incidence and effect of meloidogyne incognita (nematoda … · incognita (nematoda: meloidogyninae)...

Doctoraatsproefschrift nr. 933 aan de faculteit Bio-ingenieurswetenschappen van de K.U.Leuven

INCIDENCE AND EFFECT OF MELOIDOGYNE INCOGNITA (NEMATODA: MELOIDOGYNINAE)

ON BLACK PEPPER PLANTS IN VIETNAM

Trinh Thi Thu THUY

Promotor: Prof. Dirk De Waele, K.U.Leuven Co-promotor: Prof. Le Luong Te, HUA - Hanoi

University of Agriculture, Vietnam.

Members of the Examination Committee: Prof. B. Goddeeris, Chairman, K.U.Leuven Prof. J. Coosemans, K.U.Leuven Prof. B. Cammue, K.U.Leuven Dr. N. Viaene, ILVO, Merelbeke Prof. A. Elsen, BDB & UGent

Dissertation presented in partial fulfilment of the requirements for the degree of Doctor of Bioscience Engineering

October 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 978-90-8826-168-8 Wettelijk depot D/2010/11.109/51

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ACKNOWLEDGEMENTS

First and foremost I would like to offer my sincerest gratitude to my promotor, Professor Dirk De Waele, who has supported me throughout the duration of my study for his encouragement, guidance, patience and knowledge.

I am also heartily thankful to my co-promotor in Vietnam, Professor Le Luong Te, whose support and interest from the very beginning enabled me to complete my PhD study.

I would like to thank the Flemish University Council (VL.I.R) for the scholarship I received and for the financial support during my research and studies in Vietnam and Belgium.

I am truly grateful to the members of the examination committee, Professors Bruno Goddeeris, Bruno Cammue, Jozef Cooseman, Nicole Viaene and Annemie Elsen, for their time, remarks and advice to improve the quality of my PhD thesis.

I am grateful to Professor Rony Swennen, head of the Laboratory of Tropical Crop Improvement, Catholic University of Leuven, for allowing me to use the laboratory facilities which enabled me to complete my PhD research. I also would like to show my gratitude to the nematology team and all the staff of the laboratory for their kind assistance.

I would like to thank Dr. Duong Minh Tu from the Plant Quarantine Diagnosis Centre (PQDC) and the board of directors from the Plant Protection Department (PPD), Ministry of Agriculture and Rural Development, for their support and for providing all the necessary laboratory facilities. I would also like to express my gratitude to my colleagues from the nematology group at PQDC and PPD: Mrs. Yen, Anh Tuyet, Ngoc Anh, … without whom I could not have achieved this work.

It is a pleasure to thank those who made this PhD thesis possible: many of my colleagues from the Post-entry Plant Quarantine Centre No. 1, the Phytopathology Laboratory of the Plant Protection Research Institute in Hanoi and the Western Highlands Agro-Forestry Scientific and Technical Institute in Dac Lac for their cooperation and assistance. Many thanks to all my colleagues from the six Plant Protection Sub-departments in Quang Tri, Thua Thien Hue, Kon Tum, Gia Lai, Dac Lac and Kien Giang provinces for their support and assistance during the survey.

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I am grateful to Professors Nguyen Vu Thanh and Nguyen Ngoc Chau, from the Institute of Ecology and Biological Resources in Hanoi, for their cooperation and assistance.

My special thanks are dedicated to Mrs. Mai Chi for her knowledge and help in my studies. I owe my deepest gratitude to all my friends in Vietnam and Belgium. Their understanding and encouragement helped me to study well during my stay in Belgium.

Tôi xin cảm ơn bố, mẹ, các em và các cháu, những người thân yêu luôn bên cạnh, động viên, chia sẻ những lúc khó khăn nhất, luôn là điểm tựa vững chắc, giúp tôi vượt qua tất cả để có thành quả ngày hôm nay. Những tình cảm yêu thương nhất gửi tới con gái và chồng tôi, anh và con gái đã luôn là động lực để tôi có thể hoàn thành tốt công việc của mình.

Lastly, I offer my regards and blessings to all those who have supported me in any respect during the completion of this thesis.

Leuven, 27th October 2010

Trinh Thi Thu Thuy

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TABLE OF CONTENTS

Acknowledgements .................................................. i

Table of contents................................................... iii

List of tables ........................................................vii

List of figures ....................................................... xi

Summary .............................................................xv

CHAPTER 1. INTRODUCTION ............................................. 1

1.1. Objectives of the study and outline of the thesis ...................1

1.2. Background..................................................................5 1.2.1. The black pepper plant....................................................5

1.2.1.1. Classification (CABI, 2007) ...........................................5 1.2.1.2. Origin....................................................................5 1.2.1.3. Morphology .............................................................6 1.2.1.4. Climate and soil .......................................................9 1.2.1.5. Varieties ................................................................9 1.2.1.6. Cultivation and harvest...............................................9 1.2.1.7. Use..................................................................... 10 1.2.1.8. Production ............................................................ 11

1.2.2. Nematodes and fungi associated with black pepper plants ....... 16 1.2.2.1. Nematodes............................................................ 16 1.2.2.2. Fungi and nematodes-fungi disease complexes................. 18

CHAPTER 2. PLANT-PARASITIC NEMATODES AND YELLOWING OF LEAVES ASSOCIATED WITH BLACK PEPPER PLANTS IN VIETNAM...................21

2.1. Introduction................................................................... 21

2.2. Agro-ecological regions of Vietnam ...................................... 23 North Central Coast ........................................................... 24 Central Highlands .............................................................. 24 Southeast........................................................................ 25 Mekong River Delta ............................................................ 25

2.3. Materials and methods...................................................... 26

2.4. Results ......................................................................... 28

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2.4.1. Nematodes associated with black pepper plants in Vietnam...... 28 2.4.2. Frequency of occurrence and abundance of Meloidogyne incognita, root galling and percentage of plants with yellow leaves .... 28

2.4.2.1. North Central Coast ................................................. 31 2.4.2.2. Central Highlands.................................................... 32 2.4.2.3. Phu Quoc Island (Mekong River Delta)............................ 34 2.4.2.4. Description of root galls ............................................ 35 2.4.2.5. Description of yellow leaves ....................................... 36 2.4.2.6. Correlation between Meloidogyne incognita population density and percentage of plants with yellow leaves .............................. 37

2.4.3. Frequency of occurrence and abundance of Meloidogyne incognita, and root galling on black pepper varieties ...................... 38

2.5. Discussion......................................................................39

2.6. Conclusions ....................................................................43

CHAPTER 3. IDENTIFICATION AND COMPARATIVE STUDY OF THE MORPHOLOGY AND MORPHOMETRICS OF MELOIDOGYNE POPULATIONS FROM BLACK PEPPER PLANTS IN VIETNAM .............................. 45

3.1. Introduction ...................................................................45

3.2. Materials and methods ......................................................47 3.2.1. Nematode populations................................................... 47 3.2.2. Killing and fixing of second-stage juveniles (J2) and males ....... 48 3.2.3. Preparation of perineal patterns of females ......................... 49 3.2.4. Mounting of nematodes.................................................. 49 3.2.5. Light microscope observations ......................................... 49 3.2.6. Canonical discriminant analysis ........................................ 50

3.3. Results..........................................................................51 3.3.1. Morphological observations ............................................. 51 3.3.2. Morphometrical observations ........................................... 52

3.4. Discussion......................................................................58

3.5. Conclusions ....................................................................60

CHAPTER 4: POPULATION DYNAMICS OF MELOIDOGYNE INCOGNITA ON BLACK PEPPER PLANTS IN TWO AGRO-ECOLOGICAL REGIONS IN VIETNAM 61

4.1. Introduction ...................................................................61

4.2. Materials and methods ......................................................62 4.2.1. Description of the study sites........................................... 62 4.2.2. Sampling, nematode population and root galling assessment..... 64

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4.3. Results ......................................................................... 64 4.3.1. Population dynamics of Meloidogyne incognita on black pepper variety Vinh Linh in Cam Lo..................................................... 65 4.3.2. Population dynamics of Meloidogyne incognita on black pepper variety Vinh Linh in Buon Ma Thuot ........................................... 68 4.3.3. Comparison of the root population dynamics of Meloidogyne incognita J2 and percentage root galling on black pepper variety Vinh Linh in Cam Lo and Buon Ma Thuot............................................ 70

4.4. Discussion ..................................................................... 72

4.5. Conclusions.................................................................... 74

CHAPTER 5. FUNGI ASSOCIATED WITH BLACK PEPPER PLANTS IN QUANG TRI PROVINCE AND INTERACTION BETWEEN MELOIDOGYNE INCOGNITA AND FUSARIUM SOLANI.......................................................77

5.1. Introduction................................................................... 77

5.2. Materials and methods...................................................... 78 5.2.1. Survey.................................................................. 78

5.2.1.1. Nematode population and root galling assessment ............ 79 5.2.1.2. Assessment of percentage of yellow leaves..................... 80 5.2.1.3. Isolation and identification of fungi .............................. 80

5.2.2. Greenhouse experiment................................................. 81 5.2.2.1. Preparation of the black pepper plants.......................... 81 5.2.2.2. Preparation of nematode inoculum .............................. 82 5.2.2.3. Preparation of fungal inoculum ................................... 82 5.2.2.4. Experimental design ................................................ 83 5.2.2.5. Assessment of nematode reproduction, root galling, frequency of occurrence of Fusarium solani, percentage of plants with yellow leaves and plant growth ...................................................... 84

5.2.3. Statistical analysis ....................................................... 84

5.3. Results ......................................................................... 84 5.3.1. Survey.................................................................. 84

5.3.1.1. Fungi associated with black pepper plants in Quang Tri province ......................................................................... 84 5.3.1.2. Frequency of occurrence of Fusarium solani and percentage of plants with yellow leaves..................................................... 85 5.3.1.3. Frequency of occurrence of Fusarium solani and age of black pepper plants................................................................... 86 5.3.1.4. Incidence of Meloidogyne incognita and Fusarium solani, root galling and percentage of plants with yellow leaves in nurseries and plantations of black pepper plants.......................................... 87

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5.3.2. Greenhouse experiment............................................. 90 5.3.2.1. Effect of inoculation with Meloidogyne incognita and Fusarium solani either alone or combined on the percentage of plants with yellow leaves.................................................................... 90 5.3.2.2. Effect of inoculation with Meloidogyne incognita and Fusarium solani either alone or combined on plant growth......................... 92 5.3.2.3. Effect of inoculation with Meloidogyne incognita and Fusarium solani either alone or combined on nematode reproduction and root galling ............................................................................ 94

5.4. Discussion......................................................................96

5.5. Conclusions ....................................................................98

CHAPTER 6. HOST RESPONSE OF FIVE BLACK PEPPER VARIETIES TO INFECTION BY MELOIDOGYNE INCOGNITA UNDER GREENHOUSE CONDITIONS.......................................................................101

6.1. Introduction ................................................................. 101

6.2. Materials and methods .................................................... 102 6.2.1. Preparation of the black pepper plants..............................102 6.2.2. Preparation of nematode inoculum and inoculation ...............104 6.2.3. Assessment of nematode reproduction, root galling and plant growth .............................................................................105 6.2.4. Statistical analysis.......................................................105

6.3. Results........................................................................ 106 6.3.1. Plant growth..............................................................106 6.3.2. Percentage yellow leaves ..............................................106 6.3.3. Root galling and reproduction of Meloidogyne incognita .........109 6.3.4. Correlations between plant growth, root galling and Meloidogyne incognita reproduction .........................................................110

6.4. Discussion.................................................................... 118

6.5. Conclusions .................................................................. 119

CHAPTER 7. GENERAL CONCLUSIONS AND PERSPECTIVES..............121

References.........................................................125

Annex 1: List of publications...................................137

Annex 2: List of abbreviations .................................138

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LIST OF TABLES

Table 1.1: The harvest seasons of black pepper plants in the major pepper producing countries....................................................... 10

Table 1.2: Area planted with black pepper plants, pepper production and export in Vietnam from 1980 to 2009.................................. 13

Table 1.3: Comparison of pepper exports between 2006-2007 and 2007-2008. ........................................................................ 15

Table 2.1: List of pathogens effecting black pepper plants in Vietnam. .. 22

Table 2.2: Sampling periods, districts, provinces and agro-ecological regions sampled. .......................................................... 26

Table 2.3: Plant-parasitic nematodes associated with black pepper plants in Vietnam.................................................................. 29

Table 2.4: Frequency of occurrence and population density of Meloidogyne incognita, root galling and percentage of plants with yellow leaves in black pepper fields in two provinces in the North Central Coast, Vietnam..................................................................... 31

Table 2.5: Frequency of occurrence and population density of Meloidogyne incognita, root galling and percentage of plants with yellow leaves in black pepper fields in three provinces in the Central Highlands, Vietnam..................................................................... 33

Table 2.6: Frequency of occurrence and population density of Meloidogyne incognita, root galling and percentage of plants with yellow leaves in black pepper fields in three districts in Phu Quoc Island, Vietnam..................................................................... 34

Table 2.7: Occurrence of black pepper varieties in the North Central Coast, Central Highlands and Mekong River Delta, Vietnam................ 38

Table 2.8: Frequency of occurrence and population density of Meloidogyne incognita, root galling and percentage of plants with yellow leaves observed on black pepper varieties in Vietnam. ..................... 39

Table 3.1: Origin and population code of the seven Meloidogyne populations collected from black pepper plants in Vietnam....... 48

Table 3.2: Measurements taken for the comparative morphometrical study of the seven Meloidogyne populations collected from black pepper plants in Vietnam and their abbreviations. ........................... 50

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Table 3.3: Morphometrics of the females and males of the Meloidogyne population collected from black pepper plants in Cam Lo district................................................................................ 55

Table 3.4: Morphometrics of the second-stage juveniles (J2) of the seven Meloidogyne populations collected from black pepper plants in Vietnam. .................................................................... 56

Table 3.5: Standardised coefficients for canonical variates for the second-stage juveniles (J2) of the seven Meloidogyne populations collected from black pepper plants in Vietnam. ..................... 57

Table 4.1: Plant-parasitic nematodes associated with black pepper variety Vinh Linh in Cam Lo and Buon Ma Thuot, Vietnam................... 65

Table 4.2: Population dynamics of Meloidogyne incognita (and all plant-parasitic nematodes), percentage root galling and root galling index on black pepper variety Vinh Linh in Cam Lo, Vietnam, from November 2004 until October 2005..................................... 66

Table 4.3: Population dynamics of Meloidogyne incognita (and all plant-parasitic nematodes), percentage root galling and root galling index on black pepper variety Vinh Linh in Buon Ma Thuot, Vietnam, from April 2006 until April 2007............................. 68

Table 5.1: Fungi associated with black pepper plant roots in Quang Tri province, Vietnam......................................................... 85

Table 5.2: Percentage of black pepper plants with yellow leaves, number and percentage of black pepper plants infected with F. solani. . 86

Table 5.3: Plant age of black pepper plants, number and percentage of black pepper plants infected with F. solani.......................... 86

Table 5.4: Meloidogyne incognita population density, M. incognita and Fusarium solani frequency of occurrence, root galling and percentage of plants with yellow leaves in nurseries and plantations of black pepper plants in Quang Tri province, Vietnam................................................................................ 88

Table 5.5: Plant growth of black pepper plants 9 months after inoculation with Meloidogyne incognita and Fusarium solani either alone or combined. .................................................................. 93

Table 5.6: Reproduction of Meloidoygne incognita and root galling of black pepper plants 9 months after inoculation with M. incognita and Fusarium solani either alone or combined. ........................... 95

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Table 6.1: Spike and yield characteristics of the five black pepper varieties included in the host response study...................................102

Table 6.2: Oleoresin, piperine and essential oil content of the five black pepper varieties included in the host response study..............103

Table 6.3: Plant growth of five black pepper varieties, 5 months after inoculation with 2,000 eggs and J2 of Meloidogyne incognita. ...107

Table 6.4: Root galling and reproduction of Meloidogyne incognita on five black pepper varieties, 5 months after inoculation with 2,000 eggs and J2. .....................................................................110

Table 6.5: Correlation (Spearman's rho) between plant growth, root galling and Meloidogyne incognita reproduction in the roots of the black pepper variety Loc Ninh.................................................112

Table 6.6: Correlation (Spearman's rho) between plant growth, root galling and Meloidogyne incognita reproduction in the roots of black pepper variety Vinh Linh................................................113

Table 6.7: Correlation (Spearman's rho) between plant growth, root galling and Meloidogyne incognita reproduction in the roots of the black pepper variety La Da. ...................................................114

Table 6.8: Correlation (Spearman's rho) between plant growth, root galling and Meloidogyne incognita reproduction in the roots of the black pepper variety Phu Quoc................................................115

Table 6.9: Correlation (Spearman's rho) between plant growth, root galling and Meloidogyne incognita reproduction in the roots of the black pepper variety Trau......................................................116

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LIST OF FIGURES

Figure 1.1: Research outline of our study. .......................................4

Figure 1.2: Black pepper plant. ....................................................5

Figure 1.3: Black pepper plants. ...................................................6

Figure 1.4: Young inflorescences of a black pepper plant with flowering spikes. ..........................................................................7

Figure 1.5: Fruiting spikes of a black pepper plant. A: green berries; B: red berries. .........................................................................8

Figure 1.6: Black, white, green and red peppercorns.. ........................8

Figure 1.7: Distribution map of black pepper plants (Piper nigrum L.). . . 11

Figure 1.8: Global pepper production trend (in tons) between 1970 and 2008........................................................................... 12

Figure 1.9: Percentage share of the major pepper producing countries... 12

Figure 1.10: Black pepper cultivation in Vietnam. ............................ 14

Figure 1.11: Pepper production trend (in tons) in Vietnam.................. 15

Figure 1.12: Life and disease cycle of root-knot nematodes (Meloidogyne spp.). ......................................................................... 17

Figure 2.1: Agro-ecological regions of Vietnam and provinces surveyed. . 25

Figure 2.2: Meloidogyne incognita population density, percentage of root galling and plants with yellow leaves in black pepper fields in two provinces in the North Central Coast, Vietnam........................ 32

Figure 2.3: Meloidogyne incognita population density, percentage of root galling and plants with yellow leaves in black pepper fields in three provinces in the Central Highlands, Vietnam. ......................... 34

Figure 2.4: Meloidogyne incognita population density, percentage of root galling and plants with yellow leaves in black pepper fields in three districts in Phu Quoc Island, Vietnam. .................................. 35

Figure 2.5: Root galls caused by Meloidogyne incognita on a primary root (A) and secondary root (B) of a black pepper plant. Adult females of M. incognita inside a primary root of a black pepper plant (C & D)................................................................................. 36

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Figure 2.6: Yellow black pepper plant leaves (A). A black pepper field with plants with yellow leaves (B). ............................................ 36

Figure 2.7: Correlation between Meloidogyne incognita population density and percentage of plants with yellow leaves in 67 black pepper fields in three agro-ecological regions in Vietnam. ................... 37

Figure 3.1: Drawing of the perineal pattern of Meloidogyne species....... 47

Figure 3.2: Light microscope photographs of the female and male of the Meloidogyne population from Cam Lo district. Female. A: perineal pattern; B: stylet. Male. C: head; D: tail.............................. 51

Figure 3.3: Light microscope photographs of the heads of second-stage juveniles (J2) of the Meloidogyne populations from Cam Lo (A), Phu Loc (B), I A Chim (C), Buon Ma Thuot (D), Chu Se (E), An Phu (F) and Phu Quoc Island (G). ............................................ 53

Figure 3.4: Light microscope photographs of the tails of second-stage juveniles (J2) of the Meloidogyne populations from Cam Lo (A), Phu Loc (B), I A Chim (C), Buon Ma Thuot (D), Chu Se (E), An Phu (F) and Phu Quoc Island (G).............................................. 54

Figure 3.5: Canonical Discriminant Analysis of seven Meloidogyne populations collected from black pepper plants in Vietnam based on the 10 morphological J2 characters listed in Table 3.5. ........ 58

Figure 4.1: Sites in Vietnam where the population dynamics studies of Meloidogyne incognita on black pepper variety Vinh Linh were carried out.................................................................. 63

Figure 4.2: Population dynamics of Meloidogyne incognita and percentage root galling on black pepper variety Vinh Linh in Cam Lo, Vietnam, from November 2004 until October 2005. ............................. 67

Figure 4.3: Average monthly air temperature and average monthly rainfall in Cam Lo during the period November 2004–October 2005. ....... 67

Figure 4.4: Population dynamics of Meloidogyne incognita and percentage root galling on black pepper variety Vinh Linh in Buon Ma Thuot, Vietnam, from April 2006 until April 2007............................. 69

Figure 4.5: Average monthly temperature and average monthly rainfall in Buon Ma Thuot during the period April 2006–April 2007. ........... 70

Figure 4.6: Population dynamics of Meloidogyne incognita J2 in the roots of black pepper variety Vinh Linh during the rainy and dry seasons in Cam Lo (Quang Tri province) and Buon Ma Thuot (Dac Lac province).................................................................... 70

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Figure 4.7: “Dynamics” of the percentage root galling observed on the black pepper variety Vinh Linh during the rainy and dry seasons in Cam Lo (Quang Tri province) and Buon Ma Thuot (Dac Lac province). .................................................................. 71

Figure 5.1: Districts of Quang Tri province, Vietnam, surveyed. ........... 79

Figure 5.2: Black pepper plant (variety Vinh Linh) in the nursery. ......... 81

Figure 5.3: Fusarium solani colony on PDA medium. A: floccose, greyish-white mycelium; B: microconidia. ..................................... 82

Figure 5.4: Experiment in the greenhouse...................................... 83

Figure 5.5: Meloidogyne incognita soil and root population densities, frequency of occurrence of Fusarium solani, root galling and percentage of plants with yellow leaves in black pepper plants in nurseries and plantations in Quang Tri province, Vietnam. ........ 89

Figure 5.6: Average monthly air temperature and average monthly rainfall in Hanoi during the period January 2007–December 2005. ......... 90

Figure 5.7: Percentage of plants with yellow leaves at 3, 6 and 9 months after inoculation with Meloidogyne incognita and Fusarium solani, alone or combined.. ...................................................... 91

Figure 5.8: Root systems of black pepper plants 9 months after inoculation with Meloidogyne incognita and Fusarium solani either alone or combined. .................................................................. 94

Figure 6.1: Average monthly temperature and average monthly rainfall in Buon Ma Thuot during year 2008.......................................104

Figure 6.2: Effect of inoculation with Meloidogyne incognita on the black pepper varieties Loc Ninh (1), Vinh Linh (2) and La Da (3), before inoculation (A) and 5 months after inoculation (B).................108

Figure 6.3: Effect of inoculation with Meloidogyne incognita on the black pepper varieties Phu Quoc (4) and Trau (5), before inoculation (A) and 5 months after inoculation (B)....................................109

Figure 6.4: Correlation between the number of J2 of Meloidogyne incognita/5 g fresh roots and fresh root weight of five black pepper varieties. ..................................................................117

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SUMMARY Pepper produced by black pepper plants (Piper nigrum L.) is

popularly considered as the “king of spices”. Black pepper has become one of the most important agricultural products of Vietnam. Today, Vietnam is worldwide the leading producer and exporter of black pepper. Plant protection is important for pepper production because pathogens are a major cause of the reduction in yield and quality of pepper crops. One of these pathogens are the root-knot nematodes (Meloidogyne spp.).

In Vietnam, almost nothing is known about the incidence of root-knot nematodes on black pepper plants, their effect on plant growth and yield, their interaction with soil-borne fungi and their relationship with the occurrence of yellowing of leaves of black pepper plants. Therefore, the general objectives of our study were to elucidate the incidence and effect of Meloidogyne incognita on black pepper plants in Vietnam and their interaction and relationship with the incidence of soil-borne fungi and yellowing of leaves of black pepper plants.

In the first part of our study (Chapter 2), the plant-parasitic nematodes associated with black pepper plants in Vietnam were identified. In total 432 soil and 432 root samples were collected from black pepper plants in 19 districts in six provinces in three agro-ecological regions of Vietnam (North Central Coast, Central Highlands and Phu Quoc Island). 35 nematode species belonging to 19 genera were found. Five plant-parasitic nematodes were for the first time recorded on black pepper plants in Vietnam. Radopholus similis, the most important nematode species on black pepper plants world–wide was not found. The root-knot nematode Meloidogyne incognita was the predominant plant-parasitic nematode species on black pepper plants in Vietnam. There was no difference in frequency of occurrence of M. incognita among the three agro-ecological regions examined. The percentage of root galling averaged about 40% in the three agro-ecological regions. The same type of root galls and yellow leaves as described in the literature for black pepper plants infected with Meloidogyne species was observed. In general, a weak positive relationship between the population densities of M. incognita on black pepper plants and percentage of black pepper plants with yellow leaves was observed.

In the second part of our study (Chapter 3), the morphological and morphometrical characters of seven Meloidogyne populations collected from black pepper plants in six provinces in three agro-ecological regions of Vietnam were compared. The seven Meloidogyne populations collected from black pepper plants in Vietnam were all identified as M. incognita based on a combination of the perineal pattern of the mature females, and

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morphological and morphometrical characters of J2 and males. Based on the coefficient of variation, stylet length, body length and a ratio are the least variable morphometrical characters of the J2. The M. incognita populations examined were grouped using canonical discriminant analysis in three groups based on a combination of 10 morphological characters of the J2. There was no relationship between these three groups and the geographical origin of the populations.

