics)
mainly
and
measurements
of females and
of
morphological
males (Coomans
characters
et al., 1978).
Morphological resemblances (homologies) also formed the underlying basis of
evolutionary systematics (Coomans, 2000). The light microscope was usually used
for observing the morphology and the morphometry of these characters. But a light
microscope has its limitations and the, more or less recent, use of the scanning
electron microscope has considerably improved the accuracy of the observation
and description of, especially small, morphological characters. During the past two
decades, taxonomists have also increasingly used molecular diagnostic tools to
confirm the validity of existing species and to assist in the identification and
description of new species (De Waele & Elsen, 2007; see Chapter 4).
The genus Pratylenchus comprises 76 described species (De Waele & Elsen,
2007). Up to now, 139 original descriptions, among them four subspecies, were
published (Munera Uribe, 2008). The taxonomy of this genus has been the subject
of many publications (see for instance Sher & Allen, 1953; Loof, 1978, 1991;
Corbett, 1969; Román & Hirschmann, 1969; Corbett & Clark, 1983; Bajaj & Bhatti,
1984; Geraert & Raski, 1987; Café Fihlo & Huang, 1989; Frederick & Tarjan, 1989;
Handoo & Golden, 1989; Duncan et al., 1999; Siddiqi, 2000; Hernández et al.,
2001; Ryss, 2002; Castillo & Vovlas, 2007; Mizukubo et al., 2007). The small number
of diagnostic morphological characters at species level and the overlapping of
morphometrical characters among species are making it difficult to separate the
species. Because several Pratylenchus species are among the economically most
damaging plant-parasitic nematodes and are found on a wide variety of agricultural
crops (Duncan & Moens, 2006), and some Pratylenchus species are pathogens of
world quarantine importance (Ryss, 2002), the correct identification up to species
level is of vital importance to the prevention, locally and internationally, of their
spread and the development and success of effective nematode management
strategies.
____________________________
1
The results presented in this chapter have been submitted for publication in: Tuyet N.T.,
Elsen A., Nhi H.H. and De Waele D. Morphological and morphometrical characteristics of
Pratylenchus coffeae populations from Vietnam. Russian Journal of Nematology
(Supplement), 2010.
Chapter 3
26
Up to now, nine Pratylenchus identification keys have been published
(Sher & Allen, 1953; Corbett, 1969; Café Filho & Huang, 1989; Frederick & Tarjan,
1989; Handoo & Golden, 1989; Loof, 1991; Siddiqi, 2000; Ryss, 2002; Castillo &
Vovlas, 2007). The morphological and morphometrical characters most commonly
used to separate Pratylenchus species are (Castillo & Vovlas, 2007; Munera Uribe,
2008):
-
presence/absence of males
-
body length
-
de Man ratio’s (especially vulva position, V-value)
-
shape of head
-
number of lip annules
-
stylet length
-
shape of stylet
-
shape of stylet knobs
-
length of pharyngeal overlap
-
number of lateral field lines at vulval region
-
presence/absence of areolated bands on the lateral fields at vulval
region
-
length and structure of post-vulval uterine sac
-
shape of spermatheca
-
shape of female tail and tail tip
To identify the 10 P. coffeae populations collected from different
agricultural crops in different agro-ecological regions in Vietnam (see Chapter 2)
we used the dichotomous and tabular keys for the identification of Pratylenchus
spp. by Castillo and Vovlas (2007).
Up to now, the morphometrics of 41 Pratylenchus coffeae populations
collected from 18 different plants in 30 localities in 14 countries have been
published (Mizukubo, 1992; Pourjame, 1997a; Duncan et al., 1999; Munera Uribe,
2008; Table 3.1).
The objectives of this part of our study were to compare, on the basis of
both light microscope and scanning electron microscope observations, the
morphological and morphometrical characters of the 10 P. coffeae populations
collected from different agricultural crops in different agro-ecological regions in
Vietnam (see Chapter 2) a) among each other, b) with the Pratylenchus species
descriptions published in the literature, and c) to assess the intraspecific variability
of some of these characters.
Chapter 3
27
Table 3.1: List of the 41 Pratylenchus coffeae populations for which the morphometrics were
reported.
No. Author
Locality
Original host
1
Sher & Allen (1953)
Bangor, Indonesia
coffee
2
Loof (1960)
3
Román &Hirschmann
Florida, USA
Chinese evergreen
(1969)
4
Van den Berg (1971)
Cape, South Africa
vegetable
Aligarh, India
Chrysanthemum carinatum
5
Rashid & Khan (1976)
6
Bajaj & Bhatti (1984)
Sirsa, India
citrus
7
Inserra et al. (1989)
Djember–Java, Indonesia
coffee
Mizukubo (1992)
Takachiho, Miyazaki, Japan
Chrysanthemum morifilum
8
Artemisia feddei
9
Mizukubo (1992)
Tarami, Nagasaki, Japan
Mizukubo (1992)
Liyama, Nagano, Japan
Zea mays
10
11
Mizukubo (1992)
Kitamoto, Saitama, Japan
Ipomoea batatas
12
Mizukubo (1992)
Kitamoto, Saitama, Japan
Oryza sativa
13
Mizukubo (1992)
Nishigoshi, Kumamoto, Japan Artemisia princes
14
Florida, USA
citrus lemon
15
Florida, USA
citrus lemon
16
Florida, USA
citrus lemon
17
Pourjame (1997a)
Lahijan and Shiraz, Iran
citrus
18
Pourjame (1997b)
Honduras
citrus
Sao Paulo, Brazil
19
Duncan et al. (1999)
citrus
20
Florida, USA
Dhofar, Oman
citrus
21
Martinique
yam
22
yam
Pernambuca, Brazil
23
yam
24
Puerto Rico, USA
Kate, Ghana
banana
25
Honduras
banana
26
banana
Costa Rica
27
Malaysia
banana
28
Sao Paulo, Brazil
cocoyam
29
Diffenbachia
30
Sao Paulo, Brazil
Florida, USA
Aglaonema
31
China
ficus
32
coffee
Bangor, Indonesia
33
Sao Paulo, Brazil
coffee
34
coffee
35
Kaliwining, Indonesia
coffee
36
Dampit, Indonesia
coffee
Sumberasin, Indonesia
37
coffee
38
Wlingi, Indonesia
Kalibaru, Indonesia
coffee
39
40
(Xiuhua, 2006)
Heibei, China
Gaoyang
41
(Xiuhua, 2006)
Heibei, China
Dioscorea sp.
3.2
3.2.1 Nematode populations
The 10 P. coffeae populations collected from different agricultural crops
in different agro-ecological regions in Vietnam were used for the morphological and
morphometrical study (Table 2.6). The specimens studied were obtained from in
vitro carrot disc cultures (see Chapter 2). Carrot disc cultures with vigorous
Chapter 3
28
developing nematode populations (many active nematodes in the Petri dish) were
selected. The nematodes were collected in a test tube by rinsing the Petri dishes
with sterile water.
3.2.2 Killing and fixing of nematodes
The Seinhorst method (1959) as modified by De Grisse (1969) was used to
kill and fix the nematodes. The collected specimens were killed and fixed by
adding 4% hot (60-80oC) formaldehyde to a small drop of water in a glass cavity
vessel which contained the nematodes. The killed nematodes were transferred to
solution I (99 parts 4% formaldehyde + 1 part pure glycerine) in a deep glass dish.
This deep glass dish was placed in a closed glass vessel (desiccator) containing
about 1/10th of its volume of 96% ethanol. The desiccator was left overnight in an
oven at 37oC. The next day, the deep glass dish containing the nematodes was
removed from the desiccator and placed in an oven at 37oC. Some of the solution in
the deep glass dish was removed with a Pasteur pipet. Then, 2 to 3 ml of solution II
(95 parts 96% ethanol + 5 parts pure glycerine) were added to the deep glass dish.
This was repeated 3 to 4 times at 3-hours intervals, while the deep glass dish was
partially covered by a glass slide to allow slow evaporation. Finally, 1 to 2 ml of
solution III (50 parts 96% ethanol + 50 parts pure glycerine) were added and the
deep glass dish was left overnight at 37oC in the oven. The nematodes in pure
glycerine were used to mount on permanent slides.
3.2.3 Mounting of nematodes
Cobb slides, a simple aluminium slide holder with two coverslips (Cobb,
1917), were used for mounting. A paraffin ring was made with a heated copper
tube and paraffin. A drop of glycerine was placed in the center of the paraffin ring.
Of each P. coffeae population, about 100 females and 100 males were mounted on
slides. On one slide, eight to 10 females or males were transferred to a drop of
glycerine and covered with a cover slip. Then the slide was gently heated on a
heating plate till the paraffin ring melted and then allowed to cool down again.
3.2.4 Light microscope observations
Morphological characters studied were based on Coomans (1978) and on
the
morphological
characters
usually
used
in
the
taxonomic studies
of
Pratylenchus. The measurements taken for the comparative morphometrical study
and their abbreviations are listed in Table 3.2.
Chapter 3
29
Table 3.2: Measurements taken for the comparative morphometrical study of the 10
Pratylenchus coffeae populations from Vietnam and their abbreviations.
Character
Abbreviation used
in the tables
Body length (in µm)
L
Body length/maximum body width
a
Body length/distance from anterior end to pharyngo-intestinal
b
junction
Body length/distance from anterior end to posterior end of
b´
pharyngeal gland
Body length/tail length
c
Tail length/body width at anus (female) or cloaca (male)
c´
Stylet length (in µm)
stylet
Distance from basal knobs of stylet to dorsal pharyngeal gland orifice DGO
(in µm)
Maximum body width (in µm)
body width
Distance from anterior end to excretory pore (in µm)
excr. pore
Body length/distance from anterior end to excretory pore
L/excr. pore
Distance from head end to pharyngo-intestinal junction (in µm)
pharynx length
Distance from pharyngo-intestinal junction to posterior end of
pharyngeal overlap
pharyngeal gland (in µm)
Distance of vulva from anterior end x 100/total body length (%)
V
Distance from cloaca to anteriormost part of testis x 100/total body
T
length (%)
Distance from vulva to anteriormost part of ovary (in µm)
ovary
Distance from cloaca to anteriormost part of testis (in µm)
testis
Length of post-vulval uterine sac (in µm)
uterine sac
Tail length (in µm)
tail
Distance from vulva to anus (in µm)
vulva-anus
Lip width (in µm)
lip width
Lip height (in µm)
lip height
Median pharyngeal bulb x 100/total body width at level of the
pharyngeal bulb
median pharyngeal bulb (%)
Spicule length (in µm)
spicule
Fifteen females and 15 males of each P. coffeae population were
measured. Measurements were taken using a camera lucida drawing tube attached
to a light microscope (ZEISS Asioskop Plus 2) which was equipped with a Canon D30
digital camera for photography. The mean, standard deviation and coefficient of
variation (CV) were calculated for all characters measured. To assess the variation
in tail and tail tip shape, at least 50 females of each P. coffeae population were
examined.
3.2.5 Scanning electron microscope observations
After light microscope observation and measuring, the slides were broken
open with a scalpel and the glycerine-embedded nematodes transferred to a drop
of pure glycerine in a small embryo dish to which distilled water was added
gradually until the nematodes were almost in pure distilled water. To remove
particles adhering to the surface of the nematodes, the specimens were exposed to
ultrasonic treatment for about 10 to 15 minutes. The nematodes were then
dehydrated by passing them through a series of 25, 50, 75 and 100% ethanol at 2-
Chapter 3
30
hours intervals, and left overnight in 100% ethanol. For drying, the standard critical
point drying procedure with CO2 as drying liquid was used. Finally, the dried
specimens were mounted on stubs and coated with gold-palladium. The nematodes
were examined with a JEOL LSM-840 scanning electron microscope at 15 kV.
3.2.6 Canonical discriminant analysis
The morphometrical data were analysed statistically using GenStat
Release 9.1. Canonical discriminant analysis was used to make an objective
assessment of the relative similarity among the populations based on 14
morphometrical characters for the females and five morphometrical characters for
the males.
3.3
Results
3.3.1 Morphological observations
Female. Body rather slender in young females, thick in old ones. Body
width increases from the level of the median pharyngeal bulb towards the vulva
and decreases from the level of the vulva towards the tail tip. Cuticular annulation
fairly conspicuous.
Lip region usually bearing two distinct annules and not set off from the
rest of the body by a constriction but often showing a small discontinuity in body
outline at its base (see arrow in Fig. 3.1B). When viewed by light microscope, outer
margins of the heavily sclerotised labial framework extending into the body about
one body annule. Occasionally, three annules on one side of the lip region were
observed in some specimens of the Northwest, Red River Delta 2 and Central
Highlands populations (see arrow in Fig. 3.1I). When viewed by scanning electron
microscope, a minute or incomplete initiation of a 3rd lip annule visible in a
specimen of the Northeast 3 and Red River Delta 1 populations (see arrows in Figs
3.5H & J). En face view characterised by a complete fusion of the 1st lip annule and
the oral disc, resulting in a plain undivided en face view with no division between
the sub-median and lateral segments (Figs 3.5A, C, E, G, I, K, M, O, Q & S). There
are six small pores very close to the oral aperture. Amphid opening wide, slightly
triangular (Figs 3.5A, C, E, G, K, M, O, Q & S). The anterior surface of the oral disc
low-conical or flat with rounded edges (Figs 3.5B, D, F, H, L, N, P, R & T).
Stylet strong, basal knobs of stylet are round to oblong (Figs 3.1B, C & F).
Median pharyngeal bulb broadly oval to nearly round (Fig. 3.1A). Glandular lobe of
pharyngeal glands overlapping anterior end of the intestine ventrally and slightly
Chapter 3
31
laterally (Figs 3.1E & H). Hemizonid just anterior to the excretory pore, about two
body annules long (Fig. 3.1H). Excretory pore and canal located slightly posterior to
the pharyngo-intestinal junction except in the Red River Delta 1 population. In this
population, the excretory pore is located slightly anterior to the pharyngointestinal junction.
Lateral fields distinct with four lines forming three equal or unequal bands
(Figs 3.2E, H-I & M and Fig. 3.6). Outer lines of lateral fields crenated in the tail
region (Fig. 3.2N). Occasionally the internal band can be sculptured by striae,
oblique or parallel to the main bands (Figs 3.2E & M). When viewed by scanning
electron microscope, areolation of the outer bands was observed in all populations
(Fig. 3.6). Some specimens of the Northeast 1 and Red River Delta 2 populations
have punctuations scattered in the outer bands when viewed by light microscope
(Fig. 3.2O). When viewed by scanning electron microscope, the beginning of the
lines of the lateral fields at the 9th or 10th annule appear as two lines (Figs 3.5B, D,
F, H, L, N, P, R & T), widening to three lines at the following annules and to four
lines at the level of the median pharyngeal bulb (Fig. 3.2D) that extend to the
phasmid. Only the outer bands extend till the tail tip in all populations examined
except in the Red River Delta 2 population. At the level of the 10th annule anterior
to the tail tip, the outer bands fuse into one band (Fig. 3.7G). At the level of the
vulva, the structure of the lateral field is similar as at the mid-body level except in
the Northwest and Northeast 4 populations, that have three unequal bands at the
mid-body level and three equal bands at the level of the vulva (Figs 3.6A-B & I-J).
Vulva with well-developed lips, often protruding (Figs 3.2F & J-K and Figs
3.6B, D, F, H, J, L, O, Q & S). In young specimens, the ovary extends over 1/4th of
the body length; in old specimens, over more than one-half of the body length. The
ovary does not extend to the pharyngeal glands, except in the North Central Coast
1 population in which the ovary extends to the median pharyngeal bulb in some
specimens. Ovary consists of a single row of oocytes, except a double row near the
anterior end (Fig. 3.2A). A reflected ovary (Fig. 3.2B) was found in specimens from
the Northeast 1 and Red River Delta 1 populations. Spermatheca variable in size,
nearly round to broadly oval in all populations (Figs 3.2C, F-G & J-L), except in the
Northeast 1 population in which the spermatheca is small and round. Spermatheca
usually contains spermatozoa. Post-vulval uterine sac generally short. Sometimes
cellular tissue is present at the distal end of the post-vulval uterine sac. Phasmids
slightly posterior to the middle of the tail.
A higher frequency of tails tapering strongly with narrow tip was observed
in most populations (Figs 3.3 & 3.7), except in the Red River Delta 1 and North
Central Coast 2 populations. Tail shapes of the females examined from the Red
Chapter 3
32
River Delta 1 and North Central Coast 2 populations tapering slightly with broad tail
tip were recorded in 63 and 40.3%, respectively, (Figs 3.3Q-S & Z, A1-C1,
respectively). Tail tip variable in shape: smooth or crenated, truncated or
obliquely truncated, broadly rounded, hemispherical or sub-hemispherical, bluntly
pointed or pointed, convex, digitated, cleft or biloped (Table 3.2 and Fig. 3.4). A
smooth tail tip was more common than the other tail tip shapes. The Northwest
and Central Highlands populations were the most variable in tail tip shape with 11
out of a total of 12 tail tip shapes (Table 3.3). An obliquely truncated tail tip shape
was observed with the highest frequency (22.5-41%) in most of the populations,
except in the Red River Delta 1 and 2, and Central Highlands populations. In these
three populations, a hemispherical tail tip was observed with the highest frequency
(24-31%).
Male. Males as abundant as females in all populations examined.
Morphologically similar to the females, but body length is smaller and slender than
in the females. Stylet knobs smaller than in females (Fig. 3.1D). Reproductive
system with a single anteriorly outstretched testis, extending over about one-half
of the body length, not extending to the pharyngeal glands. Testis shorter than vas
deferens, spermatocytes in single or multiple rows. Spicules curved ventrally,
gubernaculum plain and non-protrusible (Fig. 3.1J). Male tail pointed, bursal edges
faintly crenated (Figs 3.1G & J). Bursa arising from a little anterior to the level of
the head of the spicules and surrounding the tail tip (Fig. 3.8). Phasmids slightly
posterior to the middle of the tail.
Chapter 3
33
A
B
C
D
G
E
F
H
J
I
Figure 3.1: Light microscope photographs of Pratylenchus coffeae populations from Vietnam.
A-C, F & I: anterior region females; D: anterior region male; E: pharyngo-intestinal junction
female; H: hemizonid and excretory pore female; G & J: tail region males. Scale bars: 10 µm.
Northwest (I), Northeast 2 (A, C), Northeast 3 (H), Northeast 4 (J), Red River Delta 1 (E), Red
River Delta 2 (B), North Central Coast 1 (F) and North Central Coast 2 (G) populations. Arrows
show the small discontinuity in body outline (B) and three annules on one side of the lip
region (I).
Chapter 3
A
34
B
C
D
E
F
H
I
G
J
K
L
N
M
O
Figure 3.2: Light microscope photographs of Pratylenchus coffeae populations from Vietnam.
A-B: anterior genital branch females; C: post-vulval uterine sac female; D: lateral field at
level of the median pharyngeal bulb female; E, H-I & M: lateral field at mid-body females; FG, J-L: spermatheca females. Scale bars: A: 50 µm; B-O: 10 µm. Northwest (C, H), Northeast
1 (B, N), Northeast 2 (A, G, L), Northeast 3 (M), Northeast 4 (J, K), Red River Delta 1 (E), Red
River Delta 2 (D, F, O) and Central Highlands (I) populations.
Chapter 3
35
B
C
27.6%
9.2%
A
60.5%
D
F
E
2.6%
52.3%
Northwest
J
I
58.6%
L
34.5%
63.3%
T
U
16.6%
59%
33.4%
Red River Delta 1
Z
19.3%
17.5%
N
33.8%
43%
Northeast 3
S
R
20%
M
66.2%
Northeast 2
Q
23.8%
6.3%
Northeast 1
K
6.9%
H
G
O
P
45%
11.6%
Northeast 4
V
W
7%
X
67.7%
Red River Delta 2
27.4%
Y
4.8%
North Central Coast 1
A1
B1
C1
D1
E1
F1
35.5%
4.8%
40.3%
65.5%
9.5%
25%
Central Highlands
Figure 3.3: Light microscope photographs of the tail and tail tip shapes of Pratylenchus
coffeae females from Vietnam. %: frequency of occurrence of that specific tail shape in the
population (n ≥ 50). Scale bars: 10 µm.
Chapter 3
36
Table 3.3: Frequency of occurrence (in %; n ≥ 50) of the tail tip shapes of Pratylenchus coffeae females from Vietnam.
Population
a
b
c
d
e
f
g
h
i
j
k
l
Northwest
8
31
2
16
14
16
6
2
2
1
2
0
Northeast 1
1
29
2
20
8
24
14
0
1
1
0
0
Northeast 2
4
30
0
8
5
4
3
0
20
26
0
0
Northeast 3
0
41
10
14
9
0
0
12
14
0
0
0
Northeast 4
10
26
2
12
4
12
4
0
4
26
0
0
Red River Delta 1
7
20
0
25
11
14
7
2
2
10
2
0
Red River Delta 2
6
18
5
31
13
8
8
0
10
1
0
0
4.8
22.5
3.2
9.7
12.9
17.7
6.4
4.8
12.9
4.8
0
0
20
13.3
25.3
9.3
0
8
12
8
4
0
0
0
Central Highlands
10
10
4
24
0
14
4
4
4
12
2
12
a: truncated; b: obliquely truncated; c: broadly rounded; d: hemispherical; e: sub-hemispherical; f: bluntly pointed; g: pointed; h: convex; i: igitated;
j: cleft; k: crenated; l: bilobed.
A
B
C
D
E
F
G
H
I
J
K
L
Figure 3.4: Light microscope photographs of the tail tip shapes of Pratylenchus coffeae females from Vietnam: truncated (a); obliquely truncated (b);
broadly rounded (c); hemispherical (d); sub-hemispherical (e); bluntly pointed (f); pointed (g); convex (h); digitated (i); cleft (j); crenated (k); bilobed (l).
Scale bars: 10 µm.
Chapter 3
A
C
E
37
B
D
F
Figure 3.5: En face and lateral head scanning electron microscope photographs of
Pratylenchus coffeae specimens from Vietnam. Northwest (A-B), Northeast 1 (C-D) and
Northeast 2 (E-F) populations.
Chapter 3
38
H
G
I
K
J
L
Figure 3.5: Continued. Northeast 3 (G-H), Northeast 4 (I) and Red River Delta 1 (J-L)
populations. Arrows show the incomplete initiation of a 3rd lip annule.
Chapter 3
39
N
M
O
Q
P
R
Figure 3.5: Continued. Red River Delta 2 (M-N), North Central Coast 1 (O-P) and North
Central Coast 2 (Q-R) populations.
Chapter 3
40
S
T
Figure 3.5: Continued. Central Highlands (S-T) population.
A
B
C
D
Figure 3.6: Lateral field at mid-body and at vulva level scanning electron microscope
photographs of Pratylenchus coffeae specimens from Vietnam. Northwest (A-B) and
Northeast 1 (C-D) populations.
Chapter 3
41
E
F
G
H
G
I
J
Figure 3.6: Continued. Northeast 2 (E-F), Northeast 3 (G-H) and Northeast 4 (I-J)
populations.
Chapter 3
42
K
L
M
N
M
O
Figure 3.6: Continued. Red River Delta 1 (K-L), Red River Delta 2 (M) and North Central
Coast 1 (N-O) populations.
Chapter 3
43
P
Q
S
R
Figure 3.6: Continued. North Central Coast 2 (P-Q) and Central Highlands (R-S)
populations.
C
A
B
Figure 3.7: Female tail scanning electron microscope photographs of Pratylenchus coffeae
specimens from Vietnam. Northwest (A), Northeast 1 (B) and Northeast 2 (C) populations.
C
Chapter 3
44
D
E
F
H
I
G
J
K
Figure 3.7: Continued. Northeast 3 (D), Northeast 4 (E), Red River Delta 1 (F), Red River
Delta 2 (G), North Central Coast 1 (H), North Central Coast 2 (I) and Central Highlands (KL) populations.
Chapter 3
45
A
B
F
G
E
D
C
H
I
E
J
Figure 3.8: Male tail scanning electron microscope photographs of Pratylenchus coffeae specimens from Vietnam. Northwest (A), Northeast 1 (B),
Northeast 2 (C), Northeast 3 (D), Northeast 4 (E), Red River Delta 1 (F), Red River Delta 2 (G), North Central Coast 1 (H), North Central Coast 2 (I) and
Central Highlands (K-L) populations.
J
Chapter 3
46
3.3.2 Morphometrical observations
Female. The morphometrics of the females of the P. coffeae populations
collected in Vietnam are presented in Table 3.4. The body length of the
Vietnamese females ranged from 465 to 765 µm (mean: 601 µm). The females of
the Red River Delta 1 population had the shortest average body length (553 µm)
while the females of the North Central Coast 2 population had the longest average
body length (650 µm). The a ratio of the Vietnamese females ranged from 19 to
35.6 (mean: 26.8). The females of the Northeast 4 population had the lowest
average a ratio (24.6) while the females of the Central Highlands population had
the highest a ratio (28.5).The average body width of the former population was
23.8 µm vs 19.8 µm of the latter population. The b ratio of the Vietnamese females
ranged from 4.1 to 9.7 (mean: 7.3). The females of the Red River Delta 1
population had the lowest average b ratio (6.6) while the females of the North
Central Coast 1 population had the highest average b ratio (8.1). The c ratio of the
Vietnamese females ranged from 13.1 to 26.3 (mean: 20.1). The females of the
Central Highlands population had the lowest average c ratio (18.3) while the
females of the Northeast 3 population had the highest average c ratio (21.3). The
average tail length of the former population was 31.5 µm vs 29.1 µm of the latter
population. The pharynx length of the Vietnamese females ranged from 65.5 to 130
µm (mean: 83.1 µm). The females of the Northeast 1 and North Central Coast 1
populations had the shortest average pharynx length (79.9 µm) while the females
of the Northwest population had the longest average pharynx length (85.8 µm). The
length of the pharyngeal overlap of the Vietnamese females ranged from 29.5 to
79.5 µm (mean: 51.1 µm). The females of the Central Highlands population had the
shortest average pharyngeal overlap length (41.4 µm) while the females of the
North Central Coast 2 population had the longest average pharyngeal overlap
length (62.3 µm). The V-value of the Vietnamese populations ranged from 71.5 to
92.3% (mean: 80.1%). The females of the Northeast 2 population had the lowest
average V-value (79.2%) while the females of the North Central Coast 2 population
had the highest V-value (81.9%). The stylet length of the Vietnamese females
ranged from 13.5 to 17.7 µm (mean: 15.6 µm). The shortest average stylet length
was observed in the females of the Central Highlands population (15.3 µm) while
the longest average stylet length was observed in the females of the Northwest
population (16.1 µm). The length of the post-vulval uterine sac of the Vietnamese
females ranged from 19.7 to 54.5 µm (mean: 30.1 µm). The post-vulval uterine sac
was on average the shortest in the females of the North Central Coast 2 population
(25.7 µm) and the longest in the females of the Northeast 3 (33.3 µm) population.
The distance of the excretory pore from the anterior end of the Vietnamese
Chapter 3
47
females ranged from 63.3 to 98.5 µm (mean: 84.1 µm). This distance was on
average shortest in the Red River Delta 1 population (78.9 µm) and longest in the
North Central Coast 2 population (89.9 µm).
Male. The morphometrics of the males of the P. coffeae populations
collected in Vietnam are presented in Table 3.5. The body length of the
Vietnamese males ranged from 442 to 673 µm (mean: 532.6 µm). The males of the
Red River Delta 1 population had the shortest average body length (497 µm) while
the males of the North Central Coast 1 population had the longest average body
length (551 µm). The a ratio of the Vietnamese males ranged from 20.7 to 38
(mean: 28.4). The males of the Red River Delta 2 population had the lowest
average a ratio (24.6) while the males of the North Central Coast 2 population had
the highest a ratio (32.4). The average body width of the former population was
22.2 µm vs 16.8 µm of the latter population. The b ratio of the Vietnamese males
ranged from 5.8 to 8 (mean: 6.8). The males of the Red River Delta 1 population
had the lowest average b ratio (6.4) while the males of the North Central Coast 1
population had the highest average b ratio (7.2). The c ratio of the Vietnamese
males ranged from 14.6 to 23.4 (mean: 18.4). The males of the Northeast 2
population had the lowest average c ratio (17.1) while the males of the Northeast 3
population had the highest average c ratio (19.4). The average tail length of the
former population was 29.6 µm vs 28.3 µm of the latter population. The pharynx
length of the Vietnamese males ranged from 66.2 to 94.5 µm (mean: 78.3 µm). The
males of the Northeast 2 population had the shortest average pharynx length (76.4
µm) while the longest average pharynx length of males was observed in the Red
River Delta 2 population (83.9 µm). The length of the pharyngeal overlap of the
Vietnamse males ranged from 30 to 67.3 µm (mean: 45 µm). The males of the
Northeast 1 population had the shortest average pharyngeal overlap length (37.7
µm) while the males of the North Central Coast 2 population had the longest
average pharyngeal overlap length (51.2 µm). The T-value of the Vietnamese
populations ranged from 18.9 to 59.3% (mean: 44.1%). The males of the Red River
Delta 2 population had the lowest average T-value (38%) while the males of the
Northwest population had the highest T-value (50.7%). The stylet length of the
Vietnamese males ranged from 13.1 to 16.7 µm (mean: 14.5 µm). The shortest
average stylet length was observed in the males of the North Central Coast 1
population (14 µm) while the longest average stylet length was observed in the
males of the
Northwest, Northeast 2, Northeast 4, and North Central Coast 2
populations (14.8 µm). The distance of the excretory pore from the anterior end of
the males ranged from 63.6 to 95.1 µm (mean: 77.9 µm). This distance was on
average shortest in the Red River Delta 1 population (73.2 µm) and longest in the
Chapter 3
48
North Central Coast 2 population (81.7 µm). The spicule length of the Vietnamese
males ranged from 14 to 20.7 µm (mean: 17.3 µm). The shortest average spicule
length was observed in the males of the Northeast 2 and North Central Coast 1
populations (16.7 µm) while the longest average spicule length was observed in the
males of the Northeast 1, Northeast 3 and Northeast 4 populations (17.6 µm).
