i

UDO, IDORENYIN ASUKWO

PG/Ph.D/08/48902

INTEGRATED ROOT-KNOT DISEASE MANAGEMENT ON TOMATO WITH BIOFORMULATED Paecilomyces lilacinus,

ARBUSCULAR MYCORRHIZAL FUNGI AND Mucuna species GREEN MANURES.

FACULTY OF AGRICULTURE

DEPARTMENT OF CROP SCIENCE

Ebere.omeje Digitally Signed by: Content manager’s Name

DN : CN = Webmaster’s name

O= University of Nigeria, Nsukka

OU = Innovation Centre

ii

INTEGRATED ROOT-KNOT DISEASE MANAGEMENT ON TOMATO WITH

BIOFORMULATED Paecilomyces lilacinus, ARBUSCULAR MYCORRHIZAL FUNGI AND

Mucuna species GREEN MANURES.

BY

UDO, IDORENYIN ASUKWO

PG/Ph.D/08/48902

DEPARTMENT OF CROP SCIENCE, UNIVERSITY OF NIGERIA, NSUKKA.

JUNE, 2015

iii

INTEGRATED ROOT-KNOT DISEASE MANAGEMENT ON TOMATO WITH

BIOFORMULATED Paecilomyces lilacinus, ARBUSCULAR MYCORRHIZAL FUNGI AND

Mucuna species GREEN MANURES.

A Ph.D THESIS SUBMITTED TO THE DEPARTMENT OF CROP SCIENCE,

UNIVERSITY OF NIGERIA, NSUKKA IN PARTIAL FULFILMENT OF THE

REQUIREMENTS FOR THE AWARD OF A DEGREE OF DOCTOR OF PHILOSOPHY

IN PLANT NEMATOLOGY

BY

UDO, IDORENYIN ASUKWO

PG/Ph.D/08/48902

DEPARTMENT OF CROP SCIENCE, UNIVERSITY OF NIGERIA, NSUKKA.

JUNE, 2015

iv

CERTIFICATION

Mr. UDO, IDORENYIN ASUKWO, a postgraduate student of the Department of Crop Science,

University of Nigeria, Nsukka with Registration number PG/Ph.D/08/48902 has satisfactorily

completed the requirements for research work for the award of the degree of Doctor of Philosophy

(Ph.D) in Plant Nematology. The work embodied in this thesis is original and has not been

submitted in part or full for any other diploma or degree in this or any other university.

……………………………… ……………………………… Prof. R.O. Ogbuji Prof. M.I. Uguru (Supervisor) (Supervisor)

Date…………………………. Date…………………………

……………………………… …………………………….. Prof. B.C. Echezona External Examiner (Head of Department) Date………………………. Date………………………..

v

DEDICATION

This work is dedicated to my parents, Chief and Mrs. Asukwo David Udo for their sacrifice in

seeing me through my first degree.

vi

ACKNOWLEDGEMENTS

I thank God for His divine guidance and protection throughout the course of this research

work. In moments of despair you were my source of hope. I acknowledge with great humility, my

main supervisor, Prof. R.O. Ogbuji for his unquantifiable effort in seeing me through. Fourteen

years ago, you asked me “you mean no one talked you into reading nematology?” and I answered

yes. You replied “then I will expose you to nematology” Prof, you went beyond exposure, thank

you for your constructive criticisms, patience and understanding during the course of this research.

To my second supervisor, Prof. M.I. Uguru, your thorough criticisms, encouragement and the

academic discipline you have instilled in me are highly valued. I am highly indebted to the

academic staff of Crop Science Department, University of Nigeria, Nsukka who have contributed to

the success of this work through suggestions, constructive criticisms and encouragement. The HOD

Crop Science Department, UNN, Prof.B.C Echezona, my big brother, Prof. K.P. Baiyeri, Prof. I.

Obi, Prof. J. Asiegbu, Drs. P.E. Ogbonna, K.I. Ugwuoke, C.U. Agbo, V.N. Onyia, S.C. Eze, C.C.

Onyeonagu and Dr. (Mrs.) M.N. Ndubaku are gratefully acknowledged. To my colleagues in the

University of Calabar, Prof. S.B.A. Umoetok ,Dr E.O.Osai, Dr. A.E. Uko, Dr. F.A. Nwagwu, Dr.

D. F. Uwah, Dr. G.A. Iwo, Dr. M. A. Ittah, Dr. E.B. Effa, Dr. Binang, Dr. Shiyam, Mr. E. Obok,

Mr. Ali Ibrahim, Dr. Etuk, Dr. Chris Akpan, Dr. Idiong, Dr. P.B. Okon, Miss Grace Ukoha, Dr. S.

Okweche, Mrs. Joyce Akpan, Dr. Iso, Dr. A.U. Akpanidiok, Dr. Iren and others too numerous to

mention, I say thank you for your support and encouragement. To my late friend and colleague, Dr.

Donald Ukeh, you were a source of inspiration to me, supplying me with literature materials when

you were at UK for your Ph.D study, may your gentle soul rest in peace .I will not forget to say

thank you to Mr. Godswill Bassey and Mr. Isaac Onoko for helping me in transporting the soil

from the different locations to Calabar.

I sincerely acknowledge University of Calabar for granting me a study fellowship to pursue

a Ph.D in UNN. I am also grateful to Biological Control Products South Africa (Pty) Ltd for

allowing their products to be tested in Nigeria. I do acknowledge Mrs. Giwa and Dr. O.S. Bello

who helped me in the procurement of the starter-culture of AMF used in this study. I thank IITA,

Ibadan, Nigeria for their magnanimity in providing the seeds of Mucuna used in this study. The

cooperation of Nigeria Agricultural Quarantine service in the procurement of PL GoldTM is also

acknowledged. The financial support given to me by the Society of Nematologists (SON), Forum

for Agricultural Research in Africa (FARA) and LOC 6th ICN to attend the 6th International

Congress of Nematology, May, 2014 in South Africa and present part of the findings of this work is

gratefully acknowledged.

vii

I am ever indebted to my parents, Chief and Mrs. Asukwo David Udo who denied

themselves the luxury of this life to give me the basic education. To my siblings, Mr. Aniefiok Udo

and family, David, Uwem, Ndueso, Nsikak, my only sister and in-law, Pastor and Mrs. Sunday

Ejike and my little niece Miracle Ejike, I love you all, thank you for your prayers, patience and

support. To a special family, Dr. and Mrs Ubokudom Okon and their son, Aniekemeabasi, I say

thank you for providing me with a conducive environment in Nsukka to put this piece together. My

special thanks go to the members of Udofia Obot family, Mr. Victor Akpan, Mr. David J.Akpan

,Mr. Edmond Ukpong, Mr. Archibong E. Archibong, my typist Miss Victorin Wilson and all my

well wishers which time and space may not accommodate here.

viii

ABSTRACT

Five Screenhouse experiments and one field experiment were conducted at the Teaching and Research Farm of the Department of Crop Science, Faculty of Agriculture, University of Calabar between 2008 and 2010. Experiment I evaluated the host status of five Mucuna species to Meloidogyne incognita. It was laid out in a completely randomized design (CRD) having six treatments represented by five Mucuna species (M. pruriens utilis, M. ghana, M. cochichinensis, M. jaspaeda and M. pruriens 1R2) plus a check (susceptible tomato cv. Roma VF) which were inoculated with 5,000 eggs of M. incognita / plant. In Experiments II , III, V and VI, tomato seedlings (cv. Roma VF) were inoculated with 5,000 eggs of M. incognita per plant. Experiment II consisted of five rates: 2, 4, 6, 8 and 10 t/ha on dry matter basis of each Mucuna species with fresh foliage applied as green manure and soil without amendment served as control (0 t/ha). The 26 treatments were laid out in a CRD with three replications. Experiment III was a 6 x 6 factorial laid out in a CRD with three replications. The treatments were combinations of the five species of Mucuna amendment at 8 t/ha each with five species of arbuscular mycorrhizal fungus (AMF): Glomus etunicatum, Glomus mosseae, Glomus clarum, Glomus deserticola and Gigaspora gigantea plus their respective controls. The tomato seedlings were inoculated with the AMF species at the nursery stage. Experiment IV was done in the field and was laid out as a split-plot in randomized complete block design with three replications. The main- plots were the Mucuna species planted and ploughed-in as green manures. Naturally fallowed plots served as control. AMF- inoculated tomato seedlings were transplanted to the sub- plots and uninoculated seedlings served as the control. Tomato grown in the field were naturally infected with M. incognita. In experiment V, top soils were collected from Calabar, Ikom, Obubra and Ogoja (Cross River State), Nsukka (Enugu State), Umudike (Abia state) and Uyo (Akwa Ibom state) and the experimental design was 3 x 6 factorial in CRD with three replications. Three frequencies of bioformualted Paecilomyces lilacinus application were combined with six levels of AMF species. Experiment VI was a 6 x 6 x 2 factorial laid out in CRD with three replications and the treatments included six levels each of Mucuna species and AMF species and two levels of P. lilacinus application. The tomato plants were grown to full maturity and data were collected on number of galls and eggmasses/ root system, gall index (0-5 scale), nematode larvae/200 g of soil, mycorrhizal root colonization (%), weight (g) of fresh root, dry shoot and total fresh fruit/ plant. Mineral contents of the Mucuna species were determined. Data collected were subjected to analysis of variance and means separated with Fisher’s least significant difference and Duncan’s new multiple range test at 5% probability level. Tomato responses to rates of Mucuna were tested with linear or curvilinear regression model at 1% probability level. The results obtained showed that the roots of the Mucuna spp in both Screenhouse and field trials were neither galled nor had egg masses and were rated immune to infection with a gall index (GI) = 0.00. The tomato plant (check) was highly susceptible, with GI rating of 5.00. The number of nematode larvae on tomato rhizosphere was significantly (p≤ 0.05) higher than that of Mucuna species. In all the Mucuna species, successive increase in the rate of amendment resulted in a significant (P0.05) decrease in the number of galls, eggmases,

nematode larvae but with a significant (p 0.05) enhancement in growth, dry matter, and fresh

fruit yield. Mucuna jaspaeda and M. ghana amendment produced plants with the fewest galls and eggmasses. These two Mucuna species had the lowest C:N ratio. Number of galls and fresh fruit yield responded in a highly significant (p<0.01) negative (r <- 0.80) and positive (r > 0.70) linear

ix

relationship, respectively with Mucuna amendment rate. In both Screenhouse and field, AMF inoculation and Mucuna amendment significantly (p ≤ 0.05) suppressed galling and eggmass production but enhanced growth and fruit yield of tomato compared with their respective controls. Mucuna amendment significantly (p ≤0.05) enhanced root colonization by AMF. Combined application of both control agents was more effective than in sole applications. The highest fresh fruit yield of 409.00 g/plant was obtained in plots inoculated with Gi. gigantea and amended with M. jaspaeda. Application of P. lilacinus or AMF inoculation significantly ( p ≤ 0.05) inhibited root galling and eggmass production by M. incognita in the soils from all locations with a significant ( p ≤ 0.05) enhancement in growth and fresh fruit yield of tomato. Double application of the bionematicide was significantly ( ≤.0.05) more effective than single. The most effective AMF

species in gall suppression across the soil types was G. etunicatum, while G. deserticola was the most effective in fresh fruit yield enhancement. Combined application of the three control agents significantly (p 0.05) inhibited galling with a significant (p 0.05) increase in growth and fruit

yield of tomato relative to sole applications. The highest fresh fruit yield of 139.46 and 136. 06 g/plant were obtained from G. mosseae and Gi. gigantea inoculated plants, respectively grown in M. jaspaeda amended soils with P. lilacinus applied. The trials have shown that Mucuna could be used as a short- term rotation/green manure crop in combination with early inoculation of tomato seedlings with effective AMF species and application of the bioformualted P. lilacinus to manage root- knot disease on tomato in a more sustainable and eco- friendly way.

x

TABLE OF CONTENTS Page

Certification iii

Dedication iv

Acknowledgement v

Abstract vii

Table of Contents ix

Introduction 1

Literature Review 4

Materials and Methods 13

Experiment site 13

Source of Experimental Materials 13

Building up of Nematode Population 14

Nematode Inoculum Preparation 14

Multiplication of Arbuscular Mycorrhizal Fungi Inoculum 17

Inoculation of Tomato Seedlings with Arbuscular Mycorrhizal Fungus 18

Collection of Soil Samples 18

Soil Extraction for Pre-planting population density of Nematodes 19

Soil Analysis to Determine Arbuscular Mycorrhizal Fungus (AMF) Spore Density 19

Soil Analysis for physical and chemical properties 20

Experiment 1: Evaluation of the host status of Five Mucuna spp to Meloidogyne

incognita inoculation 21

Experiment II: Effects of five species of Mucuna used as green manures in the

management of M. incognita infecting tomato 22

xi

Experiment III: Greenhouse Evaluation of the Effects of Mucuna spp Green Manure

Amendment and Arbuscular Mycorrhizal Fungi (AMF) on the Pathgogenicity of

M.incognita on tomato 24

Experiment IV: Field Evaluation of the Effects of Mucuna spp Green Manure and AMF

on the pathogenicity of M. incognita on Tomato 27

Experiment V: Evaluation of the Effects of Paecilomyces lilacinus and AMF

against M. incognita on Tomato 29

Experiment VI: Evaluation of the Effects of P. lilacinus (PL GoldTM), Arbuscular

Mycorrhizal Fungi and Mucuna Green Manure on the Pathogenicity of M. incognita on

tomato 30

Statistical Analysis 31

Results and Discussion 32

Physico-chemical properties, Arbuscular Mycorrhizal spore Density and Pre-plant

Nematode Density of the soils used for the Experiments 32

Climate Data 33

Experiment I: Evaluation of the Host Status of Five Mucuna spp to Meloidogyne

incognita inoculation 36

Experiment II: Effects of five species of Mucuna used as green manures in the

management of M. incognita infecting tomato 40

Mineral contents and carbon-to-Nitrogen Ratios of the Different Mucuna Species 48

Experiment III: Greenhouse Evaluation of the Effects of Mucuna spp Green

Manure soil Amendment and Arbuscular Mycorrhizal Fungi on the Pathogenicity

of M. incognita on Tomato 50

Experiment IV: Field Evaluation of Effects of Mucuna spp Green Manure and AMF

on the Pathogenicity of M. incognita on Tomato 59

xii

Experiment V: Evaluation of the Effects of Paecilomyces lilacinus and AMF

against M. incognita on Tomato 70

Experiment VI: Evaluation of the Effects of P.lilacinus (PL GoldTM), Arbuscular

Mycorrhizal Fungi and Mucuna Green Manure on the Pathogenicity of M. incognita

on Tomato 103

Discussion 122

Summary, Conclusion and Recommendations 133

Summary 133

Conclusion 136

Recommendations 136

References 138

xiii

LIST OF TABLES

Tables Page

1 Physico-Chemical Properties, AMF Spore Density and Pre-plant Nematode Density of the Soils used for the Experiments. 34

2 Monthly Maximum Temperature (0c) and Rainfall (mm) during the period of study (2008- 2010) in Calabar, Cross River 35

3 Number of galls and eggmasses/root system and number of nematode larvae recovered from 200 g of soil planted with Mucuna spp and susceptible tomato (Roma VF) and inoculated with M. incognita 38

4 Effects of M. incognita inoculation on fresh root weight (g)/Plant, fresh and dry above- ground weight(g)/plant of five Mucuna spp and a susceptible tomato CV. Roma VF 39

5 Effects of rates of different Mucunna spp soil amendment on number of galls/root system gall index (GI)*, number of Eggmasses/root system and Eggmass Index (EMI) of tomato inoculated with M. incognita. 42

6 Effects of different rates of Mucuna spp soil amendment on number of nematode larvae/200g soil, fresh root weight(g)/plant, shoot length (cm)/plant and dry shoot weight(g)/plant of tomato inoculated with M. incognita 44

7 Effects of different rates of Mucunna spp soil amendment on number of fruits/plant and total fresh fruit weight (g)/plant of tomato inoculated with M. incognita 46

8 Mineral content and C/N ratio of the different Mucuna species 49

9 Effects of arbuscular mycorrhizal fungi and Mucuna spp soil amendment on root galls and galls index (GI) of tomato infected with M. incognita 52

9 Effects of arbuscular mycorrhizal fungi and Mucuna spp soil amendment on number of egg masses/root system and egg mass index of tomato infected with M. incognita 53

10 Effects of arbuscular mycorrhizal fungi and Mucuna spp soil amendment on fresh

weight (g) and dry shoot weight (g)/plant of tomato infected with M. incognita 55

11 Effects of arbuscular mycorrhizal fungi and Mucuna spp soil amendment on shoot

xiv

length (cm)/ plant and AMF root colonization (%) of tomato infected with

M. incognita 56

13 Effects of arbuscular mycorrhizal fungi and Mucuna spp soil amendment on number

of fruits/plant and total fresh fruit weight (g)/plant of tomato infected M 58

14 Physico-chemical properties of soil amended with different Mucuna spp sampled

at Mid-season (6 weeks after incorporation of Mucuna green manure) 60

15 Effects of Arbuscular Mycorrhizal fungi and Mucuna Spp soil amendment

on root galls and mean gall index (MGI)* of tomato infested with M. incognita in the

field 62

16 Effects of Arbuscular Mycorrhizal fungi and Mucuna Spp soil amendment on number

of eggmasses/root system and eggmass index (EMI) of tomato infested with

M. incognita in the field 63

17 Effects of Arbuscular Mycorrhizal fungi and Mucuna spp amendments on root-knot

incidence (%) on tomato grown in field infested with M. incognita in the field 65

18 Effects of Arbuscular Mycorrhizal fungi and Mucuna Spp soil amendment on

fresh root weight (g)/ plant and root colonization by AMF (%) of tomato

grown in M. incognita infested field 66

19 Effects of Arbuscular Mycorrhizal fungi and Mucuna Spp soil amendment on shoot

length (cm)/ plant and dry shoot weight (g)/ plant of tomato grown in M.incognita

infested field 68

20 Effects of Arbuscular Mycorrhizal fungi and Mucuna Spp soil amendment on

number of fruits/plant and total fresh fruit weight (g)/plant of tomato grown in

M. incognita infested field 69

21 Effects of arbuscular mycorrhizal fungus and P. lilacinus application on number of

galls and Eggmasses per root system, gall index (GI)* and Eggmass Index* of

tomato inoculated with M. incognita in Calabar soil 72

xv

22 Effects of arbuscular mycorrhizal fungus and P. lilacinus application on fresh root

weight (g)/plant, root colonization by AMF (%), shoot length (cm)/plant and dry

shoot weight (g) /plant of tomato inoculated with M. incognita in Calabar 73

23 Effects of arbuscular mycorhizal fungus and P. lilacinus application on number of

fruits/plant and total fresh weight of fruits (g)/plant of tomato inoculated with

M. incognita in Calabar soil 74

24 Effects of arbuscular mycorrhizal fungus and P. lilacinus application on number of galls and Eggmasses per root system, gall index (GI)* and Eggmass Index

of tomato inoculated with M. incogmta in Ikom soil 76

25. Effects of arbuscular mycorrhizal fungus and P. lilacinus application on fresh root

weight (g)/plant, root colonization by AMF (%), shoot length(cm)/plant and

dry shoot weight(g)/plant of tomato inoculated with M. incognita in Ikom soil 77

26 Effects of arbuscular mycorhizal fungus and P. lilacinus application on number of

fruits and total fresh weight of fruits (g)/plant of tomato inoculated with M. incognita

in Ikom Soil 79

27 Effects of arbuscular mycorrhizal fungus and P. lilacinus application on number of galls

and Eggmasses per root system, gall index (GI)* and Eggmass Index* of tomato

inoculated with M. incogmta in Nsukka soil 80

28 Effects of arbuscular mycorrhizal fungus and P. lilacinus application on fresh root

weight (g)/plant, root colonization by AMF (%), shoot length(cm)/plant and dry

shoot weight(g)/plant of tomato inoculated with M. incognita in Nsukka soil 82

29 Effects of arbuscular mycorhizal fungus and P. lilacinus application on number of

fruits and total fresh weight of fruits (g)/plant of tomato inoculated with M.

incognita in Nsukka soil 84

30 Effects of arbuscular mycorrhizal fungus and P. lilacinus application on number of galls

and Eggmasses per root system, gall index (GI)* and Eggmass Index* of tomato

inoculated with M. incogmta in Obubra soil 85

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31 Effects of arbuscular mycorrhizal fungus and P. lilacinus application on fresh root

weight (g)/plant, root colonization by AMF (%), shoot length(cm)/plant and dry

shoot weight(g)/plant of tomato inoculated with M. incognita in Obubra soil 87

32 Effects of arbuscular mycorhizal fungus and P. lilacinus application on number of fruits per plant and total fresh weight of fruits (g)/plant of tomato inoculated with M. incognita in Obubra soil 88 33 Effects of arbuscular mycorrhizal fungus and P. lilacinus application on number of galls and Eggmasses per root system, gall index (GI)* and Eggmass Index* of tomato inoculated with M. incogmta in Ogoja soil 90 34 Effects of arbuscular mycorrhizal fungus and P. lilacinus application on fresh root weight (g)/plant, root colonization by AMF(%), shoot length(cm)/plant and dry shoot weight(g)/plant of tomato inoculated with M. incognita in Ogoja soil 91 35 Effects of arbuscular mycorhizal fungus and P. lilacinus application on number of fruits per plant and total fresh weight of fruits (g) plant of tomato inoculated with M. incognita in Ogoja soil 93 36 Effects of arbuscular mycorrhizal fungus and P. lilacinus application on number of galls and Egg masses per root system, gall index (GI)* and Eggmass Index* of tomato inoculated with M. incogmta in Umudike soil 94 37 Effects of arbuscular mycorrhizal fungus and P. lilacinus application on fresh root weight (g)/plant, root colonization by AMF(%), shoot length(cm)/plant and dry shoot weight(g)/plant of tomato inoculated with M. incognita in Umudike soil 96 38 Effects of arbuscular mycorhizal fungus and P. lilacinus application on number of fruits/plant and total fresh weight of fruits (g)/plant of tomato inoculated with M. incognita in Umudike soil 97 39 Effects of arbuscular mycorrhizal fungus and P. lilacinus application on number of galls and Eggmasses per root system, gall index (GI)* and Eggmass Index* of tomato inoculated with M. incogmta in Uyo soil 99 40 Effects of arbuscular mycorrhizal fungus and P. lilacinus application on fresh root weight (g)/plant, root colonization by AMF(%), shoot length(cm)/plant and dry shoot weight(g)/plant of tomato inoculated with M. incognita in Uyo soil 100 41 Effects of arbuscular mycorhizal fungus and P. lilacinus application on number of fruits per plant and total fresh fruit weight (g)/plant of tomato inoculated with M. incognita in Uyo soil 102

xvii

42 Effects of arbuscular mycorrhizal fungi, P. lilacinus and Mucuna spp soil amendment on number of galls/root system of tomato inoculated with M. incognita 104

43 Effects of arbuscular mycorrhizal fungi; P. lilacinus and Mucuna spp soil

amendment on root gall index (GI) of tomato inoculated with M. incognita 107

44 Effects of arbuscular mycorrhizal fungus, P. lilacinus and Mucuna spp soil amendment on number of egg masses/ root system of tomato inoculated with M. incognita 108

45 Effects of arbuscular mycorrhizal fungi, P. lilacinus and Mucuna spp soil amendment on Eggmass index (EMI) of tomato inoculated with M. incognita 110

46 Effects of arbuscular mycorrhizal fungi; P. lilacinus and Mucuna spp soil amendment on number of nematode larvae/200g soil of tomato inoculated with M. incognita 111

47 Effects of arbuscular mycorrhizal fungus, P. lilacinus and Mucuna spp soil amendment

on fresh root weight (g) /plant of tomato inoculated with M. incognita 112

48 Effects of P. lilacinus inoculation and Mucuna spp soil amendment on percentage

root colonization by arbuscular mycorrhizal fungus of tomato inoculated with

M. incognita 114

49 Effects of arbuscular mycorrhizal, P. lilacinus and Mucuna spp soil amendment on shoot length (cm/plant) of tomato inoculated with M. incognita 115 50 Effects of arbuscular mycorrhizal, P. lilacinus and Mucuna spp soil amendment on

dry shoot weight (g)/plant of tomato inoculated with M. incognita 117

51 Effects of arbuscular mycorrhizal, P. lilacinus and Mucuna spp soil amendment on number of fruits per plant of tomato inoculated with M. incognita 118 52 Effects of arbuscular mycorrhizal fungi, P.lilacinus and Mucuna spp soil amendment

on total fresh fruit of weight (g/plant) of tomato inoculated with M. incognita 120

xviii

LIST OF FIGURES

Figures Page

1 Effects of Different Rates of Mucuna spp on root galling of Tomato infected

with M. incognita 43

2 Effects of Different Rates of Mucuna spp on Total Fresh Fruit weight (g)/plant

of Tomato infected with M. incognita. 47

xix

LIST OF PLATES

Plates page

1. Seeds of different Mucuna specie 15

2. Bioformulated P.lilacinus 16

3. Some Mucuna species showing gall- free roots 37.

4. Lightly galled and heavily galled roots of tomato due to treatment effects 51

5. Lightly galled and heavily galled roots of tomato in Nsukka soil. 81

6. Lightly galled and heavily galled roots of tomato due to treatment effects 105

7. Potted tomato plants with fruits. 121

1

INTRODUCTION

The commercial tomato (Solanum lycopersicum L.) belongs to the family Solanaceae and it

is one of the most highly cherished fruit vegetables in Nigeria(Yayock et al.,1998). Tomato is

ranked 15th among the world’s food crops (Vietmeyer, 1986). The total area under tomato

production in tropical Africa is about 300,000ha with an estimated annual production of 2.3 million

tonnes (Van der Vossen et al., 2004). Nigeria is the largest producer in Tropical Africa with

126,000ha yielding 879,000 tonnes of fresh fruits annually (FAO, 2004). Tomato fruit is very rich

in vitamins A and C, providing between 20% and 40% of an adult’s requirements based on an

average consumption of 100-125g of fresh fruits (Janes, 1994). It is also a good source of thiamine,

riboflavin, niacin, potassium and sodium (Holland et al., 1991). The fruit can be eaten raw or

cooked. Large quantities are used to produce soups, juices, sauces, ketchups, purees and pastes. The

seeds extracted from the pulp contain 24% of a semi-drying edible oil (Yayock et al., 1988).