In the third part of our study (Chapter 4), the population dynamics of M. incognita on black pepper plants in two agro-ecological regions (North Central Coast and Central Highlands) of Vietnam was studied. There were significant differences in rainfall and air temperature between Cam Lo in Quang Tri province (North Central Coast) and Buon Ma Thuot in Dac Lac province (Central Highlands). The most important climatic difference was rainfall. The highest root population densities of M. incognita J2 were observed during the 1st half of the dry season on black pepper variety Vinh Linh in both study sites. The root galling index was observed the highest during the 1st half of the dry season in Cam Lo and towards the end of the rainy season in Buon Ma Thuot.

In the fourth part of our study (Chapter 5) the fungi associated with black pepper plant roots were identified and the relationship between the incidence of M. incognita, F. solani and yellowing of leaves examined in Quang Tri province. The interaction between M. incognita and F. solani alone or in combination on the percentage of black pepper plants with yellow leaves was also examined under greenhouse conditions. Nine fungal genera were isolated from the roots of black pepper plants in Quang Tri province. Fusarium solani was not found in roots of black pepper plants in the nurseries, plants younger than 5 years and plants without yellow leaves. Yellowing of leaves increased with increasing frequency of occurrence of F. solani. In the greenhouse experiment, there was a negative relationship between the inoculation with M. incognita alone or in combination with F. solani and percentage of black pepper plants with yellow leaves and plant growth. No effect of inoculation with F. solani before, at the same time, or 2 week after inoculation with M. incognita was observed.

In the fifth and last part of our study (Chapter 6), the host response of five black pepper varieties to M. incognita was evaluated under greenhouse conditions. No differences in host response to M. incognita of the five black pepper varieties were observed. All five varieties are considered equally susceptible to M. incognita. There are however indications that the variety La Da might be more sensitive and the variety Loc Ninh might be less sensitive or even tolerant to damage caused by M. incognita.

CHAPTER 1 1

CHAPTER 1. INTRODUCTION

1.1. OBJECTIVES OF THE STUDY AND OUTLINE OF THE THESIS Black pepper plants (Piper nigrum L.1) produce one of the oldest

and best-known spices in the world which is popularly considered as the “king of spices” because of its widespread use. Ground black peppercorn (dry fruit), usually referred to as “pepper2”, may be found on nearly every dinner table in many parts of the world, often alongside table salt.

Black pepper plants are mostly grown in tropical regions with a hot and humid climate. Apart from Asian countries such as India, Sri Lanka, Thailand, Malaysia, Indonesia, Vietnam and China, black pepper plants are also cultivated on other continents in countries such as Brazil and Madagascar. The eating habits of Asians ensure pepper a daily consumption. Hence, the production of pepper is important for the continued economic development of several highly-populated countries in Asia. Pepper crops are contributing to an increase in revenues for many rural families and are making a notable difference to the quality of life of millions of people.

Pepper represents about 1/3rd of the world trade in spices according to the International Pepper Community (IPC). The IPC was founded in 1972. Its members are Brazil, India, Sri Lanka, Thailand, Malaysia, Indonesia, Vietnam, Papua New Guinea and China. Together the IPC produces about 80% of the world production of pepper and accounts for about 90% of the world pepper export. Vietnam officially became a member of IPC at its 33rd session held in Bali, Indonesia, from November 29 to December 1, 2005. Today, the international pepper trade encompasses the world with the United States, Western Europe, Korea and Japan being the highest consumers. The major pepper producing countries are Vietnam, India, Brazil, Malaysia, Indonesia and Sri Lanka.

In Vietnam, agricultural products are still the country’s main export product, currently accounting for nearly 25% of the total export value. Vietnam has moved from a food importer to the world’s 3rd largest rice exporter and also emerged as a world-leading exporter of coffee,

1 Piper is the Latin word for pepper. Most of the European names for pepper were derived from the Sanskrit, pippali, a word used for the black pepper plant at least 3,000 years ago in India (Nelson & Eger, 2009). 2 Throughout our study, pepper will refer only to the spice produced by black pepper plants (P. nigrum).

CHAPTER 1 2

pepper and cashews. Today, Vietnam is the most important producer and exporter of pepper worldwide. High quality (white) pepper now accounts for 35% of the country’s total pepper export, according to the Vietnam Pepper Association (VPA). Vietnamese pepper is being exported to 80 countries around the world, mainly to the United States, the European Union and the Middle East.

Pepper is also considered as one of the major export products from Vietnam for the coming decade. For the period 2010-2020, the target is to cultivating black pepper plants on 50,000 ha producing about 120,000 tons pepper per annum and realising an export value of at least 240 million USD per year. To achieve this target and to stay on top of the international pepper trade, the improvement in yield and quality of the Vietnamese pepper crops is necessary.

Plant protection is important for pepper production because pathogens are a major cause of the reduction in yield and quality of pepper crops. For example, Phytopthora foot rot is a major disease of black pepper plants in India, Malaysia and Indonesia (Sarma et al., 1992; Holliday & Mowat, 1963; Kueh & Sim, 1992) whereas Fusarium solani f. sp. piperis causes root rot and stem blight of black pepper plants in Brazil (Duarte & Albuquerque, 1991). Anthracnose caused by fungi of the genus Colletotrichum in black pepper plants is referred to as "pollu" disease in India (which means hollow fruits) and as black berry disease in Malaysia and Indonesia (Anandaraj, 2000). Yellowing of the leaves of black pepper plants caused by nematodes (Radopholus similis and Meloidogyne spp.), either alone or in combination with fungi, have been observed (Bridge, 1978; Mustika, 1990; Ramana & Eapen, 2000).

Many nematode species have been reported from black pepper plants but the only nematodes known to cause serious damage to pepper crops are the burrowing nematode (R. similis) and root-knot nematodes (Meloidogyne spp.) (Koshy et al., 2005). Radopholus similis does not occur in Vietnam but root-knot nematodes (Meloidogyne spp.) are economically important plant pathogens in the country. Because of their wide host range and worldwide geographical distribution, Meloidogyne spp. pose a threat to agricultural crop production in many subtropical and tropical countries. They not only reduce yield but quality of crops as well.

In Vietnam, few in-depth information is available on the occurrence and importance of plant-parasitic nematodes on black pepper plants, and on their interaction with other pathogens of black pepper plants such as fungi. A slow yellowing of the leaves of black pepper plants has been observed since several years in all the major pepper production

CHAPTER 1 3

regions in Vietnam such as Quang Tri and Dac Lac provinces (Hoa et al., 2003; PPD, 2007) but the etiology of this common symptom has neither been examined nor determined.

One of the general objectives of our study was to elucidate the incidence of plant-parasitic nematodes on black pepper plants in Vietnam and their relationship with the incidence of soil-borne fungi and yellowing of the leaves of black pepper plants. Another general objective of our study, but longer term, was to improve the cash income of small-scale farmers in black pepper production regions of Vietnam through the development of a low-input and environmentally-friendly strategy for the management of root-knot nematodes on this agricultural crop.

To achieve these objectives, the frequency of occurrence and abundance of plant-parasitic nematodes on black pepper plants in the major pepper production regions in three agro-ecological regions of Vietnam and the relationship between the incidence of M. incognita, the predominant plant-parasitic nematode species found in the roots of black pepper plants in the country, with the incidence of yellow leaves of black pepper plants were examined (Chapter 2). The root-knot nematode diversity found on black pepper plants was studied (Chapter 3). The population dynamics of M. incognita on black pepper plants was followed in two agro-ecological regions of Vietnam (Chapter 4). The incidence of soil-borne fungi on black pepper plants and the relationship with the incidence of yellow leaves and M. incognita was examined in Quang Tri province while the interactions between M. incognita and F. solani, the predominant soil-borne fungus found in the roots of black pepper plants in Quang Tri province, was studied in a greenhouse experiment (Chapters 5). The host response to infection by M. incognita of five black pepper varieties commonly grown in Vietnam was evaluated in a greenhouse experiment Chapter 6). Finally, the conclusions of our study and some prospects for further research are presented (Chapter 7).

The study was conducted at the Plant Quarantine Diagnosis Centre of the Plant Protection Department (PPD), Ministry of Agriculture and Rural Development (MARD) in Vietnam. Figure 1.1 gives the research outline of our study.

CHAPTER 1 4

Figure 1.1: Research outline of our study.

Identification and comparative study of the morphology and

morphometrics of Meloidogyne populations from black pepper

plants in Vietnam (Chapter 3)

Host response of five black pepper varieties to infection

by Meloidogyne incognita under greenhouse conditions

(Chapter 6)

Fungi associated with black pepper plants in Quang Tri province and interaction

between Meloidogyne incognita and Fusarium solani

(Chapter 5)

Plant-parasitic nematodes and yellowing of leaves associated

with black pepper plants in Vietnam

(Chapter 2)

Population dynamics of Meloidogyne incognita on black

pepper plants in two agro-ecological regions in Vietnam

(Chapter 4)

Introduction

(Chapter 1)

Conclusions and

perspectives

(Chapter 7)

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5

1.2. BACKGROUND

1.2.1. THE BLACK PEPPER PLANT

1.2.1.1. Classification (CABI, 2007)

Kingdom: Viridiplantae Phylum: Spermatophyta Class: Dicotyledonae Order: Piperales Family: Piperaceae Genus: Piper Species: P. nigrum L.

Figure 1.2: Black pepper plant. Source of the picture: http://en.wikipedia.org/wiki/Black_pepper.

1.2.1.2. Origin

Black pepper plants originated in the monsoon forests along the coast of Malabar in southwestern India (Purseglove et al., 1981; De Waard, 1986). Today, this region belongs to the Indian state of Kerala. Black pepper plants are cultivated since millennia. The wild form has not been unambiguously identified but closely related pepper plant species occur in South India and Myanmar.

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6

Until well after the Middle Ages, virtually all of the pepper found in Europe, the Middle East and North Africa travelled there from Malabar. By the 16th century, black pepper plants were also being grown in Madagascar, Malaysia, Indonesia and elsewhere in Southeast Asia but these areas traded mainly with China or used the pepper locally (Dalby, 2002). In the last decades of the 20th century, pepper production increased dramatically as new plantations were established in Sri Lanka, Thailand, Vietnam and China.

1.2.1.3. Morphology

The black pepper plant is a perennial climbing and branching vine. It grows up to 4 m high on supporting trees, poles or trellises (Fig. 1.3). The black pepper plant attaches itself to these support structures by means of aerial roots and is thus not a parasitic plant. The leaves are oblong, pointed at the tip and arranged alternately. Leaves range in size from 13 to 25 cm.

Figure 1.3: Black pepper plants. Source of the picture: own picture.

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7

Black pepper plants have a shallow root system. There are usually a few main roots that can penetrate the soil to a depth of 2 m and feeder roots in the upper 60 cm of the soil (concentrated at 15 to 40 cm) which form an extensive dense mat.

The black pepper plant has three types of aerial stems (runners). The main stem (primary runner) forms the permanent stem from which other runners develop. The secondary runners are round, long shoots with lengthy internodes. They climb to a considerable height and later droop downwards. Tertiary runners are shorter, sturdier branches that spread horizontally from axils of primary and secondary runners.

Elongated, slender spikes (1.6 to 2 cm in length) bear minute, white, mainly hermaphroditic flowers (Fig. 1.4). The flower spikes always develop opposite a leaf and only on tertiary wood. A single stem usually bears 20 to 30 flower spikes.

Figure 1.4: Young inflorescences of a black pepper plant with flowering spikes. Source of the picture: own picture.

Each flower spike produces 50 to 60 berries (fruit). The berries are round, 4 to 10 mm in diameter. Each berry has a single spherical seed, 3 to 4 mm in diameter, encapsulated by the fruit flesh. The spikes lengthen from 7 to 15 cm as the berries mature (Fig. 1.5). The plant bears fruit from the 4th or 5th year onwards. Full production is realised 7 to 8 years after planting. Plants are most productive at 8 to 20 years of age but can continue bearing fruit for 30 years.

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8

Figure 1.5: Fruiting spikes of a black pepper plant. A: green berries; B: red berries. Source of the pictures: own pictures.

Pepper is unique in the spice world as the pepper fruit is being marketed in four different forms. It can be processed to give either black, white, green or red peppercorn (Fig. 1.6). By choosing the time of harvest and post processing methods carefully, all four forms can, in principle, be produced from the same black pepper plant (Karzer, 2002; De Witt, 2005; CABI, 2007).

Figure 1.6: Black, white, green and red peppercorns. Source of the picture: http://homecooking.about.com/od/foodhistory/a/pepperhistory.htm.

A B

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9

Black peppercorn is the most common form. The berries are harvested in their unripe, green stage and dried until they become wrinkled and black. White peppercorn is black peppercorn without its dark outer skin (pericarp). Green peppercorn consists of the unripe berries which are either pickled or preserved in brine. Red peppercorn is the rarest form. It is the dried form of berries in their red stage.

1.2.1.4. Climate and soil

Black pepper is a plant originating from the hot and humid tropics and, therefore, it requires adequate temperature and rainfall (Nelson & Eger, 2009). It thrives from sea level up to 600 m above sea level. The plant prefers temperatures from 22 to 30°C. It can tolerate temperatures between 10 and 40°C but it cannot tolerate frost. A total annual rainfall of 600 to 2,000 mm, evenly distributed over a long rainy season, is considered ideal for the cultivation of black pepper plants but black pepper plants can also grow well in wet soils. A short dry season favors fruit ripening and harvesting. Black pepper plants prefer soils rich in humus with acidic pH (5.5-6), good drainage and aeration. The most favourable soil types are deep, well-drained soils rich in organic matter and of medium texture.

1.2.1.5. Varieties

A majority of the black pepper varieties are monoecious (male and female flower on the same spike) through variation in sex ranging from complete male to complete female can be found. The diversity of the black pepper varieties cultivated is high. In India, for example, about 75 black pepper varieties are commonly being grown. The varieties can be distinguished from each other by differences in leaf shape and size, flowering and fruiting characteristics, etc.

In Vietnam, the common black pepper varieties grown are Vinh Linh, Loc Ninh, Tieu Se, LaDa Belangtoeng, Trau, Phu Quoc and An Do. These black pepper varieties originated in different regions. For example, variety Vinh Linh originated from Quang Tri province in Vietnam, variety An Do from India, variety LaDa Belangtoeng from Malaysia while variety Phu Quoc originated from Cambodia (Tiem, 2007).

1.2.1.6. Cultivation and harvest

Black pepper plants are usually propagated by rooted stolon cuttings. Land is cleared, tilled and hoed, and hardwood supports are placed at intervals in the ground. Cuttings, once rooted, are planted close

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10

to the supports. As the stems grow, they climb the supports. After almost 3 years, the plants are over 2 m tall and bushy. They start flowering at the onset of rains. Ripe berries may be picked about 9 months after flowering. The berries are picked by hand and are harvested 6 to 8 times each season at 2 weeks intervals.

The harvest season of black pepper plants varies throughout the world. Vietnamese pepper is usually harvested from March until June, a few months after the January-February harvest season in India and just before the Malaysian and Indonesian harvest seasons. The harvest seasons in Malaysia and Indonesia are usually from June to July and from July to October, respectively. In Brazil, the harvest season is from August to September (Table 1.1).

A full-grown (7 to 8 years old) well-developed black pepper plant can yield about 1.8 to 2.3 kg of dried berries each season (Nelson & Eger, 2009). Per hectare about 11,230 kg of green berries can be produced which converts to 3,930 kg/ha of dried black pepper or 3,140 kg/ha dried white pepper. Yield can vary significantly among production regions.

Table 1.1: The harvest seasons of black pepper plants in the major pepper producing countries.

Month

Country

Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

India

Vietnam

Sri Lanka

Malaysia

Indonesia

Brazil

Source: Anon. (2008).

1.2.1.7. Use

Black pepper is one of the earliest spices known and is probably the most widely used spice in the world today. Spices are increasingly being used to improve the taste and colour of foods. They are appreciated as natural ingredients. Pepper is widely used in most cuisines of the world. In West Asia, it is used cautiously but the cuisines use more spices when

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11

one moves east. In its native home, pepper is used abundantly. The Arabs had monopolised the pepper trade for millennia. It is thus not surprising that pepper is also popular in Arab cooking. Black pepper has found friends in the New World also and, subsequently, entered traditional cooking styles in Latin America. In Europe, pepper is still popular and is a main constituent of the French cuisine (Karzer, 2002).

Pepper has also a large number of medicinal uses (see Nelson & Eger, 2009) and produces piperine, an alkaloid, that has insecticidal activity against houseflies.

1.2.1.8. Production

Although black pepper plants can nowadays been found in many countries on both sides of the equator (Fig. 1.7), the production of pepper is restricted to relatively few countries, mainly in South Asia and Southeast Asia, and in Brazil.

Figure 1.7: Distribution map of black pepper plants (Piper nigrum L.). •: national record. Source of the map: CABI (2007).

India and Indonesia have for a very long time dominated the production of pepper in the world and the international pepper trade but lost this dominant position between 1997 and 2003 when the world production of pepper increased dramatically from 189,000 tons to 341,000 tons per year, an increase of 12% per annum (IPC) (Fig. 1.8). This increase in production is mostly due to Vietnam’s emergence as a major pepper producer and exporter although in most other producing countries pepper production also increased substantially during this period.

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12

Figure 1.8: Global pepper production trend (in tons) between 1970 and 2008. Source of the graph: Anon. (2008).

Today (production data from 2008), Vietnam has replaced India and Indonesia as the largest producer and exporter of pepper (Fig. 1.9). Vietnam now produces about 1/3rd of the pepper production worldwide. India and Indonesia together still produce somewhat more than 1/4th of the pepper production worldwide but during the early 1990’s, before the arrival of Vietnam as a major producer, India and Indonesia together produced about 50% of the pepper production worldwide. India (19%) is followed by Brazil (13%) as a major pepper producer. In Brazil, black pepper plants are mainly grown along the Amazon River in the State of Para and named after Belém, its main port (Katzer, 2002).

Figure 1.9: Percentage share of the major pepper producing countries. Source of the graph: Anon. (2008).

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13

In Vietnam, in 1980, only 500 ha were planted with black pepper plants. In 2009, the land planted with black pepper plants has increased 100-fold to 50,000 ha (Table 1.2). Over the same period, annual pepper production increased from 600 to 105,000 tons, a 175-fold increase. Production per ha increased from 1.2 tons in 1980 to 2.1 tons in 2009. Over a time span of almost 20 years (1990-2009), the export of pepper increased 14 times. In 2005, pepper export earned Vietnam about 150 million USD.

Table 1.2: Area planted with black pepper plants, pepper production and export in Vietnam from 1980 to 2009.

Year Area (1,000 ha)

Production (1,000 tons)

Export (1,000 tons)

1980 0.5 0.6

1985 2.2 1.3

1990 9.2 8.6 9.0

1995 7.0 9.3 17.9

2000 27.9 39.2 36.4

2001 31.6 44.4 56.5

2002 47.9 46.8 78.4

2003 50.5 68.5 74.6

2004 50.8 73.4 98.5

2005 49.1 77.0 96.2

2006 50.1 100.0 116.6

2007 50.0 90.0 82.9

2008 48.0 87.0 80.0

2009 50.0 105.0 128.0 Source: Vietnam General Statistics Office (2009).

In Vietnam, black pepper plants are mainly being grown from the

North Central Coast to the Southeast (Fig. 1.10). In the Mekong River Delta, cultivation is limited to the west of this region and Phu Quoc Island.

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14

Figure 1.10: Black pepper cultivation in Vietnam. ●●: Provincial record. Source of the map: Vietnam Pepper Association (VPA).

As can been seen on the figures with the global pepper production

trend (Fig. 1.8) and the pepper production trend in Vietnam (Fig. 1.11), and the table with a comparison of pepper exports between 2006-2007 and 2007-2008 (Table 1.3), pepper production may vary considerable from year to year. This is attributed to a combination of factors including erratic rain, poor crop management resulting in exhausted black pepper plants and the outbreak of diseases.

North Central Coast

Central Highlands

Southeast

Mekong River Delta

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15

Figure 1.11: Pepper production trend (in tons) in Vietnam. Source of the graph: Anon. (2008).

Table 1.3: Comparison of pepper exports between 2006-2007 and 2007-2008 in tons).

Country 2006-2007 2007-2008 % change

Vietnam 116,670 83,000 -28.9

Brazil 42,194 36,000 -14.7

India 26,377 30,000 13.7

Malaysia 16,602 19,000 14.4

Indonesia 20,000 16,300 -18.5

Sri Lanka 8,190 8,500 4.0

China 10,000 3,000 -70.0

Thailand 1,400 1,500 7.1

Source: International Pepper Community (IPC).

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16

1.2.2. NEMATODES AND FUNGI ASSOCIATED WITH BLACK PEPPER PLANTS

1.2.2.1. Nematodes

The first report of a nematode (Meloidogyne sp.) attacking black pepper plants was made by Zimmerman in Java, Indonesia, in 1901 (Winoto, 1972). Since then, many plant-parasitic nematode species have been found associated with black pepper plants. Koshy et al. (2005) list 28 species belonging to 17 genera but the number of nematode species attacking black pepper plants is much higher as demonstrated by Chau and Thanh (1993b) who identified 49 nematode species associated with black pepper plants in Quang Tri province, Vietnam, alone.

Only two nematode taxa are considered to cause serious damage to pepper crops: R. similis and Meloidogyne spp. (Koshy et al., 2005).

Association of the burrowing nematode R. similis with the so-called “yellow(s) disease” of black pepper plants was first reported in 1936 in Indonesia. This disease caused the loss of 22 million black pepper plants within 20 years in Bangka Island, Indonesia (Christie, 1957, 1959). Later, R. similis was also reported on black pepper plants in India, Sri Lanka, Malaysia and Thailand (Koshy et al., 2005). In India, it was shown that R. similis is involved in the so-called “slow wilt disease” of black pepper plants (Ramana et al., 1987) which is almost identical to the yellow(s) disease in Indonesia (Van der Vecht, 1950; Mohandas & Ramana, 1987). Radopholus similis was most probably introduced in South Asia and Southeast Asia with infected root cuttings of black pepper plants (Koshy et al., 2005).

Association of root-knot nematodes (Meloidogyne spp.) with black pepper plants was first reported in the beginning of the 1990’s in India (Koshy et al., 2005). Later, M. incognita and Meloidogyne javanica were reported on black pepper plants in India, Thailand, Malaysia, Cambodia, Vietnam, Indonesia, Brunei, the Philippines and Brazil while Meloidogyne arenaria was reported on black pepper plants in Sri Lanka (Koshy et al., 2005). In 2000, a new species on black pepper plants, Meloidogyne piperi, was described (Sahoo et al., 2000).

Meloidogyne spp. are obligate sedentary endoparasites. Mobile second-stage juveniles (J2) emerge from the eggs and attack nearby roots. They usually penetrate the root tips. The feeding of the J2 on protoxylem and protophloem inside the root induces these cells to differentiate into specialised feeding cells, the so-called “giant cells”. At the same time, galling of the roots occurs. The J2 feed on these giant cells and moult

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17

three times to form the adult females that enlarge rapidly. Reproduction is parthenogenetic. The J2 (the infective stage) and males are the stages of Meloidogyne spp. that can be found freely in soil. A high percentage of males are produced only in adverse conditions. The eggs are laid within a gelatinous matrix to form an external egg mass. A single egg mass can contain several hundred eggs (Fig. 1.12). The life cycles of the most common Meloidogyne spp. are well studied and differ little among the major species (De Guiran & Ritter, 1979).

Figure 1.12: Life and disease cycle of root-knot nematodes (Meloidogyne spp.). Source of the figure: The American Pathological Society (2003).

On black pepper plants, a gradual decline characterised by unthrifty growth and yellowing of the leaves is the most prominent symptom caused by Meloidogyne spp. Infected leaves exhibit dense yellowish discoloration of the interveinal areas, making the leaf veins quite prominent with a deep green colour (whereas leaves of black pepper

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18

plants infected with R. similis show uniform pale yellow or whitish discoloration and typical drooping) (Ramana et al., 1994). Roots of black pepper plants infected with Meloidogyne spp. become heavily galled and the adult females are generally enclosed deep within the root tissues (Ramana, 1992; Ramana et al., 1994). Koshy et al. (1979) reported that an initial population of 10 M. incognita J2 per rooted cutting reduced growth of black pepper plants by 16% while a maximum of 50% reduction was observed at an inoculum level of 100,000 M. incognita J2 over a period of 1 year in sterile soil under potted conditions. Meloidogyne incognita was also found highly pathogenic at 100-10,000 J2 per black pepper seedling (Freire & Bridge, 1985; Mohandas & Ramana, 1991). In Indonesia, yellow symptoms appeared on the leaves of black pepper plants infected with Meloidogyne spp. at population levels of 47 J2/100 g soil and 305 J2/10 g roots (Mustika, 1978).

1.2.2.2. Fungi and nematodes-fungi disease complexes

Several diseases caused by soil-borne fungi, such as foot rot caused by Phytophthera spp. and root rot caused by Fusarium spp., can cause important pepper crop losses. Information on the interaction between these soil-borne fungi, R. similis and Meloidogyne spp. on black pepper plants is limited and sometimes contradictory.