In the females of the Vietnamese P. coffeae populations, the coefficient
of variation was the lowest for the V-value (3.1%) followed by stylet length (4.6%),
the pharyngeal bulb value (7.8%), distance of the excretory pore from the anterior
end (7.9%), the lip width (8.3%), body length/distance from the anterior end to the
excretory pore (8.4%), body length (8.6%) and pharynx length (9.3%). The
coefficient of variation was the highest for the length of the ovary (22.1%) followed
by the distance from the anterior end to the dorsal pharyngeal gland orifice
(17.8%), pharyngeal overlap length (17.1%), length of the post-vulval uterine sac
(17%) and lip height (15.1%).
In the males of the Vietnamese P. coffeae populations, the coefficient of
variation was the lowest for the stylet length (4.8%) followed by the pharynx length
(5.9%), body length and body length/distance from the anterior end to the
excretory pore (both 6.1%), b ratio (6.3%), distance of the excretory pore from the
anterior end (6.6%) and spicule length (6.8%). The coefficient of variation was the
highest for the T-value and the length of the testis (both 16.4%) followed by the
pharyngeal overlap length (15.9%), distance from the anterior end to the dorsal
pharyngeal gland orifice (14.6) and lip height (13.9%).
Chapter 3
49
Table 3.4: Morphometrics of the females of the Pratylenchus coffeae populations collected in Vietnam (n = 15).
Population
NW
L*
a
b
b’
c
c’
stylet*
DGO*
605.6 ± 43.9
26.4 ± 1.9
7.1 ± 0.3
4.2 ± 0.3
19.1 ± 1.3
2.4 ± 0.4
16.1 ± 0.7
2.9 ± 0.4
(519-664)
(21.9-29.0)
(6.6-7.8)
(3.8-4.7)
(17.1-22.2)
(1.9-3.2)
(14.6-17.7)
(2.1-3.9)
NE1
630.3 ± 36.4
27.6 ± 1.8
7.9 ± 0.5
5.2 ± 0.4
20.8 ± 2.0
2.1 ± 0.2
15.4 ± 0.8
3.1 ± 0.4
(580-706)
(22.8-29.5)
(7.2-8.8)
(4.4-5.8)
(17.0-25.2)
(1.7-2.4)
(13.5-17.1)
(2.6-3.6)
NE2
582.5 ± 55.0
25.6 ± 2.3
7.0 ± 1.0
4.5 ± 0.4
19.1 ± 1.8
2.1 ± 0.4
15.6 ± 0.7
2.6 ± 0.6
(467-760)
(22.7-31.6)
(4.1-7.9)
(3.9-5.2)
(16.4-22.0)
(1.4-2.6)
(14.5-16.7)
(1.6-4.2)
NE3
607.6 ± 40.9
28.1 ± 1.8
7.4 ± 0.7
4.6 ± 0.4
21.4 ± 2.1
2.0 ± 0.2
15.5 ± 0.7
3.0 ± 0.6
(501-676)
(23.6-30.5)
(6.3-8.8)
(4.1-5.3)
(17.6-25.9)
(1.8-2.7)
(14.5-16.5)
(2.1-4.2)
NE4
573.4 ± 29.0
24.6 ± 3.3
7.1 ± 0.3
4.2 ± 0.3
19.9 ± 1.9
2.2 ± 0.2
15.4 ± 0.6
3.1 ± 0.4
(534-633)
(19.0-30.3)
(6.5-7.7)
(3.8-4.9)
(16.3-22.4)
(1.9-2.6)
(14.1-16.7)
(2.6-3.6)
RRD1
552.9 ± 39.4
26.1 ± 2.9
6.6 ± 0.5
4.0 ± 0.3
20.2 ± 2.2
1.9 ± 0.2
15.4 ± 0.6
3.1 ± 0.4
(465-610)
(22.0-30.9)
(5.7-7.4)
(3.6-4.5)
(17.2-26.3)
(1.6-2.3)
(14.0-16.4)
(2.6-3.6)
RRD2
588.8 ± 42.9
26.4 ± 3.8
6.9 ± 0.8
4.6 ± 0.3
20.0 ± 3.1
2.2 ± 0.3
15.7 ± 0.7
2.9 ± 0.4
(534-664)
(21.5-34.6)
(5.8-8.5)
(4.2-5.1)
(13.4-24.6)
(1.7-3.0)
(14.5-17.0)
(2.3-3.6)
NCC1
646.9 ± 51.0
27.3 ± 2.0
8.1 ± 0.7
4.9 ± 0.5
20.6 ± 2.6
2.3 ± 0.2
15.4 ± 0.6
2.7 ± 0.5
(567-765)
(24.4-31.2)
(7.0-9.7)
(3.8-5.8)
(15.8-24.9)
(1.8-2.7)
(14.5-16.4)
(1.9-3.1)
NCC2
649.6 ± 34.6
26.9 ± 3.1
7.6 ± 0.3
4.4 ± 0.2
21.3 ± 1.9
2.2 ± 0.3
15.9 ± 0.6
3.0 ± 0.7
(571-706)
(21.8-32.7)
(6.9-8.0)
(4.0-4.8)
(18.4-25.4)
(1.6-2.9)
(14.6-16.7)
(2.1-5.2)
CH
568.7 ± 42.9
28.5 ± 3.3
6.8 ± 0.6
4.6 ± 0.5
18.3 ± 2.5
2.3 ± 0.3
15.3 ± 0.8
3.2 ± 0.5
(508-647)
(23.4-35.6)
(5.5-7.8)
(3.8-5.2)
(13.1-22.4)
(1.7-2.8)
(13.8-16.5)
(2.6-4.2)
CV
8.6
10.7
10.3
10.7
11.7
14.3
4.6
17.8
*: measurements in µm. Mean ± standard deviation; (range).
For the list with the abbreviations of the nematode population codes see Table 2.6. For the other abbreviations see Table 3.1.
CV: coefficient of variation (%).
body width*
excr. pore*
23.0 ± 2.1
(20.2-27.8)
22.9 ± 2.1
(20.2-27.0)
22.8 ± 2.0
(18.3-26.5)
21.7 ± 2.0
(17.0-25.2)
23.8 ± 3.9
(18.7-30.3)
21.3 ± 2.2
(18.3-25.2)
22.3 ± 3.2
(18.3-29.3)
23.8 ± 2.4
(19.8-27.7)
24.4 ± 2.6
(19.7-27.3)
19.8 ± 1.8
(17.6-22.6)
12.1
89.3 ± 6.4
(76.3-98.5)
80.5 ± 5.8
(71.2-89.1)
83.7 ± 7.1
(71.2-97.0)
84.6 ± 4.2
(76.9-91.7)
84.3 ± 5.0
(75.8-93.4)
78.9 ± 4.9
(69.0-86.0)
84.4 ± 5.3
(77.4-92.6)
84.4 ± 9.4
(63.3-92.6)
89.9 ± 5.2
(81.8-98.0)
80.7 ± 3.5
(72.5-87.6)
7.9
Chapter 3
50
Table 3.4: continued.
Population
NW
Pharynx
length*
Pharyngeal
overlap*
L/excr.
pore
V
ovary*
post-vulval
uterine sac*
tail*
vulva-anus*
lip width*
85.8 ± 5.9 58.1 ± 8.9 6.7 ± 0.2
80.0 ± 1.6 238.1 ± 46.2
31.2 ± 3.5
31.9 ± 3.2 90.5 ± 12.5
7.8 ± 0.5
73.7-96.0 41.1-69.5
(6.4-7.1) (77.3-83.0) (171.7-314.9) (24.2-35.4) (26.3-36.4) (60.6-121.2) (6.8-8.3)
NE1
79.9 ± 4.9 42.1 ± 7.4 7.8 ± 0.6
80.6 ± 1.7 218.8 ± 57.4
30.8 ± 7.9
30.7 ± 3.2 92.3 ± 10.4
7.6 ± 0.6
71.8-87.5 32.9-58.1
(6.8-8.8) (78.5-84.8) (149.4-382.9) (22.2-54.5) (23.0-36.5) (76.2-107.1) (6.5-9.1)
79.2 ± 3.8 197.1 ± 50.6
29.9 ± 4.0
30.9 ± 3.9 89.7 ± 10.0
8.2 ± 0.4
NE2
83.3 ± 16.3 49.9 ± 10.4 7.0 ± 0.5
68.7-130
35.2-66.2
(6.2-8.1) (73.0-85.7) (102.5-279.1) (24.2-37.9) (21.2-35.9) (74.5-102.7) (7.3-8.9)
NE3
82.7 ± 5.5 48.5 ± 9.2 7.2 ± 0.4
80.1 ± 1.1 211.1 ± 42.9
33.3 ± 5.3
29.1 ± 3.5
87.0 ± 8.1
8.2 ± 0.6
71.8-94.5 29.7-61.6
(6.5-7.9) (77.9-82.0) (156.8-284.1) (27.3-41.4) (24.7-35.4) (73.7-106.6) (7.3-9.4)
NE4
81.1 ± 3.8 55.6 ± 9.3 6.8 ± 0.3
81.2 ± 3.3 265.4 ± 47.2
29.2 ± 5.2
27.5 ± 2.9 81.2 ± 17.2
8.2 ± 1.0
75.8-88.4 36.8-71.4
(6.1-7.4) (78.4-92.3) (165.5-333.5) (24.2-39.4) (22.9-33.8) (24.2-99.0) (6.8-10.4)
RRD1
83.9 ± 7.1 52.7 ± 5.6 7.0 ± 0.6
81.2 ± 3.3 265.4 ± 47.2
31.2 ± 3.8
27.5 ± 2.9 81.2 ± 17.2
8.2 ± 1.0
73.7-103.1 39.5-59.8
(5.9-7.9) (78.4-92.3) (165.5-333.5) (27.7-41.9) (22.9-33.8) (24.2-99.0) (6.8-10.4)
80.6 ± 1.7 186.7 ± 45.6
RRD2
85.6 ± 10.2 43.8 ± 7.4 6.9 ± 0.4
28.3 ± 4.1
30.0 ± 4.6
88.1 ± 9.3
7.9 ± 0.5
72-114.7
34.7-66.1
(6.1-7.4) (71.5-82.6) (49.4-255.6)
(22.2-36.4) (23.3-40.0) (75.0-105.2) (7.3-8.9)
NCC1
79.9 ± 7.7 53.9± 13.8 7.8 ± 0.9
30.5 ± 4.6
31.8 ± 4.2 98.3 ± 15.1
7.8 ± 0.9
80.4 ± 1.9 227.8 ± 35.6
65.5-91.9 39.0-79.5 (6.8-10.1) (75.7-83.1) (179.1-290.2) (21.7-38.9) (23.3-40.9) (74.9-125.7) (6.2-9.4)
25.7 ± 4.1
30.7 ± 3.1
88.8 ± 8.2
8.4 ± 0.7
NCC2
85.3 ± 4.9 62.3 ± 5.4 7.2 ± 0.3
81.9 ± 1.3 212.0 ± 33.5
77.3-93.9 52.4-72.5
(6.6-7.6) (78.9-84.0) (151.9-281.6) (19.7-34.8) (23.2-35.4) (77.8-110.6) (7.3-9.9)
CH
83.7 ± 6.3 41.4 ± 7.3 7.1 ± 0.6
81.5 ± 3.4 199.3 ± 41.1
30.9 ± 4.5
31.5 ± 3.4
88.9 ± 9.0
8.0 ± 0.5
73.7-96.1 29.5-58.7
(5.8-8.1) (78.0-89.6) (129.7-249.5) (24.2-37.9) (23.7-38.7) (75.6-105.8) (7.3-8.9)
CV%
9.3
17.1
8.4
3.1
22.1
17.0
12.0
13.1
8.3
lip height* pharyngeal
bulb
2.4 ± 0.3
(2.1-3.1)
2.3 ± 0.3
(1.9-2.9)
2.3 ± 2.1
(2.1-2.6)
2.3 ± 0.3
(2.0-3.1)
2.3 ± 0.6
(1.6-2.3)
2.3 ± 0.6
(1.6-2.3)
2.2 ± 0.3
(1.9-2.6)
2.1 ± 0.2
(1.7-2.6)
2.5 ± 0.4
(1.9-3.1)
2.3 ± 0.3
(1.9-2.9)
15.1
0.58 ±0.03
(0.4-0.7)
0.56 ±0.05
(0.5-0.6)
0.51 ±0.05
(0.4-0.6)
0.55 ± 0.03
(0.5-0.6)
0.53 ± 0.03
(0.5-0.6)
0.53 ± 0.03
(0.5-0.6)
0.53 ± 0.03
(0.5-0.6)
0.58 ± 0.03
(0.5-0.6)
0.59 ± 0.05
(0.5-0.7)
0.55 ± 0.03
(0.5-0.6)
7.8
Chapter 3
51
Table 3.5: Morphometrics of the males of the Pratylenchus coffeae populations collected in Vietnam (n = 15).
Population
NW
L*
a
b
b’
c
c’
stylet*
DGO*
540.2 ± 22.1
29.0 ± 2.1
7.0 ± 0.4
4.4 ± 0.3
17.5 ± 1.8
2.7 ± 0.4
14.8 ± 0.5
2.4 ± 0.2
(498.6-571.3)
(24.6-32.3)
(6.3-7.7)
(3.8-5.0)
(15.6-23.4)
(2.0-3.3)
(14.1-15.7)
(2.0-2.5)
NE1
530.1 ± 17.0
27.6 ± 2.0
6.8 ± 0.5
4.6 ± 0.3
19.1 ± 0.9
2.5 ± 0.2
14.1 ± 0.4
2.7 ± 0.2
(508.0-567.0)
(23.4-30.0)
(6.3-7.8)
(4.1-5.1)
(17.1-20.0)
(2.2-2.8)
(13.5-15.1)
(2.5-2.8)
NE2
503.2 ± 35.8
26.8 ± 2.4
6.6 ± 0.4
4.2 ± 0.4
17.1 ± 1.6
2.6 ± 0.3
14.8 ± 0.6
2.6 ± 0.3
(441.5-561.0)
(22.6-30.3)
(5.8-7.4)
(3.6-4.8)
(14.6-20.4)
(1.9-3.0)
(13.2-15.7)
(2.2-3.2)
NE3
541.7 ± 16.4
29.9 ± 3.5
6.8 ± 0.3
4.4 ± 0.3
19.4 ± 2.3
2.6 ± 0.4
14.2 ± 0.6
3.0 ± 0.2
(515.0-574.0)
(22.9-36.0)
(6.1-7.4)
(3.9-4.9)
(15.5-22.7)
(2.0-3.4)
(13.2-14.8)
(2.8-3.2)
NE4
543.3 ± 21.9
29.2 ± 3.3
7.0 ± 0.3
4.4 ± 0.4
18.8 ± 1.2
2.7 ± 0.2
14.8 ± 0.5
2.3 ± 0.3
(503.8-576.5)
(25.3-34.9)
(6.5-7.6)
(3.8-5.1)
(17.3-20.6)
(2.3-2.9)
(13.6-15.2)
(1.5-2.5)
RRD1
497.1 ± 23.0
27.1 ± 2.1
6.4 ± 0.2
4.0 ± 0.3
18.5 ± 1.5
2.4 ± 0.2
14.4 ± 0.7
2.7 ± 0.6
(468.0-554.0)
(23.6-30.2)
(6.0-6.9)
(3.5-4.5)
(16.0-20.7)
(2.0-2.7)
(13.2-15.7)
(2.0-3.8)
RRD2
540.8 ± 32.3
24.6 ± 2.5
6.5 ± 0.4
4.2 ± 0.3
18.4 ± 1.0
2.5 ± 0.2
14.2 ± 0.5
2.7 ± 0.2
(468.0-580.0)
(20.7-28.9)
(5.9-7.1)
(3.6-4.7)
(15.8-20.2)
(2.0-2.9)
(13.2-15.1)
(2.5-3.2)
27.5 ± 2.5
7.2 ± 0.6
4.5 ± 0.3
18.9 ± 1.9
2.5 ± 0.2
14.0 ± 0.5
2.9 ± 06
NCC1
551.0 ± 34.4
(502.0-633.0)
(24.1-33.2)
(6.3-8.0)
(4.1-5.2)
(15.7-21.8)
(2.1-2.9)
(13.2-14.8)
(1.9-3.7)
NCC2
542.3 ± 23.2
32.4 ± 2.4
7.1 ± 0.3
4.3 ± 0.3
17.6 ± 1.2
2.7 ± 0.3
14.8 ± 1.2
2.6 ± 0.2
(472.7-571.3)
(28.5-38.0)
(6.4-7.5)
(3.9-5.0)
(15.5-19.6)
(2.2-3.0)
(13.1-16.7)
(2.5-3.0)
CH
534.9 ± 45.4
29.5 ± 3.0
6.7 ± 0.2
4.4 ± 0.4
18.5 ± 1.6
2.7 ± 0.2
14.5 ± 0.6
2.4 ± 0.4
(482.0-673.0)
(26.1-37.5)
(6.3-7.1)
(3.9-5.1)
(16.3-21.0)
(2.2-3.1)
(13.5-15.8)
(1.9-3.2)
CV
6.1
11.5
6.3
8.1
9.1
11.0
4.8
14.6
body width*
excr. pore*
18.7 ± 1.2
(16.1-20.7)
19.3 ± 1.5
(17.6-22.0)
19.0 ± 1.5
(15.5-20.7)
18.4 ± 2.2
(15.4-23.7)
18.8 ± 1.6
(16.1-20.7)
18.3 ± 1.7
(15.7-20.8)
22.2 ± 1.9
(17.6-25.2)
20.2 ± 1.6
(18.3-23.3)
16.8 ± 1.0
(15.0-18.1)
18.3 ± 1.8
(13.9-20.8)
10.9
80.8 ± 4.5
(72.2-88.9)
75.8 ± 4.2
(67.4-81.3)
76.7 ± 3.0
(73.7-83.8)
75.8 ± 3.1
(70.6-83.2)
81.5 ± 4.1
(74.2-88.4)
73.2 ± 4.4
(66.2-81.8)
78.2 ± 5.1
(64.3-84.1)
78.6 ± 5.4
(65.5-88.0)
81.7 ± 2.3
(78.8-86.4)
76.3 ± 7.1
(63.6-95.1)
6.6
Chapter 3
52
Table 3.5: continued.
Population
NW
L/excr.
pore
Pharynx
length
Pharyngeal
overlap
T
testis*
tail*
lip width*
6.7 ± 0 .3
77.7 ± 4.3
44.8 ± 6.9
50.7 ± 4.4
271.7 ± 25.3
31.2 ± 3.3
6.2 ± 0.5
(6.0-7.2)
66.2-84.3
34.6-57.3
(43.3-56.5)
(225-304)
(21.7-36.2)
(5.6-7.1)
NE1
7.0 ± 0.5
77.8 ± 5.0
37.7 ± 3.7
40 ± 10.4
210.3 ± 50.9
27.7 ± 1.1
6.7 ± 0.3
(6.3-8.4)
68.0-85.7
31.8-43.9
(18.9-57.5)
(107-296)
(25.8-30.2)
(6.3-7.0)
NE2
6.6 ± 0.4
76.4 ± 4.9
44.8 ± 6.3
43.6 ± 7.5
218.8 ± 35.1
29.6 ± 3.1
6.6 ± 0.4
(5.9-7.2)
70.7-89.5
35.2-55.3
(32.6-58.1)
(153-278)
(23.3-34.6)
(6.1-7.2)
NE3
7.1 ± 0.3
79.93 ± 4.8
42.2 ± 8.1
43.8 ± 5.2
237.6 ± 30.0
28.3 ± 3.5
6.8 ± 0.6
(6.6-7.5)
74.3-94.5
30.0-56.7
(34.8-57.3)
(193-314)
(23.3-36.2)
(6.1-7.5)
NE4
6.7 ± 0.3
77.9 ± 4.2
47.1 ± 8.2
46.3 ± 4.2
251.4 ± 23.9
29.1 ± 1.4
6.3 ± 0.4
(6.2-7.2)
71.2-86.4
31.6-67.3
(39.3-54.4)
(222-294)
(26.8-31.8)
(5.6-7.1)
RRD1
6.8 ± 0.4
77.3 ± 4.0
48.4 ± 8.9
44.9 ± 9.0
223.1± 45.2
26.8 ± 2.4
7.1 ± 0.7
(6.2-7.9)
73.0-86.9
30.4-61.4
(24.6-59.3)
(120-280)
(23.9-32.8)
(6.1-8.2)
RRD2
6.9 ± 0.3
83.9 ± 3.1
46.3 ± 5.7
38 ± 6.6
204.8 ± 38.0
29.4 ± 1.7
7.1±0.7
(6.4-7.3)
78.1-90.4
36.4-55.4
(21.4-50.3)
(113-275)
(27.0-32.1)
(6.1-7.7)
77.5 ± 4.9
44.4 ± 6.9
43.9 ± 6.2
241.9 ± 37.7
29.3 ± 2.3
7.4 ± 0.4
NCC1
7.0 ± 0.7
(6.0-8.3)
69.9-86.9
32.1-53.5
(29.0-53.3)
(159-299)
(25.0-35.0)
(6.6-7.9)
NCC2
6.6 ± 0.3
76.5 ± 2.6
51.2 ± 7.6
44.3 ± 4.9
239.8 ± 23.1
30.9 ± 2.7
6.4 ± 0.5
(5.5-7.1)
73.2-81.8
38.6-63.8
(36.0-53.0)
(196.4-289)
(27.3-35.9)
(5.6-7.6)
CH
7.0 ± 0.3
78.9 ± 4.4
41.5 ± 7.1
45.1 ± 4.7
241.5 ± 28.6
29.1 ± 2.7
7.2 ± 0.8
(6.4-7.6)
70.6-87.6
30.4-50.6
(36.4-52.6)
(192-288)
(23.9-33.4)
(6.1-8.2)
CV
6.1
5.9
15.9
16.4
16.4
9.5
9.1
lip height*
spicule
length*
1.6 ± 0.2
(1.5-2.0)
1.7 ± 0.2
(1.5-1.9)
1.7 ± 0.2
(1.5-2.0)
1.8 ± 0.2
(1.5-1.9)
1.5 ± 0.2
(1.0-1.5)
1.6 ± 0.2
(1.4-2.0)
1.8 ± 0.2
(1.5-2.0)
2.0 ± 0.2
(1.5-2.4)
1.5 ± 0.2
(1.3-2.0)
1.7 ± 0.2
(1.3-2.0)
13.9
17.3 ± 1.5
(14 - 18.6)
17.6 ± 1.0
(16.6 - 19.7)
16.7 ± 1.1
(15.5 - 19.2)
17.6 ± 1.2
(15.5 - 19.7)
17.6 ± 1.5
(15 - 20.7)
17.5 ± 0.8
(16.1 - 18.6)
17 ± 1.1
(15 - 19.2)
16.7 ± 1.0
(15.5 - 18.1)
17 ± 1.3
(14.5 - 19.2)
17.2 ± 0.7
(16.1 - 18.6)
6.8
Chapter 3
53
The results of the canonical discriminant analysis are presented in Table
3.6. Using a combination of 14 morphometrical characters for the females, it was
not possible to separate the 10 Pratylenchus populations from Vietnam by
canonical discriminant analysis (Fig. 3.9A). However, a combination of five
morphometrical characters for the males (Table 3.6) enabled the separation of the
10 P. coffeae populations from Vietnam in three groups by canonical discriminant
analysis (Fig. 3.9B). One group (on the right on Fig. 3.9B) includes the Northwest,
Northeast 4 and North Central Coast 1 populations (populations originally isolated
from banana) and the North Central Coast 2 population (a population originally
isolated from coffee). Another group (on the middle on Fig. 3.9B) clusters the other
P. coffeae populations with the exception of the Red River Delta 1 population (a
population originally isolated from banana) which stands out from the two other
groups. The body length, the b ratio and the distance from the anterior end to the
excretory pore were the best morphometrical male characters for the separation of
the populations.
Table 3.6: Standardised coefficients for canonical variates of females and males of the
Pratylenchus coffeae populations from Vietnam.
Females
Males
Root 1
Root 2
Root 1
Root 2
% of variation
44.7
22.1
81.57
9.62
Selected characters
Vector loadings
L
0.9690
0.2831
-1.3796
-1.2341
A
0.0784
0.8185
B
1.7399
0.5770
c’
0.2800
-0.4652
Stylet
0.1019
-0.1957
0.2669
0.5761
V
0.3434
0.3977
Spicule length
0.0394
0.2695
Post-vulval uterine sac
-0.0340
0.5899
Tail
-0.2947
0.6838
DGO
-0.1586
-0.1068
Lip width
0.2933
0.0928
Lip height
-0.0940
-0.3505
Pharyngeal bulb
0.8123
0.1580
Excr. pore
0.5492
-0.7876
2.0118
-0.2585
Pharynx length
0.9582
-0.2664
Pharyngeal overlap
0.0413
-0.1260
For the list with the abbreviations of the selected characters see Table 3.1.
Chapter 3
54
A
B
Figure 3.9: Canonical discriminant analysis of 10 Pratylenchus coffeae populations from
Vietnam for females (A) and males (B) performed with 14 female and five male
morphometrical characters (Table 3.5). The circles display 95% confidence regions. For the
list with the abbreviations of the nematode population codes see Table 2.6.
Chapter 3
3.4
55
Discussion
In the 10 P. coffeae populations from Vietnam examined the presence of
substantial variability in morphology and morphometry within and between the
populations was observed.
En face view of the head (= 1st (lip) annule). Observed by scanning
electron microscope, the en face view of the head (= 1st (lip) annule) is similar in
all P. coffeae populations from Vietnam examined: complete fusion of the 1st (lip)
annule with the oral disc resulting in an undivided en face view with no division
between the lateral and median (sub-dorsal and sub-ventral) segments of the 1st lip
annule. According to Geraert (2006), the en face views of the head in the genus
Pratylenchus as observed by scanning electron microscope are characterised by the
fusion of the median segments with each other and the oral disc. The large lateral
segments are either also fused with the median segments and the oral disc or are
completely separated. The en face view of the P. coffeae populations from
Vietnam is similar to the en face views of P. coffeae populations from citrus in
Florida, USA, described and illustrated by Corbett and Clark (1983). In contrast, the
en face views of a P. coffeae population from coffee (type host) in Java (near the
type locality), Indonesia, show a dsitinct oral disc separated from the lateral and
median segments of the 1st (lip) annule (Corbett & Clark, 1983; Golden et al.,
1992; Inserra et al., 1998).
Pratylenchus coffeae was described in 1898 by Zimmermann from coffee
in Java, Indonesia. Zimmermann did not designate a type location when he
described P. coffeae, specifying only that it caused a decline of coffee in eastern
Java. In 1953, the type material was re-examined by Sher and Allen and P. coffeae
re-described.
They
synonomised
Pratylenchus
musicola
from
banana
and
Pratylenchus mahogani from mahogany with P. coffeae. Subsequently, lesion
nematodes found on coffee, banana, citrus, yam and other crops have been
identified as P. coffeae. Zimmermann, Sher and Allen were unable to use a
scanning electron microscope to examine the en face views of the heads of the
type specimens of P. coffeae. The only type material of P. coffeae preserved is a
neotype designated by Sher and Allen in 1953 but there is ambiguity about the
geographical origin of this neotype (Duncan et al., 1999). In addition to this
neotype, specimens collected in 1952 from coffee in Djember near the type
locality are also available in two permanent slides. This neotype and the Djember
specimens have a divided en face view (Inserra et al., 1998). In contrast,
Pratylenchus populations identified as P. coffeae from citrus in Florida (USA),
Brazil and Oman, from yam in Puerto Rico (USA), Martinique and Brazil, from
banana in Costa Rica, Honduras, Ghana and Malaysia, from cocoyam and
Chapter 3
56
Diffenbachia (both in Brazil), Aglaonema (Florida, USA) and ficus (China), and from
coffee in Brazil and Indonesia have an undivided en face view (Duncan et al.,
1999). In fact, of the 25 P. coffeae populations examined by Duncan et al. (1999),
only the preserved neotype of P. coffeae has a divided en face view. Among these
25 P. coffeae populations were five populations collected from coffee in five
different provinces of eastern Java which is the type locality of P. coffeae. Corbett
and Clark (1983) suggested that this morphological character is a reliable
taxonomic character for the identification of Pratylenchus populations at species
level and this is confirmed by our observations. In 2002, Ryss presented a tabular
multi-entry key to the genus Pratylenchus and a computerised multi-entry imageoperating key developed on the basis of the stepwise computer diagnostic system
BIKEY-PICKEY using 26 morphological and morphometrical characters. However, the
en face view of the head was not included as a diagnostic morphological character.
Number of head annules. With the exception of some specimens of the
Red River Delta 2 and Central Highlands populations, which have three annules on
one side and two annules on the other side, all specimens of all P. coffeae
populations from Vietnam examined have two lip annules. Román and Hirschmann
(1969) also observed that in Pratylenchus species characterised by the presence of
two head annules, such as P. coffeae, individuals were sometimes found with three
annules on one side and two annules on the other side. Corbett and Clark (1983)
remarked that in the genus Pratylenchus the number of head annules varied
between species and somewhat within species but that the variability was never so
large as to invalidate the number of head annules as a reliable diagnostic
character. The number of head annules was included as a diagnostic morphological
character in the multi-entry keys of Ryss (2002).
