Root endophytic fungi of tomato and their role as biocontrol agents of root-knot nematodes Meloidogyne incognita (Kofoid and white) Chitwood and growth promotion in tomato plants (Lycopersicon esculentum Mill):

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I declare that this thesis titled “Root Endophytic Fungi of Tomato and Their Role as

Biocontrol Agents of Root-knot Nematodes Meloidogyne incognita (Kofoid and White)

Chitwood and Growth Promotion in Tomato Plants Lycopersicon

esculentum (Mill)” was

entirely completed by myself with resourceful help from the Department of Plant

Protection, Bogor Agricultural University. Information and quotes which were sourced

from journals and books have been acknowledged and mentioned where they appear in this

thesis, all complete references are given at the end of the paper.

Bogor, April 2010

Bruce Ochieng’ Obura

Reg. No. A352088061

BRUCE OCHIENG’ OBURA. Cendawan Endofit Asal Akar Tanaman Tomat dan

Peranannya Sebagai Agen Biokontrol Terhadap Nematoda Puru Akar

Meloidogyne

incognita

(Kofoid and White) Chitwood

serta Pemacu Pertumbuhan pada Tanaman

Tomat (

Lycopersicon

esculentum

Mill). Dibimbing oleh SUPRAMANA dan SURYO

WIYONO

Nematoda puru akar, (Meloidogyne

incognita) adalah salah satu OPT utama tomat

(Lycopersicon esculentum Mill) di seluruh dunia. Tujuan penelitian ini adalah untuk (1)

mengeksplorasi cendawan endofit (2) melihat pengaruh cendawan endofit terhadap

nematoda puru akar (Meloidogyne incognita) serta pemacu pertumbuhan pada tanaman

tomat. (3) menginvestigasi mekanisme cendawan endofit menekan nematoda puru akar

secara in-vitro.

Pada penelitian ini, pengaruh 12 isolat cendawan endofit asal akar tomat yaitu

Nigrospora sp, isolate XP9, Fusarium

oxysporum,

Fusarium

chlamydosporum,

Chrysosporium sp, Trichoderma

hamatum, Trichoderma pseudokoningii, Sterile black 1,

Torula sp, Sterile black 2, Ulocladium sp dan Fusarium sp 3 terhadap preferensi inang oleh

Meloidogyne

incognita serta pengaruh terhadap pemacu pertumbuhan tanaman tomat

dilaksanakan di Laboratorium Nematoda Departemen Proteksi Tanaman Fakultas

Pertanian, Institut Pertanian Bogor (IPB), dan rumah kaca IPB Cikabayan Bogor.

Percobaan ini dilaksanakan mulai dari bulan Februari sampai Agustus 2009.

Hasil penelitian mengindikasikan bahwa dari 12 isolat cendawan endofit yang diuji, 9

isolat yaitu isolate XP9, Nigrospora sp, Chrysosporium sp, Fusarium oxysporum, Fusarium

chlamydosporum,

Trichoderma hamatum,

Trichoderma pseudokoningii, Sterile black 1,

Sterile black 2 berpotensi untuk menekan nematoda puru akar, baik pada penelitian secara

in vivo maupun in vitro dan juga berpotensi untuk meningkatkan pertumbuhan tanaman

tomat.

Kata kata kunci: Tomat, nematoda puru akar, Meloidogyne incognita, cendawan

endofit, biokontrol

BRUCE OCHIENG’ OBURA. Root Endophytic Fungi of Tomato and Their Role as

Biocontrol Agents of Root-knot Nematodes

Meloidogyne

incognita

(Kofoid and White)

Chitwood

and Growth Promotion in Tomato Plants (

Lycopersicon

esculentum

Mill).

Supervised by SUPRAMANA and SURYO WIYONO

Root knot nematode, (Meloidogyne

incognita) is a major constraint to tomato

(Lycopersicon esculentum Mill) production in the whole world. The aim of this research

was (1) exploration of endophytic fungi from highland and lowland areas in healthy and

nematode infected tomato plants, (2) to assess the potential of endophytic fungi of tomato

to suppress of root knot nematodes as well as in growth promotion of tomato plants (3) ) to

investigate the mechanisms by which endophytic fungi suppress root knot nematodes in

vitro

In this research, the effect of 12 tomato root endophytic fungi isolates that is

Nigrospora sp, isolate XP9, Fusarium

oxysporum,

Fusarium

chlamydosporum,

Chrysosporium sp, Trichoderma

hamatum, Trichoderma pseudokoningii, Sterile black 1,

Torula sp, Sterile black 2, Ulocladium sp dan Fusarium sp 3 against host preference by root

knot nematode (Meloidogyne incognita) as well as growth promotion in tomato plants. This

research was conducted in Nematology Laboratory, Department of Plant Protection,

Faculty of Agriculture, Bogor Agricultural University, and in the greenhouse at the

University Farm in Cikabayan Bogor from February to August 2009.

The result of this research indicated that from the 12 isolates of endophytic fungi

tested, 9 isolates that is isolate XP9, Nigrospora

sp, Chrysosporium sp,

Fusarium

oxysporum, Fusarium chlamydosporum,

Trichoderma hamatum,

Trichoderma

pseudokoningii, Sterile black 1, Sterile black 2 had the potential to reduce root knot

nematode both in vivo and in vitro, and they also had the potential to increase growth of the

tomato plants

Keywords: Tomato plants, root-knot nematode, Meloidogyne incognita, root

endophytic fungi, biocontrol

BRUCE OCHIENG’ OBURA. A35208861. Root endophytic fungi of tomato and their

role as biocontrol agents of root-knot nematodes

Meloidogyne

incognita

(Kofoid and

White) Chitwood

and growth promotion in tomato plants (

Lycopersicon

esculentum

Mill). Supervised by SUPRAMANA and SURYO WIYONO

Biological control is an environmentally friendly way of controlling plant pests and

diseases. Biological control of root-knot nematodes using endophytic fungi has been

conducted in many studies, hence arises the need to research on the potential of endophytic

fungi against root-knot nematodes in tomato plants. Endophytic fungi appear to be

ubiquitous in healthy plant tissues and much evidence suggests that endophytes of some

plants help hosts tolerate adverse abiotic and biotic factors including pathogens, this

suggests the use of endophytes as biocontrol agents. This study describe issues regarding

endophytes associated with tomato plants, with dual goals of how they suppress pathogens

as well as promoting plant growth performance, understanding the abundance and diversity

of endophytes associated with this host, and of assessing those endophytes for use in

biocontrol.

This study was divided into three major sections (1) exploration of endophytic fungi

from healthy and nematode infected tomato plants in highland areas (Puncak) and lowland

areas (Tegallega Central Bogor Indonesia) which resulted to a total of 12 potential isolates

of endophytic fungi.

(2)

in vivo trials were conducted to assess the effect of endophytic

fungi treatments on suppression of root knot nematodes as well as growth promotion of

tomato plants and 9 out of the 12 endophytic fungi isolates used that is: isolate XP9,

Nigrospora

sp, Chrysosporium sp,

Fusarium oxysporum, Fusarium chlamydosporum,

Trichoderma hamatum,

Trichoderma pseudokoningii, Sterile black 1, Sterile black 2

showed significant effect in suppression of root knot nematodes and egg mass formation as

well as growth promotion in tomato plants (3) in vitro trials were conducted to assess the

antagonistic mechanisms of endophytic fungi isolates against juveniles of the root-knot

nematodes and 9 out of the 12 endophytic fungi isolates used that is: isolate XP9,

Nigrospora

sp, Chrysosporium sp,

Fusarium oxysporum, Fusarium chlamydosporum,

Trichoderma hamatum,

Trichoderma pseudokoningii, Sterile black 1, Sterile black 2

showed significant antagonistic effect against root-knot nematode juveniles.

Keywords: Tomato plants, root-knot nematode, Meloidogyne incognita, root

endophytic fungi

© Copyright of Bogor Agricultural University, year 2010

Copy right reserved

1.

No part or whole of this thesis may be excerpted without inclusion or

mentioning the sources.

a.

Excerption only for research and educational use, writing for

scientific papers, reporting, critical writing or reviewing of a

problem.

b.

Excerption does not inflict a financial loss in the proper interest of

Bogor Agricultural University.

2.

No part or all of this thesis may be transmitted or reproduced in any form

without a written permission from Bogor Agricultural University.

BIOCONTROL AGENTS OF ROOT-KNOT NEMATODES

Meloidogyne incognita (Kofoid and White) Chitwood AND GROWTH

PROMOTION IN TOMATO PLANTS (Lycopersicon esculentum Mill)

BRUCE OCHIENG OBURA

Thesis

Submitted in partial fulfillment of

Master of Science

Major Entomology/Phytopathology

GRADUATE SCHOOL

BOGOR AGRICULTURAL UNIVERSITY

BOGOR

White) Chitwood and Growth Promotion in Tomato Plants

(Lycopersicon esculentum Mill).