According to Holliday and Mowat (1963) Meloidogyne spp. do not significantly enhance the sensitivity3 of black pepper plants to foot rot caused by Phytophthora palmivora. However, in Malaysia, Winoto (1972) reported increased sensitivity of M. incognita- and M. javanica-infected black pepper plants (cv. Kuching) to Phytopthora infection while in India Ramana et al. (1992) and Anandaraj et al. (1996a,b) reported that black pepper plants showed wilting symptoms faster when Meloidogyne spp. and R. similis were inoculated first followed by Phytophthora capsici.

3 For description of the host plant/pathogen (nematode) relationships, this thesis will follow the terminology of Bos and Parlevliet (1995). Resistance/susceptibility on the one hand and tolerance/sensitivity on the other hand are defined as independent, relative qualities based on comparison between genotypes. A host plant may either suppress (resistance) or allow (susceptibility) nematode development and reproduction; it may suffer either little injury (tolerance), even when quite heavily infected with nematodes, or much injury (sensitivity), even when relatively lightly infected with nematodes.

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19

Yellowing of the leaves of black pepper plants caused by R. similis and Meloidogyne spp. has been described and it has been speculated that this symptom is caused by a nematode-fungus complex involving either R. similis or Meloidogyne spp. and Fusarium spp. or possibly other fungi (Koshy et al., 2005). This is the case for the yellow(s) disease and the gradual decline characterised by unthrifty growth and yellowing of leaves. There are indeed several reports of the joint occurrence of Meloidogyne spp. and Fusarium spp. in the roots of black pepper plants. Meloidogyne incognita and F. solani were found associated with black pepper plants in Brazil. Infested plants showed wilting, yellowing of leaves, rotting of roots and stems, and cracking of stems (Koshy et al., 2005). Mustika (1984) showed that M. incognita and Fusarium sp. caused severe necrosis in the stellar part of black pepper plants in Indonesia resulting in the formation of tyloses that blocked the xylem. But although there are several reports that Meloidogyne spp. and Fusarium spp. together cause more damage to black pepper plants than either of them alone (Lopes & Lordello, 1979; Sheela & Venkitesan, 1990; Mustika, 1991, 1992) the existence of nematodes-fungi disease complexes in black pepper plants remain unclear.

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20

CHAPTER 2 21

CHAPTER 2. PLANT-PARASITIC NEMATODES AND YELLOWING OF LEAVES ASSOCIATED WITH BLACK PEPPER PLANTS IN

VIETNAM4

2.1. INTRODUCTION Black pepper (Piper nigrum L.) is considered, together with rice,

coffee, rubber, cashew, vegetables and fruit, as one of the major agricultural products in Vietnam. During 2007-2010, the Ministry of Agricultural and Rural Development undertook action to stabilise the area for pepper production at about 50,000 ha and the pepper production itself at about 120,000 tons per annum. To achieve this objective, Vietnam maintains and expands the cultivation of black pepper plants only in areas endowed with a favourable climate. Extension officers help farmers inter alia with the identification of areas ecologically suitable for pepper production, new high-yielding black pepper varieties and knowledge in identifying and controlling pathogens.

Plant protection is an especially important part of pepper production because pathogens are considered a major cause of the reduction in yield and quality of pepper crops. In Indonesia, for example, complete fields with black pepper plants were destroyed by pathogens such as fungi (Phytophthora spp.) and the burrowing nematode (Radopholus similis) (Sitepu & Mustika, 2000). In 2007, the Plant Protection Department (PPD) recorded 17 pathogens affecting black pepper plants in Vietnam (Table 2.1). Among the symptoms commonly observed were stunting, quick wilt, leaf blight, anthracnose and slow yellowing of the leaves (PPD, 2007). In Vietnam, farmers are not very well aware of the occurrence of plant-parasitic nematodes in their fields. They often confuse symptoms caused by nematodes with those caused by other plant pathogens, resulting in control measures not being applied properly or not being applied at all.

4 The results presented in this chapter have been accepted for publication in: Thuy, T.T.T., Yen, N.T., Tuyet, N.T.A., Te, L.L. and De Waele, D. Plant parasitic nematodes associated with black pepper in Vietnam. Russian Journal of Nematology. (in press)

CHAPTER 2 22

Table 2.1: List of pathogens effecting black pepper plants in Vietnam.

No Name/Scientific name Plant parts affected Occurrence

1 Collectotrichum gloeosporioides Leaves +++

2 Corticium salmonicolor Stems ++

3 Fusarium solani Stem and roots +++

4 Fusarium spp. Stem and roots ++

5 Lasiodiplodia theobromae Roots ++

6 Phytophthora capsici Whole plants +++

7 Phytophthora nicotianae Whole plants +

8 Phytophthora cinnamomi Whole plants +

9 Phytophthora palmivora Whole plants +++

10 Pythium sp. Stem and roots ++

11 Rhizoctonia solani Roots ++

12 Rosellia sp. Roots ++

13 Sclerotium rolfsii Roots +

14 Red algae Leaves +

15 Stunted disease Whole plants ++

16 Slow wilt disease Whole plants +++

17 Nematodes Roots +++

+++: common; ++: less common but not rare; +: rare.

Plant-parasitic nematodes can also cause substantial damage to

black pepper plants. Yellowing of the leaves of black pepper plants caused by R. similis and Meloidogyne spp. has been described. Radopholus similis can cause the so-called “yellow(s) disease” and “slow wilt disease” of black pepper plants (Koshy et al., 2005). The first disease has been mainly studied in Indonesia where R. similis is notorious for being associated with the loss of 22 million black pepper plants within 20 years in Bangka Island. The second disease has been mainly studied in India. Both diseases look very similar. It has been speculated that the yellow(s) disease in Indonesia is caused by a nematode-fungus complex involving R. similis, Fusarium spp. and possibly other fungi (Bridge, 1978) but there is little evidence to support this hypothesis. A gradual decline characterised by unthrifty growth and yellowing of the leaves of black pepper plants caused by Meloidogyne spp. has also been reported (Koshy et al., 2005). Also in this

CHAPTER 2 23

case it has been speculated that the yellowing of the leaves is caused by a nematode-fungus complex involving Meloidogyne species and fungi (Fusarium spp., Phytophthora spp.) but there is also little evidence to support this hypothesis. The difference between the yellow leaves symptoms caused by R. similis and Meloidogyne spp. is that R. similis causes a uniform pale yellow or whitish discoloration while Meloidogyne spp. cause a dense yellowish discoloration of the interveinal areas making the leaf veins quite prominent with a deep green colour (Koshy et al., 2005).

In Vietnam, Chau and Thanh (1993b) listed 49 nematode species associated with black pepper plants in Quang Tri province alone. Meloidogyne spp. were found in almost all pepper production regions. Root-knot nematodes are regarded as the most damaging and serious nematode threat to pepper production in Vietnam (Bien, 1989; Chau et al., 1990; PPD, 2007) but more precise information on their incidence on black pepper plants and their relationship with the incidence of yellow leaves of black pepper plants is missing.

The objectives of this part of our study were 1) to identify the predominant plant-parasitic nematode species associated with black pepper plants in the major pepper production regions of Vietnam, 2) to study the frequency of occurrence and abundance of Meloidogyne spp. on black pepper plants, 3) to examine the incidence of yellow leaves on black pepper plants, and 4) to investigate if a there is a relation between the incidence of Meloidogyne spp. on the one hand and the incidence of yellow leaves on black pepper plants on the other hand. The study was carried out in three agro-ecological regions of Vietnam so that the effect of the environment on the incidence of plant-parasitic nematodes and yellow leaves on black pepper plants could also be studied.

2.2. AGRO-ECOLOGICAL REGIONS OF VIETNAM Lying on the eastern part of the Indochinese peninsula, Vietnam is

a strip of land shaped like the letter “S”. China borders it to the north, Laos and Cambodia to the west, the South China Sea to the east and the Pacific Ocean to the east and south. It covers a land area about 330,000 km2, extending from latitude 9 to 23°N and longitude 102 to 110°E with a 3,000 km long coastline.

Vietnam has a tropical monsoon climate, with an air humidity averaging 84% throughout the year, especially during the rainy season. 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

CHAPTER 2 24

distinguished. The annual rainfall is substantial in all regions and torrential in some, 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 generally higher in the south than in the north, and in the lowlands than in the mountains and plateaus. In the north, temperatures range from a low of 5°C in December and January, the coolest months, to more than 37°C in July and August, the hottest months. Seasonal differences are more clearly marked in the north than in the south of the country, where, except in the highlands, seasonal temperatures vary only a few degrees, usually in the 21 to 28°C range. Based mainly on differences in climate and topography, eight agro-ecological regions can be distinguished in Vietnam (Fig. 2.1).

Mountains, hills and plateaus cover two-thirds of the mainland. Areas over 500 m above sea level account for 70% of the mainland. The most grandiose and highest mountain ranges lie in the west and northwest of the country. In the southwest part of Central Vietnam, there is a huge “mountain-highland” over 1,000 m above sea level, covered by basalt soil ideal for producing tropical industrial crops such as tea, coffee, cacao, black pepper and rubber. The major pepper producing agro-ecological regions are the North Central Coast, the Central Highlands, the Southeast and the Mekong River Delta (Phu Quoc Island).

North Central Coast

This region has a cold winter lasting from December to February with an average monthly temperature below 20°C (about 16 to 19°C). In July, the average temperature is 28 to 29°C. 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.

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 22°C. The warmest months are March and April, the coldest month is January. Rainfall is low during the dry season from 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.

CHAPTER 2 25

Southeast The Southeast has an average annual temperature of about 21°C.

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 part of the region and from 1,400 to 1,600 mm in the coastal part of the region.

Figure 2.1: Agro-ecological regions of Vietnam and provinces surveyed. 1: Quang Tri; 2: Thua Thien Hue; 3: Kon Tum; 4: Gia Lai; 5: Dac Lac; 6: Phu Quoc.

Mekong River Delta

This region has a high average annual temperature (26 to 27°C). The daily temperatures range from 17 to 34°C in January and from 22 to 33°C in July. The rainfall is very variable: from the western parts of this region, with a rainy season of 8 months, the annual rainfall gradually decreases towards the eastern part of this region from more than 2,000 mm to 1,400 mm.

Phu Quoc Island is located in the Gulf of Thailand and is part of Kien Giang province (Mekong River Delta). The island covers an area of 567 km² and is about 50 km long north to south, and 28 km at its widest point (west to east) in the north. The island's monsoonal sub-equatorial climate is characterised by a distinct rainy (June to November) and a distinct dry

CHAPTER 2 26

season (December to May). The average annual rainfall is about 2,900 m and the average annual temperature is 27°C.

2.3. MATERIALS AND METHODS The sampling was carried out during 2004-2006. The samples were

collected in three agro-ecological regions: the North Central Coast, the Central Highlands and the Mekong River Delta (Phu Quoc Island) (Table 2.2 and Fig. 2.1).

Table 2.2: Sampling periods, districts, provinces and agro-ecological regions sampled.

Sampling period District Province Agro-ecological region

July, November 2004 and

June 2005

Cam Lo Quang Tri North Central Coast

July, November 2004 and

June 2005

Gio Linh Quang Tri North Central Coast

June 2005 Huong Tra Thua Thien Hue North Central Coast

June 2005 Huong Thuy Thua Thien Hue North Central Coast

June 2005 Phu Loc Thua Thien Hue North Central Coast

May 2005 Buon Ma Thuot Dac Lac Central Highlands

May 2005 and August 2006 CMgar Dac Lac Central Highlands

May 2005 and August 2006 Buon Don Dac Lac Central Highlands

May 2005 and August 2006 EaHleo Dac Lac Central Highlands

May 2005 Dac Doa Gia Lai Central Highlands

May 2005 Chu Se Gia Lai Central Highlands

May 2005 Chu Prong Gia Lai Central Highlands

May 2005 An Phu Gia Lai Central Highlands

May 2005 Dac Ha KonTum Central Highlands

May 2005 SaThay KonTum Central Highlands

May 2005 I A Chim KonTum Central Highlands

May 2006 Duong To Phu Quoc Island Mekong River Delta

May 2006 Cua Duong Phu Quoc Island Mekong River Delta

May 2006 Cua Can Phu Quoc Island Mekong River Delta

In July and November 2004 and in June 2005, samples were taken

in the North Central Coast. In this region, the dry season is from April to August and the rainy season from September to March. In May 2005 and August 2006, samples were taken in the Central Highlands. In this region,

CHAPTER 2 27

the dry season is from October to March and the rainy season from April to September. In May 2006, samples were taken in Phu Quoc Island. On this island, the dry season is from December to May and the rainy season from June to November.

In each agro-ecological region sampled, one to three provinces were visited. In each province, one to three districts were visited. In each district, samples were taken from three fields with black pepper plants. Per field, at least five soil and root samples were collected at random. Sampling depth was 25 cm. Roots and soil of three black pepper plants constituted one sample. Samples were stored in plastic bags in a coolbox until nematode extraction was carried out.

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 with 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 200, 160 and 25 μm apertures. The extracted nematodes were examined under a stereomicroscope for the presence of plant-parasitic nematodes.

The nematode population densities in the soil and the roots were defined as the number of second-stage juveniles (J2) per 100 g soil and 5 g roots for Meloidogyne spp. and as the number of vermiform nematodes per 100 g soil and 5 g roots for the other plant-parasitic nematodes.

Root galling was assessed by examining the roots on the presence of galls. The root galling index used was based on the percentage of galled roots (Speijer & De Waele, 1997):

0 = no galling 1 = a few small galls 2 = < 25% of the roots galled 3 = 25–50% of the roots galled 4 = 50–75% of the roots galled 5 = > 75% of the roots galled.

The percentage of plants with yellow leaves was calculated based on the number of black pepper plants with yellow leaves out of 100 black pepper plants per field.

CHAPTER 2 28

2.4. RESULTS

2.4.1. NEMATODES ASSOCIATED WITH BLACK PEPPER PLANTS IN VIETNAM In total, 432 soil and 432 root samples were collected from black

pepper plants in 19 districts belonging to six provinces in Vietnam. A list of all the plant-parasitic nematode species associated with black pepper plants found during the surveys is given in Table 2.3.

Thirty-five plant-parasitic nematode taxa belonging to 19 genera and 11 families were identified. Five taxa of plant-parasitic nematodes were present in all provinces surveyed: Meloidogyne spp., Rotylenchulus reniformis, Tylenchus sp., Aphelenchus avenae and Ditylenchus ausafi. Meloidogyne spp. were found in almost every root sample (98.4%) while R. reniformis was found in only one out of five root samples (20.8%). Based on nematode population densities, Meloidogyne spp. was the most abundant taxon present. All the Meloidogyne populations collected were identified as M. incognita (see Chapter 3). Twelve nematode taxa were found in only one province: Aphelenchoides dubius, Criconemella curvata, Criconemella magnifica, Hemicriconemoides cocophilus, Helicotylenchus cornurus, Helicotylenchus crenacauda, Hoplolaimus seinhorsti, Pratylenchus brachyurus, Seinura sp., Scutellonema sp., Tylenchulus semipenetrans and Xiphinema insigne.

Some plant-parasitic nematode species occurred only in one agro-ecological region. For example, Aphelenchoides bicaudatus and Longidorus elongatus were only found in the North Central Coast while A. cylindricaudatus and Pratylenchus coffeae were only found in the Central Highlands. In Phu Quoc Island only eight out of the 35 nematode taxa identified were found. Spiral nematodes (Helicotylenchus and Hoplolaimus spp.) and root-lesion nematodes (Pratylenchus spp.) were only found in 9.6% and 9.8%, respectively, of the samples examined and were not found in Phu Quoc Island.

2.4.2. FREQUENCY OF OCCURRENCE AND ABUNDANCE OF MELOIDOGYNE INCOGNITA, ROOT GALLING AND PERCENTAGE OF PLANTS WITH YELLOW LEAVES

The frequency of occurrence and abundance of M. incognita, root galling and the percentage of plants with yellow leaves in the three agro-ecological regions surveyed are presented in the Tables 2.4-2.6 and the Figs 2.2-2.5.

CHAPTER 2 29

Table 2.3: Plant-parasitic nematodes associated with black pepper plants in Vietnam.

Population density

NCCoast Central Highlands Mekong River Delta No. Scientific name n

Frequency of occurrence

(%) QT TTH DL GL KT PQ

1* Aglenchus sp. 14 3.2 + + + +

2 Filenchus sp. 15 3.5 + + + +

3* Tylenchus sp. 128 29.6 + + ++ ++ + +

4 Aphelenchoides bicaudatus (Imamura) Filipjev & Sch. Stekhoven 20 4.6 + +

5 A. curiolis Gritzenko 10 2.3 + +

6* A. dubius Wasilewska 4 0.9 +

7* A. singhi Das 9 2.1 + + +

8* A. trivialis Franklin & Siddiqi 10 2.3 + + +

9 Aphelenchoides sp. 35 8.1 + + + + +

10 Aphelenchus avenae Bastian 382 88.4 ++ ++ ++ ++ ++ +

11 A. cylindricaudatus (Steiner) Steiner 10 2.3 + + +

12 Criconemella curvata (Raski) de Grisse & Loof 7 1.6 +

13 Criconemella magnifica (Eroshenko & Nguyen V.T.) Raski & Luc 8 1.9 +

15 Discocriconemella limitanea (Luc) De Grisse & Loof 14 3.2 ++ + + +

16 Ditylenchus ausafi Husain & Khan 134 31.0 + + + + + ++

17 Ditylenchus myceliophagus Goodey 11 2.6 + +

18 Helicotylenchus certus Eroshenko & Nguyen V.T. 10 2.3 + + +

CHAPTER 2 30

Continued (Table 2.3) n (%) QT TTH DL GL KT PQ

19 H. coffeae Eroshenko & Nguyen V.T. 3 0.7 +

20 H. cornurus Anderson 4 0.9 +

21 H. crenacauda Sher 4 0.9 +

22 H. dihystera (Cobb) Sher 20 4.6 + + + + +

23 Hemicriconemoides cocophilus (Loos) Chitwood & Birchfield 4 0.9 +

24 Hoplolaimus seinhorsti Luc 1 0.2 +

25 Meloidogyne spp. 425 98.4 +++ +++ +++ +++ +++ +++

26 Pratylenchus coffeae (Zimmerman) Filipjev & Sch. Stekhoven 37 6.6 + + +

27 Pratylenchus brachyurus (Godfrey) Filipjev & Sch. Stekhoven 14 3.2 +

28 Rotylenchulus reniformis Linford & Oliveira 90 20.8 ++ ++ ++ ++ + +

29 Seinura sp. 2 0.5 +

30 Scutellonema sp. 3 0.7 +

31 Tylenchulus semipenetrans Cobb 5 1.2 +

32 Xiphinema americanum Cobb 24 5.5 + + + + +

33 Xiphinema elongatum Sch. Stekhoven & Teunissen 17 3.9 + + + +

34 Xiphinema insigne Loos 2 0.5 +

35 Longidorus elongatus de Man 3 0,7 + + +

QT: Quang Tri; TTH: Thua Thien Hue; DL: Dac Lac; GL: Gia Lai; KT: KonTum; PQ: Phu Quoc; NCCoast: North Central Coast; n: number of samples examined;

+: < 50 J2 or vermiform nematodes/100 g soil or 5 g roots; ++: from 50 to 150 J2 or vermiform nematodes/100 g soil or 5 g roots; +++: > 150 J2 or vermiform

nematodes/100 g soil or 5 g roots ; * for the first time found on black pepper plants in Vietnam.

CHAPTER 2 31

2.4.2.1. North Central Coast

One hundred and twenty three soil and 123 root samples were collected in two provinces in the North Central Coast. The presence of plants with yellow leaves was examined in 21 black pepper fields. The results are shown in Table 2.4 and Fig. 2.2.

Table 2.4: Frequency of occurrence and population density of Meloidogyne incognita, root galling and percentage of plants with yellow leaves in black pepper fields in two provinces in the North Central Coast, Vietnam.

Frequency of

occurrence (%)

Population density Province

District n

Soil Roots Soil

(J2/100 g)

Roots

(J2/5 g)

Root

galling

(%)

Yellow

leaves

(%)

Gio Linh 40 100 100 119 110 44.8 10.3

Cam Lo 32 100 100 122 96 28.5 32.7

Quang Tri

(n=72)

Average 100 100 121 103 36.6 21.5

Huong

Thuy

18 94.4 88.9 56 167 21.7 7.7

Phu Loc 15 100 100 275 752 72.0 37.7

Huong Tra 18 100 100 545 135 30.0 33.3

Thua

Thien Hue

(n=51)

Average 98.2 96.3 292 352 41.3 26.2

All

districts

(n=123)

Average 99.0 98.2 207 227 39.5 24.3

n: number of samples examined.

Meloidogyne incognita was found in every root sample collected in Quang Tri and 96.3% of the roots samples collected in Thua Thien Hue province. On average, the number of J2 in the soil and roots was higher in Thua Thien Hue than in Quang Tri province (292 J2/100 g soil and 352 J2/5 g roots vs 121 J2/100 g soil and 103 J2/5 g roots, respectively). On average, percentage root galling was about 40%. Percentage root galling was only slightly higher in Thua Thien Hue than in Quang Tri province (41.3 vs 36.6%). The highest average percentage of root galling (72%) was

CHAPTER 2 32

observed in Phu Loc district while the lowest average percentage of root galling (21.7%) was observed in Huong Thuy district.

On average, yellow leaves were observed in almost one out of every four black pepper plants examined (24.3%). The percentage of black pepper plants with yellow leaves was highest in Phu Loc district (37.7%) and lowest in Huong Thuy district (7.7%).

Figure 2.2: Meloidogyne incognita population density, percentage of root galling and plants with yellow leaves in black pepper fields in two provinces in the North Central Coast, Vietnam.

2.4.2.2. Central Highlands

Two hundred and forty soil and 240 root samples were collected in three provinces in the Central Highlands. The presence of plants with yellow leaves was examined in 36 black pepper fields. The results are shown in Table 2.5 and Fig. 2.3.

Meloidogyne incognita was found in 95% of all root samples examined. The frequency of occurrence was similar in the three provinces ranging from 92.8 (Dac Lac) to 98.4% (Kon Tum). On average, the number of J2 in the soil and roots was highest in Gia Lai (391 J2/100 g soil and 663 J2/5 g roots) and lowest in Dac Lac province (158 J2/100 g soil and 43 J2/5 g roots). On average, percentage root galling was about 40%. Percentage root galling was quite similar in the three provinces, ranging on average from 37.7 to 45.8%. The highest average percentage of root galling (60%)

0

50

100

150

200

250

300

350

400

Quang Tri Thua Thien Hue

Province

Num

ber

of J

2

0

5

10

15

20

25

30

35

40

45

Perc

enta

ge (

%)

Number of J2 in 100 g soil Number of J2 in 5 g fresh rootsYellow leaves (%)Root galling (%)

CHAPTER 2 33

was observed in ChuProng district while the lowest average percentage of root galling (23.3%) was observed in Dac Doa district.

On average, yellow leaves were observed in almost one out of every five black pepper plants examined (22.9%). The percentage of black pepper plants with yellow leaves was highest in Gia Lai (36.9%) and lowest in Dac Lac province (11.8%). In Dac Ha district, yellow leaves were observed in only 2% out of the black pepper plants examined. In An Phu district, yellow leaves were observed in almost half of the black pepper plants examined (48.3%).

Table 2.5: Frequency of occurrence and population density of Meloidogyne incognita, root galling and percentage of plants with yellow leaves in black pepper fields in three provinces in the Central Highlands, Vietnam.

Frequency of

occurrence (%)

Population density

Province

District

n

Soil Roots Soil

(J2/100 g)

Roots

(J2/5 g)

Root

galling

(%)

Yellow

leaves

(%)

Cu Mgar 24 91.7 91.7 90 19 54.3 13.0

Buon Ma

Thuot

15 100 94.1 196 71 31.6 15.0

Buon Don 30 100 90.0 210 25 43.0 11.0

EaHleo 21 100 95.2 137 84 42.6 8.0

Dac Lac

(n=90)

Average 97.9 92.8 158 43 42.9 11.8

Dac Doa 19 100 94.7 118 36 23.3 33.3

Chu Se 24 100 100 108 299 55.5 33.3

ChuProng 29 100 100 333 642 60.0 32.7

An Phu 10 80.0 80.0 1,004 1,675 44.5 48.3

Gia Lai

(n=82)

Average 95.0 93.7 391 663 45.8 36.9

Dac Ha 21 95.2 95.2 59 24 24.1 2.0

Sa Thay 19 100 100 175 28 34.6 12.0

IA Chim 32 100 100 412 97 54.4 46.0

Kon Tum

(n=72)

Average 98.4 98.4 216 50 37.7 20.0

All

districts

(n=244)

Average 97.1 95.0 255 253 42.5 22.9


CHAPTER 2 34

Figure 2.3: Meloidogyne incognita population density, percentage of root galling and plants with yellow leaves in black pepper fields in three provinces in the Central Highlands, Vietnam.

2.4.2.3. Phu Quoc Island (Mekong River Delta)

Sixty-five soil and 65 root samples were collected in Phu Quoc Island. The presence of plants with yellow leaves was examined in 10 black pepper fields. The results are shown in Table 2.6 and Fig. 2.4.