Structure of the lateral field. In all the P. coffeae populations from
Vietnam examined the basic pattern of the structure of the lateral field can be
described as follows: beginning at the 9th or 10th annule as two lines, widening to
three lines at the following annules and to four lines at the level of the median
pharyngeal bulb that extend to the phasmid; only the outer bands extend until the
tail tip where they fuse to one band; outer lines crenate in the tail region;
occasionally the internal band sculptured by striae; viewed with the light
microscope in some specimens punctuations scattered in the outer bands were
observed; viewed with the scanning electron microscope in all populations
areolation of the outer bands was observed. Similar deviations from the basic
pattern were reported by Román and Hirschmann (1969). They studied six
Pratylenchus species (Pratylenchus penetrans, Pratylenchus vulnus, Pratylenchus
scribneri, Pratylenchus zeae, Pratylenchus brachyurus and P. coffeae) but found
Chapter 3
57
punctuations scattered in the outer bands of the lateral field only in P. coffeae. In
Tylenchida, the structure of the lateral field is considered a usefull taxonomic
character (Siddiqi, 2000). But in the genus Pratylenchus, variability especially of
the ornamentation of the lateral field was found in so many species as to make the
structure of the lateral field an unreliable diagnostic character according to Román
and Hirschmann (1969), and Corbett and Clark (1983). Nevertheless, Ryss (2002)
included three features of the lateral field (lateral field incisures at mid-body;
lateral field areolation; lateral field incisures between phasmid and tail tip) in his
multi-entry keys. At mid-body, all P. coffeae populations from Vietnam examined
in our study had four lines.
Shape of the spermatheca. According to Ryss (2002), the best diagnostic
morphological character to separate species of the genus Pratylenchus is the shape
of the spermatheca in females. In his view, the shape of the spermatheca
represents a sequence of (five) stages of reduction which corresponds with the
transition of Pratylenchus species from an amphimictic to a parthenogenic mode of
reproduction. Parthenogenesis is typical for more than 60% of the Pratylenchus
species. Pratylenchus coffeae is amphimictic. Ryss (2002) distinguishes the
following five stages: a) spermatheca filled with sperm, round, b) spermatheca
filled with sperm, oval, c) spermatheca without sperm, distinct, offset, with round
or oval cavity, d) spermatheca without sperm, distinct, offset, with slit-like cavity
and e) spermatheca indistinct, not offset from outline of female genital branch.
Separation of Pratylenchus loosi from P. coffeae was only based on the difference
of the structure of the spermatheca between these two species (Pourjame et al.,
1997a). For P. loosi, the spermatheca was described as long, oval to almost
rectangular, filled with rounded sperms. In the P. coffeae populations from
Vietnam examined the shape of the spermatheca varied from small rounded to
large oval, usually filled with sperms.
Shape of female tail and tail tip. In some Pratylenchus species, including
P. coffeae, the intraspecific variability in tail and, especially, tail tip shape can be
very high (see for instance Román & Hirschmann, 1969; Tarjan & Frederick, 1978;
Corbett & Clark, 1983; Bajaj & Bhatti, 1984). Ryss (2002) included the shapes of
the tail tip and the tail tip annulation as two different morphological characters of
the tail in his multi-entry keys. In our study of the P. coffeae populations from
Vietnam we distinguish 12 types (see Table 3.3) based on a combination of the tail
tip and tail tip shape following the descriptions by Román and Hirschmann (1969),
Tarjan and Frederick (1978), Corbett and Clark (1983) and Bajaj and Bhatti (1984).
When comparing the morphometrics of Pratylenchus pseudocoffeae
populations with those of Pratylenchus gutierrezi populations, Inserra et al. (1998)
Chapter 3
58
observed that the ranges of all morphometrical characters measured overlapped
except the length of the pharyngeal overlap. The populations from P.
pseudocoffeae have females with a longer pharyngeal overlap (> 60 µm) than those
of P. gutierrezi (< 60 µm). According to Inserra et al. (1998), the lenght of the
pharyngeal overlap is the only morphological character of diagnostic value which
allows the separation of P. pseudocoffeae females from those of P. gutierrezi. In
the females of the P. coffeae populations from Vietnam examined the length of the
pharyngeal overlap varies on average from 41.4 to 62.3 µm. The coefficient of
variation is 17%.
Based on the coefficient of variation (CV), the position of the vulva (Vvalue; CV = 3.1%) in the females and the stylet length in both females (4.6%) and
males (4.8%) of the P. coffeae populations from Vietnam examined are the least
variable morphometrical characters. This observation confirms earlier reports
(Román & Hirschmann, 1969; Tarjan & Frederick, 1978; Bajaj & Bhatti, 1984; Café
Filho & Huang, 1989) that in P. coffeae the intraspecific variability of the position
of the vulva and the stylet length is low and stable and thus of diagnostic value.
The V-value observed in the P. coffeae populations from Vietnam examined ranged
on average from 79.2 to 81.9%. The stylet length of the P. coffeae populations
from Vietnam examined ranged on average from 15.3 to 16.1 µm (females) and
from 14 to 14.8 µm (males). These values are similar to values reported for P.
coffeae populations from the Americas (Duncan et al. 1999; Inserra et al. 2001),
Africa (Van den Berg, 1971; Duncan et al. 1999) and Asia (Sher & Allen, 1953;
Rashid & Khan, 1976; Bajaj & Bhattti, 1984; Inserra et al. 1998; Xiuhua, 2006).
Based on the coefficient of variation (CV), the length of the ovary in
females (CV = 22.1%) and the length of the testis in males (T-value; CV = 16.4%) of
the P. coffeae populations from Vietnam examined are the highest variable
morphometrical characters. This is not unexpected since the length of these
structures will depend upon the stage of reproductive development of the
nematodes measured. In the females, the coefficient of variation of the length of
the post-vulval uterine sac was also very high (CV = 17%) and ranged on average
from 25.5 to 31.2 µm. This observation confirms earlier reports (Román &
Hirschmann, 1969; Tarjan & Frederick, 1978; Bajaj & Bhatti, 1984).
All other morphometrical characters, including those commonly used in
nematode taxonomy, have relatively high coefficients of variation.
Canonical discriminant analysis enabled the separation of the 10 P.
coffeae populations from Vietnam in three groups based on a combination of five
morphological characters for the males but there was no relationship nor between
Chapter 3
59
these groups and their geographic origin or between these groups and their host
plants.
The best morphometrical male characters for the separation of the
populations (body length, b ratio and distance from the anterior end to the
excretory pore) correspond partly with the male morphometrical characters with
the lowest coefficient of variation and partly with the morphometrical characters
most commonly used to separate Pratylenchus species. de La Peña et al. (2007)
reported that canonical discriminant analysis enabled discrimination of four
Pratylenchus species: Pratylenchus pratensis, Pratylenchus dunensis, Pratylenchus
brzeskii and P. penetrans. For the females and males 62.8% and 65.2%,
respectively, of the variation could be explained by differences in 12
morphometrical characters. However, in their analysis, body length and distance
from the anterior end to the excretory pore were not included. Also in their
analysis, the b ratio was an important discriminating morphometrical character.
3.5
Conclusions
Our detailed comparative morphological and morphometrical observations
of the 10 P. coffeae populations from Vietnam established in vitro on carrot discs
reveal the presence of substantial variability in morphology and morphometry
within and between these populations. However, these differences fall within the
range of the morphological and morphometrical variabilty described previously in
P. coffeae populations from other parts of the world. Our scanning electron
microscope observations confirm that in P. coffeae there is a complete fusion of
the 1st (lip) annule with the oral disc resulting in an undivided en face view with no
division between the lateral and median (sub-dorsal and sub-ventral) segments of
the 1st (lip) annule. Canonical discriminant analysis enabled the separation of the
10 P. coffeae populations from Vietnam examined in three groups based on a
combination of five morphological characters for the males but there was no
relationship nor between these groups and their geographic origin or between
these groups and the host plants from which they were originally isolated.
Chapter 3
60
Chapter 4
61
Chapter 4: Molecular characterisation of
Pratylenchus coffeae populations from Vietnam
4.1
Introduction
Due to the low number of morphological characters that can be observed
with a microscope and the high intraspecific variability of some of these
characters, it is very difficult to separate the species of the genus Pratylenchus
based on their morphology and morphometrics alone (Román & Hirschmann, 1969;
Tarte & Mai, 1976; Corbett & Clark, 1983; Handoo et al., 2001). During the past
two decades, several molecular tool boxes have been developed for the
identification and comparison of nematode genera, species and populations
including the polymerase chain reaction (PCR) to amplify, detect and compare DNA
fragments, PCR-restriction fragment length polymorphism (PCR-RFLP) to reveal
variation in sequences in PCR products obtained by restriction endonuclease
digestion, random amplified polymorphic DNA (RAPD) and sequencing (Subbotin &
Moens, 2006). For the taxonomy of Pratylenchus species, DNA-based techniques
have been used since the early 1990’s (Castillo & Vovlas, 2007).
In nematology, often rDNA and especially the variability of the internal
transcriber spacer (ITS) region has been examined. The rDNA repeat consists of
three genes (18S, 28S and 5.8S), internal and external transcribed spacer (ITS, ETS)
regions, and an external nontranscribed spacer region (NTS; Fig. 4.1). In
nematodes, the sequences of the rDNA genes are highly conserved whereas there is
less conservation within the ITS regions and little homology is found in the NTS
regions. The more conserved sequences are most useful for classification at higher
taxonomic levels (genus to phylum) whereas the ITS sequences are more useful for
diagnostic purposes at species level (Hyman & Powers, 1991).
D2/D3 expansion segment
D2/D3 expansion segment
Figure 4.1: Structure of the ribosomal DNA gene family in nematodes. Coding regions of the
18S small subunit (SSU), 5.8S and 28S large subunit (LSU); non-coding regions of the internal
transcribed spacer (ITS) and external transcribed spacer (ETS) regions; external
nontranscribed spacer (NTS) region.
Chapter 4
62
In Pratylenchus, a remarkable ITS region size difference exists:
approximately 350 bp separate the smallest and largest amplified ITS region among
the species studied so far (Castillo & Vovlas, 2007). In addition, intraspecific ITS
region variation has been observed in this genus, particularly within populations of
Pratylenchus coffeae and Pratylenchus vulnus (Orui, 1996; Uehara et al., 1998;
Waeyenberge et al., 2000; Mizukubo et al., 2003).
The RAPD method uses an arbitrary primer, about ten nucleotides long,
for the random amplification of any fragment of any part of the genome thus
creating a genomic fingerprint. Amplified DNA fragments obtained using different
random primers can be separated on gels and compared. Random amplified
polymorphic DNA results from the fact that if a primer hybridisation site in a
genome differs by even a single nucleotide, the change can lead to elimination of a
specific amplification product. The resulting individual bands are considered as
equivalent independent characters. The band polymorphism can be binary scored
and the data matrix can be used for calculating the genetic distance among the
populations under study and then represented as a dendrogram (Subbotin & Moens,
2006).
Random amplified polymorphic DNA PCR analysis has shown its potential
usefulness in distinguishing seven populations of P. vulnus from one population of
Pratylenchus neglectus, the patterns of the amplified DNA bands of the P.
neglectus population being clearly different from those of the P. vulnus
populations (Pinochet et al., 1994). However, a high level of polymorphism was
observed among the P. vulnus populations. To estimate the phylogenetic
relationship among 15 populations of P. coffeae, Pratylenchus pseudocoffeae,
Pratylenchus guttierezi and Pratylenchus loosi, Duncan et al. (1999) constructed a
dendrogram based on 227 RAPD bands obtained with 18 primers. The authors
observed a low level of similarity among the populations suggesting that the
Pratylenchus genome
is
highly
variable
and that
therefore
phylogenetic
relationships among Pratylenchus species and populations cannot be based solely
on RAPD data.
Although molecular approaches based on PCR amplification are frequently
applied in diagnostic studies, this method can lead to erroneous conclusions
because samples may be contaminated with a wide range of diverse DNA
sequences. Thus, DNA sequencing provides a valuable alternative which may
eliminate false positives due to contamination (Volossiouk et al., 2003; Castillo &
Vovlas, 2007).
Chapter 4
63
In nematology, especially the sequence of the D2/D3 expansion segment
of the 28S rDNA gene has been examined and has been shown to be an important
molecular tool for the taxonomy of plant-parasitic nematode taxa including
Pratylenchus (see for instance Handoo et al., 2001; Subbotin et al., 2006; de la
Peña et al., 2007). Duncan et al. (1999) sequenced the DNA of the D2/D3 28S rDNA
expansion segment of 19 populations of P. coffeae and closely related species (P.
pseudocoffeae, P. guttierezi and P. loosi) collected worldwide. The DNA sequence
of the D2/D3 expansion segment indicated a close relationship among the P.
coffeae populations isolated from coffee in Indonesia and from citrus, yam, banana
and miscellaneous plants although the sequence identity was not absolute: the
sequences of some populations varied 1 to 5 nucleotides. The same study also
showed that P. coffeae and P. loosi could be distinguished based on the DNA
sequence of the D2/D3 expansion segments. As mentioned in Chapter 3, the
morphology and morphometry of these two nematode species are almost identical,
the only difference being the shape of the spermatheca (Pourjame et al., 1997a).
Also, PCR-RFLP of the ITS regions could not separate the two species (Uehara et
al., 1998; Pourjame et al., 1997b). De Luca et al. (2004) sequenced a specific
portion of DNA (the D3 expansion segment of the 26S rDNA gene) of several
Pratylenchus species (but not P. coffeae) and compared these sequences with
similar sequences available in databases. The results suggested that one of the
species studied (Pratylenchus penetrans) may represent a species complex. In the
three populations of another species (Pratylenchus neglectus) intraspecific
heterogeneity among the D3 26S rDNA expansion segments was observed which may
be due to intraspecific variability. In contrast, the specific conservation of some
nucleotides in the five populations of another species (Pratylenchus thornei)
included in the study indicated their stability.
The objectives of this part of our study were to compare the intraspecific
genomic variability of the 10 P. coffeae populations collected from different
agricultural crops in different agro-ecological regions in Vietnam (see Chapter 2)
based a) on RAPD analysis of the complete genome and b) on sequencing of the
D2/D3 expansion segments of the 28S rDNA gene, and c) to compare these with the
sequences available in the GenBank database.
4.2
The 10 P. coffeae populations collected from different agricultural crops
in different agro-ecological regions in Vietnam and one P. coffeae population
Chapter 4
64
originally isolated from banana in Ghana (Table 2.6) were included in our study.
The specimens studied were obtained from in vitro carrot disc cultures (see
Chapter 2). Carrot disc cultures with vigorous developing nematode populations
(many active nematodes in the Petri dish) were selected. The nematodes were
collected in a test tube by rinsing the Petri dishes with sterile water. The
nematodes were immediately used for DNA extraction.
4.2.2 Random amplified polymorphism DNA (RAPD)
4.2.2.1
DNA extraction
Approximately 1,000 nematodes of each P. coffeae population obtained
from the in vitro carrot disc cultures were transferred into 1.5 ml Eppendorf tubes
and centrifugated at 13,000 rpm for 20 min (minutes). After centrifugation, the
supernatans was discarded and the tubes stored at –80oC for at least 30 min. Then,
the pellets were crushed with a sterile plastic stick. 275 µl digestion solution
master mix (Promega Benelux, Leiden, The Netherlands) were added to each tube
and the tubes were incubated during 16-18 h (hours) in a 55oC heat block. After
incubation, 250 µl wizard SV lysis buffer (Promega Benelux, Leiden, The
Netherlands) were added to each tube and the content of the tubes mixed by
vortexing. Purification of the genomic DNA of the lysate was done by
microcentrifugation. According to the protocol by Promega (Promega Benelux,
Leiden, The Netherlands), the lysate in the 1.5 ml Eppendorf tubes was transferred
to a wizard SV minicolumn placed in centrifugation tubes and centrifugated 13,000
rpm for 3 min to bind the genomic DNA to the minicolumn. After centrifugation,
the liquid in the collection tube was discarded, 650 µl wizard SV wash solution
(diluted with 95% ethanol) added to the minicolumn placed in centrifugation tubes,
the mixture centrifugated at 13,000 rpm for 1 min and the liquid in the collection
tube again discarded. This washing was repeated 4 times. Then, the minicolumn
assembly was centrifugated at 13,000 rpm for 2 min to dry the binding matrix. The
contents of the minicolumns were transferred to 1.5 ml Eppendoft tubes and 250 µl
nuclease-free water added, the mixture incubated for 2 min at room temperature
and centrifugated at 13,000 rpm for 1 min. Then, 250 µl nuclease-free water was
added again, the mixture incubated for 2 min at room temperature and
centrifugated again at 13,000 rpm for 1 min. The purified DNA was stored at -20oC.
4.2.2.2
RAPD-PCR reaction
The extracted DNA was used for PCR. Ten random selected primers, 10
oligonucleotides long, were used (Table 4.1). RAPD-PCR was performed as follows:
5 µl extracted DNA suspension were added to the PCR reaction mixture containing
Chapter 4
65
4 µl 10X PCR Rxn buffer (MgCl2), 1.5 µl 50 mM MgCl2, 1 µl (10 mM) of each dNTP, 1
µl (0.2 µM) of a primer, 2U Taq DNA polymerase recombinant (Invitrogen,
Merelbeke, Belgium) and double distilled water (ddH2O) to a final volume of 35 µl.
The PCR reaction mixture was placed in a thermocycler (PTC-200 Biozym,
Landgraaf, The Netherlands) preheated at 95oC. The DNA amplification profile
consisted of 7 min at 95oC, 44 cycles of 1 min at 92oC, 1 min at 36oC and 3 min at
72oC. A final step of 10 min at 72oC completed the DNA amplification.
Following DNA amplification, 5 µl of each PCR product was loaded on a 1%
agarose gel. A 100 bp and 1 Kb DNA mass ladder (Promega Benelux, Leiden, The
Netherlands) were included as size markers. After electrophoresis for 45 min at 100
V, 100 mA, 10 W, the DNA bands were stained with 0.003% ethidium bromide (0.02
µg/ml) for 10 min. The gel was viewed on a UV-transilluminator and photographed.
Ten independent reactions were performed 3 times for each nematode
population/primer combination.
Table 4.1: Oligonucleotides used for the RAPD-PCR study.
Number
1
2
3
4
5
6
7
8
9
10
4.2.2.3
Primer sequence (5’ to 3’)
GGC ACT GAGG
GAG CCC TCCA
GTG CCT AACC
AGG GCC GTCT
CAG CTC ACGA
TGC CCG TCGT
CTC TCC GCCA
AGC GTC CTCC
GTC AGG GCAA
CCG AAG CCCT
Code
OPG2
OPG3
OPG6
OPG10
OPG11
OPG12
OPG13
OPG16
OPG19
SC10-30
Genetic data analysis and phylogeny
The DNA bands which were retained after the process described above
(repeated 3 times) were scored visually as present (1) or absent (0). The data were
gathered in a matrix file and analysed using PAUP version 4.0b10 (Swofford, 1998)
to construct a Neighbour-Joining (NJ) tree. The genetic distances among the P.
coffeae populations were calculated by means of the pair group method using
arithmetic averages (UPGMA) according to Dice’s coefficient. In addition, a
bootstrap analysis generating 1,000 random data sets from the original data was
carried out to check the support for the groupings within the NJ tree.
Chapter 4
66
4.2.3 D2/D3 sequencing
4.2.3.1
DNA extraction
DNA extraction was done as described by Waeyenberge et al. (2000). Of
each P. coffeae population, one female obtained from the in vitro carrot disc
cultures was transferred to 20 µl worm lysis buffer (Sigma, Bornem, Belgium).
Nematodes were cut into two or more pieces with a sterilised scalpel. Eight µl
worm lysis buffer (with the nematode fragments) were transferred into a 0.2 ml
Eppendorf tube containing 10 µl ddH2O. Two µl of proteinase K (600 µg/ml; Sigma,
Bornem, Belgium) were added to each tube now containing a total volume of 20 µl.
The mixture was homogenised using a vibromixer. The tubes were stored at -80oC
for at least 10 min. After deepfreezing, the lysate was incubated in a thermocycler
(PTC-200 Biozym, Landgraaf, The Netherlands) at 65oC for 1 h. At this temperature,
proteinase K cut all proteins into individual amino acids. Before centrifugation (2
min at 13,000 rpm) to collect the supernatans (extracted DNA), the tubes were
incubated for 10 min at 95oC to denature the proteinase K. Finally, the extracted
DNA was cooled to 4oC and stored at -20oC.
4.2.3.2
D2/D3 rDNA PCR
The extracted DNA was used for PCR. The forward primer D2A 5’-ACA AGT
ACC GTG AGG GAA AGT TG-3’ and the reverse primer D3B 5’-TCG GAA GGA ACC
AGC TAC TA-3’ (De Ley et al., 1999) were used for amplifying the D2/D3 expansion
segment of the 28S rDNA gene. Five µl of the extracted DNA were added to the PCR
reaction mixture containing 5 µl 10X DNA Taq polymerase incubation buffer with 2
µl MgCl2 (15 mM), 1 µl dNTP mixture (200 µM of each dNTP), 0.3 µl (0.3 µg) of each
primer (synthesised by Invitrogen, Merelbeke, Belgium), 0.4 µl Taq polymerase
(5U/µl), and ddH2O to a final volume of 50 µl. All chemicals were obtained from a
Taq PCR Core Kit (Qiagen, Crwaley, West Sussex, UK). The PCR reaction mixture
was incubated in a thermocycler (PTC-200 Biozym, Landgraaf, The Netherlands)
preheated for 5 min at 92oC. The PCR amplification temperature and time
conditions were 40 cycles of 30 sec (seconds) at 94oC, 45 sec at 55oC and 1 min at
72oC. A 7 min polymerisation period at 72oC followed the last cycle.
Following DNA amplification, 5 µl of each PCR product were mixed with 1
µl 6X loading dye (Promega, Leiden, The Netherlands) and loaded in 1X TAE buffer
(Sambrook et al., 1989) on a 1% agarose gel. A low DNA mass ladder (Invitrogen,
Merelbeke, Belgium; Hartley & Xu, 1994) in 5 µl volume was also loaded on the gel
as size marker. After electrophoresis for 45 min at 100 V, 100 mA, 10 W, the DNA
Chapter 4
67
bands were stained with 0.003% ethidium bromide (0.02 µg/ml) for 10 min. The gel
was viewed on a UV-transilluminator and photographed.
4.2.3.3
Purification of the PCR products
The PCR reaction mixture was loaded on a 2% agarose gel in 1X TAE
buffer. Electrophoresis was done until the DNA band of interest was isolated from
adjacent contaminating fragments, primer-dimers and left-overs. The bands were
identified by staining the gel with 0.003% ethidium bromide (0.02 µg/ml) for 10
min. The DNA bands of interest were cut from the gel by using an ethanol-cleaned
scalpel on a UV light box. The excised agarose gel slices were placed in 1.5 ml
microcentrifuge tubes. Gel mass was determined by first pre-weighting the tubes
and then re-weighting the tubes with the excised gel slice. 300 µl of a binding
buffer (MSB Spin PCRapase Kit, Invitek, Berlin, Germany) were added for every 100
mg agarose gel slice to the microcentrifuge tube. To release the DNA, the agarose
gel slices were dissolved by vortexing the microcentrifuge tubes for 20 sec to
resuspend the gel slice in the binding buffer. The suspension was incubated for 10
min at 56oC with 3 times of vortexing during incubation. The DNA was recovered on
a high pure filter tube (MSB Spin PCRapase Kit, Invitek, Berlin, Germany) placed in
a collection tube by centrifugation at maximum speed (13,000 rpm) for 60 sec.
Thirty µl elution buffer (MSB Spin PCRapase Kit, Invitek, Berlin, Germany) were
added to the upper reservoir of the filter tubes and the tubes centrifuged 1 min at
maximum speed (13,000 rpm) to collect the purified DNA.
4.2.3.4
Quantification of the purified PCR products
Five µl purified PCR product was loaded on a 1% agarose gel in 1X TAE
buffer. A low DNA mass ladder was also loaded on the gel to estimate the mass of
the purified DNA. After electrophoresis for 45 min at 100 V, the gel was stained
with 0.003% ethidium bromide (0.02 µg/ml) for 10 min. The DNA bands were
visualised on a UV-transilluminator and photographed. The concentration of
purified PCR products was estimated visually by comparing the brightness of their
bands with the DNA bands of the low DNA mass ladder.
4.2.3.5
Cloning of the PCR products
Preparation of Luria-Britani (LB) medium antibiotic plates was as follows:
15 g of agar were added to 1 liter LB medium (10 g bacto®-tryptone, 5 g bacto®yeast extract, 5 g NaCl and deionsed water added to 1 liter). pH was adjusted to
7.5 with 10N NaOH and the solution was autoclaved at 120oC for 20 min. The
container with the medium was cooled to 55oC. Filter-sterilised ampicillin (100 mg
Chapter 4
68
dissolved in 1 ml ddH2O; Sigma, Bornem, Belgium) was added to the LB medium
and mixed. Thirty ml of this medium were poured into 9-cm-diameter Petri dishes.
The agar was left overnight to solidify and then stored for a maximum of 1 month
at 4oC. Before use, 100 µl isopropyl β-D-thiogalactopyranoside (IPTG) stock solution
(Promega Benelux, Leiden, The Netherlands) and 50 µl X-Gal (50 mg/ml; Promega
Benelux, Leiden, The Netherlands,) were spread on the plates. The medium was
allowed to absorb the chemicals during 30 min at 37oC. The preparation of IPTG
stock solution (0.1 M) was as follows: 1.2 g IPTG was added to deionised water to a
50 ml filter-sterilised (0.2 µm) final volume and stored at 4oC. This stock solution
was available from Promega (Cat. # V3951). The preparation of the X-Gal solution
(50 mg/ml) was as follows: 100 mg 5-bromo-4-chloro-3-indolyl β-D-galactoside (XGal) dissolved in 2 ml of N, N’-dimethylformamide. This stock solution was
available from Promega (Cat. # V3941).
Purified DNA was Ligated into pGEM®-T Vector (pGEM®-T Vector System II, Promega
Benelux, Leiden, the Netherlands) in the presence of 2x ligation buffer and T4 DNA
ligase according to the manufacturer’s instructions. Ligation reactions were set up
as described by the Promega protocol (Protocols and applications guide, third
edition, 1996, Promega Corporation, Madison, USA):
Standard
Positive
Background
reaction
control
control
Ligase 2X Buffer
5 µl
5 µl
5 µl
pGEM®-T Vector (50 ng)
1 µl
1 µl
1 µl
PCR product (5-15 ng)
xµl*
__
__
Control Insert DNA
__
2 µl
__
T4 DNA Ligase (3 Weiss units/µl)
1 µl
1 µl
1 µl
Nuclease-Free Water
yµl°
1 µl
3 µl
Final volume
10 µl
10 µl
10 µl
*Molar ratio of PCR product: pGEM®-T Vector may require optimization
°Volume depending on amount of PCR-product used
High efficiency competent cells (> 1 x 108cfu/µg DNA) were used for
transformation. Two LB/ampicillin/ITPG/X-Gal plates were prepared for each
ligation reaction. The procedure for the transformation of the competent cells with
the prepared pGEM®-T Vector (pGEM-T Vector System II, Promega Benelux, Leiden,
The Netherlands) was as described by the Promega protocol: the tubes containing
the ligation reactions were centrifuged to collect the contents at the bottom of the
tubes. Two µl of each ligation reaction were added to sterile 1.5 ml
microcentrifuge tubes on ice. High efficiency JM109 competent cells (Escherichia
ecoli) were removed from their –70oC storage place and placed in an ice bath until
just thawed (about 5 min). The tubes were mixed by gently flicking. Because the
cells are extremely fragile, 50 µl of the cells were carefully aliquot into each tube
Chapter 4
69
containing 2 µl of ligation reaction. The tubes were mixed by gently flicking and
placed on ice for 20 min. Then, the cells were heat-shocked for 45 to 50 sec at
exactly 42oC in a water bath without shaking. The tubes were immediately placed
on ice for 2 min. 950 µl SOC medium (at room temperature) were added to the
tubes containing cells transformed with ligation reactions and 900 µl SOC medium
(at room temperature) were added to the tubes containing the positive
transformation control (supercoiled plasmid DNA). These tubes were incubated for
1.5 h at 37oC while shaked (150 rpm). 100 µl of each transformation culture were
plated onto duplicate LB/antibiotic plates. These plates were incubated overnight
at 37oC. After incubation, the plates were checked: white and blue colonies were
observed in the samples and the positive control, the negative control contained
only blue colonies. From each sample, 10 individual white colonies were recultured in tubes containing liquid LB/ampicilin. With a sterile plastic 1,000 µl tip,
each colony was picked up and transferred to a tube containing 1,000 µl of liquid
LB/ampicillin medium. These tubes were incubated at 37oC while shaked (150 rpm)
overnight.
PCR was done for each selected colony to control whether the correct
product was ligated into the vector and transformation was successful. Finally, one
selected colony of each population was sequenced as described below (see
4.2.3.6). Two PCR vector-primers were used: M13 forward (TGT AAA ACG ACG GCC
AGT) and pGEM-T reverse (CAGGAAACAGCTATGAC). The PCR products were
checked on an agarose gel after electrophoresis as described before. Only the
colonies which produced a PCR band of approximately 1 Kb (PCR product of
approximately 800 bp + part of the vector approximately 200 bp) were used for
sequencing.
DNA
purification
of
the
cloned
PCR
product
was
done
by
microcentrifugation. According to the protocol by Promega (Promega Benelux,
Leiden, The Netherlands), an equal volume of membrane binding solution was
added to each PCR product and mixed by pipetting. Afterward, the PCR product
was transferred to a wizard SV minicolumn assembly, incubated at room
temperature for 1 min and centrifugated at 13,000 rpm for 1 min. After
centrifugation, the liquid in the collection tube was discarded. Then, 700 µl of
membrane wash solution (diluted with 95% ethanol) was added to the minicolumn
placed in centrifugation tubes, the mixture centrifugated for 1 min at 13,000 rpm
and the liquid in the collection tube again dsicarded. The washing was repeated
with 500 µl of membrane wash solution and centrifugated for 5 min at 13,000 rpm.