Name : Bruce Ochieng’ Obura

Registration Number: A352088061

Dr. Ir. Supramana. M.Si

Chairman

Coordinator of Major Phytopathology

Dr.Ir. Sri Hendrastuti Hidayat, M.Sc

Examination Date: 7 April 2010

Dr. Ir. Suryo Wiyono. M.Sc. Agr

Member

Dean of Graduate School

Approved

Approved

Advisory Committee

Prof. Dr.Ir. Khairil Anwar Notodiputro, M.S

Thanks be to Almighty God for guiding, strengthening and for His endless blessings

that has seen this research work entitled “Root Endophytic Fungi of Tomato Plants and

Their Role as Biocontrol Agents of Root-knot Nematodes Meloidogyne incognita (Kofoid

and White) Chitwood and Growth Promotion in Tomato Plants (Lycopersicon esculentum

Mill)” completed.

Sincere thanks goes to my research advisory committee Dr. Ir. Supramana, MSi, and

Dr. Ir. Suryo Wiyono, MSc. Agr, who accorded me invaluable guidance and direction in

conducting this research despite their committed time schedule. Lots of thanks also goes to

my external thesis examiner Dr. Ir. Abdul Munif, MSc. Agr. for the beneficial suggestions

and comments given that shaped the outlook of this thesis.

Special thanks to the Department of Plant Protection for the full support given that

enabled successful completion of this research. Extended thanks goes to Mr. Gatot the

Nematology Laboratory assistant and Ms. Ita of Plant Clinic for all the assistance they

accorded me during the research period, not to forget all the energetic and invaluable

lecturing staff members in the Department of Plant Protection who have in one way or

another imparted knowledge that I acknowledge with gratitude.

Lots of thanks to my sponsor KNB (Kemitraan Negara Berkembang) who provided

for my scholarship throughout my masters course. This work would have not reached this

end without the support from KNB.

Further, heartfelt gratitude and thanks goes to my dad, mum, brothers and sisters

Geoffrey, Ismael, Ibrahim, Judith, Afya, Effy and Mercy for all love, care, guidance,

assistance, support, prayers and endless support kindly given during the course of the

research. Special thanks to Uncle John Ong’any Opiyo and his family for all the endless

support, love, prayers kindly given during the course of the research.

I’m very much indebted to appreciate the contributions of Mr. Francis Wanaswa,

Mrs. Sally Wanaswa, Mrs. Christine Kisenga and Mrs. Catherine who contributed

immensely in shaping the outlook of this work as they always provided for academic,

social, moral and psychological support, they always stood by me during the difficult times

of academic work.

Acknowledgement

also

goes

to

my

fellow

colleagues

in

Entomology/Phytopathology major and the KNB family, for the support and assistance

during the course of Masters Degree study at Bogor Agricultural University. Special

mention of close friends Ir. Netti Tinaprilla. MMA, Joseph Obado, Nurjanah, Weni Willia,

Rika Meliansyah, Donnarina Simanjuntak, Eva Dwi, Heri Harti, Wartono, Linda Henuk,

TriMaryono, Wawan, Fitrianiangrum Kurniawati, Nildayanti, Pras, Aceu, Diana, Apri, Peni

Lestari, Wage, Nurul, Afdhol, Shoni and Kiki, for the contributions, support and advices

provided that contributed towards the production of this work.

family members, friends and fellow students, may this work help us turn the world into a

better place than we found it. May God bless us all.

Bogor , April 2010

The writer, Bruce Ochieng Obura was born on the 31

st

of December, 1979 in Kenya

to Mr Joseph Obura and The Late Mrs. Risper Achieng from Kenya. In 1994, the writer

Completed Primary School from Ezra Gumbe Primary School and continued secondary

education at Nyabondo High school in Nyando district Kenya, after which he was admitted

to Jomo Kenyatta University of Agriculture and Technology where he graduated with

Bachelors of Science Degree studies in Horticulture on 27

th

July 2006. After Bachelors

degree level, the writer was directly employed by the Department of Plant Protection in

Kenya where he was in charge of production of Phytoseiulus persimilis a predaceous mite

for biological control of red spider mites on vegetables and ornamental crops, he also

worked for Bayer Crop Science as a researcher on tropical pests and diseases . In 2007,

Bruce Ochieng’ Obura was accepted for scholarship under the Developing countries

partnership program KNB (Kemitraan Negara Berkembang) awarded by the Indonesian

Government to take Masters Degree in Phytopathology at Bogor Agricultural University.

TABLE OF CONTENTS

Page

LIST OF TABLES ... xi

LIST OF FIGURES ... xii

LIST OF APPENDICES ... xiii

I. INTRODUCTION ... 1

1.1 Background ... 1

1.2 Research Objectives ... 3

1.3 Hypothesis ... 3

1.4 Research Benefits ... 3

II. LITERATURE REVIEW ... 5

2.1 Root-knot Nematode ... 5

2.2 Mechanisms of Root Infection by Root-knot Nematodes ... 6

2.3 Taxonomy of Root-knot Nematode (Meloidogyne incognita) ... 6

2.4 Morphology of Root-knot Nematode (Meloidogyne incognita) ... 7

2.4.1 Larva ... 7

2.4.2 Male adult ... 7

2.4.3 Female adult ... 7

2.5 Life-cycle of Root-knot Nematode ... 7

2.6 Anatomy of Root-knot Nematodes ... 9

2.7 Factors Influencing Development of Root-knot Nematodes ... 9

2.7.1 Temperature ... 9

2.7.2 Host suitability ... 10

2.7.3 Soil moisture ... 10

2.7.4 Nutrition availability ... 10

2.8 Root-knot Nematodes as Pest of Tomato Plants ... 11

2.9 Symptoms of Root-knot Nematodes in Tomato Plants ... 12

2.9.1 Above ground symptoms ... 12

2.9.2 Underground symptoms ... 12

Page

2.10 Possibility of Biocontrol by Endophytic Fungi ... 15

2.11 Plant Tissue Colonisation Process by Endophytic Fungi ... 18

2.12 Interaction between Endophytic Fungi and Plant Parasitic Nematodes ... 20

2.13 Antagonistic mechanisms of Endophytic Fungi Against RKN... 20

2.13.1 Antibiosis ... 20

2.13.2 Change in host physiology ... 21

2.13.3 Induced resistance ... 22

2.13.4 Competition ... 22

III. MATERIALS AND METHODS ... 23

3.1 Time and Location of Study ... 23

3.2 Exploration of Endophytic Fungi ... 23

3.2.1 Isolation and identification of endophytic fungi ... 23

3.2.2 Selection of endophytic fungi based on pathogenicity test .... 23

3.2.3 Inoculation of seeds with suspension spores ... 24

3.2.4 Re-inoculation of tomato plants with suspension spores ... 25

3.3 Colonisation Test ... 25

3.4 Meloidogyne incognita Egg Mass Inoculation ... 26

3.4.1 Root-knot nematode extraction and inoculation ... 26

3.4.2 Plant management practices ... 27

3.5 Antibiosis In vitro Test ... 27

3.6 Parameter Observation and Data Analysis ... 28

3.6.1 Assessment of damage in tomato plant roots by RKN ... 28

3.6.2 Assessment of plant growth parameters ... 28

3.6.3 Experimental design and data analysis ... 28

IV. RESULTS AND DISCUSSION ... 30

4.1 Results ... 30

4.1.1 Exploration of Endophytic Fungi ... 30

4.1.1.1 Isolation of endophytic fungi ... 30

4.1.1.2 Pathogenicity test ... 31

Page 4.1.3 Antagonistic Effect of Endophytic Fungi against RKN

in planta ... 32

4.1.4 Effect of Endophytic Fungi on Plant Growth ... 33

4.1.4.1 Effect on height and stem diameter of RKN inoculated plants ... 33

4.1.4.2 Effect on plant fresh and dry weight of RKN inoculated plants ... 36

4.1.4.3 Effect on number of fruit and root length of RKN inoculated plants ... 37

4.1.4.4 Effect on height and stem diameter of RKN free plants ... 38

4.1.4.5 Effect on plant fresh and dry weight of RKN free plants ... 41

4.1.4.6 Effect on number of fruit and root length of RKN free plants ... 41

4.1.5 Antibiosis In vitro Test ... 42

4.2 Discussion ... 48

4.2.1 Exploration of Endophytic Fungi ... 48

4.2.2 Colonisation Test ... 49

4.2.3 Antagonistic Effect of Endophytic Fungi against RKN In planta ... 50

4.2.4 Effect of Endophytic Fungi on Plant Growth ... 51

4.2.5 Antibiosis In vitro Test ... 52

4.2.6 Correlation Pattern between In vitro Test and In planta Tests ... 53

V. CONCLUSION AND RECOMMENDATIONS ... 54

5.1 Conclusion ... 54

5.2 Recommendation ... 54

VI. LIST OF REFERENCES ... 55

LIST OF TABLES

Page 1. Endophytic fungi isolated from healthy and nematode infected

roots ... 30

2. Colonisation ability of endophytic fungi... 32

3. Effect of endophytic fungi on number of root galls and egg masses ... 33

4. Effect of endophytic fungi on plant height of RKN inoculated plants .. 34

5. Effect of endophytic fungi on stem diameter of RKN inoculated plants 35

6. Effect of endophytic fungi on plant fresh and dry weight ... 36

7. Effect of endophytic fungi on number of fruits and root length ... 37

8. Effect of endophytic fungi on plant height of RKN free plants ... 39

9. Effect of endophytic fungi on stem diameter of RKN free plants ... 40

10. Effect of endophytic fungi on fresh and dry weight ... 41

11. Effect of endophytic fungi number of fruits and root length ... 42

12. Effectiveness of different culture filtrate concentration and time of exposure on juvenile mortality rate ... 46

LIST OF FIGURES

Page 1. Flow chart diagram on the steps followed in this research ... 4

2. Illustration of lifecycle of root-knot nematodes Meloidogyne incognita in

tomato plant roots ... 8

3. Light micrograph of stained endophytic mycelium inside plant tissues showing intercellular colonization of plant tissues by the fungal

endophytes ... 19

4. Pathogenicity test based on germination of tomato seeds on pure

isolates of endophytic fungi and on PDA medium as control ... 31

5. Colonisation test ... 32

6. RKN juvenile percentage mortality rate in different culture

filtrate concentrations ... 45

7. General correlation showing effect of endophytic fungi treatments on reduction of root gall and egg mass formation in comparison to juvenile mortality rate at 30% culture filtrate concentration after