The production of tomato in the tropics is highly constrained by a vast array of pathogenic

organisms including the plant parasitic nematodes. Over 60 species representing 19 genera of plant

parasitic nematodes attack tomato and the root-knot nematode (Meloidogyne spp) is the most

destructive (Valdez, 1979). The most widespread and devastating species are M. incognita, M.

javanica and M. arenaria (IITA, 1992). M. incognita and M. arenaria are more common in

southern Nigeria while M. javanica is more prevalent in northern Nigeria (Olowe, 2004). Root-knot

nematode is an obligate sedentary endoparasite with visible symptoms of attack as root galls, early

senescence, chlorosis, wilting, unthrifty growth, stunted appearance, fruit splitting, reduction in fruit

number and size and general susceptibility to rot and wilt-inducing pathogens (Ogbuji, 1978,

Sasser, 1980). Galled tomato roots are inefficient in nutrient and water uptake (Meon et al., 1978).

Low photosynthetic rate has been attributed to poor carbon (iv) oxide assimilation, poor partitioning

and translocation of photo assimilates in tomatoes attacked by M. incognita and M. javanica

(Meon et al., 1978; Khan and Khan, 1987). Although accurate information on yield losses

2

attributable to root-knot nematode in Nigeria is unavailable, conservative estimates indicated more

than 50% losses depending on the cultivar, population density of the nematode species, cultural

practices and environmental conditions (Olowe, 2005; Udo et al., 2008).

Over the years, different control methods have been employed in the management of root-

knot disease. Chemical control with synthetic nematicides has proved to be the most effective.

However, it is uneconomic, has detrimental effects on beneficial non-target organisms, pollutes

ground water, high mammalian toxicity, etc. Of late, emphasis is laid on environmentally sound

approaches to pest management. The use of resistant crop cultivars is one of such approaches. In

tomato, breeders have identified and incorporated the Mi resistance gene to commercial cultivars

with applauded success in root-knot nematode control (Williamson, 1998; Sorribas et al., 2005).

However, the emergence of virulent resistance-breaking pathotypes have been reported in some

species of Meloidogyne (Tzortzakakis and Gowen, 1996; Castagnone-Sereno, 2002), thus

constraining this method of control too. In recent times, there has been a worldwide swing to the

use of eco-friendly methods for protecting crops from pests and diseases. The use of potential

harmful chemical spray is viewed with contempt in many countries. The removal of some effective

chemical nematicides (Methyl bromide, Ethyldibromide, Dibromochloropropane, etc.) from the

pesticide market has spurred research on alternative management strategies of root-knot disease.

The use of biological control agents, crop rotation with antagonistic crops and green

manure/organic amendments of soils are some of the alternatives (Rodriguez-Kabana and Morgan-

Jones, 1987; Queneherve et al., 1998; Hashem and Abo-Elyousr, 2011).

Some fungi are nematophagous. Paecilomyces lilacinus parasitizes the egg of root-knot

nematodes and has been reported by many researchers to reduce the damage caused by this pest on

several crops (Jatala, 1979, Oclarit and Cumagun, 2009). Arbuscular mycorrhizal fungi (AMF)

have been reported to be effective in reducing the malady caused by Meloidogyne spp on several

crops too (Diederichs, 1987; Shreenivasa et al., 2007; Odeyemi et al., 2010). Velvetbean (Mucuna

3

spp) is a tropical leguminous plant used as a rotation, forage and green manure crop in many

countries. Most findings have reported it to be effective in reducing root-knot nematode population

in the soil and its associated damage on many crops when used as a rotation and/or green manure

crop (McSorley and Dickson, 1995; Queneherve et al., 1998).

Despite the promise for economic control of root-knot nematode with biological entities and

their combination, little work has been done in Nigeria. Considering the fact that the efficacy of

green manure varies with species, rate of application and soil types, there is need to investigate

native species of Mucuna and AMF and their combinations with established biocontrol agents under

different soils in Nigeria for root-knot disease management. In addition, the variation in the results

from combination of factors in root-knot nematode management calls for further investigation. On

the bases of these considerations, the present study was initiated with the following objectives.

i) To ascertain the host status of Mucuna species on Meloidogyne incognita

ii) To evaluate the effects of five species of Mucuna used as green manures in the management

of M. incognita infecting tomato.

iii) To evaluate the efficacy of five species of arbuscular mycorrhizal fungus (AMF) against M.

incognita in selected soil types of southeastern Nigeria.

iv) To evaluate the efficacy of bioformulated P. lilacinus against M. incognita in selected soil

types of southeastern Nigeria.

v) To evaluate the combined effects of bioformulated P. lilacinus, Mucuna green manure

amendment and arbuscular mycorrhizal fungi in the management of root-knot disease on

tomato.

4

LITERATURE REVIEW

There are reports that plant parasitic nematodes including Meloidogyne spp can be

controlled biologically (Jatala, 1986; Sayre, 1986; Hashem and Abo-Elyousr, 2011). A biological

control agent colonizes the rhizosphere, the site requiring protection and leaves no toxic residues as

opposed to chemicals. The fungus, Paecilomyces lilacinus (Thom) Samson has been reported as a

potential biological control agent for root-knot nematodes and other plant parasitic nematodes

(Jatala, 1979, 1986; Oclarit and Cumagun, 2009; Hashem and Abo-Elyousr, 2011). Paecilomyces

lilacinus is a common soil hyphomycete, closely related to Penicillium (Samson, 1975). It is a

facultative parasitic fungus that infects the egg and sometimes the other life stages of root-knot and

cyst nematodes (Dunn et al., 1982; Freire and Bridge, 1985). P. lilacinus is lilac to purple coloured

soil hyphomycete, producing smooth to rough conidia endogenously on small groups of unclumped

phialides borne conidiophores (Esser and El-Gholl, 1993). The fungus belongs to the phylum

Ascomycota and family Trichocomaceae. The fungus has been considered to have the greatest

potential for application as a biocontrol agent in sub-tropical and tropical agricultural soils

(Morgan-Jones et al., 1984). P. lilacinus has an almost worldwide distribution occurring most

frequently in warmer climates (Dunn et al., 1982). The fungus occurs naturally in soil, in egg

clusters contained in the gelatinous egg mass of root-knot nematodes, and in cysts of Globodera spp

and Heterodera spp (Esser and El-Gholl, 1993). Modern technology has enhanced the formulation

of some biocontrol agents into forms that could be easily handled and applied as conventional

pesticides (Kiewnick, 2001). Bioformulation containing P. lilacinus strain-251 has been licensed by

biotech companies in South Africa and Germany, and has been registered for the European and

USA markets (Bruckner, 2004; Kiewnick, 2004). The efficacy of P. lilacinus as a biocontrol agent

in the management of root-knot and other nematode diseases on several crops in greenhouse, micro

plots and field experiments has been evaluated by many researchers (Jatala, 1979, Davide and

5

Zorilla, 1983; Dube and Smart, 1987; Cabanillas et al., 1988, 1989; Cabanillas and Barker, 1989;

Walters and Barker, 1994; Kiewnick and Sikora, 2003; Kiewnick and Sikora, 2006; Nasresfahani

and Ansari Pour, 2006; Oclarit and Cumagun, 2009; Hashem and Abo-Elyousr, 2011 ;Singh et

al.,2013). In most of these trials, the efficacy of the fungus in the control of root parasitic

nematodes was reported to be influenced by some factors which include; the antagonist itself (age,

virulence, longevity, inoculum level, method of establishment), the environment (soil type, fertility,

amendments, organic matter, temperature, moisture, pH, rhizosphere microbial interaction) and host

susceptibility (genotype, age). Reduction in root galling and egg mass production, inhibition of

giant cell formation and egg hatch, increased crop growth and yields have been reported by many

authors with inoculation of P. lilacinus against uninoculated plots (Jatala, 1979; Davide and Zorilla,

1983; Kiewnick and Sikora, 2006; Oclarit and Cumagun, 2009; Sing et al.,2013 ). Cabanillas and

Barker (1989) observed greater parasitism of root-knot nematode eggs at higher fungal inoculum

rate and recommended early plus midseason application for effective nematode control and

increased tomato fruit yield. Bruckner (2004) recommended a split application programme for

vegetable crop which involved soil treatment with P. lilacinus strain 251 bioformulated product 7

days before planting, then drenching of seedling plugs a day prior to transplanting and followed by

additional field treatments at 4-6 week intervals. He observed that a total dose of 10-14 kg/ha of the

product provided effective control and was considered economical. Nasresfahani and Ansari Pour

(2006) were of the same opinion as they recommended simultaneous inoculation of both the

nematode and the biocontrol agent or the biocontrol agent preceeding the nematode in a sequential

inoculation. Oclarit and Cumagun (2009) observed that the reduction in root galling in tomato was

more effective at a moderate inoculum level (7.92 x 106 spores/ml) than higher level (3.96 x 108

spores/ml). Singh et al. (2013) evaluated the efficacy of 24 indigenous isolates of P. lilacinus

against M .incognita on tomato in india.They observed that application of isolate HYBDP-04 at the

rate of 10 kg/ ha with compost (1.5t/ha) was the most effective in causing juvenile mortality,

6

inhibition of egg hatch, egg production and galling and in the enhancement of marketable fruit

yield. The mechanism of egg parasitism by P. lilacinus involves enzyme and toxic metabolic

secretions (Serine, Protease, Chitinase, Leucinotoxin and acetic acid) which ultimately lead to

arborted embryonic development through a cascade of physiological disorders (Park et al., 2004;

Khan et al., 2004).

Albert Bernard Frank in 1885 (Siddiqui and Mahmood, 1995a) introduced the Greek word

“mycorrhiza”, which literally means “fungus roots”. The arbuscular mycorrhizal symbiosis is a

mutualistic association formed between plants and a wide variety of fungi from the phylum

Glomeromycota (Sieverding and Oehl, 2006) which has 4 orders and 9 families. The genera

Glomus and Gigaspora belong to the family of Glomeraceae and Gigasporaceae, respectively. The

endotrophic arbuscular mycorrhizal fungi (AMF) are ubiquitous soil microbes constituting an

integral component of terrestrial ecosystems. They form symbiotic associations with plant root

systems of over 80% of all terrestrial plant species, including many important horticultural plants

(Bagyaraj, 1991; Smith and Read, 2008). In general, the symbionts trade nutrients and AMF obtain

carbon from the plant while providing the plant with additional nutrients that are relatively

immobile in the soil such as P, Ca, Cu, Mn and Zn (Azcon-Aguliar and Barea, 1996; Turk et al.,

2006). Many researchers have reported the effectiveness of AMF as a potential biocontrol agent of

root-knot nematode and other nematodes in many cultivated plants and in different regions of the

world (Bagyaraj et al., 1979; Hussey and Roncadori, 1982; Cooper et al., 1986; Diederichs, 1987;

Carling et al., 1989; Siddiqui and Mahmood, 1995b; Calvet et al., 2001; Forge et al., 2001;

Masadeh et al., 2004; Shreenivasa et al., 2007; Zhang et al., 2008,2009; Odeyemi et al., 2010).

In most of these trials, the efficacy of the mycorrhizal fungi was reported to be influenced by the

fungal species, host plant, edaphic factors (physico-chemical and micro-flora), the nematode

species, time of inoculation and mycorrhizal inoculum level (Gera Hol and Cook, 2005). For

instance, biological enhancement of nursery seedlings through inoculation with AMF has been

7

reported to increase its efficacy against direct field application (Oyekanmi et al., 2007). The

mechanism involved in nematode suppression by AMF is still topical. Induced systemic

resistance/tolerance due to improved host’s nutrition has been advocated (Linderman, 1985; Smith,

1988, Gosling et al., 2006). Also, changes in the root morphology of the host to the detriment of the

nematode partner has been suggested (Dehne, 1982; Tahat et al., 2008). Histopathological changes

induced by AMF root colonization with a resulstant impairment of giant cell formation leading to

the reduction in nematode population vis-à-vis plant damage have been reported (Suresh, 1985;

Masadeh et al., 2004). Arbuscular mycorrhizal fungi can also induce some physiological and

biochemical changes within the host plant to the detriment of plant parasitic nematodes (Umesh et

al., 1988; Singh et al., 1990; Morandi, 1996). Morandi (1996) reported higher concentration of

phytoalexins and phenolic compounds in the roots of mycorrhizal plants compared with the non-

mycorrhizal plants. Similarly, increase in lignin, phenols, phenylalanine and serine concentration in

the roots of tomato and banana were observed in mycorrhizal plants and were associated with

reduced nematode reproduction (Suresh, 1985; Umesh et al., 1988; Singh et al., 1990).

Soil amendment with different organic materials for nematode control is fast gaining wide

acceptance as an alternative control method (Ogbuji, 1981; McSorley and Gallaher, 1992; Agu,

2008; Udo and Ugwoke, 2010; Oka, 2010). However, the scarcity and high transportation cost

associated with the movement of some of these materials to the farm are serious impediments. Of

late, many cover/green manure crops that are non-host and antagonistic to root-knot nematodes

have been tested and found to be effective in reducing nematode damage to crops and nematode

population in the soil (Ritzinger and McSorley, 1998; Ploeg, 2002; Stirling and Stirling, 2003).

Velvet bean (Mucuna spp), Brassica, Sunhemp, Sesame, Castor and Marigold are some of the short-

term rotation crops evaluated (McSorley et al., 1994; Queneherve et al., 1998; Ploeg, 2002; Stirling

and Stirling, 2003; Marla et al., 2008). Velvet bean (Mucuna spp) is a vigorous African annual

legume whose primary functions are soil fertility maintenance, soil protection and weed suppression

8

(Buckles, 1995). It can achieve nearly 100% ground cover in two months (Casky et al., 1998). It

has been reported that in some parts of the tropics with bimodal rainfall pattern, the crop can

produce between 7 and 10 tonnes dry matter/ha (Rodriguez-Kabana et al., 1992; Vissoh et al.,

1998). The efficacy of velvet bean used as a rotation and/or green manure crop in the management

of various species of plant parasitic nematodes have been reported (Queneherve et al., 1998;

McSorley and Gallaher, 1992; Weaver et al., 1993, 1998; McSorley and Dickson, 1995). In most

of these trials, the nematode suppressing ability of velvet bean has been linked to its decomposition

products, stimulation of microbial antagonists within the rhizosphere, its nematicidal constituents, it

being a poor host to many nematode genera and its general improvement of soil properties and soil

health (Rodriguez-Kabana et al., 1992; Kloepper et al., 1991, Vincente and Acosta, 1987, Vargas

et al., 1996). Nogueira et al. (1996) evaluated the nematicidal hydrocarbons in Mucuna aterrima.

They extracted two bioactive natural products (aliphatic ester triacontyl tetracosanate and aliphatic

alcohol I-triacontaanol) that were tested in vitro and in vivo and found to be

nematostatic/nematicidal against M. incognita race 3. In the same vein, Barbosa et al. (1999)

isolated several chemical constituents from Mucuna aterrima and observed that (β-sitosterol +

stigmasterol, an unknown alcohol and KNO3 + NaNO3) were highly nematicidal against M.

incognita at 5μg ml-1, while others (fatty acids, allantoin, daucosterol + stigmasterol D-glycoside

and L-Dopa) were more toxic to Heterodera glycines. However, when tested at 50μg ml-1, all the

compounds isolated from the stems and roots of M. aterrima caused greater than 97% mortality of

M. incognita. Tian et al. (1995) found 6% N, 4% polyphenols and 16.8% lignin in Mucuna spp.

leaves and petioles and observed its decomposition rate to be the highest among a group of ten

herbaceous and woody species. Also, Ritzinger and McSorley (1998) reported a high macro and

micro-element composition of Mucuna deeringiana with a low C:N ratio of 8.68. Velvet bean

(Mucuna spp) is of tropical African origin (Buckles, 1995) and has so many species. Mucuna

pruriens var utilis was previously known by different names; Styzolobium aterrima, Mucuna

9

aterrima, Mucuna deeringiana and Stizolobium deeringianum (Buckles, 1995, Queneherve et al.,

1998). A critical review of literature revealed that most of the trials evaluated M. pruriens utilis for

root-knot nematode management. As observed by Ploeg (1999), (2002) and Marla et al. (2008),

there exist species and varietal differences in Marigold (Tagetes spp) and Sunhemp (Crotalaria

juncea) used as rotational and/or green manure crops in nematode control. It is likely possible that

different species of Mucuna may vary in their nematocidal/nematotoxic properties. Rodriguez-

Kabana et al. (1987) observed that the efficacy of an organic amendment in suppressing nematode

population is influenced by its chemical composition and the rate of application. Those with narrow

C:N ratio and high protein or amine type of N content were more potent. Higher rate of application

of such materials however could be phytotoxic. Although Ritzinger and McSorley (1998) did not

observe severe phyto toxicity with higher rates of Mucuna deeringiana amendment on tomato, they

reported a general decrease in plant growth and yield of tomato which was best described by a

curvilinear relationship. Thus, there is need to determine the optimal rate of application of a

particular organic material in a particular crop genotype.

In the past, it was a general rule to use a single biocontrol agent for the control of plant

disease caused by a single pathogen (Wilson and Backman, 1999). This could have accounted in

part for the slow and inconsistent performance of biocontrol agents as observed by some authors

(Guetsky et al., 2001). A single agent is not active in all soil environments or against all pathogens

that attack a host plant. Of late, many researchers have laid emphasis on the use of biological

formulations that contain a mixture of biocontrol agents (Meyer and Roberts, 2002; Masadeh et al.,

2004; Oyekanmi et al., 2007; Akhtar and Siddiqui, 2008; Hashem and Abo-Elyousr, 2011) in the

management of nematodes. The general opinion of these authors is that, mixtures of micro

organisms may adapt better to environmental changes that occur throughout the growing season and

protect against a wider spectrum of pathogens. Also, mixtures of microorganisms may increase the

genetic diversity of biocontrol systems that may persist longer in the rhizosphere and utilize a wider

10

array of biocontrol mechanisms (Akhtar and Siddiqui, 2008). Al-Raddad (1995) evaluated the

interaction effect of P. lilacinus formulated on a chicken layer manure and Glomus mosseae

against M. javanica on tomato. The two biocontrol agents acted synergistically in suppressing root-

knot nematode population and damage to the tomato crop. In contrast, Rumbos et al. (2006) did not

observe synergistic interaction between P. lilacinus strain 251 and Glomus intraradices in root-knot

nematode suppression with concomitant application but however reported that early inoculation of

seedlings with P. lilacinus as pre-planting soil treatment at 4 and 1 week before transplanting

resulted in the highest nematode control and crop yield. Siddiqui and Mahmood (1995)

recommended the simultaneous use of P. lilacinus, Verticillium chlamydosporium and AMF

(Gigaspora margarita) in the management of wilt disease complex of pigeon pea caused by

Heterodera cajani and Fusarium udum.Although Masadeh et al. (2004) supported the combined

use of G. intraradices and a nematode antagonist; Trichoderma viride in the management of root-

knot disease of tomato, they also did not observe synergistic interaction between the two beneficials

in nematode control. However, they concluded that nematode control was highly influenced by the

host genotype. Oyekanmi et al. (2007) reported improved root-knot nematode management in

soybean through the combined application of Bradyrhizobium japonicum, Trichoderma

pseudokoningii and AMF (Glomus mosseae). Similarly, root-rot disease complex of chickpea

induced by M. incognita and Macrophomia phaseolina was reported to be effectively managed

through synergistic interaction among Glomus intraradices, Rhizobium spp and Pseudomonas

straita (Akhtar and Siddiqui, 2008). Anastasiads et al. (2008) recommended the combination of P.

lilacinus with Bacilus firmus for the management of Meloidogyne spp but not with soil solarization.

On the other hand, Hashem and Abo-Elyousr (2011) warned against the combination of P. lilacinus

with a cyanobacterium (Calothrix parietina) as it reduces the efficacy of the biocontrol activities of

the fungus but however recommended the combined use of P. lilacinus with the bacterium

(Pseudomonas fluorescens) and yeast (Pichia gulliermondii) for root-knot disease control in

11

tomato.Also, recently,Flor-Peregrin et al. (2014) observed that the AMF Funneliformis mosseae

was more effective than Rhizophagus irregularis when combined with the bacterium (Pasteuria

penetrans) in the reduction in galling and root-knot nematode reproduction in tomato.Some fungi

(Fusarium oxysporum f.sp.lycopersici and Trichoderma harzianum) have been reported lately to

inhibit tomato root colonization by some Glomus species thereby reducing the growth enhancing

ability of these AMF species( Singh et al., 2014).