Table 2.6: Frequency of occurrence and population density of Meloidogyne incognita, root galling and percentage of plants with yellow leaves in black pepper fields in three districts in Phu Quoc Island, Vietnam.

Frequency of

occurrence (%)

Population density Island District n

Soil Roots Soil

(J2/100 g)

Roots

(J2/5 g)

Root

galling

(%)

Yellow

leaves

(%)

Duong To 20 90.0 95.0 59 13 34.0 7.7

Cua Duong 17 100 82.4 191 10 32.0 21.8

Phu

Quoc

Cua Can 28 100 100 344 96 48.0 21.3

All

districts

(n=65)

Average 98.4 98.4 216 50 38.0 16.9


0

100

200

300

400

500

600

700

Kon Tum Gia Lai Dac Lac

Province

Num

ber

of J

2

0

5

1015

20

25

30

3540

45

50

Perc

enta

ge (%)


CHAPTER 2 35

Figure 2.4: Meloidogyne incognita population density, percentage of root galling and plants with yellow leaves in black pepper fields in three districts in Phu Quoc Island, Vietnam.

Meloidogyne incognita was found in almost every root sample examined (98.4%). The frequency of occurrence ranged from 82.4 (Cua Duong) to 100% (Cua Can district). On average, the number of J2 in the soil and roots was highest in Cua Can district (344 J2/100 g soil and 96 J2/5 g roots). On average, percentage root galling was about 40%. The highest average percentage of root galling (48%) was observed in Cua Can district while the lowest average percentage root galling (32%) was observed in Cua Duong district.

On average, yellow leaves were observed in almost one out of every five black pepper plants examined (16.9%). The percentage of black pepper plants with yellow leaves was highest in Cua Duong (21.8%) and lowest in Duong To district (7.7%).

2.4.2.4. Description of root galls

Root galls on the thick primary roots were elongated (Fig. 2.5A) while root galls on the fibrous secondary roots were knot-like (Fig. 2.5B). In the thick primary roots, a single elongated gall or root thickening contained usually several adult females with egg masses (Figs 2.5C & D).

0

50

100

150

200

250

300

350

400

Duong To Cua Duong Cua Can

District

Num

ber

of J

2

0

10

20

30

40

50

60

Perc

enta

ge (

%)


CHAPTER 2 36

Figure 2.5: Root galls caused by Meloidogyne incognita on a primary root (A) and secondary root (B) of a black pepper plant. Adult females of M. incognita inside a primary root of a black pepper plant (C & D). Source of the pictures: own pictures.

2.4.2.5. Description of yellow leaves

Yellow leaves exhibited a dense yellowish discoloration of the interveinal areas, making the leaf veins prominent with a deep green colour (Fig. 2.6A). Plants with yellow leaves were stunted and tended to wilt easily even when soil moisture was adequate (Fig. 2.6B).

Figure 2.6: Yellow black pepper plant leaves (A). A black pepper field with plants with yellow leaves (B). Source of the pictures: own pictures.

A B

D C

B A

CHAPTER 2 37

2.4.2.6. Correlation between Meloidogyne incognita population density and percentage of plants with yellow leaves

The correlation between M. incognita population density and percentage of plants with yellow leaves in 67 black pepper fields in three agro-ecological regions in Vietnam is shown in Fig. 2.7.

Figure 2.7: Correlation between Meloidogyne incognita population density and percentage of plants with yellow leaves in 67 black pepper fields in three agro-ecological regions in Vietnam.

There was a weak positive relationship (r² = 0.36; r² = 0.43; P ≤ 0.05) between the number of M. incognita J2/100 g soil and J2/5 g roots, respectively, and percentage of plants with yellow leaves.

0

10

20

30

40

50

60

70

80

0 300 600 900 1200 1500 1800 2100

Average number of J2/100 g soil

Yellow

lea

ves

(%)

0

10

20

30

40

50

60

70

80

0 300 600 900 1200 1500 1800 2100 2400 2700 3000

Average number of J2/5 g fresh roots

Yellow

lea

ves

(%)

CHAPTER 2 38

2.4.3. FREQUENCY OF OCCURRENCE AND ABUNDANCE OF MELOIDOGYNE INCOGNITA, AND ROOT GALLING ON BLACK PEPPER VARIETIES

In the black pepper fields surveyed, Vinh Linh was the most common black pepper variety cultivated (Table 2.7). This variety was grown in every province visited in the North Central Coast and the Central Highlands but not in Phu Quoc Island. The other six black pepper varieties that could be identified were only cultivated in one province each.

Table 2.7: Occurrence of black pepper varieties in the North Central Coast, Central Highlands and Mekong River Delta, Vietnam.

Varieties North Central Coast Central Highlands Mekong River Delta

Quang Tri

Thua Thien Hue

Dac Lac Gia Lai Kon tum

Phu Quoc Island

Vinh Linh + + + + + -

Loc Ninh - - - + - -

Trau - - + - - -

LaDa - - + - - -

Tieu Se - - - + - -

Ha Tien - - - - - +

Phu Quoc - - - - - +

Others - - + + + -

+: present; -: absent.

Every root sample of the black pepper varieties Loc Ninh, Tieu Se and Phu Quoc was infected with M. incognita (Table 2.8). Of the variety LaDa, 80% of the root samples were infected with M. incognita. Nematode soil population densities ranged from 14 J2/100 g soil (variety Loc Ninh) to 279 J2/100 g soil (variety Trau). Nematode root population densities ranged from 18 J2/5 g roots (variety Phu Quoc) to 267 J2/5 g roots (variety Vinh Linh).

Root galling was observed in all the black pepper varieties examined. The average percentage root galling was highest (52.6%) in Tieu Se and lowest (39%) in Loc Ninh.

CHAPTER 2 39

Table 2.8: Frequency of occurrence and population density of Meloidogyne incognita, root galling and percentage of plants with yellow leaves observed on black pepper varieties in Vietnam.

Frequency of occurrence (%)

Population density Variety n

Soil Roots Soil

(J2/100 g) Roots

(J2/5 g)

Root galling

(%)

Yellow leaves

(%)

Vinh Linh 274 97.0 97.8 261 267 44.8 30.6

Loc Ninh 7 85.7 100 14 123 39.0 18.0

Trau 26 100 92.0 279 28 44.0 9.3

LaDa 5 100 80.0 248 46 46.6 12.0

Tieu Se 10 90.0 100 114 42 52.6 36.5

Ha Tien 55 100 92.7 235 58 42.5 17.0

Phu Quoc 10 90.0 100 166 18 42.0 21.0

Others 45 97.2 93.3 93 28 23.8 23.7

Average

(n=432)

95.0 94.5 176 76 41.7 21.0


Yellow leaves were observed in all the black pepper varieties

examined. On average, the percentage of black pepper plants with yellow leaves was highest in the variety Tieu Se (36.5%) and lowest in the variety Trau (9.3%).

2.5. DISCUSSION In comparison with most other agricultural crops, the number of

nematode species that can infect black pepper plants is high. In their reviews of plant-parasitic nematodes associated with black pepper plants, Sundararaju et al. (1979) listed 48 nematode species belonging to 29 genera, Ramana and Eapen (1998) listed 54 nematode species belonging to 30 genera while Koshy et al. (2005) listed 28 nematode species belonging to 17 genera. Also in our study, many nematode species were found on black pepper plants: 35 nematode species belonging to 19 genera. In comparison, preliminary surveys in black pepper fields in the south of Vietnam listed about 10 to 15 nematode genera (Khuong, 1983; Bien, 1989)

CHAPTER 2 40

while a detailed survey of nematodes associated with black pepper plants in Quang Tri province alone listed 49 nematode species belonging to 19 genera (Chau & Thanh, 1993b).

According to the literature, the predominant nematode species associated with black pepper plants may differ from country to country. In India, the predominant nematode species associated with black pepper plants are Meloidogyne spp., R. similis, Trophotylenchus piperis, Helicotylenchus sp. and R. reniformis (Jacob & Kuriyan, 1979, 1980; Ramana & Mohandas, 1987, 1989). In Thailand, Meloidogyne spp., T. semipenetrans and R. reniformis are the predominant nematode species associated with black pepper (Sher et al., 1969). In Indonesia, Malaysia and Sri Lanka, only Meloidogyne spp. and R. similis are of common occurrence on black pepper plants (Lamberti et al., 1983; Gnanapragasam et al., 1985; Sitepu & Mustika, 2000). Outside Asia, Meloidogyne spp., Xiphinema sp., Helicotylenchus sp. and Macroposthonia onoensis are more prevalent on black pepper plants in Brazil (Freire & Monteiro, 1978). In our study, based on their frequency of occurrence and abundance, the most predominant nematode species were M. incognita, A. avenae, D. ausafi, Tylenchus sp. and R. reniformis.

Eight out of the 28 nematode species associated commonly with black pepper plants worldwide (Koshy et al., 2005) were also found during our study in Vietnam: Discocriconemella limitanea, Helicotylenchus dihystera, H. seinhorsti, M. incognita, Pratylenchus coffeae, R. reniformis, T. and Xiphinema elongatum. Of these eight nematode species, only M. incognita and R. reniformis occurred in more than 20% of the samples investigated in Vietnam (98.4 and 20.8%, respectively).

Aglenchus sp., Tylenchus sp., Aphelenchoides dubius, A. singhi and A. trivialis were for the first time found associated with black pepper plants in Vietnam. With the exception of Tylenchus sp. (29.6%), the frequence of occurrence of these species was very low (<5%).

Although many nematode taxa have been reported on black pepper plants only two are known to cause serious damage to pepper crops: R. similis and Meloidogyne spp. (Koshy et al., 2005).

Although R. similis has been found associated with black pepper plants in India, Indonesia, Malaysia, Thailand, Sri Lanka (but not in Brazil), it has not been found associated with black pepper plants in Vietnam neither during our study nor during previous surveys (Khuong, 1983; Bien, 1989; Chau & Thanh, 1993b). The results of our study reconfirm that R. similis does not occur in Vietnam (Khuong, 1983, Chau et al., 1997, Van den Bergh, 2002). The absence of R. similis in Vietnam is surprising since

CHAPTER 2 41

this species is widespread in almost every tropical and subtropical country especially when bananas are being produced in that country (Bridge, 1993) which is the case in Vietnam. As to why this species is absent in Vietnam is unknown. The worldwide distribution of R. similis is considered relatively recent (beginning of the 19th century) and is attributed to the transfer of infected planting material, especially of bananas, from country to country for commercial purposes (Gowen et al., 2005). The widespread occurrence of R. similis in black pepper plantations in South Asia and Southeast Asia is probably also caused by infected cuttings which are used as planting material (Koshy et al., 2005). Recently, two new Radopholus species, Radopholus duriophilus and Radopholus arabocoffeae, were described from durian and coffee, respectively, in the Western Highlands of Vietnam (Chau et al., 2003; Phap et al., 2004). These two Radopholus species are morphologically and morphometrically very similar to R. similis. It would be interesting to investigate if R. duriophilus and R. arabocoffeae can infect black pepper plants and cause damage to the pepper crops.

Four Meloidogyne species can be found on black pepper plants: M. incognita, M. javanica, M. arenaria and M. piperi (Koshy et al., 2005). Meloidogyne incognita and M. javanica have the largest geographical distribution (as well in South Asia, Southeast Asia as Brazil) while M. arenaria and M. piperi have only been recorded on black pepper plants in India and Sri Lanka, and India, respectively. Meloidogyne incognita is present in North, Central and South Vietnam (Chau et al., 1997) and is, together with M. javanica, the most common Meloidogyne species infecting agricultural crops in Vietnam (Khuong, 1983). Almost every soil and root sample of black pepper plants examined during our study was infected with M. incognita. No difference in frequency of occurrence of M. incognita among the three agro-ecological regions surveyed was observed. A similar high frequency of occurrence (90%) of Meloidogyne spp. associated with black pepper plants was also observed in Kerala, India (Ramana & Mohandas, 1987; Ramana et al., 1987) and Para, Brazil (Ichinohe, 1975).

Black pepper roots infected with M. incognita showed the same type of galls as described in the literature (Ramana & Eapen, 2000). Infection leads to the development of elongated swellings on the thick primary roots and typical knots or galls on the fibrous secondary roots. In the thick primary roots, several adult females with egg masses can be present in a single elongated gall or root thickening. Also the same type of yellow leaves as described in the literature (Koshy et al., 2005) for black

CHAPTER 2 42

pepper plants infected with Meloidogyne species was observed in our study.

The soil population density of M. incognita was similar in the three agro-ecological regions surveyed, averaging about 200 to 250 J2/100 g soil. The root population density of M. incognita was on average about 225 to 250 J2/5 g roots in the North Central Coast and Central Highlands which was about 5 times higher than the average root population density of M. incognita in Phu Quoc Island (50 J2/5 g roots). Large differences in root population density of M. incognita were observed in different districts within the same agro-ecological region. For example, in the Central Highlands, the average root population density of M. incognita was 19 J2/5 g roots in Cu Mgar district vs 1,675 J2/5 g roots in An Phu district.

The average percentage of root galling was very similar in the three agro-ecological regions ranging from 38 (Mekong River Delta) over 39.5% (North Central Coast) to 42.5% (Central Highlands).

The percentage of black pepper plants with yellow leaves was on average about 20 to 25% in the North Central Coast and Central Highlands which was somewhat higher than the average percentage of black pepper plants with yellow leaves in Phu Quoc Island (16.9%). Large differences in percentage of black pepper plants with yellow leaves were observed in different districts within the same agro-ecological region. For example, in the Central Highlands, the percentage of black pepper plants with yellow leaves was on average 2% in Dac Ha district vs 48.3% in An Phu district.

In general, we observed a weak positive relationship between the soil and root population densities of J2 of M. incognita and percentage of black pepper plants with yellow leaves: in districts with a high nematode population density always a high percentage of plants with yellow leaves was observed. However, in Quang Tri district a relatively low nematode population density was associated with a high percentage of plants with yellow leaves.

Several factors may have caused the differences in population density of M. incognita and percentage of black pepper plants with yellow leaves observed among different districts in the same agro-ecological regions. One of these factors is the black pepper variety cultivated. Although the frequency of occurrence of M. incognita on the black pepper varieties in our study was very high and no differences were observed among these varieties, differences in nematode soil and root population densities, and percentage of plants with yellow leaves were observed which may indicate that some varieties might be less susceptible to M. incognita or more sensitive to yellowing of the leaves. Differences in

CHAPTER 2 43

susceptibility to M. incognita and the damage this pathogen can cause have been reported from India, Malaysia and Sri Lanka (Ramana & Eapen, 2000). Further research is necessary to find out if different levels in susceptibility or sensitivity to M. incognita are also present in the black pepper varieties cultivated in Vietnam.

Several experiments were conducted to establish population damage threshold levels of M. incognita on black pepper plants but most experiments were conducted in the greenhouse with potted plants so that the actual loss in yield or quality of the pepper crop could not be established. Experiments in India (Koshy et al., 1979; Jacob & Kuriyan, 1980; Mohandas & Ramana, 1991), Sri Lanka (Lamberti et al., 1983) and Brazil (Freire & Bridge, 1985) showed that an inoculum level of 100 to 1,000 J2 per seedling can already cause a considerable reduction in growth of black pepper seedlings. Following a pathogenicity experiment conducted under simulated field conditions, Mohandas and Ramana (1991) reported that plants inoculated with 10,000 to 100,000 J2 per plant yielded about 1/3rd to 50% less.

2.6. CONCLUSIONS The diversity of plant-parasitic nematodes that can infect black pepper plants in Vietnam is very high: 35 nematode species belonging to 19 genera were extracted from 432 soil and 432 root samples collected in 19 districts in six provinces in the North Central Coast, Central Highlands and Phu Quoc Island. Based on our study, the predominant nematode species associated with black pepper plants in Vietnam is M. incognita: almost every soil and every root sample examined during our study was infected with this root-knot nematode species. No difference in frequency of occurrence of M. incognita among the three agro-ecological regions surveyed was observed. However, the root population density of M. incognita in black pepper plants was on average about 5 times higher in the North Central Coast and Central Highlands than in Phu Quoc Island. Large differences in root population density of M. incognita on black pepper plants were observed in different districts within the same agro-ecological region. Black pepper roots infected with M. incognita showed the same type of galls as described in the literature. The percentage of root galling averaged about 40% in all the three agro-ecological regions. The percentage of black pepper plants with yellow leaves was on average about 20 to 25% in the North Central Coast and Central Highlands, which was somewhat higher compared to Phu Quoc Island (16.9%). Large differences in percentage of

CHAPTER 2 44

black pepper plants with yellow leaves were observed in different districts within the same agro-ecological region. The same type of yellow leaves as described in the literature for black pepper plants infected with Meloidogyne species was observed in our study. Our data suggest that, in general, there is a relationship between population densities of M. incognita on black pepper plants and percentage of plants with yellow leaves. However, in one district (Quang Tri) a relatively low nematode population density was associated with a high percentage of plants with yellow leaves. Five nematode species were for the first time recorded on black pepper plants in Vietnam. Our study reconfirms previous reports that R. similis does not occur in Vietnam.

CHAPTER 3 45

CHAPTER 3. IDENTIFICATION AND COMPARATIVE STUDY OF THE MORPHOLOGY AND MORPHOMETRICS OF MELOIDOGYNE POPULATIONS FROM BLACK PEPPER PLANTS IN VIETNAM

3.1. INTRODUCTION Root-knot nematodes are members of the genus Meloidogyne. Some

species of this group of highly adapted obligate plant parasites are worldwide economically very important pathogens that can attack and damage nearly every agricultural crop (Moens et al., 2009). The genus Meloidogyne comprises 97 described species (Hunt & Handoo, 2009). The taxonomy of the genus has been the subject of many publications (for an overview see Hunt & Handoo, 2009). 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. Meloidogyne species can differ in pathogenicity and therefore accurate identification at species level is essential for the development of efficient and sustainable management strategies. Moreover, some Meloidogyne species are pathogens of world quarantine importance so that correct identification up to species level is of vital importance to prevent, locally, regionally or internationally, their spread with infected planting material (Moens et al., 2009).

In nematology in general, and thus also in the taxonomy of the genus Meloidogyne, species identification has been based primarily on light microscope observations (morphology) and measurements of morphological characters (morphometrics) mainly of females and males (Coomans et al., 1978; Eisenback & Hunt, 2009). Since the mid-1970s, scanning electron microscopy (SEM) is also being used to study in more detail the morphology of females, males and second-stage juveniles (J2) of Meloidogyne species (for an overview see Eisenback & Hunt, 2009). During the past two decades, taxonomists have also increasingly used biochemical and molecular diagnostic tools to confirm the validity of existing Meloidogyne species and to assist in the identification and description of new species (for an overview see Blok & Powers, 2009).

Four Meloidogyne species can be found on black pepper plants: M. incognita, M. javanica, M. arenaria and M. piperi (Koshy et al., 2005). As mentioned in Chapter 2, M. incognita and M. javanica have the largest geographical distribution (as well in South Asia, Southeast Asia as Brazil) while M. arenaria and M. piperi have only been recorded on black pepper plants in India and Sri Lanka, and India, respectively. Meloidogyne incognita

CHAPTER 3 46

is present in North, Central and South Vietnam (Chau et al., 1997) and is, together with M. javanica, the most common Meloidogyne species infecting agricultural crops in Vietnam (Khuong, 1983). Almost every soil and root sample of black pepper plants examined during our study was infected with M. incognita (see Chapter 2).

The morphological and morphometrical characters most commonly used to separate Meloidogyne species are (Hunt & Handoo, 2009):

Female: - shape of body - labial region - stylet length - shape of stylet cone, shaft and basal knobs - nature of perineal pattern, including form of dorsal arch - lateral field - striae - tail terminus - excretory pore/stylet length ratios

Male: - size, height and shape of labial cap - number of annulations - diameter of the labial region as compared with the 1st body

annule - stylet length - shape of stylet cone, shaft and basal knobs - distance of the dorsal gland orifice from the stylet base - spicule length and shape

J2: - body and stylet length - labial region - shape of stylet basal knobs - location of the hemizonid in relation to the excretory pore - distance of dorsal gland orifice from the stylet base - number of lines in the lateral field - length of the tail and hyaline terminus

The morphological character most frequently used for the identification of Meloidogyne species is the perineal pattern, which is located in the posterior body region of the adult females (Fig. 3.1). The basic features of the perineal pattern, i.e. the perineum (vulva-anus area), phasmid, tail terminus, lateral line and surrounding striae, wing and dorsal arch, are already present in the early adult stage. However, around the

CHAPTER 3 47

vulva and anus, the incisions forming the striae are closer together and deeper in most adults.

Figure 3.1: Drawing of the perineal pattern of Meloidogyne species. Source of the drawing: Eisenback and Triantaphillou (1991).

The objectives of this part of our study were 1) to identify at

species level seven Meloidogyne populations collected from black pepper plants in three different agro-ecological regions in Vietnam and 2) to compare the morphological and morphometrical characters of these seven Meloidogyne populations a) among each other, b) with descriptions published in the literature and c) to assess the intraspecific variability of some of these characters.

3.2. MATERIALS AND METHODS

3.2.1. NEMATODE POPULATIONS Seven Meloidogyne populations collected during our study described

in Chapter 2 were included in the morphological and morphometrical study (Table 3.1).

Second-stage juveniles (J2) and males were extracted from the soil using a tray method modified from the Baermann funnel method (Hooper et al., 2007) and from roots by maceration and sieving (Speijer & De Waele, 1997; see Chapter 2). The females were handpicked from infected black pepper roots using a scalpel and needle.

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Table 3.1: Origin and population code of the seven Meloidogyne populations collected from black pepper plants in Vietnam.

No District Province Agro-ecological region Population code

1 Cam Lo Quang Tri North Central Coast Me1

2 Phu Loc Thua Thien Hue North Central Coast Me3

3 I A Chim KonTum Central Highlands Me5

4 Chu Se Gia Lai Central Highlands Me4

5 An Phu Gia Lai Central Highlands Me7

6 Buon Ma Thuot Dac Lac Central Highlands Me2

7 Phu Quoc Island Kien Giang Mekong River Delta Me6

3.2.2. KILLING AND FIXING OF SECOND-STAGE JUVENILES (J2) AND MALES The J2 and males were killed and fixed according to the method

described by Seinhorst (1959) and modified by De Grisse (1969). The collected specimens were killed and fixed by adding 4% hot (60-80°C) formaldehyde to a small drop of water in a glass cavity vessel which contained the nematodes. The fixed nematodes were transferred to solution I (99 parts of 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 at least 12 hours in an oven at 37-40°C. Then, the deep glass dish containing the nematodes was removed from the desiccator and placed in an oven at 37-40°C. Some of the excess ethanol in the deep glass dish was removed with a pipette. Then, 2 to 3 ml of solution II (95 parts of 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 of 96% ethanol + 50 parts pure glycerine) were added and the deep glass dish was left overnight at 37-40°C in the oven. The nematodes in pure glycerine were used to mount on permanent slides. Second-stage juveniles and males were mounted on separate slides.

CHAPTER 3 49

3.2.3. PREPARATION OF PERINEAL PATTERNS OF FEMALES The method described by Hartman and Sasser (1985) was used to

prepare the perineal patterns of the females. The cuticles of the females were ruptured near the neck with a scalpel and the body contents gently pushed out. Then, 10 to 20 cuticles were placed in a drop of 45% lactic acid in a Petri-dish. After 30 minutes, the cuticles were cut in half with a scalpel. The halfs with the perineal patterns were removed from the 45% lactic acid and trimmed to a square. Finally, the cuticles were placed back in the 45% lactic acid to be mounted later on permanent slides.

3.2.4. MOUNTING OF NEMATODES Microscope glass slides with a coverslip were used for mounting. A

paraffin ring was made by heating one end of copper tube, dipping the hot end of the copper tube in paraffin and pressing the melted paraffin in the center of the glass slide resulting in a solid paraffin ring after cooling down. A drop of pure glycerine was placed in the center of each paraffin ring. In each glycerine drop, five to 10 J2, males or perineal cuticle halfs of females were placed. A coverslip was gently placed on top of the glycerine drop. Then, the glass slide was gently heated on a heating plate until the paraffin ring melted and allowed to cool down to form a solid paraffin ring again.

3.2.5. LIGHT MICROSCOPE OBSERVATIONS Morphological characters studied were based on Eisenback et al.

(1981) and Eisenback (1985). The measurements and ratio’s taken for the comparative morphometrical study and their abbreviations are listed in Table 3.2.

Twenty females and males (if available), and 30 J2 of each of the seven Meloidogyne populations were measured. Measurements were taken using a camera lucida drawing tube attached to a light microscope which was equipped with a digital camera for photography. The mean, minimum and maximum (range), standard deviation and coefficient of variation were calculated for all morphological characters measured.

CHAPTER 3 50

Table 3.2: Measurements taken for the comparative morphometrical study of the seven Meloidogyne populations collected from black pepper plants in Vietnam and their abbreviations.