24 hours ... 53

LIST OF APPENDICES

Page 1. Comparison between percentage root colonization rate, percentage

root gall reduction and egg mass reduction by endophytic fungi ... 68 2. Photos showing effect of endophytic fungi on root-knot nematodes in

tomato plants ... 69 3. Photos showing effect of RKN on negative control (endophytic fungi free) and positive control treatments (treated with carbofuran) ... 71 4. Photos shwing effect of endophytic fungi on tomato plants four weeks after re-inoculation ... 72 5. Photos showing effects of endophytic fungi on tomato plant roots four weeks after re-inoculation ... 74 6. Photos showing effect of endophytic fungi on tomato plant roots... 75 7. Photo showing roots of control treatments (without endophytic fungi) .. 77 8. Macroscopic photos of endophytic fungi colony on PDA media

and microscopic photos (Magnifications ×40) ... 78 9. Root-knot nematode attached egg mass perineal pattern of

Meloidogyneincognita and pear shaped adult female root-knot

nematode ... 81

I.

INTRODUCTION

1.1 Background

Plant parasitic nematodes cause significant damage and losses to most agricultural crops in the tropics and subtropics (Luc et al. 2005). The need to control and manage nematode population to acceptable levels remains a big concern for nematologists. The need to reduce dependent on chemical control using nematicides and the increased pressure to use pest control measures that do not pollute or degrade the environment has provided the impetus for more research geared towards the search and exploitation of potential biological control agents of plant parasitic nematodes (Cook 1988). Biological control involves the reduction of inoculum potential of a disease causing pathogen or parasite in its active or dormant state by one or more organisms accomplished naturally or by manipulation of environment, host or antagonists or by mass introduction of one or more antagonists (Baker & Cook 1974). Stirling (1991) defined biological control of nematodes as “the reduction of nematode population through the action of living organisms other than the nematode resistant host plant, and which occurs naturally, or through manipulation of the environment or manipulation of antagonists.”

Nematodes have long been known to have numerous antagonists (Kerry 1987). Several organisms have been described and exploited for the management of plant parasitic nematodes in agricultural crops. A large number of organisms including fungi, bacteria, viruses, predatory nematodes, insects and mites have been found to parasitize on the vermiform stages of nematodes or female eggs of root-knot nematodes or cyst nematodes (Stirling 1991).

More recently the use of endophytic microorganisms resident within plant tissues for the protection of plants against pests and diseases has been exploited, the most studied is the grass endophyte association in which endophytic fungi associated with grasses have been shown to protect grasses against pest and diseases, most grass endophytes are members of the Ascomycetes family Clavicipitaceae (Clay 1991). Biological control with endophytes has mostly emphasized residents or mutualistic fungi of grasses which renders hosts

unpalatable to herbivores and insects (Clay 1988, 1989) detrimental effects of grass endophytes on fungal pathogens has also been demonstrated. For example isolates of Acremonium lolii Link ex Fries, and A. coenophialum Morgan-Jones and W. Gams showed antibiosis against a range of fungal plant pathogens in culture (White & Cole 1985). Research on grass endophytes has clearly demonstrated the nature and extent of protection afforded to the host plants by the interactions, with mutualistic associations between grasses and endophytic fungi benefiting the host plants in most circumstance (Clay 1990). In mutualistic association, endophyte-infected plants are protected from attack by some species of insects, nematodes and fungi while in return, the endophyte is provided with shelter and nutrition by the host plant (Latch 1993; Saikkonen et al. 1998; Schardl et al. 2004).

Although most reports on host plant infection by endophytes concern grass endophytes, symptomless infection of other plants by endophytic fungi belonging to diverse taxonomic groups have been known for many years (Carroll 1988). The presence of endophytes has been demonstrated in many plants, including important crops such as banana (Brown et al. 1998; Pereira et al. 1999; Cao et al. 2004a; Cao et al. 2004b; Cao et al. 2005), maize Zea mays L. (Fisher et al. 1992), rice Oryza sativa L. (Fisher & Petrini 1992), and tomato Lycopersicon esculentum Mill. (Hallmann & Sikora 1994c; Cao et al. 2004a). Some principle groups of root colonizing plant beneficial fungi, which have developed symbiotic relationship with the host plants belong to the Fusarium sp and Trichoderma sp (Haas & Defago 2005).

In this review the role of endophytic fungi in the management of plant parasitic nematodes as well as plant growth improvement in agricultural crops is discussed, since limited information is available on the use of endophytic fungi to control root-knot nematodes Meloidogyne incognita in tomatoes, this review focused on existing literature between endopyhtes and plant parasitic nematodes in grasses and other crops, highlighting the implication of plant infection by endophytic fungi, and discussed the beneficial effects of endophytic fungi in the management of plant parasitic nematodes as well as promoting plant growth.

1.2Research Objectives

1. Exploration of root endophytic fungi of tomato.

2. To obtain potential endophytic fungi of tomato that can reduce population of root- knot nematode and improve plant growth.

3. To investigate the mechanisms by which endophytic fungi suppress root-knot nematodes.

1.3Hypothesis

1. Treatment of tomato plants with endophytic fungi increases induced resistance of tomato plants against infection by root-knot nematodes. 2. Treatment of tomato plants with endophytic fungi increases growth

performance of tomato plants.

1.4 Research Benefits

Findings in this study are important from the point of view of environmental pollution likely to be caused while using chemical nematicides to control root-knot nematodes in tomato plants. The future prospects looks bright for identifying endophytic fungi to replace the synthetic dangerous and expensive chemicals used at present.

Figure 1. Flow chart diagram on the steps followed in this research EXPERIMENT 1

Exploration of endophytic -Isolation of endophytic fungi. -Identification

-Diversity index analysis -Similarity analysis -Pathogenicity test

EXPERIMENT 5

In vitro test to evaluate the effect of endophytic fungi culture filtrate

on root-knot nematode juvenile mortality rate.

 EXPERIMENT 2 Colonisation test

 EXPERIMENT 3 and 4 In planta test to evaluate the effect of

endophytic fungi on root-knot nematodes as well as their effect on

plant growth promotion.

 

II.

LITERATURE REVIEW

2.1 Root-knot Nematode

Root knot nematode had already been reported by 1885 to cause damage to various plant species, majorly in the tropical and subtropical regions. According to Chitwood (1949) root-knot nematodes consist of four main species based on the perineal morphology pattern of the female adult nematodes and other morphological characteristics, the four species are Meloidogyne javanica, M. arenaria, M. incognita, M. hapla. By the year 1988 as much as 61 species of Meloidogyne had been noted (Einsenback & Triantaphyllou 1991). The root-knot nematode forms the most important plant parasitic nematode with wide host range, that is around 2000 plant species (Agrios 2005) and most of these crops are cultivated crops (Jensen 1972). In Indonesia root-knot nematodes of Meloidogyne incognita has a wide distribution area with 45.4% prevalence and M. arenaria has 38.6% prevalence (Hadisoeganda 1989).

Root knot nematodes has been known as a disease of vegetable crops since 1855, when (Berkeley 1855) in England first described the disease on cucumber Cucumis sativus L. roots, (Eisenback & Triantaphyllou 1991). The causal organism was described as Heterodera radicicola. From 1884 to 1949, root knot nematodes were considered a single species in combination with cyst nematodes and referred to by a number of designations (Johnson & Fassuliotis 1984). Chitwood (1949) described morphological differences among populations, and re-assigned the root knot nematode to the genus Meloidogyne. At this time Meloidogyne incognita, M. arenaria, M. javanica, M. hapla and M. exiqua were recognized primarily on the basis off perineal pattern and other morphological characteristics.

Initially all root knot nematodes were considered to one extremely phylophagous species, Heterodera marioni until (Chitwood 1949) re-established the genus Meloidogyne, although 51 species of Meloidogyne have been described to date (Jepson 1987), four species are of particular economic importance to vegetable production, Meloidogyne incognita, Meloidogyne javanica,

  Meloidogyne arenaria, and Meloidogyne hapla. Out of the 1000 root knot population collected from 75 countries 52% were identified as Meloidogyne incognita, 30% as Meloidogyne javanica, 8% as Meloidogyne arenaria, 8% as Meloidogyne hapla and 2% as Meloidogyne exigua or other species (Taylor & Sasser 1978). M. incognita consists of four races; M. arenaria

has two races, M. javanica and M. hapla show no clearly defined races M. incognita, M. javanica M. arenaria and M. hapla have the widest host ranges.