The minicolumn assembly was centrifugated at 13,000 rpm for 1 min to allow the
residual ethanol to evaporate completely. Then, 40 µl nuclease-free water was
Chapter 4
70
pippeted to the center of the minicolumn, the mixture incubated for 1 min at room
temperature and centrifugated again at 13,000 rpm for 1 min. The microcentrifuge
tube containing the eluted DNA was stored at -20oC.
4.2.3.6
Sequencing
Cycle sequencing
After purifying and quantifying the colony-PCR product as described
above, the following products were mixed: 3 µl terminator ready reaction mix, 30
to 90 ng purified colony-PCR product, 1 µl primer (3.2 pmol) and ddH2O to a total
volume of 20 µl. The cycle sequencing reaction profile consisted of 25 cycles each
composed of 10 sec at 96oC, 5 sec at 50oC and 4 min at 60oC (ABI Prism® BigDye
Terminator v3.1 Ready Reaction Cycle Sequencing Kit, AB Applied Biosystems,
Lennik, Belgium 2002).
Purification of the sequenced products
After finishing the cycle sequencing, 16 µl of ddH2O and 64 µl of nondenatured 95% ethanol were added to each tube. After leaving the tubes at room
temperature for 15 min to precipitate the extension products, the tubes were
placed in a microcentrifuge with fixed orientations and spun for 20 min at
maximum speed (13,000 rpm). The supernatans was removed and 150 µl of 70%
ethanol were added and mixed briefly with the pellet to wash away nonincorporated nucleotides. After a second wash, the tubes were then centrifuged for
10 min at 13,000 rpm. Finally, the tubes were dried for 10 min at 50oC.
Sequence alignments and analyses
The sequences were created using an ABI Prism 310 Genetic Analyser. ABI
Prism DNA Sequencing Analysis Software version 3.7 was used to analyse the results
(Applied Biosystems, Foster City, CA, USA). Due to cycle sequencing and
electrophoresis limitations, only about 700 bp could be sequenced. Therefore, from
all the samples, both DNA-strands were sequenced by using one of the vectorprimers to be able to construct the complete sequence of the cloned PCR products
which
are
longer
than
1
Kb.
Software-package
Chromas
version
1.45
(Technelysium, Helensvale, QLD, Australia) was used to visualise the sequences on
the computer. Both sequences from the same sample (forward sequence and
inverted complementary of the reverse sequence) were exported together in one
text file in order to find the overlapping sequence. After removing this overlapping
part in one of the sequences and the sequence part belonging to the plasmid in
both sequences, one complete sequence was generated for each population and
saved as a text file. ClustalX 2.0 (Thompson et al., 1997) was used to make an
Chapter 4
71
alignment of all complete sequences together with 10 P. coffeae sequences from
GenBank (Table 4.2). The D2/D3 28S rDNA expansion segment sequences were
obtained with BLAST (basic local alignment search tool) from GenBank
(http://www.ncbi.nlm.nih.gov).
Table 4.2: Origin, accession numbers, codes and number of base pairs of Pratylenchus
accessions from Genbank (deposited by Duncan et al., 1999) used for comparison with the
Pratylenchus
species
Origin
Accession
number
Code
Number of base pairs
(D2/D3 sequence size)
1
P. coffeae
Florida, USA
AF170428
C4
745
2
3
4
5
6
7
8
9
10
11
12
13
P. coffeae
P. coffeae
P. coffeae
P. coffeae
P. coffeae
P. coffeae
P. coffeae
P. coffeae
P. coffeae
P. jaehni
P. jaehni
P. loosi
Dhofar, Oman
Martinique
Pernambuco, Brazil
Puerto Rico, USA
Ghana
Honduras
Sao Paulo, Brazil
Florida, USA
Kaliwining, Indonesia
Sao Paulo, Brazil
Sao Paulo, Brazil
USA
AF170429
AF170430
AF170431
AF170432
AF170433
AF170434
AF170435
AF170436
AF170443
AF170426
AF170427
AF170437
C6
Y1
Y2
Y3
B1
B2
M1
M3
K6
C1
C2
N1
735
730
741
726
754
731
731
750
735
750
756
738
4.2.3.7
Phylogenetic analyses
The phylogenetic trees were constructed based on the D2/D3 28S rDNA
expansion segment sequences with Maximum Parsimony (MP) algorithms as
implemented in PAUP version 4.0b10 (Swofford, 1998). Bootstrap analysis with 100
replicates was performed to assess the degree of support for each clade on the
tree. Radopholus similis accession number GQ281474 from GenBank was used as an
outgroup taxon.
4.3
Results
4.3.1 Random amplified polymorphism DNA (RAPD)
The 10 10-oligonucleotide-long primers used generated a total of 152
polymorphic DNA bands, ranging from 0.2 to 2.2 Kb in size (Fig. 4.2). All primers
produced complex patterns but at least one band of the same size was observed in
all P. coffeae populations. Primer OPG3 amplified a fragment of approximately
1,200 bp for all P. coffeae populations from Vietnam but not for the P. coffeae
population from Ghana. In contrast, a fragment of approximately 600 bp was
Chapter 4
72
amplified only in the P. coffeae population from Ghana. A similar observation was
made for the primers OPG11, OPG12 and SC10.30. These primers amplified
fragments of approximately 350, 190, and 750 bp, respectively, for all P. coffeae
populations from Vietnam examined but not for the P. coffeae population from
Ghana. In contrast, fragments of approximately 1700, 2200, 1400 bp were
amplified in the P. coffeae population from Ghana but not in the P. coffeae
populations from Vietnam.
The primer OPG3 amplified a band of approximately 950 bp in all P.
coffeae populations from Vietnam except in the Northeast 1 population. Primer
OPG6 amplified one band of approximately 280 bp in all P. coffeae populations
except in the North Central Coast 1 population. And one band of approximately 450
bp was observed only in the Northwest, Northeast 2, Red River Delta 1, North
Central Coast 1 and Central Highlands populations while a band of approximately
410 bp was only generated in the Central Highlands population by primer OPG11.
All the results obtained with the 10 10-oligonucleotide-long primers are
summarised in a NJ tree that shows the genetic distance among the 10 P. coffeae
populations from Vietnam and the P. coffeae population from Ghana based on 152
DNA bands (Table 4.3 and Fig. 4.3). Table 4.3 shows that the pairwise distance
between the P. coffeae populations from Vietnam and the P. coffeae population
from Ghana was the highest, ranging from 0.41 to 0.57. The pairwise distance
among the P. coffeae populations from Vienam ranged from 0.23 to 0.42. In this NJ
tree, the P. coffeae populations from Vietnam were separated into two groups with
less than 50 bootstrap value support. The Northeast 1 and 4 populations (originally
isolated from coffee and banana roots, respectively) and the Red River Delta 2
population (originally isolated from the roots of an onarmental tree) were
clustered together. This group is separated from another cluster including all the
other P. coffeae populations from Vietnam, which were originally isolated either
from banana or coffee. Within the latter group, the Red River Delta 1 and
Northeast 3 populations, both originally isolated from banana, clustered into a
subgroup with 81 bootstrap value support.
Chapter 4
73
Primer OPG3
Primer OPG2
3 Kb
bpb
1 Kb
bpb
500 bp
bpbpp
M
1
2 3 4
5 6 7 8 9 10 11 L
L 1
2 3
Primer OPG6
4
5
6 7
8 9 10 11 M
Primer OPG10
1.5 Kb
bpbp
p
1 Kb
bpb
500 bp
bpbpp
L 1
2 3 4
5 6 7 8
9 10 11 M
L 1
4 5
6
7 8 9 10 11 L
8 9 10 11 M
Primer OPG12
Primer OPG11
M 1 2 3
2 3 4 5 6 7
L 1
2 3 4 5 6 7 8 9 10 11 M
Figure 4.2: RAPD bands generated by 10 10-oligonucleotide-long primers for 11 Pratylenchus
coffeae populations. L: 100 bp DNA ladder; M: 1 Kb DNA ladder; 1: Northwest; 2: Northeast 1;
3: Northeast 2; 4: Northeast 3; 5: Northeast 4; 6: Red River Delta 1; 7: Red River Delta 2; 8:
North Central Coast 1; 9: North Central Coast 2; 10: Central Highlands population; 11:
population from Ghana. Arrows show the fragments of approximately 600, 950 and 1,200 bp
generated by primer OPG3; 280 and 450 bp generated by primer OPG6; 350, 410 and 1,700 bp
generated by primer OPG11; 190 and 2,200 bp generated by primer OPG12.
Chapter 4
74
Primer OPG11
M 1 2
3
4
5 6
7
Primer OPG11
8 9 10 11 L
L
1
2 3
Primer OPG19
L 1
2 3
4 5 6 7
4 5 6
7 8 9 10 11 M
Primer SC10.30
8 9 10 11 M
L 1
2 3 4 5 6 7 8 9 10 11 M
Figure 4.2: Continued. Arrows show the fragments of approximately 750 and 1,400
bp generated by primer SC10.30.
Table 4.3: Pairwise distances between 10 Pratylenchus coffeae populations from Vietnam and
a P. coffeae population from Ghana based on RAPD bands generated by 10 10-oligonucleotidelong primers.
NW
NE1
RRD2
NCC1
NCC2
CH
Gha
-
0.336 0.342 0.283 0.349 0.342 0.322
0.269
0.316
0.362
0.526
NE1
51
0.296 0.408 0.263 0.362 0.237 0.303
0.362 0.355
NE2
52
45
0.388 0.296 0.355 0.269 0.309
0.329 0.362
NE3
43
62
59
0.382 0.257 0.395 0.329
0.388 0.368
NE4
53
40
45
58
0.388 0.250 0.342
0.401 0.368
RRD1 52
55
54
39
59
0.296 0.309
0.421 0.335
RRD2 49
36
41
60
38
45
0.263
0.335 0.355
NCC1 41
46
47
50
52
47
40
0.335 0.382
NCC2 48
55
50
59
61
64
51
51
0.388
CH
55
54
55
56
56
51
54
58
59
Gha
80
63
70
87
65
88
67
69
74
75
Below diagonal: total character differences.
Above diagonal: mean character differences (adjusted for missing data).
For the list with the abbreviations of the nematode population codes see Table 2.6.
0.414
0.460
0.572
0.427
0.579
0.441
0.454
0.487
0.493
-
NW
NE2
NE3
NE4
RRD1
Chapter 4
75
Figure 4.3: Neighbour-Joining tree of 10 Pratylenchus coffeae populations from Vietnam and a
P. coffeae population from Ghana based on RAPD bands generated by 10 10-ologonucleotidelong primers. A bootstrap value > 50% is given in the appropriate clade. For the list with the
abbreviations of the nematode population codes see Table 2.6.
4.3.2 D2/D3 sequencing
The comparison of the sequences of the D2/D3 28S rDNA expansion
segments of 10 P. coffeae populations from Vietnam among themselves and with
those of 10 P. coffeae and two Pratylenchus jaehni populations obtained from
GenBank is presented in Figure 4.4. A total of 759 bp was analysed. Among the 10
P. coffeae populations from Vietnam examined, the D2/D3 28S rDNA expansion
segment sequences of the Red River Delta 2 and Northeast 4 populations were
identical while between the North Central Coast 1 population and the Red River
Delta 1 and Northeast 1 populations these sequences varied at 10 sites. The D2/D3
28S rDNA expansion segments sequences of the P. coffeae populations from
Vietnam differed at least at 1 to 23 sites compared with those of the P. coffeae
populations obtained from GenBank with the exception of the North Central Coast
2 population of which the sequences were identical with those of the P. coffeae
Chapter 4
76
populations from Honduras (B2-AF170434), Brazil (M1-AF170435), USA (M3AF170436) and Martinique (Y1-AF170430). The D2/D3 28S rDNA expansion segments
sequences of the P. coffeae population from Ghana (B1-AF170533) differed from
those of all other P. coffeae populations at 18 to 23 sites.
The sequence divergence of the D2/D3 28S rDNA expansion segments
between the P. coffeae populations was low. Among the P. coffeae populations
from Vietnam, the North Central Coast 1 population showed the highest difference
in pairwise distance (> 0.006; Table 4.4). All sequences of the D2/D3 28S rDNA
expansion segments of the 10 P. coffeae populations from Vietnam matched the
sequences obtained from GenBank with a similarity higher than 99% except with
those of the P. coffeae population from Ghana (B1-AF170433) and the two P.
jaehni populations (C1-AF170426 and C2-AF170427). The pairwise distance between
the 10 P. coffeae populations from Vietnam and the P. coffeae population from
Ghana was higher than 0.024. The D2/D3 28S rDNA expansion segments sequences
of the Northeast 4, Red River Delta 2 and North Central Coast 2 population had a
similarity of almost 100% with those of the P. coffeae populations from Honduras
(B2-AF170434), Brazil (M1-AF170435), the USA (M3-AF170436) and Martinique (Y1AF170430).
The maximum parsimony tree constructed on the basis of the D2/D3 28S
rDNA expansion segment sequences is shown in Figure 4.5. Eight out of ten P.
coffeae populations from Vietnam are placed in two exclusive groups. The first
group contains the Northwest, Red River Delta 2, Northeast 4 and Central Coast 1
populations with a bootstrap value of 63%. The second group contains the
Northeast 1, 2 and 3 populations and the Red River Delta 1 population with a
bootstrap value of 65%. These two groups are in turn part of a larger group with a
bootstrap value of 99% that includes the other two P. coffeae populations from
Vietnam (North Central Coast 2 and Central Highlands) and the other P. coffeae
populations obtained from GenBank with the exception of the P. coffeae
population from Ghana (B1-AF170433). The two other Pratylenchus species (P.
jaehni and P. loosi) included in the analysis are clearly separated from all the P.
coffeae populations (bootstrap value 59%).
Chapter 4
77
K6- AF170443
B1- AF170433
NW
M1- AF170435
Y1- AF170430
B2- AF170434
NE1
NCC2
NE3
Y2- AF170431
RRD1
M3- AF170436
Y3- AF170432
NE4
NCC1
RRD2
C1- AF170426
C2- AF170427
C6- AF170429
C4- AF170428
CH
NE2
10
....|....|
GCACTTTGAA
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
20
....|....|
GAGAGAGTTA
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
30
....|....|
AAGAGGACGT
..........
..........
..........
..........
..........
..A.......
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
40
....|....|
GAAACCGATG
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
50
....|....|
AGATGGAAAC
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
60
....|....|
GGACAGAGCT
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
K6- AF170443
B1- AF170433
NW
M1- AF170435
Y1- AF170430
B2- AF170434
NE1
NCC2
NE3
Y2- AF170431
RRD1
M3- AF170436
Y3- AF170432
NE4
NCC1
RRD2
C1- AF170426
C2- AF170427
C6- AF170429
C4- AF170428
CH
NE2
70
....|....|
AGCGTATCTG
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
.......A..
..........
..........
..........
........C.
........C.
..........
..........
..........
..........
80
....|....|
GCCTGCATTC
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
A.T.......
A.T.......
..........
..........
..........
..........
90
....|....|
AGCTTGTGCG
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
.........A
.........A
..........
..........
..........
..........
100
....|....|
GTCGCTACCG
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
......G..C
......G..C
..........
..........
..........
..........
110
....|....|
ATGAATCGCT
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
120
....|....|
GATCTCCAGA
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..C.......
..C.......
..........
..........
..........
..........
Figure 4.4: Alignment of the D2/D3 28S rDNA expansion segments sequences of 10
Pratylenchus coffeae populations from Vietnam and 10 P. coffeae populations as well as two
Pratylenchus jaehni populations obtained from GenBank. For the list with the abbreviations
of the population codes see Tables 2.6 and 4.2.
Chapter 4
78
K6- AF170443
B1- AF170433
NW
M1- AF170435
Y1- AF170430
B2- AF170434
NE1
NCC2
NE3
Y2- AF170431
RRD1
M3- AF170436
Y3- AF170432
NE4
NCC1
RRD2
C2- AF170427
C1- AF170426
C6- AF170429
C4- AF170428
CH
NE2
130
140
150
160
170
180
....|....| ....|....| ....|....| ....|....| ....|....| ....|....|
TTGGGACTGT TGACTAGTCG GTCGGTGGCT GTATAGTGCA TTTGCAGGTG GAGTGCGTCG
.......... .......... .......... .......... .......... ..........
.......... .......... .......... .......... .......... ..........
.......... .......... .......... .......... .......... ..........
.......... .......... .......... .......... .......... ..........
.......... .......... .......... .......... .......... ..........
.......... .......... .......... .......... .......... ..........
.......... .......... .......... .......... .......... ..........
.......... .......... .......... .......... .......... ..........
.......... .......... ........G. .......... .......... ..........
.......... .......... .......... .......... .......... ..........
.......... .......... .......... .......... .......... ..........
.......... .......... .......... .......... .......... ..........
.......... .......... .......... .......... .......... ..........
.......... .......... .......... .......... .......... ..........
.......... .......... .......... .......... .......... ..........
.....G.... ........G. .C.......G ..GC...... ......A... ..........
.....G.... ........G. .C.......G ..GC...... ......A... ..........
.......... .......... ...A...... .......... .......... ..........
.......... .......... ...A...... .......... .......... ..........
.......... .......... .......... .......... .......... ..........
.......... .......... .......... .......... .......... ..........
K6- AF170443
B1- AF170433
NW
M1- AF170435
Y1- AF170430
B2- AF170434
NE1
NCC2
NE3
Y2- AF170431
RRD1
M3- AF170436
Y3- AF170432
NE4
NCC1
RRD2
C1- AF170426
C2- AF170427
C6- AF170429
C4- AF170428
CH
NE2
190
....|....|
AGGCATCCGG
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
......T...
......T...
..........
..........
..........
..........
Figure 4.4. Continued.
200
....|....|
GATGGCGGCA
A.........
..........
..........
..........
..........
..........
..........
..........
..........
.G........
..........
..........
..........
..........
..........
........A.
........A.
..........
..........
..........
..........
210
....|....|
TGAACTCAGC
...G......
..G.......
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
......GG..
......GG..
....A.....
..........
..........
..........
220
....|....|
TTTGAGGCCA
..........
..........
..........
..........
..........
....G....G
..........
..........
..........
C.........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
230
....|....|
GCTTG----C
.....----.
.....----.
.....----.
.....----.
.....----.
.....----.
.....----.
.....----.
.....----.
.....----.
.....----.
.....----.
.....----.
.....----.
.....----.
.....----.
.....----.
.....----.
.....----.
.....----.
.....----.
240
....|....|
TGGTACCCGG
..........
..........
..........
..........
..........
..........
..........
..........
..........
..C.......
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
Chapter 4
79
K6- AF170443
B1- AF170433
NW
M1- AF170435
Y1- AF170430
B2- AF170434
NE1
NCC2
NE3
Y2- AF170431
RRD1
M3- AF170436
Y3- AF170432
NE4
NCC1
RRD2
C1- AF170426
C2- AF170427
C6- AF170429
C4- AF170428
CH
NE2
250
....|....|
GCTGGGGGAC
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
...T.....T
...T.....T
..........
..........
..........
..........
260
....|....|
TTTGTGCTGT
...A......
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..----....
..----....
..........
..........
..........
..........
270
....|....|
TCGTTCTGGG
..A.......
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
280
....|....|
TGTTCACACA
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
.....C....
.....C....
..........
..........
..........
..........
290
....|....|
CAAT-TGAAG
..T.-..GGC
....-.....
....-.....
....-.....
....-.....
....-.....
....-.....
....-.....
....-.....
....-.....
....-.....
....-.....
....-.....
....-.....
....-.....
..T.A..G.C
..T.A..G.C
....-.....
....-.....
....-.....
....-.....
300
....|....|
GGGGCTTTGAA.......C
........................................T.................T.................T.................................................AA.......C
AA.......C
...............................T....-
K6- AF170443
B1- AF170433
NW
M1- AF170435
Y1- AF170430
B2- AF170434
NE1
NCC2
NE3
Y2- AF170431
RRD1
M3- AF170436
Y3- AF170432
NE4
NCC1
RRD2
C1- AF170426
C2- AF170427
C6- AF170429
C4- AF170428
CH
NE2
310
....|....|
-------GTA
GGGTTTG...
-------...
-------...
-------...
-------...
-------...
-------...
-------...
-------...
-------...
-------...
-------...
-------...
-------...
-------...
GAGTTGG.CT
GAGTTGG.CT
-------...
-------...
-------...
-------...
320
....|....|
GGGTGCCGAG
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
.......-.A
.......-.A
..........
..........
..........
..........
330
....|....|
CTGGGTGTC..........................................................................................T
..................................................A........A.......................................-
340
....|....|
GGTGGCGGTC
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
A.........
..........
..........
..........
..........
..........
..........
..........
350
....|....|
GCTTGC-GAC
......C...
......-...
......-...
......-...
......-...
......-...
......-...
......-...
......-...
......-...
......-...
......-...
......-...
..C...-...
......-...
..A...-...
..A...-...
......-...
......-...
......-...
......-...
360
....|....|
ACGTACTGTG
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
Chapter 4
80
K6- AF170443
B1- AF170433
NW
M1- AF170435
Y1- AF170430
B2- AF170434
NE1
NCC2
NE3
Y2- AF170431
RRD1
M3- AF170436
Y3- AF170432
NE4
NCC1
RRD2
C1- AF170426
C2- AF170427
C6- AF170429
C4- AF170428
CH
NE2
370
....|....|
CACACAGTTC
..AT......
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
.CTCTG...T
.CTCTG...T
..........
..........
..........
..........
380
....|....|
GGTCCTTGCC
.........T
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
......G.T.
......G.T.
..........
..........
..........
..........
390
....|....|
GAGCTCACTC
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
A.....CAC.
A.....CAC.
..........
..........
..........
..........
400
....|....|
CAT-----CT
..CATGTC..
...-----A.
...-----..
...-----..
...-----..
...-----..
...-----..
...-----..
...-----..
...-----..
...-----..
...-----..
...-----..
T..-----..
...-----..
TC.--GTT.G
TC.--GTT.G
...-----..
...-----..
...-----..
...-----..
410
....|....|
ATCTCGGCGT
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
CG....A...
CG....A...
..........
..........
.........A
..........
420
....|....|
AAAAGCTGGT
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
K6- AF170443
B1- AF170433
NW
M1- AF170435
Y1- AF170430
B2- AF170434
NE1
NCC2
NE3
Y2- AF170431
RRD1
M3- AF170436
Y3- AF170432
NE4
NCC1
RRD2
C1- AF170426
C2- AF170427
C6- AF170429
C4- AF170428
CH
NE2
430
....|....|
CATCTTTCCG
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..G.......
..........
..........
..........
..........
..........
440
....|....|
ACCCGTCTTG
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
450
....|....|
AAACACGGAC
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
460
....|....|
CAAGGAGTTT
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
470
....|....|
AT-CGTGT-G
..-.....-.
..-.....-.
..-.....-.
..-.....-.
...-.......-.....-T
..-.....-.
..-.....-.
..-.....-.
..-.....-.
..-.....-.
..-.....-.
..-.....-.
..-.....-.
..-.....-.
..T...T.T.
..-.....GC
..-.....-.
..-T..T.T.
..-.....-.
..-.....-.
480
....|....|
CGCGAGTCAT
........T.
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..G.......
G.........
..........
..........
..........
..........
Chapter 4
81
K6- AF170443
B1- AF170433
NW
M1- AF170435
Y1- AF170430
B2- AF170434
NE1
NCC2
NE3
Y2- AF170431
RRD1
M3- AF170436
Y3- AF170432
NE4
NCC1
RRD2
C1- AF170426
C2- AF170427
C6- AF170429
C4- AF170428
CH
NE2
490
....|....|
TGGGTGTTCA
...AC.....
....C.....
....C.....
....C.....
....C.....
....C.....
....C.....
....C.....
....C.....
....C.....
....C.....
....C.....
....C.....
....C.....
....C.....
....C...G.
....C...G.
....C.....
....C.....
....C.....
....C.....
500
....|....|
AAACCCAAAG
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
510
....|....|
GCGTAATGAA
...C......
...C......
...C......
...C......
...C......
...C......
...C......
...C......
...C......
...C......
...C......
...C......
...C......
...A......
...C......
...C......
...C......
...C......
...C......
...C......
...C......
520
....|....|
AGTGAACGTT
.........A
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
530
540
....|....| ....|....|
TCCGC-TAG GAGCCAACGTG
....T-.... ..........
....T-.... ..........
....T-.... ..........
....T-.... ..........
....T-.... ..........
....T-.... ..........
....T-.... ..........
....T-.... ..........
....T-.... ..........
....T-.... ..........
....T-.... ..........
....T-.... ..........
....T-.... ..........
....T-.... ..........
....T-.... ..........
...ATA.C.. ....G.....
...ATA.C.. ....G.....
....T-.... ..........
....T-.... ..........
....T-.... ..........
....T-.... ..........
K6- AF170443
B1- AF170433
NW
M1- AF170435
Y1- AF170430
B2- AF170434
NE1
NCC2
NE3
Y2- AF170431
RRD1
M3- AF170436
Y3- AF170432
NE4
NCC1
RRD2
C1- AF170426
C2- AF170427
C6- AF170429
C4- AF170428
CH
NE2
550
....|....|
CGATCCTGGT
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
560
....|....|
CACCACGGTG
....G.....
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
....G.....
....G.....
..........
..........
..........
..........
570
....|....|
GCCGGGCGCA
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
...A......
...A......
..........
..........
..........
.........G
580
....|....|
GCATGGCCCC
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
C.........
590
....|....|
ATCCTGACTG
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
....C.....
....C.....
..........
..........
..........
..........
600
....|....|
CTTGCAGTGG
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
Chapter 4
K6- AF170443
B1- AF170433
NW
M1- AF170435
Y1- AF170430
B2- AF170434
NE1
NCC2
NE3
Y2- AF170431
RRD1
M3- AF170436
Y3- AF170432
NE4
NCC1
RRD2
C1- AF170426
C2- AF170427
C6- AF170429
C4- AF170428
CH
NE2
K6- AF170443
B1- AF170433
NW
M1- AF170435
Y1- AF170430
B2- AF170434
NE1
NCC2
NE3
Y2- AF170431
RRD1
M3- AF170436
Y3- AF170432
NE4
NCC1
RRD2
C1- AF170426
C2- AF170427
C6- AF170429
C4- AF170428
CH
NE2
82
610
....|....|
GGTGGAGGAA
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
620
....|....|
GAGCGTACGC
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
630
....|....|
GGTGAGACCC
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
.A........
.A........
..........
..........
..........
..........
640
....|....|
GAAAGATGGT
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
650
....|....|
GAACTATTCC
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
670
....|....|
TGAGCAGGAT
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
.........C
.........C
..........
..........
..........
..........
680
690
700
710
720
730
....|....| ....|....| ....|....| ....|....| ....|....| ....|....|
AAAGCCAGAG GAAACTCTGG TGGAAGTCCG AAGCGATTCT GACGTGCAAA TCGATCGTCT
.......... .......... .......... .......... .......... ..........
.......... .......... .......... .......... .......... ..........
.......... .......... .......... .......... .......... ..........
.......... .......... .......... .......... .......... ..........
.......... .......... .......... .......... .......... ..........
.......... .......... .......... .......... .......... ..........
.......... .......... .......... .......... .......... ..........
.......... .......... .......... .......... .......... ..........
.......... .......... .......... .......... .......... ..........
.......... .......... .......... .......... .......... ..........
.......... .......... .......... .......... .......... ..........
.......... .......... .......... .......... .......... ..........
.......... .......... .......... .......... .......... ..........
.......... .......... .......... .......... .......... ..........
.......... .......... .......... .......... .......... ..........
G......... .......... .......... .......... .......... ..........
G......... .......... .......... .......... .......... ..........
.......... .......... .......... .......... .......... ..........
.......... .......... .......... .......... .......... ..........
.......... .......... .......... .......... .......... ..........
.......... .......... .......... .......... ....................
Chapter 4
K6- AF170443
B1- AF170433
NW
M1- AF170435
Y1- AF170430
B2- AF170434
NE1
NCC2
NE3
Y2- AF170431
RRD1
M3- AF170436
Y3- AF170432
NE4
NCC1
RRD2
C1- AF170426
C2- AF170427
C6- AF170429
C4- AF170428
CH
NE2
83
740
....|....|
GACTTGGGTA
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
.......-..
..........
..........
750
760
....|....| ....|....|
TAGGGGCGAA AGACTAATC
........... ........
........... ..A.....
........... ........
........... .-......
.. ........ ........
........... ........
........... ........
........... ........
........... ........
........... A.......
........... ........
........... ........
........... ..A.....
........... ..A.....
........... ..A.....
........... ..-.....
........... ..-.....
........... ........
.....-..... ........
........... ........
........... ........
84
Chapter 4
Table 4.4: Pairwise distances between 10 Pratylenchus coffeae populations from Vietnam, 10 P.
from GenBank based on D2/D3 28S rDNA expansion segments sequences.