Character Abbreviation used in

the tables

Body length (µm) L

Body length/maximum body width a

Body length/tail length c

Tail length/body width at anus (female) or

cloaca (male)

c´

Stylet length (µm) stylet

Stylet cone length (µm)

Neck length* (µm)

stylet cone

neck length

Neck width* (µm) neck width

Head region height (µm) head height

Head region width (µm) head width

Maximum body width body width

Body width at anus (µm) ** Tail length

body width anus

tail

Length hyaline tail terminus*** h

Length hyaline tail terminus/tail length*** (%) h%

Distance of dorsal gland orifice from the stylet

base

Distance from cloaca to anteriormost part of

testis/body length** (%)

DGO

T

Spicule length** (µm) spicule

Gubernaculum length** (µm) gubernaculum

* female specimens only; ** male specimens only; *** J2 specimens only.

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 seven Meloidogyne populations based on 10 morphometrical characters of the J2.

CHAPTER 3 51

3.3. RESULTS

3.3.1. MORPHOLOGICAL OBSERVATIONS Females (population from Cam Lo district). Body shape saccate to

globose, soft, pearl-white in colour. Neck protruding anteriorly. Stylet robust; stylet cone longer than shaft and knobs, pointed, tapering gradually toward tip, distinctly curved dorsally; shaft cylindrical, broadening at base; knobs rounded, offset from shaft (Fig. 3.2B). Perineal pattern oval to rounded, typically with high, squarish dorsal arch and no lateral line or weakly demarcated by forked striae (Fig. 3.2A).

Figure 3.2: Light microscope photographs of the female and male of the Meloidogyne population from Cam Lo district. Female. A: perineal pattern; B: stylet (scale bar: 5 μm). Male. C: head; D: tail (scale bars C & D: 10 μm).

A B

C D

CHAPTER 3 52

Males (population from Cam Lo district). Body shape vermiform, tapering to bluntly rounded ends with head narrower than tail end. Head cap narrow, distinctly offset from head region with rounded labial disc. Stylet length 23.1 µm; basal knobs offset, rounded (Fig. 3.2C). Spicules curved ventrally, tapering toward tip (Fig. 3.2D).

Second-stage juveniles (all populations). Body slender, tapering anteriorly but more so posteriorly. Cuticle with fine transverse annulations, increasing in size and becoming irregular in posterior tail region. Lateral field with four incisures. Head skeleton weak, truncate, slightly offset from body; head cap narrower than head region (Figs 3.3A-E,G) or not (Fig. 3.3F). Stylet often slender; stylet cone sharply pointed, increasing in width gradually posteriorly; shaft cylindrical, widening slightly posteriorly; knobs rounded and offset from shaft. Tail long, conical, tapering to fine with rounded tip (Figs 3.4A-G). Hyaline tail terminus distinct and variable in length.

3.3.2. MORPHOMETRICAL OBSERVATIONS Females and males (population from Cam Lo district). The

morphometrics of the females and males of the population from Cam Lo are listed in Table 3.3.

The average body width of the females was 419 µm, neck length 134 µm, neck width 118 µm. The stylet length was 11.2 µm with stylet cone 6.7 µm.

The average body length of the males was 856 µm. The stylet length was 23.1 µm. Head region height 4 µm and head region width 7.4 µm. Distance of the dorsal gland orifice from the stylet base was 2.6 µm. Spicules were 26.7 µm long, with gubernaculum 6.7 µm.

CHAPTER 3

53

Figure 3.3: Light microscope photographs of the heads of second-stage juveniles (J2) of the Meloidogyne populations from Cam Lo (A), Phu Loc (B), I A Chim (C), Buon Ma Thuot (D), Chu Se (E), An Phu (F) and Phu Quoc Island (G). Scale bars: 20 µm.

A C D E G B F

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54

Figure 3.4: Light microscope photographs of the tails of second-stage juveniles (J2) of the Meloidogyne populations from Cam Lo (A), Phu Loc (B), I A Chim (C), Buon Ma Thuot (D), Chu Se (E), An Phu (F) and Phu Quoc Island (G). Scale bars: 20 μm.

A B C D E G F

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55

Table 3.3: Morphometrics of the females and males of the Meloidogyne population collected from black pepper plants in Cam Lo district.

Character Males Females

n 3 20

Body length (µm) 855.9 ± 93.1 -

Maximum body width (µm) 30.1 ± 1.1 418.9 ± 82.1

Neck length (µm) - 134.0 ± 58.1

Neck width (µm) - 118.0 ± 35.5

Body width at anus (µm) 11.3 ± 1.5 -

Stylet length (µm) 23.1 ± 1.0 11.2 ± 1.3

Stylet cone length (µm) 13.1 ± 0.6 6.7 ± 0.7

DGO 2.6 ± 0.7 -

Head region height (µm) 4.0 ± 0.1 -

Head region width (µm) 7.4 ± 0.4 -

Tail length (µm) 5.9 ± 0.5 -

Spicule length (µm) 26.7 ± 1.5 -

Gubernaculum length (µm) 6.7 ± 0.7 -

a 28.5 ± 3.7 -

c 165.0 ± 28.8 -

c’ 0.5 ± 0.2 -

n: number of specimens measured. Mean ± standard deviation. For the abbrevations see Table 3.2.

Second-stage juveniles (J2). The morphometrics of the second-stage

juveniles (J2) of the seven Meloidogyne populations are listed in Table 3.4.

CHAPTER 3

56

Table 3.4: Morphometrics of the second-stage juveniles (J2) of the seven Meloidogyne populations collected from black pepper plants in Vietnam.

n : number of specimens measured. Mean ± standard deviation (range). * measurements in μm. CV: coefficient of variation (%). For the abbrevations see Table 3.2.

Gia Lai Character Quang Tri Thua Thien Hue Kon Tum

An Phu Chu Se

Dac Lac Phu Quoc CV

n 30 30 30 26 19 30 30 Body length* 277.9 ± 17.5

(246-313) 383.3± 29.8 (332-482)

355.8 ± 28.1 (295-420)

328.8 ± 36.5 (257-396)

400 ± 54.7 (319-477)

348.2 ± 16.1 (310-311)

359.2 ± 35.8 (295-482)

14.0

Maximum body width* 9.6 ± 1.1 (7.7-12.3)

14.9 ± 2.1 (11.5-2.1)

14.0 ± 2.1 (10.8-15.4)

13.7 ± 2.3 (9.5-17.5)

15.3 ± 0.8 (14.2-16.9)

15.3 ± 2.8 (10.8-21.6)

14.6 ± 2.2 (10.8-18.9)

20.6

Body width at anus* 6.6 ± 0.8 (5.4-8.7)

8.7 ± 1.3 (6.7-12.8)

9.4 ± 1.5 (7.2-13.8)

7.7 ± 1.1 (5.6-9.7)

9 ± 1 (7.7-10.8)

8.9 ± 1.6 (6.2-12.8)

8.9 ± 1.1 (7.2-10.8)

18.7

Stylet length* 9.9 ± 0.5 (8.7-10.8)

13.9 ± 1.0 (12.3-16.4)

13.8 ± 1.2 (10.8-15.4)

13.1 ± 1.3 (10.3-15.5)

12.9 ± 1.2 (10.3-14.4)

13.7 ± 1.0 (11.3-15.1)

13.5 ± 1.2 (15.4-10.8)

13.6

Stylet cone length* 6.6 ± 0.4 (5.9-7.4)

- 9.4 ± 0.4 (8.7-10.3)

7.4 ± 0.3 (7.2-7.7)

8.4 ± 0.8 (6.9-9.7)

9.2 ± 0.7 (7.4 -10.3)

9.2 ± 0.8 (7.2-10.3)

15.8

Tail length* 34.5 ± 2.6 (25.6-39.7)

46.8 ± 5.0 (37.2-55.9)

47.7 ± 5.4 (40.0-62.6)

53.5 ± 6.7 (40.5-65.6)

48.4 ± 8.1 (36.4-59.7)

47.4 ± 4.6 (38.5-57.4)

46.1 ± 5.0 (37.2-57.4)

16.7

Length hyaline tail terminus*

8.2 ± 1.3 (5.1-10.5)

10.8 ± 2.8 (6.9-16.9)

10.2 ± 1.6 (6.2-12.8)

13.8 ± 3.8 (8.7-20.5)

12.6 ± 3.1 (7.7-10.8)

11.8 ± 2.7 (7.7-18.5)

10.0 ± 2.2 (6.2-16.4)

27.8

a 29.3 ± 2.8 (21.5-35.0)

26.2 ± 3.9 (19.7-35.9)

25.8 ± 3.5 (19.7-34.7)

24.4 ± 3.0 (18.0-29.5)

26.2 ± 3.5 (19.8-33.6)

23.4 ± 3.9 (16.6-30.2)

25.0 ± 3.4 (19.7-34.7)

14.9

c 8.1 ± 0.8 (7.4-11.8)

8.3 ± 1.1 (6.7-10.5)

7.6 ± 1.2 (5.6-10.4)

6.3 ± 0.8 (5.0-7.8)

8.3 ± 1.4 (6.3-11.9)

7.4 ± 0.6 (6.2-8.5)

7.9 ± 1.3 (5.6-10.5)

15.2

c’ 5.3 ± 0.7 (2.9-6.5)

5.4 ± 0.8 (3.6-7.5)

5.2 ± 0.8 (4.0-6.8)

7 ± 0.9 (5.3-8.4)

5.4 ± 0.8 (4.3-7.0)

5.4 ± 0.7 (4.4-7.4)

5.2 ± 0.8 (4.1-6.8)

16.8

h% 23.7 ± 4.0 (14.7-32.0)

23.2 ± 5.7 (14.1-37.2)

21.7 ± 4.8 (13.5-31.6)

24.3 ± 7.2 (14.2-38.0)

26.3 ± 6.9 (14.5-38.9)

25 ± 5.8 (17.0-40.9)

21.8 ± 5.4 (13.5-34.0)

23.8

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Body length was on average the longest in the population from Chu Se and the shortest in the population from Cam Lo (400 vs 278 µm). Maximum body width was on average the widest in the population from Chu Se and the smallest in the population from Cam Lo (15.3 vs 9.6 µm). The average a ratio ranged from 23.4 (Buon Ma Thuot population) to 29.3 (Cam Lo population). Stylet length was on average the longest in the population from Phu Loc and the shortest in the population from Cam Lo. Tail length was on average the longest in the population from An Phu and the shortest in the population from Cam Lo (53.5 vs 34.5 µm). The average c ratio ranged from 6.3 (An Phu population) to 8.3 (Phu Loc and Chu Se populations). The average length of the hyaline tail terminus ranged from 8.2 μm (Cam Lo population) to 13.8 μm (An Phu population).

In the J2 of the seven M. incognita populations collected from black pepper plants in Vietnam, the coefficient of variation was the lowest for stylet length (13.6%) followed by body length (14%) and a ratio (body length/maximum body width; 14.9%). The coefficient of variation was the highest for length of the hyaline tail terminus (27.8%) followed by h% (length hyaline tail terminus/tail length x 100; 23.8%) and maximum body width (20.6%).

The results of the canonical discriminant analysis are presented in Table 3.5.

Table 3.5: Standardised coefficients for canonical variates for the second-stage juveniles (J2) of the seven Meloidogyne populations collected from black pepper plants in Vietnam.

Root 1 Root 2

% of variation 75.2 14.0

Selected characters Vector loading

Body length -0.0558 -1.0289

Maximum body width -0.0274 0.0865

Stylet length -0.5147 -0.1114

Tail length -0.0572 -0.1108

Length hyaline tail terminus 0.7572 -0.2338

Body width at anus -0.7308 -0.7794

a 0.5542 -0.0537

c -0.6975 -2.9509

c' -1.3436 -2.1990

h% 0.0375 0.4245

For the abbreviations see Table 3.2.

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Using a combination of 10 morphological characters for the J2, the seven Meloidogyne populations collected from black pepper plants in Vietnam were clustered in three groups by canonical discriminant analysis (Fig. 3.5). One group includes only the Cam Lo population from Quang Tri province (North Central Coast; Me1 in Fig. 3.5), another group clusters the other J2 populations with the exception of the An Phu population from Gia Lai province (Central Highlands; Me 7 in Fig. 3.5) which stands out from the two other groups. The body length, c ratio (body length/tail length) and c' ratio (tail length/body width at cloaca) were the best J2 morphometrical characters for the separation of the populations.

Figure 3.5: Canonical Discriminant Analysis of seven Meloidogyne populations collected from black pepper plants in Vietnam based on the 10 morphological J2 characters listed in Table 3.5. The circles display 95% confidence regions. For the list with the abbreviations of the Meloidogyne populations see Table 3.1.

3.4. DISCUSSION Based on a combination of the perineal pattern of the mature

females, various morphological and morphometric characters of the J2 and males, the seven Meloidogyne populations collected from black pepper plants in Vietnam were positively identified as Meloidogyne incognita. The perineal pattern of the mature females corresponds with the typical "incognita" pattern: striae closely spaced, very wavy to zig-zag, especially

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dorsally and laterally; dorsal arch high, rounded; lateral field unclear, sometimes marked by breaks in the striae, broken ends often forked, pattern merging into body striae (CABI, 2007). The morphological and morphometric characters of the Meloidogyne population from Cam Lo district are similar with previous descriptions of M. incognita populations from around the world. Some variability, especially in morphometrics of the J2, was observed among the seven M. incognita populations but this variability is in line with the variability reported before (Hunt & Handoo, 2009).

Based on the coefficient of variation (CV), stylet length, body length and a ratio (body length/maximum body width) are the least variable morphometrical characters of the J2 of the seven M. incognita populations collected from black pepper plants in Vietnam. This observation confirms earlier reports that in M. incognita the intraspecific variability of stylet length and body length is low and thus of diagnostic value (Hunt & Handoo, 2009).

Based on the coefficient of variation (CV), length of the hyaline tail terminus and h% (length hyaline tail terminus/tail length x 100) are the highest variable morphometrical characters of the J2 of the seven M. incognita populations collected from black pepper plants in Vietnam. This observation is somewhat surprising because these characters are some of the most commonly used to separate Meloidogyne species (Hunt & Handoo, 2009).

Canonical discriminant analysis enabled the clustering of the seven M. incognita populations collected from black pepper plants in Vietnam in three groups based on a combination of 10 morphological characters of the J2. The population from Cam Lo district is in general smaller than the other six populations and this may explain why this population was separated from the other populations. The population from An Phu differs from the other populations in a longer tail resulting in lower c ratio (body length/tail length) and higher c’ ratio (tail length/body width at cloaca) compared with the other populations and this may explain why this population was separated from the other six populations. There was no relationship between these three groups and the geographic origin (agro-ecological region) of the populations.

The best morphometrical J2 characters used for the separation of the M. incognita populations (body length, c and c’ ratios) correspond only partially with the most commonly used morphometrical characters to separate Meloidogyne species which usually do not include the c and c’ ratio’s (Hunt & Handoo, 2009).

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3.5. CONCLUSIONS Based on a combination of the perineal pattern of the mature

females, various morphological and morphometrical characters of the J2 and males, the seven Meloidogyne populations collected from black pepper plants in Vietnam were positively identified as M. incognita. Based on the coefficient of variation, stylet length, body length and a ratio (body length/maxumum body width) are the least variable morphometrical characters of the J2 of the seven M. incognita populations examined. This observation confirms earlier reports that in M. incognita the intraspecific variability of stylet length and body length is low and thus of diagnostic value. Canonical discriminant analysis enabled the clustering of the seven M. incognita populations collected from black pepper plants in Vietnam in three groups based on a combination of 10 morphological characters of the J2. There was no relationship between these three groups and the geographic origin (agro-ecological region) of the populations.

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CHAPTER 4: POPULATION DYNAMICS OF MELOIDOGYNE INCOGNITA ON BLACK PEPPER PLANTS IN TWO AGRO-

ECOLOGICAL REGIONS IN VIETNAM

4.1. INTRODUCTION Black pepper plants (Piper nigrum L.) are mostly grown in tropical

regions with a hot and humid climate. Apart from Asian countries such as India, Sri Lanka, Thailand, Malaysia, Indonesia, Vietnam and China, black pepper plants are also cultivated in countries such as Brazil and Madagascar. Today, Vietnam has become one of the most important producers and exporters of pepper worldwide. In 2009, Vietnam held an export market share of about 50%, according to the Vietnam Pepper Association. Vietnamese pepper is being exported to 80 countries around the world, mainly to the United States, the European Union and the Middle East.

Black pepper is a plant originating from the hot and humid tropics and therefore it requires adequate temperature and rainfall (Nelson & Eger, 2009). It thrives from sea level up to 600 m above sea level. The plant tolerates temperatures between 10 and 40°C but it prefers temperatures from 22 to 30°C. An evenly over a long rainy season distributed total annual rainfall of 600 to 2,000 mm is considered ideal for black pepper cultivation. Black pepper plants prefer a deep, well-drained soil rich in organic matter and of medium texture.

Vietnam lies in the tropics, subtropics and monsoon zone where the environmental conditions are favourable for the cultivation of black pepper plants. Most pepper production is concentrated in four agro-ecological regions: the North Central Coast, Central Highlands, Southeast and Mekong River Delta (Phu Quoc Island) (see Fig. 1.10 in Chapter 1).

Plant protection is important for pepper production because pathogens are a major cause of the reduction in yield and quality of pepper crops. Many nematode species have been reported from black pepper plants but the only nematodes known to cause serious damage to the crop are Radopholus similis and root-knot nematodes (Meloidogyne spp.) (Koshy et al., 2005). Radopholus similis does not occur in Vietnam but Meloidogyne spp. are economically important plant pathogens in the country. Examination of 432 soil and 432 root samples of black pepper plants in three agro-ecological regions in Vietnam showed that Meloidogyne incognita is the predominant Meloidogyne species attacking

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this agricultural crop in the most important production regions in Vietnam (see Chapter 2).

So far, no study has been undertaken to investigate the effect of local environmental conditions on the population dynamics of M. incognita and the damage this pathogens causes on black pepper plants in Vietnam. The term population dynamics is used to express the changes (in quantity) within a population over time. Nematode populations are under the influence of both abiotic (for example climate and soil type) and biotic (for example plant host status and competition with other micro-organisms) factors.

It was the objective of this part of our study to examine the effect of local environmental conditions, especially air temperature and rainfall, in two agro-ecological regions in Vietnam 1) on the population dynamics of M. incognita on black pepper plants and 2) on the damage this pathogen causes to this crop. Two field studies were conducted in North Central Coast and the Central Highlands to follow the population dynamics of M. incognita and percentage root galling over time in relation to air temperature and rainfall.


4.2.1. DESCRIPTION OF THE STUDY SITES The two study sites were situated in Cam Lo, Quang Tri province

(North Central Coast) and Buon Ma Thuot, Dac Lac province (Central Highlands) (Fig. 4.1).

Quang Tri province is situated in the southern part of the North Central Coast. Climate-wise, this specific area is in fact a transitional area between the two major climatic zones of Vietnam: the cold, “winter”-like climate in the north and the warm all year round “summer”-like climate in the South. In Quang Tri, two seasons can be distinguished. A dry season which usually lasts from March to August, with a limited amount of rainfall, bright sunlight and a southwest wind causing droughts. A rainy season which usually lasts from September to February, with heavy rains from September to December causing floods. The average annual temperature is 24.1°C and the average total annual rainfall is 2,400 mm. Quang Tri province is situated at about 400 m above sea level.

Dac Lac province is situated in the central part of the Central Highlands. It consists of relative flat terrain at 500 to 800 m above sea level. Dac Lac has a temperate climate with an average annual

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temperature of 24°C. The dry season usually lasts from November to April. The rainy season usually lasts from May to October. The climate, subject to both tropical monsoons and highland conditions, is highly suitable for the cultivation of a wide range of perennial crops such as black pepper, coffee, cashew nuts, cotton and rubber.

Both study sites, Cam Lo and Buon Ma Thuot, are situated at more or less the same altitude (400 and 500 m above sea level, respectively), and the same soil (a red basaltic soil with a loamy-clay texture). At both study sites, the same black pepper variety (Vinh Linh) was grown and black pepper plants sampled had the same age (6 to 8 years).

Figure 4.1: Sites in Vietnam where the population dynamics studies of Meloidogyne incognita on black pepper variety Vinh Linh were carried out. 1: Quang Tri province; 2: Dac Lac province.

Buon Ma Thuot

Cam Lo

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4.2.2. SAMPLING, NEMATODE POPULATION AND ROOT GALLING ASSESSMENT In Cam Lo, soil and root sampling of black pepper plants started in

November 2004 while in Buon Ma Thuot, sampling started in April 2006. Sampling was done on a monthly basis during 1 year. In each study site, samples were taken from three black pepper fields. In each field, nine black pepper plants were sampled at random. Sampling depth was 25 cm. Soil and roots of three black pepper plants constituted one sample. Samples were stored in plastic bags in a coolbox until nematode extraction was carried out.

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 with 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 200, 160 and 25 μm aperture. The extracted nematodes were examined under a stereomicroscope for the presence of plant-parasitic nematodes.

The M. incognita population densities in the soil and the roots were defined as the number of second-stage juveniles (J2) per 100 g soil and 5 g roots.



4.3. RESULTS The plant-parasitic nematodes associated with black pepper plants

in the study sites are listed in Table 4.1. Thirteen plant-parasitic nematode taxa belonging to twelve genera

were identified. Ten of these taxa were present in both study sites. Helicotylenchus certus, Hemicriconemoides cocophilus and Pratylenchus coffeae were only found in Buon Ma Thuot. Based on nematode population

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densities, M. incognita was the most abundant taxon present (Tables 4.2 and 4.3).

Table 4.1: Plant-parasitic nematodes associated with black pepper variety Vinh Linh in Cam Lo and Buon Ma Thuot, Vietnam.

Scientific name Population density

Cam Lo

(Quang Tri)

North Central Coast

Buon Ma Thuot

(Dac Lac)

Central Higlands

Aglenchus sp. + +

Tylenchus sp. + +

Aphelenchoides bicaudatus (Imamura)

Filipjev & Sch. Stekhoven

+ +

Aphelenchus avenae Bastian + +

Discocriconemella limitanea (Luc) De

Grisse & Loof

+ +

Ditylenchus myceliophagus Goodey + +

Helicotylenchus certus Eroshenko &

Nguyen V.T.

+

H. dihystera (Cobb) Sher + +

Hemicriconemoides cocophilus (Loos)

Chitwood & Birchfield

+

Meloidogyne incognita (Kofoid &

White) Chitwood

+++ +++

Pratylenchus coffeae (Zimmerman)

Filipjev & Sch. Stekhoven

+

Rotylenchulus reniformis Linford &

Oliveira

++ +

Xiphinema elongatum Sch. Stekhoven

& Teunissen

+ +

+: < 50 J2 or vermiform nematodes/100 g soil or 5 g roots; ++: from 50 to 150 J2 or

vermiform nematodes/100 g soil or 5 g roots; +++: > 150 J2 or vermiform

nematodes/100 g soil or 5 g roots.

4.3.1. POPULATION DYNAMICS OF MELOIDOGYNE INCOGNITA ON BLACK PEPPER VARIETY VINH LINH IN CAM LO

The sampling was done from November 2004 until October 2005. The average monthly soil and root population densities (J2) of M.

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incognita, percentage root galling and root galling index are presented in Table 4.2 and Fig. 4.2.

Table 4.2: Population dynamics of Meloidogyne incognita (and all plant-parasitic nematodes), percentage root galling and root galling index on black pepper variety Vinh Linh in Cam Lo, Vietnam, from November 2004 until October 2005.

Month

Population density M. incognita

Population density all plant-parasitic

nematodes*

Root galling

Soil (J2/100 g)

Roots (J2/5 g)

Soil (/100 g)

Roots (/5 g)

% Index**

Nov 04 91 34 141 37 31.7 3

Dec 04 245 36 295 39 35.1 3

Jan 05 1,933 224 2,085 226 90.9 5

Feb 05 1,797 567 1,877 736 90.1 5

Mar 05 721 271 987 297 90.3 5

Apr 05 731 197 905 202 65.2 4

May 05 74 8 153 8 29.9 3

Jun 05 60 52 82 62 38.6 3

Jul 05 44 5 45 26 49.5 3

Aug 05 91 1 106 2 40.9 3

Sep 05 88 33 166 33 41.9 3

Oct 05 87 24 146 31 46.2 3

* Including J2 of Meloidogyne incognita. ** For the root galling index see 4.2.2.

Soil and root population densities of M. incognita followed a same

trend during the duration of the study in Cam Lo. Nematode populations were low during November and December. Between December and January (soil) and February (roots) they increased to their highest level. Then, they decreased slowly towards May (soil and roots) and remained very low until the end of the study in October. Percentage root galling followed the same trend. Between January and March, root galling averaged about 90%. Between May and October, root galling averaged from 30 to about 45%.

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Figure 4.2: Population dynamics of Meloidogyne incognita and percentage root galling on black pepper variety Vinh Linh in Cam Lo, Vietnam, from November 2004 until October 2005.

The average monthly air temperature and average monthly rainfall

in Cam Lo during the study are shown in Fig. 4.3.

Figure 4.3: Average monthly air temperature and average monthly rainfall in Cam Lo during the period November 2004–October 2005. Source: Meteorological Centre of Quang Tri province.