M. incognita and M. javanica are commonly found in the tropics, while M. hapla is a species commonly found in the temperate regions and occasionally in the cooler upland tropics.

2.2 Mechanism of Infection by Root-knot Nematodes Meloidogyne incognita The direct mechanical injury inflicted by nematodes while feeding causes only slight damage to plants. Most of the damage is caused by secretion of saliva injected into the plant while the nematodes are feeding. The nematodes puncture cell walls using their stylet, inject saliva into the cells, withdraw cell contents, they remain sedentary at their feeding site for the whole of their life while feeding at the site.

The feeding process causes the affected plant cells to react, resulting to dead or devitalized root tips, lesion forming and tissue break down, swelling and gall formation, these are caused by the dissolution of the infected tissues by nematode enzymes which causes tissue disintegration and death of the cells, others are caused by abnormal cell enlargement (hypertrophy) by suppression of cell division, or by stimulation of cell division proceeding in a controlled manner and resulting in the formation of galls, or large number of lateral roots at or near the point of infection.

2.3 Taxonomy of Root-knot Nematode (Meloidogyne incognita)

Kingdom animalia, Phylum Nematoda, Class: Secernentea, Order Tylenchida, Family Heteroderidae, Genus Meloidogyne, Species: M. incognita (Franklin 1982).

  2.4 Morphology of Root-knot Nematode (Meloidogyne incognita)

Meloidogyne incognita like other a plant parasitic nematode has a colourless body that is cylindrical in shape (Wallace 1963). Adult female, adult male and the larva can be differentiated based on their body form.

2.4.1 Larva. First instar (L1) has a blunt tail and molts within the egg, the second instar larva (L2) is hatched and live freely in the soil and look for a host, according to (Walker 1975) the length of the (L2) is between 375-500 µm with a diameter of 12-15 µm. The third and fourth larval instas develop within the host plant tissues.

2.4.2 Male adult. Meloidogyne incognita adult males are stretched cylindrical and are threadlike with the length 1.2-1.5 mm (Agrios 2005). The male head is composed of head cap and head region provides many good diagnostic features. The head cap includes labial disk surrounded by lateral and medial lips, a centrally located prestoma leads to a slit like stoma, four sensory organs terminate on medial lips (cephalic sensilia), and the head region may or may not be set off from the remainder of the body.

2.4.3 Female adults. Name of the genus Meloidogyne originated from greek language with the meaning that literally means apple and female because the body form of the female nematode is apple or pear shaped, with the length of 0.40-1.30 mm and diameter of 0.27-0.75mm (Walker 1975; Agrios 2005) with the neck of 0.15-0.24 mm wide (Walker 1975), the name Meloidogyne was given for the first time by Goeldi in the year 1887 (Franklin 1982).

2.5 Lifecycle of the Root-knot Nematode

Root-knot nematodes display marked sexual dimorphism i.e. the females are pyriform or saccate, the males vermiform. These general differences in the body form between male and female become established during the post embryonic development of Meloidogyne incognita. The embryonic development results in the first stage juvenile which molts once in the egg and hatches as a second stage

  juvenile. This motile vermiform, infective stage migrates through the soil and enters roots of the suitable host plant, it moves through the plant tissue to a preferred feeding site and establishes a complex host parasite relationship with the plant. The second stage juvenile becomes sedentary and as it feeds on special nurse cells (giant cells), it undergoes more morphological changes, and become flask-shaped, without further feeding it molts three times into third and fourth stage juveniles and finally becomes an adult. Shortly after last molt the saccate adult female resumes feeding and continues to feed for the remainder of her life, during this post embryonic development, the reproductive system develops and grows into functional gonads, the sexes can be differentiated based on the number of gonads (females have two gonads; males only one gonad). The change in shape from saccate male juvenile to vermiform adult male takes place during the fourth juvenile stage. The adult male does not feed it will leave the root and move freely through the soil. Depending on type and mode of reproduction, of a particular species, amphimixis or parthenogenesis, males may search for females and mate or remain in the soil and finally die. Length of life cycle of root-knot nematodes is greatly influenced by temperature, for Meloidogyne incognita is about 29°C, the first adult females appear 13-15 days after root penetration. The lifespan of egg-producing females may extend from 2-3 months and they lay upto 2000 eggs, but that of males maybe shorter.

Figure 2. Lifecycle of the root-knot nematodes Meloidogyne incognita (Source; The American Phytopathological Society 2003)

  2.6 Anatomy of Root-knot Nematodes

The nematode body is more or less transparent; it is covered by a colourless cuticle that molts when the nematode goes through successive juvenile stages. The cuticle is produced by the hypodermis which consists of living cells and extends into the body cavity as four chords separating four bands of longitudinal muscles, the muscles enable the nematode to move.

The body cavity contains fluid through which circulation and respiration takes place, the digestive system is a hollow tube extending from the mouth through the esophagus, rectum and anus. Lips usually six in number, surrounds the mouth. Most plant parasitic nematodes have a hollow stylet or spear that they use to puncture holes in plant cells and through which to withdraw nutrients from the cell.

The reproductive system of the nematodes is well developed, females have one or two ovaries followed by an oviduct terminating in a vulva. The male reproductive structure is similar to that of the females, but there is a testis, seminal vesicle, and a terminus in a common opening with the intestine. A pair of protrusible and, copulatory spicules is also present in males. Reproduction in plant parasitic nematodes is through eggs and may be sexual or parthenogetic to the species that lack males.

2.7 Factors Influencing Development of Root knot Nematodes

Many factors limits the growth development of root-knot nematodes, however there are two major important factors that is temperature and host suitability (Chrystie 1959).

2.7.1 Temperature. Meloidogyne incognita is sedentary endoparasite and completes its lifecycle in 20-25 days within the root cortex at a temperature of 27°C (Agrios 2005). Between 27°C-30°C the development of female root-knot nematodes begins from infective larva up to egg hatching going on for 17 days, at temperature of 24°C, egg hatching goes for 31 days, the longest development takes place at around 15.4°C and it takes up to 54 days. However in environments

  with temperatures below 15.4°C and above 33.5°C the development in root-knot nematodes will fail to take place up to adult stage.

2.7.2 Host suitability. In suitable host plants, eggs produced by the Meloidogyne incognita are many, the more suitable the host plant the more eggs produced (Chrystie 1959). Occurrence of continuous infection is influenced by the host suitability. When the larva has already entered the non-suitable host tissues, in about 4-6 days this infective larva will leave that plant tissue and invade another plant, or stay in the latter plant tissues with its life development experiencing disturbance (Dropkin 1980).

2.7.3 Soil moisture. This will influence the development of the Meloidogyne incognita by determining the time taken for the start of egg hatching. Egg hatching will be impeded in dry conditions with low moisture levels (Chrystie 1959). Sufficient soil moisture content (in the field capacity), forms the best condition for the development of root-knot nematodes, but flooded soils also will have bad consequences, or even cause death. According to Dropkin (1980) best conditions for the development of root-knot nematodes is in the soils with little sand, and not good in clay soils.

Water availability will really determine the life process and at the same time it is an important media for movement of root-knot nematodes in the soil (Norton 1978). Low moisture conditions will influence mobility acceleration of Meloidogyne incognita but has not resulted to death, it only changes physiological mechanisms of root-knot nematodes. Soil moisture content best for the existence of the root-knot nematodes ranges between 40-60% from field capacity (Wallace 1963).

2.7.4 Nutrition availability: Total nutrient availability shows much influence in the population ratio between males and females. According to Norton (1978) host plants tissue that gives abundant nutrition leads to increased development of the larva to female while host plant tissue that gives less plant nutrition, leads to increased development of the larva mostly into males.

  Based on the experimental results it is known that giving of mineral nutrients to plants is influential to nematode development. Giving solutions of N, P and K to tomato and potato host plants increases the production of root-knot nematode eggs (Dropkin 1980). In plants with much nitrogen, nematode development also increases, however on the contrary in plants with less nitrogen availability development of Meloidogyne incognita is impeded (Dropkin 1980).

Existence of excess potassium like in cucumber shows increase in the development of Meloidogyne incognita although this case does not occur with M. hapla and M. javanica. In pea plant with excess potassium hatching of the first egg takes place on the 16th day after inoculation, very different from plants with less potassium, hatching of eggs takes place 40 days after inoculation (Chrystie 1959).

2.8 Root Knot Nematodes as Pests of Tomato Plants

The species of root knot nematodes found to be most detrimental to tomato plants are those involved in the destruction of primary roots, disrupting the anchorage system and divitalization of the root tips and eventually death of the

plant in severe cases. The most wide spread and important are Meloidogyne incognita, it is found worldwide in tropical and sub-tropical regions

and occurs wherever tomatoes are grown (Bridge & Gowen 1993). Areas where the nematode is known to occur on tomatoes include Africa, parts of Asia, Central and South America, Cuba, Australia and several countries in Southern Europe.