C2 C1 NW
NCC1 NE4 RRD2 NE2
RRD1 NE3 NE1
C4
CH
NCC2
C2
0.01 0.09 0.09
0.08
0.08
0.09
0.09
0.09
0.09
0.09
0.09
0.08
C1
6
0.09 0.09
0.09
0.09
0.09
0.09
0.09
0.09
0.08
0.09
0.09
NW
63 64
0.008 0.003 0.003 0.008 0.011 0.006 0.011 0.009 0.006 0.004
NCC1 62 63
6
0.006 0.006 0.011 0.014 0.008 0.014 0.011 0.008 0.007
NE4 61 62
2
4
0.00
0.005 0.008 0.003 0.003 0.003 0.003 0.001
RRD2 61 62
2
4
0
0.003 0.008 0.003 0.003 0.003 0.003 0.001
NE2 64 65
6
8
4
4
0.008 0.003 0.082 0.009 0.005 0.004
RRD1 66 67
8
10
6
6
6
0.005 0.011 0.011 0.008 0.007
NE3 62 63
4
6
2
2
2
4
0.005 0.005 0.003 0.001
NE1 65 67
8
10
6
6
6
8
4
0.011 0.008 0.007
C4
65 62
6
8
4
4
6
8
4
8
0.005 0.004
CH
62 63
4
6
2
2
4
6
2
6
4
0.001
NCC2 61 62
3
5
1
1
3
5
1
5
3
1
M3
61 62
3
5
1
1
3
5
1
5
3
1
0
C6
62 63
5
7
3
3
5
7
3
7
3
3
2
Y3
62 63
4
6
2
2
4
6
2
6
4
2
1
Y2
62 63
4
6
2
2
4
6
2
6
4
2
1
B2
61 62
3
5
1
1
3
5
1
5
3
1
0
Y1
61 62
3
5
1
1
3
5
1
5
3
1
0
M1
61 62
3
5
1
1
3
5
1
5
3
1
0
K6
63 64
6
7
4
4
6
8
4
8
6
4
3
B1
67 68
21
23
19
19
21
23
19
23
21
19
18
Below diagonal: total character differences.
Above diagonal: mean character differences (adjusted for missing data).
For the list with the abbreviations of the population codes see Tables 2.6 and 4.2.
coffeae and two Pratylenchus jaehni populations obtained
M3
0.08
0.09
0.004
0.007
0.001
0.001
0.004
0.007
0.001
0.007
0.004
0.001
0.000
2
1
1
0
0
0
3
18
C6
0.09
0.09
0.007
0.01
0.004
0.004
0.007
0.009
0.004
0.01
0.004
0.004
0.003
0.003
3
3
2
2
2
5
20
Y3
0.09
0.09
0.006
0.008
0.003
0.003
0.005
0. 008
0.003
0.008
0.005
0.003
0.001
0.001
0.004
2
1
1
1
4
19
Y2
0.09
0.09
0.006
0.008
0.003
0.003
0.005
0.008
0.003
0.007
0.005
0.003
0.001
0.001
0.004
0.003
1
1
1
4
19
B2
0.08
0.09
0.004
0.007
0.001
0.001
0.004
0.007
0.001
0.007
0.004
0.001
0.000
0.000
0.003
0.001
0.001
0
0
3
18
Y1
0.08
0.09
0.004
0.007
0.001
0.001
0.004
0.007
0.001
0.007
0.004
0.001
0.000
0.000
0.003
0.001
0.001
0.000
0
3
18
M1
0.08
0.09
0.004
0.007
0.001
0.001
0.004
0.007
0.001
0.007
0.004
0.001
0.000
0.000
0.003
0.001
0.001
0.000
0.000
3
18
K6
0.09
0.09
0.008
0.01
0.005
0.005
0.008
0.011
0.005
0.01
0.008
0.005
0.004
0.004
0.007
0.005
0.005
0.004
0.004
0.004
21
B1
0.09
0.09
0.029
0.031
0.026
0.026
0.028
0.03
0.026
0.03
0.029
0.026
0.024
0.025
0.023
0.026
0.026
0.025
0.025
0.025
0.029
-
Chapter 4
85
P. coffeae (NW)
P. coffeae (RRD2)
P. coffeae (NE4)
P. coffeae (NCC1)
P. coffeae (NE2)
P. coffeae (NE1)
P. coffeae (NE3)
P. coffeae (RRD1)
P. coffeae (Florida, C4)
P. coffeae (Oman, C6)
P. coffeae (CH)
P. coffeae (Indonesia, K6)
P. coffeae (Brazil, M1)
P. coffeae (Martinique, Y1)
P. coffeae (Honduras, B2)
P. coffeae (Brazil, Y2)
P. coffeae (Puerto Rico, Y3)
P. coffeae (Florida, M3)
P. coffeae (NCC2)
P. coffeae (Ghana, B1)
P. jaehni (Brazil, C2)
P. jaehni (Brazil, C1)
P. loosi (Florida, N1)
Radopholus similis (C1)
Figure 4.5: Single maximum parsimony tree of 10 Pratylenchus coffeae populations from
Vietnam, 10 P. coffeae, two Pratylenchus jaehni, one Pratylenchus loosi and one Radopholus
similis population obtained from GenBank based on the sequence alignment of the D2/D3 28S
rDNA expansion segments. Bootstrap values > 50% are given in the appropriate clades. For the
list with the abbreviations of the population codes see Tables 2.6 and 4.2.
4.4
Discussion
RAPD bands analysis of genome clustered the 10 P. coffeae populations
from Vietnam examined in two groups. The largest group consisted of seven
nematode populations. This group included populations collected in all the
different agro-ecological regions of Vietnam: the only population collected in the
Central Highlands clustered together with both populations collected in the North
Chapter 4
86
Central Coast and four populations collected in the north. The smallest group
consisted of the remaining three nematode populations, all collected in the two
most northeastern situated agro-ecological regions (Red River Delta and
Northeast). These results indicate that genomic similarity as determined by RAPD
bands analysis of genome did not correspond with geographic proximity. Similar
results were obtained when Pinochet et al. (1994) compared the PCR-RAPD bands
of seven P. vulnus populations from different geographical areas (USA, Argentina,
France, Spain and Italy).
Mizukubo et al. (2003) examined the genomic similarity of 20 P. coffeae
populations isolated from 10 different agricultural crops in Japan using PCR-RFLP
analysis. Based on this analysis, they suggested the existence of at least three
groups of P. coffeae in Japan which they designated as A, B and C according to the
digestion pattern using Hinf I, Alu I, Dde I and Hha I restriction endonucleases.
Although the largest group included five out of the six P. coffeae
populations originally isolated from banana, our results indicate that genomic
similarity as determined by RAPD bands analysis of genome did also not correspond
with host plant origin. In the largest group also two P. coffeae populations
originally isolated from coffee were included while in the smallest group
populations originally isolated from banana, coffee and the only population
originally isolated from the roots of an ornamental tree were included. In contrast,
Siciliarno-Wilcken et al. (2002; abstract of a poster) reported that, based on PCRRAPD bands analysis, seven P. coffeae populations isolated from different host
plants and regions of Brazil could be grouped in four clusters according to the host
plants.
Our results show that the genetic differences among the P. coffeae
populations from Vietnam examined are low which indicates a low divergence.
Moreover, the sequences of the D2/D3 28S rDNA expansion fragments of all the P.
coffeae populations from Vietnam examined were closely related with the
sequences of the P. coffeae populations obtained from GenBank. Subbotin et al.
(2008) considered P. coffeae to be monophyletic but divided in three rDNA groups
based on their 28S Bayesian inference trees. In the study of Subbotin et al. (2008)
also the P. coffeae population originally isolated from Ghana was included and the
authors suggested that taxonomic clarification of this population is required. In our
study, based on RAPD bands analysis of genome, all the 10 P. coffeae populations
from Vietnam examined were to some extent genetically different from the P.
coffeae population originally isolated from banana in Ghana.
Partial or complete sequence analysis of D2/D3 28S rDNA expansion
segments have been used to separate different Pratylenchus species including P.
Chapter 4
87
coffeae (Duncan et al., 1999; Inserra et al., 2007; Subbotin et al., 2008). Our
sequence analysis clearly supports the separation of the 10 P. coffeae populations
from Vietnam examined from P. jaehni and P. loosi.
Interestingly, two out of three P. coffeae populations originally isolated
from coffee in Vietnam (one collected in the North Central Coast, the other one
collected in the Central Highlands) appears to be more related with the P. coffeae
populations obtained from GenBank which were all originally isolated from coffee
from a wide range of countries from the Americas (USA, Honduras, Brazil), the
Caribean (Puerto Rico, Martinique), Africa (Ghana) and Asia (Oman, Indonesia).
4.5
Conclusions
RAPD bands analysis of the complete genome clustered the 10 P. coffeae
populations from Vietnam examined in two groups. However, genomic similarity
did not correspond nor with geographic origin or the host plants from which the
populations were originally isolated. Comparison and phylogenetic analysis of the
sequences of the D2/D3 28s rDNA expansion segments of the 10 P. coffeae
populations from Vietnam examined among themselves and with 10 P. coffeae
populations obtained from Genbank showed low divergence. Our observations
further indicate that the P. coffeae populations from Vietnam examined are
genetically divergent from the P. coffeae population originally isolated from
banana in Ghana.
Chapter 4
88
Chapter 5
89
Chapter 5: Effect of temperature on the in vitro
reproductive fitness of Pratylenchus coffeae
populations from Vietnam
5.1
Introduction
The biological diversity of a pathogen is also characterised by its
pathogenicity, defined as the ability of an organism to cause disease on host plants
(Inomoto et al., 2007). One of the major components of pathogenicity, in addition
to virulence, is the reproductive fitness (Shaner et al., 1992), which is defined as
the ability of a species or population to multiply on a specific host plant (Sarah &
Fallas, 1996; Inomoto et al., 2007). To compare the reproductive fitness of
Pratylenchus populations the use of in vitro monoxenic cultures, such as carrot disc
cultures, offers a suitable approach since this technique provides homogenous
environmental conditions including a constant temperature. In Radopholus similis,
a direct relationship was found between the in vitro reproductive fitness on carrot
discs of populations and their in vivo pathogenicity on banana roots: the higher the
reproductive fitness on carrot discs, the higher the pathogenicity on banana roots
(Sarah et al., 1993; Fallas et al., 1995). However, in other R. similis studies, high
in vivo reproductive fitness was not always related with high pathogenicity (Tarté
et al., 1981; Hahn et al., 1996).
Intraspecific differences in in vitro reproductive fitness on carrot discs
have been reported for several Pratylenchus species. This was the case for seven
populations of Pratylenchus vulnus from Argentina, USA, France, Spain and Italy
(originally isolated from rose, apple, apricot, walnut and olive; Pinochet et al.,
1994) and three populations of Pratylenchus sudanesis originating from Uganda
(originally isolated from yam tubers and roots; Mudiope et al., 2004). Pinochet et
al. (1994) found no relationship between the in vitro reproductive fitness on carrot
discs of the P. vulnus populations they examined and the host plants from which
the populations were originally isolated.
Stoffelen et al. (1999) compared the in vitro reproductive fitness on
carrot discs of three P. coffeae populations, all originally isolated from banana in
Honduras, Ghana and Vietnam. They observed that 11 weeks after inoculation with
25 females at 28oC, the density of the population from Honduras was about twice
as high as the densities of the populations from Ghana and Vietnam (a 827-fold
increase vs a 375.4 and 466.5-fold increase, respectively).
Chapter 5
90
Although data on intraspecific differences in in vitro reproductive fitness
in the genus Pratylenchus are rather scarce, the above mentioned observations
may indicate that within a Pratylenchus species different biotypes exist which
could result in differences in damage potential among populations of the same
Pratylenchus species.
So far, little is known about the effect of temperature on the in vitro
reproductive fitness of P. coffeae. In vivo, the optimum temperature for
penetration, colonisation and development of P. coffeae is 25-30oC (Yokoo &
Kukoda, 1966, cited by Radewald et al., 1971b; Ascota & Malek, 1979). In vitro, on
carrot discs, Pinochet et al. (1995) observed that the P. coffeae populations
examined (originally isolated from coffee in Guatemala) barely multiplied at 16oC
and that optimum population increase was recorded at 25oC, the highest
temperature used (the third temperature used was 21oC).
The objective of this part of our study was to examine the in vitro
reproductive fitness on carrot discs of the 10 P. coffeae populations collected from
different agricultural crops in different agro-ecological regions in Vietnam (see
Chapter 2). In a first experiment, the in vitro reproductive fitness of all 10 P.
coffeae populations from Vietnam was compared among each other and with the in
vitro reproductive fitness of a P. coffeae population from Ghana originally isolated
from banana (Stoffelen et al., 1999). In a second experiment, the effect of
temperature on the in vitro reproductive fitness on carrot discs of three selected
P. coffeae populations from Vietnam (collected in three different agro-ecological
regions: Northwest, North Central Coast and Central Highlands) was compared over
time.
5.2
Ten P. coffeae populations originally isolated from banana, coffee and the
roots of an ornamental tree collected in different agro-ecological regions in
Vietnam and one P. coffeae population from Ghana originally isolated from banana
(Table 2.6) were studied. The population from Ghana has previously been studied
(Stoffelen et al., 1999). The nematode populations were maintained in vitro on
monoxenic carrot discs at 25oC in the dark (Speijer & De Waele, 1997).
Nematode inoculum was obtained from in vitro carrot disc cultures with
vigorous developing nematode populations (many active nematodes in the Petri
dish). Under a laminar flow, the Petri dishes were rinsed with sterile water and the
Chapter 5
91
nematodes collected in a sterile test tube. When the nematodes had settled on the
bottom of the tube, the sterile water on the top was removed with a sterile
pipette. To surface-sterilise the nematodes, 1 ml of 6,000 ppm streptomycin
sulphate was added to 2 ml of nematode suspension to obtain a final concentration
of 2,000 ppm streptomycin sulphate. The test tube was shaken and kept at 25oC in
the dark for one night. Then, the streptomycin sulphate solution was removed by
three rinses in sterile water. The surface-sterilised nematodes were used for
inoculation on the carrot discs.
5.2.2 Experimental set-up
Two separate experiments were carried out.
In the first experiment, the in vitro reproduction of all 10 P. coffeae
populations from Vietnam and a population from Ghana (Table 2.6) was determined
on carrot discs 10 weeks after inoculation with 25 mature females. For each
population, 10 Petri dishes each containing one carrot disc (considering each Petri
dish as one replicate) were incubated at 25oC in the dark.
In the second experiment, the effect of temperature on the reproduction
of three selected P. coffeae populations from Vietnam (Northwest, North Central
Coast 2 and Central Highlands) was determined on carrot discs 4, 8, 12 weeks after
inoculation with 25 mature females at 15, 20, 25 and 30oC. For each population, 30
Petri dishes, each containing one carrot disc (considering each Petri disc as one
replicate) were incubated in the dark.
Preparation of the carrot discs was as described in section 2.7 of Chapter 2.
The mature females were individually collected with a micropipette and
placed in a drop of sterile water on the surface of the carrot discs. Before
incubation, the Petri dishes were sealed with parafilm. The Petri dishes were
arranged in a completely randomised design.
5.2.3 Assessment of the nematode reproduction
Nematodes were extracted from the carrot discs by the macerationsieving technique as described by Speijer and De Waele (1997). The Petri dishes
and carrot discs were rinsed with distilled water to collect the nematodes that had
migrated out of the carrot discs. To extract the nematodes from inside the carrot
discs, each carrot disc was macerated in a kitchen blender with distilled water
three times during 10 sec (seconds) with 5-sec intervals. The suspension was passed
through a series of 106, 25 and 5 µm-pore sieves. The residue on the sieves was
Chapter 5
92
rinsed with tap water. Carrot disc debris collected on the 106 µm sieve was
discarded. The nematodes on both the 25 µm and 5 µm sieves were collected.
With a light microscope, the number of eggs, juveniles, females and males
were counted separately in 2 ml subsamples. The sum of the number of eggs,
juveniles, females and males was considered the final nematode population density
(Pf). Reproduction factor (RF) = Pf/Pi (Pi: initial nematode population density =
inoculum).
5.2.4 Data analysis
Data were analysed with the STATISTICA® package (Anonymous, 1997).
Average
female:male
ratio
and
final nematode
population
density
(first
experiment) and nematode reproduction factor (first and second experiment) were
analysed by an one-way ANOVA. Before analysis of variance (ANOVA), nematode
data were log10(x+1) transformed to meet the assumptions of ANOVA (i.e. normality
and homogenity of variances). Subsequently, the means were separated using
Tukey’s Honestly Significant Difference test (P ≤ 0.05).
The transformed data of the final nematode population densities (Pf) were
subjected to a quadratic model. Regression analysis was used to understand the
statistical dependence of nematode population density on temperature and time.
This statistical analysis can show what proportion of variance between variables is
due to the dependent variables and what proportion is due to the independent
variables. The relationship between the variables can be illustrated graphically or
described as an equation. Coefficients of determination (R2) were used to
determine appropriateness of the model (Hicks, 1973; Draper & Smith, 1998).
5.3
Results
5.3.1 In vitro reproductive fitness of 10 Pratylenchus coffeae
populations from Vietnam and a P. coffeae population
from Ghana
Table 5.1 summarises the results of the first experiment: the in vitro
reproduction on carrot discs of the 10 P. coffeae populations from Vietnam and the
P. coffeae population from Ghana, 10 weeks after inoculation with 25 mature
females, incubated at 25oC.
All populations completed their life cycle and multiplied very well as
indicated by a reproduction factor (RF) > 500. The population from Ghana had
increased most (2,334.1-fold) while the Red River Delta 2 and North Central Coast
Chapter 5
93
1 populations had increased least (518.1 and 614.8-fold, respectively) compared
with all other populations. Half of the populations from Vietnam has increased
significantly (P ≤ 0.05) less than the population from Ghana. The Red River Delta 2
and North Central Coast 1 populations had significantly (P ≤ 0.05) lower RF than
three other populations (Northeast 3, North Central Coast 2 and Central Highlands).
In all the populations, the majority of the populations consisted of eggs,
varying from 49% (North Central Coast 1 population) to 72% (Central Highlands
population). The percentage of juveniles ranged from 20% (Central Highlands
population) to 33% (North Central Coast 2 population).
With the exception of the Red River Delta 2 population, the number of
females and males was about similar, with a female:male ratio ranging from 0.8 to
1.2. In the Red River Delta 2 population, the female:male ratio was 1.5 and this
value was significantly (P ≤ 0.05) different from all other populations except the
North Central Coast 1 population which had a female:male ratio of 1.2. In all
populations, except the North Central Coast 1 population, the percentage of
females and males combined ranged from 8% (Central Highlands population) to 15%
(Northwest population). In the North Central Coast 1 population, the percentage of
females and males combined was 21%.
Chapter 5
94
Table 5.1: Comparative in vitro reproduction on carrot discs of 10 Pratylenchus coffeae populations from Vietnam and one P. coffeae population from
Ghana, 10 weeks after inoculation with 25 females, incubated at 25oC in the dark.
Population
Eggs
Juveniles
Northwest
13,075
(55)*
7,384
(30)*
Northeast 1
13,539
(62)
6,243
(27)
Northeast 2
20,745
(69)
6,660
Northeast 3
34,462
(66)
Northeast 4
18,818
Red River Delta 1
Females
1,728
Males
Female:male ratio
Pf
RF
(7)*
1,769
(8)*
1.0 : 1 a
23,956
958.3 ab
993
(4)
1,263
(6)
0.8 : 1 a
22,038
881.5 ab
(23)
1,410
(5)
1,323
(4)
1.0 : 1 a
30,138
1205.5 abc
11,294
(23)
2,750
(5)
3,088
(6)
1.0 : 1 a
51,594
2063.8 bc
(66)
7,518
(24)
1,425
(5)
1,429
(5)
0.9 : 1 a
29,189
1167.6 abc
16,301
(65)
5,430
(22)
1,571
(6)
1,709
(7)
1.0 : 1 a
25,010
1000.4 ab
Red River Delta 2
8,235
(62)
3,263
(26)
904
(8)
551
(5)
1.5 : 1 b
12,953
518.1 a
7,843
(49)
4,623
(32)
1,567
(11)
1,337
(10)
1.2 : 1 ab
15,370
614.8 a
25,626
(57)
13,029
(33)
1,881
(5)
2,319
(6)
0.8 : 1 a
42,855
1714.2 bc
Central Highlands
31,374
(72)
9,183
(20)
1,773
(4)
1,623
(4)
1.0 : 1 a
43,953
1758.1 bc
Ghana
38,354 (66)
13,962 (24)
2,998 (5)
3,038 (5)
1.0 : 1 a
Pf: final nematode population density (sum of the number of eggs, juveniles, females and males).
RF: reproduction factor = Pf/Pi (Pi: initial nematode population density = inoculum).
n: number of replicates.
*: numbers between parentheses are percentages of Pf.
Means in the same column followed by the same letter do not differ significantly according to Tukey’s test (P ≤ 0.05).
58,352
2334.1
c
Chapter 5
95
5.3.2 Effect of temperature on the reproductive fitness of three
Pratylenchus coffeae populations from Vietnam
Table 5.2 summerises the results of the second experiment: the in vitro
reproduction on carrot discs of three P. coffeae populations from Vietnam, 4, 8 and
12 weeks after inoculation with 25 mature females, incubated at 15, 20, 25 and
30oC.
At 15oC, the three populations had multiplied by a RF > 1 except the
Central Highlands population at 12 weeks after inoculation. At 12 weeks after
inoculation, the RF of this population was significantly (P ≤ 0.05) lower compared
with the Northwest population (0.6 vs 2.4, respectively).
From 20oC onwards, the RF was always higher than 1 and increased with
increasing temperature and increasing time except at 30oC at 12 weeks after
inoculation when all three populations decreased. At 20oC, at 8 weeks after
inoculation, the RF of the Central Highlands population was significantly (P ≤ 0.05)
lower compared with the Northwest population (9.6 vs 27.7, respectively). At 30oC,
at 4 weeks after inoculation, the RF of the Central Highlands population was
significantly (P ≤ 0.05) lower compared with both the Northwest and North Central
Coast 2 populations (52.2 vs 113.6 and 108.2, respectively). At this temperature, at
8 weeks after inoculation, the Northwest population has increased about 2,500-fold
while the North Central Coast 2 and Central Highlands populations had increased
about 1,500-fold. At 30oC, at 12 weeks after inoculation, the RF of all three
populations decreased substantially but this decrease was more outspoken in the
Northwest and Central Highlands populations compared with the North Central
Coast 2 population (25.1 and 13.5 vs 163.5, respectively; P ≤ 0.05).
At 15oC, the number of females extracted was always lower than 25 in all
three populations but sometimes a few (< 10) males were observed. At 20oC (and
higher temperatures), from 8 weeks after inoculation onwards, the number of
females extracted was always higher than 25 and this number increased with
increasing temperature and increasing time. This trend was also observed for the
males. However, at 30oC, at 12 weeks after inoculation, the number of both
females and males decreased.
Chapter 5
96
Table 5.2: Effect of time and temperature on the in vitro reproduction factor (RF) on carrot discs of three Pratylenchus coffeae populations from Vietnam
after inoculation with 25 females, incubated at 15, 20, 25 and 30oC.
RF (4 WAI)
RF (8 WAI)
RF (12 WAI)
Temperature
___________________________
________________________________
___________________________
(oC)
NW
NCC2
CH
NW
NCC2
CH
NW
NCC2
CH
15
1.5
1.3
1.6 n.s.
3.4
2.5
2.6 n.s.
2.4 b
1.3 ab
0.6 a
20
5.1
5.9
4.1 n.s.
27.7 b
18.1 ab
9.6 a
63.0
86.1
80.1 n.s.
25
11.4 ab
14.3 b
8.4 a
192.0
195.0
187.4 n.s.
755.8
798.2
709.8 n.s.
30
113.6 b
108.2 b
52.2 a
2507.3 b
1474.0 a
1663.6 ab
25.1 a
163.5 b
13.5 a
Temperature
Female (4 WAI)
Female(8 WAI)
Female (12 WAI)
_____________________________________________________________
______________________________________________________________________
______________________________________________________________
o
( C)
NW
NCC2
CH
NW
NCC2
CH
NW
NCC2
CH
15
11
12
10 n.s.
14
7
11
8
0
4
20
16
16
11 n.s.
62
65
46 n.s
132
175
144 n.s
25
37
50
36 n.s.
370
402
347 n.s
1515
1761
1485 n.s
30
179
117
123 n.s.
4156 b
2450 ab
2155 a
106 a
1344 b
59 a
Temperature
Male (4 WAI)
Male(8 WAI)
Male (12 WAI)
______________________________________________________________
_____________________________________________________________________
______________________________________________________________
o
( C)
NW
NCC2
CH
NW
NCC2
CH
NW
NCC2
CH
15
2
0
0
5
3
3
8
0
2
20
6b
2a
4 ab
65 b
62 ab
21 a
157
162
94 n.s
25
41 b
50 b
11 a
390 ab
485 b
266 a
1140
1461
1670 n.s
30
174 b
141 b
71 a
3150
2487
1855 n.s.
109 b
587 c
22 a
RF: reproduction factor = Pf/Pi (Pf: final nematode population density (sum of the number of eggs, juveniles, females and males)/Pi: initial nematode
population density = inoculum).
Means in rows at 4, 8 and 12 weeks after inoculation followed by the same letter do not differ significantly according to Tukey’s test (P < 0.05).
n.s. indicates no significant diference according to the analysis of variance (ANOVA) P < 0.05).
WAI: weeks after inoculation.
NW: Northwest population; NCC2: North Central Coast 2 population; CH: Central Highlands population.
Chapter 5
97
Figure 5.1 illustrates the results of the second experiment. It is clear that
the temperature affected the population dynamics of the three P. coffeae
populations from Vietnam in a very similar way: at 15oC, almost no reproduction;
at 20 and 25oC, the reproduction increased throughout the duration of the
experiment; at 30oC, the reproduction increased until 8 weeks after inoculation but
decreased between 8 and 12 weeks after inoculation.
5
Northwest population
Log (Pf + 1)
4
3
2
1
0
0
2
4
6
8
10
12
14
10
12
14
10
12
14
Weeks after inoculation
5
North Central Coast 2 population
Log (Pf + 1)
4
3
2
1
0
0
2
4
6
8
We e ks afte r inoculation
5
Central Highlands population
Log (Pf + 1)
4
3
2
1
0
0
2
4
6
8
We eks after inoculation
Figure 5.1: Population dynamics of three Pratylenchus coffeae populations from Vietnam at
different temperatures (15, 20, 25 and 30oC). Pf: final nematode population density. ♦: 15oC;
■: 20oC; ▲: 25oC and ○: 30oC.
Chapter 5
98
Figure 5.2 illustrates the composition of the three P. coffeae populations
observed during the second experiment. At 15oC, the populations consisted mainly
of eggs. With increasing temperature, from 15 to 20oC and from 20 to 25oC, the
percentage of juveniles increased. At 30oC, at 4 and 8 weeks after inoculation, the
populations consisted mainly of eggs. At this temperature, at 12 weeks after
inoculation, apparently less eggs were laid while the percentage of males
increased.
The quadratic equation log10(x+1) = at2 + bt + c describes the effect of
temperature on the increase of the nematode population density over time, in
which x is the final population and t is time after inoculation (weeks). Based on
this quadratic equation, the optimum time after inoculation was determined to be
7, 8 and 7 weeks at 30oC at which the maximum final population density of the
Northwest, North Central Coast 2 and Central Highlands populations was obtained,
respectively (Table 5.3). At 25oC, the maximum final population density of the
North Central Coast 2, Northwest and Central Highlands populations was reached
much later compared with incubation at 30oC. At 25oC, maximum final population
density would be reached after 21, 23 and 32 weeks, respectively. Due to the low
nematode reproduction at 15 and 20oC, no regression analysis was performed for
these temperatures.
Table 5.3: Regression analysis of the population dynamics at different temperatures of three
P. coffeae population
Northwest
Noth Central Coast 2
Central Highlands
o
C
Quadratic equation
R2
Weeks*
25
Log10(x+1) = -0.0069t2 + 0.3232t + 1.3743
0.99
23
30
Log10(x+1) = -0.0631t2 + 0.8904t + 1.2741
0.93
7
25
Log10(x+1) = -0.0079t2 + 0.3367t + 1.3866
0.99
21
30
Log10(x+1) = -0.0475t2 + 0.7532t + 1.3516
0.98
8
25
Log10(x+1) = -0.0047t2 + 0.2999t + 1.3509
0.98
32
30
Log10(x+1) = -0.0588t2 + 0.8236t + 1.2457
0.89
7
*: time when the maximum final population was or could be reached was determined by
interpolation of the regression equations. It can be computed by the following formula: t = (b/2a). R2: coefficients of determination.
Chapter 5
99
80%
Males
60%
Females
40%
Juveniles
Eggs
20%
0%
NW
NCC2
CH
NW
NCC2
4 WAI
CH
NW
8 WAI
NCC2
Final nematode population (Pf)
100%
100%
80%
Males
60%
Females
Juveniles
40%
Eggs
20%
0%
NW
CH
NCC2
CH
NW
4 WAI
12 WAI
CH
NW
8 WAI
15
C
Ato15oC
NCC2
CH
12 WAI
o
C
At20
20oC
80%
60%
Males
Females
40%
Juveniles
Eggs
20%
100%
100%
Final nemtode population (Pf)
NCC2
80%
60%
Males
Females
40%
Juveniles
Eggs
20%
0%
0%
NW
NCC2
4 WAI
CH
NW
NCC2
8 WAI
o
C
At25
25oC
CH
NW
NCC2
12 WAI
CH
NW
NCC2
4 WAI
CH
NW
NCC2
8 WAI
CH
NW
NCC2
CH
12 WAI
o
At3030oC
C
Figure 5.2: Effect of time and temperature on the in vitro population composition on carrot discs of three Pratylenchus coffeae populations from Vietnam
after inoculation with with 25 females, incubated in the dark.
WAI: weeks after inoculation.
NW: population from Northwest region; NCC2: population from North Central Coast; CH: population from the Central Highlands.
Chapter 5
5.4
100
Discussion
Few differences in in vitro reproductive fitness on carrot discs were
observed among the P. coffeae populations collected from different agroecological regions in Vietnam with the exception of the Red River Delta 2 and North
Central Coast 1 populations which had a significantly lower reproduction factor
compared with the other populations from Vietnam examined. Interestingly, the
Red River Delta 2 population was the only population that was not originally
isolated from neither banana nor coffee but from the roots of an ornamental tree.