The study in Cam Lo was initiated at the end of the rainy season

(November) when the average monthly rainfall was 664.2 mm and the

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average monthly air temperature 23.1°C. The rainfall dropped to less than 72 mm between November and December when the dry season started. The dry season lasted until July when the rainy season started again. In October 2005, the average monthly rainfall was 800 mm. Air temperature was lower during the 1st half of the dry season but increased to its highest level during the 2nd half of the dry season when it reached about 30°C during May and June. The beginning of the rainy season caused a decline in air temperature to about 25°C.

4.3.2. POPULATION DYNAMICS OF MELOIDOGYNE INCOGNITA ON BLACK PEPPER VARIETY VINH LINH IN BUON MA THUOT

The sampling was done from April 2006 until April 2007. The average monthly soil and root population densities (J2) of M. incognita, percentage root galling and root galling index are presented in Table 4.3.

Table 4.3: Population dynamics of Meloidogyne incognita (and all plant-parasitic nematodes), percentage root galling and root galling index on black pepper variety Vinh Linh in Buon Ma Thuot, Vietnam, from April 2006 until April 2007.

Population density M. incognita

Population density all plant-parasitic

nematodes*

Root galling Month

Soil (J2/100 g)

Roots (J2/5 g)

Soil (/100 g)

Roots (/5 g)

% Index**

Apr 06 312 73 332 74 19.5 2

May 06 850 22 968 77 25.9 3

Jun 06 266 4 288 10 22.2 2

Jul 06 425 33 465 40 47.0 3

Aug 06 5,447 30 5,721 63 71.0 4

Sep 06 4,213 214 5,469 261 75.5 5

Oct 06 3,122 363 3,390 373 61.6 4

Nov 06 14,799 327 15,522 385 50.0 3

Dec 06 10,423 854 11,063 892 28.8 3

Jan 07 9,596 106 10,315 111 35.9 3

Feb 07 2,336 36 3,257 37 21.7 2

Mar 07 5,136 14 5,344 23 14.6 2

Apr 07 2,449 5 2,575 6 15.9 2

* Including J2 of Meloidogyne incognita. ** For the root galling index see 4.2.2.

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Meloidogyne incognita soil populations were low during April until July. Then they increased again between July and August, decreased, increased again between October and November to decrease again towards the end of the study in April. Compared with the soil population densities, the root population densities remained low during the duration of the study. Percentage root galling was lowest (on average about 20 to 25%) between April and May. Between June and September, root galling steadily increased until it reached on average about 75%. Then, root galling decreased to on average about 15% during March and April.

Figure 4.4: Population dynamics of Meloidogyne incognita and percentage root galling on black pepper variety Vinh Linh in Buon Ma Thuot, Vietnam, from April 2006 until April 2007.

The average monthly air temperature and total monthly rainfall in Buon Ma Thuot during the study are shown in Fig. 4.5.

The study in Buon Ma Thuot was inititated in the beginning of the rainy season when the average total monthly rainfall was already 2,084 mm and the average monthly air temperature 25.6°C. The rainy season lasted until October when the rainfall started to drop. From November onwards, the dry season started. From April onwards, the rainy season started again. During the duration of the study, air temperature fluctuated between 20 and 25°C. It started to decrease at the beginning of the rainy season but continued to decrease until the very end of the dry season.

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Figure 4.5: Average monthly temperature and average monthly rainfall in Buon Ma Thuot during the period April 2006–April 2007. Source: Meteorological Centre of Dac Lac province.

4.3.3. COMPARISON OF THE ROOT POPULATION DYNAMICS OF MELOIDOGYNE INCOGNITA J2 AND PERCENTAGE ROOT GALLING ON BLACK PEPPER VARIETY VINH LINH IN CAM LO AND BUON MA THUOT

The population dynamics of M. incognita J2 per 5 g roots during the rainy and dry seasons in Cam Lo and Buon Ma Thuot are shown in Fig. 4.6.

Figure 4.6: Population dynamics of Meloidogyne incognita J2 in the roots of black pepper variety Vinh Linh during the rainy and dry seasons in Cam Lo (Quang Tri province) and Buon Ma Thuot (Dac Lac province).

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Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar 06 06 06 06 06 06 06 06 06 07 07 07

Dry season Rainy season

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In Cam Lo, the rainy season lasted 5 months vs 7 months in Buon Ma Thuot. In both study sites, the M. incognita J2 root population density followed a similar pattern: In Cam Lo, the highest root population density (854 J2/5 g roots) was reached 3 months after the end of the rainy season while in Buon Ma Thuot, the highest root population density (567 J2/5 g roots) was reached 2 months after the end of the rainy season. In both study sites, the root population densities decreased towards the end of the dry season remained low during the rainy season. In Buon Ma Thuot, the study site with the longest rainy season, the M. incognita J2 root population densities were higher compared with Cam Lo, the study site with the shortest rainy season.

The “dynamics” of the percentage root galling during the rainy and dry seasons in Cam Lo and Buon Ma Thuot are shown in Fig. 4.7.

Figure 4.7: “Dynamics” of the percentage root galling observed on the black pepper variety Vinh Linh during the rainy and dry seasons in Cam Lo (Quang Tri province) and Buon Ma Thuot (Dac Lac province).

In Cam Lo, the study site with the shortest rainy season, the highest percentage root galling (about 90%) was observed in the middle of the dry season. In Buon Ma Thuot, the study site with the longest rainy season, the highest percentage root galling (75%) was observed at the end of the rainy season.

Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct04 04 05 05 05 05 05 05 05 05 05 05

Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar 06 06 06 06 06 06 06 06 06 07 07 07

Rainy season Dry season

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4.4. DISCUSSION Rainfall and air temperature differed significantly between Cam Lo

(Quang Tri province) and Buon Ma Thuot (Dac Lac province). In Cam Lo, the rainy season started in July after a dry season of about 7 months. In Buon Ma Thuot, the rainy season started in April after a dry season of about 5 months. During the rainy season, the rainfall was much higher in Buon Ma Thuot than in Cam Lo. In Buon Ma Thuot, the air temperature fluctuated between 20 and 25°C while in Cam Lo the air temperature was a few degrees Celsius higher reaching 30°C during the dry season. Black pepper plants are usually cultivated in hot, humid areas with a high rainfall. Ideal is an annual rainfall of 600 to 2,000 mm with uniform distribution throughout the rainy season. In this respect, Cam Lo and Buon Ma Thuot are suitable for black pepper cultivation. Although black pepper plants tolerate temperatures between 10 and 40°C, ideal is a temperature from 22 to 30°C. In this respect, Cam Lo and Buon Ma Thuot are also suitable for black pepper cultivation although the higher air temperature recorded in Cam Lo appears to be somewhat more favourable compared to Buon Ma Thuot. Growth of black pepper plants will stop at temperatures below 10°C and above 40°C but these temperatures were not recorded at our two study sites. According to the literature, black pepper plants do not tolerate excessive dryness. In Cam Lo and Buon Ma Thuot, the duration of the dry season is 7 and 5 months, respectively, and this may have had an influence on yield. In Cam Lo and Buon Ma Thuot, the soil population densities of M. incognita fluctuated considerably throughout the duration of our study. In contrast, in both study sites the nematode root population densities fluctuated much less throughout the duration of our study remaining even more or less constant during most months especially during the rainy season. The highest root population densities of M. incognita were in both sites observed during the 1st half of the dry season. The optimum temperature for invasion and development of M. incognita is 25 to 30°C (Griffin et al., 1996) which corresponds with the prevailing temperatures throughout the year recorded both in Cam Lo and Buon Ma Thuot. In our opinion, the observed fluctuations of the nematode population densities, especially in the soil, are therefore not caused by temperature but by differences in rainfall between the dry and rainy seasons. Although occupying many ecological niches, nematodes are essentially aquatic animals (Luc et al., 1990). Several studies have shown that rainfall appears to be the main factor that modulates nematode populations (see for example Vilardebo, 1976; Quénéhervé, 1989a,b; Van den Bergh et al.,

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2005). It is known that most Meloidogyne species are most active in the soil at 40 to 60% field capacity (Karssen & Moens, 2006). Most studies of the population dynamics of Meloidogyne species have shown a decline in population during the rainy season (see for example Souza et al., 2008). Excess of water results in an oxygen deficit and many nematode species succumb to these conditions. In contrast, too little water inhibits the hatching of eggs and limits the movement of nematodes in the soil. According to our study, during the dry season in both Cam Lo and Buon Ma Thuot, the soil moisture content during the 1st half of the dry season was ideal for nematode invasion of the black pepper plants, nematode development and reproduction. During the 2nd half of the dry seasons the soil became too dry inhibiting hatching of eggs and limiting movement of the infective J2 in the soil toward the roots.

Soil population densities of M. incognita in Buon Ma Thuot where much higher compared with Cam Lo. For example, the highest average soil population density recorded in Buon Ma Thuot was 14,799 J2/100 g soil vs 1,933 J2/100 g soil in Cam Lo. However, this was not the case for the root population densities. For example, the highest average root population density recorded in Buon Ma Thuot was 854 J2/5 g roots vs 567 J2/5 g roots in Cam Lo. The different climates could have caused this difference. In both sites, the highest root population densities were observed about 1 month after the highest soil population densities were recorded.

In Cam Lo, the “dynamics” of percentage root galling followed closely the nematode population dynamics in the soil and in the roots. During the 1st half of the dry season, the highest percentage root galling was observed, averaging about 90% during 3 consecutive months. During the rainy season, when the nematode population densities were lowest, the percentage root galling was also lowest but still averaged from 30 to about 45%. In Buon Ma Thuot, the “dynamics” of percentage root galling also followed the nematode population dynamics in the soil and in the roots but the highest percentage root galling was observed 2 and 3 months before the highest soil and root nematode population densities, respectively. In Cam Lo, the highest percentage root galling was observed during the same month that the highest soil nematode population density was recorded and 1 month before the highest root nematode population was observed. Root galling is induced by Meloidogyne spp. from the moment the J2 have penetrated the roots and before the J2 have developed into reproductive females and this may explain the asynchronisation between percentage of root galling and nematode population dynamics. In Buon Ma Thuot, in contrast with Cam Lo, the

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highest percentage of root galling was observed during the 2nd half of the rainy season, declining to about 10% during the dry season. During the rainy season, when the nematode population densities were lowest, the percentage root galling was also lowest but still averaged from 30 to about 45%. Our results show that the behaviour of the nematodes in terms of population dynamics was similar in both sites. At both sites, nematode population densities were highest during the 1st half of the dry season. Estimating the population levels of M. incognita, for instance to decide on the application of a management strategy, should therefore be done during the 1st half of the rainy season. Assessment of the number of J2 in the soil will yield a better estimation of the nematode population levels than assessment of the number of J2 in the roots. Although the nematode population densities were much lower in Cam Lo compared with Buon Ma Thuot the percentage of root galling was much higher in Cam Lo than in Buon Ma Thuot. There is no direct explanation for this difference. The same black pepper variety (Vinh Linh) was grown at both sites so that a difference in host plant response to M. incognita could be excluded as a cause of the observed differences. It may be that the pathogenicity of the M. incognita populations is different. As mentioned before, in Cam Lo the dry season lasts longer significantly longer compared to Buon Ma Thuot. Less rain might have resulted in black pepper plants that are weaker and therefore more sensitive to infection by M. incognita resulting in higher root galling. But this is only a hypothesis. Whatever the cause is, our results indicate that predicting damage to black pepper plants based only on assessment of nematode population densities either in the soil or roots or both might be misleading if other environmental features (such as rainfall) are also not considered.

4.5. CONCLUSIONS Rainfall and air temperature differed significantly between the two study sites, Cam Lo in Quang Tri province (North Central Coast) and Buon Ma Thuot in Dac Lac province (Central Highlands). The most important climatic difference was rainfall: in Buon Ma Thuot, the rainy season lasted 2 months longer and the monthly rainfall during the rainy season was much higher compared to Cam Lo. Although this difference resulted in some differences in the population dynamics of M. incognita in the soil and roots of the black pepper variety Vinh Linh, the highest root population densities were in both study sites observed during the 1st half of the dry season. In Cam Lo, the highest percentage root galling was

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observed during the 1st half of the dry season. In Buon Ma Thuot, the highest percentage root galling was observed towards the end of the rainy season.

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CHAPTER 5. FUNGI ASSOCIATED WITH BLACK PEPPER PLANTS IN QUANG TRI PROVINCE AND INTERACTION BETWEEN

MELOIDOGYNE INCOGNITA AND FUSARIUM SOLANI

5.1. INTRODUCTION Black pepper plants can be affected by several diseases caused by

mycoplasma’s, viruses, bacteria, fungi and nematodes, besides nutritional disorders, in all countries where this crop is being cultivated (Anandaraj, 2000). Crop losses caused by diseases are a major production constraint in all pepper producing countries.

The major diseases on black pepper plants are quick wilt, anthracnose, mosaic disease, Fusarium wilt and slow wilt (Anandaraj, 2000).

Quick wilt is caused by several Phytophthora species (for instance P. capsici and P. palmivora var. piperis) that infect either the soil or aerial parts of black pepper plants resulting in foot and collar rot (soil parts), blight, spike shedding, defoliation, die back and at times death of plants (aerial parts). Anthracnose on black pepper plants is referred to as “pollu” disease in India, which means hollow fruits, and as black berry disease in Malaysia and Indonesia. The fungus Colletotrichum necator is the cause of this disease. Cucumber mosaic virus (CMV) causes a leaf disease referred to as mosaic disease in India, stunted disease in Indonesia, wrinkled leaf disease in Malaysia and mosaic disease or little leaf disease in Sri Lanka.

Fusarium wilt of black pepper plants in Brazil is caused by Fusarium solani f. sp. piperis. The symptom of Fusarium infection is root rot leading to flaccidity and yellowing of the leaves. The affected plants are killed within a short period (Anandaraj, 2000). Fusarium infection in a plantation is reported to reduce the economic life of the plantation from 20 to 6-8 years and the productivity per plant from 3 to 1.5 kg (Duarte & Albuquerque, 1991)

Slow wilt5 of black pepper plants is a debilitating disease where the affected plants survive for several years and death of the plants occurs gradually over a period of 3 to 4 years. This disease has been referred to under different names: slow wilt or slow decline in India, yellow or yellows diseases in Indonesia. Studies on the etiology of slow wilt have shown that the burrowing nematode Radopholus similis is associated with this disease.

5 In contrast, quick wilt refers to foot rot caused by Phytophthora species.

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Yellowing of the leaves of black pepper plants, different from the yellowing of leaves observed during Fusarium wilt and slow wilt, has also been observed and it has been speculated that this symptom is caused by a nematode-fungus complex involving Meloidogyne spp. and Fusarium spp. or possibly other fungi (Koshy et al., 2005). There are indeed several reports of the joint occurrence of Meloidogyne spp. and Fusarium spp. in the roots of black pepper plants. Meloidogyne incognita and F. solani were found associated with black pepper plants in Brazil. Infected plants showed wilting, yellowing of leaves, rotting of roots and stems, and cracking of stems (Koshy et al., 2005). Mustika (1984) showed that M. incognita and Fusarium sp. caused severe necrosis in the stellar part of black pepper plants in Indonesia together with the formation of tyloses that blocked the xylem. But although there are several reports that Meloidogyne spp. and Fusarium spp. together cause more damage to black pepper plants than either of them alone (Lopes & Lordello, 1979; Sheela & Venkitesan, 1990; Mustika, 1991, 1992) information on the Meloidogyne-Fusarium disease complex in black pepper plants remains scarce.

In Vietnam, quick wilt and mosaic disease occur on black pepper plants (PPRI, 2007). Quick wilt caused heavy yield losses in black pepper plantations in Quang Tri, Gia Lai and Kien Giang provinces (Thanh et al., 2004; Ton, 2005; Loang, 2007). Slow wilting of black pepper plants has also been observed in Vietnam but never studied in more detail.

The objectives of this part of our study were 1) to identify the predominant fungi associated with black pepper plants in Quang Tri province, one of the major pepper production regions of Vietnam, 2) to examine the relationship between the incidence of F. solani, M. incognita and yellowing of leaves in this province and 3) to investigate the interaction between M. incognita and F. solani alone or in combination on the percentage of black pepper plants with yellow leaves under greenhouse conditions.


5.2.1. SURVEY The survey was carried out during January 2007 in Quang Tri

province, Vietnam (Fig. 5.1). Quang Tri is situated in the southern part of the North Central Coast agro-ecological region. In Quang Tri, two seasons can be distinguished: a dry season which usually lasts from March to August and a rainy season which usually lasts from September to February.

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Samples were taken in three districts (Cam Lo, Gio Linh and Vinh Linh; Fig. 5.1) during January 2007 when the climate was cool with an average daily temperature of about 21oC and a monthly rainfall of more than 200 mm (Institute of Meteorology, Hydrology and the Environment, 2007). The soil type in all these districts is a red basalt (loamy clay). In all districts, samples were taken from the black pepper variety Vinh Linh. To obtain samples from black pepper plants differing in age, samples were taken both in nurseries and plantations.

In each district, samples were taken from three black pepper fields. Per field, at least five soil and root samples were collected at random. Sampling depth was 25 cm. Roots and soils of one black pepper plant constituted one sample. Samples were stored in plastic bags in a coolbox until nematode extraction and fungal isolation were carried out.

Figure 5.1: Districts of Quang Tri province, Vietnam, surveyed. 1: Cam Lo; 2: Gio Linh; 3: Vinh Linh.

5.2.1.1. Nematode population and root galling assessment

Nematodes were extracted from the soil using a tray method modified from the Baermann funnel method (Hooper et al., 2005) and from 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 200, 160 and 25 μm apertures. The extracted samples were examined under a stereomicroscope for the presence of plant-parasitic nematodes.

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The nematode population densities in the soil and the roots were defined as the number of second-stage juveniles (J2) per 100 g soil and 5 g fresh roots.

For counting the egg-laying females (ELF), roots were cut into 1 to 2 cm long pieces. Then, a sample of 5 g roots was taken at random and stained with phloxine B for stereomicroscopic observation of the red stained egg masses (Hooper et al., 2005).



5.2.1.2. Assessment of percentage of yellow leaves

The percentage of plants with yellow leaves was calculated based on the number of black pepper plants with yellow leaves out of 100 black pepper plants per field.

5.2.1.3. Isolation and identification of fungi

The roots were washed to remove the adhering soil. Main and adventitious roots were collected at random and cut into small segments. These segments were again washed, air-dried, and then surface-sterilised with 70% ethanol. After repeated washing in distilled water, the root segments were placed on potato dextrose agar medium (PDA) in Petri dishes (five root segments/dish) in a laminar flow. Per root sample five Petri dishes were used. Monoconidial fungal cultures of each fungus isolated were maintained on PDA at room temperature. Cultures were stored on PDA slants at 10oC and transferred every 4 weeks.

The appearance of the fungal colonies, the vegetative and reproductive structures of the fungi were described after 7 days incubation. Spores were suspended in sterile water using a sterile needle. Length and width were measured for 30 conidia, and conidial shape was recorded using a light microscope.

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Pure cultures of the fungal isolates were grown on standard media and a few selective media, and identified using the fungal keys provided by Domsch et al. (1980), Sinaga and Quimio (1987), Singh et al. (1991), Quimio and Hanlin (1999) and Quimio (2001). The fungi isolated were deposited at the collection of the Phytopathology Laboratory of the Plant Protection Research Institute in Hanoi, Vietnam.

In vitro colony growth of F. solani was moderately rapid, 4-4.5 cm diameter in 4-6 days. Colour of aerial mycelium was white to cream to grayish-white. Hyphae were septate and hyaline, branched straight or with slightly oblique angle and diameter of 3-8 µm. Microconidia were hyaline, cylindrical to oval, 1 to 2 celled, wed-shaped or allantoid, 12-21 x 3-5 µm. Macroconidia were moderately curved, thick-walled, typically 3-5 septated, 33-51 x 3-5 µm, often slightly wider towards the apex and with a well-marked foot.

5.2.2. GREENHOUSE EXPERIMENT The experiment was carried out in a greenhouse of the Post-entry Plant Quarantine Centre No. 1 in Hanoi, Vietnam.

5.2.2.1. Preparation of the black pepper plants

The black pepper variety Vinh Linh was used. The black pepper plants were grown from seeds. Red fresh seeds were collected from healthy plants in Quang Tri province and sown in trays in sterilised soil. After 5 months, the plants were transferred to plastic pots (15 cm-diameter) filled with a mixture of sterilised soil and humus (ratio 3:1) (Fig. 5.2).

Figure 5.2: Black pepper plant (variety Vinh Linh) in the nursery. Source of the picture: own picture.

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5.2.2.2. Preparation of nematode inoculum

The M. incognita population used in the greenhouse experiment was originally isolated from the roots of black pepper plants in Quang Tri province and maintained on tomato roots in pots in soil in the greenhouse. To obtain the nematode inoculum, heavily infected tomato roots were washed, cut into small pieces (0.5 cm), put in a 0.25% NaOCl solution and macerated in a kitchen blender (three 10 seconds periods separated by 5 seconds intervals). The macerated plant tissues with the nematodes were passed through a sieve with 40 μm aperture, rinsed with tap water, and then passed through a sieve with 25 μm aperture to recover the egg masses and J2 (fraction 1). The residue on the sieve with 40 μm aperture was placed on a Baermann tray to separate the living J2 from the dead J2 and the fine root tissues (fraction 2). Both fractions were then mixed in a beaker. The number of eggs and J2 in the suspension was counted using a stereomicroscope and the suspension was diluted to obtain a nematode inoculum density of 2,000 eggs and J2 per 10 ml.

5.2.2.3. Preparation of fungal inoculum

A culture of F. solani, isolated from the roots of black pepper plants in Quang Tri province, was grown on PDA in Petri dishes and incubated for 6 days at 28 ± 2oC. The filtrate was diluted with distilled water to obtain a fungal inoculum of 105 microconidia per ml (10 ml/plant).

Figure 5.3: Fusarium solani colony on PDA medium. A: floccose, greyish-white mycelium; B: microconidia. Source of the pictures: own pictures.

A B

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5.2.2.4. Experimental design

To inoculate the plants, three holes were made in the soil near to the stem base of each plant.

Six treatments were included in the experiment: 1. Uninoculated control plants. 2. Plants inoculated with F. solani only. 3. Plants inoculated with M. incognita only (2,000 eggs + J2).

4. Plants inoculated with F. solani and M. incognita simultaneously.

5. Plants inoculated with F. solani, after 2 weeks followed by an inoculation with M. incognita.

6. Plants inoculated with M. incognita, after 2 weeks followed by an inoculation with F. solani.

The pots were placed according to a randomised complete

block design with 10 replications. Each replication consisted of one black pepper plant (Fig. 5.4).

Figure 5.4: Experiment in the greenhouse. Source of the picture: own picture.

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5.2.2.5. Assessment of nematode reproduction, root galling, frequency of occurrence of Fusarium solani, percentage of plants with yellow leaves and plant growth

The experiment was terminated at 9 months after inoculation and the following parameters measured:

- plant growth: plant height, number of leaves and branches, fresh shoot and root weight;

- nematode reproduction: number of J2/100 g soil, J2/5 g roots and J2/root system, number of egg-laying females (ELF);6

- root galling;2

- percentage of plants with yellow leaves. Percentage of plants with yellow leaves was also noted at 3 and 6

months after inoculation.

5.2.3. STATISTICAL ANALYSIS The distribution and the homogeneity of the data were tested with

the Kolmogorov-Simirnov and Levene's test, respectively. For normally distributed and homogenous data, ANOVA was used to analyse the data. Mean separation was performed with the Duncan test. For non-normally distributed and non-homogenous data, the non-parametric Tamhane's T2 test was used to analyse the data. Mean separation was also performed with the Tamhane's T2 test. The combined confidence level of all the paired tests was at least 0.95 (combined confidence coefficient α = 0.05).

5.3. RESULTS

5.3.1. SURVEY

5.3.1.1. Fungi associated with black pepper plants in Quang Tri province

In total, 82 soil and 82 root samples were collected from black pepper plants in three districts in Quang Tri province in Vietnam. A list of the fungi isolated from the roots of the black pepper plants during the survey is given in Table 5.1.

6 For the methodology used see 5.2.1.1.

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Table 5.1: Fungi associated with black pepper plant roots in Quang Tri province, Vietnam.

No. Fungus No. of roots

infected

Frequency of

occurrence

(%)

1 Aspergillus niger van Tieghem 41 50.0

2 Fusarium solani (Martius) Saccardo 19 23.2

3 Fusarium spp. 17 20.7

4 Penicillium sp. 17 20.7

5 Rhizopus sp. 22 26.8

6 Trichoderma sp. 7 8.5

7 Pythium sp. 5 6.1

8 Lasiodiplodia sp. 2 2.4

9 Sclerotium rolfsii Sacc. 12 14.6

Total number of root samples examined = 82.

Aspergillus niger was the most common fungus isolated from the roots of the black pepper plant. It was present in 50% of the root samples examined. Its frequency of occurrence was about twice the frequency of occurrence of Rhizopus sp., the second-most frequent fungus isolated. Other more or less common fungi were F. solani (23.2%), other Fusarium species (20.7%) and Penicillium sp. (20.7%). Pythium sp. and Lasiodiplodia sp. were found in only 6.1% and 2.4%, respectively, of the root samples examined.

5.3.1.2. Frequency of occurrence of Fusarium solani and percentage of plants with yellow leaves

The frequency of occurrence of F. solani in roots of black pepper plants growing in fields differing in percentage of plants with yellow leaves is shown in Table 5.2.