The root-knot nematode second stage juveniles are short (400-600µm) the

cephalic framework is weakly sclerotized, and has indistinct knobs. The esophageal gland lobe overlaps the intestine ventrally, and tail tapers to a

pointed tip with a clear terminus. The males of root-knot nematodes are 1.0 to 2.0 mm long, the stylet is about 18-24µm and has distinct knobs. The esophageal gland lobe overlaps the intestine ventrally. The tail is short and rounded and lacks bursa. The spicules of root-knot male nematodes open a short distance from the tail tip, unlike those of the cyst nematodes which opens near the terminus. The females of the Meloidogyne incognita are swollen and pear shaped, pearly white, and sedentary. They deposit all of their eggs in gelatinous mass that usually

  protrude from the galled root tissues, unlike cyst nematodes the female root-knot nematodes usually remain completely endoparasitic.

The species has a pronounced sexual dimorphism in which males are warm like vermiform and about 1.0 to 2.0 mm long by 30 to 36 micrometer in diameter (Agrios 2005). Each female lays approximately 2000 eggs in a gelatinous substance the first stage juveniles develop inside each egg, the second stage juvenile emerges from the egg into the soil and this is the only infective stage of the nematode, if it reaches a susceptible host the juvenile enters the roots become sedentary and grows thick like a sausage (Agrios 2005). Meloidogyne incognita is a sedentary endoparasite and completes its life cycle in 20-25 days within the root cortex at a temperature of 27°C (Agrios 2005). Females lay 20-30 eggs per day for a period of two weeks (Niere 2001). The eggs hatch in 8-10 days and the juvenile stages are completed in 10-13 days, the nematode cannot survive more than six months in soil deficient of the host (Ssango & Speijer 1997).

2.9 Symptoms of Root-knot Nematodes in Tomato Plant

Root-knot nematode infection of plants results in appearance of symptoms, typical symptoms of nematode injury can involve both above ground and below ground plant parts.

2.9.1 Above ground symptoms: Infected plants will shows inhibited growth (stunting), yellowing (chlorosis) of leaf, reduced yield, poor quality and quantity of crop products like the tomato fruits, premature leaf fall, erratic stands, wilting during the day.

2.9.2 Underground symptoms: Infected plants will show excessive branching of secondary roots, overall development of root galls, injured root tips and egg masses on the root surface, rough root surfaces with club appearance, infected roots are small and show necrosis.

Interactions involving fungal plant pathogens and plant parasitic nematodes

have been reviewed previously (Powell 1971a; Webster 1985; Mai & Abawi 1987; Rowe et al. 1987; Evans & Haydock 1993; Francle &

  Wheeler 1993). Interaction between Meloidogyne incognita and Fusarium wilt fungi have received special attention and were documented in 20 crop species. Interactions of these pathogens were especially obvious when the root knot nematode infection preceded those of the Fusarium wilt pathogens by 3 to 4 weeks. Majority of studies have established that the presence of root-knot nematodes increases the incidence and rate of development and severity of wilt or the mortality of the Fusarium-susceptible and tolerant crops. However the role of root-knot nematodes in the breakdown or alteration of the monogenic type of resistance to Fusarium wilt fungi (such as tomato cultivars with the dormant I-genes against F. oxysporum f.sp. lycopersici) remains controversial and requires further investigation (Mai & Abawi 1987).

Many example of disease complexes are known (Pitcher 1963; Powell 1971a; Powell 1971b; Taylor 1979; Webster 1985). Tomato plants wilt more quickly and can be killed when Fusarium oxysporium is simultaneously present along side with nematodes, resistance of tomato cultivars to fungal wilt caused by Fusarium oxysporum f.sp. lycopersici was reduced in the presence of Meloidogyne incognita (Jenkins & Coursen 1957; Sidhu & Webster 1977). Damage to the root system caused by root knot nematode attack has been considered responsible for the increase in the intensity of bacterial wilt caused by Pseudomonus solanacearum (Valdez 1978) and bacterial canker caused by Corynebacterium michiganense (Moura et al. 1975). Several of the viruses that are transmitted by the nematodes cause significant economic losses on major food crops such as tomato and tobacco ring spot virus. Meloidogyne incognita race 1

was shown to increase wilt caused by both R. solanacearum and F. oxysporum f.sp. lycopersici on resistant tomato cultivars when inoculated

simultaneously (Chindo et al. 1991). Van Gundy et al. (1977) demonstrated that leaching of nematode infected plants applied to tomato inoculated with Rhizoctonia sp resulted in the appearance of severe rots. The presence of root-knot nematodes play a major role in increasing the incidence and severity of bacterial wilt diseases caused by Pseudomonas solanacearum on various crops including tomato, potato, egg plants and tobacco.

  Yield loss in tomatoes due to root-knot nematodes in the world has been

estimated to be approximately $ 100 billion world wide annually (Sasser & Freckman 1987). The root knot nematodes have a worldwide

distribution but are more abundant in warm temperate and tropical soils. Losses due to Meloidogyne incognita in tomatoes can be as high as 50% (Niere 2001). In addition to the direct crop damage caused by the nematodes, many nematode species have also been shown to predispose plants to various infections by fungal or bacterial pathogens, or to transmit virus diseases.

Several control methods are available for the control of the tomato root-knot nematodes. The most important cultural control method is use of clean planting materials, only seedlings with roots free of galls should be selected for transplanting, the nurseries should also be free from root knot nematodes and seed beds should be selected on sites where previously there were no host plants. Crop rotation reduces the impact of root knot nematodes in tropical region and it’s the main management strategy to regulate the population of nematodes but its success is often limited because of the wide host range of most root knot nematodes species and the frequent occurrence of infestation composed of more than one species (Sikora et al. 1988), galled roots remaining in the field after harvest should be eliminated by uprooting and destruction, other control practices include bare fallowing and flooding.

Nematicides are widely used by growers producing fruits for export trade, a number of organophosphates and carbamates are used. However, their use is often prohibitive for many resources poor small scale farmers, registered products are highly toxic, expertise is required for application and most of them have been phased out of the market. The pesticide usually inactivates the nematode within the plant tissue or in the soil, which after microbial degradation the nematode recovers and damage continues (Sikora & Pocasangre 2004).

Possible agents for biological control of root knot nematodes are fungal antagonists that include nematode trapping or predacious fungi, endoparasitic fungi, parasites of nematode eggs, and fungi that produce enzymes and metabolites toxic to the nematode (Coosemans 1993). Research on way of exploiting these agents on field scale has yielded little success. Development of

  root knot nematode resistant cultivars would substitute the toxic nematicides currently in use and permit cultivation where farmers could not previously afford nematode control (Sikora et al. 2003). Research on the control of root knot nematodes suggests that no single control strategy will provide complete control (Sikora et al. 2003). A broad integrated pest management (IPM) approach including new components of pest control is necessary to safe guard sustainable tomato production. The biological enhancement of tomato plants with mutualistic fungal endophytes is a new approach that seems as a strong option for sustainable and ecologically sound nematode control. Inoculation of tomato plants with endophytes has resulted in reduced nematode reproduction, numbers and damage in pot experiments by more than 30% over controls (Sikora et al. 2003). Conducted research on ability of the fungal endophytes to persist in the tissues of inoculated plants and the interactions between the fungal endophytes and tomato plants has yielded conclusive results (Paparu et al. 2004; Niere et al. 1999).

2.10 Possibility of Biocontrol by Endophytic Fungi

Several definitions of endophytism have been proposed (Carroll 1988; Clay 1990), for the purpose of this research, the term endophyte

refers to fungi or bacteria, which for all or part of their life cycle invade and live inside tissues of living plants without causing any disease symptom or any apparent injury to the host (Petrini 1991; Wilson 1995), while epiphytes are bacteria or fungi that colonize plant surface tissues, in contrast to the epiphytes, endophytes are contained entirely within plant tissues, are asymptomatic and may be described as mutualistic (Clay 1990), fungi associated with root rhizospheres of the plants are called plant growth promoting fungi (PGPF). Some of the important PGPF belong to the genus Trichoderma and Gliocladium and the arbuscular mycorrhizal fungi (AMF), which form symbiotic association with plant

roots and are also capable of colonizing the roots of their hosts (Gera Hol & Cook 2005).

Estimated yield loss due to plant parasitic nematodes range from 20-80% in tomato production systems (Swennen & Vulysteke 2001). The control methods for root knot nematodes are not completely effective in subsistence farming

  system (Niere 2001). The strategic use of naturally occurring antagonistic organisms to control pest population and increase crop production represents a viable option (Marshall et al. 1999). Endophytic fungi are potentially effective

biological control agents for plant parasitic nematodes management (Niere et al. 1999; Sikora et al. 2003). A wide diversity of endophytic fungi has

been isolated from healthy tomato tissues with majority of isolates being from the genus non pathogenic Fusarium sp (Niere 2001; Pocasangre et al. 2000).

Culture filtrates for a number of isolates of non-pathogenic Fusarium oxysporum screened for in vitro activity against the root knot nematodes have shown high nematicidal activity causing mortality rates of 82-100%. Production of secondary metabolites by endophytic fungi is thought to be one of the mechanisms leading to plant pest control, these has been shown for grass endophytes but has not been elucidated for endophytes of crop plants (Niere et al. 2002).

The use of endophytes for control of plant parasitic nematodes is relatively a new approach. Since endophytes spend most of their life cycle inside plant tissues they are less exposed to the environment factors, hence they don’t entirely

depend on the environment for multiplication and survival (Siddiqui & Shaukat 2003a). Endophytes occupy a similar niche as pests and thus

are in close contact with the pest which make them an edge over other biological control agents (Hallmann et al. 1996b; Hallmann et al. 1997b). Inside the plant tissues the host plant provides relatively uniform and protected environment enabling the endophytes to avoid microbial competition and extreme environmental conditions such as fluctuations of temperature and moisture (Ramamoorthy et al. 2001).