This indicates that the (unknown) ornamental tree is a less suitable host plant of P.
coffeae compared with banana and coffee since all but one of the other
populations from Vietnam were originally isolated either from banana or coffee.
Also interesting is the observation that the Red River Delta 2 population has a
significantly higher female to male ratio (1.5) compared with eight out of the 10 P.
coffeae populations from Vietnam examined. This observation suggests that the
host plant may affect the population composition of P. coffeae. In R. similis, a
population originally isolated from black pepper in Indonesia had significantly the
lowest in vitro reproductive fitness on carrot discs but a significantly different
female to male ratio compared with the four other R. similis populations examined
(Elbadri et al., 2001). But it is risky to make comparisons between P. coffeae and
R. similis as in the latter species there are much less males than females, the
females having the ability to reproduce without males.
The observation that the reproduction factors of five out of the 10 P.
coffeae populations from Vietnam examined were not statistically different from
the reproduction factor of the P. coffeae population from Ghana indicates that the
in vitro reproductive fitness on carrot discs of the P. coffeae populations from
Vietnam is similar to other P. coffeae populations found in other continents. A
similar observation was made by Stoffelen et al. (1999) when comparing another P.
coffeae population from Vietnam with the same P. coffeae population from Ghana
used in our study. This observation may also suggest that the pathogenicity of the
P. coffeae populations from Vietnam is similar to the pathogenicity of other P.
coffeae populations found in other countries.
The observation that the P. coffeae populations from Vietnam that did not
differ significantly in in vitro reproductive fitness on monoxenic carrot discs from
the P. coffeae population from Ghana were originally isolated as well from banana
(three populations) as from coffee (two populations) indicates that there is no
difference in reproductive fitness between P. coffeae populations originally
isolated from either banana or coffee. The P. coffeae population from Ghana was
originally isolated from banana.
Chapter 5
101
Obviously, P. coffeae is temperature-dependent. All three populations
from Vietnam examined reproduced at 15oC. However, at this temperature the
percentage of eggs was higher than the percentage of juveniles which may indicate
that the temperature was too low for normal hatching of the eggs. At 15oC at 4
weeks after inoculation only exceptionally males were observed. This may indicate
that cool temperature inhibits the development of males. However, over time at
15oC all development stages were observed which indicates that P. coffeae may
survive at this temperature. This tolerance to low temperatures (16 to 20oC) in P.
coffeae was also mentioned by Pinochet et al. (1995) and may explain why P.
coffeae can survive the cooler climates in the northern part of Vietnam.
Although the Northwest and North Central Coast 2 populations may have
been subjected to lower temperatures (in the Northwest region, the minimum
temperature may be 0oC during the winter; in the North Central Coast region, the
winter temperature ranges from 16 to 19oC) these populations have clearly not
adapted to cool conditions as has been shown for R. similis populations collected in
Europe (Elbadri et al., 2001). However, the observation that at 15oC (at 12 weeks
after inoculation) and at 20oC (at 8 weeks after inoculation), the reproduction
factor of the Northwest population was significantly higher compared with the
reproduction factor of the Central Highlands population may indicate that some
adaptation to cooler conditions might have taken place. In the Central Highlands of
Vietnam the average temperature is 22oC.
The highest population increase at 4 and 8 weeks after inoculation was
observed at 30oC. This observation is in line with earlier reports that P. coffeae
prefers a high temperature (Yokoo & Kukoda, 1966, cited by Radewald et al.,
1971b; Acosta & Malek, 1979; Duncan, 2005). The decrease of the reproduction
factors at 12 weeks after inoculation at 30oC in all three populations examined can
be explained by an exhaustion of the food source. Such a decline has been
observed in other in vitro carrot discs experiments and depends upon the initial
nematode population density, duration of the experiment and/or temperature (see
for instance Stoffelen et al., 1999).
The effect of temperature over time on the population dynamics of the
three P. coffeae populations from Vietnam examined was similar. Fallas and Sarah
(1995) reported that the population dynamcics of seven R. similis populations
which were originally isolated from banana in seven production areas worldwide
were in a similar way affected by temperatures over time. They considered this
behaviour as a character inherent to R. similis which seems to have resisted
modifications despite divergent evolution under different environmental conditions
at different geographical locations. Based on the regression analysis, the North
Chapter 5
102
Central Coast P. coffeae population seems to be more adaptive to temperature
than the other two P. coffeae populations from Vietnam examined. The reason for
this may be that this population has better adapted to cooler temperature than the
populations collected more north (Northwest) and more south (Central Highlands).
Based on the number of females observed, the life cycle of all three P.
coffeae populations examined was not completed at all at 15oC. At 20oC, the life
cycle was clearly completed within 8 weeks after inoculation while at 25 and 30oC,
the life cycle was already completed at 4 weeks after inoculation. A similar life
cycle duration at 25-30oC has been reported before (Gotoh, 1964, cited by Siddiqi,
1972; Thompson et al., 1973).
5.5
Conclusions
Our observations reveal few major differences in in vitro reproductive
fitness on carrot discs among the 10 P. coffeae populations from Vietnam examined
(with the exception of one population originally isolated from the roots of an
unidentified ornamental tree and one population originally isolated from banana),
and between these populations and a P. coffeae population originally isolated from
banana in Ghana. Our observations further indicate that although the optimum
temperature for reproduction of three P. coffeae populations from Vietnam
examined is 25 to, at least, 30oC, these populations are also tolerant to low
temperatures (15 to 20oC) enabling them to survive the low temperatures which
occur during the winter in the northern and central parts of Vietnam.
Chapter 6
103
Chapter 6: Host range characterisation, in vivo
reproduction and pathogenicity of Pratylenchus
coffeae populations from Vietnam
6.1
Introduction
Pratylenchus coffeae is one of the root-lesion nematodes that are
considered important plant pathogens. It has a worldwide distribution and a wide
host plant range (Siddiqi, 1972; Castillo & Vovlas, 2007). Over the years, when
more and more P. coffeae populations were found, differences in host range, in
vivo reproduction and pathogenicity on agricultural crops among some of these
populations were observed.
Populations of this species were found as an important pathogen of yams
in Uganda and the Pacific but did not invade the surrounding banana plants. In
contrast, in Ghana, a population of P. coffeae was found which damaged both yams
and plantains (Bridge et al. 1997). Edwards and Wehunt (1973) demonstrated that
P. coffeae populations from Panama can infect maize but those from Honduras not.
Silva and Inomoto (2002) reported that different populations of P. coffeae can have
different host ranges and suggested the existence of biotypes of P. coffeae based
on differences in in vivo reproduction on coffee and citrus between two P. coffeae
populations.
Other studies on bananas (Bridge et al., 1997), on sweet potato
(Mizukubo, 1995; Mizukubo & Sano, 1997) and on coffee (Kubo et al., 2003; Villain
et al., 2002 cited by Campos & Villain, 2005; Inomoto et al. 2007) showed
differences in pathogenicity among P. coffeae populations originating from
different geographical regions. Inoculation of seven different host plants with a P.
coffeae population originally isolated from coffee revealed differences in in vivo
reproduction and pathogenicity (Kumar & Viswanathan, 1972). Based on the in vivo
reproduction of P. coffeae populations on different agricultural crops and on
susceptible and resistant sweet potato cultivars, Mizukubo (1995) suggested the
presence of physiological races among Japanese P. coffeae populations.
In Vietnam, P. coffeae has been reported as the most common and
widespread Pratylenchus species (Chau & Thanh, 2002). In this country, it is found
on many crops including banana, coffee, ginger, sugarcane, pineapple, etc. (Chau
et al., 1997). Although it is known that P. coffeae can cause considerable damage
to several agricultural crops worldwide (Bridge et al., 1997), its impact on
agricultural crops in Vietnam is largely unknown. In fact, only damage caused by P.
Chapter 6
104
coffeae on bananas (Van den Bergh et al., 2006) and coffee, in relation with
yellow-leaf disease (Nghi et al., 1996; Trung et al., 2000; Sung et al., 2001), has
been studied.
The characterisation of intraspecific differences with respect to the host
range, in vivo reproduction and pathogenicity on agricultural crops among
populations of the same nematode species is very important for the development
of efficient and sustainable nematode management strategies such as crop rotation
(Bakker et al. 1993). In addition, this characterisation provides additional
information on the biodiversity of this nematode species. Therefore, the objectives
of this part of our study were a) to establish the host range of the 10 P. coffeae
populations collected from different agro-ecological regions in Vietnam and b) to
compare the in vivo reproduction and pathogenicity of these P. coffeae populations
on selected agricultural crops.
6.2
In total eight greenhouse experiments were conducted from March 2004 to
November 2006.
During these experiments, the soil temperature was measured. A
thermometer was put 8 cm deep in the soil in the pots and the temperature
recorded at the hottest time of the day, i.e. between 14:00 and 16:00. The
monthly minimum and maximum soil temperature in the greenhouse ranged from
20 to 32.5oC (Figure 6.1).
2004
2005
2006
Temperature (oC)
35
30
25
20
15
10
5
N
ov
.
O
ct
.
S
ep
.
A
ug
.
Ju
l.
Ju
n.
M
ay
A
pr
.
M
ar
.
0
Month
Figure 6.1: Average monthly soil temperature in the soil in the pots from March 2004 to
November 2006.
Chapter 6
105
6.2.1 Host range experiment set-up
To study the host range of the P. coffeae populations from Vietnam, 13
agricultural crops commonly grown in Vietnam were included in four experiments
(Table 6.1): soybean (Glycine max var. VX 93), groundnut (Arachis hypogea var.
V79), tomato (Solanum lycopersicum), banana (Musa cv. Ngop Dui Duc BBB), sweet
potato (Ipomoea batatas cv. Hoang Long), coffee (Coffea arabica var. Catimor),
ginger (Zingiber officinali cv. Rose), pinapple (Ananas comosus cv. Cayen), sesame
(Sesamum orientale
var. V67), upland rice (Oryza sativa
var. CIRAD 141),
sugarcane (Saccharum officinarum var. ROC 20), maize (Zea mays var. LVN 10) and
citrus (Citrus nobilis Lour. var. nobilis).
Table 6.1: Experiments carried out to study the host range, in vivo reproduction and
pathogenicity of 10 Pratylenchus coffeae populations from Vietnam on 13 agricultural crops
commonly grown in Vietnam.
Crop
Pratylenchus coffeae populations
NW
NE1
NE2
NE3
RRD1 RRD2 NCC1 NCC2
CH
Soybean
x
x
x
x
x
Groundnut
x
x
x
x
x
x
Tomato
x
x
x
x
x
x
Exp. 1
Banana
x
x
x
x
x
x
Sweet potato
x
x
x
x
x
x
Coffee
x
x
x
x
x
x
Coffee
x
x
x
x
x
x
x
x
x
Ginger
x
x
x
x
x
x
x
x
x
Pinapple
x
x
x
x
x
x
x
x
x
Exp. 2
Sesame
x
x
x
x
x
x
x
x
x
Banana
x
x
x
x
x
x
x
x
Rice
x
x
x
x
x
x
x
Sugarcane
x
x
x
x
x
x
x
x
x
Exp. 3
Maize
x
x
x
x
x
x
x
x
x
Banana
x
x
x
x
x
x
x
x
x
Exp. 4
Citrus
x
x
x
x
x
x
x
x
x
x: tested; -: not tested.
For the list with the abbreviations of the population codes see Table 2.6.
Seedlings were grown in plastic pots, each of which contained 2,000 ml of
a sterilised compost mixture of alluvial soil:composted manure:rice scale (6:1:2).
For coffee and citrus, seeds were first sown in trays containing sterilised sand and
seedlings with two fully expanded cotyledons were individually transplanted to the
plastic pots. For banana, sugarcane and pineapple, in vitro produced plantlets
were first transplanted to trays containing sterilised sand. After 4 weeks (for
banana and sugarcane) and 8 weeks (for pineapple), the plantlets were individually
transplanted to the plastic pots. For ginger, ginger tubers were first planted into
trays containing sterilised sand. After 1 month, uniformly sized plantlets were
individually transplanted to the plastic pots. For sweet potato, three-node stem
cuttings were individually planted in the plastic pots. For maize, sesame, tomato,
Chapter 6
106
upland rice, groundnut and soybean, seeds were directly sown in plastic pots: one
seed for maize, tomato, groundnut and soybean, two seeds for sesame and five
seeds for upland rice. The plants were watered as needed.
The banana, pineapple and sugarcane plantlets were inoculated 4 weeks
after transplanting. The sweet potato plantlets were inoculated 10 days after
planting of the nodal cuttings. Maize, sesame, tomato and rice seedlings were
inoculated 2 weeks after emergence of the seeds. The groundnut and soybean
seedlings were inoculated 10 days after the emergence of the seeds. The coffee
plants were inoculated 8 months after sowing when the seedlings were with eight
to 10 leaves (first experiment) and 2 months after sowing when two leaves were
expanded (second experiment). The citrus plantlets were inoculated 6 weeks after
emergence of the seeds.
The experiments were designed as completely randomised blocks with
nine replicates for experiment 1 and six replicates for the experiments 2, 3 and 4.
Each pot was considered a replicate. In the experiments 2 and 3, the banana plants
were used as the reference (control) host plant to confirm the viability of the
inoculum and the effectiveness of the inoculation.
6.2.2 Pathogenicity experiment set-up
To study the pathogenicity of the P. coffeae populations from Vietnam,
four agricultural crops commonly grown in Vietnam were included in four
experiments: banana (Musa cv. Ngop Dui Duc BBB), coffee (Coffea arabica var.
Catimor), sugarcane (Saccharum officinarum var. ROC 20) and maize (Zea mays
var. LVN 10). The preparation of the seedlings as well as the time of inoculation
and the experimental conditions were similar to those of the host range
experiments mentioned above.
The experiments were designed as completely randomised blocks with
seven replicates for banana, five replicates for coffee and six replicates for
sugarcane and maize.
6.2.3 Preparation of nematode inoculum and inoculations
The nematode inoculum was obtained from in vitro carrot disc cultures
with vigorous developing nematode populations (many active nematodes in the
Petri dish, see Chapter 2). Nematodes were extracted from the carrot discs by the
maceration-sieving technique as described by Speijer and De Waele (1997).
Suspensions of all vermiform developmental stages were used as inoculum for the
experiments. Each pot was inoculated with 1,000 vermiforms. Three holes were
Chapter 6
107
made in the soil around every plant, 5 ml of the nematode suspension was added
with a pippette and the holes were recovered with soil.
6.2.4 Assessment of the nematode reproduction
Fourteen weeks after inoculation, the plants were removed from the pots,
and the entire root systems and 200 ml of soil sampled. The nematode population
densities were assessed both in the soil and roots. For the extraction of the
nematodes from the plant roots, roots were cut into 1-cm-long pieces and
macerated in a kitchen blender for 30 sec (seconds; three 10-sec periods separated
by 5-sec intervals). The suspension was passed through 260, 106 and 40 µm-pore
sieves, rinsed with tap water and the nematodes from the 40 µm-pore sieve
collected and counted using a stereomicroscope. For the extraction of the
nematodes from the soil, the modified Baermann dish method was used (Hooper et
al., 2005). Two hundred ml of soil were placed on a sieve in a dish containing 300
ml of distilled water and left at room temperature for 48 hours. Then the
suspension in the dish was collected and the nematodes counted using a
stereomicroscope. The number of nematodes in the soil in the pots was determined
based on the number of nematodes in 200 ml of soil.
6.2.5 Assessment of the host plant range
The final nematode population density (Pf) was calculated as the number
of vermiform nematodes in both root and soil. The reproduction factor (RF =
Pf/Pi1) was used to determine the host plant response to P. coffeae. When RF > 1
the host plant was considered a good host. When RF < 1 but > 0.5 the host plant
was considered a poor host. When RF < 0.5 the host plant was considered a non
host (Pinochet & Duarte, 1986; Robinson & Percival, 1997; Bell & Watson, 2001).
6.2.6 Assessment of the pathogenicity
To assess the pathogenicity of the P. coffeae populations from Vietnam on
banana, coffee, sugarcane and maize, plant height, shoot and root fresh weights of
plants inoculated with the P. coffeae populations and uninoculated plants were
recorded at 14 weeks after inoculation.
For banana, the root necrosis was determined by following the
methodology of Speijer and De Waele (1997). Five 10 cm-long pieces of roots were
collected randomly and cut lengthwise. The percentage of necrosis was scored for
one half of each of the five roots. The maximum root necrosis per root half was
20% giving a maximum root necrosis of 100% for the five root halves together. For
_____________________________
1
Reproduction factor (RF) = final nematode population density (Pf)/initial nematode
population density (Pi = inoculum).
Chapter 6
108
sugarcane, 10 roots were collected randomly to assess the damage caused by P.
coffeae. The percentage of necrosis on the surface of each root was scored. The
maximum root necrosis per root was 10% giving a maximum root necrosis of 100%
for the 10 roots together.
6.2.7 Data analysis
For the statistical analysis of the results, the STATISTICA® package
(Anonymous, 1997) was used. The Shapiro-Wilk’s test was used to evaluate whether
the dependent variable was normally distributed within groups. The homogeneity
of the variances of the groups was tested with the Levene’s test. The nematode
population densities were log10(x+1) transformed before analysis. The root necrosis
data were arcsin(x/100) transformed before analysis. When less than 10 replicates
per group were available, the outliers were determined by calculating the
standardised residuals. Outliers were defined as data with a standardised residual
falling outside the range from -2 to 2.
One-way ANOVA was used to analyse the data. The means were separated
using Tukey’s Honestly Significant Difference test (P < 0.05).
6.3
Results
6.3.1 Host range of Pratylenchus coffeae populations from
Vietnam
The results of the four host range experiments are presented in Table
6.2. In the three experiments in which banana was included as one of the host
plants, all the P. coffeae populations examined reproduced very well on banana: in
the first experiment, the reproduction factor (RF) on banana ranged on average
from 16 to 27.2, in the second experiment from 28.9 to 37.7 and in the third
experiment from 29.4 to 42.5. The RF on banana among the P. coffeae populations
examined was more or less similar.
All P. coffeae populations examined reproduced on rice (with the
exception of the Red River Delta 1 population), sugarcane and maize. On these
three crops, RF between 2 and 5 were observed.
Chapter 6
109
Table 6.2: Reproduction factors (RF: final nematode population density/initial nematode population density) for 10 Pratylenchus coffeae populations from
Vietnam on 13 selected agricultural crops, 14 weeks after inoculation with 1,000 vermiforms/plant.
Crop
Pratylenchus coffeae population
Central
Northwest
Northeast 1
Northeast 2
Northeast 3
Red River
Red River
North
North
Highlands
Delta 1
Delta 2
Central
Central
Coast 1
Coast 2
Soybean
0.9
1.0
1.1
0.7
2.5
Groundnut
0
0
0
0
0
0
Exp. 1
Tomato
0.1
< 0.1
< 0.1
0.1
0.1
< 0.1
Banana
27.2
16.0
20.8
17.9
27.2
25.2
Sweet potato
0.1
0.1
0.1
0.3
0.1
0.1
Coffee
< 0.1
< 0.1
0
< 0.1
< 0.1
< 0.1
Coffee
0.1
< 0.1
0
0.1
0
< 0.1
< 0.1
0.2
< 0.1
Ginger
0
0
0
0
0
0
0
0
0
Pinapple
0
0
0
0
0
0
0
0
0
Exp. 2
Sesame
0.1
< 0.1
< 0.1
< 0.1
0
0.5
0.2
< 0.1
0
Banana
29.7
37.2
30.5
28.9
31.6
36.9
37.7
29.6
Rice
3.0
2.9
3.2
0.2
5.4
5.3
2.3
Exp. 3
Sugarcane
4.5
3.5
2.8
3.6
3.3
5.1
3.6
4.7
2.6
Maize
2.8
3.7
3.3
2.2
2.0
4.1
3.5
3.4
4.5
Banana
33.9
29.4
32.7
33.7
34.2
35.2
38.4
37.7
42.5
Exp. 4
Citrus
0
0
0
0
< 0.1
0
0.4
0.1
0
-: missing.
Chapter 6
110
On soybean, the RF of all the P. coffeae populations examined fluctuated
on average around 1 (0.7 to 2.5) while in the other eight crops included in the
experiments the RF was less than 1. No nematodes were extracted from the roots
of groundnut, ginger and pineapple.
6.3.2 In vivo reproduction and pathogenicity of Pratylenchus
coffeae from Vietnam
6.3.2.1
In vivo reproduction and pathogenicity of Pratylenchus
coffeae on banana
The results of the in vivo reproduction experiment on banana cv. Ngop Dui
Duc are presented in Table 6.3. Differences were observed in in vivo reproduction
on this banana cultivar among the 10 P. coffeae populations from Vietnam
examined. The highest average nematode population density observed per root
system (32,209, North Central Coast 1 population, originally isolated from banana)
was 7 times higher (P < 0.05) than the lowest average nematode population density
observed per root system (4,457, North Central Coast 2 population, originally
isolated from coffee). The RF of the Northwest, Northeast 4 and North Central
Coast 1 populations were on average significantly (P < 0.05) higher than the RF of
the Northeast 2, Red River Delta 2, North Central Coast 2 and Central Highlands
populations. The highest average nematode population density observed per 10 g
fresh roots (13,793, North Central Coast 1 population) was 15.4 times higher (P <
0.05) than the lowest average nematode population density observed per 10 g
fresh roots (893, North Central Coast 2 population). The average nematode
population densities of the population originally isolated from the roots of an
ornamental tree (Red River Delta 2) per root system and per 10 g fresh roots were
7,520 and 1,512, respectively, which was not significantly different from the
Northeast 2, Red River Delta 1, North Central Coast 2 and Central Highlands
populations which were originally isolated from banana and coffee.
Chapter 6
111
Table 6.3: In vivo reproduction and percentage root lesions caused by 10 Pratylenchus
coffeae populations from Vietnam on banana cv. Ngop Dui Duc, 14 weeks after inoculation
with 1,000 vermiforms/plant (n = 7).
P. coffeae
Average number of nematodes
RF
Root lesion
population
(%)
per 10 g fresh roots per root system
in soil
NW
10,280
e
31,561 c
3,745 35.3
d
36.4
d
NE1
4,547 cd
13,731 abc
3,782 17.5 bcd
25.0 bc
NE2
3,760 bcd
12,000 abc
2,837 14.8 abc
19.9 abc
NE3
3,940 cd
17,817 bc
3,622 21.4 bcd
20.4 abc
NE4
7,794
de
29,894 c
8,042 37.9
d
29.4 cd
RRD1
3,453 bc
9,518 ab
14,211
23.8 bcd
28.7 cd
RRD2
1,512 ab
7,520 ab
702
8.2 a
14.0 ab
NCC1
13,793
e
32,209 c
3,620 35.8
d
28.9 cd
NCC2
893 a
4,457 a
5,140
9.7 ab
9.7 a
CH
2,824 bc
14,939 abc
1,454 16.4 ab
21.3 bc
RF: final nematode population density/initial nematode population density (= inoculum).
Means in the same column followed by the same letter do not differ significantly according to
Tukey’s test (P < 0.05).
The relationship between the average nematode population densities per
10 g fresh roots and the percentage root necrosis is illustrated in Fig. 6.2. In
general, the nematode populations with the highest root population densities also
caused the highest percentage of root necrosis. The North Central Coast 2
population which had the lowest nematode population densities per root system
and per 10 g fresh roots still caused about 10% root necrosis, a percentage which
was not significantly different from the root necrosis caused by several other
20.0
45
18.0
40
16.0
35
14.0
30
12.0
25
10.0
20
8.0
15
6.0
4.0
10
2.0
5
0.0
0
NW
NE1
NE2
NE3
NE4
RRD1
RRD2
NCC1
NCC2
R o o t n e cro sis ( % )
N u m b e r o f n e m a to d e s x 1 , 0 0 0
populations such as the Northeast 2, Northeast 3 and Red River Delta 2 populations.
CH
Figure 6.2: Nematode population density per 10 g fresh roots and percentage root necrosis
caused by 10 Pratylenchus coffeae populations from Vietnam on banana cv. Ngop Dui Duc, 14
weeks after inoculation with 1,000 vermiforms/plant.
■: number of nematodes per 10 g fresh roots, ♦: percentage root necrosis.
Each point and bar is the mean ± standard error of 7 replicates.
Chapter 6
112
The results of the pathogenicity experiment on banana cv. Ngop Dui Duc
are presented in Table 6.4. The plant height, shoot and root fresh weights of this
banana cultivar were decreased by all the 10 P. coffeae populations from Vietnam
examined with 13.4-38.7%, 7.4-50.7% and 3.4-42.4%, respectively, compared with
the uninoculated control plants. Significant (P < 0.05) differences were observed in
reduction in plant height and shoot fresh weight among the populations examined.
For instance, the Northwest and Northeast 4 populations decreased these two plant
growth parameters significantly (P < 0.05) more than the Northeast 3, North
Central Coast 2 and Central Highlands populations. No differences were observed in
pathogenicity among the populations originally isolated from banana and coffee.
Also the Red River Delta 2 population originally isolated from the roots of an
ornamental tree caused a reduction in plant growth similar to most of the other
populations examined. Only the North Central Coast 1 population significantly (P <
0.05) decreased root fresh weight compared with the uninoculated control plants.
The lowest reduction (3.4%) in this plant growth parameter was observed in the
banana plants inoculated with the Red River Delta 2 population. In contrast, this
population reduced the plant height and shoot fresh weight with 30.8 and 32.6%,
respectively.
Table 6.4: Plant height, shoot and root fresh weights of banana cv. Ngop Dui Duc inoculated
with 10 Pratylenchus coffeae populations from Vietnam, 14 weeks after inoculation with
1,000 vermiforms/plant (n = 7).
P. coffeae
Plant height
Shoot weight
Root weight
population
(cm)
% change
(g)
% change
(g)
% change
NW
21.9 a
(-38.7)
63.7 a
(-49.9)
30.7 ab
(-35.5)
NE1
24.0 ab
(-32.8)
81.6 abc
(-35.8)
37.2 ab
(-21.8)
NE2
24.1 ab
(-32.5)
73.6 ab
(-42.1)
35.5 ab
(-25.4)
NE3
29.3 bcd
(-17.9)
112.5 bcd
(-11.5)
41.6 ab
(-12.6)
NE4
22.7 a
(-36.4)
71.6 a
(-43.7)
39.1 ab
(-17.9)
RRD1
23.7 ab
(-33.6)
62.7 a
(-50.7)
29.7 ab
(-37.6)
RRD2
24.7 abc
(-30.8)
85.7 abc
(-32.6)
46.0 b
(-3.4)
NCC1
26.4 abcd
(-26.1)
71.0 a
(-44.1)
27.4 a
(-42.4)
NCC2
30.9
de (-13.4)
116.7 cd
(-8.2)
41.5 ab
(-12.8)
CH
30.3 cde (-15.1)
117.7 cd
(-7.4)
41.0 ab
(-13.9)
Control
35.7
e
(0.0)
127.1
d
(0.0)
47.6 b
(0.0)
(% change compared with the uninoculated control plants).
6.3.2.2
coffeae from Vietnam on coffee
The results of the in vivo reproduction experiment on coffee var. Catimor
are presented in Table 6.5. The RF of all 10 P. coffeae populations examined was
on average less than 1. The highest average nematode population density observed
Chapter 6
113
per root system (212, Northeast 1 population, originally isolated from coffee) was
5.9 times higher (P < 0.05) than the lowest average nematode population density
observed per root system (36, North Central Coast 1 population, originally isolated
from banana). The RF of the Northeast 1 and North Central Coast 2 populations
were on average significantly (P < 0.05) higher than the RF of the Northwest, Red
River Delta 1, and North Central Coast 1 populations. The highest average
nematode population density observed per 1 g fresh roots (330, North Central Coast
2 population, originally isolated from coffee) was 7.7 times higher (P < 0.05) than
the lowest average nematode population density observed per 1 g fresh roots (43,
North Central Coast 1 population, originally isolated from banana). The average
nematode population densities of the population originally isolated from the roots
of an ornamental tree per root system and per 1 g fresh roots were 44 and 98,
respectively, which was not significantly different from several other populations
which were originally isolated from banana and coffee.
Table 6.5: In vivo reproduction of 10 Pratylenchus coffeae populations from Vietnam on
coffee cv. Catimor, 14 weeks after inoculation with 1,000 vermiforms/plant (n = 5).
P. coffeae
RF
population
per 1 g fresh roots
per root system
in soil
NW
65 ab
52 ab
10
0.1 ab
NE1
307 bc
212 b
80
0.3 c
NE2
109 abc
84 ab
80
0.1 abc
NE3
58 a
40 a
50
0.1 abc
NE4
150 abc
48 ab
50
0.1 abc
RRD1
78 ab
48 a
30
0.1 ab
RRD2
98 abc
44 a
70
0.1 abc
NCC1
43 a
36 a
10
< 0.1 ab
NCC2
330 c
100 ab
130
0.2 c
CH
107 abc
88 ab
70
0.2 bc
The results of the pathogenicity experiment on coffee var. Catimor are
presented in Table 6.6. At 14 weeks after inoculation, the root fresh weight of the
uninoculated control plants was on average 3.1 g while the root fresh weight of the
plants inoculated with the P. coffeae populations was on average less than 1. The
plant height, shoot and root fresh weights of this coffee variety were decreased by
all the 10 P. coffeae from Vietnam examined with 31.1-35.4%, 53.8-61.5% and 7190.3%, respectively, compared with the uninoculated control plants. No significant
differences were observed in reduction in plant height, shoot and root fresh
weights among the populations examined. Also, the Red River Delta 2 population
originally isolated from the roots of an ornamental tree caused a reduction in plant
growth similar to most of the other populations examined.