The judicious combination of different methods for plant parasitic nematodes control with

the aim of protecting the environment as well, is an acceptable management option world-wide. The

combined use of soil amendment and beneficial biocontrol agents in nematode management have

been recommended by many authors (Rodriguez-Kabana and Morgan-Jones, 1987; Goswami et al.,

2007; El-Sherif and Ismail, 2009; Serfoji et al., 2010). Goswami et al. (2007) observed that

amendment of soil with farmyard manure and karanj oil seed cake and inoculation with AMF

(Glomus fasciculatum) significantly reduced the wilt disease complex of pigeonpea caused by M.

incognita and Fusarium udum with the greatest improvement in growth and yield attributes. They

also noted that the combination of the two organic amendments provided an excellent substrate for

the proliferation and colonization of the test plant by the AMF.Similarly, inoculation of tomato

plants with Glomus intraradices and Pseudomonas putida in combination with composted cow

manure was more effective in gall inhibition and growth enhancement than individual application of

all the control agents (Siddiqui and Akhtar, 2008a). However, in another trial by the same authors

(Siddiqui and Akhtar,2008b), the combination of P.lilacinus with composted cow manure was more

effective than P. putida and other antagonists in root –knot disease suppression and yield

enhancement of tomato. Concomitant application of camel manure, dried leaf powder of marigold,

Trichoderma harzianum filtrate and Bacillus thuringiensis were found to suppress M. incognita

population and enhanced soybean growth more than single application of the above listed control

agents. (El-Sherif and Ismail, 2009). The combination of neem cake and AMF (Glomus mosseae)

12

was found to be more effective in reducing root-knot disease on tomato than single application of

either control agent (Rao et al., 1995). Recently, Serfoji et al. (2010) recommended the combined

use of vermicompost, AMF (Glomus aggregatum) and mycorrhiza helper bacterium (Bacillus

coagulans) in the management of M. incognita on a susceptible tomato Cv. Pusa Ruby in a sandy

loam acid soil. In most cases, soil organic amendments do promote the proliferation and

colonization of the biocontrol agents through some induced changes in the host’s rhizosphere

(Muthukumar and Udaiyan, 2002; Vestberg et al., 2005).

13

MATERIALS AND METHODS

Experimental Site

Five Screenhouse experiments and one field experiment were conducted at the Teaching and

Research Farm of the University of Calabar, Cross River State. Calabar lies in the tropical high

rainforest agroecology of the Equitorial climatic belt of Nigeria (Latitude 5o00’ and 5o40’N,

Longitude 8o04’ and 8o62’E) and about 70m above sea level (Iwena, 2008). It has a bimodal annual

rainfall distribution that ranges from 2500-3500mm with a mean annual temperature range of

22.2oC to 38.2oC and a relative humidity that ranges from 75-90%.

Source of Experimental Materials

a) Arbuscular Mycorrhizal Fungi (AMF):

Starter cultures of five species of arbuscular mycorrhizal fungi inocula with their respective

INVAM accession numbers, namely; Glomus etunicatum KE118, Glomus mosseae FR113, Glomus

clarum ML108, Gigaspora gigantea VA105 and Glomus deserticola FL912 were obtained from the

Soil Microbiology unit of the Department of Agronomy, University of Ibadan, Ibadan, Oyo State.

The accessions were collected from Kenya, France, Mali, Florida and Virginia and cultured by the

International Institute of Tropical Agriculture, Ibadan, Nigeria,

b) Planting materials:

Seeds of five species of Mucuna namely; M. pruriens var utilis, M. ghana, M.

cochichinensis, M. jaspaeda and M. pruriens IR2 were sourced from International Institute of

Tropical Agriculture (IITA), Ibadan, Oyo State, as shown in plate 1. Seeds of a susceptible cultivar

of tomato (cv. Roma VF) to root-knot nematode (M. incognita) were obtained from National

Horticultural Research Institute (NIHORT) Ibadan, Oyo State.

c) Bioformulated P. lilacinus:

14

A bioformulation containing P. lilacinus as the active ingredient with a trade name PL

GoldTM was obtained from the Biological Control Products, South Africa (Pty) Ltd. with

registration number L7698 Act No. 36/1947, as indicated in plate 2. According to the manufacturer,

it is a wettable powder spore concentrate of Paecilomyces lilacinus, a fungal nematicide with an

active ingredient of 4 x 109 spores/gramme used with a Gold starter (fungal spore activator). The

product was imported into Nigeria with the permission of Nigeria Agricultural Quarantine.

Building up of Nematode Population

An indigenous population of Meloidogyne incognita initially isolated from infected cowpea

plants growing in a field in the Teaching and Research Farm of the University of Calabar served as

a source of inoculum for all the screenhouse experiments. A stock culture of this population

maintained on Begonia plants was multiplied on Celosia agentia. Seedlings of Celosia were raised

in a heat-sterilized nursery soil. Top soil (0-15cm) was sterilized by heating in an earthern pot to a

temperature of 100oC and maintained for an hour. The seedlings were transplanted to plastic pots

containing sterile soil and inoculated with chopped galled tissues of Begonia plants. The Celosia

plants were allowed to grow for three months to ensure nematode multiplication and heavy root

gallings.

Nematode Inoculum Preparation

Heavily galled roots of Celosia agentia plants were uprooted and washed thoroughly under

a flowing tap water. Root-knot nematode eggs were extracted from the gall tissues using the method

of Hussey and Barker (1973). The galled tissues were cut into pieces of 1-2cm length and placed in

1000ml measuring cylinder. A 200 ml solution of 0.5 % sodium hypochlorite (household bleach)

poured into the cylinder which was then tightly capped.

15

PLATE 1: Seeds of different Mucuna species

16

PLATE 2: Bioformulated P. lilacinus

17

The mixture was shaken vigorously for 4mins to dissolve the gelatinous egg mass. The mixture

was then poured through 200 mesh sieve nested in a 500-mesh sieve. The eggs trapped in the 500-

mesh sieve were washed off residual sodium hypochlorite under a slow stream of cold tap water.

The eggs were then rinsed into a 2-L flask. This procedure was repeated and the number of eggs/ml

of the suspension was determined using a multiple tally counter under a stereoscopic microscope.

The inoculum density was adjusted to 500 eggs/ml.

Multiplication of Arbuscular Mycorrhizal fungi inoculum

A starter-culture of each AMF consisting of chopped roots of the trapping plant, spores,

chlamydospores and soil was multiplied in a sterilized soil. Fifty grammes of the inoculum was

applied to a plastic bucket filled with 10 kg of sterilized sandy soil and sown with 4 seeds of maize

after being surface sterilized with 0.5% sodium hypochlorite. Hoagland’s solution (half strength

low in phosphorus) was prepared as an additional source of nutrient. The Hoagland’s solution was

constituted thus:

Constituent g/litre

KNO3 25.250

MgSo4. 7H2O 24.600

Na Fe EDTA 1.835

KH2 Po4 0.348

H2BO3 0.620

Na2 Mo O4. 2H2O 0.500

Zn SO4. 7H2O 0.291

Mn Cl2. 4H2O 0.390

Cu So4. 5H2O 0.120

Ni So4 0.050

18

A 0.91ml of HCl3N was added per litre of the above constituents. In addition, 59g of Ca

(NO3)2 was dissolved separately in one litre of distilled water to prevent precipitation of other ions

in the mixture. The two solutions were further diluted ten times before use. The pots were watered

moderately for three months with the solution and afterwards, subjected to drought stress to

stimulate abundant spore production by AM fungus. The top part of the maize plant was cut off the

soil medium and roots including the fungal hyphae and spores were stored in a cool dry place until

needed for inoculation in the nursery.

Inoculation of tomato seedlings with arbuscular mycorrhizal fungus

Sandy soil was mixed with poultry manure in the ratio of 3:1 by volume and heat sterilized

in earthern pot as stated earlier. Four kilogrammes of the sterilized soil mixture was used to fill

plastic baskets. Two hundred and fifty grammes (250g) of the arbuscular mycorrhizal inoculum was

added to the 4 kg of soil mixture (Oyekanmi et al., 2007). Seeds of Roma VF tomato were surface

sterilized with 0.5% sodium hypochlorite and rinsed three times in distilled water. The seeds were

drilled in each basket and seedlings thinned to 40 per basket after emergence. Watering was done

moderately on daily basis and the seedlings kept for four weeks. Seedlings raised in baskets without

arbuscular mycorrhizal fungus served as the control.

Collection of soil samples

Top soil (0-15cm) was collected, respectively, from a fallow land in Ogoja, Ikom, Obubra,

and Calabar (Cross River State), Umudike (Abia State), Uyo (Akwa Ibom State) and Nsukka

(Enugu State). According to FAO/ UNESCO soil classification (1974), the soils belong to the order

Ultisols and are strongly acidic with low nutrient status. In each location, soil samples were

collected from 10 points in a marked out area (30m x 30m) using a soil auger and bulked to form a

composite sample. Top soil was then collected with a shovel and put into sacks with appropriate

19

labels. The soils were transported to the Screenhouse of Faculty of Agriculture, University of

Calabar.

Soil extraction for pre-planting population density of nematodes

The composite soil sample collected from each location was subjected to extraction tray

method or modified Baerman technique as outlined by Coyne et al. (2007) for pre-plant nematode

density estimation. Two hundred grammes of soil sifted through a 2mm sieve was poured into a

plastic basket lined with a paper napkin and placed on a plastic tray. Water was gently poured into

the plastic tray to just wet the soil but not covering it. The extraction apparatus was left

undisturbed on laboratory tables for a period of 48hours. Water was added to ensure that the soil

in the plastic basket (sieve) did not dry out. After this period, excess water was drained off the soil

and sieve into the plastic tray. Water collected in the plastic tray which contained the active second

stage larvae of nematodes was emptied into a labeled beaker. Wash bottle was used to rinse the

plastic tray into the beaker. The nematodes in the beaker were allowed to settle for some hours and

excess water decanted to concentrate the nematode larvae in the suspension. The nematodes in the

beaker were poured into a nematode counting dish and the number ascertained with the aid of a

stereoscopic microscope.

Soil analysis to determine arbuscular mycorrhizal fungus (AMF) spore density

The composite soil sample collected from each location was analyzed for AMF spore

density by wet-sieving and decanting method of Gerdemann and Nicolson (1963). A 100g sample

of the composite sample was weighed and poured into a 2-litre container and 1 litre of tap water

added. The soil-water mixture was shaken vigorously to free spores from soil. The suspension was

allowed to settle for about 20 seconds and the supernatant decanted through a 425-μm sieve over a

45-μm sieve. The spores/chlamydospores collected on the smaller size sieve were washed into 50ml

20

centrifuge tubes with a wash bottle. The opposing tubes were balanced by filling with equal volume

of distilled water. Centrifugation was done at 1200-1300 x g in a swinging-bucket rotor for 3mins.

The centrifuge was allowed to stop without breaking. The supernatant was carefully decanted and

the soil particles in the bottom of the tube were suspended with a chilled 1.17M solution of sucrose.

It was mixed and centrifuged immediately at 1200-1300 x g for 1.5mins and the centrifuge was

stopped by applying brake. The supernatant was poured through a small mesh sieve and spores

washed off the sieve into a counting dish with wash bottle. Spores were counted by scanning the

dish under a dissecting microscope.

Soil Analysis for physical and chemical properties

The composite soil samples collected from the different locations were air dried in the

laboratory for 4 days after collection and then ground with a soil roller. It was sifted with a 2mm

sieve. Particle size fractions were separated using Bouyoucos hydrometer method after dispersion

with sodium hexameta phosphate (calgon) as described by Udo (1986). Soil pH was determined

with the aid of a glass electrode connected to a pH meter in 1:2.5 (soil : water) ratio. Organic carbon

was determined using Walkley and Black Wet oxidation method as described by Allison (1965).

Total nitrogen was determined by micro Kjedhal method as outlined by Bremmer and Mulvaney

(1982). Available phosphorus was determined using Bray and Kurts (1945) method. Exchangeable

acidity was determined by extraction method using potassium chloride and result obtained through

the formula of Peech (1965). Exchangeable bases (Ca, Mg, K and Na) were extracted from the soil

using ammonium acetate solution. The concentration of Ca and Mg were determined by Versenate

(0.1M EDTA) titration method while that of Na and K were determined using flame photometer.

Effective cation exchange capacity (ECEC) was obtained as the summation of total exchangeable

bases and exchangeable acidity. Climatic data (temperature and rainfall.) were obtained from the

21

Nigerian Meteorological Agency (NIMET), Margaret Ekpo International Airport, Calabar, Cross

River State during the period of each study.

Experiment I: Evaluation of the host status of five Mucuna spp to Meloidogyne incognita inoculation

This experiment was carried out in the Screenhouse between October, 2008 and

January,2009 with an average maximum temperature of 32oC . Seeds of five species of Mucuna

namely M. pruriens var utilis, M. ghana, M. cochichinensis, M. jaspaeda and M. pruriens IR2 were

surface sterilized in 0.5% NaOCl and used for the study. A susceptible tomato (cv. Roma VF) to M.

incognita was included as a check. Top soil was collected from the Teaching and Research Farm of

University of Calabar and heat sterilized as stated earlier. Thirty plastic pots of diameter 15cm and

depth 25cm were filled with 3kg of the sterilized top soil. Three seeds of each Mucuna species were

planted per pot. Tomato seedlings were raised for four weeks in a sterilized top soil-poultry manure

mixture. Two weeks after emergence, the Mucuna plants were thinned to one stand per pot and

inoculated with 5000 eggs of M. incognita. Inoculation was accomplished by making three 5cm

deep holes around each stand and pouring 10 ml of the prepared nematode inoculum. At the same

time, tomato seedlings were transplanted and inoculated with the same inoculum density per pot.

The pots were arranged in a completely randomized design (CRD) fashion on the Screenhouse

benches. The treatments consisted of five species of Mucuna and the susceptible tomato as a check

with five replications giving a total of 30 experimental units. The Mucuna plants were grown for a

period of 3 months (before flowering) and the tomato 2 months. The following data were collected

at the end of the experiment:

1) Number of galls per root system

2) Root gall index (GI)

3) Number of egg masses per root system

22

4) Egg mass index (EMI)

5) Number of nematode larvae/200g of soil

6) Fresh weight of root (g)/plant

7) Fresh weight of leaf + vine (g)/plant

8) Dry weight of leaf + vine (g)/plant

For root gall assessment, plants were carefully uprooted and the root system washed under

flowing tap water. The number of galls was determined through counting. The number of egg mass

was determined by staining 1g of fresh root sample with a solution of phloxine B obtained by

dissolving 0.15g of phloxine B in 1 litre of distilled water. The egg masses stained red were counted

under a stereoscopic microscope. The total number of egg masses was extrapolated for the entire

root system (Daykin and Hussey, 1985). Root gall or egg mass index was determined on a 0-5 scale

rating according to Taylor and Sasser (1978); 0 = 0, 1 = 1-2, 2 = 3-10, 3 = 11-30, 4 = 31-100 and 5

= more than 100 galls or egg masses per root system. A composite soil sample was collected from

each treatment. A 200g sample was subjected to modified Baerman technique as outlined by Coyne

et al. (2007) for nematode extraction. The root system and the vine + leaf were separated and

weighed with the help of an electronic weighing balance. They were packed separately in a well

labeled envelope and oven dried in a hot air oven at 70oC for 48hrs and dry weight obtained by

weighing.

Experiment II: Effects of five species of Mucuna used as green manures in the management of M. incognita infecting tomato

This experiment was done in the Screenhouse between October, 2008 and January of 2009.

The test crop was a highly susceptible tomato cultivar (Roma VF) to M. incognita. Five species of

Mucuna (M. pruriens utilis, M. ghana, M. cochichinensis, M. jaspaeda and M. pruriens IR2) were

planted in the vicinity of the Screenhouse in plots measuring 1.0m x 1.0m and the plants were

23

allowed to grow for 3 months and were harvested just before flowering. Five rates of each Mucuna

spp were evaluated viz: 2, 4, 6, 8 and 10t/ha. Each rate was replicated three times. Three pots

without Mucuna amendment served as the control (0t/ha). Thus, there were a total of 78

experimental units. The rate of Mucuna soil amendment was on dry weight basis. As the Mucuna

species varied in their dry matter contents, 100g fresh (leaf + vine) matter were harvested and oven

dried in a hot air oven at 105oC for 24hrs and dry matter percentage calculated for each species.

Equivalent fresh matter weight to give a particular rate of amendment on dry matter basis was

determined for each Mucuna species. For M.pruriens utilis, 15.3 g, 30.6 g , 45.9 g, 61.2 g and 76.5

g ,M. ghana : 17.75 g, 35.5 g, 53.25 g, 71.01 g and 88.75 g, M.cochichinensis: 16.76 g, 33.52 g ,

50.28 g, 67.04 g and 83.80 g, M. jaspaeda :16.95 g, 33.90 g, 50.85 g , 67.80 g, and83.80 g, M.

pruriens IR2 : 17.14 g, 34.29 g, 51.43 g, 68.57 g and 85.71 g were the equivalent fresh matter used

for rates stated above, respectively.The experimental design was a completely randomized design

(CRD). Plastic pots (15cm diameter and 25cm deep) were filled with 3 kg of unsterilized top soil

obtained from the Teaching and Research Farm of University of Calabar. Tomato seedlings were

raised in a heat sterilized top soil : poultry manure: sharp sand (3:2:1 V/V) mixture for four weeks.

The pots were amended with appropriate quantity of fresh chopped leaf + vine of each Mucuna spp

two weeks before transplanting. Four-week-old tomato seedlings were transplanted one per pot and

inoculated with 5000 eggs of M. incognita as described previously. The number of nematode

larvae in the composite sample from the unsterilized soil was determined based on modified

Baerman technique as outlined by Coyne et al. (2007).

24

The tomato plants were grown to full maturity and the following data were collected at the end of

the trial:

1) Number of galls/root system

2) Gall index (GI) on a 0-5 scale

3) Number of egg masses/root system

4) Egg mass Index (EI) on a 0-5 scale.

5) Number of nematode larvae/200g of soil

6) Plant height (cm)/plant

7) Fresh root weight (g)/plant

8) Dry shoot weight (g)/plant

9) Number of fruits/plant

10) Total weight of fresh fruits/plant

Also, leaf + vine of the different Mucuna spp were harvested just before flowering (3

months after planting) and oven dried at 70oC for 48hrs. The samples

were ground to powder in a porcelain mortar and used for N, P and C analysis. Another portion of

the dried sample was dry-ashed at 500oC for 4 hrs and used for the analysis of other elements.

Macro and micro elements were determined according to procedures described by Tel and Rao

(1982). Total N was determined by Kjeldahl approach, P by Vanadomolybdate Colorimetry, K by

flame photometer and Ca, Cu, Mg, Mn and Zn by atomic absorption spectro-photometry. Analysis

was done in triplicates. The ratio of carbon to nitrogen was calculated.

Experiment III: Greenhouse evaluation of the effects of Mucuna spp green manure soil amendment and arbuscular mycorrhizal fungi (AMF) on the pathogenicity of M. incognita on tomato

This experiment was carried out in the Screenhouse from January to April, 2009. The five

species of Mucuna were cultivated in plots around the vicinity of the Screenhouse as described in

25

Experiment II. However, the optimum rate of amendment of each species of Mucuna based on the

outcome of experiment II was chosen for this trial. The optimum rate of amendment chosen was 8 t/

ha for all the Mucuna species. Tomato cv .Roma VF which is highly susceptible to M. incognita

was used as the test plant. The experiment was laid out as a 6 x 6 factorial in a completely

randomized design (CRD) with three replications. Five species of velvet bean (M. pruriens utilis,

M. ghana, M. cochichinensis, M. jaspaeda and M. pruriens IR2) used as green manure soil

amendment plus unamended soil (control) were factorially combined with five species of AMF

(Glomus etunicatum, Glomus mosseae, Glomus clarum, Glomus deserticola and Gigaspora

gigantea) plus uninoculated control to give 36 treatment combinations. Unsterilized top soil (0-

15cm) collected from the Teaching and Research Farm of University of Calabar was used to fill 108

plastic pots at the rate of 3kg per pot. Composite soil sample was analyzed for pre-plant nematode

density and AMF spore density as described by Coyne et al. (2007) and Gardemann and Nicolson

(1963), respectively. Each pot was amended with the chosen quantity of each Mucuna spp on dry

weight basis, equivalent to 8 t/ h as described in experiment II. Two weeks after amendment, four-

week-old tomato seedlings biologically enhanced with the different species of AMF through

nursery inoculation were transplanted to velvet bean amended soil. Each seedling was inoculated

with 5000 eggs of M. incognita as described in previous experiments. Plants were watered

appropriately throughout the period of growth. Plants were grown to full maturity and the following

data were collected:

1) Number of galls/root system

2) Gall index on a 0-5 scale

3) Number of egg masses/root system

4) Egg mass index on a 0-5 rating scale

5) Fresh root weight (g)/plant

6) Plant height (cm)/plant

26

7) Dry shoot weight (g)/plant

8) Percentage root mycorrhizal colonization.

9) Relative mycorrhizal effectiveness (RME) %

10) Number of fruits/plant

11) Total weight of fresh fruits (g)/plant

The relative mycorrhizal effectiveness (RME) was calculated based on Vestberg et al.

(2005) formula:

RME (%) = Ymyc+ - Ymyc- x 100

Ymyc+

Where Ymyc+ and Ymyc- are the shoot dry weights of the mycorrhizal and non-mycorrhizal

plant, respectively. Percentage root mycorrhizal colonization was determined after staining root

samples with 0.05% trypan blue in lactic acid after Phillips and Hayman (1970) as modified by

Koske and Gemma (1989). Representative feeder root samples (0.5g each) were washed, fixed and

stored in 50% ethanol. Fixed roots were placed in 10% aqueous solution of KOH for 24 hours

under room temperature, after which the roots were rinsed in several changes of water. The roots

were then bleached in freshly prepared solution of alkaline hydrogen peroxide (H2O2) until the

tissues in the roots became transparent. The solution of H2O2 was washed off the roots using tap

water and the roots were acidified over night in 2% HCl. The acidified roots were stained in a

solution of 0.05% trypan blue in 70% glycerol. The stained roots were destained in acidified

glycerol at room temperature for mycorrhizal colonization assessment. Mycorrhizal root

colonization were evaluated using the grid-line intersect technique of Giovannetti and Mosse

(1980). Stained root samples were evenly spread in a petridish with grid lines of uniform distances

apart on the bottom to form 12.7cm2. Vertical and horizontal lines were scanned at 15-45X

magnification with a dissecting microscope. The total number of root intersections with the grid as

27

well as the number of intersects with colonized roots (hyphae, vesicles or arbuscles) were recorded.

Percentage root colonization (PRC) was calculated as follows:

PRC = Number of gridline with colonization x 100

Total number of gridlines intersects counted

Experiment IV: Field evaluation of the effects of Mucuna spp green manure and AMF on the pathogenicity of M. incognita on tomato.