The endophytic fungi are easy to culture in vitro and can be applied as seed treatments or on transplants, reducing the inoculums levels required (Sikora 1992; Sikora & Schuster 1999). Another advantage is that once developed, farmers will not need to apply the control products themselves as this may be done by public or private organizations engaged in commercial tissue culture production. Also fungal endophytes can easily be inoculated into tomato plants hence leading to production of naturally occurring biocontrol into one

  strategy. The use of endophytic fungi from both environmental and economic point of view has a major advantage over other biological control agents that are applied directly to the soil. The latter, due to the high levels of inoculums is needed to treat the soil, are more costly, have to be applied more frequently, and their efficacy is often strongly influenced by environmental factors. Another advantage is that endophytic fungi live in plant tissue, thereby reducing the risk of side effects on non-target organisms (Niere et al. 2002). Once the endophytic fungi has established and colonized the plant tissues they can be used as biocontrol agents potential for controlling the root knot nematode in the tomato plants.

In spite of these advantages of endophytes over other biological control agents, the potential of fungal endophytes in pest and disease management in crops remains largely unexplored. Mutualistic endophytic fungi (MEF) can therefore be defined as fungus that live some time in their lifecycle in a plant tissues without producing symptoms of a disease, but simultaneously demonstrate antagonistic activity towards one or more pest or disease affecting the root system (Sikora et al. 2003). It is assumed that mutualistic endophytes have evolved from plant pathogenic fungi and that most if not all higher plants host endophytic fungi (Isaac 1992). Majority of endophytic species which have been successfully identified are Ascomycetes, Deutromycetes with few Basidiomycetes and Oomycetes (Isaac 1992). Among the best studied endophytes are intercellular symbionts in the family Clavicipitaceae found in many cool season grasses which are known to benefit the host with improved tolerance to heavy metals, increased drought resistance, systemic resistance to pests and pathogens and enhanced growth (Arnold et al. 2003).

Endophytes are known to confer resistance to their host against pathogens through a number of mechanisms that include competitive exclusion, parasitism, metabolites production and induced resistance. Due to this, they are potential pest control tools and scientists are using beneficial endophytes as biological control agents against crop pest such as nematodes, borers and plant pathogenic fungi (IITA 1998). Their presence has been proven in all plants investigated such as rice, maize, tomato and banana (Niere et al. 2002).

  Mutualistic endophytic fungi have been shown to biologically control root knot nematodes of tomatoes (IITA 1998). The root knot nematodes attack tomato plants through the roots, therefore biological enhancement of the tomato plant using mutualistic fungal endophytes will increase plant resistance to infection (Sikora & Pocasangre 2004). Endophytes are well adapted to the life inside the plant and share the same ecological niche with endoparasitic nematodes, thus they

are effective at the exact site of the pest or disease attack (Sikora & Pocasangre 2004).

2.11 Plant Tissue Colonization Process by Endophytic Fungi

The process of colonization of plant tissues by endophytic fungi are complex and include host recognition, spore germination, penetration and colonization. Endophytes penetrate their host plants through natural openings or

wounds or actively using hydrolytic cellulases and pectinases (Hallmann et al. 1997b), forming inconspicuous infection within healthy plant

tissues for all or part of their life cycles. Plant wounding induced by biotic factors such as plant-parasitic nematodes also constitute a major factor for the entry of the endophytic microorganisms (Hallmann et al. 1998).

For many years endophytic microorganisms colonizing plant tissues have been thought to be weekly virulent pathogens (Sinclair & Cerkauskas 1996). The distinction between endophytic infection and latent infection is that in latent infections, the host plant does not show any symptoms, with the infection persisting latently until symptoms are prompted to appear by environmental or nutritional stress conditions. The state of host plant and the pathogen may also provide signals for symptom expression. Since the production of disease symptoms is dependent upon the interaction between the host, parasite and the environment over time endophytic colonization is considered not to cause any disease (Sinclair & Cerkauskas 1996).

To detect endophyte colonization of plants, several methods for in situ detection of fungal endophytes in plant tissues have been developed. A simple method involves microscopic examination of differentially stained samples of endophyte infected plants (Saha et al. 1988). This method is however time

  consuming and less reliable since histological staining is not endophyte specific (Hahn et al. 2003). Other methods for in situ detection of endophytes include the use of monoclonal antibodies (Hiat et al. 1997; Hiat et al. 1999) tissue printing

immunoblotting (Gwinn et al. 1991) tissue print immunoassay (Hahn et al. 2003), electron microscopy (Sardi et al. 1992) and autoradiography (You et al. 1995).

Figure 3. Light micrographs of stained endophytic mycelium inside plant tissue showing intercellular colonization by endophytic fungi. A, B. Mycelium(arrow) running along the host vascular bundle (VB) x1000. PM: palisade mesophyll, SM: spongy mesophyll, T: tracheids Bars = 10μm. (Source; Review Iberoam Micology 2007)

Majority of endophytic fungi isolated from healthy tomato tissues belong to the genus Fusarium, followed by Acremonium, others include soil fungi belonging to the genera Penicillium, Aspergillus and Gongronella also Trichoderma which has biological potential is usually isolated (Niere et al. 2002). The most dominant species is Fusarium oxysporum, which has been reported as an endophyte of many crop plants including banana, tomato, rice and maize and is an effective colonizer of plant roots (Niere et al. 2002). However, Fusarium sp are also notorious as causal agent of Fusarium wilt of many crops these are distinguished as specialised forms and physiological races, but majority of isolates of F. oxysporum are non-pathogenic (Niere et al. 2002). Two fungal endophytes F. oxysporum and Fusarium solani when added to tissue culture plants were found to be highly effective in immobilizing root knot nematodes (IITA 1998).

  2.12 Interaction between Endophytic Fungi and Plant Parasitic Nematodes

Inhibitory effects against some species of migratory and sedentary endoparasites occur in grasses infected by Neotyphodium endophytes (West et al. 1988; Kimmons et al. 1990). Neotyphodium species infect aerial tissues, not roots. Therefore the inhibitory effects observed in the infected plants were interpreted as a result of fungal alkaloids being translocated to roots.

Non pathogenic Fusarium oxysporum isolated from roots are other groups of endophytic fungi known to be implicated in the antinematode activity. Culture filtrates of F. oxysporum have an inhibitory effect on Meloidogyne incognita suggesting that fungal toxins could be the mechanism of interaction (Hallmann & Sikora 1996). However the mechanism of Fusarium inhibition of nematodes appears to be more complex than toxin operated system.

2.13 Antagonistic Mechanisms of Endophytic Fungi Against RKN

Various mechanisms of action by endophytic fungi have been suggested (Clay 1987). Antibiosis which is the production of toxic metabolites

(Hallmann & Sikora 1994a; Hallmann & Sikora 1994b; Siddiqui & Ehteshamul-Haque 2001; Li et al. 2002), changes in the host plant

physiology (West et al. 1988; Assuero et al. 2000; Elmi et al. 2000) and the induction of the general plant defense responses (Kimmons et al. 1990; Fuchs et al. 1999; Siddiqui and Shaukat 2003b).

2.13.1 Antibiosis: The production of toxic compounds is an important mechanism of action of beneficial endophytic organisms against plant parasitic nematodes. Grass endophytes mainly those belonging to Neotyphodium sp

produces a wide range of metabolites both in culture and in plants. The production of alkaloids toxic to both insects and herbivores by grass

endophytes has been documented (Breen 1994). These toxins have been isolated successfully from pure cultures of grass endophytes. Infection of tall fascue plants by N. coenophialum resulted in both qualitative and quantitative differences in the production of volatile compounds between endophyte-infected and endophyte free plants (Yue et al. 2001).

  The ability of the endophyte infected plants to produce biologically active compounds depends on the location and concentration of endophyte in plants. Distribution of these compounds in the plant may also vary depending on the compound itself and the season. Toxins produced in endophyte-infected plants may be translocated elsewhere and exuded into the surrounding soil, affecting the nematode population.

Although toxic metabolites produced by most endophytic fungi in culture may show antagonistic activity against nematodes in vitro, the role of these compounds in nematode reduction in plants can only be shown if they are present in detectable concentrations in plant tissues. Secondary metabolites from endophytic isolates obtained from tomato cultivars have been shown to have inactivating or killing effects on the root knot nematode and mortality rates of up to 80-90% have been recorded (Niere et al. 2002). Majority of isolates that produce nematoxic or entomotoxic metabolites are F. oxysporum, others include F. solani, F. concentricum and Acremonium sp (Niere et al. 2002).

Both the type and quantity of secondary metabolites produced in endophyte infected plants might depend on the fungal genotype. For example tall fescue endophytes grown in vitro differed in the production of ergot alkaloid

(Bacon 1988). Hill et al. (1990) also found that different isolates of A. coenophialum from tall fescue plants differed in the amounts and types of

ergopeptine alkaloids produced. The host plant may also affect the production and concentration of the secondary metabolites and therefore its very important to determine a compatible host-endophyte-genotype combinations inorder to maximize the benefits of the association (Hill et al. 1990; Breen 1994; Siddiqui & Shaukat 2003a).