Chapter 6
114
Table 6.6: Plant height, shoot and root fresh weights of coffee cv. Catimor inoculated with 10
Pratylenchus coffeae populations from Vietnam, 14 weeks after inoculation with 1,000
vermiforms/plant (n = 5).
P. coffeae
Plant height
Shoot weight
Root weight
population
(cm)
% change
(g)
% change
(g)
% change
NW
14.0 a
(-33.0)
2a
(-61.5)
0.9 a
(-71.0)
NE1
14.1 a
(-32.5)
2.4 a
(-53.8)
0.9 a
(-71.0)
NE2
14.4 a
(-31.1)
2.3 a
(-55.8)
0.8 a
(-74.2)
NE3
14.3 a
(-31.6)
2.1 a
(-59.6)
0.8 a
(-74.2)
NE4
13.5 a
(-35.4)
2.0 a
(-61.5)
0.5 a
(-83.9)
RRD1
13.6 a
(-34.9)
2.0 a
(-61.5)
0.7 a
(-77.4)
RRD2
14.2 a
(-32.1)
2.2 a
(-57.7)
0.6 a
(-80.6)
NCC1
13.9 a
(-33.5)
2.1 a
(-59.6)
0.9 a
(-71.0)
NCC2
13.7 a
(-34.4)
2.2 a
(-57.7)
0.3 a
(-90.3)
CH
14.0 a
(-33.0)
2.1 a
(-59.6)
0.8 a
(-74.2)
Control
20.9 b
(0.0)
5.2 b
(0.0)
3.1 b
(0.0)
6.3.2.3
coffeae from Vietnam on sugarcane
The results of the in vivo reproduction experiment on sugarcane var.
ROC20 are presented in Table 6.7. Few differences were observed in in vivo
reproduction on this sugarcane variety among the 10 P. coffeae populations from
Vietnam examined. The highest average nematode population density observed per
root system (3,513, Red River Delta 2 population, originally isolated from the roots
of an ornamental tree) was 2.4 times higher (P < 0.05) than the lowest average
nematode population density observed per root system (1,448, Red River Delta 1
population, originally isolated from banana). The RF of the Red River Delta 2
population was significantly (P < 0.05) higher than the RF of the Central Highlands
population. The highest average nematode population density observed per 1 g
fresh roots (1,552, Red River Delta 2 population) was 4 times higher (P < 0.05) than
the lowest average nematode population density observed per 1 g fresh roots (391,
Red River Delta 1 population).
Chapter 6
115
Table 6.7: In vivo reproduction and percentage root lesions caused by 10 Pratylenchus
coffeae populations from Vietnam on sugarcane var. ROC20, 14 weeks after inoculation with
1,000 vermiforms/plant (n = 6).
P. coffeae
RF
Root lesion
population
(%)
per 1 g fresh roots
Per root system
in soil
NW
839 bcd
2,226 ab
3,500
4.5 ab
49.2 abc
NE1
692 abc
2,166 ab
1,393
3.6 ab
39.2 ab
NE2
473 ab
1,650 a
1,350
2.8 ab
25.8 a
NE3
889 bcd
2,326 ab
2,960
3.7 ab
35.8 a
RRD1
391 a
1,448 a
2,060
3.3 ab
29.2 a
RRD2
1,552
d
3,513 b
1,915
5.1 b
60.8 bc
NCC1
695 abc
1,986 ab
1,591
3.6 ab
62.5
c
NCC2
1,289 cd
2,873 ab
2,387
4.7 ab
62.5 c
CH
704 abc
1,530 a
1,086
2.6 a
28.3 a
The results of the pathogenicity experiment on sugarcane var. ROC20 are
presented in Table 6.8. All three plant growth parameters measured, plant height,
shoot and root fresh weights of this sugarcane variety were not significantly
decreased by the 10 P. coffeae populations from Vietnam examined, compared
with the uninoculated control plants. A few differences in pathogenicity were
observed among the P. coffeae populations examined. The North Central Coast 2
and Central Highlands populations caused a decrease in plant height of 16 and
18.1%, respectively, while the North Central Coast 1 population caused an increase
in plant height of 7.1% (P < 0.05). The Central Highlands population caused a
decrease in shoot fresh weight of 22.3% while the Red River Delta 2 population
caused an increase in shoot fresh weight of 13.3% (P < 0.05). The Central Highlands
population caused a decrease in root fresh weight of 30% while the Northeast 2 and
Red River Delta 1 populations caused an increase in root fresh weight of 10% (P < 0.05).
Table 6.8: Plant height, shoot and root fresh weights of sugarcane var. ROC20 inoculated with
10 Pratylenchus coffeae populations from Vietnam, 14 weeks after inoculation with 1,000
P. coffeae
Plant height
Shoot weight
Root weight
population
(cm)
% change
(g)
% change
(g)
% change
NW
37.3 ab
(-14.6)
38.7 ab
(-17.1)
2.6 abc
(-13.3)
NE1
39.7 ab
(-9.2)
39.2 ab
(-16.1)
3.2 bc
(6.7)
NE2
38.0 ab
(-13.0)
38.9 ab
(-16.7)
3.3
c
(10.0)
NE3
37.5 ab
(-14.2)
42.2 ab
(-9.6)
2.6 abc
(-13.3)
RRD1
41.2 ab
(-5.7)
39.7 ab
(-15.0)
3.3
c
(10.0)
RRD2
38.6 ab
(-11.7)
52.9 b
(13.3)
2.4 abc
(-20.0)
NCC1
46.8 b
(7.1)
47.8 ab
(2.4)
2.8 abc
(-6.7)
NCC2
36.7 a
(-16.0)
44.3 ab
(-5.1)
2.3 ab
(-23.3)
CH
35.8 a
(-18.1)
36.3 a
(-22.3)
2.1 a
(-30.0)
Control
43.7 ab
(0.0)
46.7 ab
(0.0)
3.0 abc
(0.0)
Chapter 6
116
The relationship between the average nematode population densities per
1 g fresh roots and the percentage root necrosis is illustrated in Fig. 6.3. In
general, the nematode populations with the highest root population densities also
caused the highest percentage of root necrosis. However, the North Central Coast 1
population which had one of the lowest nematode population densities per 1 g
fresh roots caused 62.5% root necrosis which was similar to the percentage root
necrosis caused by the North Central Coast 2 population from which almost twice
2000
80
1800
70
1600
60
1400
1200
50
1000
40
800
30
600
20
400
Root necrosis (%)
Number of nematodes
as much nematodes per 1 g roots were extracted.
10
200
0
0
NW
NE1
NE2
NE3
RRD1
RRD2
NCC1
NCC2
CH
Figure 6.3: Nematode population density per 10 g fresh roots and percentage root necrosis
caused by 10 Pratylenchus coffeae populations from Vietnam on sugarcane var. ROC20, 14
weeks after inoculation with 1,000 vermiforms/plant (n = 6).
■: number of nematodes per 10 g roots, ♦: percentage root necrosis.
Each bar and point are the mean ± standard error of 6 replicates.
6.3.2.4
coffeae from Vietnam on maize
The results of the in vivo reproduction experiment on maize var. LVN10
are presented in Table 6.9. Few differences were observed in in vivo reproduction
on this maize cultivar among the 10 P. coffeae populations from Vietnam
examined. For seven out of the nine P. coffeae populations examined, the average
nematode population density per root system ranged from about 1,000 to 1,800
while for eight out of the nine P. coffeae populations examined, the average
nematode population density per 10 g fresh roots ranged from about 500 and 1,000.
The highest average nematode population density observed per root
system (1,806, Red River Delta 2 population, originally isolated from the roots of
an ornamental tree) was 5.2 times higher (P < 0.05) than the lowest average
nematode population density observed per root system (346, Red River Delta 1
Chapter 6
117
population, originally isolated from banana). There was no significant difference in
RF among the populations examined. The highest average nematode population
density observed per 10 g fresh roots (1,074, Central Highlands population,
originally isolated from coffee) was 5.2 times higher (P < 0.05) than the lowest
average nematode population density observed per 10 g fresh roots (205, Red River
Delta 1 population).
Table 6.9: Reproduction of 10 Pratylenchus coffeae populations from Vietnam on maize var.
LVN10, 14 weeks after inoculation with 1,000 vermiforms/plant (n = 6).
P. coffeae
RF
population
per 10 g fresh roots
per root system
in soil
NW
535 ab
846 ab
2,000
2.8
NE1
616 ab
1,093 b
2,430
3.7
NE2
558 ab
1,186 b
2,086
3.3
NE3
507 ab
1,150 b
1,312
2.2
RRD1
205 a
346 a
2,300
2.0
RRD2
970 b
1,806 b
2,313
4.1
NCC1
689 ab
1,532 b
2,193
3.5
NCC2
848 b
1,410 b
2,392
3.4
CH
1,074 b
1,793 b
2,800
4.5
n.s.
n.s. indicates no significant diference according to the analysis of variance (ANOVA; P < 0.05).
The results of the pathogenicity experiment on maize var. LVN10 are
presented in Table 6.10. All three plant growth parameters measured, plant
height, shoot and root fresh weights, of this maize variety were not significantly
decreased by the 10 P. coffeae populations from Vietnam examined, compared
with the uninoculated control plants. A few differences in pathogenicity were
observed among the P. coffeae populations examined. The Red River Delta 2
population caused a decrease in plant height of 16.6% while the Northwest and
Northeast 3 populations caused an increase in plant height of 9.7 and 10.2%,
respectively (P < 0.05).
Chapter 6
118
Table 6.10: Plant height, shoot and root fresh weights of maize var. LVN10 inoculated with 10
Pratylenchus coffeae populations from Vietnam, 14 weeks after inoculation with 1,000
P. coffeae
Plant height
Shoot weight
Root weight
population
(cm)
% change
g)
(g)
NW
117.7 b
(9.7)
40.8
19.7
NE1
106.2 ab
(-1.0)
46.0
18.6
NE2
112.3 ab
(4.7)
54.1
22.2
NE3
118.2 b
(10.2)
45.7
21.6
RRD1
103.2 ab
(-3.8)
43.1
18.0
RRD2
89.5 a
(-16.6)
37.4
19.3
NCC1
110.8 ab
(3.3)
37.8
21.2
NCC2
105.8 ab
(-1.4)
41.9
16.1
CH
111.8 ab
(4.2)
41.3
16.1
Control
107.3 ab
(0.0)
46.3
21.1
n.s.
n.s.
n.s. indicates no significant diference according to the analysis of variance (ANOVA; P < 0.05).
Damage caused by P. coffeae on the roots of banana cv. Ngop Dui Duc,
coffee var. Catimor, sugarcane var. ROC20 and maize var. LVN10, 14 weeks after
inoculation with 1,000 vermiforms/plant is illustrated in Fig. 6.4.
A
C
B
D
Figure 6.4: Damage caused by Pratylenchus coffeae on the roots of banana (A), coffee (B),
sugarcane (C) and maize (D), 14 weeks after inoculation with 1,000 vermiforms/plant.
Chapter 6
6.4
119
Discussion
Soil temperature is an important environmental factor affecting the
reproduction of plant-parasitic nematodes including Pratylenchus spp. According to
Radewald et al. (1971b), Acosta and Malek (1979) and Gowen (2000), the optimum
temperature for development and reproduction of P. coffeae is 25 to 30oC. This is
precisely the temperature at which our experiments were carried out.
Out of the 13 agricultural crops included in our study, the reproduction
factor of all the P. coffeae populations from Vietnam examined was always > 1 on
banana, rice (with the exception of one population: Red River Delta 1), sugarcane
and maize and these crops can be considered as good hosts of this nematode
species. Compared with the latter three crops, banana is a much better host. The
host response of rice, sugarcane and maize was similar. On. No reproduction was
observed on the other eight crops. Thus, soybean, the reproduction factor
fluctuated around 1 (0.7 to 2.5) and therefore we consider this crop as a poor host,
groundnut, tomato, sweet potato, coffee, ginger, sesame, pinapple and citrus can
be considered as very poor hosts or non hosts of P. coffeae.
Our results confirm that banana is a good host of P. coffeae (Gowen et al.,
2005). The banana variety used in our experiments belongs to the BBB genome
group which was previously demonstrated as one of the most susceptible banana
varieties in Vietnam to P. coffeae (Van den Bergh, 2002). The good reproduction of
all the P. coffeae populations from Vietnam examined on this crop also confirms
the viability of the P. coffeae populations and effectiveness of the inoculation
method used in our experiments.
According to our results, rice, sugarcane and maize are good hosts of all
the P. coffeae populations from Vietnam examined while sweet potato, coffee and
citrus are very poor hosts. This observation is, in general, in contrast with the
nematological literature in which rice, sugarcane and maize are usually not
reported as good hosts of P. coffeae in contrast to sweet potato, coffee and citrus
which are usually reported as (very) good hosts of P. coffeae (Castillo & Vovlas,
2007). Interestingly, Silva and Inomoto (2002) made a more or less similar
observation when they characterised the host range of two P. coffeae populations
from Brazil and observed that coffee, citrus (Citrus limonia) and also banana were
not among the better host plants of these two populations but rather rice and
maize were the best host plants of the P. coffeae population originally isolated
from coffee. As mentioned in the Introduction above, also differences in in vivo
reproduction on Musa spp. (Bridge et al., 1997), maize (Edward & Wehunt, 1973;
Chapter 6
120
Silva & Inomoto, 2002), rice and sesame (Silva & Inomoto, 2002) among P. coffeae
populations have been reported before.
The contradictions among these studies suggest that the host status
classification of an agricultural crop based on the study of one cultivar or variety
only of this crop cannot be generalised. As emphasised by Jacobsen et al. (2009),
this classification might be influenced by several factors including host plant
response differences among cultivars and varieties, differences in pathogenicity
among nematode populations and methodological differences among studies. As
mentioned in the Introduction above, differences in pathogenicity among P.
coffeae populations originating from different geographical regions have been
reported. In the experiments conducted by Silva and Inomoto (2002), one of the
two P. coffeae populations from Brazil used was originally isolated from coffee
plants in the field but maintained on alfalfa callus before the host range
experiments were carried out. Culturing plant-parasitic nematodes in vitro on
monoxenic plant tissue cultures (such as alfalfa callus or carrot discs) might have
influenced the reproduction fitness, virulence and/or pathogenicity of the cultured
nematodes although we are not aware of any report in this respect. Finally,
methodological differences such as the time of sampling and the extraction method
used may also have contributed to the observed differences in host status among
the published studies.
Although three out of the 10 P. coffeae populations from Vietnam used in
our experiments were originally isolated from coffee plants in the field, none of
the nematode populations examined did reproduce well on the coffee variety used
in our experiments. This indicates that coffee var. Catimor is a poor host of P.
coffeae. In Brazil, Silva and Inomoto (2002) observed low reproduction (RF < 2.5)
on coffee 10 weeks after inoculation with 1,000 nematodes of a P. coffeae
population originally isolated from coffee roots in the field.
In their experiments with the two P. coffeae populations from Brazil, Silva
and Inomoto (2002) also observed that peanut was a poor host (RF < 1) which is in
agreement with our results while soybean was a good host (RF ranging from 2 to 3).
In our study, RF of soybean fluctuated around 1 (RF = 0.7 to 2.5).
The in vivo reproduction of all the P. coffeae populations from Vietnam
examined on the 13 agricultural crops was, in general, very similar. In some rare
cases differences were observed. On banana, reproduction of the Northwest,
Northeast 4 and North Central Coast 1 populations was about 4.5 times higher than
reproduction of the Red River Delta 2 population in one experiment but in another
experiment no differences in reproduction on banana among these populations
Chapter 6
121
were observed. On sugarcane, only the reproduction of two P. coffeae populations
(Red River Delta 2 and Central Highlands populations) was significantly different
from each other but the difference was small (RF = 5.1 vs 2.6). On rice, the Red
River Delta 1 population was the only population out of seven populations included
in the experiment that did not reproduce on this crop (RF = 0.2) but the
reproduction factor of the other six populations was also not very high ranging from
2.3 to 5.4. On maize, no differences in reproduction were observed among the
populations examined.
The results of the pathogenicity experiments carried out in our study
indicate that the P. coffeae populations from Vietnam examined were able to
cause considerable damage to the vegetative growth of banana and coffee but not
of sugarcane and maize. Our results confirm many earlier observations on the
percentage root lesion and damage P. coffeae can cause to banana (Gowen et al.,
2005) while there are, to our knowledge, no reports on damage caused by P.
coffeae to sugarcane (Cadet & Spaull, 1985, 2005) and maize (McDonald & Nicol,
2005).
Remarkably is the damage caused by all P. coffeae populations included in
the experiment on coffee in spite of the very low reproduction of these populations
(RF < 1). Compared with the uninoculated control plants, infection with P. coffeae
caused about 33% (31.1-35.4%) decrease in plant height, about 60% (53.8-61.5%)
decrease in shoot fresh weight and about 70 to 90% decrease in root fresh weight.
Fourteen weeks after inoculation, the root fresh weight of the uninoculated control
plants was only 3.1 g and the fragility of these roots may have caused a high
sensitivity to damage by P. coffeae. But it can also not be excluded that the coffee
var. Catimor is resistant to P. coffeae or that the P. coffeae populations are
extremely virulent on this coffee variety. Our results resemble results recently
published by Inomoto et al. (2007) who inoculated two coffee cultivars (Mundo
Novo and Catuai) with 8,000 vermiform nematodes of a P. coffeae population
originally isolated from coffee roots in the field and maintained on alfalfa callus
prior to inoculation. About 37 weeks after inoculation and in spite of a low
reproduction (RF < 1.5) they observed a 72 and 61% reduction in plant weight, 95
and 89% reduction in shoot fresh weight and 93 and 86% reduction in root fresh
weight, respectively. The average root fresh weight at 37 weeks after inoculation
of the uninoculated control plants was around 10 g. Our results thus confirm the
highly destructive nature of P. coffeae on coffee, especially coffee seedlings.
Pratylenchus coffeae has been reported as a very destructive nematode to coffee
in South America and Asia (Campos & Villain, 2005). This nematode species may
cause the destruction of the whole root system resulting in production losses up to
Chapter 6
122
80%, decay of coffee seedlings and trees leading to their death and even the
abandonment of coffee fields.
The in vivo pathogenicity of all the P. coffeae populations from Vietnam
examined on banana, coffee, sugarcane and maize was, in general, very similar. As
was the case for the in vivo reproduction, differences were only observed in some
very rare cases. Exceptionally, the effect on vegetative plant growth of one or a
few P. coffeae populations were significantly different either compared with the
uninoculated control plants or compared with the other P. coffeae populations.
Finally, our results did not indicate any relationship between host plant
range, in vivo reproduction and pathogenicity on the one hand and geographical
origin and host plant from which the P. coffeae populations were originally isolated
on the other hand.
6.5
Conclusions
Our observations indicate that of the 13 agricultural crops examined
banana, sugarcane, maize and upland rice are good hosts of the P. coffeae
populations from Vietnam examined. Soybean is a poor host while groundnut,
tomato, sweet potato, ginger, sesame, pineapple and citrus are very poor hosts or
non hosts. On coffee, the in vivo reproduction was very low but this is considered
the result of the high pathogenicity of the P. zeae populations on this crop
resulting in very few surviving roots. The in vivo reproduction on the 13 agricultural
crops examined and the in vivo pathogenicity on banana, coffee, sugarcane and
maize was, in general, very similar among the P. coffeae populations from Vietnam
examined.
Chapter 7
123
Chapter 7: General conclusions and perspectives
During 2000-2005, in total 95 soil and 95 root samples were collected from
21 agricultural crops in seven agro-ecological regions in Vietnam and examined for
the presence of Pratylenchus coffeae. About 25% of the root samples examined was
infected with this nematode species. Our observations confirm that P. coffeae is
the most common plant-parasitic nematode species associated with banana in
Vietnam, that it is common on coffee in this country and that its geographical
distribution is apparently restricted to North and Central Vietnam. The reason for
this remarkable geographical distribution remains unknown. More surveys in the
southern part of Vietnam shoud be carried out to confirm this geographical
distribution pattern. If P. coffeae is indeed absent in this part of Vietnam its
introduction should be prevented by inspecting planting material (for instance of
banana) that is being moved from North and Central Vietnam to South Vietnam for
contamination with P. coffeae. Ten of the collected P. coffeae populations,
isolated from banana, coffee and the roots of an (unidentified) ornamental tree,
were successfully in vitro established on carrot discs.
Although a detailed comparative morphological and morphometrical study
of the 10 P. coffeae populations from Vietnam in vitro established on carrot discs
revealed the presence of substantial variability in morphology and morphometry
within and between these populations, we conclude that these differences fall
within the range of the morphological and morphometrical variability described
previously in P. coffeae populations from other parts of the world. Our scanning
electron microscopy observations further confirm that in P. coffeae there is a
complete fusion of the 1st (lip) annule with the oral disc resulting in an undivided
en face view with no division between the lateral and median (sub-dorsal and subventral) segments of the 1st (lip) annule. This is an important morphological
character typical for this nematode species and a small number of related
Pratylenchus species. Although canonical discriminant analysis enabled the
separation of the 10 P. coffeae populations from Vietnam examined in three groups
based on a combination of five morphological characters for the males, there was
no relationship nor between these groups and their geographic origin or between
these groups and the host plants from which they were originally isolated.
As determined by RAPD bands analysis of the complete genome, genomic
similarity did not correspond nor with geographic or original host plant origin. As
determined by sequence analysis of the D2/D3 28S rDNA expansion fragments, all
10 P. coffeae populations from Vietnam examined were closely related among each
other and with the P. coffeae populations of which the D2/D3 28S rDNA expansion
Chapter 7
124
fragments sequences were obtained from GenBank. Based on these observations we
(also) conclude that P. coffeae is monophyletic. Interestingly, both our RAPD bands
analysis of the complete genome and our sequence analysis of the D2/D3 28S rDNA
expansion fragments indicate genetic divergence between the 10 P. coffeae
populations from Vietnam examined on the one hand and the P. coffeae population
from Ghana on the other hand, supporting previous suggestions made in the
nematological literature that taxonomic clarification of this population is required.
Few major differences in in vitro reproductive fitness on carrot discs were
observed among the 10 P. coffeae populations from Vietnam examined with the
exception of one population originally isolated from the roots of an (unidentified)
ornamental tree and one population originally isolated from banana (the other
eight P. coffeae populations we examined were originally isolated from either
banana or coffee). The results of these in vitro experiments, may suggest that the
host status (for instance poor host) of the plant from which a P. coffeae population
was originally isolated might have an effect on the in vitro reproductive fitness of
that population on carrot discs (for instance low reproduction). Further
experiments should be carried out to test this suggestion and, if this is indeed the
case, to examine if this effect is being maintained over time.
The in vitro reproductive fitness on carrot discs of five out of the 10 P.
coffeae populations from Vietnam examined were not statistically different from
the reproductive fitness of the P. coffeae population from Ghana. This observation
enables us to suggest that the pathogenicity of the P. coffeae populations from
Vietnam might be similar to the pathogenicity of the other P. coffeae populations
found in other countries. Also, no difference was observed in the in vitro
reproductive fitness on carrot discs between P. coffeae populations originally
isolated from either banana or coffee.
Pratylenchus coffeae is
temperature-dependent and the
optimum
temperature for reproduction of this nematode species is 25 to, at least, 30oC. On
the basis of our in vitro temperature experiments, we can conclude that the three
P. coffeae populations examined are tolerant to low temperatures (15 to 20oC) and
that this enables the P. coffeae populations in Vietnam to survive the low
temperatures which occur during the winter in the northern and central parts of
the country. We found some preliminary indication that there might be differences
in adaptability to low temperatures among the three P. coffeae populations
examined but this observation requires more investigation.
In general, the in vivo reproduction of all the 10 P. coffeae populations
from Vietnam on the 13 agricultural crops included in our experiments was very
Chapter 7
125
similar. This supports our finding based on the in vitro reproductive fitness on
carrot discs experiments that the 10 P. coffeae populations from Vietnam
examined might belong to the same biotype.
Based on our in vivo greenhouse experiments, we can confirm that banana
is a good host of the P. coffeae populations from Vietnam examined. Surprisingly,
rice, sugarcane and maize appeared also to be good hosts of P. coffeae while sweet
potato, coffee and citrus are very poor hosts. This observation is, in general, in
contrast with the nematological literature in which rice, sugarcane and maize are
usually not reported as good hosts of P. coffeae in contrast to sweet potato, coffee
and citrus which are usually reported as (very) good hosts of this nematode
species. Additional experiments should be carried out to clarify these contradictory
findings. It is possible that the host plant specificity is (plant) genotype-dependent.
In general, the in vivo pathogenicity on banana, coffee, sugarcane and
maize of all the 10 P. coffeae populations from Vietnam examined was very
similar. These populations were able to cause considerable damage to the
vegetative growth of banana and coffee but not of sugarcane and maize. In view of
the low reproduction on coffee, the extensive damage the P. coffeae populations
from Vietnam examined caused on this agricultural crop is surprising and illustrates
the high damage potential of P. coffeae on coffee.
Based on all our observations we conclude that all 10 Vietnamese
Pratylenchus populations included in our study belong to the same species: P.
coffeae.
Chapter 7
126
References
127
References
Acosta N. and Ayala A. 1975. Pathogenicity of Pratylenchus coffeae, Scutellonema
bradys, Meloidogyne incognita and Rotylenchulus reniformis on Dioscorea
rotundata. Journal of Nematology 7: 1-6.
Acosta N. and Ayala A. 1976. Effect of Pratylenchus coffeae and Scutellonema
bradys alone and in combination on Guinea Yam (Dioscorea rotundata).
Journal of Nematology 8: 315-317.
Acosta N. and Malek R.B. 1979. Influence of temperature on population
development of eight species of Pratylenchus on soybean. Journal of
Nematology 11: 229-232.
Adiko A. 1988. Plant-parasitic nematodes associated with plantain, Musa
paradisiaca (AAB), in the Ivory Coast. Revue de Nématologie 11: 109-113.
Agrios G.N. 2005. Plant pathology (5th edition). Academic Press, San Diego, USA,
922 p.
Andrés M.F., Pinochet J., Hernández-Dorrego A. and Delibes A. 2000. Detection and
analysis of inter- and intraspecific diversity of Pratylenchus spp. using isozyme
markers. Plant Pathology 49: 640-649.
Anonymous. 1997. STATISTICA® Release 5. Statsoft Inc., Tulsa, USA.
Anonymous. 2008. Taro pest: an illustrated guide to pests and diseases of taro in
the South Pacific. Australian Centre for International Agricultural Research:
60-61.
Bajaj H.K. and Bhatti D.S. 1984. New and known species of Pratylenchus Filipjev,
1936 (Nematoda: Pratylenchidae) from Haryana, India, with remarks on
interspecific variations. Journal of Nematology 16: 360-367.
Bakker J., Folfertsma R.T., Rouppe van der Voort J.N.A.M., de Boer J.M. and
Gommers F.J. 1993. Changing concepts and molecular approaches in the
management of virulence genes in potato cyst nematodes. Annual Review of
Phytopathology 31: 169-190.
Baldridge G., O’Neill N. and Samac D. 1998. Alfalfa (Medicago sativa L.) resistance
to the root-lesion nematode, Pratylenchus penetrans: defence-response gene
mRNA and isoflavonoid phytoalexin levels in roots. Plant Molecular Biology 38:
999–1010.
References
128
Bell N.L. and Watson R.N. 2001. Identification and host range assessment of
Paratylenchus nanus (Tylenchida: Tylenchulidae) and Paratrichodorus minor
(Triplonchida: Trichodoridae). Nematology 3: 483-490.
Brentu C.F., Speijer P.R., Green K.R., Hemeng B.M.S., De Waele D. and Coyne D.L.
2004. Micro-plot evaluation of the yield reduction potential of Pratylenchus
coffeae, Helicotylenchus multicinctus and Meloidogyne javanica on plantain
cv. Apantu-pa (Musa spp., AAB-group) in Ghana. Nematology 6: 455-462.
Bridge J. 1988. Plant nematode pest of banana in East Africa with particular
reference to Tanzania. In: Nematodes and the bore weevil in bananas: present
status of research and outlook, Proceedings of a Workshop, International
Network for the Improvement Banana and Plantain (INIBAP), Burundi, 7-11
Dec. 1987: 35-39.
Bridge J., Coyne D.L. and Kwoeseh C.K. 2005. Nematode parasites of tropical root
and tuber crops (excluding potatoes). In: Luc M., Sikora R.A. and Bridge J.
(eds), Plant-parasitic nematodes in subtropical and tropical agriculture (2nd
edition). CAB International, Wallingford, UK: 221-258.
Bridge J., Fogain R. and Speijer P. 1997. The root lesion nematodes of banana:
Pratylenchus coffeae (Zimmermann, 1898) Filipjev and Schuurmans Stekhoven
1941. Pratylenchus goodeyi Sher and Allen, 1953. Musa Pest Fact Sheet No. 2.
International Network for the Improvement of Banana and Plantain (INIBAP),
Montpellier, France, 4 p.
Bridge J., Hunt D.J. and Hunt P. 1996. Plant-parasitic nematodes of crops in Belize.
Nematropica 26: 111-119.
Bridge J., Price N.S. and Patrick K. 1995. Plant-parasitic nematodes of plantain and
other crops in Cameroon, West Africa. Fundamental and Applied Nematology
18: 251-260.
CABI/EPPO, 2007. Pratylenchus coffeae. Distribution Maps of Plant Diseases, Map
No. 816, CAB International, Wallingford, UK.
Cadet P. and Spaull V.W. 1985. Studies on the relationship between nematodes and
sugarcane in South and West Africa: plant cane. Revue de Nématologie 8: 131142.
Cadet P. and Spaull V.W. 2005. Nematode parasites of sugarcane. In: Luc M., Sikora
R.A. and Bridge J. (eds), Plant-parasitic nematodes in subtropical and tropical
agriculture (2nd edition), CAB International, Wallingford, UK: 645-674.