This experiment was carried out in the Teaching and Research Farm of the Faculty of

Agriculture, University of Calabar from May to December of 2009. It confirmed the findings of

experiment III in the field. The area used for the trial was planted with crops known to be

susceptible to root-knot nematode (i.e. okra, garden egg, cucumber, etc.) for the past three years

which guaranteed natural infestation. An area of land measuring 46m x 34m (0.156ha) was marked

out for the trial. Soil samples were taken randomly from 12 points with the help of a soil auger to

the depth of 15cm prior to land preparation. It was bulked and a composite sample taken for

physico-chemical properties analysis, pre-plant nematode density and AMF spore density

determination as described in the previous sections. The experiment was laid out as a split-plot in

randomized complete block design (RCBD) with 3 replications. The main-plot was represented by

the five species of Mucuna (M. pruriens utilis, M. ghana, M. cochichinensis, M. jaspaeda and M.

pruriens IR2) and control (natural fallow or no amendment). The sub-plots consisted of the five

AMF species as stated in experiment III plus control (non-mycorrhizal tomato). The test crop was a

highly susceptible tomato cultivar (Roma VF) to root-knot nematode. Thus, there were 36 treatment

combinations replicated three times which gave a total 108 sub-plots or experimental units. The

vegetation was cleared manually and the land tilled with a disc plough. It was then harrowed with a

disc harrow, thrash and stump removed to provide a fine tilth. The land was divided into three

blocks perpendicular to the slope. A block measured 32m x 14m with a footpath of 2m separating

them. Each block was divided into 6 main plots each measuring 10m x 6.5m and separated with a

28

foot path of 1m. The five species of Mucuna were randomly assigned to the main plots while a

natural weed fallow served as the control. Seeds of each Mucuna spp were planted to the respective

main-plot at a spacing of 0.50m x 0.50m, two seeds per stand. Two weeks after emergence, it was

thinned to one per stand giving a population of 260 plants/main-plot (40,000 plants/ha). After 3

months (just before flowering) of growth, the leaf + vine of the velvet bean were harvested

randomly and oven dried for dry matter determination for each species as described earlier. In each

main-plot, the velvet bean was slashed and ploughed into the 15cm soil depth based on the chosen

dry matter rate in experiment II. M. pruriens utilis, M. ghana, M .cochichinensis, M. jaspaeda and

M. pruriens IR2 were applied at 265.30, 307.70, 290.17, 293.80 and 297.10 kg / main- plot as fresh

green manure equivalent to 8 t/ ha application on dry matter basis. For the natural weed fallow, a

quadrate was used to determine the predominant weed flora species. The natural fallow was

cleared, packed and tilled. Each main plot was split into 6 sub-plots to accommodate the AMF

species. A sub-plot measured 3m x 3m with a footpath of 0.5m separating it from another. Four-

week-old tomato seedlings inoculated with the respective arbuscular mycorrhizal fungus species at

the nursery stage as described previously were transplanted at a spacing of 60cm between rows and

40cm within a row into each sub plot (≈ 38 plants/sub-plot ≡ 41,667 plants/ha). Non-myccorhizal

seedlings served as control. Weeding was done manually with a weeding hoe as and when

necessary. Inorganic fertilizer and synthetic pesticides were not applied. Plants were grown to full

maturity and 20 tomato plants were randomly uprooted and ten mature females were teased out of

their galls, stained and then fixed for species identification through perineal pattern examination.

Prior to slashing and ploughing in the Mucuna in situ, 20 stands of the Mucuna plants in each

main-plot were randomly uprooted and evaluated for root galling and egg mass production. Also,

composite soil samples were obtained from each main-plot at midseason (four weeks after

transplanting of tomato or 6 weeks after incorporation of Mucuna green manure) for physico-

29

chemical properties analysis as stated earlier. Data were obtained as in experiment III plus the

following:

1. Percentage emergence of Mucuna spp in each main-plot (two weeks after planting).

2. Gall index and Egg mass index for each Mucuna spp in the main-plot.

3. Root-knot incidence on tomato (%).

4. Mean Gall Index (MGI) at 0-5 scale.

Mean gall index (MGI) per sub-plot was calculated as the product of the rating scale (Gall

index) and the frequency product of the plants (Vito et al., 1996) and root-knot incidence as the

ratio of the sum of infected plants to the entire plant population for sub-plot.

Experiment V: Evaluation of the effects of Paecilomyces lilacinus and AMF against M. incognita on tomato

Separate Screenhouse experiments were carried out on soils obtained from the different

locations in southeastern Nigeria namely: Ogoja, Obubra, Ikom, Calabar (Cross River State), Uyo

(Akwa Ibom State), Umudike (Abia State) and Nsukka (Enugu State).The experiments were

conducted from June to October of 2010. The test plant was a highly susceptible tomato cultivar

(Roma VF) to M. incognita. The experiment was laid out as a 3 x 6 factorial in CRD with 3

replications. The formulated biocontrol agent containing P. lilacinus (PL GoldTM) was applied at 3

frequencies. No application (control), applied once at transplanting and applied twice as

recommended by the manufacturer (i.e. at transplanting and later at 2 weeks after transplanting).

Five arbuscular mycorrhizal species used in the previous experiments (Glomus etunicatum, G.

mosseae, G. clarum, G. deserticola and Gigaspora gigantea) plus an uninoculated control were

combined in a factorial fashion with the bionematicide application frequency to give a total of 18

treatment combinations replicated thrice to give a total of 54 experimental units (pots) per soil type.

Fifty grammes (50g) of the spore powder was weighed out and mixed with 50 ml of the spore

activator resulting in a mixture ratio of 1:1. It was allowed to stand for an hour before further

30

dilution with 30litres of distilled water. This mixture was sufficient to treat 1000 tomato seedlings

(i.e. 0.05g spore powder/plant ≡ 2 x 108 spores/plant). Tomato seedlings raised in a heat-sterilized

soil inoculated with the different AMF were transplanted at four weeks and inoculated with 5,000

eggs of M. incognita per stand as previously described in pots filled with 3kg of soil from each

location. Prior to bionematicide application, each seedling was irrigated lightly with tap water, then

inoculated with 30 ml of the spore mixture. The spores were later flushed down the 15cm depth of

the root zone with excess irrigation water. Application was repeated two weeks after the first

application for treatments that required two applications. Plants were allowed to grow up to full

maturity and data were collected as in experiment III.

Experiment VI: Evaluation of the effects of P. lilacinus (PL GoldTM), arbuscular mycorrhizal fungi and Mucuna green manure on the pathogenicity of M. incognita on tomato

This experiment was conducted in the Screenhouse of the Faculty of Agriculture, University

of Calabar from June to October of 2010. It was laid out as a 2 x 6 x 6 factorial experiment in a

completely randomized design (CRD) with three replications. The test plant was a highly

susceptible tomato cultivar (Roma VF) to M. incognita. The bioformulated product of P. lilacinus

was applied at two frequencies (no application, double application: at transplanting and at two

weeks later). Also, the five species of Mucuna used in experiment IV were grown, harvested 3

months after planting and applied at the same rate( 8 t/ha) on dry matter basis as a green manure

soil amendment. Pots uninoculated with bionematicide and not amended with velvet bean served as

the control. The two treatments were combined in a factorial fashion with five arbuscular

mycorrhizal fungi (Glomus etunicatum, G. mosseae, G. clarum, G. deserticola and Gigaspora

gigantea) plus non-mycorrhizal plants (control) to give 72 treatment combinations with three

replications resulting in 216 experimental units (pots). Unsterilized top soil (0-15cm) collected from

the Teaching and Research Farm of the University of Calabar was used to fill 216 plastic pots at

31

the rate of 3 kg per pot. Each pot was amended with the chosen quantity of Mucuna species on dry

weight basis as described in experiment III. Two weeks after amendment, four-week-old tomato

seedlings raised in sterile soil inoculated with the various AMF were transplanted to the Mucuna

amended soil. Each seedling was inoculated with 5,000 eggs of M. incognita as described in the

previous experiments. The spore powder was prepared as described in experiment V. Each seedling

was inoculated with 30 ml of the spore mixture during transplanting and two weeks after the first

application. The pots were labeled appropriately. Seedlings were watered appropriately and grown

to full maturity. Data were collected as in experiment III.

STATISTICAL ANALYSIS

Data obtained on nematode counts were transformed using Log10(x+1) prior to analysis. For

experiments I and II, data were subjected to analysis of variance (ANOVA) in a completely

randomized design (CRD) and mean separations were achieved through Duncan’s New Multiple

Range Test (DNMRT) at 5% level of probability according to Obi (1986). However, for experiment

II, plant responses to different rates of Mucuna amendments were tested with linear or curvilinear

models (Ritzinger and McSorley, 1998). For experiments III and V, data obtained were subjected

to ANOVA for a two-factor factorial experiment in CRD as outlined by Steel and Torrie (1980) and

significant differences among means were detected by Fisher’s Least Significant Difference (F-

LSD) according to Obi (1986). For experiment IV, data collected were analyzed through ANOVA

for a split-plot in Randomized Complete Block Design (RCBD) and significant means separated

with F-LSD at 5% level of probability. For experiment VI, the data obtained were subjected to

ANOVA for a three-factor factorial experiment in CRD and means separated with the aid of F-LSD

at 5% probability level. All statistical analyses were carried out using GenStat Release 7.11 (7th

edition) software.

32

RESULTS AND DISCUSSION

Physico-chemical Properties, Arbuscular Mycorrhizal Fungi Spore Density and Pre-plant Nematode Density of the Soils used for the Experiments.

The results of the physico-chemical and microbiological properties of soils from different

locations used for the experiments are presented in Table 1. Generally, the soils from most of the

locations were strongly acidic with the exception of Obubra, Uyo and Umudike soils that were

slightly acidic. Calabar soil was moderately acidic. The organic matter contents varied among the

locations. It was rated medium in Uyo soil, low in Calabar and Obubra soils but very low in the

other soil locations. The total N content of the soils was very low in some locations but was rated

medium in Uyo soil, low in Calabar and Obubra soils. The available P was low in the soils from

some locations. However, it was high in Uyo soil and medium in Calabar and Umudike soils. The

amount of exchangeable K in the soil from all the locations was very low with the exception of

Nsukka soil that was rated low.

Exchangeable calcium varied among the soil locations. It was low in Ikom, Ogoja and

Umudike soils, medium in Calabar, Obubra and Nsukka soils but high in Uyo soil. The

exchangeable Mg was low in most of the locations, high in Ikom and Obubra soil but very high in

Nsukka soil. Soils from all the locations were very low in exchangeable Na. The soils varied in their

effective cation exchange capacity (ECEC). The soils were generally low in ECEC with the

exception of Uyo soil that was rated medium. The ECEC of Umudike soil was very low. The

percentage base saturation of the soil from all the locations was rated very high with the exception

of Ogoja soil that was rated high. The soils also varied in texture. Ogoja soil had the highest sand

content followed by Umudike, Uyo and Calabar. Thus Uyo, Umudike and Calabar soils were loamy

sand in texture, Ogoja soil was sandy, Obubra and Nsukka soils were sandy loam, while Ikom soil

was sandy clay loam (Table1). The AMF spore density and pre-plant nematode density varied

among the soils from different location. The highest mycorrhizal spore density was recorded in

33

Nsukka and Obubra soil while the least was found in Ogoja soil. The nematode density was high in

Calabar, Obubra and Ogoja soils.

Climatic Data

Table 2 presents the monthly maximum temperatures and monthly rainfall data during the duration

of the trial (2008-2010). The highest temperatures were recorded in the dry months (December –

March), while the lowest temperatures were recorded in the wet months (June – July) for the study

period. The highest amount of rainfall was recorded in the months of July-September in 2008, July-

August in 2009 and June in 2010. However, it is worth noting the sharp decline in the amount of

rainfall from August to September, 2009 which was the year in which the field experiment was

conducted. There was no rainfall in any of the days in the month of December 2009.

34

Table 1: Physico-Chemical Properties, AMF Spore Density and Pre-plant Nematode Density

of the Soils used for the Experiments.

Soil property Location of soil

Calabar Ikom Obubra Ogoja Nsukka Umudike Uyo

pH (H20) 5.90 5.30 6.50 5.40 5.50 6.30 6.50

Org. matter (%) 2.58 1.79 3.52 1.17 1.49 1.48 4.07

Total N(%) 0.12 0.08 0.17 0.05 0.07 0.05 0.20

AV.P (mg/kg) 43.00 8.25 2.25 3.00 6.00 24.75 73.00

Exch. K (Cmol/Kg) 0.14 0.14 0.18 0.10 0.22 0.10 0.19

Exch. Ca (Cmol/Kg) 6.00 3.20 9.40 3.00 6.80 3.40 18.00

Exch. Mg (Cmol/Kg) 0.80 3.40 3.80 0.60 31.20 0.40 1.00

Exch Na (Cmol/Kg) 0.09 0.08 0.10 0.07 0.12 0.06 0.11

Exch. Al3+ (Cmol/Kg) 0.00 0.00 0.20 0.00 0.16 0.00 0.00

Exch. H+ (Cmol/Kg) 0.64 0.72 0.50 1.54 0.72 0.64 0.64

ECEC (Cmol/Kg) 7.39 7.54 14.00 5.31 14.00 4.60 19.94

BS (%) 98.00 90.00 96.00 71.00 96.00 86.00 97.00

Clay (%) 10.20 23.40 20.00 2.00 3.00 7.00 5.00

Silt (%) 5.70 10.20 12.00 6.70 13.00 5.70 9.70

Sand (%) 84.30 66.40 68.00 91.30 64.00 87.30 85.30

Texture Loamy sand Sandy

clay loam

Sandy

loam

Sandy

soil

Sandy

loam

Loamy

sand

Loamy

sand

AMFspore

density/100g soil

195.00 190.00 243.00 97.00 255.00 110.00 125.00

Pre-plant Nematode

density/200g soil

245.00 115.00 214.00 205.00 163.00 125.00 183.00

35

Table 2: Monthly Maximum Temperature (0c) and Rainfall (mm) during the period of study

(2008-2010) in Calabar, Cross River State.

Max Temperature (0c) Rainfall (mm)

Month 2008 2009 2010 2008 2009 2010

January 31.10 32.30 33.80 151.00 897.00 318.00

February 34.10 32.80 33.10 10.00 385.00 882.00

March 32.20 33.30 33.00 1,081.00 875.00 636.00

April 31.50 32.10 33.10 2,169.00 1,505.00 1,304.00

May 31.10 31.60 31.50 2,868.00 3,089.00 3,065.00

June 29.40 30.00 29.80 4,370.00 2,184.00 6,113.00

July 28.50 28.90 28.80 5,977.00 5,774.00 3,840.00

August 28.40 28.10 28.20 5,092.00 5,073.00 4,067.00

September 29.70 29.70 28.90 5,179.00 2,739.00 4,513.00

October 30.50 30.00 31.70 3,150.00 1,481.00 2,696.00

November 31.40 31.40 30.80 1,051.00 1,269.00 2, 721.00

December 33.00 33.00 31.70 771.00 0.00 562.00

Mean 30.80 31.10 31.20 2,406.00 2,106.00 2,550.00

(Source: Nigerian Meterological Agency (NIMET, 2012) Margaret Ekpo International Airport,

Calabar)

36

Experiment I: Evaluation of the Host status of five Mucuna spp to Meloidogyne incognita

inoculation

The results of the response of five Mucuna species to infection by M. incognita are shown in Table

3. The roots of all the Mucuna spp were not galled and no eggmass was found on their roots, as

shown in Plate 3. All the Mucuna spp were rated immune to M. incognita infection with a gall

index (GI) = 0.00 and Eggmass index (EMI) = 0.00. However, the test crop (tomato cv. Roma VF)

used as a check was heavily galled with GI = 5.00 and with more than 100 eggmasses per root

system. It was rated highly susceptible. Compared with the Mucuna spp, the number of nematode

larvae in the tomato rhizosphere was significantly higher. Among the Mucuna spp, M. pruriens IR2

rhizosphere haboured significantly (P≤0.05) the highest nematode larvae, followed by M.

cochichinensis. The rhizosphere of M. jaspaeda and M. ghana haboured significantly the least

nematode population (Table 3). There were significant (P≤0.05) variations in fresh root weight

among the Mucuna spp. M. cochichinensis had significantly the heaviest root mass followed by M.

ghana while M. pruriens IR2 had the least (Table 4). Almost a similar trend was observed with the

above ground fresh matter. However, M. pruriens Utilis had the least above- ground fresh weight.

For the above- ground dry weight, the trend was different .M. pruriens Utilis had significantly

(P≤0.05) the highest above-ground dry matter M. ghana and M. pruriens IR2 accumulated the least

above ground dry matter (Table 4). In comparison, the check plant (tomato) had significantly

(P≤0.05) lower below-ground biomass than any of the Mucuna spp.

37

PLATE 3: Some Mucuna species showing Gall-free roots.

38

Table 3: Number of galls and eggmasses/root system and number of nematode larvae recovered from 200 g of soil planted with Mucuna spp and susceptible tomato (Roma VF) and inoculated with M. incognita.

Mucuna spp No. Galls/root system

Gall Index* No. Eggmasses/root system

Eggmass Index* No. nematode larvae/200g soil

M. pruriens utilis 0.00(0.71)** 0.00 0.00(0.71)** 0.00 581.00(13.81)***

M. Ghana 0.00(0.71) 0.00 0.00(0.71) 0.00 356.00(12.75)

M. cochichinensis 0.00(0.71) 0.00 0.00(0.71) 0.00 690.00(14.19)

M. jaspaeda 0.00(0.71) 0.00 0.00(0.71) 0.00 316.20(12.50)

M. pruriena IR2 0.00(0.71) 0.00 0.00(0.71) 0.00 1,000.80(15.00)

Tomato (check) 206.60(14.39) 5.00 125.00(11.19) 5.00 11,615.20(20.32)

F-LSD (0.05) 0.20 0.35 0.04

* 0 = Immune, 1 = Highly resistant, 2= Resistant, 3=Moderately Susceptible, 4=Susceptible, 5 = Highly Susceptible

** Figures in parentheses are square root transformed data ( ) to which F-LSD apply

*** Figures in parentheses are Logx+1 transformed data to which F-LSD apply.

39

Table 4: Effects of M. incognita inoculation on fresh root weight (g)/Plant, fresh and dry above-ground weight (g) / plant of five Mucuna spp and a susceptible tomato CV. Roma VF.

Mucuna spp Fresh root weight (g)/plant

Above ground fresh weight (g)/plant

Above-ground dry weight (g) / plant

M. pruriens utilis 29.02 196.81 38.53

M. Ghana 30.28 202.85 35.08

M. cochichinensis 31.94 207.19 36.97

M. jaspaeda 29.28 199.92 36.36

M. pruriens IR2 27.77 199.14 35.46

Tomato (check) 19.45 86.31 18.16

F-LSD (0.05) 0.94 4.20 1.06

40

Experiment II: Effects of five species of Mucuna used as Green Manures in the

management of M. incognita infecting tomato.

The results of soil amendment with the different rates of Mucuna spp on nematode

infectivity and egg production are presented in Table 5. Amendment of soil with any Mucuna spp

irrespective of the rate significantly (P≤0.05) inhibited root galling by M. incognita on tomato

compared with unamended (control) soil. However, the efficacy of gall inhibition differed

significantly (P≤ 0.05) among the Mucuna species even at the same rate as well as in a particular

Mucuna species at different rates. Thus, in all the Mucuna species, successive increase in the rate of

amendment resulted in a significant decrease in the number of galls produced per root system.

Among all the Mucuna species tested, at the highest rate of amendment (10t/ha), M. jaspaeda and

M. ghana amended soil had significantly (P≤ 0.05) plants with the fewest number of galls,

followed by M. pruriens utilis.

However, M. pruriens IR2 amended soil had the highest number of galls per root system. For root-

gall severity rating among the Mucuna species, only M. ghana and M. jaspaeda amendment at the

lowest rate (2t/ha) significantly (P≤0.05) reduced the GI to 4.00 compared with the unamended soil

(control) with GI = 5.00 (Table 5). For M. cochichinensis and M. pruriens IR2, increase in the

amendment rate up to 4t/ha did not effect a significant change in GI compared with the control

(unamended) soil. Amendment of soil with M. jaspaeda and M. ghana at the highest rate (10t/ha)

caused a significant (P ≤0.05) change in the gall rating of the tomato cultivar with GI = 5.00

(unameded soil) to GI = 2.33 and 2.67, respectively. For the other Mucuna spp, the gall index (GI)

was changed from 5.00 to 4.00. Eggmass production almost followed the trend of root galling. Soil

amended with Mucuna irrespective of the rate or species significantly (P≤0.05) inhibited egg

production by M. incognita compared with the unamended soil (control).

41

However, the efficacy of eggmass production inhibition varied significantly among the Mucuna spp

when compared at similar rate of amendment. At all rates of amendment, M. jaspaeda and M.

ghana significantly (P≤0.05) reduced eggmass production more than other species of Mucuna

.Increase in the rate of application of amendment significantly (P ≤0.05) reduced egg production by

all the Mucuna spp with the exception of M. jaspaeda and M. ghana ,where there was no significant

(P≤0.05) difference between 8 and 10 t/ha rates. The fewest eggmass was observed in roots of

tomato where the soil was amended with 10t/ha of M. jaspaeda and M. ghana. Amendment with

Mucuna significantly (P≤0.05) reduced the eggmass index (EMI) relative to the unamended soil.

However, there were no significant (P≤0.05) differences among the rates in M. cochichinensis and

M. pruriens IR2. The least EI of 2.00 was obtained in plants where the soil was amended with

10t/ha of M. jaspaeda and M. ghana. The results of the regression analysis between number of galls

and amendment rates of Mucuna are presented in Fig.1. For all the Mucuna species tested, the

response clearly depicted a strong and highly significant (P≤0.01) inverse linear relationship with

‘r’ values > - 0.80. The coefficient of determination ‘r2’ was high for all the species of Mucuna

indicating that the response fits a linear model.

The result of the effects of soil amendment with different Mucuna spp at various rates on

nematode larval population, growth and biomass of tomato plant are presented in Table 6. Soil

amendment with Mucuna significantly (P≤0.05) reduced soil nematode population compared with

the unamended soil (control). In each Mucuna species, there was a significant decrease in nematode

population with successive increase in the rate of amendment. Soil amended with 10t/ha of M.

jaspaeda had significantly (P≤0.05) the lowest nematode population followed by M. ghana at the

same rate. The fresh root biomass of tomato increased significantly with Mucuna amendment

relative to the unamended soil (control).

42

Table 5: Effects of rates of different Mucunna spp soil amendment on number of galls/root system gall index (GI)*, number of Eggmasses/root system and Eggmass Index (EMI) of tomato inoculated with M. incognita.

Mucuna spp/rate(t/ha) No. Galls/root system

Gall Index No. Eggmasses/root system

Eggmass Index

Control/ (0.00) 235.00a** 5.00a 181.00a 5.00a 2.00 111.67cd 5.00a 82.00b 4.00b 4.00 95.00efg 4.33ab 59.33cd 4.00b M. pruriens 6.00 74.33ijkl 4.00b 46.67efg 4.00b Utilis 8.00 65.00klmn 4.00b 37.00fghi 4.00b 10.00 45.00o 4.00b 25.00jk 3.00d 2.00 89.67fgh 4.00b 46.67efg 4.00b M. Ghana 4.00 76.00ijk 4.00b 35.67hi 4.00b 6.00 55.67n 4.00b 28.00ijk 3.00d 8.00 28.67p 3.33c 15.00lm 3.00d 10.00 12.33qr 2.67cd 9.00m 2.00f 2.00 143.33b 5.00a 77.33b 4.00b 4.00 112.67cd 5.00a 63.33cd 4.00b M. cochichinensis 6.00 99.00ef 4.67a 53.67de 4.00b 8.00 80.00hij 4.00b 40.00fgh 4.00b 10.00 62.33mn 4.00b 34.67hij 4.00b 2.00 69.67jklm 4.00b 36.67ghi 4.00b 4.00 64.33lmn 4.00b 31.33hij 3.67c M. jaspaeda 6.00 43.67o 4.00b 23.00kl 3.00d 8.00 22.33pq 3.00c 12.33m 2.67e 10.00 10.67r 2.33d 6.00m 2.00f 2.00 121.67c 5.00a 82.33b 4.00b 4.00 104.33de 5.00a 67.67c 4.00b M.pruriens IR2 6.00 100.00ef 4.00b 65.67c 4.00b 8.00 85.00ghi 4.00b 54.67de 4.00b 10.00 72.00jklm 4.00b 37.67fghi 4.00b

**Means followed by the same letter within a column are not significantly different according to Duncan’s new multiple Range Test at 5% probability level.