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Table 5.2: Percentage of black pepper plants with yellow leaves, number and percentage of black pepper plants infected with F. solani.

Percentage of plants

with yellow leaves

n No. of roots infected

with F. solani

No. of roots infected

with F. solani (%)

No yellow leaves 14 0 0

Yellow leaves ≤ 30% 39 3 7.7

Yellow leaves > 30% 29 16 55.2

Total 82 19 23.2

n: number of root samples examined.

Fusarium solani was not isolated from the roots of black pepper

plants that did not have yellow leaves. In black pepper fields in which more than 30% of the plants had yellow leaves, F. solani was isolated from more than 50% of the roots examined. In contrast, in black pepper fields in which less than 30% of the plants had yellow leaves, F. solani was isolated from less than 10% of the black pepper plants.

5.3.1.3. Frequency of occurrence of Fusarium solani and age of black pepper plants

The frequency of occurrence of F. solani in roots of black pepper plants differing in age is shown in Table 5.3.

Table 5.3: Plant age of black pepper plants, number and percentage of black pepper plants infected with F. solani.

Plant age n No. of roots infected

with F. solani

No. of roots infected

with F. solani (%)

< 3 years (nurseries) 22 0 0

3–5 years 12 0 0

6–8 years 29 12 41.4

> 10 years 19 7 36.8

Total 82 19 23.2

n: number of root samples examined.

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Fusarium solani was not isolated from the roots of young black pepper plants growing in nurseries or younger than 5 years. In the roots of 6 to 8 years old black pepper plants and plants older than 10 years, F. solani was isolated from 41.4% and 36.8%, respectively, of the roots examined.

5.3.1.4. Incidence of Meloidogyne incognita and Fusarium solani, root galling and percentage of plants with yellow leaves in nurseries and plantations of black pepper plants

The frequency of occurrence and soil and root population densities of M. incognita, frequency of occurrence of F. solani, root galling and percentage of plants with yellow leaves in nurseries and plantations of black pepper plants is given in Table 5.4.

Soil and root samples were collected from black pepper plant nurseries in Cam Lo and Vinh Linh, and from black pepper plant plantations in Cam Lo, Vinh Linh and Gio Linh. In the nurseries sampled, the age of the black pepper plants was less than 3 years. In the plantations sampled, the age of the black pepper plants was more than 3 years.

Meloidogyne incognita was found in the roots of all the black pepper plants examined in the nurseries and in 87% of the roots of the black pepper plants examined in the plantations. The frequency of occurrence of M. incognita was lowest in plantations in Cam Lo district (81.8% and 72.7% for the soil and root samples, respectively).

The soil and root population densities of M. incognita varied considerable but the differences were higher in the soil compared with the roots. For example, in nurseries, the nematode soil population density was on average 161 J2/100 g in Cam Lo district vs 3,971 J2/100 g in Vinh Linh district (1:25) while the nematode root population density was on average 92 J2/5 g vs 637 J2/5 g in Cam Lo district (1:7). On average, the root and soil populations of M. incognita were 1.9 times and 4 times higher, respectively, in the nurseries than in the plantations.

The average percentage of root galling observed was about 10% higher on the roots of black pepper plants grown in nurseries (42.6%) compared with black pepper plants grown in plantations (32.5%)

Fusarium solani was not isolated from the roots of the black pepper plants grown in the nurseries. In contrast, it was isolated from about 30% of the roots of the black pepper plants grown in the plantations. No major differences in frequence of occurrence of F. solani in the plantations was observed among the three districts surveys.

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Table 5.4: Meloidogyne incognita population density, M. incognita and Fusarium solani frequency of occurrence, root galling and percentage of plants with yellow leaves in nurseries and plantations of black pepper plants in Quang Tri province, Vietnam.

Population density Frequency of occurrence (%)

Meloidogyne incognita

Plant age

District

n Soil

(J2/100 g) Roots

(J2/5 g) Soil Roots

Fusarium solani

Root galling

(%)

Yellow leaves

(%)

Nursery Cam Lo 12 161 637 83.3 100 0 40.8 21.7

(n=22) Vinh Linh 10 3,971 92 100 100 0 44.0 68.3

Average 2,066 383 91.7 100 0 42.6 45.0

Plantation Cam Lo 22 980 79 81.8 72.7 36.4 24.8 50.7

(n=60) Vinh Linh 21 2,227 177 100 100 28.6 60.2 43.7

Gio Linh 17 95 32 100 88.2 29.4 8.2 10.3

Average 1,101 96 93.9 87.0 31.5 32.5 34.9


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89

The percentage of plants with yellow leaves averaged 45% in the nurseries vs 34.9% in the plantations. The percentage of plants with yellow leaves varied considerable among the districts surveyed. For example, in nurseries in Cam Lo district 21.7% of the black pepper plants examined had yellow leaves vs 68.3% in Vinh Linh district (1:3) while in plantations in Gio Linh district 10.3% of the black pepper plants had yellow leaves vs 50.7% in Cam Lo district (1:5).

The relationship between the root and soil population densities of

M. incognita, the frequency of occurrence of F. solani, percentage of root galling and percentage of plants with yellow leaves in the black pepper nurseries and plantations is presented in Fig. 5.5.

Figure 5.5 indicates that the percentage of plants with yellow leaves is positively correlated with the soil (and root) population densities of M. incognita: the higher the nematode population density the higher the percentage of plants with yellow leaves. However, in the nurseries nematode population densities in the soil and roots, percentage of root galling and percentage of plants with yellow leaves were higher compared with the plantations but F. solani was not isolated from black pepper plants grown in the nurseries.

Figure 5.5: Meloidogyne incognita soil and root population densities, frequency of occurrence of Fusarium solani, root galling and percentage of plants with yellow leaves in black pepper plants in nurseries and plantations in Quang Tri province, Vietnam.

0

500

1000

1500

2000

2500

Nursery Plantation

Num

ber

of J

2

0

5

10

15

20

25

30

35

40

45

50

Perc

enta

ge (%)

Number of J2 per 100 g soil Number of J2 per 5 g fresh roots Fusarium (%)Root galling (%)Yellow leaves (%)

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90

5.3.2. GREENHOUSE EXPERIMENT The average monthly air temperature and average monthly rainfall

in Hanoi during the greenhouse experiment are shown in Fig.5.6.

Figure 5.6: Average monthly air temperature and average monthly

rainfall in Hanoi during the period January 2007–December 2005.

Source: Meteorological Institute of Hanoi.

The black pepper plants were inoculated with M. incognita and/or F. solani in March 2007 at the beginning of the rainy season when the air temperature started to rise. The air temperature was highest during July (30.4°C). From July onwards, when the rainfall was high, the air temperature slowly dropped to 20°C. Rainfall was highest during July-September. The dry season started in November.

5.3.2.1. Effect of inoculation with Meloidogyne incognita and Fusarium solani either alone or combined on the percentage of plants with yellow leaves

The effect of inoculation with M. incognita and F. solani alone or combined on the percentage of plants with yellow leaves over time is shown in Figure 5.7.

0

50

100

150

200

250

300

350

Jan07

Feb07

Mar07

Apr07

May07

Jun07

Jul07

Aug07

Sep07

Oct07

Nov07

Dec07

Time (month/year)

Rai

nfal

l (m

m)

0

5

10

15

20

25

30

35

Tem

pera

ture

(°C

)

Rainfall (mm)

Temperrature (°C)

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91

Figure 5.7: Percentage of plants with yellow leaves at 3, 6 and 9 months after inoculation with Meloidogyne incognita and Fusarium solani, alone or combined. C: Uninoculated control plants; Fs: F. solani alone; Mi: M. incognita alone; Fs+Mi: F. solani and M. incognita inoculated simultaneously; Fs→Mi: M. incognita inoculated 2 weeks after inoculation with F. solani; Mi→Fs: F. solani inoculated 2 weeks after M. incognita.

The percentage of plants with yellow leaves was lowest in the uninoculated control plants. In this treatment at the end of the experiment about 8% of the plants had yellow leaves. In all treatments, the percentage of plants with yellow leaves increased over time. At 3 and 6 months after inoculation, the percentage of plants with yellow leaves was significantly (P ≤ 0.05) higher in the treatments in which the plants were either inoculated with F. solani followed by inoculation with M. incognita 2 weeks later or vice versa. The percentage of plants with yellow leaves was also significantly (P ≤ 0.05) higher in the former treatment compared with the uninoculated control plants at the end of the experiment when about 30% of the plants inoculated with F. solani followed by inoculation with M. incognita 2 weeks later had yellow leaves vs about 8% in the uninoculated control plants.

a

aa

ab

abab

ab

b

abab

b

ab

bb

b

ab

b

b

0

5

10

15

20

25

30

35

3 months 6 months 9 months

Treatment

Yel

low

lea

ves

(%)

C Fs MiFs+Mi Fs→Mi Mi→Fs

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92

5.3.2.2. Effect of inoculation with Meloidogyne incognita and Fusarium solani either alone or combined on plant growth

The effect of inoculation with M. incognita and F. solani either alone or combined on plant growth is shown in Table 5.5.

The different treatments had no effect on plant height. The number of leaves and branches, however, were significantly (P ≤ 0.05) reduced in the treatment in which the plants were inoculated with M. incognita alone (with 46.7% and 41.2%, respectively) compared with the uninoculated control plants. For both these plant growth parameters a significant (P ≤ 0.05) reduction was also observed when M. incognita was inoculated first followed 2 weeks later by inoculation with F. solani. Simultaneous inoculation with M. incognita and F. solani significantly (P ≤ 0.05) reduced the number of leaves (with 37%) compared with the uninoculated control plants but not the number of branches. Inoculation with M. incognita alone significantly (P ≤ 0.05) reduced fresh shoot and root weight (with 19.7% and 31.2%, respectively) compared with the uninoculated control plants. Also inoculation with F. solani alone and inoculation with M. incognita followed by inoculated with F. solani 2 weeks later resulted in a significant (P ≤ 0.05) reduction of fresh root weight (with 43.7% and 43%, respectively) compared with the uninoculated control plants.

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93

Table 5.5: Plant growth of black pepper plants 9 months after inoculation with Meloidogyne incognita and Fusarium solani either alone or combined.

Treatment n Plant height

(m)

Number of

leaves

Number of

branches

Fresh shoot

weight (g)

Fresh root

weight (g)

Control 10 2.6 a 136.7 a 13.6 a 221.4 a 45.8 a

Fusarium solani 10 2.4 a 104.6 ab 12.4 a 206.8 ab 25.8 b

Meloidogyne incognita 10 2.2 a 72.9 b 8.0 b 177.7 b 31.5 b

Fusarium solani + M. incognita 10 2.3 a 86.1 b 12.1 a 203.9 ab 39.5 ab

Fusarium solani → M.incognita

(after 2 weeks)

10 2.2 a 96.8 ab 12.9 a 180.8 ab 36.6 ab

M. incognita → Fusarium solani

(after 2 weeks)

9 2.5 a 88.3 b 9.0 b 205.0 ab 30.7 b

n: number of plants.

Means in the same column followed by the same letter do not differ significantly according to the Duncan test (α = 0.05).

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94

The effect of the different treatments on the root systems of the black pepper plants was visually more obvious than the root weight data indicated (Fig. 5.8). Roots inoculated with M. incognita either alone or in combination with F. solani were shorter and less dense. Root galls were larger in size when M. incognita was inoculated alone.

Figure 5.8: Root systems of black pepper plants 9 months after

inoculation with Meloidogyne incognita and Fusarium solani either alone

or combined. Control: Uninoculated plants; I: F. solani alone; II: M.

incognita alone; III: F. solani and M. incognita inoculated simultaneously;

IV: M. incognita inoculated 2 weeks after inoculation with F. solani; V: F.

solani inoculated 2 weeks after inoculation with M. incognita.

5.3.2.3. Effect of inoculation with Meloidogyne incognita and Fusarium solani either alone or combined on nematode reproduction and root galling

The effect of inoculation with M. incognita and F. solani either alone or combined on nematode reproduction and root galling is shown in Table 5.6.

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95

Table 5.6: Reproduction of Meloidoygne incognita and root galling of black pepper plants 9 months after inoculation with M. incognita and Fusarium solani either alone or combined.

Treatment n Root

galling

index

Number

of ELF

J2/100 g soil J2/5 g roots J2/root system

Control 10 - - - - -

Fusarium solani 10 - - - - -

Meloidogyne incognita 10 4.0 a 3. 1 a 10,854 a 791 a 4,984 ab

Fusarium solani + M. incognita 10 3.7 a 2.6 a 10,361 a 2,402 a 18,976 b

Fusarium solani → M. incognita

(after 2 weeks)

10 3.6 a 2.9 a 11,902 a 1,061 a 7,767 ab

M. incognita → Fusarium solani

(after 2 weeks)

9 3.9 a 3.2 a 10,385 a 564 a 3,463 a

n: number of plants.

For the root galling index see 5.2.1.1.

ELF: number of egg-laying females. 0: no egg masses; 1 = 1-2 egg masses; 2 = 3-10 egg masses; 3 = 11-30 egg masses; 4 = 31-100 egg

masses; 5 = >100 egg masses.

Means in the same column followed by the same letter do not differ significantly according to the Duncan test (α = 0.05).

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96

No significant effect of simultaneous, pre- or postinoculation with F. solani on the number of egg-laying females of M. incognita and nematode soil and root population densities (per 100 g soil and 5 g roots) were observed. Inoculation with 2,000 eggs and J2 of M. incognita resulted in an average nematode population of about 8,800 J2/root system after 9 months which is a 4.4 times increase. Simultaneous inoculation with M. incognita and F. solani resulted in average nematode population of about 19,000 J2/root system after 9 months which is a 9.5 times increase. This number was significantly (P ≤ 0.05) lower (about 3,500 J2/root system) when M. incognita was inoculated first followed by F. solani 2 weeks later.

5.4. DISCUSSION As could be expected several fungi were isolated from the roots of

black pepper plants during our survey. In Quang Tri province we found nine fungal taxa. This number compares very well with other studies of fungi associated with the roots of black pepper. For example, Noveriza and Quimio (2004) isolated 14 fungal genera from the roots of black pepper plants in the Philippines. They found more or less the same fungal genera as we found during our survey. In our survey, Aspergillus niger was the most common fungus isolated from the roots of black pepper plants. This fungus is not considered pathogenic to black pepper plants (Anandaraj, 2000). Usually, A. niger is associated with seed decay of agricultural crops (Agrios, 2005). In Brazil also Freire et al. (2000) reported that A. niger was the predominant fungus associated with black pepper plants.

During our survey in Quang Tri province, F. solani was found in about one out of four black pepper root samples examined. This frequency of occurrence is lower than the frequency of occurrence (77%) on black pepper plants in the Central Highlands reported by Hoa et al. (2003). In Brazil, F. solani f. sp. piperis is considered the major root pathogen of black pepper plants and the cause of severe Fusarium wilt of black pepper plants (Duarte & Albuquerque, 1991; Anandaraj, 2000). The presence of this F. solani isolate has so far not been established in Asia where the role of F. solani alone as a root pathogen of black pepper plants remains uncertain.

During our survey in Quang Tri province, we observed that F. solani was not isolated from the roots of black pepper plants that did not had yellow leaves and that the frequency of occurrence of F. solani increased with increasing percentage of plants with yellow leaves. This observation suggests that there may be a relationship between the frequency of

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occurrence of F. solani in the roots of black pepper plants and the incidence of yellow leaves. But, during the same survey, we also observed that in the black pepper plant nurseries where F. solani was not isolated about one out of two black pepper plants examined had yellow leaves. This observation suggest that another pathogen must be the initial cause of the yellowing of the leaves of black pepper plants and that F. solani may play a role in this disease in a later stage of the development of the plants . A likely pathogenic candidate is M. incognita because every single black pepper plant examined in the nurseries was infected with this nematode species.

Also during our survey in Quang Tri, we observed that F. solani was not isolated from the roots of black pepper plants growing in nurseries (and younger than 3 years) and black pepper plants younger than 5 years growing in plantations. Our observation is in agreement with the results of a study carried out by the Plant Protection Research Institute in Dac Lac province in Vietnam in which was reported that the percentage of black pepper plants in plantations infected with fungi was higher compared with black pepper plants in nurseries (7.2, 19.6 and 23.4% in plants growing 2, 5 and over 6 years, respectively) (PPRI, 2007). This indicates that the nurseries examined during our survey were either not infested with F. solani or that F. solani only infects older roots of black pepper plants.

In the nursery in Cam Lo district about 20% of the black pepper plants had yellow leaves. In these nurseries on average 161 and 637 J2 of M. incognita/100 g soil and 5 g roots, respectively, were extracted from the roots of black pepper plants. Since F. solani was not isolated from these nurseries, these M. incognita population densities may indicate that a relative low number of nematodes may result in a high percentage of black pepper plants with yellow leaves. In Indonesia, yellow symptoms appeared on the leaves of black pepper plants infected with Meloidogyne spp. at population levels of 74 J2/100 g soil and 305 J2/10 g roots (Mustika, 1978).

In the greenhouse experiment, at the end of the experiment (i.e. at 9 months after inoculation) 8 % of the uninoculated control plants had yellow leaves. This observation suggests that, at least under our experimental conditions, black pepper plants are prone to yellowing of the leaves. Nevertheless, the results of the greenhouse experiment shows, in general, an increase in the percentage of black pepper plants with yellow leaves in the treatments in which the plants were inoculated with M. incognita alone or in combination with F. solani. This effect was most obvious at 6 months after inoculation when all the treatments in which M.

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incognita was inoculated were significantly different from the uninoculated control plants and the plants inoculated only with F. solani.

The results of the greenhouse experiment also show the negative effect M. incognita can have on the growth of black pepper plants since inoculation with M. incognita alone resulted at 9 months after inoculation in significantly less number of leaves and branches, and a significant reduction in fresh shoot and root weights compared with the uninoculated control plants. For the other treatments these suppressive effects could not be consistently demonstrated except in the treatment in which M. incognita was inoculated first followed by inoculation with F. solani 2 weeks later. Also in this treatment, a significantly lower number of leaves and branches, and a significant reduction in fresh shoot and root weights compared with the uninoculated control plants were observed. Mustika (1990) also showed that infection with M. incognita 2 weeks prior to infection F. solani caused wilting and death of black pepper plants earlier than when the two pathogens were inoculated simultaneously.

Inoculation with F. solani before, at the same time, or 2 weeks after inoculation with M. incognita did not affect root galling, number of egg-laying females of M. incognita, and nematode population densities in the soil and roots of the black pepper plants. The significant suppressive effect inoculation with F. solani 2 weeks after inoculation with M. incognita had on the average number M. incognita per root system can be explained by the fact that this treatment also significantly reduced the fresh root weight of the black pepper plants.

5.5. CONCLUSIONS During a survey of nurseries and plantations of black pepper plants

in Quang Tri province in Vietnam during the rainy season of 2007, nine fungal taxa were isolated from the roots of the black pepper plants. Fusarium solani was found in about one out of four black pepper root samples examined but not in the nurseries and also not from black pepper plants younger than 5 years growing in plantations. Since in these nurseries about one out of two black pepper plants examined had yellow leaves, this observation suggests that another pathogen must be the initial cause of the yellowing of the leaves. A likely pathogenic candidate is M. incognita which was extracted from every single black pepper plant examined in the nurseries.

During the same survey we also observed that F. solani was not isolated from the roots of black pepper plants that did not had yellow

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leaves and that the percentage of black pepper plants with yellow leaves increased with increased frequency of occurrence of F. solani. This observation indicates that F. solani plays a role in the yellowing of the leaves of black pepper plants in a later stage of the development of the plants.

The results of the greenhouse experiment show the negative effects inoculation with M. incognita alone or in combination with F. solani may have on the percentage of black pepper plants with yellow leaves and on plant growth.

No effect of inoculation with F. solani before, at the same time, or 2 weeks after inoculation with M. incognita on root galling and nematode reproduction was observed.

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CHAPTER 6

101

CHAPTER 6. HOST RESPONSE OF FIVE BLACK PEPPER VARIETIES TO INFECTION BY MELOIDOGYNE INCOGNITA UNDER

GREENHOUSE CONDITIONS

6.1. INTRODUCTION Many nematode species have been reported from black pepper

plants but the only nematodes known to cause serious damage to pepper crops are the burrowing nematode (R. similis) and root-knot nematodes (Meloidogyne spp.) (Koshy et al., 2005).

Many consider natural resistance, when available, to be the best option for nematode management because of its cost effectiveness, compatibility with other management practices and because natural resistance is environmentally benign (Starr & Mercer, 2009). Natural resistance can also be a powerful tool for the management of root-knot nematodes and there are several examples of crop varieties bred specifically for resistance to one or more Meloidogyne species that have made important contributions to nematode management such as in tomato (Williamson & Roberts, 2009).

Differences in host response of black pepper varieties to M. incognita have been reported (Koshy et al., 2005). However, according to these reports, resistance to M. incognita in Piper nigrum germplasm is difficult to find. For example, a total of 101 cultivars, 74 accessions of wild Piper sp. and 140 intercultivar hybrids were screened against M. incognita by several nematologists in India but only one cultivar (CLTP-812) was found resistant (Koshy et al., 2005). This cultivar was later released under the name “Pournami” for cultivation in areas infested with M. incognita.

The objective of this part of our study was to evaluate the host response of five black pepper varieties commonly grown in Vietnam to M. incognita. The evaluation was undertaken under greenhouse conditions.

As mentioned in Chapter 1, resistance to nematodes is most often defined based on the ability of a host plant to suppress nematode reproduction, with resistant or less susceptible plants supporting lower levels of nematode reproduction than nematode reproduction on susceptible plants.

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6.2. MATERIALS AND METHODS The experiment was carried out in a greenhouse of the Western Highlands Agro-Forestry Scientific and Technical Institute (WASI) in Dac Lac province, Vietnam.

6.2.1. PREPARATION OF THE BLACK PEPPER PLANTS Five black pepper varieties were included in the study: Vinh Linh,

Loc Ninh, Trau, Lada Belangtoeng (La Da) and Phu Quoc. The planting material was received from the black pepper varieties field collection of WASI in Dac Lac province, Vietnam.

The Vinh Linh, Loc Ninh and Trau varieties were collected in Vietnam, La Da in Indonesia and Phu Quoc in Cambodia (Tiem et al., 2007).

The spike and yield characteristics of the five black pepper varieties are given in Table 6.1. These characteristics can be very different among the varieties. Spike length ranged from 7.5 cm (Phu Quoc) to 9.7 cm (Trau). Fruit of La Da is small and light in weight compared with Vinh Linh.

Table 6.1: Spike and yield characteristics of the five black pepper varieties included in the host response study.

Variety Spike length

(cm)

No. of

berries/spike

Fruit fresh

volume

1,000 berries

(ml)

Fruit fresh

weight

1,000 berries

(g)

Loc Ninh 9.0 ± 0.5 37.8 ± 2.3 114.0 ± 0.3 114.3 ± 0.8

Vinh Linh 8.1 ± 0.4 43.3 ± 2.3 161.0 ± 0.3 180.9 ± 0.2

La Da 8.5 ± 0.3 46.3 ± 3.7 100.7 ± 0.2 103.6 ± 0.2

Phu Quoc 7.5 ± 0.3 46.4 ± 2.1 104.7 ± 0.4 114.3 ± 0.2

Trau 9.7 ± 0.5 50.1 ± 3.9 135.0 ± 0.3 148.0 ± 0.3

n = 30.

The oil-content characteristics of the five black pepper varieties are given in Table 6.2. The chemistry of pepper can be broadly classified into a) compounds contributing to its pungency and b) compounds imparting its characteristic aroma. The pungency is due to the alkaloid

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piperine. The aroma is due to the essential oil present in the fruits. The commercial product oleoresin, extracted from pepper powder, contains both pungency and aroma contributing compounds. These characteristics can be different among the varieties. Piperine content ranged from 4.8% (Trau) to 5.8% (La Da), essential oil content ranged from 1.6% (Trau) to 2.6% (Phu Quoc) and oleoresin content from 9.4% (Trau) to 16.7% Vinh Linh.

Table 6.2: Oleoresin, piperine and essential oil content of the five black pepper varieties included in the host response study.

Variety Oleoresin (%) Piperine (%) Essential oil (%)

Loc Ninh 16.4 5.2 2.2

Vinh Linh 16.7 5.2 2.1

La Da 10.9 5.8 2.1

Phu Quoc 11.9 5.4 2.6

Trau 9.4 4.8 1.6

Source: The Western Highlands Agro-Forestry Scientific and Technical Institute

(WASI).

The black pepper plants were grown from cuttings. Healthy cuttings of each variety were planted in pots in a steam-sterilised sandy loam soil. When shoot heights were 15 to 30 cm, 10 plants of each variety were transferred to plastic pots (15-cm-diameter) filled with a mixture of sterilised soil and humus (ratio 3:1). These plastic pots were placed in the greenhouse. This experiment was carried out in year 2008 at WASI in Buon Ma Thuot district, in Dac Lac province.

The average monthly air temperature and total monthly rainfall in Buon Ma Thuot during the study are shown in Fig. 6.1.