References
129
Café Filho A.C. and Huang C.S. 1989. Description of Pratylenchus pseudofallax n.
sp. with a key to species of the genus Pratylenchus Filipjev, 1936 (Nematoda:
Pratylenchidae). Revue de Nématologie 12: 7-15.
Campos V.P. and Villain L. 2005. Nematode parasites of coffee and cocao. In: Luc
M., Sikora R.A. and Bridge J. (eds), Plant-parasitic nematodes in subtropical
and tropical agriculture (2nd edition), CAB International, Wallingford, UK: 529580.
Castillo P. and Vovlas N. 2007. Pratylenchus, (Nematoda, Pratylenchidae):
diagnosis, biology, pathogenicity and management. Nematology Monographs
and Perspectives 6, 529 p.
Chau N. N., N. V. Thanh, D. De Waele D and E. Geraert 1997. Plant-parasitic
nematodes associated with banana in Vietnam. International Journal of
Chau N.N. and Thanh N.V. 2000. Plant-parasitic nematodes. The Fauna in Vietnam
No. 4, The Science and Technique Publishing House, 401 p. (In Vietnamese).
Coomans A. 2000. Nematode systematics: past, present and future. Nematology 2:
3-7.
Coomans A. 2002. Present status and future of nematode systematics. Nematology
4: 573-582.
Coomans A., De Coninck L. and Heip C. 1978. Data to be considered in descriptions
of new species or redescriptions of poorly known species. Annals of the Royal
Zoological Society of Belgium 108: 119-122.
Corbett D.C.M. 1969. Pratylenchus pinguicaudatus n. sp. (Pratylenchinae,
Nematoda) with a key to the genus Pratylenchus. Nematologica 15: 550-556.
Corbett D.C.M. and Clark S.A. 1983. Surface characters in the taxonomy of
Pratylenchus species. Revue de Nématologie 6: 85-98.
Cuc N.T.T., Phien T.V., Hieu T.V., Hau T.V. and Bien L.V. 1990. Review of the
nematode situation on coffee in Tien Giang, Ben Tre and Hau Giang. Plant
Protection Institution 3: 14-16. (in Vietnamese).
De Grisse A.T. 1969. Rédescription ou modification de quelques techniques
utilisées
dans
l’étude
des nématodes phytoparasitaires.
Rijksfakulteit Landbouwwetenschappen Gent 34: 351-369.
Mededelingen
References
130
de la Peña E., Karssen G. and Moens M. 2007. Distribution and diversity of rootlesion nematodes (Pratylenchus spp.) associated with Ammophila arenaria in
coastal dunes of Western Europe. Nematology 9: 881-901.
De Ley P., Felix M.A., Frisse L.M., Nadler S.A., Sternberg P.W. and Thomas W.K.
1999. Molecular and morphological characterisation of two reproductively
isolated species with mirror-image anatomy (Nematoda: Cephalobidae).
De Luca F., Fanelli E., di Vito M., Reyes A. and de Giorgi C. 2004. Comparison of
the sequences of the D3 expansion of the 26S ribosomal genes reveals different
degrees of heterogeneity in different populations and species of Pratylenchus
from the Mediterranean region. European Journal of Plant Pathology 110: 949957.
De Waele D. and Elsen A. 2007. Challenges in tropical plant nematology. Annual
Review of Phytopathology 45: 457-485.
De Waele D. and Elsen A. 2002. Migratory endoparasites: Pratylenchus and
Radopholus species. In: Starr J.L., Cook R., and Bridge J. (eds), Plant
Resistance to Parasitic Nematodes, CABI International, Wallingford, UK: 175–
206.
Díaz-Silveira M.F. and Herrera J.O. 1998. An overview of nematological problems in
Cuba. Nematropica 28: 151-163.
Draper N.R. and Smith H. 1998. Applied regression analysis (3rd edition). New York:
A Wiley-Interscience Publication, USA, 706 p.
Duncan L.W. 2005. Nematode parasites of citrus. In: Luc M., Sikora R. A. and Bridge
J. (eds), Plant parasitic nematodes in subtropical and tropical agriculture (2nd
edition), CAB International, Wallingford, UK: 437-466.
Duncan L.W. and Moens M. 2006. Migratory endoparasitic nematodes. In: Perry R.N.
and Moens M. (eds), Plant nematology, CAB International, Wallingford, UK:
123-152.
Duncan L.W., Inserra R.N., Thomas W.K., Dunn D., Mustika I., Frisse L.M., Mendes
M.L., Morris K. and Kaplan D.T. 1999. Molecular and morphological analysis of
isolates of Pratylenchus coffeae and closely related species. Nematropica 29:
61-80.
References
131
Edwards D.I. and Wehunt E.J., 1973. Hosts of Pratylenchus coffeae with additions
from Central American banana producing areas. Plant Disease Reporter 57: 4750.
Elbadri G.A.A., De Waele D. and Moens M. 2001. Reproduction of Radopholus
similis isolates after inoculation of carrot disks with one or more females.
Esser R.P. 1969. Data summary. Pratylenchus coffeae (Mimeo). Florida Department
of Agriculture and Consumer Services, Division of Plant Industry, Gainesville,
32 p.
Fallas G. and Sarah J. 1995. Effect of temperature on the in vitro multiplication of
seven Radopholus similis populations from different banana producing zones of
the world. Fundamental and Applied Nematology 18: 445-449.
Fallas G.A., Sarah J.L. and Fargette M. 1995. Reproductive fitness and
pathogenicity of eight Radopholus similis populations on banana plants (Musa
AAA cv. Poyo). Nematropica 25: 135-141.
Frederick J.J. and Tarjan A.C. 1989. A compendium of the genus Pratylenchus
Filipjev, 1936 (Nemata: Pratylenchidae). Revue de Nématologie 12: 243-256.
Geraert E. 2006. Functional and detailed morphology of the Tylenchida
(Nematoda). Nematology Monographs and Perspectives 4, 215 p.
Geraert E. and Raski D.J. 1987. A reappraisal of Tylenchina (Nematoda). 3. The
family Tylenchidae Örley, 1880. Revue de Nématologie 10: 143-161.
Golden A.M., Róger López Ch. and Hernán Vilchez R. 1992. Description of
Pratylenchus gutierrezi n. sp. (Nematoda: Pratylenchidae) from coffee in
Costa Rica. Journal of Nematology 24: 298-304.
Gowen S. Quénéhervé, P. and Fogain, R. 2005. Nematode parasites of bananas and
plantains. In: Luc M., Sikora R.A. and Bridge J. (eds), Plant-parasitic
nematodes in subtropical and tropical agriculture (2nd edition), CAB
International, Wallingford, UK: 431-460.
Gowen S. R. 2000. Nematode pathogens: root-lesion nematodes. In: Jones D. R.
(ed.), Diseases of banana, abaca and ensete, CABI Publishing, Singapore: 303306.
Hahn M.L., Sarah J.L., Boisseau M., Vines N.J., Wright D.J. and Burrows P.R. 1996.
Reproductive fitness and pathogenicity of selected Radopholus populations on
two banana cultivars. Plant Pathology 45: 223-231.
References
132
Handoo Z.A. and Golden M. 1989. A key and diagnostic compendium to the species
of the genus Pratylenchus Filipjev, 1936 (lesion nematodes). Journal of
Handoo Z.A.; Carta L.K., and Skantar A.M. 2001. Morphological and molecular
characterisation of Pratylenchus arlingtoni n. sp., P. convallariae and P. fallax
(Nematoda: Pratylenchidae). Nematology 3: 607-618.
Hartley J.L. and Xu L. 1994. DNA mass ladder: estimation of PCR products in gels.
Focus 16: 52-53.
Hernández M., Jordana R., Goldaracena A. and Pinochet J. 2001. SEM observations
of nine species of the genus Pratylenchus Filipjev, 1936 (Nematoda:
Pratylenchidae). Journal of Nematology, Morphology and Systematics 3: 165174.
Hicks C.R. 1973. Fundamental concepts in the design of experiments. Hold,
Rinehart and Winston, USA, 349 p.
Hooper D.J.J., Hallmann and S.A. Subbotin. 2005. Methods for extraction,
processing and detection of plant and soil nematodes. In: Luc M., Sikora R. A.
and Bridge J. (eds), Plant parasitic nematodes in subtropical and tropical
agriculture, CABI Publishing, Wallingford, UK: 53-86.
Hunt D.J., Luc M. and Manzanilla-López R.H. 2005. Identification, morphology and
biology of plant parasitic nematodes. In: Luc M., Sikora R.A. and Bridge J.
(eds), Plant parasitic nematodes in subtropical and tropical agriculture (2nd
edition), CAB International, Wallingford, UK: 11-52.
Hyman B.C. and Powers T.O. 1991. Integration of molecular data with systematics
of plant parasitic nematodes. Annual Review of Phytopathology 29: 89-107.
Inomoto M.M. and Oliveira C.M.G. 2008. Coffee-associated Pratylenchus spp.ecology and interactions with plants. In: Souza R.M. (ed.), Plant-parasitic
nematodes of coffee. Springer Science Business Media B.V.: 51-64.
Inomoto M.M., Oliveira C.M.G., Mazzafera P. and Goncalves W. 1998. Effects of
Pratylenchus brachyurus and P. coffeae on seedlings of Coffea arabica.
Inomoto M.M., Kubo R.K., Silva R.A., Oliveira C.M.G., Tomazini D. and Mazzafera P.
2007. Pathogenicity of two Pratylenchus coffeae populations from Brazil on
coffee plants. Nematology 9: 853-858.
References
133
Inserra R.N. and O’Bannon J.H. 1975. Rearing migratory endoparasitic nematodes
in citrus callus and roots produced from citrus leaves. Journal of Nematology
7: 261-263.
Inserra R.N., Duncan L.W., Dunn D., Kaplan D. and Porazinska D. 1998.
Pratylenchus pseudocoffeae from Florida and its relationship with P. gutierrezi
and P. coffeae. Nematologica 44: 683-712.
Inserra R.N., Duncan L.W., Troccoli A., Dunn D., Maia dos Santos J., Kaplan D. and
Vovlas N. 2001. Pratylenchus jaehni sp. n. from citrus in Brazil and its
relationship with P. coffeae and P. loosi (Nematode: Pratylenchidae).
Inserra R.N., Troccoli A., Gozel U., Bernard E.C., Dunn D. and Duncan. L.W. 2007.
Pratylenchus hippeastri sp. n. (Nematode: Pratylenchidae) from amaryllis in
Florida with notes on P. scribneri and P. hexincisus. Nematology 9: 25-42.
Jackson G.V.H., Ruabete T.K. and Wright J.G. 2003. Burrowing and lesion
nematodes of banana. In: Pest advisory leaflet No.5, Secretariat of the Pacific
Community, 4 p.
Jacobsen K., Maes L., Norgrove L., Mouassom H., Hauser S. and De Waele D. 2009.
Host status of twelve commonly cultivated crops in the Cameroon Highlands
for the nematode Pratylenchus goodeyi. International Journal of Pest
Management 55: 293-298.
Kubo R.K., Silva R.A., Tomazini M.D., Oliveira C.M.G., Mazzafera P. and Inomoto
M.M. 2003. Patogenicidade de Pratylenchus coffeae em plântulas de cafeeiro
cv. Mundo Novo. Fitopatologia Brasileira 28: 41-48.
Kumar A.C. and Viswanathan P.R.K. 1972. Study on physiological races of
Pratylenchus coffeae. Journal of Coffee Research 2: 10-15.
Loof P.A.A. 1978. The genus Pratylenchus Filijev, 1936 (Nematoda: Pratylenchida):
a
review
of
its
anatomy,
morphology,
distribution,
systematics
and
identification. Vaxtskyddsrapporter 5, 50 p.
Loof P.A.A. 1991. The family Pratylenchidae Thorne, 1949. In: Nickle W.R. (ed.),
Manual of agricultural nematology. Marcel Dekker, New York, USA: 126-134.
McDonald A.H. and Nicol J.M. 2005. Nematode parasites of cereals. In: Luc M.,
Sikora R.A. and Bridge J. (eds), Plant-parasitic nematodes in subtropical and
tropical agriculture (2nd edition), CAB International, Wallingford, UK: 131-192.
References
134
McDonald A.H. and Nicol J.M 2005. Nematode parasitees of cereals. In: Luc M.,
Sikora R.A. and Bridge J. (eds), Plant-parasitic nematodes in subtropical and
tropical agriculture (2nd edition), CAB International, Wallingford, UK: 131-192.
Mizukubo T. 1992. Morphological and statistical differentiation of Pratylenchus
coffeae complex in Japan (Nematoda: Pratylenchidae). Applied Entomology
and Zoology 27: 213-224.
Mizukubo T. 1995. Evidence for Pratylenchus coffeae races in differential
reproduction on fifteen cultivars (Nematoda: Pratylenchidae). Japanese
Mizukubo T. and Sano Z. 1997. Research paper: Pratylenchus coffeae virulent races
in sweetpotato. Sweetpotato Research Front 4: 2p.
Mizukubo T., Orui Y., Hanada K. and Sano Z. 2003. Microevolutionary trend in
Pratylenchus coffeae sensu stricto (Nematoda: Pratylenchidae): the diversity
in PCR-RFLP phenotype, compatibility on host plants and reproductive
segregation. Japanese Journal of Nematology 3: 57-76.
Mizukubo T., Sugimura K. and Uesugi K. 2007. A new species of the genus
Pratylenchus from chrysanthemum in Kyushu, western Japan (Nematoda:
Pratylenchidae). Japanese Journal of Nematology 37: 63-74.
Moens T., Araya M., Swennen R. and De Waele D. 2006. Reproduction and
pathogenicity of Helicotylenchus multicinctus, Meloidogyne incognita and
Pratylenchus coffeae, and their intraction with Radopholus similis on Musa.
Mudiope J., Coyne D.L., Adipala E. and Sikora R.A. 2004. Monoxenic culture of
Pratylenchus sudanensis on carrot disks, with evidence of differences in
reproductive rates between geographical isolates. Nematology 6: 617-619.
Munera Uribe G.E. 2008. Biodiversity of phytoparasitic nematodes associated with
Musaceae and fruit crops in Colombia (PhD thesis), Universiteit Gent, Belgium,
209 p.
Nghi N.S., Phong T.A., Toan B.Q. and Linh N.V. 1996. The coffee tree in Vietnam.
The Hanoi Agricultural Publishing House, Hanoi, Vietnam: 156-157 (in
Vietnamese).
O´Bannon J.H. and Tomerlin A.T. 1973. Citrus tree decline caused by Pratylenchus
coffeae. Journal of Nematology 5: 311-316.
References
135
Orui Y. 1996. Discrimination of the main Pratylenchus species (Nematoda:
Pratylenchidae) in Japan by PCR-RFLP analysis. Applied Entomology and
Zoology 31: 505-514.
Pinochet J. 1978. Histopathology of the root lesion nematode, Pratylenchus
coffeae, on plantains, Musa AAB. Nematologica 24: 331-340.
Pinochet J. 1998. A review of banana attacking nematodes in the subtropics with
emphasis on Pratylenchus goodeyi in the Canary Islands. Acta Horticulturae
490: 353-359.
Pinochet J. and O. Duarte. 1986. Additional list of ornamental foliage plants host of
the lesion nematode Pratylenchus coffeae. Nematropica 16: 11-19.
Pinochet J., C. Fernández and J. L. Sarah 1995. Influence of temperature on the in
vitro reproduction of Pratylenchus coffeae, P. goodeyi, and Radopholus
similis. Fundamental and Applied Nematology 18: 391-392.
Pinochet P., Cenis J.L., Fernandez C., Doucet M. and Marull J. 1994. Reproductive
fitness and random amplified polymorphic DNA variation among isolates of
Pratylenchus vulnus. Journal of Nematology 26: 271-277.
Pourjame E., Kheiri A. and Geraert E. 1997a. The genus Pratylenchus Filip’Jev,
1936 (Tylenchida: Pratylenchidae) from North of Iran. Mededelingen Faculteit
Landbouwwetenschappen Universiteit Gent 62/3a: 741-756.
Pourjame E., Waeyenberge L., Moens M. and Geraert E. 1997b. Morphological,
morphometrical and molecular study of Pratylenchus coffeae and P. loosi
(Nematoda:
Pratylenchidae).
Mededelingen
Faculteit
Landbouwwetenschappen Universiteit. Gent 64/3a: 391-401.
Powers T. 2004. Nematode molecular diagnostics: from bands to barcodes. Annual
Review of Phytopathology 42: 367-383.
Radewald J.D., O´Bannon J.H. and Tomerlin A.T. 1971a. Anatomical study of Citrus
jambhiri roots infected by Pratylenchus coffeae. Journal of Nematology 3:
409-416.
Radewald J.D., O’Bannon J.H. and Tomerlin A.T. 1971b. Temperature effects on
reproduction and pathogenicity of Pratylenchus coffeae and P. brachyurus and
survival of P. coffeae in roots of Citrus jambhiri. Journal of Nematology 3:
390-394.
References
136
Rashid A. and Khan A.M. 1976. Morphometric studies on Pratylenchus coffeae with
description of Pratylenchus typicus Rashid, 1974. Indian Journal of Nematology
6: 63-72.
Robinson A.F. and Percival A.E. 1997. Resistance to Meloidogyne incognita race 3
and Rotylenchulus reniformis in wild accessions of Gossypium hirsutum and G.
barbadense from Mexico. Journal of Nematology 29: 746-755.
Román J. and Hirschmann H. 1969. Morphology and morphometrics of six species of
Pratylenchus. Journal of Nematology 1: 363-385.
Román J. and Triantaphyllou A.C. 1969. Gametogenesis and reproduction of seven
species of Pratylenchus. Journal of Nematology 1: 357-362.
Ryss A.Y. 2002. Phylogeny and evolution of the genus Pratylenchus according to
morphological data (Nematoda: Tylenchida). Zoosystematica Rossica 10: 257273.
Sambrook J., Fritsch E.F., Maniatis T. 1989. Composition of the electrophoresis
buffer. In: Forsol, N., Nolan C., Ferguson M. and Ockler M. (eds). Molecular
cloning, a laboratory manual. New York, USA, Cold Spring Harbor Laboratory
Press.
Sarah J.L. and Fallas G. 1996. Biological, biochemical and molecular diversity of
Radopholus similis. In: Frison E.A., Horry J.P. and De Waele D. (eds), New
frontiers in resistance breeding for nematode, fusarium and sigatoka.
Proceeding of the workshop, held in Kuala Lumpur, Malaysia, 2-5 October
1995. International Network for the Improvement of Banana and Plantain
(INIBAP), France: 50-57.
Sarah J.L., Sabatini C. and Boisseau M. 1993. Differences in pathogenicity to
banana (Musa sp., cv. Poyo) among populations of Radopholus similis from
different production areas of the world. Nematropica 23: 75-79.
Sasser J.N. and Freckman D.W. 1987. A world perspective on nematology: the role
of the society. In: Veech J.A. and Dickerson D.W. (eds), Vistas on nematology.
Society of Nematologists, Hyattsville, Maryland: 7-14.
Scurrah M.I., Niere B. and Bridge J. 2005. Nematode parasites of solanum and
sweet potatoes. In: Luc M., Sikora R.A. and Bridge J. (eds), Plant parasitic
nematodes in subtropical and tropical agriculture (2nd edition), CAB
International, Wallingford, UK: 193-220.
References
137
Shaner G., Stromberg L., Lacy G.H., Barker K.R. and Pirone T.P. 1992.
Nomenclature and concepts of pathogenicity and virulence. Annual Review of
Phytopathology 30: 47-66.
Sher S.A. and Allen M.W. 1953. Revision of the genus Pratylenchus (Nematoda:
Tylenchidae). University of California Publications, Zoology 57: 441-470.
Siciliarno-Wilcken S.R.S., Inomoto M.M., Ferraz L.C.C.B. and Oliveira C.M.G. 2002.
Morphometry of Pratylenchus populations from coffee, banana, ornamental
plant and citrus in Brazil. Nematology 4: 248.
Siddiqi M.R 2000. Tylenchida parasites of plants and insects (2nd edition), CAB
International, Wallingford, UK, 833 p.
Siddiqi M.R. 1972. Pratylenchus coffeae. C.I.H. Descriptions of Plant-parasitic
Nematodes. Set 1, No. 6, CAB International, Wallingford, UK, 3 p.
Silva R.A. and Inomoto M.M. 2002. Host-range characterization of two Pratylenchus
coffeae populations from Brazil. Journal of Nematology 34: 135-139.
Sipes, B. S., E. P. Cashwell-Chen, J. L. Sarah and W. J. Apt 2005. Nematode
parasites of pineapple. In: M. Luc, R. A. Sikora and J. Bridge (eds), Plant
parasitic nematodes in subtropical and tropical agriculture, CAB International
Wallingford, UK: 709-731.
Soriano I.R., Riley I.T., Potter M.J. and Bowers W.S. 2004. Phytoecdysteroids: A
novel defense against plant-parasitic nematodes. Journal of Chemical Ecology
30: 1885-1899.
Souza R.M. 2008. Plant-parasitic nematodes of coffee. Springer Science Besiness
Media B.V., 340 p.
Speijer P. R. and D. De Waele 1997. Screening of Musa germplasm for resistance
and tolerance to nematodes. INIBAP Technical Guidelines 1. International
Network for the Improvement of Banana and Plantain, Montpellier, France, 47
p.
Speijer P.R., Rotimi M.O. and De Waele D. 2001. Plant parasitic nematodes
associated with plantain (Musa spp., AAB-group) in southern Nigeria and their
relative importance compared to other biotic constraints. Nematology 3: 423436.
Stoffelen R., Jimenez M. I., Dierckxsens C., Tam V.T.T., Swennen R. and De Waele
D. 1999. Effect of time and inoculum density on the reproductive fitness of
References
138
Pratylenchus coffeae and Radopholus similis populations on carrot disks.
Subbotin S.A. and Moens M. 2006. Molecular taxonomy and phylogeny. In: Perry
R.N. and Moens M. (eds), Plant nematology, CABI Publishing, Wallingford, UK:
33-58.
Subbotin S.A., Ragsdale E.J., Mullens T., Roberts P.A., Mundo-Ocampo M. and
Baldwin J.G. 2008. A phylogenetic framework for root lesion nematodes of the
genus Pratylenchus (Nematoda): evidence from 18S and D2-D3 expansion
segments of 28S ribosomal RNA genes and morphological characters. Molecular
Phylogenetics and Evolution 48: 491-505.
Subbotin S.A., Sturhan D., Chizhov V.N., Vovlas N. and Baldwin G. 2006.
Phylogenetic analysis of Tylenchida Thorne, 1949 as inferred from D2 and D3
expansion fragments of the 28S rRNA gene sequences. Nematology 8: 455-474.
Sung P. Q. 1976. Study results of nematode diseases on coffee in Phu Quy, Nghe An
Province. Phu Quy Tropical Crops Reseach Centre, 2 p. (in Vietnamese).
Sung P. Q., H. M. Trung, H. T. Tiem, T. K. Loang, T. D. Minh, C. T. Tuan Nam, T.
Hong, L. N. Bau, N. T. Chat, N. V. Tuat, N. V. Vien N. V. Van 2001.
Investigation of the yellow-leaf symptom on coffee trees and control
meassures. Ministry of Sciences, Technology and the Environment, Hanoi,
Vietnam, 165 p. (in Vietnamese).
Swofford D.L 1998. PAUP* Phylogenetic analysis using parsimony. Version 4.
Sunderland, Massachusetts, Sinauer Associates, 128 p.
Tarjan A.C. and Frederick J.J. 1978. Intraspecific morphological variation among
populations of Pratylenchus brachyurus and P. coffeae. Journal of Nematology
10: 152-160.
Tarjan A.C. and O’Bannon J.H. 1969. Observations on meadow nematodes
(Pratylenchus spp.) and their relation to decline of citrus in Florida. Plant
Disease Reporter 58: 683-686.
Tarte R. and Mai W.F. 1976. Morphological variation in Pratylenchus penetrans.
Tarté R., Pinochet J., Gabrielli C. and Ventura O. 1981. Differences in population
increase, host preferences and frequencies of morphological variants among
isolates of the banana race of Radopholus similis. Nematropica 11: 43-52.
References
139
Thomas W.K., Vida J.T., Frisse L.M., Mundo M., and Baldwin J.G. 1997. DNA
sequences
from
formalin-fixed
nematodes:
integrating
molecular
and
morphological approaches to taxonomy. Journal of Nematology 29: 250-254.
Thompson A.K., Been B.O. and Perkins C. 1973. Nematodes in stored yams.
Experimental Agriulture 9: 281-286.
Thompson J.D., Gibson T.J., Plewniak F., Jeanmougin F. and Higgins D.G. 1997.
The ClustalX windows interface: flexible strategies for multiple sequence
alignment aided by quality analysis tools. Nucleic Acids Research 24: 4876-4882
Timmer L. W. and Graham J. H. 2002. Plagas y enfermedades de los cítricos (2nd
edition), 95 p.
Trung H.M., Vien N.V., Hanh T.H., Ly N.T., Thuan T.T., Quan H.A., Thang P.H.,
Chau N.N., Huan N.T., Khoa N.V. and Ha An N.T. 2000. Results of a study of
the yellow-leaf symptom on coffee trees and control measures. Journal of
Agriculture and Food Industry 3: 106-109. (in Vietnamese).
Uehara T., Mizukubo T., A. Kushida and Y. Momota. 1998. Identification of
Pratylenchus coffeae and P. loosi using specific primers for PCR amplification
of ribosomal DNA. Nematologica 44: 357-368.
Vaast Ph., Caswell-Chen E.P. and Zasoski R.J. 1998. Effects of two endoparasitic
nematodes (Pratylenchus coffeae and Meloidogyne konaensis) on ammonium
and nitrate uptake by Arabica coffee (Coffea arabica L.). Applied Soil Ecology
10: 171-178.
Volossiouk T., Robb E.J and Nazar R.N. 2003. Avoiding false positives in PCR-based
identification methods for nonsterile plant pathogens. Canadian Journal of
Plant Pathology 25, 192-197.
Van den Berg E. 1971. The root lesion nematodes of South Africa (genus
Pratylenchus, family Hoplolaimidae). Department of Agricultural Technical
Services. Technical Communication No.99. Plant Protection Research Institute,
Pretoria, 13 p.
Van den Bergh I, Nguyet D.T.M., Tuyet N.T., Nhi H.H. and De Waele D. 2006.
Influence of Pratylenchus coffeae and Meloidogyne spp. on plant growth and
yield of banana (Musa spp.) in Vietnam. Nematology 8: 265-271.
Van den Bergh I. 2002. Host-plant response of Vietnamese bananas (Musa spp.) to
plant-parasitic nematodes. Dissertationes de Agricultura 547, Catholic
University of Leuven, 156 p.
References
140
Waeyenberge L., Ryss A., Moens M., Pinochet J. and Vrain T.C. 2000. Molecular
charaterisation of 18 Pratylenchus species using rDNA restriction fragment
length polymorphism. Nematology 2: 135-142.
Wehunt E.J. and Edwards D.I. 1971. Research notes: intra-uterine egg development
of Pratylenchus coffeae (Zimmerman) Filipjev and Schuurmans Stekhoven.
Whitehead A.G. 1969. Nematodes attacking coffee, tea and cocao and their
control. In: Peachey J.E. (ed.), Nematodes of tropical crops. Technical
communication No. 40. Commenwealth Bureaux of Helminthology. St Albans,
Herts, England: 238-250.
Xihua Li. 2006. Morphological characterization of various Pratylenchus populations
(Nematoda: Tylenchina). Master Thesis, Gent, Belgium, 87p.
List of publications
141
List of accepted publications in international
refereed journals
Elsen A., Stoffelen R., Tuyet N.T., Baimey H., Dupre de Boulois H. and De Waele D.
2002. In vitro screening for resistance to Radopholus similis in Musa spp. Plant
Science 163: 407-416.
Tuyet N.T., Nhi H.H., Van den Bergh I., Elsen A. and De Waele D. 2008. Occurrence
of Pratylenchus coffeae on agricultural crops in Vietnam. International Journal
of Nematology 18: 174-180.
Van den Bergh I., Nguyet D.T.M., Tuyet N.T., Nhi H.H. and De Waele D. 2002.
Screening of Vietnamese Musa germplasm for resistance to root knot and root
lesion nematodes in the greenhouse. Australasian Plant Pathology 31: 363-371.
Van den Bergh I., Nguyet D.T.M., Tuyet N.T., Nhi H.H. and De Waele D. 2002. Host
plant response of Vietnamese Musa genotypes to Meloidogyne spp. under field
conditions. Nematology 4: 917-923.
Van Den Bergh I., Nguyet D.T.M., Tuyet N.T., Nhi H.H. and De Waele D. 2006.
Influence of Pratylenchus coffeae and Meloidogyne spp. on plant growth and
yield of banana (Musa spp.) in Vietnam. Nematology 8: 265-271.
Van den Bergh I., Tuyet N.T., Nguyet D.T.M., Nhi H.H. and De Waele D. 2005.
Population dynamics of Pratylenchus coffeae on banana in North Vietnam.

a comparative polyphasic study of 10 pratylenchus coffeae

Transcription

Similar documents

Good Morning Vietnam! - Broadcast Electronics

Federico A. Subervi-VeIez, Maria Denney, Anthony Ozuna, and

Hibiscus cravenii - Northern Territory Government

#TVforHE – Pick of the Week – Storyville: Last Days of Vietnam

Reducing Milk Thistle (Silybum marianum) to Zero Density - Cal-IPC

Ecosystem Connections: who, what, where, when Remember

Le Hong Phong Forest Vicinity

Semi-annual Newsletter

Understanding and Identifying Target Populations for

INVITATION