43

Figure 1: Effects of different rates of Mucuna spp on root galling of tomato infected with M. incognita

0

25

50

75

100

125

150

175

200

225

250N

umbe

r of

Gal

ls p

er r

oot s

yste

m

-2 0 2 4 6 8 10 12M. pruriens utiliis (t / ha)

Y = 183.67 - 15.87x r = -0.87** R^2 = 0.76

Regression Plot

0

25

50

75

100

125

150

175

200

225

250

275

Num

ber

of G

alls

per

roo

t sys

tem

-2 0 2 4 6 8 10 12M. ghana (t /ha)

Y = 176.67 - 18.81 r= -0.88** R^2 = 0.77

Regression Plot

40

60

80

100

120

140

160

180

200

220

240

260

Num

ber

of G

alls

per

roo

t sys

tem

-2 0 2 4 6 8 10 12M. cochichinensis (t / ha)

Y = 198.27 - 15.24 r = -0.91** R^2 = 0 .83

Regression Plot

0

25

50

75

100

125

150

175

200

225

250

275

Num

ber

of G

alls

per

roo

t sys

tem

-2 0 2 4 6 8 10 12M . jaspaeda (t ha)

Y = 166.02 - 8.34 r =-0.84** R^2 = 0.70

Regression Plot

60

80

100

120

140

160

180

200

220

240

260

Num

ber

of G

alls

per

roo

t sys

tem

-2 0 2 4 6 8 10 12M. pruriens IR 2 (t / ha)

Y = 186.05 - 13.28 r = -0.84 ** R^2 = 0.70

Regression Plot

44

Table 6: Effects of different rates of Mucuna spp soil amendment on number of nematode

larvae/200g soil, fresh root weight (g)/plant, shoot length (cm)/plant and dry shoot

weight(g)/plant of tomato inoculated with M. incognita

Mucuna spp/rate(t/ha) No. larvae/200g soil Fresh root weight(g)/plant

Shoot length (cm)/plant

Dry shoot weight(g)/plant

Control (0.00) 12,310.00 (4.09a)** 16.80l* 50.00m 16.44l 2.00 10,606.67 (4.03bc) 19.38k 65.00kl 18.40k M. pruriens 4.00 8,412.33 (3.93fg) 21.01ij 71.67ij 20.17j Utilis 6.00 7,497.00 (3.87h) 22.41hi 77.33fgh 22.49h 8.00 5,096.67 (3.71j) 24.43cdefg 83.33cde 23.58g 10.00 3,600.00 (3.55n) 23.83defgh 79.33efg 21.89h 2.00 6,193.33 (3.79i) 23.49fgh 71.67ij 26.18e 4.00 4,638.33 (3.67l) 24.35cdefg 80.00efg 28.30d M. Ghana 6.00 3,935.33 (3.59m) 26.16b 85.33bcd 30.65c 8.00 3,145.00 (3.50o) 29.32a 90.00ab 31.97b 10.00 1,792.00 (3.25q) 28.83a 90.33ab 31.49bc 2.00 11,083.33 (4.04b) 20.24jk 62.00l 20.15j M. cochichinensis 4.00 10,071.67 (4.00cd) 21.07ij 65.33kl 21.72hi 6.00 8,790.00 (3.94ef) 23.75efgh 71.17ij 23.73fg 8.00 6,700.33 (3.83i) 25.48bcd 78.33efgh 25.73e 10.00 4,993.33 (3.70jkl) 25.03bcdef 75.33ghi 24.67f 2.00 5,231.66 (3.72j) 21.47ij 73.67hi 27.42d 4.00 3,925.33 (3.59m) 24.47cdefg 81.00def 29.31c M. jaspaeda 6.00 3,080.00 (3.49o) 25.96bc 86.33bcd 31.29bc 8.00 2,710.00 (3.43p) 29.44a 92.00a 33.03a 10.00 948.00 (2.99r) 29.20a 87.00abc 31.97b 2.00 10,784.00 (4.03bc) 21.15ij 65.67kl 20.07j 4.00 9,436.66 (3.97de) 22.50hi 68.33jk 20.81ij M.pruriens IR2 6.00 7,933.33 (3.90gh) 23.27gh 86.00bcd 22.38h 8.00 6,760.00 (3.83i) 25.81bc 88.00abc 23.71fg 10.00 4,906.67 (3.69kl) 25.28bcde 83.00cde 23.45g

*Means followed by the same letter within a column are not significantly different according to Ducan’s New Multiple Range Test at 5% probability level. **Figures in parantheses are Logx+1 transformed data to which F-LSD applies.

45

In most of the Mucuna spp, increase in the rate of amendment caused a significant increase in fresh

root weight. However, in all the Mucuna spp, there was a non significant decrease as the rate was

increased from 8 to 10 t/ha. Soil amendment with Mucuna significantly (P≤0.05) enhanced tomato

growth compared with unamended soil. In most of the Mucuna spp, increase in the amendment rate

significantly increased plant height. However, at 10t/ha rate, there was a decline in growth. Tallest

plants were obtained with M. jaspaeda and M. ghana at 8t/ha rate.

Dry shoot matter accumulation followed the trend of shoot length (Table 6). However, with

the exception of M.ghana and M. pruriens IR2, increase in amendment rate from 8 to 10t/ha

significantly inhibited shoot dry matter accumulation. There was no significant difference (P>0.05)

between Mucuna amended soil and the unamended soil in the number of fruit set with the exception

of M. jaspaeda amended at 8t/ha (Table 7). All rates of Mucuna spp amendment significantly (P≤

0.05) increased fresh fruit weight compared with the unamended soil. With the exception of M.

jaspaeda and M. ghana, successive increase in amendment rate did not cause a significant (P>0.05)

increase in fresh fruit yield. Increase in amendment rate from 8 to 10t/ha significantly inhibited fruit

yield in M. jaspaeda and M. ghana. M. jaspaeda amendments produced significantly the highest

fresh fruit yield at 8t/ha amendment (Table 7). The regression analysis results between Mucuna

rates and total fresh fruit yield indicates a strong positive highly significant (P≤ 0.01) linear

relationship (Fig.2). The coefficient of correlation ‘r’ values were high (r > 0.70). The r2 values

were also high with the exception of M. ghana which was moderate, indicating that the plant

response to Mucuna amendment rates fits into a linear regression model.

46

Table 7: Effects of different rates of Mucunna spp soil amendment on number of fruits/plant and total fresh fruit weight (g)/plant of tomato inoculated with M. incognita Mucuna spp/rate(t/ha) No. of fruits/plant Total fresh fruit weight (g)/plant Control/ (0.00) 1.00b 16.21m 2.00 1.00b 24.70ijk M. pruriens 4.00 1.00b 26.39hij Utilis 6.00 1.33ab 29.87fg 8.00 1.33ab 30.95f 10.00 1.67ab 30.38fg 2.00 1.00b 34.55e M. ghana 4.00 1.00b 36.16de 6.00 1.00b 38.59cd 8.00 1.67ab 41.02c 10.00 1.67ab 38.06d 2.00 1.00b 22.65k 4.00 1.00b 25.16ijk M. cochichinensis 6.00 1.00b 26.65hij 8.00 1.33ab 29.82fg 10.00 1.67ab 29.07fgh 2.00 1.00b 37.52d 4.00 1.00b 41.23c M. jaspaeda 6.00 1.33ab 46.14b 8.00 2.00a 50.26a 10.00 1.67ab 47.33b 2.00 1.00b 22.88k 4.00 1.00b 23.61jk M.pruriens IR2 6.00 1.00b 27.55ghi 8.00 1.33ab 30.92f 10.00 1.33ab 30.05fg *Means followed by the same letter within a column are not significantly different according to Ducan’s New Multiple Range Test at 5% probability level.

47

Figure 2: Effects of different rates of Mucuna spp on total fresh fruit weight (g)/ plant of tomato infected with M. incognita

12.5

15

17.5

20

22.5

25

27.5

30

32.5

35

37.5

Tot

al F

resh

Fru

it W

eigh

t (g

/ Pla

nt)

-2 0 2 4 6 8 10 12M. pruinirns utiliis (t / ha)

Y = 19.85 + 1.26 r =o.86** R^2 = 0.73

Regression Plot

10

15

20

25

30

35

40

45

Tot

al F

resh

Fru

it W

eigh

t (g

/ Pla

nt)

-2 0 2 4 6 8 10 12M. ghana (t / ha)

Y = 24.73 + 1.87 r = 0.76** R^2 = 0.58

Regression Plot

14

16

18

20

22

24

26

28

30

32

34

Tot

al F

resh

Fru

it W

eigh

t (g

/ Pla

nt)

-2 0 2 4 6 8 10 12M. cochichinensis ( t / ha)

Y = 18.69 + 1.25 r =0.91** R^2 = 0.83

Regression Plot

14

16

18

20

22

24

26

28

30

32

34

Tot

al F

resh

Fru

it W

eigh

t (g

/ Pla

nt)

-2 0 2 4 6 8 10 12M . jaspaeda ( t / ha)

Y = 25.58 + 2.84 r = 0.85 ** R^2 = 0.73

Regression Plot

14

16

18

20

22

24

26

28

30

32

34

Tot

al F

resh

Fru

it W

eigh

t (g

/ Pla

nt)

-2 0 2 4 6 8 10 12M. Pruriens IR 2 (t / ha)

Y = 18.31 + 1.39 r= 0.94** R^2 = 0.88

Regression Plot

48

Mineral contents and carbon-to-nitrogen ratio of the different Mucuna species

The above-ground tissue content of both macro and micro elements for the different species

of Mucuna is presented in Table 8. The Mucuna spp differed significantly (p<0.05) in their mineral

contents. M. ghana had significantly the highest concentration of N followed by M. jaspaeda while

M.pruriens IR2 had the least. The P , K and Ca contents of M. pruriens utilis were significantiy

(p<0.05) higher than that of the other Mucuna species.For K and Ca,M. cochichinensis followed M.

pruriens utilis. M. jaspaeda and M. pruriens IR2 had significantly the lowest P and K

concentration. Also,M .pruriens utilis and M.cochichinensis had significantly the highest amount of

Mg while M. pruriens IR2 had the least.M.cochichinensis had significantly(p<0.05) the highest

percentage of carbon , closely followed by M.pruriens IR2 while M. ghana had the least.M.pruriens

utilis had significantly the highest concentration of Fe and Cu followed by M.ghana while

M.pruriens IR2 had the least.The highest concentration of Zn and Mn was obtained in M.pruriens

utilis and M.cochichinensis, respectively, and the least was obtained in M. jaspaeda and M.pruriens

IR2.The carbon-to-nitogen ratio value was generally narrow in all the Mucuna species excepting M.

pruriens IR2 (21.28:1). However, M. ghana and M. jaspaeda had the lowest value (<10:1).

49

Table 8: Mineral content and C/N ratio of the different Mucuna species

*Means followed by the same letter within a column are not significantly different according to Duncan’s New Multiple Range Test at 5% probability level.

Macro Elements (%) Micro Element (Mg/kg)

Mucuna spp N P K Ca Mg C Zn Mn Fe Cu C:N

Ratio

M.pruriens utilis 3.50d* 1.33a 1.10a 2.56a 0.24a 39.90d 48.47a 27.24b 387.30a 24.35a 11.40

M. ghana 4.76a 0.51b 0.96c 1.76e 0.17b 34.31e 43.62c 24.38c 359.90b 22.35b 7.21

M.cochichinensis 3.78c 0.38c 1.04b 2.24b 0.22a 48.29a 40.39d 37.40a 347.80c 20.32c 12.78

M. jaspaeda 4.20b 0.33d 0.86d 1.92d 0.14c 41.89c 36.95e 28.30b 331.60d 19.46c 9.97

M. pruriens IR2 2.10e 0.33d 0.84d 2.08c 0.10d 44.68b 45.55b 22.80d 204.80e 17.35d 21.28

50

Experiment III: Greenhouse Evaluation of the Effects of Mucuna spp Green Manure

Soil Amendment and Arbuscular Mycorrhizal Fungi on the Pathogenicity of M. incognita

on Tomato.

The results of soil amendment with Mucuna spp and AMF inoculation on root galling and gall

index (GI) of tomato infected with M. incognita are presented in Table 9. The number of galls

was significantly (P<0.05) reduced with soil amendment and AMF inoculation compared with

their respective control. The lowest number of galls was obtained with Gi. gigantea inoculation

followed by G. mosseae. The greatest gall inhibition occurred in soil amended with M. jaspaeda

followed by M. ghana. Interaction between Mucuna amendment and AMF was significant. The

least G1 of 2.00 was obtained when the soil was amended with M. jaspaeda and in combination

with all the AMF species excepting G. deserticola and G. clarum. Root galling was significantly

(P<0.05) inhibited when Gi.gigantea or G. mosseae was combined with all the Mucuna spp

relative to other AMF species, this is illustrated in Plate 4. Eggmass production almost followed

the trend of root galling (Table10). Egg production differed significantly among the AMF

species as well as soil amendment with the Mucuna species. Egg mass count was very low in Gi.

gigantea and G. mosseae inoculated plants. Also, egg production was more significantly deterred

in soil amended with M. jaspaeda or M. ghana than others. The interaction between Mucuna

spp amendment and AMF inoculation was significant (P <0.05).

51

M4V4: Gi gigantea + M. jaspaeda

M0V0: CONTROL

PLATE 4: Lightly galled and heavily galled roots of Tomato plants due to treatment effects.

52

Table 9: Effects of arbuscular mycorrhizal fungi and Mucuna spp soil amendment on root galls and galls index (GI) of tomato infected with M. incognita

No. of Galls/Root system

Mycorrhizal fungus

Mucuna spp

Control M. pruriens utilis

M. ghana

M. cochichinensis

M. jaspaeda

M. pruriens IR2

Mean

Control 165.00 41.67 30.00 37.67 13.33 87.33 62.50

G. etunicatum 76.00 22.33 22.00 22.33 8.33 60.00 35.17

G. mosseae 69.00 13.33 17.67 18.33 6.67 50.00 29.17

G. clarum 91.00 22.00 22.33 27.67 12.33 48.00 37.22

Gi. Gigantean 56.00 10.00 12.00 13.33 4.67 30.33 21.06

G. deserticola 87.33 32.00 20.00 25.67 12.00 65.00 40.33

Mean 90.72 23.56 20.67 24.17 9.56 56.78

Gall index (GI)*

Control 5.00 4.00 3.00 3.00 3.00 4.00 3.67

G. etunicatum 4.00 3.00 3.00 3.00 2.00 4.00 3.17

G. mosseae 4.00 3.00 3.00 3.00 2.00 4.00 3.17

G. clarum 4.00 3.00 3.00 3.00 2.67 4.00 3.28

Gi. Gigantean 4.00 2.33 2.67 3.00 2.00 3.33 2.89

G. deserticola 4.00 3.67 3.00 3.00 3.00 4.00 3.44

Mean 4.17 3.17 2.94 3.00 2.44 3.89

No of Galls GI

LSD(0.05) Mycorrhiza (M) Mean = 2.74 0.14

LSD (0.05) Mucuna (V) Mean = 2.74 0.14

LSD (0.05) (MxV) Interaction Mean = 6.70 0.35 *0= Immune, 1=Highly resistant; 2=Resistant; 3= Moderately susceptible; 4= Susceptible; 5=Highly susceptible

53

54

Table 10: Effects of arbuscular mycorrhizal fungi and Mucuna spp soil amendment on number of egg masses/root system and egg mass index of tomato infected with M. incognita

No. of Eggmasses/Root system

Mycorrhizal fungus

Mucuna spp

Control M. pruriens utilis

M. ghana

M. cochichinensis

M. jaspaeda

M. pruriens IR2

Mean

Control 130.00 25.67 15.67 22.00 7.33 53.66 42.39

G. etunicatum 50.67 12.67 13.00 13.00 5.00 32.00 21.06

G. mosseae 47.33 7.00 10.00 10.00 2.67 27.67 17.44

G. clarum 62.00 10.00 13.00 14.00 8.33 31.67 23.17

Gi. gigantea 38.67 5.00 8.00 8.00 1.67 17.67 13.17

G. deserticola 61.67 18.00 12.00 13.00 6.00 35.33 24.33

Mean 65.06 13.06 11.94 13.33 5.17 33.00

Eggmass index (EMI)*

Control 5.00 3.00 3.00 3.00 2.00 4.00 3.33

G. etunicatum 4.00 3.00 3.00 3.00 2.00 3.67 3.11

G. mosseae 4.00 2.00 2.33 2.33 1.67 3.00 2.56

G. clarum 4.00 2.33 3.00 3.00 2.00 3.67 3.00

Gi. gigantea 4.00 2.00 2.00 2.00 1.00 3.00 2.33

G. deserticola 4.00 3.00 3.00 3.00 2.00 4.00 3.17

Mean 4.17 2.56 2.72 2.72 1.78 3.56

No of Eggmass EMI

LSD(0.05) Mycorrhiza (M) Mean = 1.28 0.16

LSD (0.05) Mucuna (V) Mean = 1.28 0.16

LSD (0.05) (MxV) Interaction Mean = 3.13 0.38

*0= Immune, 1=Highly resistant; 2=Resistant; 3= Moderately susceptible; 4= Susceptible; 5=Highly susceptible

55

Generally, inoculation with Gi. gigantea or G. mosseae in combination with soil amendment

with the various species of Mucuna significantly inhibited egg production more than other AMF

species. Soil amended with M. jaspaeda had plants with the least EMI of < 2 in combination

with all the AMF species (Table 10). Also, Gi. gigantea and G. mosseae had EMI of about 2.00

when combined with all the Mucuna spp excepting M. pruriens 1R2.

There was a significant (P<0.05) increase in fresh root weight when the soil was amended

with Mucuna as green manure compared with unamended soil (Table 11). Soil amended with M

jaspaeda and M. ghana had plants with the heaviest root weights. Mycorrhizal plants had

significantly (P<0.05) greater fresh root weights than non-mycorrhizal plants. Interaction

between the two factors was significant. The fresh root weight of tomato was always

significantly (P < 0.05) higher in soils amended with M. jaspaeda or M. ghana in combination

with Gi gigantea and G. mosseae inoculation than other treatment combinations.The effects of

soil amendment with Mucuna spp and AMF inoculation on dry shoot matter accumulation

almost followed the trend of fresh root weight(Table 11). Soil amended with M. jaspaeda or

M.ghana and inoculated with Gi. gigantea and G. mosseae produced plants with the highest

dry shoot weight. Also, the highest relative mycorrhizal effectiveness was obtained with Gi

gigantea inoculation (36.21%) followed by G. mosseae (27.75%).

Effects of AMF inoculation and soil amendment with Mucuna on shoot length and root

colonization by AMF are presented in Table 12. Soil amendment with Mucuna, significantly

(P.<0.05) enhanced tomato growth relative to the control. The same was true for AMF

inoculation. However, the tallest plants were obtained when the soil was amended with either M.

jaspaeda or M. ghana in combination with Gi gigantea and G. mosseae.

56

Table 11: Effects of arbuscular mycorrhizal fungi and Mucuna spp soil amendment on fresh root weight (g) and dry shoot weight (g)/plant of tomato infected with M. incognita

Fresh root weight (g)/plant

Mycorrhizal fungus

Mucuna spp

Control M. pruriens utilis

M. Ghana

M. cochichinensis

M. jaspaeda

M. pruriens IR2

Mean

Control 21.45 24.78 29.90 26.96 31.45 25.40 26.66

G. etunicatum 25.08 27.44 31.26 28.75 33.57 27.53 28.94

G. mosseae 27.31 30.27 33.65 31.09 36.45 32.43 31.87

G. clarum 23.52 26.43 31.54 28.71 33.01 28.03 28.54

Gi. gigantea 27.66 31.50 34.33 31.95 38.12 33.64 32.87

G. deserticola 24.19 26.78 32.11 28.61 32.73 27.94 28.72

Mean 24.87 27.86 32.13 29.35 34.22 29.16

Dry shoot weight (g)/plant

Control 18.54 22.25 31.24 24.86 30.74 22.02 24.94

G. etunicatum 21.77 25.60 32.84 28.72 34.61 27.38 28.49 (14.23)*

G. mosseae 24.59 29.02 39.01 29.92 38.91 30.43 31.86 (27.75)

G. clarum 20.70 22.68 33.08 26.10 34.31 25.48 27.06 (8.50)

Gi. gigantea 26.59 29.86 40.08 36.20 39.68 31.43 33.97 (36.21)

G. deserticola 23.64 23.87 32.93 27.56 36.66 27.74 28.73 (15.20)

Mean 22.64 25.55 34.86 28.90 35.70 27.41

Fresh root wt Dry shoot wt.

LSD (0.05) Mycorrhiza (M) Mean = 0.53 0.53

LSD (0.05) Mucuna (V) Mean = 0.53 0.53

LSD (0.05) (MxV) Interaction Mean = 1.30 1.30

*Relative mycorrhizal effectiveness (RME) percentage (%)

57

Table 12: Effects of arbuscular mycorrhizal fungi and Mucuna spp soil amendment on shoot length (cm)/ plant and AMF root colonization (%) of tomato infected with M. incognita

Shoot length (cm)/ plant

Mycorrhizal fungus

Mucuna spp

Control M. pruriens utilis

M. Ghana

M. cochichinensis

M. jaspaeda

M. pruriens IR2

Mean

Control 55.12 63.76 74.34 66.36 73.29 66.37 66.54

G. etunicatum 62.37 72.10 75.24 71.51 78.01 69.24 71.41

G. mosseae 66.73 75.60 82.39 74.34 85.88 76.25 76.86

G. clarum 60.40 67.53 78.14 70.26 79.20 70.09 70.94

Gi. gigantea 68.88 75.40 86.35 78.25 92.26 75.82 79.50

G. deserticola 63.24 62.44 76.89 71.70 79.29 70.01 70.58

Mean 62.79 69.47 78.89 72.07 81.31 71.31

AMF Root Colonization (%)

Control 15.00 19.67 22.00 21.00 22.67 20.00 20.06

G. etunicatum 55.33 60.33 62.00 60.00 59.67 58.67 59.33

G. mosseae 65.33 70.00 72.00 74.00 75.00 70.00 71.06

G. clarum 58.33 61.00 60.67 61.00 63.67 61.67 61.06

Gi. gigantea 70.00 73.67 74.67 77.33 78.67 71.67 74.33

G. deserticola 55.67 59.67 61.67 61.67 60.00 58.33 59.50

Mean 53.28 59.39 58.83 59.17 59.94 56.72

Shoot length AMF Root Colonization

LSD (0.05) Mycorrhiza (M) Mean = 0.77 1.30

LSD (0.05) Mucuna (V) Mean = 0.77 1.30

LSD (0.05) (MxV) Interaction Mean = 1.88 NS

58

Tomato root colonization was significantly higher in the inoculated plants than the uninoculated

plants (Table 12). Among the AMF species, the highest colonization rates were obtained in Gi

gigantea and G. mosseae .Soil amendment with Mucuna significantly (P≤0.05) enhanced root

colonization by AMF relative to unamended soil. Although interaction between AMF and

Mucuna amendment was not significant, the combination of Gi. gigantea or G. mosseae with all

the Mucuna spp always resulted in higher colonization rate than the other AMF species.

Significant (P≤0.05) increase in the number of fruits set was observed only when soil

was amended with M. jaspaeda, M. pruriens IR2 and M. ghana compared with unamended soil

(Table 13). Also, significant enhancement in fruit set was obtained with Gi gigantea and G.

mosseae inoculation relative to the uninoculated control. Although interaction between the two

factors was not significant, combination of either Gi. gigantea or G. mosseae with the Mucuna

spp produced higher fruit setting compared with other AMF species.

Fresh fruit yield was significantly (P≤0.05) enhanced with inoculation of AMF relative to non-

mycorrhizal plants. The highest fresh fruit yield was obtained with Gi gigantea followed by G.

mosseae inoculated plants. Soil amendment with Mucuna also significantly produced greater

fruit yield compared with the unamended soil with the exception of M. cochichinensis. The

highest fruit yield was obtained from soil amended with M. jaspaeda followed by M. ghana.

Although the interaction between the two treatments was not significant (P>0.05); inoculation

with Gi gigantea in combination with soil amendment with the various Mucuna spp produced the

highest fresh fruit yield followed by G. mosseae (Table 13).