2.13.2 Changes in host physiology: Endophyte infected plants have improved physiological responses to nematode parasitism; endophyte infected tall fescue plants has been associated with enhanced root growth and osmotic adjustments in growing points of the plant, thereby reducing the effects of drought on the host plant (Elmi et al. 2000).

  Endophytes have also been shown to influence photosynthesis rate in host plants as seen in tall fescue plants infected by N. coenophialum photosynthesised faster and flowered earlier than the non-infected ones (Newman et al. 2003), also endophyte infected tall fescue plants exhibited higher survival and flowering frequency (Hill et al. 1991). Such attributes of endophyte infection confer an ecological advantage to the endophyte infected plants, enabling their survival and dorminance over endophyte free plants.

2.13.3 Induced resistance: Induction of systemic resistance by non-pathogenic microorganisms against pests and diseases is well documented phenomenon (Rammamorthy et al. 2001; Compant 2005a). For example

non-pathogenic F. oxysporum isolates induced resistance in tomato plants to F. oxysporum f.sp. lycopersici Jarvis et Shoem, when inoculated prior to infection

by the pathogen. Induced systemic resistance (ISR) can be defined as the resistance in plants induced by localized infection or treatment with microbial

components or their products, or chemicals compounds (Rammamorthy et al. 2001). ISR can be differed from systemic acquired

resistance (SAR). SAR develops in plants in response to both biotic (pathogen attack) and abiotic factors (Chemicals) and depends on the accumulation of the salicylic acid (Van Loon et al. 1998), the onset of SAR is characterized by the expression of the genes for the PR-proteins such as PR-1, PR-2, Chitinase and peroxidase (M’Piga et al. 1987; Rammamorthy et al. 2001; Jeun et al. 2004). ISR on the other hand is dependent on the jasmonic acid and phenylpropanoid pathways (Pieterse et al. 1998; Van Loon et al. 1998; Rammamorthy et al. 2001) ISR leads to the synthesis of plant defence products including peroxidases, polyphenol oxidases and phenylalanine ammonia-lyases (PAL). Polyphenol oxydase catalyses the formation of lignin through polymerization of phenols while PAL are involved in synthesis of phytoalexins and phenolic compounds.

2.13.4 Competition: Competition for plant space and resources may occur between resident endophytes and incoming plant pathogens this could eventually lead to the reduction of various pathogens in plants by the endophytes.

 

III. MATERIALS AND METHODS

3.1 Time and Location of Study

This research was conducted in Nematology Laboratory Department of Plant Protection, Faculty of Agriculture and in greenhouse at Cikabayan, Bogor Agricultural University, Bogor West Java Province, Indonesia from February to August 2009.

3.2 Exploration of Endophytic Fungi

3.2.1 Isolation and identification of endophytic fungi

Healthy and nematode infected tomato plant samples were collected for isolation of endophytic fungi from tomato plant roots in highland and lowland areas. The method used for isolation of endophytic fungi was that proposed by (Rodrigues 1994) that has already been modified. Tomato plant roots were washed thoroughly using flowing water to remove all the soil particles from the roots. Sterilization of the root surface was done by dipping the roots in 70% ethanol for one minute, and then to 1% NaOCl for three minutes after which the roots were rinsed three times with sterile water then dried on dry sterile blotting paper. The roots were then cut into small pieces and placed on petri dishes already filled with PDA under laminar airflow, in each petri dish three root pieces were placed and replicated three times then incubated at room temperature and observed for a period of one week, after which the fungal colonies that developed from the cut root tissues were purified on new PDA media and the isolates that developed were tested for their pathogenicity potential then identified based on colony colour and morphology as well as observation of microscopic features using identification keys according to (Watanabe 2002).

3.2.2 Selection of endophytic fungi based on pathogenicity test

To ensure that the isolated endophytic fungi were not pathogenic and produce disease symptoms to the host plants later after inoculation then there was high need to test for the pathogenicity potential of the isolates to be used, this was

  done by growing tomato seeds on the petri dish containing pure colonies of the isolated fungi. The fungal colonies where tomato seeds grew were proved to be non pathogenic while colonies where there were no growth at all or growth was inhibited were proved to be potential pathogenic isolates, selection of endophytic and pathogenic isolates was based on the pathogenicity test, the seeds were also grown on a control petri dish filled only with PDA. At this stage 12 out of the 20 isolates were proved to be potential isolates of endophytic fungi and were used for further inoculation. The resulting isolates of endophytic fungi included both sporulating and non-sporulating fungi. These isolates were further used for in planta test to assess their effect against root-knot nematodes as well as growth promotion in tomato plants, and in vitro test to estabilish their antagonistic mechanism against the RKN.

3.2.3 Inoculation of seeds with suspension spore

The potential endophytic fungi selected that were preserved in test-tube agar slopes in the refrigerator were re-cultured on 60 mm diameter petri dishes filled with PDA enriched with 50 mg streptomycine-sulphate per litre to prevent bacterial contamination all these were incubated under laboratory conditions with natural photoperiod of 12 hours daylight and darkness for 7 days. Spore suspension for inoculating tomato seeds were produced in 500-ml erlenmeyer flasks containing 200 ml of half strength potato dextrose broth (PDB). Half strength PDB was prepared by dissolving 12 g of PDB per litre of sterile distilled water the flasks containing PDB were sterilized by autoclaving at 121°C for 15 minutes then allowed to cool down. Mycelia blocks of each endophytic fungi isolate were cut from 1 week old culture on PDA and aseptically transferred to PDB under laminar airflow. Two replicate flasks were used for each endophytic fungi isolate. Two flasks containing non-inoculated PDB were used as controls. Flasks were incubated under laboratory conditions using rotary shaker for two week to allow for fungal sporulation and to disperse spores throughout the PDB medium. The fungal spores were harvested by filtering the suspension through a sterile cheese cloth to remove mycelial fragments. The spore density of sporulating fungi was then estimated using the heamocytometer and the

  suspension was standardized to provide a final spore concentration of 1.5 × 106 spores/ml. Tomato seeds were soaked in the already made spore suspension culture of each endophytic fungi for 12 hours after which the seeds were planted on the sterilized soil media that was already prepared on planting trays. The seeds were left for germination and routine cultural practices were carried out.

3.2.4 Re-inoculation of tomato plants with suspension spores

At transplanting stage tomato seedlings were selected based on their uniformity in size and growth vigour, the selected seedlings were transplanted followed by immediate reinoculation whereby suspension spore of each particular endophytic fungi isolate were watered at the holes made around the roots of the tomato plants. The flasks containing the suspension spores were shaken occasionally to resuspend the spores in the solution. The plants were then maintained in the greenhouse conditions with frequent cultural practices carried out on daily basis.

3.3 Colonisation Test

To detect endophytic fungi colonization of plants, several methods for in-situ detection of endophytic fungi in plant tissues have been developed, microscopic examination of differentially stained samples of endophyte infected plants (Saha et al. 1988), other methods for in-situ detection of endophytes include the use of monoclonal antibodies (Hiat et al. 1997; Hiat et al. 1999),

tissue printing immunoblotting (Gwinn et al. 1991), tissue print immunoassay (Hahn et al. 2003), electron microscopy (Sardi et al. 1992) and autoradiography (You et al. 1995).

In this research re-isolation was done to determine the ability of the endophytic fungi to colonise tomato plant tissues. Re-isolation was done from the roots three weeks after re-inoculation of endophytic fungi to tomato plants at

transplanting stage, where by the roots of seven tomato plants to be used for re-isolation per endophytic fungi treatment were selected at random from each

treatment, surface sterilised by dipping the roots in 70% ethanol for one minute, then to 1% NaOCl for three minutes to eliminate root epiphytic microorganisms

  after which the roots were rinsed three times with sterile distilled water then dried on dry sterile blotting paper. The sterilized roots were cut into small pieces under laminar airflow and plated on PDA medium in 60 mm diameter petri dishes, from each tomato plant root, three root segments were plated per petri dish and the petri dishes incubated for seven days under laboratory conditions. Frequency of colonization was determined by observing the total number of roots samples per treatment where colonies of endophytic fungi emerged.

3.4 Meloidogyne incognita Egg Mass Inoculation

3.4.1 Root-knot nematode extraction and inoculation

Identification of the root-knot nematodes upto the species level forms the basic important prerequisite needed for controlling the specific species of the nematodes. In this research the method used in the identification of root-knot nematodes was perineal pattern of the female root knot nematodes of the Meloidogyne incognita species according to (Chitwood 1949; Thorne 1961), and also based on the morphology of the adult male root-knot nematode (Einsenback & Hirschmann 1981).

Root-knot nematode heavily infected tomato plants were identified from the field, the female root-knot nematodes were extracted from the roots and identified using morphological and perineal pattern methods, the egg masses were then extracted from the roots and cultured in two week old tomato plants to act as source of inoculum, after two months RKN egg masses were extracted from the roots and inoculated into potted tomato tomato plants already treated with endophytic fungi.

Before inoculation a rough estimate of number of juveniles from ten sampled egg masses was made. The egg masses obtained from the inoculum source were placed in 1% NaOCl to expose RKN juveniles from the egg mass for counting, a mean number of 350 juveniles were obtained from the 10 sampled egg masses. Three holes of 3 cm deep were made in the soil at the base of tomato plant around the root using a 0.5 cm diameter stick, care was taken not to damage the plant roots while drilling the holes where three egg masses were inoculated at the holes at the base of each tomato plant.