Meloidogyne incognita were inoculated on black pepper plants in the rainy season (June) when the average total monthly rainfall was already 1,169 mm and the average monthly air temperature 25.3°C. The rainy season lasted until October when the rainfall started to drop. From November onwards, the dry season started. During the duration of the study, air temperature fluctuated between 21 and 25°C.

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Figure 6.1: Average monthly temperature and average monthly rainfall in Buon Ma Thuot during year 2008. Source: Meteorological Centre of Dac Lac province.

6.2.2. PREPARATION OF NEMATODE INOCULUM AND INOCULATION The M. incognita population used in the greenhouse experiment

was originally isolated from black pepper roots in Dac Lac province and maintained on tomato roots in pots in soil in the greenhouse. To obtain the nematode inoculum, heavily infected tomato roots were washed, cut into small pieces (0.5 cm), put in a 0.25% NaOCl solution and macerated in a kitchen blender (three 10 seconds periods separated by 5 seconds intervals). The macerated plant tissues with the nematodes were passed through a sieve with 40 μm aperture, rinsed with tap water, and then passed through a sieve with 25 μm aperture to recover the egg masses and J2 (fraction 1). The residue on the sieve with 40 μm aperture was placed on a Baermann tray to separate the living J2 from the dead J2 and the fine root tissues (fraction 2). Both fractions were then mixed in a beaker. The number of eggs and J2 in the suspension was counted using a stereomicroscope and the suspension was diluted to obtain a nematode inoculum density of 2,000 eggs and J2 per 10 ml.

Five months after the black pepper cuttings were planted in the plastic 15-cm-diameter pots and placed in the greenhouse, 10 plants of each black pepper variety were inoculated with 2,000 eggs and J2. To

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

Jan

08

Feb

08

Mar

08

Apr

08

May

08

Jun

08

Jul

08

Aug

08

Sep

08

Oct

08

Nov

08

Dec

08

Time (month/year)

Rain

fall

(mm

)

0

5

10

15

20

25

30

Air

tem

pera

ture

(°C

)

Rainfall (mm)Air temperature (°C)

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inoculate the plants, three holes were made in the soil near to the stem base of each plant.

The pots were placed according to a randomised complete block design with 10 replications. Each replication consisted of one black pepper plant.

6.2.3. ASSESSMENT OF NEMATODE REPRODUCTION, ROOT GALLING AND PLANT GROWTH

The experiment was terminated at 5 months after nematode inoculation and the following parameters measured:

- plant growth: plant height, number of leaves and branches, fresh shoot and root weight;

- nematode reproduction: number of J2/100 g soil, J2/5 g roots and J2/root system, number of egg-laying females (ELF);7

- root galling;1

6.2.4. STATISTICAL ANALYSIS The distribution and the homogeneity of the data were tested with

the Kolmogorov-Simirnov and Levene's test, respectively. For normally distributed and homogenous data, ANOVA was used to analyse the data. Mean separation was performed with the Duncan test. For non-normally distributed and non-homogenous data, the non-parametric Tamhane's T2 test was used to analyse the data. Mean separation was also performed with the Tamhane's T2 test. The combined confidence level of all the paired tests was at least 0.95 (combined confidence coefficient α = 0.05). Prior to analysis, nematode reproduction data were log(x+1) transformed.

The Spearman correlation coefficient was used for data that did not satisfy the normality assumption. This coefficient is based on the ranks rather than on the actual values. Values of the two coefficients range from -1 to +1. The sign of the coefficient indicates the direction of the relationship and its absolute value indicates the strength, with larger values indicating stronger relationships (Van den Bergh, 2002).

7 For the methodology used see Chapter 5.2.1.1.

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106

6.3. RESULTS

6.3.1. PLANT GROWTH Plant growth of the five varieties included in the host response

experiment at harvest (i.e. at 5 months after inoculation) is shown in Table 6.3.

No significant differences in plant height, number of leaves and branches, and fresh shoot weight were observed among the five varieties included in the experiment. Only the fresh root weights of the variety Vinh Linh and Phu Quoc were significantly (P ≤ 0.05) different. The fresh root weight of Vinh Linh was about twice the fresh root weight of Phu Quoc (24 vs 11.6 g, respectively).

6.3.2. PERCENTAGE YELLOW LEAVES It was not possible to count the number of plants with yellow leaves because the foliar discolouration was very faint and difficult to measure in an objective way. However, during the experiment, we observed that inoculation with M. incognita resulted in inward held leaves together with upward or stiff drooping of these leaves (Figs 6.2.B & 6.3B).

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107

Table 6.3: Plant growth of five black pepper varieties, 5 months after inoculation with 2,000 eggs and J2 of Meloidogyne incognita.

Variety n Plant height

(cm)

Number of

leaves

Number of

branches

Fresh shoot

weight (g)

Fresh root

weight (g)

Loc Ninh 10 47.0 a 9.8 a 2.5 a 49.2 a 17.5 ab

Vinh Linh 10 43.9 a 9.9 a 3.4 a 51.5 a 24.0 b

La Da 10 40.7 a 9.2 a 2.8 a 44.8 a 20.1 ab

Phu Quoc 10 35.2 a 8.5 a 3.0 a 35.2 a 11.6 a

Trau 10 42.2 a 8.2 a 2.5 a 46.5 a 21.9 ab

n: number of plants. Mean in the same column followed by the same letter do not differ significantly according to the Duncan test (α = 0.05).

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108

Figure 6.2: Effect of inoculation with Meloidogyne incognita on the black pepper varieties Loc Ninh (1), Vinh Linh (2) and La Da (3), before inoculation (A) and 5 months after inoculation (B).

1A 1B

2A 2B

3A 3B

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109

Figure 6.3: Effect of inoculation with Meloidogyne incognita on the black pepper varieties Phu Quoc (4) and Trau (5), before inoculation (A) and 5 months after inoculation (B).

6.3.3. ROOT GALLING AND REPRODUCTION OF MELOIDOGYNE INCOGNITA Root galling and reproduction of M. incognita on the five black

pepper varieties included in the host response experiment are shown in Table 6.

Root galling was significantly (P ≤ 0.05) higher in the variety Trau compared with the varieties Loc Ninh and Phu Quoc. No significant differences in the number of egg-laying females, J2/5 g fresh roots and J2/root system were observed among the five varieties. The number of J2/100 g soil was significantly (P ≤ 0.05) lower in the variety La Da variety

4A 4B

5A 5B

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110

compared with the other four varieties (1,219 vs more than 2,000 J2, respectively).

Table 6.4: Root galling and reproduction of Meloidogyne incognita on five black pepper varieties, 5 months after inoculation with 2,000 eggs and J2.

Variety n Root

galling

index

Number

of ELF

J2/100 g

soil

J2/5 g

roots

J2/root

system

Loc Ninh 10 1.2 a 1.8 a 2,081 b 126 a 441 a

Vinh Linh 10 1.6 ab 2.3 a 2,560 b 114 a 548 a

La Da 10 1.4 ab 1.5 a 1,219 a 121 a 487 a

Phu Quoc 9 1.2 a 2.0 a 2,494 b 173 a 402 a

Trau 8 2.1 b 2.5 a 2,130 b 115 a 504 a

n: number of plants

For the root galling index see Chapter 5.2.1.1.

ELF: number of egg-laying females. 0: no egg masses; 1 = 1-2 egg masses; 2 = 3-10

egg masses; 3 = 11-30 egg masses; 4 = 31-100 egg masses; 5 = >100 egg masses.

Means in the same column followed by the same letter do not differ significantly

according to the Duncan test (number of J2 in soil and roots) or Tamhane's T2 test

(root galling index and number of ELF) (α = 0.05).

6.3.4. CORRELATIONS BETWEEN PLANT GROWTH, ROOT GALLING AND MELOIDOGYNE INCOGNITA REPRODUCTION

In the Tables 6.5-6.9, the correlations between plant growth, root galling and M. incognita reproduction on the five black pepper varieties examined in the host response experiment are shown.

In general, very few significant negative correlations between root galling and M. incognita reproduction on the one hand and plant growth on the other hand could be observed. Only in the variety La Da there was a significant (P ≤ 0.05) negative relationship between M. incognita reproduction and fresh shoot weight. Also a very few significant positive correlations between root galling and M. incognita reproduction on the one hand and plant growth on the other hand could be observed. For instance,

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111

in the variety Loc Ninh there was a significant (P ≤ 0.05) positive relationship between M. incognita reproduction and number of branches.

In the black pepper varieties Vinh Linh, La Da, Phu Quoc and Trau a weak negative correlation was observed between the number of J2/5 g fresh roots and fresh root weight (Figure 6.4). In contrast, in the black pepper variety Loc Ninh, fresh root weight increased with increasing number of J2/5 g fresh roots.

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112

Table 6.5: Correlation (Spearman's rho) between plant growth, root galling and Meloidogyne incognita reproduction in the roots of the black pepper variety Loc Ninh.

Plant

height

(cm)

Number of

leaves

Number of

branches

Fresh

shoot

weight

(g)

Fresh root

weight

(g)

Root

galling

(%)

Number of

egg-laying

females

Number of

J2/5 g

roots

Plant height (cm) 1

Number of leaves 0.77** 1

Number of branches 0.06 .21 1

Fresh shoot weight (g) .70* .71* .28 1

Fresh root weight (g) .02 .27 .37 .51 1

Root galling (%) .22 .35 .22 .52 .35 1

Number of egg-laying females .09 .43 .81** .49 .37 .63* 1

Number of J2/5 g roots .05 .40 .74* .45 .40 .70* .97** 1 Number of plants: 10. * Correlation significant at the 0.05 level. ** Correlation significant at the 0.01 level.

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113

Table 6.6: Correlation (Spearman's rho) between plant growth, root galling and Meloidogyne incognita reproduction in the roots of black pepper variety Vinh Linh.

Plant

height

(cm)

Number of

leaves

Number of

branches

Fresh

shoot

weight (g)

Fresh root

weight (g)

Root

galling (%)

Number of

egg-laying

females

Number of

J2/5 g

roots

Plant height (cm) 1

Number of leaves 0.77** 1

Number of branches 0.51 .59 1

Fresh shoot weight (g) .37 .10 .22 1

Fresh root weight (g) -.48 -.64 -.62 .33 1

Root galling (%) .30 .11 .17 .07 -.20 1

Number of egg-laying females -.37 -.28 -.06 -0.41 -.05 -.48 1

Number of J2/5 g roots -.29 -.11 -.02 -0.30 -.04 -.42 .93** 1 Number of plants: 10. * Correlation significant at the 0.05 level. ** Correlation significant at the 0.01 level.

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114

Table 6.7: Correlation (Spearman's rho) between plant growth, root galling and Meloidogyne incognita reproduction in the roots of the black pepper variety La Da.

Plant

height

(cm)

Number of

leaves

Number of

branches

Fresh

shoot

weight (g)

Fresh root

weight (g)

Root

galling (%)

Number of

egg-laying

females

Number of

J2/5 g

roots

Plant height (cm) 1 Number of leaves .71* 1 Number of branches .11 .61 1 Fresh shoot weight (g) .60 .52 .53 1 Fresh root weight (g) .65* .59 .46 .66* 1

Root galling (%) -.16 .33 .60 .05 -.02 1

Number of egg-laying females -.32 -.32 -.45 -.68* -.48 -.10 1

Number of J2/5 g roots -.29 -.36 -.57 -.73* -.46 -.27 .97** 1 Number of plants: 10. * Correlation significant at the 0.05 level.** Correlation significant at the 0.01 level.

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115

Table 6.8: Correlation (Spearman's rho) between plant growth, root galling and Meloidogyne incognita reproduction in the roots of the black pepper variety Phu Quoc.

Plant

height

(cm)

Number of

leaves

Number of

branches

Fresh

shoot

weight (g)

Fresh root

weight (g)

Root

galling (%)

Number of

egg-laying

females

Number of

J2/5 g

roots

Plant height (cm) 1 Number of leaves .93** 1 Number of branches .64* .68* 1 Fresh shoot weight (g) .32 .53 .57 1 Fresh root weight (g) .07 .21 .18 .74* 1

Root galling (%) .62 .70* .77* .25 .03 1

Number of egg-laying females .11 -.19 -.11 .03 -.21 -.48 1 Number of J2/5 g roots .03 -.28 .04 -.23 -.40 -.39 .69* 1

Number of plants: 9. * Correlation significant at the 0.05 level. ** Correlation significant at the 0.01 level.

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116

Table 6.9: Correlation (Spearman's rho) between plant growth, root galling and Meloidogyne incognita reproduction in the roots of the black pepper variety Trau.

Plant

height

(cm)

Number of

leaves

Number of

branches

Fresh

shoot

weight (g)

Fresh root

weight (g)

Root

galling (%)

Number of

egg-laying

females

Number of

J2/5 g

roots

Plant height (cm) 1 Number of leaves .83** 1 Number of branches .38 .68* 1 Fresh shoot weight (g) .42 .56 .75* 1 Fresh root weight (g) .25 .18 -.33 .20 1

Root galling (%) -.08 -.25 -.34 -.25 -.08 1

Number of egg-laying females .00 .20 .75 .80 -.50 -.26 1 Number of J2/5 g roots -.11 .14 .67 .61 -.50 -.17 .96* 1

Number of plants: 8. * Correlation significant at the 0.05 level. ** Correlation significant at the 0.01 level.

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117

Figure 6.4: Correlation between the number of J2 of Meloidogyne incognita/5 g

fresh roots and fresh root weight of five black pepper varieties (♦: fresh root

weight).

Loc Ninh

0

5

10

15

20

25

30

35

0 20 40 60 80 100

Number of J2

Root

wei

ght

(g)

Vinh Linh

0

10

20

30

40

50

60

0 20 40 60Number of J2

Root

wei

ght

(g)

LA DA

0

5

10

15

20

25

30

0 20 40 60 80

Number of J2

Root

wei

ght

(g)

Phu Quoc

0

5

10

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20

0 50 100 150 200 250

Number of J2

Root

wei

ght

(g)

Trau

0

10

20

30

40

50

0 10 20 30 40Number of J2

Root

wei

ght

(g)

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118

6.4. DISCUSSION In general, the host response to M. incognita reproduction of the

five black pepper varieties included in the greenhouse experiment was similar. Although M. incognita reproduction was not very high, all five varieties can be considered susceptible to this nematode species. The density of J2 of M. incognita in the rhizosphere of the variety La Da was significantly lower compared to the other four varieties but still relatively high. Since the density of egg-laying females and J2 of M. incognita in the roots of the variety La Da were not significantly different from the other four varieties we consider this variety as equally susceptible to this nematode species.

Because no uninoculated control plants were included in this greenhouse experiment it is difficult to compare the sensitivity to M. incognita damage of the five black pepper varieties examined. The low fresh root weight of the inoculated variety Phu Quoc plants may indicate that this variety is more sensitive to M. incognita damage than the other four varieties but it is also possible that also uninfected plants will have a low fresh root weight. Also, no significant negative correlations were observed between M. incognita reproduction on this variety on the one hand and plant growth on the other hand. In contrast, in the variety La Da a significant negative correlation was observed between M. incognita reproduction on the one hand and plant growth on the other hand and this indicates that this variety is more sensitive to M. incognita damage than the other varieties.

The observation that in the variety Loc Ninh M. incognita reproduction is significantly positively correlated with the number of branches and that the root fresh weight of inoculated variety Loc Ninh plants increases with increasing nematode root population density may indicate that this black pepper variety is less sensitive or even tolerant to M. incognita damage. Root galling was also lowest on this variety.

Differences in sensitivity to damage by M. incognita and in root galling among black pepper varieties have been reported (Koshy et al., 2005). Although a tolerant agricultural crop variety may not limit the build up of a nematode population in the soil and consequently in the roots this ability of the plant may result in a higher crop quality and yield. Since black pepper is a perennial plant and cultivated in regions which are enduring a dry season during which nematode populations are suppressed because of the climate, black pepper varieties tolerant to M. incognita may be as effective as resistant varieties.

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To confirm these preliminary observations, the host plant response to M. incognita damage of the varieties La Da and Loc Ninh should be further investigated. If our observations are confirmed this may lead to advise the black pepper farmers not to plant the variety La Da in fields heavily infested with M. incognita but to plant the variety Loc Ninh in stead.

6.5. CONCLUSIONS No differences in host response to M. incognita reproduction of the

five black pepper varieties included in the greenhouse experiment were observed. All these five varieties are considered equally susceptible to M. incognita. However, differences in host repsonse to M. incognita damage were observed. Our results suggest that the variety La Da is more sensitive and the variety Loc Ninh less sensitive or even maybe tolerant to damage caused by this nematode species

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CHAPTER 7. GENERAL CONCLUSIONS AND PERSPECTIVES

During 2004-2006, in total 432 soil and 432 root samples were collected from black pepper plants in 19 districts belonging to six provinces in three agro-ecological regions in Vietnam and examined for the presence of plant-parasitic nematodes. Percentage of root galling was also determined and the percentage of black pepper plants with yellow leaves in the fields surveyed noted. 35 nematode species belonging to 19 genera were identified confirming previous reports that the biodiversity of the nematofauna associated with the roots of black pepper plants is high. Almost every soil and root sample examined was infected with Meloidogyne incognita indicating that this root-knot nematode species is the predominant plant-parasitic nematode species associated with black pepper plants in Vietnam (and probably in most black pepper growing regions in Asia). No difference in frequency of occurrence of M. incognita among the three agro-ecological regions surveyed was observed. However, the root population density of M. incognita was on average 5 times higher in the North Central Coast and Central Highlands than in Phu Quoc Island. The percentage of black pepper plants with yellow leaves was on average about 20 to 25% in the North Central Coast and Central Highlands which was somewhat higher compared to Phu Quoc Island. Further research should clarify if M. incognita and yellow leaves pose a larger problem in the North Central Coast and Central Highlands than in Phu Quoc Island. Large differences in root population density of M. incognita and percentage of black pepper plants with yellow leaves were observed in different districts within the same agro-ecological region which may indicate that local differences are higher than regional differences. The percentage of root galling averaged about 40% in all the three agro-ecological regions. Black pepper roots infected with M. incognita showed the same type of galls and yellow discoloration of the leaves as described in the literature for black pepper plants infected with M. incognita. In general, a positive relationship between soil and root population densities of M. incognita on black pepper plants and percentage of plants with yellow leaves was observed. Again Radopholus similis has not been found in Vietnam although this nematode species is common on black pepper plants in South Asia and Southeast Asia. Recently two Radopholus species (R. duriophilus and R. arabocoffeae) morphologically very similar to R. similis were described from the Western Highlands in Vietnam. It would be interesting to investigate if these two Radopholus species can infect black

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pepper plants and can cause damage to pepper crops. If this is the case, quarantine measures should be taken to prevent their spread from durian and coffee, respectively, to fields with black pepper plants.

A detailed comparative morphological and morphometrical study

of seven M. incognita populations isolated from black pepper plants in three agro-ecological regions in Vietnam was made. Although some variability in morphometry among these populations was observed we conclude that these differences fall within the range of morphometrical variability described previously in M. incognita populations from other agricultural crops and parts of the world. Although canonical discriminant analysis enabled the clustering of the seven M. incognita populations collected from black pepper plants in Vietnam in three groups based on a combination of 10 morphological characters of the second-stage juveniles (J2), there was no relationship between these three groups and their geographic origin (agro-ecological region).

During 2004-2007, the population dynamics of M. incognita on the

black pepper variety Vinh Linh was studied during 1 year at two sites: Cam Lo situated in the North Central Coast and Buon Ma Thuot situated in the Central Highlands. Percentage of root galling was also determined. The major climatic difference between the two sites was that in Cam Lo the rainy season lasted 5 months only vs 7 months in Buon Ma Thuot. Interestingly, in both sites, the root population density of M. incognita followed a similar pattern: the highest nematode root population densities were reached at 2 (Buon Ma Thuot) to 3 (Cam Lo) months after the end of the rainy season. In both study sites, the root population densities of M. incognita decreased towards the end of the dry season and remained low during the rainy season. Based on this information, sampling of roots of black pepper plants to assess the nematode population densities should thus be done a few months after the end of the rainy season. Our results further indicate that root galling may not be a good and easy indicator for the assessment of the population densities of M. incognita since the “dynamics” of the percentage root galling did not always follow the nematode population dynamics.

During 2007, in total 82 soil and 82 root samples were collected

from black pepper plants in three districts in Quang Tri province in the southern part of the North Central Coast in Vietnam to study in the field the joint occurrence of root fungi and M. incognita. Percentage of root

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galling was also determined and the percentage of black pepper plants with yellow leaves in the fields surveyed noted. Nine fungal taxa were isolated. Fusarium solani was isolated from about one out of every four black pepper plants. The observations that F. solani was not isolated from the roots of black pepper plants that did not had yellow leaves and that the frequency of occurrence of F. solani increased with increasing percentage of plants with yellow leaves suggest that there may be a relationship between the frequency of occurrence of F. solani in the roots of black pepper plants and the incidence of yellow leaves. But, the observation that in the black pepper plant nurseries where F. solani was not isolated about one out of two black pepper plants examined had yellow leaves suggests that another pathogen must be the initial cause of the yellowing of the leaves of black pepper plants and that F. solani may play a role in this disease in a later stage of the development of the plants. Furthermore, F. solani was not isolated from the roots of black pepper plants growing in nurseries (and younger than 3 years) and black pepper plants younger than 5 years growing in plantations. This indicates that the nurseries examined during our survey were either not infested with F. solani or that F. solani only infects older roots of black pepper plants. This aspect of the infection cycle of F. solani on black pepper plants should be investigated further. We also observed that relatively low population densities of M. incognita are can be associated with a high percentage of black pepper plants with yellow leaves which confirms the high pathogenicity of this nematode species on black pepper plants as reported in the literature.

The high pathogenicity of M. incognita on the growth of black

pepper plants was also observed during a greenhouse experiment. Inoculation with 2,000 eggs and J2 of M. incognita per plant resulted at 9 months after inoculation in significantly less number of leaves and branches, and a significant reduction in fresh shoot and root weights compared with the uninoculated control plants. The results of the greenhouse experiment also showed that infection with M. incognita alone could result in a high percentage of black pepper plants with yellow leaves thus confirming our observations made in the nurseries in which F. solani was absent. Based on our results we cannot conclude that infection with F. solani together with M. incognita had a synergistic effect on the percentage of black pepper plants with yellow leaves. Further research during which also the colonisation of the roots of the black pepper plants

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by F. solani is quantified is necessary to clarify the interaction between M. incognita and F. solani.

The host response to M. incognita reproduction and damage of five

black pepper varieties commonly grown in Vietnam was evaluated in a greenhouse experiment. All five varieties were susceptible to this nematode species. However, our results suggest that the variety La Da might be highly sensitive while the variety Loc Ninh might be less sensitive (even tolerant) to M. incognita damage. To confirm these preliminary observations, the host plant response to M. incognita damage of these varieties should be further investigated. If our observations are confirmed this may lead to advise the black pepper farmers in Vietnam not to plant the variety La Da in fields heavily infested with M. incognita but to plant the variety Loc Ninh in stead.

One of the general objectives of our study (as formulated in

Chapter 1) was to elucidate the incidence of plant-parasitic nematodes on black pepper plants in Vietnam and their relationship with the incidence of soil-borne fungi and yellowing of the leaves of black pepper plants. Although our study has certainly contribute to this objective it has also become clear that the relationship between the incidence of, in particular, M. incognita and F. solani on black peppers plants and the yellow leaves syndrome is very complex and in need of further research. We think that our study has pointed out, at least, some aspects of this complex interaction which need to be investigated in more depth.

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ANNEX

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ANNEX 1: LIST OF PUBLICATIONS Nguyet, D.T.M., Thuy, T.T.T., Tuyet, N.T., Tu, D.M., Yen, N.T. and Nhi,

H.H. 2003. Occurrence of Pratylenchus coffeae and occurrence, damage and reproduction of Radopholus similis in the Northern and Central Highlands of Vietnam. In: Proceedings of the training workshop towards management of Musa nematodes in Asia and the Pacific (F.S. dela Cruz Jr, I. Van den Bergh, D. De Waele, D.M. Hautea and A.B. Molina, eds). University of Philippines Los Banos, Laguna, Philippines, 1-5 December 2003: 65-77.

Elsen, A., Ferrandis Vallterra, S., Van Wauwe, T., Thuy, T.T.T., Swennen,

R., De Waele, D. and Panis, B. 2007. Cryopreservation of Radopholus similis, a tropical plant-parasitic nematode. Cryobiology 55:148-157.

Thuy, T.T.T., Yen, N.T., Tuyet, N.T.A., Te, L.L. and De Waele D. In print.

Plant parasitic nematodes associated with black pepper plants in Vietnam. Russian Journal of Nematology.

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ANNEX 2: LIST OF ABBREVIATIONS

CABI Commonwealth Agricultural Bureau International

HUA Hanoi University of Agriculture

IAS Institute of Agricultural Science for Southern Vietnam

IMHEN Institute of Meteorology, Hydrology and Environment

IPC International Pepper Community

MARD Ministry of Agriculture and Rural Development

PPD Plant Protection Department

PPRI Plant Protection Research Institute

VPA Vietnam Pepper Association

WASI Western Highlands Agro-Forestry Scientific and Technical Institute

incidence and effect of meloidogyne incognita (nematoda … · incognita (nematoda: meloidogyninae)...

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