59

Table 13: Effects of arbuscular mycorrhizal fungi and Mucuna spp soil amendment on number of fruits/plant and total fresh fruit weight (g)/plant of tomato infected with M. incognita

No. of fruits/plant

Mycorrhizal

fungus

Mucuna spp

Control M.

pruriens

utilis

M.

Ghana

M.

cochichinensis

M.

jaspaeda

M.

pruriens

IR2

Mean

Control 1.00 1.33 1.33 1.00 1.67 1.33 1.28

G. etunicatum 1.67 1.33 1.33 1.33 2.00 1.67 1.56

G. mosseae 1.67 2.00 2.33 1.67 2.67 2.33 2.11

G. clarum 1.33 1.33 1.67 1.33 2.00 1.67 1.56

Gi. gigantea 2.00 2.33 2.67 2.00 3.00 2.67 2.44

G. deserticola 1.33 1.33 1.67 1.33 2.00 1.67 1.56

Mean 1.50 1.61 1.83 1.44 2.22 1.89

Total fresh weight (g)/plant

Control 19.15 25.79 31.23 20.72 43.42 29.99 28.38

G. etunicatum 29.99 28.19 41.07 26.52 51.65 36.99 35.74

G. mosseae 31.46 42.42 55.14 33.87 60.48 51.37 45.79

G. clarum 25.44 26.56 41.74 27.80 51.03 30.65 33.87

Gi. gigantea 41.49 50.37 58.32 53.02 65.35 54.01 53.76

G. deserticola 26.73 26.70 41.40 32.86 50.73 30.26 34.78

Mean 29.04 33.34 44.81 32.40 53.78 38.88

No. of fruits/plant Total fresh fruit

LSD (0.05) Mycorrhiza (M) Mean = 0.33 3.47

LSD (0.05) Mucuna (V) Mean = 0.33 3.47

LSD (0.05) (MxV) Interaction Mean = NS NS

60

Experiment IV: Field Evaluation of Effects of Mucuna spp Green Manure and AMF on the Pathogencity of M. incognita on tomato

The predominant weed flora species found in the natural weed fallow of the Teaching and

Research Farm, Faculty of Agriculture University of Calabar were Sida acuta, Aspilia africana,

Tridax procumbens, Chromolaena, odorata and Axonopus compressus. The ten adult females

Meloidogyne teased out of the galled roots of infected tomato plants after fixing and staining,

indicated perineal pattern features of Meloidogyne incognita with high squarish dorsal archs,

distinct whorl and without a lateral line. The results of the physico-chemical properties of the soil

sampled mid-season (six weeks after Mucuna spp incorporation in situ as green manure) are

presented in Table 14. Although there was a slight increase in the pH of the soil amended with

Mucuna, both amended and unamended soils were moderately acidic in reaction. There was also

a slight increase in organic matter content and total N with Mucuna amendment relative to the

control. There was an appreciable increase in available P when the soil was amended with M.

pruriens utilis, M. ghana and M. jaspaeda. Although the exchangeable K, Ca and Mg contents of

both amended and unamended soils were rated low, the amount of these nutrient elements was

higher in amended soil. Conversely, there was a slight decrease in exchangeable Na with

Mucuna amendment. There was a great increase in effective cation exchange capacity ( ECEC)

of the soil amended with the Mucuna spp relative to the unamended soil excepting M. ghana and

M. jaspaeda. The base saturation for the amended soil was higher than that of the control soil.

The texture of both Mucuna amended and unamended soil was loamy sand.

The percentage emergence of the Mucuna species differed significantly (P ≤ 0.05). M.

pruriens utilis, M. ghana, M. cochichinensis and M. pruriens IR2 had 73.72%, 75.90%, 72.05%

and 73.97%, respectively which differed significantly (P ≤ 0.05) from M. jaspaeda with 63.08%

emergence.

61

Table 14: Physico-chemical properties of soil amended with different Mucuna spp sampled at Mid-season (6 weeks after incorporation

of Mucuna green manure)

Soil Property

Mucuna spp PH (%)

Org

matter

(%)

Total

N

(mg/Kg)

AV.P

K

Ca

Cmol/kg

Mg Na Al3+

H+

ECEC

(%)

BS

(%)

Clay

(%)

Silt

(%)

sand

Texture

Control (No

amendment

5.60 2.21 0.11 50.00 0.09 3.80 0.60 0.10 0.00 0.80 5.39 85.00 10.00 4.70 85.30 Loamy sand

M. pruriens utilis 5.90 2.47 0.14 68.75 0.15 5.23 0.80 0.06 0.00 0.56 6.80 91.00 11.00 6.70 82.30 Loamy sand

M. Ghana 5.80 2.42 0.14 63.50 0.10 4.10 0.70 0.07 0.00 0.56 5.53 90.00 9.00 7.70 83.30 Loamy sand

M. cochichinensis 5.80 2.53 0.13 58.33 0.11 5.60 0.90 0.07 0.00 0.64 7.32 91.00 11.00 7.70 81.30 Loamy sand

M. jaspaeda 5.90 2.46 0.14 60.15 0.12 4.20 0.80 0.70 0.00 0.56 5.75 90.00 11.00 6.70 82.30 Loamy sand

M. pruriens IR2 5.90 2.49 0.12 56.28 0.12 4.95 0.80 0.08 0.00 0.56 6.51 91.00 11.00 7.70 81.30 Loamy sand

62

None of the Mucuna species uprooted and examined for galling and eggmass was found to be

galled nor with any eggmass as was the case with the greenhouse trial. All the species of Mucuna

were rated immune to M. incognita infection in the field.

The results of the effect of AMF inoculation and Mucuna amendment as green manure on

root galling and mean gall index (MGI) of tomato infested with M. incognita are presented in

Table 15. Incorporation of Mucuna as green manure significantly (P ≤ 0.05) inhibited root

galling compared with the control plots. M. jaspaeda and M. ghana were the most potent species.

Similarly, seedlings biologically enhanced with AMF significantly (P ≤ 0.05) impaired galling

compared with the non mycorrhizal seedlings. Gi. gigantea and G. mosseae were the most

efficient species. In most of the Mucuna species amended soil, inoculation with Gi gigantea or

G. mosseae resulted in a significantly fewer galls than other AMF species. The least MGI was

obtained in plots with G. gigantea inoculated plants amended with M. jaspaeda or M.pruriens

IR2. G. mosseae inoculated plants responded in a similar way when planted in M. pruriens utilis,

M. jaspeade or M. pruriens IR2 amended plots.

Eggmass production differed significantly (P ≤ 0.05) among the AMF species as well as

Mucuna spp amendment (Table 16). Gi gigantea and G. moseae were the most inhibitive in egg

production inhibition. M. jaspaeda and M. ghana were also the most efficient species in egg

production impairment. The interaction between Mucuna amendment and AMF inoculation was

significant. There was no significant difference (P>0.05) between mycorrhizal and non

mycorrihizal plants in egg production in plots amended with either M. ghana or M. jaspaeda.

The least number of eggmasses were recorded in roots of plants inoculated with either G.

mosseae or Gi. gigantea and planted in plots amended with M. jaspaeda. The least EMI of 1.00

was recorded in G. mosseae or Gi gigantea inoculated plants in combination with M. jaspaeda

amendment.

63

Table 15: Effects of Arbuscular Mycorrhizal fungi and Mucuna Spp soil amendment on

root galls and mean gall index (MGI)* of tomato infested with M. incognita in the field

AV. No. of Galls/Root System

Mucuna Spp

Mycorrhizal fungus

Control M.

pruriens

Utilis

M.

ghana

M.

cochichinensis

M.

jaspaeda

M.

pruriens

IR2

Mean

Control 78.83 10.64 5.97 15.24 5.89 16.97 21.42

G. etunicatum 30.14 6.68 4.38 10.44 5.40 10.19 11.21

G. mosseae 16.39 5.50 2.45 6.16 2.47 4.79 6.29

G. clarum 39.75 8.42 5.24 10.03 4.89 8.22 12.76

G. gigantea 12.39 4.03 2.32 5.40 1.74 2.14 4.67

G. deserticola 40.78 10.15 5.25 7.74 4.61 7.61 12.69

Mean 35.55 7.57 4.27 9.17 4.17 8.32

Mean Gall Index (MGI)

Control 2.86 1.64 1.47 1.87 1.60 2.14 1.93

G. etunicatum 2.50 1.49 1.30 1.93 1.49 2.11 1.82

G. mosseae 1.98 1.36 1.11 1.55 1.14 1.50 1.44

G. clarum 2.50 1.92 1.47 1.97 1.47 1.89 1.87

G. gigantea 1.83 1.19 1.43 1.47 1.01 1.11 1.34

G. deserticola 2.75 1.89 1.35 1.83 1.50 1.75 1.85

Mean 2.42 1.58 1.36 1.77 1.37 1.75

No. of Galls/root system MGI LDS (0.05) for comparing Mycorrhizal (M) Means = 1.55 0.15 LDS (0.05) for comparing Mucuna (V) Means = 1.35 0.14 LDS (0.05) for comparing (MXV) interaction Means = 3.65 0.36 LDS (0.05) for comparing (MXV) interaction Means with the same level of V = 3.79 0.37

* 0= Immune, 1 = Highly resistant, 2 = Resistant, 3 = Moderately susceptible, 4 = Susceptible, 5

= Highly Susceptible

64

Table 16: Effects of Arbuscular Mycorrhizal fungi and Mucuna Spp soil amendment on

number of eggmasses/root system and eggmass index (EMI) of tomato infested with M.

incognita in the field

No. Eggmasses/root system

Mucuna Spp

Mycorrhizal fungus

Control M.

pruriens

Utilis

M.

ghana

M.

cochichinensis

M.

jaspaeda

M.

pruriens

IR2

Mean

Control 104.00 13.33 5.00 15.67 5.67 20.00 27.28

G. etunicatum 41.67 7.67 5.00 9.67 4.00 10.33 14.83

G. mosseae 15.67 5.67 2.00 4.33 1.67 5.00 5.72

G. clarum 46.00 9.00 3.33 7.00 4.33 8.33 13.00

G. gigantea 13.00 4.67 2.33 3.33 1.67 2.33 4.56

G. deserticola 28.00 8.33 3.67 6.67 4.00 8.33 9.83

Mean 41.39 8.11 5.33 7.78 3.56 9.06

Eggmass Index (EMI)

Control 4.33 3.00 2.00 3.00 2.00 3.00 2.89

G. etunicatum 4.00 2.00 2.00 2.00 2.00 2.33 2.39

G. mosseae 3.00 2.00 1.33 2.00 1.00 2.00 1.89

G. clarum 4.00 2.00 2.00 2.00 2.00 2.00 2.33

Gi. gigantea 3.00 2.00 1.33 2.00 1.00 1.33 1.78

G. deserticola 3.33 2.00 2.00 2.00 2.00 2.00 2.22

Mean 3.61 2.17 1.78 2.17 1.67 2.11

No. Eggmass/root system EMI LDS (0.05) for comparing Mycorrhizal (M) Means = 3.62 0.16 LDS (0.05) for comparing Mucuna (V) Means = 4.91 0.17 LDS (0.05) for comparing (MXV) interaction Means = 9.21 0.38 LDS (0.05) for comparing (MXV) interaction Means with the same level of V = 8.88 0.39

* 0= Immune, 1 = Highly resistant, 2 = Resistant, 3 = Moderately susceptible, 4 = Susceptible,

5 = Highly Susceptible

65

Table 17 presents the incidence of root knot disease on tomato inoculated with different AMF

species and plots amended with different species of Mucuna as green manure. Although not

significant, G. etunicatum and G. clarum inoculated plants had higher root- knot incidence than

the non mycorrhizal plants. However, G. mosseae inoculated plants had significantly lower

incidence of root- knot than G. clarum and G. etunicatum inoculated plants. Plots amended with

M. cochichinensis and M. pruriens IR2 had higher incidence of root knot than the unamended

plots. However, M. ghana and M. pruriens utilis amended plots had significantly lower root-knot

incidence compared with some other Mucuna spp amended plots.

Soil amendment with Mucuna significantly (P ≤0.05) produced plants with heavier root

mass than unamended soil (Table 18). Fresh root weight of plants obtained from M. jaspaeda

and M. ghana amended plot was significantly heavier than those from other Mucuna spp

amended soil. Mycorrhizal plants had significantly (P ≤0.05) higher fresh root weights than non

mycorrhizal plants. The fresh root weight of tomato was always significantly higher in soils

amended with M. jaspaeda or M. ghana in combination with Gi. gigantea inoculation than other

treatment combinations.

Amendment of soil with Mucuna as green manure significantly (P ≤0.05) enhanced root

colonization by AMF (Table 18) relative to unamended plot M. jaspaeda and M. cochichinensis

amendment significantly enhanced mycorrhizal colonization than the other species. The AMF

species differed significantly in their root colonizing ability. Gi. gigantea and G. mosseae had

significantly higher colonization percentage. The uninoculated (control) plants were lightly

colonized by indigenous soil AMF species. Although interaction between AMF and Mucuna

amendment was not significant, inoculation with Gi gigantean or G. mosseae in combination

with all the Mucuna spp always had the highest root colonization.

66

Table 17: Effects of Arbuscular Mycorrhizal fungi and Mucuna spp amendments on root-

knot incidence (%) on tomato grown in field infested with M. incognita in the field

Mucuna Spp

Mycorrhizal fungus

Control M.

pruriens

Utilis

M.

ghana

M.

cochichinensis

M.

jaspaeda

M.

pruriens

IR2

Mean

Control 75.00 61.10 63.90 66.70 63.90 66.70 66.20

G. etunicatum 72.00 55.60 61.10 72.20 72.20 80.40 69.00

G. mosseae 55.60 63.90 55.60 66.70 52.80 66.70 60.20

G. clarum 69.40 69.40 63.90 77.80 77.80 75.00 72.20

G. gigantea 61.10 55.60 58.30 72.20 69.40 69.40 64.40

G. deserticola 69.40 66.70 61.10 75.00 61.10 66.70 66.70

Mean 67.10 62.00 60.60 71.80 66.20 70.80

LDS (0.05) for comparing Mycorrhizal (M) Means = 7.57 LDS (0.05) for comparing Mucuna (V) Means = 7.06 LDS (0.05) for comparing (MXV) interaction Means = NS LDS (0.05) for comparing (MXV) interaction Means with the same level of V = NS

67

Table 18: Effects of Arbuscular Mycorrhizal fungi and Mucuna Spp soil amendment on

fresh root weight (g)/ plant and root colonization by AMF (%) of tomato grown in M.

incognita infested field

Fresh Root Weight

Mucuna Spp

Mycorrhizal fungus

Control M.

pruriens

Utilis

M.

ghana

M.

cochichinensis

M.

jaspaeda

M.

pruriens

IR2

Mean

Control 25.42 29.90 34.36 28.22 37.61 30.23 30.95

G. etunicatum 31.88 39.57 41.40 36.40 42.46 33.01 37.45

G. mosseae 34.07 40.01 44.16 38.38 44.25 36.76 39.60

G. clarum 32.03 37.84 43.52 37.02 40.92 32.45 37.30

G. gigantea 36.51 42.27 46.38 40.78 47.62 38.50 42.01

G. deserticola 31.32 38.40 40.71 37.34 41.18 37.69 37.77

Mean 31.87 38.00 41.75 36.36 42.34 34.77

AMF Root Colonization

Control 15.67 20.00 21.33 20.00 23.33 20.00 20.06

G. etunicatum 49.33 55.33 57.67 57.00 56.00 53.00 54.72

G. mosseae 54.33 61.67 62.67 65.33 64.67 60.33 61.50

G.clarum 47.67 54.00 57.33 57.33 57.67 55.00 54.83

Gi. gigantea 54.33 62.67 65.33 64.00 66.67 62.33 62.56

G. deserticola 44.00 53.00 55.33 57.67 54.00 55.33 53.22

Mean 44.22 51.11 53.28 53.56 53.72 51.00

Fresh Rt. Weight AMF Root Colonization LDS (0.05) for comparing Mycorrhizal (M) Means = 1.17 1.42 LDS (0.05) for comparing Mucuna (V) Means = 0.91 2.37 LDS (0.05) for comparing (MXV) interaction Means = 2.74 NS LDS (0.05) for comparing (MXV) interaction Means with the same level of V = 2.87 NS

68

Effects of AMF inoculation and soil amendment on shoot length and dry shoot matter of tomato

infected with M. incognita are presented in Table 19. Soil amendment with Mucuna significantly

(P ≤0.05) enhanced tomato growth relative to the unamended soil.

The same was true for AMF inoculation. Gi gigantea inoculated plants were significantly taller

than other AMF species inoculated plants. Interaction between the factors was significant.

However, the tallest plants were obtained when soil was amended with either M. jaspaeda or M.

ghana in combination with either Gi. gigantea or G. mosseae . Dry shoot matter accumulation

was significantly (P ≤0.05) increased with Mucuna soil amendment compared with the

unamended soil (Table 19). M. ghana and M. jaspaeda amended plots produced plants with

significantly higher dry shoot weight. Similarly, mycorrhizal inoculation significantly (P ≤0.05)

enhanced dry matter production in shoot compared with the control. The highest relative

mycorrhizal effectiveness was obtained with Gi gigantea inoculation followed by G. mosseae.

Generally, plots amended with M. jaspaeda or M. ghana and in combination with Gi. gigantea

and G. mosseae produced plants with the greatest dry shoot matter.

The results of the effect of Mucuna amendment and AMF inoculation on the number of

fruits and fresh fruit yield of tomato infested with M. incognita are presented in Table 20. Soil

amendment with Mucuna and mycorrhizal inoculation significantly (P ≤0.05) promoted fruit set

relative to the control plots. Gi gigantea and G. mosseae inoculated plants had the highest

number of fruits per plant. The same was observed for plots amended with M. ghana and M.

jaspaeda. In both Mucuna amended and unamended plots, Gi gigantea and G. mosseae

inoculated plants produced significantly the highest number of fruits. The effects of treatments

on total fresh fruit weight per plant followed the trend of number of fruit (Table 20). The

interaction between Mucuna amendment and AMF inoculation was significant.

69

Table 19: Effects of Arbuscular Mycorrhizal fungi and Mucuna Spp soil amendment on

shoot length (cm)/ plant and dry shoot weight (g)/ plant of tomato grown in M. incognita

infested field

Shoot length (cm)/Plant

Mucuna Spp

Mycorrhizal fungus

Control M.

pruriens

Utilis

M.

ghana

M.

cochichi-

nensis

M.

jaspaeda

M.

pruriens

IR2

Mean

Control 53.50 80.17 92.17 83.83 92.50 75.00 79.53

G. etunicatum 67.67 91.77 96.00 88.00 104.33 79.33 87.85

G. mosseae 70.67 104.83 101.39 98.50 101.61 86.00 93.83

G. clarum 64.33 83.00 96.00 93.44 98.17 83.67 86.44

G. gigantea 80.00 99.33 109.00 99.33 125.33 90.33 100.56

G. deserticola 66.00 93.83 97.67 90.17 97.33 81.33 87.72

Mean 67.03 92.16 98.70 92.21 103.21 82.61

Dry shoot weight(g)/plant

Control 25.88 43.40 48.53 40.24 44.73 41.61 40.73

G. etunicatum 38.22 50.92 49.86 44.79 48.51 43.13 45.90 (12.69)*

G. mosseae 42.85 53.03 57.19 48.10 56.28 47.51 50.83 (24.80)

G.clarum 35.95 45.48 49.78 43.54 50.53 45.04 45.05 (10.61)

Gi. gigantea 47.50 51.97 61.93 49.73 64.26 52.10 54.58 (34.00)

G. deserticola 35.51 47.32 49.11 47.02 50.30 45.30 45.76 (12.35)

Mean 37.65 48.69 52.73 45.57 52.44 45.76

Shoot length Dry shoot wt. LDS (0.05) for comparing Mycorrhizal (M) Means = 2.37 0.83 LDS (0.05) for comparing Mucuna (V) Means = 3.61 0.71 LDS (0.05) for comparing (MXV) interaction Means = 6.20 1.94 LDS (0.05) for comparing (MXV) interaction Means with the same level of V = 5.80 2.04

*Relative Mycorrhizal Effectiveness (RME) (%)

70

Table 20: Effects of Arbuscular Mycorrhizal fungi and Mucuna Spp soil amendment on

number of fruits/plant and total fresh fruit weight (g)/plant of tomato grown in M.

incognita infested field

No. Fruits/plant

Mucuna Spp

Mycorrhizal fungus

Control M.

pruriens

Utilis

M.

ghana

M.

cochichinensis

M.

jaspaeda

M.

pruriens

IR2

Mean

Control 3.33 5.33 6.33 5.00 6.00 4.67 5.11

G. etunicatum 5.33 7.67 8.33 6.67 7.67 6.33 7.00

G. mosseae 7.67 10.00 9.33 8.33 8.33 9.00 8.78

G. clarum 4.67 6.67 7.33 6.33 7.33 7.33 6.61

G. gigantea 8.00 10.67 11.33 9.33 12.00 10.67 10.33

G. deserticola 4.33 7.00 7.33 7.00 7.67 6.33 6.61

Mean 5.56 7.89 8.33 7.11 8.17 7.39

Total fresh fruit Weight

Control 77.33 156.00 202.67 155.67 185.00 135.00 151.94

G. etunicatum 135.00 199.67 273.00 201.00 250.33 183.67 207.11

G. mosseae 181.33 259.00 316.00 259.67 285.67 242.33 257.33

G.clarum 143.33 224.33 260.33 213.33 242.33 204.67 214.72

Gi. gigantea 188.00 293.33 409.00 295.00 383.67 330.33 316.56

G. deserticola 131.00 208.00 254.00 212.00 234.67 188.00 204.61

Mean 142.67 223.39 285.83 222.78 263.61 214.00

No. fruits Total fresh fruit LDS (0.05) for comparing Mycorrhizal (M) Means = 0.39 5.70 LDS (0.05) for comparing Mucuna (V) Means = 0.49 6.31 LDS (0.05) for comparing (MXV) interaction Means = 0.98 13.92 LDS (0.05) for comparing (MXV) interaction Means with the same level of V = 0.96 13.97

71

In all the Mucuna amended plots, Gi gigantea inoculation significantly (P ≤0.05) enhanced fruit

yield compared with other AMF species. This was followed by G. mosseae inoculated plants. Gi.

gigantea inoculation in combination with M. ghana amendment produced significantly the

highest fruit yield.

Experiment V: Evaluation of the Effects of Paecilomyces lilacinus and AMF against

M. incognita on Tomato

The results of Paecilomyces lilacinus and AMF inoculation on root galling and egg production

by M. incognita infected tomato in Calabar soil are presented in Table 21. Mycorrhizal

inoculation significantly (P ≤0.05) reduced the severity of root galling by M. incognita

compared with the non-mycorrhizal plants. Gall index (GI) was reduced from 4 to less than 4

with AMF inoculation.Double application of the bionematicide was significantly (P ≤0.05) more

efficient in gall inhibition than single application. The least GI of 2.33 was obtained when G.

etunicatum, G. mosseae and G. deserticola were combined with double application of P.

lilacinus. Eggmass production almost followed the trend of root galling. Egg production differed

significantly (P ≤0.05) among the AMF species as well as application frequency of the

bionematicide. Eggmass count was significantly low in G. mosseae and Gi gigantea inoculated

plants. The interaction between the two bicontrol agents was significant. The least egg

production was observed when G. mosseae and G. etunicatum were combined with P. lilacinus.

Similarly, the least eggmass index (EI) of 2.00 was obtained in plants that were double treated

with the bionematicide.