  The plants were left unwatered until 24 hours after inoculation to enable the juveniles that hatch from the inoculated eggs to identify the roots via the root exudates and infect the roots, after juvenile inoculation the plants were maintained in the green house for eight weeks and watered daily, this time permitted for at least two generations of root-knot nematodes.

3.4.2 Plant management practices

Plant management practices that were carried out included, daily watering, physical removal of weeds and insects, fertilizer application N; P; K 15: 15: 15 at the rate of 1 g/plant, fertilizer application was done after every 4 weeks.

3.5 Antibiosis In vitro Test

Endophytic fungi were re-cultured on PDA media on petri-dish for one week, PDB was sterilized at 121°C for 15 minutes, the PDB medium was allowed to cool then asceptically inoculated with seven days old colonies of endophytic fungi, inoculated flasks were placed on a rotary shaker at 120 rpm to disperse the fungal spores in the medium for forteen days at laboratory conditions. The fungal spores and mycelia were removed from the spore suspension using a sterile cheese cloth and the culture filtrate formed was made into three different concentrations for each particular treatment (30%, 50% and 90%), 90% culture filtrate concentration was made by adding 10% volume of sterile distilled water to the original stock solution at ratio of 90ml original stock solution : 10 ml distilled water, 50% culture filtrate concentraton was made by adding 50% volume of sterile distilled water to the original stock solution at a ratio of 50 ml original stock solution : 50ml distilled water, 30% culture filtrate concentration was made by adding 70% volume of sterile distilled water to the original stock solution at the rate of 30 ml original stock soln: 70 ml distilled water. 100 root knot nematode juveniles were placed in the culture filtrate at different concentration and their in vitro mortality rates due to the antagonistic activity of the culture filtrates concentrations was monitored. For control experiments 100 root knot nematode juveniles were placed in potato dextrose broth PDB and sterile distilled water. Three replicate petri dishes were used for each endophytic fungi isolate and

  the control treatments the experiment was arranged in a completely randomized design in the laboratory.

3.6 Parameter Observations, Data Collection and Analysis 3.6.1 Assessment of damage in tomato plant roots by RKN

Four weeks after nematode inoculation the experiment was terminated and the intensity of nematode damage on the roots was determined by checking on the number of root-galls formed and number of egg masses on each root. Then the percentage reduction in infection by each particular endophytic fungi treatment was determined using the following formular:

RI = Where:

¾ Nc - the intensity of RKN infection in control plants.

¾ Nt - the intensity of RKN infection in endophytic fungi treated plants. ¾ RI - the percentage reduction in infection

3.6.2 Assessment of plant growth parameters

Plant growth parameters were also measured to determine the effect of the endophytic fungi on growth performance of the tomato plants. The following parameters were measured; plant height, stem diameter, number of fruits formed, plant fresh weight, plant dry weight, root length. Plant growth parameters were taken on a weekly basis for the whole duration of the experiment. Plant height was measured as the distance from the point where the youngest leaf emerges from the shoot tip to the base of the plant while the stem diameter was taken across the stem.

3.6.3 Experimental design and data analysis

. All the experiments were designed in a Randomized Complete Block Design in similar green house. There were twelve treatments with three plants per

  treatment replicated four times. This experiment lasted for a period of 7 months to the end.

Data was analysed using Microsoft Excel and Statistical Analysis System (SAS) software version 9.1. ANOVA was used to determine the difference among treatment means, while the significance difference between each treatment means was done further using DMRT at 5% significance level.

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West CP, Izekore E, Oosterhuis DM, Robbins RT. 1998. The effect of Acremonium coenophialum on growth of nematode infestation of tall fescue. Plant Soil 112: 3-6.

White JF, Cole GT. 1985. Endophyte-hosts Associations in Forage Grasses. In vitro inhibition of fungi by A. coenophialum. Mycology 77: 487-489.

Webster JM. 1985. Interaction of Meloidogyne with fungi on crop plants. In the book: Sasser JN and Carter CC (eds). An Advanced Treatise on Meloidogyne Volume I. Biology and Control. Raleigh, North Carolina, U.S.A, North Carolina University Press. pp 183-192.

Wilson D. 1995. Endophyte-the evolution of a term, and classification of its use and definition. Oikos 73: 274-276.

Yue Q, Wang C, Gianfag TJ, Meyer WA. 2001. Volatile compounds of endophyte-free and infected tall fescue (Festuca arundinaceae Schreb). Phytochemistry 58: 935-941.

You CB, Lin M, Fang XJ, Song W. 1995. Attatchment of alcaligenes to rice roots. Soil Biology and Biochemistry 27: 463-466.

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DECLARATION

I declare that this thesis titled “Root Endophytic Fungi of Tomato and Their Role as

Biocontrol Agents of Root-knot Nematodes Meloidogyne incognita (Kofoid and White)

Chitwood and Growth Promotion in Tomato Plants Lycopersicon esculentum (Mill)” was

entirely completed by myself with resourceful help from the Department of Plant Protection, Bogor Agricultural University. Information and quotes which were sourced from journals and books have been acknowledged and mentioned where they appear in this thesis, all complete references are given at the end of the paper.

Bogor, April 2010

Bruce Ochieng’ Obura

ACKNOWLEDGEMENT

Thanks be to Almighty God for guiding, strengthening and for His endless blessings that has seen this research work entitled “Root Endophytic Fungi of Tomato Plants and Their Role as Biocontrol Agents of Root-knot Nematodes Meloidogyne incognita (Kofoid

and White) Chitwood and Growth Promotion in Tomato Plants (Lycopersicon esculentum

Mill)” completed.

Sincere thanks goes to my research advisory committee Dr. Ir. Supramana, MSi, and Dr. Ir. Suryo Wiyono, MSc. Agr, who accorded me invaluable guidance and direction in conducting this research despite their committed time schedule. Lots of thanks also goes to my external thesis examiner Dr. Ir. Abdul Munif, MSc. Agr. for the beneficial suggestions and comments given that shaped the outlook of this thesis.

Special thanks to the Department of Plant Protection for the full support given that enabled successful completion of this research. Extended thanks goes to Mr. Gatot the Nematology Laboratory assistant and Ms. Ita of Plant Clinic for all the assistance they accorded me during the research period, not to forget all the energetic and invaluable lecturing staff members in the Department of Plant Protection who have in one way or another imparted knowledge that I acknowledge with gratitude.

Lots of thanks to my sponsor KNB (Kemitraan Negara Berkembang) who provided

for my scholarship throughout my masters course. This work would have not reached this end without the support from KNB.

Further, heartfelt gratitude and thanks goes to my dad, mum, brothers and sisters Geoffrey, Ismael, Ibrahim, Judith, Afya, Effy and Mercy for all love, care, guidance, assistance, support, prayers and endless support kindly given during the course of the research. Special thanks to Uncle John Ong’any Opiyo and his family for all the endless support, love, prayers kindly given during the course of the research.

I’m very much indebted to appreciate the contributions of Mr. Francis Wanaswa, Mrs. Sally Wanaswa, Mrs. Christine Kisenga and Mrs. Catherine who contributed immensely in shaping the outlook of this work as they always provided for academic, social, moral and psychological support, they always stood by me during the difficult times of academic work.

Acknowledgement also goes to my fellow colleagues in

Entomology/Phytopathology major and the KNB family, for the support and assistance during the course of Masters Degree study at Bogor Agricultural University. Special mention of close friends Ir. Netti Tinaprilla. MMA, Joseph Obado, Nurjanah, Weni Willia, Rika Meliansyah, Donnarina Simanjuntak, Eva Dwi, Heri Harti, Wartono, Linda Henuk, TriMaryono, Wawan, Fitrianiangrum Kurniawati, Nildayanti, Pras, Aceu, Diana, Apri, Peni Lestari, Wage, Nurul, Afdhol, Shoni and Kiki, for the contributions, support and advices provided that contributed towards the production of this work.

I would also like to express my sincere gratitude to Flavian Odingo, whose advices, care and support contributed immensely to this work. With that, this work is dedicated to family members, friends and fellow students, may this work help us turn the world into a better place than we found it. May God bless us all.

Bogor , April 2010

BIOGRAPHY

The writer, Bruce Ochieng Obura was born on the 31st of December, 1979 in Kenya to Mr Joseph Obura and The Late Mrs. Risper Achieng from Kenya. In 1994, the writer Completed Primary School from Ezra Gumbe Primary School and continued secondary education at Nyabondo High school in Nyando district Kenya, after which he was admitted to Jomo Kenyatta University of Agriculture and Technology where he graduated with Bachelors of Science Degree studies in Horticulture on 27th July 2006. After Bachelors degree level, the writer was directly employed by the Department of Plant Protection in Kenya where he was in charge of production of Phytoseiulus persimilis a predaceous mite for biological control of red spider mites on vegetables and ornamental crops, he also worked for Bayer Crop Science as a researcher on tropical pests and diseases . In 2007, Bruce Ochieng’ Obura was accepted for scholarship under the Developing countries

partnership program KNB (Kemitraan Negara Berkembang) awarded by the Indonesian