Inoculation of AMF significantly (P ≤0.05) increased fresh root weight in Calabar soil (Table

22) compared with non-mycorrhizal plants. Also, application of the bionematicide significantly

72

(P ≤0.05) increased fresh root weight of the tomato plants compared with the control. Double

application of the bionematicide to G. deserticola inoculated plants produced the highest fresh

root weight. There were significant (P ≤0.05) differences among the species of AMF in their

rates of tomato root colonization in Calabar soil (Table 22). The highest colonization was

obtained in Gi. gigantea and G. mosseae inoculated plants. Uninoculated plants were lightly

colonized by indigenous soil AMF species. Application of P. lilacinus did not have any

significant (P>0.05) effect on mycorrhizal colonization in Calabar soil. Tomato growth was

significantly (P ≤0.05) enhanced with mycorrhizal inoculation relative to non-mycorrhizal

plants. Application of the bionematicide also significantly improved tomato growth. Double

application of the bionematicide in combination with the AMF inoculation produced

significantly taller plants than other treatment combinations. The tallest plant was obtained with

G. mosseae inoculation combined with double application of the bionematicide. Dry shoot

matter accumulation was significantly (P ≤0.05) enhanced with both AMF inoculation and

bionematicide application compared with their respective controls (Table 22). Plants that

received double application of the bionematicide in combination with mycorrhizal inoculation

had higher dry shoot matter than others. The tomato plants were highly responsive to AMF

inoculation with more than 30% RME. However, Gi gigantea and G. mosseae were the most

efficient species. The effects of the two biocontrol agents on fruit set and fresh fruit yield of

tomato in Calabar soil are presented in Table 23. There was a significant (P ≤0.05) increase in

the number of fruit set and fresh fruit weight with inoculation of AMF and bionematicide

compared with their respective control. However, significantly higher fruit yield was obtained

with double application of P. lilacinus compared with the other application frequencies. The

highest fresh fruit yield was obtained from plants inoculated with Gi gigantea in combination

with double application of the bionematicide. This was closely followed by G. mosseae and

G.derserticola inoculated plants.

73

Table 21: Effects of arbuscular mycorrhizal fungus and P. lilacinus application on number of galls and Eggmasses per root system, gall index (GI)* and Eggmass Index* of tomato inoculated with M. incognita in Calabar soil

Mycorrhizal fungus

No. of Galls

P. lilacinus

Gall index

P. lilacinus

Control Single application

Double application

Mean Control Single application

Double application

Mean

Control 119.33 50.00 22.33 63.89 5.00 4.00 3.00 4.00

G. etunicatum 80.67 25.67 10.33 38.89 4.00 3.00 2.33 3.11

G. mosseae 45.00 28.67 9.33 27.67 4.00 3.33 2.33 3.22

G. clarum 57.67 36.00 14.33 36.00 4.00 4.00 3.00 3.67

Gi. gigantea 40.87 31.00 12.00 27.96 4.00 3.00 2.67 3.22

G. deserticola 66.67 26.67 10.00 34.45 4.00 3.30 2.33 2.21

Mean 68.36 33.00 13.05 4.17 3.44 2.61

No. of Eggmasses Eggmass Index

Control 92.33 15.00 7.33 38.22 4.33 3.00 2.00 3.11

G. etunicatum 50.00 10.00 4.00 21.33 4.00 2.33 2.00 2.78

G. mosseae 22.33 11.00 3.67 12.33 3.00 2.33 2.00 2.44

G. clarum 32.33 12.67 9.00 18.00 3.67 2.67 2.33 2.89

Gi. gigantea 19.66 10.67 7.00 12.44 3.00 2.33 2.00 2.44

G. deserticola 47.33 13.00 7.00 22.44 4.00 2.67 2.00 2.89

Mean 44.00 12.06 6.33 3.67 2.56 2.06 No. of Galls Gall Index No of Eggmasses Eggmass index

LSD (0.05) for P. lilacinus (F) Means = 6.11 0.20 1.17 0.26 LSD (0.05) for Mycorrhizal (M) Means = 8.64 0.29 1.66 0.37 LSD (0.05) for (FXM) interaction Means = 14.97 NS 2.87 NS

*0 = immune, 1 = Highly Resistant, 2 = Resistant, 3 = Moderately Susceptible, 4 = Susceptible,

5 = Highly Susceptible.

74

Table 22: Effects of arbuscular mycorrhizal fungus and P. lilacinus application on fresh root weight (g)/plant, root colonization by AMF (%), shoot length (cm)/plant and dry shoot weight (g) /plant of tomato inoculated with M. incognita in Calabar soil

Mycorrhizal fungus

Fresh root weight

P. lilacinus

AMF root colonization

P. lilacinus

Control Single app[ication

Double application

mean Control Single application

Double application

Mean

Control 11.20 13.40 15.25 13.28 20.33 19.33 20.67 20.11

G. etunicatum 13.27 14.18 15.33 14.26 58.67 59.33 59.67 59.22

G. mosseae 13.24 15.12 16.30 14.89 81.00 79.62 80.00 80.21

G. clarum 13.58 14.99 16.50 15.02 67.67 68.67 70.00 68.78

Gi. gigantea 12.74 16.18 17.59 15.50 81.67 81.33 85.33 82.78

G. deserticola 12.66 17.25 18.13 16.01 80.33 71.33 79.67 79.11

Mean 12.78 15.19 16.52 64.95 64.27 65.89

Shoot length Dry shoot weight

Control 59.69 71.33 78.33 69.78 11.68 15.55 18.08 15.10

G. etunicatum 68.67 71.67 82.33 74.22 14.43 18.82 20.37 17.87*(18.34)

G. mosseae 71.67 83.67 90.00 81.18 18.97 20.62 22.17 20.59(36.36)

G. clarum 64.33 76.00 82.33 74.22 16.97 20.51 21.79 19.76(30.86)

Gi. gigantea 65.00 73.00 78.33 72.11 19.30 21.35 22.38 21.01(39.14)

G. deserticola 63.33 69.67 75.67 69.56 16.08 19.30 21.21 18.86(24.90)

Mean 65.45 74.22 81.17 16.24 19.36 21.00 Fresh Rt. Wt. Root Colonization Shoot length Dry shoot wt.

LSD (0.05) for P. lilacinus (F) Means = 0.52 NS 1.87 0.61 LSD (0.05) for Mycorrhizal (M) Means = 0.74 2.20 2.67 0.87 LSD (0.05) for (FXM) interaction Means = 1.28 NS 4.92 NS * Relative Mycorrhizal Effectiveness (RME)%

75

Table 23: Effects of arbuscular mycorhizal fungus and P. lilacinus application on number of fruits/plant and total fresh weight of fruits (g)/plant of tomato inoculated with M. incognita in Calabar soil Mycorhizal fungus

Number of fruits/plant P. lilacinus

Mean

Control Single application

Double application

Control 1.00 1.33 2.33 1.89 G. etunicatum 2.00 2.67 3.67 2.78 G. mosseae 1.67 3.37 3.33 2.56 G. clarum 2.67 2.33 3.67 3.22 G. gigantean 2.33 3.33 3.33 2.67 G. deserticola 2.67 3.37 4.00 3.41 Mean 2.06 2.83 3.39

Total Fresh Weight of Fruit

Control 25.40 42.00 55.24 40.88 G. etunicatum 38.10 53.05 56.29 49.15 G. mosseae 49.37 53.09 67.15 56.54 G. clarum 42.80 48.77 58.38 49.98 G. gigantean 53.19 57.32 74.24 61.58 G. deserticola 47.92 56.23 67.16 57.10 Mean 42.80 51.74 63.08

No of fruits Total fresh Wt. fruit

LSD (0.05) for P. lilacinus (F) Means = 0.39 4.51 LSD (0.05) for Mycorrhizal fungus (M) = 0.55 6.38 LSD (0.05) for (FXM) Interaction Means = 0.96 NS

76

The effects of AMF inoculation and bionematicide application on root galling and eggmass

production on tomato infected with M. incognita in Ikom soil are presented in Table 24. The

severity of root galling was significantly (P ≤0.05) reduced with AMF inoculation and

bionematicide relative to their controls. G. etunicatum and Gi. gigantea had the least GI = 300.

Double application of the bionematicide was more efficient in gall inhibition than single

application . The least GI of 2.00 was obtained with Gi. gigantea and G. etunicatum inoculated

plants in combination with double application of the bionematicide. Egg production was

significantly (P ≤0.05) inhibited with mycorrhizal inoculation as well as bionematicide

application. The trend followed root galling. Combination of either Gi. gigantea or G. etunicatum

with double application of the bionematicide resulted in the lowest number of eggmasses, with

eggmass index (EMI) of 1.33 (Table 24).

Fresh root biomass of tomato was significantly (P ≤0.05) increased with AMF inoculation as

well as bionematicide application compared with the control in Ikom soil (Table 25). G.

etunicatum or Gi. gigantea inoculated plants when planted in soils where bionematicide was

double applied significantly produced plants with the highest fresh root weight. Mycorrhizal

colonialization of tomato root differed significantly (P ≤0.05) among the AMF species.

However, G. mosseae and Gi. gigantea had higher colonization rate than others. The

bioformulated nematicide had no significant effect on root colonization by the AMF species. The

uninoculated plants were lightly colonized. Inoculation of AMF and bionematicide application

significantly (P ≤0.05) enhanced growth of tomato plants grown in Ikom soil when compared

with their respective controls (Table 25). Double application of the bionematicide significantly

enhanced tomato growth more than single application. Gi. gigantea inoculated plants in

combination with double application of P. lilacinus produced the tallest plants followed by G.

etunicatum.

77

Table 24: Effects of arbuscular mycorrhizal fungus and P. lilacinus application on number of galls and Eggmasses per root system, gall index (GI)* and Eggmass Index* of tomato inoculated with M. incognita in Ikom soil

Mycorrhizal fungus

No. of Galls

P. lilacinus

Gall index

P. lilacinus

Control Single application

Double application

Mean Control Single application

Double application

Mean

Control 78.33 27.67 14.33 40.11 4.00 3.00 2.67 3.22

G. etunicatum 41.00 21.00 7.67 23.22 4.00 3.00 2.00 3.00

G. mosseae 41.00 22.33 13.33 29.00 4.00 3.00 3.00 3.33

G. clarum 51.33 22.00 13.00 30.22 4.00 3.00 2.67 3.22

Gi. gigantea 55.67 15.00 6.00 20.00 4.00 3.00 2.00 3.00

G. deserticola 39.00 22.33 9.67 27.78 4.00 3.00 2.33 3.11

Mean 52.78 21.72 10.67 4.00 3.00 2.44

No. of Eggmasses Eggmass Index

Control 42.67 10.00 7.00 19.89 4.00 2.33 2.00 2.78

G. etunicatum 25.67 7.00 2.33 11.67 3.00 2.00 1.33 2.11

G. mosseae 32.00 8.33 4.33 14.89 3.67 2.00 2.00 2.56

G. clarum 39.67 9.67 5.33 17.89 4.00 2.33 2.00 2.78

Gi. gigantea 22.33 8.67 2.33 11.11 3.00 2.00 1.33 2.11

G. deserticola 31.00 9.33 5.00 15.11 3.67 2.33 2.00 2.67

Mean 32.22 8.83 4.22 3.67 2.17 1.78 No. of Galls Gall Index No of Eggmasses Eggmass index LSD (0.05) for P. lilacinus (F) Means = 2.40 0.16 1.38 0.24 LSD (0.05) for Mycorrhizal (M) Means = 43.40 0.23 1.95 0.34 LSD (0.05) for (FXM) interaction Means = 5.89 0.39 3.38 NS *0 = Immune, 1 = Highly Resistant, 2= Resistant, 3= Moderately susceptible 4 = Susceptible, 5 = Highly susceptible

78

Table 25: Effects of arbuscular mycorrhizal fungus and P. lilacinus application on fresh root weight (g)/plant, root colonization by AMF (%), shoot length (cm)/plant and dry shoot weight (g)/plant of tomato inoculated with M. incognita in Ikom soil

Mycorrhizal fungus

Fresh root weight

P. lilacinus

AMF root colonization

P. lilacinus

Control Single application

Double application

mean Control Single application

Double appliction

Mean

Control 4.22 6.05 7.16 5.81 18.00 20.00 20.33 19.44

G. etunicatum 6.36 9.87 11.09 9.11 53.67 55.00 52.67 53.78

G. mosseae 5.99 7.88 10.16 8.01 59.67 60.00 60.33 60.00

G. clarum 5.75 8.12 9.49 7.78 51.33 52.67 52.00 52.00

Gi. gigantea 5.63 10.31 11.13 9.03 61.67 60.33 62.00 61.33

G. deserticola 5.23 7.89 9.77 7.63 49.00 50.33 50.67 50.00

Mean 5.53 8.35 9.80 48.89 49.72 49.67

Shoot length Dry shoot weight

Control 44.33 50.33 57.67 50.78 4.93 7.55 10.09 7.53

G. etunicatum 57.33 62.00 65.33 61.56 7.09 10.45 12.11 9.88(31.21)*

G. mosseae 50.00 57.33 60.67 56.00 6.27 9.58 11.28 9.04(20.05)

G. clarum 50.00 58.00 63.33 57.11 6.19 9.20 10.43 8.60(14.21)

Gi. gigantea 58.67 64.33 70.33 64.44 6.71 12.29 12.97 10.66(41.57)

G. deserticola 52.33 59.33 64.00 58.56 5.79 9.46 10.46 8.57(13.81)

Mean 52.11 58.56 63.56 6.17 9.75 11.22 Fresh Rt. Wt. Root Colonization Shoot length Dry shoot wt.

LSD (0.05) for P. lilacinus (F) Means = 0.37 NS 1.44 0.39 LSD (0.05) for Mycorrhizal (M) Means = 0.52 1.68 2.03 0.55 LSD (0.05) for (FXM) interaction Means = 0.89 NS NS 0.95

* % Relative Mycorrhizal Effectiveness (RME)

79

Dry shoot matter accumulation was also significantly (P ≤0.05) increased in mycorrhizal as

well as bionematicide treated plots compared with their respective controls. Plants inoculated

with either Gi. gigantea or G. etunicatum in combination with double application of

bionematicide accumulated the highest dry matter in their shoots. The highest relative

mycorrhizal effectiveness was observed in Gi. gigantea inoculated plants followed by G.

etunicatum (Table 25). Fruit set and total fresh weight of tomato fruit were significantly (P

≤0.05) enhanced in Ikom soil with inoculation of seedlings with AMF and application of

bionematicide compared with the control (Table 26). The highest fresh fruit yield was obtained

when Gi. gigantea or G. etunicatum plants was combined with double application of P. lilacinus.

The effects of AMF inoculation and application of a bioformulated nematicide on root galling

and eggmass production by M. incognita on tomato in Nsukka soil are presented in Table 27.

Mycorrhizal plants had significantly (P ≤0.05) fewer galls than the non-mycorrhizal plants.

Also, double application of the bionematicide was more efficient in gall reduction than single

application. The least GI of 2.00 was obtained when G. etunicatum was combined with P.

lilacinus, as shown in Plate 5. Eggmass production almost followed the trend of root galling. Egg

production differed significantly among the AMF species and application frequency of the

bionematicide. Significantly fewer eggs were produced when G. etunicatum was combined with

double application of the bionematicide. This treatment combination also gave the least EMI of

1.33. Fresh root weight of tomato was significantly (P ≤0.05) increased with mycorrhizal

inoculation and application of P. lilacinus compared with their respective control. However, G.

deserticola in combination with double application of the bionematicide produced the highest

fresh root biomass (Table 28). There were significant differences (P ≤0.05) among the AMF in

their colonizing ability of roots. G. clarum and G. mosseae had significantly higher root

colonization rate.

80

Table 26: Effects of arbuscular mycorhizal fungus and P. lilacinus application on number of fruits and total fresh weight of fruits (g)/plant of tomato inoculated with M. incognita in Ikom soil Mycorhizal fungus

Number of fruits/plant P. lilacinus

Mean

Control Single application

Double application

Control 1.00 1.33 1.67 1.33 G. etunicatum 2.00 2.67 3.00 2.56 G. mosseae 1.67 2.33 2.67 2.22 G. clarum 1.67 2.33 3.00 2.33 G. gigantean 2.33 3.00 3.00 2.78 G. deserticola 1.67 2.33 2.67 2.22 Mean 1.72 2.33 2.67

Total Fresh Weight of Fruit

Control 9.48 14.16 16.62 13.42 G. etunicatum 19.34 38.68 45.05 34.36 G. mosseae 17.18 29.79 38.31 28.43 G. clarum 17.26 28.50 35.32 27.03 G. gigantean 20.89 40.50 50.86 37.42 G. deserticola 17.17 36.45 36.75 30.12 Mean 16.89 31.55 37.15

No of fruits Total fresh Wt. fruit LSD (0.05) for P. lilacinus (F) Means = 0.32 1.71 LSD (0.05) for Mycorrhizal fungus (M) = 0.45 2.41 LSD (0.05) for (FxM) Interaction Means = NS 4.18

81

Table 27: Effects of arbuscular mycorrhizal fungus and P. lilacinus application on number of galls and Eggmasses per root system, gall index (GI)* and Eggmass Index* of tomato inoculated with M. incognita in Nsukka soil

Mycorrhizal fungus

No. of Galls

P. lilacinus

Gall index

P. lilacinus

Control Single application

Double application

Mean Control Single application

Double application

Mean

Control 85.00 35.67 20.00 46.89 4.00 4.00 3.00 3.67

G. etunicatum 39.00 12.33 7.67 19.67 4.00 2.67 2.00 2.89

G. mosseae 56.00 31.00 20.00 35.67 4.00 3.33 3.00 3.44

G. clarum 36.67 26.33 16.00 26.33 4.00 3.67 3.00 3.56

Gi. gigantea 55.00 35.00 17.67 35.89 4.00 4.00 3.00 3.67

G. deserticola 45.00 17.67 17.33 26.67 4.00 3.00 3.00 3.33

Mean 52.78 26.33 16.45 4.00 3.45 2.83

No. of Eggmasses Eggmass Index

Control 49.33 11.00 7.67 22.67 4.00 2.33 2.00 2.78

G. etunicatum 10.67 4.33 2.33 5.78 2.67 2.00 1.33 2.00

G. mosseae 32.33 10.33 8.00 16.89 3.67 2.33 2.00 2.67

G. clarum 9.67 9.00 5.67 8.11 2.33 2.00 2.00 2.11

Gi. gigantea 31.67 11.00 8.00 16.89 3.67 2.33 2.00 2.67

G. deserticola 16.33 9.00 8.67 11.33 3.00 2.00 2.00 2.33

Mean 25.00 9.11 6.72 3.22 2.17 1.89

No. of Galls Gall Index No of Eggmasses Eggmass index LSD (0.05) for P. lilacinus (F) Means = 2.85 0.17 1.11 0.26 LSD (0.05) for Mycorrhizal (M) Means = 4.03 0.23 1.56 0.37 LSD (0.05) for (FXM) interaction Means = 6.99 0.41 2.71 0.64

82

M0F0: Control

M1F2: G. etunicatum + Bionematicide application twice Plate 5: Lightly galled and heavily galled roots of tomato in Nsukka soil.

83

Table 28: Effects of arbuscular mycorrhizal fungus and P. lilacinus application on fresh root weight (g)/plant, root colonization by AMF (%), shoot length(cm)/plant and dry shoot weight(g)/plant of tomato inoculated with M. incognita in Nsukka soil

Mycorrhizal fungus

Fresh root weight

P. lilacinus

AMF root colonization

P. lilacinus

Control Single application

Double application

mean Control Single application

Double application

Mean

Control 9.13 11.34 12.79 11.09 37.67 38.67 37.67 38.00

G. etunicatum 13.25 14.10 14.97 14.11 80.33 85.00 84.67 83.33

G. mosseae 12.65 13.70 14.23 13.53 88.67 86.33 85.00 86.67

G. clarum 13.76 14.15 15.06 14.32 90.00 88.00 86.67 88.22

Gi. Gigantean 12.72 13.94 14.41 13.69 81.67 84.00 84.00 83.22

G. deserticola 13.70 15.37 16.28 15.12 85.67 86.00 82.33 84.67

Mean 12.54 13.77 14.62 77.34 78.00 76.72

Shoot length Dry shoot weight

Control 47.00 66.67 69.67 61.11 9.99 13.13 15.66 12.90

G. etunicatum 59.00 69.67 73.00 67.22 13.73 15.43 18.60 15.92*(23.41)

G. mosseae 63.33 67.33 75.00 68.55 14.14 16.63 18.45 16.41(27.21)

G. clarum 57.67 65.00 70.33 64.33 14.60 15.92 17.63 16.05(24.42)

Gi. Gigantean 52.00 60.00 67.67 59.89 13.15 15.99 17.07 15.40(19.38)

G. deserticola 58.67 70.00 73.67 67.45 14.47 19.43 19.99 17.96(39.22)

Mean 56.28 66.45 71.56 13.35 16.09 17.90

Fresh Rt. Wt. Root Colonization Shoot length Shoot dry wt. LSD (0.05) for P. lilacinus (F) Means = 0.42 NS 1.79 0.56 LSD (0.05) for Mycorrhizal (M) Means = 0.60 2.40 2.54 0.79 LSD (0.05) for (FXM) interaction Means = NS NS 4.39 1.38 *Relative Mycorrhizal Effectiveness (RME) (%)

84

The bionematicide did not exert any significant effect on root colonization by the AMF species.

Tomato growth was significantly (P ≤0.05) enhanced with mycorrhizal inoculation of seedlings

and bionematicide application compared with their respective control in Nsukka Ultisol (Table

28). Generally plants that received double application of the bionematicide were taller than

others. The tallest plant was obtained with the combination of G. mosseae and double application

of the bionematicide. Dry shoot matter was significantly (P ≤0.05) increased with AMF

inoculation and as well as successive increase in the frequency of the bionematicide application

relative to the control plants. However, G. deserticola inoculated plants in combination with

double application of the bionematicide produced plants with significantly higher dry shoot

weight than other treatment combinations. G. deserticola was the most efficient AMF species

with respect to relative mycorrhizal effectiveness followed by G. mosseae (Table 28).

Inoculation of the tomato seedlings with AMF significantly (P ≤0.05) increased the number of

fruits set and fresh fruit weight relative to uninoculated plants in Nsukka Ultisol (Table 29). G.

deserticola inoculated plants had the highest number of fruit per plant and significantly the

greatest fruit yield. Significantly (P ≤0.05) higher fresh fruit yield was obtained when G.

deserticola was combined with double application of the bionematicide compared with other

AMF species. Results of the effect of AMF inoculation and application of bioformulated P.

lilacinus on root galling and eggmass production by M. incognita in Obubra soil are presented in

Table 30. Inoculation of AMF caused a significant (P ≤ 0.05) inhibition of root galling compared

with the control excepting Gi. gigantea. G. etunicatum was the most efficient species. Repeated

application of the bionematicide significantly reduced root galling more than applying once. The

least galling was obtained when G. etunicatum and G. deserticola were combined with double

application of the bionematicide. Also, the least gall index (GI) of 3 was obtained when

seedlings were inoculated with AMF and double treated with P. lilacinus. Eggmass production

significantly declined in mycorrhizal plants compared with non-mycorrhizal plants (Table 30).

85

Table 29: Effects of arbuscular mycorhizal fungus and P. lilacinus application on number of fruits and total fresh weight of fruits (g)/plant of tomato inoculated with M. incognita in Nsukka soil Mycorhizal fungus

Number of fruits/plant P. lilacinus

Mean

Control Single application

Double application

Control 1.33 2.00 2.67 2.00 G. etunicatum 2.33 3.00 3.67 3.00 G. mosseae 2.00 2.33 3.33 2.55 G. clarum 2.67 3.67 4.00 3.45 G. gigantea 2.33 4.00 4.33 3.55 G. deserticola 3.00 4.67 4.67 4.11 Mean 2.28 3.28 3.78 Total Fresh Weight of Fruit Control 16.38 28.04 53.09 34.17 G. etunicatum 44.87 56.34 68.74 56.65 G. mosseae 40.29 46.71 63.74 50.25 G. clarum 50.05 69.19 78.66 65.97 G. gigantea 58.19 92.02 96.07 82.09 G. deserticola 66.03 104.27 112.23 94.18 Mean 45.97 66.10 78.76

No of fruits Total fresh Wt. fruit LSD (0.05) for P. lilacinus (F) Means = 0.47 4.37 LSD (0.05) for Mycorrhizal fungus (M) = 0.68 6.68 LSD (0.05) for (FxM) Interaction Means = NS 11.57

86

Table 30: Effects of arbuscular mycorrhizal fungus and P. lilacinus application on number of galls and Eggmasses per root system, gall index (GI)* and Eggmass Index* of tomato inoculated with M. incognita in Obubra soil

Mycorrhizal fungus

No. of Galls

P. lilacinus

Gall index

P. lilacinus

Control Single application once

Double application twice

Mean Control Single application

Double application

Mean

Control 116.67 55.00 37.67 69.78 4.67 4.00 4.00 4.22

G. etunicatum 41.33 25.00 17.67 28.00 4.00 3.33 3.00 3.44

G. mosseae 65.00 45.00 24.33 44.78 4.00 3.67 3.00 3.56

G. clarum 113.33 27.33 21.33 54.00 5.00 3.33 3.00 3.78

Gi. gigantea 92.33 71.67 27.33 63.78 4.00 4.00 3.00 3.67

G. deserticola 65.00 33.00 15.67 37.89 4.00 4.00 3.00 3.67

Mean 82.28 42.83 24.00 4.28 3.72 3.17

No. of Eggmasses Eggmass Index

Control 96.67 23.33 12.00 44.00 4.33 3.00 2.67 3.33

G. etunicatum 27.67 10.67 8.67 15.67 3.00 2.67 2.00 2.56

G. mosseae 30.00 18.67 11.00 19.89 3.00 3.00 2.33 2.78

G. clarum 70.00 12.33 8.33 30.22 4.00 2.67 2.00 2.89

Gi. gigantea 62.67 30.00 8.00 33.56 4.00 3.00 2.00 3.00

G. deserticola 26.67 10.33 7.00 14.67 3.00 2.33 2.00 2.44

Mean 52.28 17.56 9.17 3.56 2.78 2.17 No. of Galls Gall Index No of Eggmasses Eggmass index

LSD (0.05) for P. lilacinus (F) Means = 5.86 0.18 1.52 0.23 LSD (0.05) for Mycorrhizal (M) Means = 8.24 0.26 2.15 0.32 LSD (0.05) for (FXM) interaction Means = 14.27 0.45 3.72 0.55 *0 = Immune, 1 = Highly Resistant, 2= Resistant, 3= Moderately susceptible 4 = Susceptible, 5 = Highly susceptible

87

The highest egg production inhibition was obtained with G. deserticola and G. etunicatum

inoculation. The least number of eggmasses was produced when bionematicide was applied

twice in combination with all the AMF species excepting G. mosseae. Eggmass index (EMI) was

reduced from 4.33 in the control plant to 2.00 in plants inoculated with AMF and treated with the

bionematicide twice. Mycorrhizal plants had significantly (P ≤0.05) higher fresh root weight

than non-mycorrhizal plants (Table 31). The greatest increase in fresh root weight was obtained

when plants were inoculated with AMF and treated twice with the bionematicide. There were

significant differences among the species of AMF in their root colonization rates. G. etunicatum

and G. deserticola had the highest colonization of more than 80%. Uninoculated plants were

lightly colonized by indigenous soil AMF species. Application of P. lilacinus had no significant

(P >0.05) effect on tomato root colonization by AMF. Arbuscular mycorrhizal fungi differed in

their ability to enhance tomato growth (Table 31). The tallest plants were found in G. deserticola

and G. mosseae inoculated plants. Growth enhancement was greater with the double application

of the bionematicide combined with all the AMF species compared with only one application.

Mycorrhizal plants significantly (P ≤ 0.05) accumulated more dry matter than their non-

mycorrhizal counterparts (Table 31) G. deserticola inoculated plants had the highest dry shoot

weight. Application of the bionematicide twice in combination with the AMF, significantly

increased dry shoot weight compared with no application G. deserticola inoculated plants had

the highest relative mycorrhizal effectiveness value followed by G. mosseae. There was a

significant (P≤ 0.05) increase in fruit-set and fresh fruit weight with AMF inoculation relative to

the uninoculated plants (Table 32). G. deserticola inoculated plants had the highest number of

fruits and the greatest total fresh fruit weight. P. lilacinus inoculation significantly (P ≤ 0.05)

enhanced fruit yield. G. etunicatum and G. deserticola in combination with application of

bionematicide twice produce significantly the highest fresh fruit yield.

88

Table 31: Effects of arbuscular mycorrhizal fungus and P. lilacinus application on fresh root weight (g)/plant, root colonization by AMF (%), shoot length (cm)/plant and dry shoot weight(g)/plant of tomato inoculated with M. incognita in Obubra soil

Mycorrhizal fungus

Fresh root weight

P. lilacinus

AMF root colonization

P. lilacinus

Control Single application

Double application

mean Control Single application

Double application

Mean

Control 8.29 11.66 13.02 10.99 16.00 17.33 17.33 16.89

G. etunicatum 12.70 14.20 15.11 14.01 84.00 79.67 83.67 82.45

G. mosseae 12.13 13.89 14.39 13.47 76.33 76.00 74.67 75.67

G. clarum 10.76 15.47 17.42 14.55 63.33 63.33 62.33 63.00

Gi. gigantea 11.78 13.27 15.97 13.67 70.00 70.33 70.33 70.22

G. deserticola 14.15 16.37 18.66 16.39 84.00 84.00 87.00 85.00

Mean 11.63 14.14 15.76 65.61 65.11 65.89

Shoot length Dry Shoot weight

Control 42.33 57.33 66.33 55.33 9.26 13.50 14.70 12.49

G. etunicatum 60.00 67.33 72.00 66.44 14.26 15.42 16.41 15.36(22.98)*

G. mosseae 62.00 67.67 71.67 67.11 14.97 16.85 17.65 16.49(32.03)

G. clarum 60.33 65.00 71.67 65.67 12.64 15.62 18.36 15.54(24.42)

Gi. gigantea 61.33 65.67 71.33 66.11 14.09 15.09 18.08 15.75(26.10)

G. deserticola 62.00 70.00 74.33 68.78 15.76 15.73 20.48 17.32(38.67)

Mean 58.00 65.50 71.22 13.50 15.37 17.61 Fresh Rt. Wt. Root Colonization Shoot length dry shoot wt.

LSD (0.05) for P. lilacinus (F) Means = 0.49 NS 1.71 0.59 LSD (0.05) for Mycorrhizal (M) Means = 0.69 1.67 2.42 0.84 LSD (0.05) for (FXM) interaction Means = 1.20 NS 4.19 1.47 *Relative Mycorrhizal Effectiveness (RME) (%)

89

Table 32: Effects of arbuscular mycorhizal fungus and P. lilacinus application on number of fruits per plant and total fresh weight of fruits (g)/plant of tomato inoculated with M. incognita in Obubra soil Mycorhizal fungus

Number of fruits/plant P. lilacinus

Mean

Control Single application

Double application

Control 1.00 2.33 2.33 1.89 G. etunicatum 2.00 2.67 3.67 2.78 G. mosseae 1.67 3.67 3.33 2.55 G. clarum 2.67 2.33 3.67 3.22 G. gigantea 2.33 3.33 3.33 2.67 G. deserticola 2.67 3.67 4.00 3.41 Mean 2.06 2.83 3.39

Total Fresh Weight of Fruit

Control 30.80 52.35 58.29 47.15 G. etunicatum 39.97 61.19 77.15 59.44 G. mosseae 43.50 58.12 69.82 57.15 G. clarum 36.22 52.82 65.65 51.56 G. gigantea 45.80 55.06 63.10 54.65 G. deserticola 51.48 65.60 75.77 64.28 Mean 41.30 57.52 68.30

No of fruits Total fresh Wt. fruit LSD (0.05) for P. lilacinus (F) Means = 0.55 2.32 LSD (0.05) for Mycorrhizal fungus (M) = 0.39 3.28 LSD (0.05) for (FXM) Interaction Means = 0.96 5.68

90

The results of the effect of AMF inoculation and application of P. lilacinus on root galling and

eggmass production on tomato infected with M. incognita in Ogoja soil are shown in Table 33.

AMF inoculation and application of P. lilacinus significantly (P ≤ 0.05) reduced number of galls

formed on the tomato root compared with their respective control. Gi. gigantea and G.

etunicatum were the most efficient AMF species. Application of the bionematicide twice was

more effective in gall inhibition than when applied once. The least number of galls was obtained

when G. etunicatum was combined with double application of the bionematicide. This treatment

combination also gave the least GI of 2.33. There was a change in the gall rating of the control

plants with GI = 5.00 (highly susceptible) to moderately susceptible GI = 3.00 when plants were

inoculated with AMF and the bionematicide double applied. Inoculation with AMF significantly

(P ≤ 0.05) deterred egg production relative to the non-mycorrhizal plants (Table 33). G.

etunicatum and Gi. gigantea were the most efficient. Double application of the bionematicide

twice significantly reduced eggmass more in most of the AMF inoculated plants compared with

single application. Generally EMI was reduced from 5.00 in the control plants to 2.00 in plants

inoculated with AMF and double applied with P. lilacinus twice. Inoculation of tomato seedlings

with AMF significantly (P ≤ 0.05) increased fresh root weight in Ogoja soil relative to the non-

mycorrhizal excepting G. clarum inoculated plants (Table 34). Increase in the frequency of P.

lilacinus application significantly enhanced fresh root weight. Double application of the

bionematicide in combination with AMF inoculation resulted in higher root biomass. AMF

species differed significantly (P≤0.05) in their ability to colonize tomato roots. However, Gi.

gigantea and G. etunicatum had significantly higher colonization rate. Application of

bionematicide induced an increase in the rate of root colonization by AMF in Ogoja soil.

91

Table 33: Effects of arbuscular mycorrhizal fungus and P. lilacinus application on number of galls and Eggmasses per root system, gall index (GI)* and Eggmass Index* of tomato inoculated with M. incognita in Ogoja soil

Mycorrhizal fungus

No. of Galls

P. lilacinus

Gall index

P. lilacinus

Control Single application

Double application

Mean Control Single application

Double application

Mean

Control 191.00 42.33 17.67 83.67 5.00 4.00 3.00 4.00

G. etunicatum 55.00 21.33 10.00 28.78 4.00 3.00 2.33 3.11

G. mosseae 62.00 33.33 15.33 36.89 4.00 3.67 3.00 3.56

G. clarum 76.00 35.00 14.33 41.78 4.00 4.00 2.67 3.56

Gi. gigantea 51.00 20.67 15.00 28.89 4.00 3.00 3.00 3.33

G. deserticola 59.00 22.00 16.33 32.44 4.00 3.00 3.00

Mean 82.33 29.11 14.78 4.17 3.44 2.83

No. of Eggmasses Eggmass Index

Control 110.00 22.00 10.33 47.44 5.00 3.00 2.33 3.44

G. etunicatum 30.00 10.00 6.00 15.33 3.33 2.33 2.00 2.56

G. mosseae 32.67 18.67 9.67 20.33 3.67 3.00 2.33 3.00

G. clarum 55.00 19.33 8.67 27.67 4.00 3.00 2.33 3.11

Gi. gigantea 23.00 11.33 7.67 14.00 3.00 2.67 2.00 2.56

G. deserticola 25.00 11.00 8.67 14.89 3.00 2.67 2.00 2.56

Mean 45.94 15.39 8.50 3.67 2.78 2.17 No. of Galls Gall Index No of Eggmasses Eggmass index

LSD (0.05) for P. lilacinus (F) Means = 2.67 0.16 1.90 0.26 LSD (0.05) for Mycorrhizal (M) Means = 3.78 0.23 2.69 0.37 LSD (0.05) for (FXM) interaction Means = 6.55 0.39 4.65 0.64 *0 = Immune, 1 = Highly susceptible, 2= Resistant, 3= Moderately susceptible 4 = Susceptible, 5 = Highly susceptible

92

Table 34: Effects of arbuscular mycorrhizal fungus and P. lilacinus application on fresh root weight (g)/plant, root colonization by AMF (%), shoot length (cm)/plant and dry shoot weight(g)/plant of tomato inoculated with M. incognita in Ogoja soil

Mycorrhizal fungus

Fresh root weight

P. lilacinus

AMF root colonization

P. lilacinus

Control Single application

Double application

mean Control Single application

Double application

Mean

Control 5.29 9.96 10.54 8.60 15.00 18.00 16.67 16.56

G. etunicatum 7.63 10.10 13.17 10.30 64.33 67.33 68.67 66.78

G. mosseae 8.79 11.16 12.21 10.72 60.00 61.00 60.67 60.56

G. clarum 5.93 8.88 10.94 8.58 57.00 58.67 57.33 57.67

Gi. gigantea 9.79 10.88 12.09 10.92 70.33 71.00 70.33 70.56

G. deserticola 7.42 10.20 11.15 9.59 60.00 60.67 60.33 60.33

Mean 7.48 10.20 11.68 54.44 56.11 55.67

Shoot length Dry shoot weight

Control 50.00 60.33 69.33 59.89 6.74 10.69 12.85 10.09

G. etunicatum 62.33 70.33 77.67 70.11 10.19 13.08 17.67 13.65(35.28)*

G. mosseae 62.00 67.67 72.33 67.33 9.69 12.49 15.94 12.71(25.97)

G. clarum 58.33 63.33 70.33 64.00 8.04 10.83 14.46 11.11(10.11)

Gi. gigantea 62.67 72.33 78.33 71.11 12.59 14.60 18.09 15.10(49.65)

G. deserticola 60.33 70.00 74.67 68.33 8.84 11.16 15.00 11.67(15.66)

Mean 59.28 67.33 73.78 9.35 12.14 15.67

Fresh Rt. Wt. Root Colonization Shoot length Dry shoot wt.

LSD (0.05) for P. lilacinus (F) Means = 0.45 1.02 1.50 0.47 LSD (0.05) for Mycorrhizal (M) Means = 0.64 1.44 2.11 0.67 LSD (0.05) for (FXM) interaction Means = 1.11 NS NS NS *Relative Mycorrhizal Effectiveness (RME) (%)

93

However, this increase was significant only when it was applied once. Growth enhancement by

AMF inoculation was significant (P≤0.05) when compared with the non-mycorrhizal plants. Gi.

gigantea and G. etunicatum were the most efficient species. Double application of P. lilacinus

twice significantly (P≤ 0.05) increased shoot length than single application. Shoot dry matter

accumulation was significantly (P≤ 0.05) enhanced with AMF inoculation relative to

uninoculated plants. Gi. gigantea and G. etunicatum inoculated plants accumulated the greatest

shoot dry matter (Table 34). Double application of the bionematicide induced the highest dry

shoot weight relative to other frequencies of applications. The most efficient AMF species was

Gi. gigantea with the highest relative mycorrhizal effectiveness value followed by G.

etunicatum. There was a significant (P ≤ 0.05) increase in the number of fruits set and the

weight of fresh fruits with inoculation of AMF and application of bionematicide in Ogoja soil

compared with the control plants (Table 35). However, for the AMF species, Gi gigantea and G.

etunicatum were the most efficient. Double application of the bionematicide in combination with

Gi gigantea and G. etunicatum produced significantly the highest fruit yield.

The results of the effect of AMF inoculation and application of bioformulated P. lilacinus on

root galling and egg production by M. incognita on tomato in Umudike soil are presented in

Table 36. There was a significant (P ≤ 0.05) reduction in the severity of root galling and

eggmass production by M. incognita with AMF inoculation and bionematicide application

compared with their respective control. Gall and eggmass were more significantly (P ≤ 0.05)

reduced with double application of the bionematicide than single. For both variables, Gi gigantea

and G. mosseae were the most efficient AMF species. Combination of all the AMF species with

double application of the bionematicide significantly (P ≤ 0.05) inhibited egg production

relative to single application excepting G. mosseae and Gi. gigantea.

94

95

Table 35: Effects of arbuscular mycorhizal fungus and P. lilacinus application on number of fruits per plant and total fresh weight of fruits (g) plant of tomato inoculated with M. incognita in Ogoja soil Mycorhizal fungus

Number of fruits/plant P. lilacinus

Mean

Control Single application

Double application

Control 1.00 1.67 2.33 1.67 G. etunicatum 2.00 3.00 4.33 3.11 G. mosseae 2.00 2.67 3.67 2.78 G. clarum 1.67 2.33 3.33 2.44 G. gigantea 2.33 2.33 4.33 3.33 G. deserticola 2.00 2.67 3.67 2.78 Mean 1.83 2.61 3.61 Total Fresh Weight of Fruit Control 8.60 23.22 36.55 22.79 G. etunicatum 33.70 42.38 69.45 48.51 G. mosseae 28.76 40.38 56.44 41.86 G. clarum 20.42 33.98 48.58 34.33 G. gigantea 37.50 46.26 75.71 53.16 G. deserticola 28.01 46.27 53.42 42.57 Mean 26.16 38.75 56.69

No of fruits Total fresh Wt. fruit LSD (0.05) for P. lilacinus (F) Means = 0.33 2.59 LSD (0.05) for Mycorrhizal fungus (M) = 0.47 3.66 LSD (0.05) for (FxM) Interaction Means = NS 6.35

96

Table 36: Effects of arbuscular mycorrhizal fungus and P. lilacinus application on number of galls and Egg masses per root system, gall index (GI)* and Eggmass Index* of tomato inoculated with M. incognita in Umudike soil

Mycorrhizal fungus

No. of Galls

P. lilacinus

Gall index

P. lilacinus

Control Single application

Double application

Mean Control Single application

Double application

Mean

Control 121.67 41.00 28.00 63.56 4.67 4.00 3.33 4.00

G. etunicatum 59.67 31.00 15.67 35.44 4.00 3.33 3.00 3.44

G. mosseae 47.00 21.00 10.00 26.00 4.00 3.00 2.33 3.11

G. clarum 55.00 26.67 12.00 31.22 4.00 3.00 2.67 3.22

Gi. gigantea 45.00 15.00 10.00 23.33 4.00 3.00 2.33 3.11

G. deserticola 57.33 20.33 12.67 30.11 4.00 3.00 3.00 3.33

Mean 64.28 25.83 14.72 4.11 3.22 2.78

No. of Eggmasses Eggmass Index

Control 87.33 26.33 11.67 41.78 4.00 3.00 2.67 3.22

G. etunicatum 40.33 18.33 9.33 22.67 4.00 3.00 2.33 3.11

G. mosseae 32.00 11.67 7.33 17.00 3.67 2.67 2.00 2.78

G. clarum 31.67 17.33 7.67 18.89 3.67 3.00 2.00 2.89

Gi. gigantea 23.00 9.67 6.33 13.00 3.00 2.33 2.00 2.44

G. deserticola 34.00 15.67 9.00 19.67 4.00 3.00 2.00 3.00

Mean 41.44 16.50 8.56 3.72 2.83 2.17 No. of Galls Gall Index No of Eggmasses Eggmass index

LSD (0.05) for P. lilacinus (F) Means = 4.81 0.23 2.15 0.23 LSD (0.05) for Mycorrhizal (M) Means = 6.81 0.32 3.04 0.32 LSD (0.05) for (FXM) interaction Means = 11.79 NS 5.27 NS *0 = Immune, 1 = Highly Resistant, 2= Resistant, 3= Moderately susceptible 4 = Susceptible, 5 = Highly susceptible

97

The lowest number of galls and eggmasses were obtained with the combination of Gi. gigantea

or G. mosseae and double application of the bionematicide. Gall index and eggmass index

followed the trend of number of galls and eggmasses per root system. In mycorrhizal plants,

double application of P. lilacinus twice reduced the EMI from 4.00 to 2.00.

Fresh root weight was significantly (P ≤ 0.05) increased with AMF inoculation as well as

application of the bioformulated P. lilacinus in Umudike soil (Table 37) relative to their

respective control. Gi. gigantea and G. mosseae inoculated plants had the heaviest root mass.

Double application of the bionematicide twice significantly (P ≤ 0.05) increased root weight

over single application. The rate of root colonization by AMF species varied significantly. Gi

gigantea and G. mosseae had significantly higher colonization rate. The uninoculated plants

were lightly colonized by indigenous AMF species. Application of P. lilacinus did not

significantly (P>0.05) affect root colonization by the AMF species. Tomato growth and shoot

dry matter accumulation were significantly (P ≤ 0.05) enhanced with AMF inoculation and

bionematicide application in Umudike soil compared with the control treatments. Double

application of the bionematicide significantly (P ≤ 0.05) enhanced growth and dry matter

production relative to single application. The tallest plants with the highest dry shoot matter were

obtained with the combination of G. gigantea or G. mosseae and double application of the

bionematicide. There was a very high response to mycorrhizal inoculation in Umudike soil.

However, Gi gigantea and G. mosseae had the highest relative mycorrhizal effectiveness (RME)

values (Table 37). There was a significant increase in the number of fruits produced and the

weight of fresh fruit when tomato seedlings were inoculated with AMF and bionematicide

applied in Umudike soil (Table 38). More fruits were set and the fresh fruit weight was

significantly increased with double application of the bionematicide compared with application

once. The most efficient species in yield enhancement were Gi. gigantea and G. mosseae.

98

Table 37: Effects of arbuscular mycorrhizal fungus and P. lilacinus application on fresh root weight (g)/plant, root colonization by AMF (%), shoot length(cm)/plant and dry shoot weight (g)/plant of tomato inoculated with M. incognita in Umudike soil

Mycorrhizal fungus

Fresh root weight

P. lilacinus

AMF root colonization

P. lilacinus

Control Single application

Double application

mean Control Single application

Double application

Mean

Control 5.16 8.25 9.10 7.50 19.00 20.00 20.33 19.78

G. etunicatum 8.07 10.24 12.06 10.12 61.67 61.33 60.33 61.11

G. mosseae 9.33 11.21 13.53 11.36 65.00 65.00 64.67 64.89

G. clarum 8.08 12.71 11.65 10.81 55.00 55.33 56.33 55.56

Gi.gigantea 10.22 12.79 14.83 12.61 66.33 68.33 70.00 68.22

G. deserticola 9.03 10.63 12.83 10.83 60.00 61.00 61.00 60.67

Mean 8.32 10.97 12.33 54.50 55.17 55.44

Shoot length Dry shoot weight

Control 52.00 62.33 67.67 60.67 7.58 11.96 13.18 10.91

G. etunicatum 61.00 65.33 72.67 66.33 10.21 13.16 17.01 13.46*(23.37)

G. mosseae 65.33 71.00 75.33 70.56 11.98 15.90 18.69 15.52(42.25)

G. clarum 62.00 66.67 71.00 66.56 9.68 13.14 16.56 13.12(20.26)

Gi. Gigantean 68.67 74.00 77.67 73.44 13.20 16.69 19.71 16.53(51.51)

G. deserticola 60.00 65.33 71.33 65.56 10.64 14.84 17.47 14.32(31.26)

Mean 59.28 67.33 72.61 10.55 14.28 17.10 Fresh Rt. Wt. Root Colonization Shoot length Dry shoot wt.

LSD (0.05) for P. lilacinus (F) Means = 0.96 NS 1.00 0.35 LSD (0.05) for Mycorrhizal (M) Means = 1.36 1.46 1.42 0.49 LSD (0.05) for (FXM) interaction Means = NS NS 2.46 0.85 *Relative Mycorrhizal Effectiveness (RME) (%)

99

Table 38: Effects of arbuscular mycorhizal fungus and P. lilacinus application on number of fruits/plant and total fresh weight of fruits (g)/plant of tomato inoculated with M. incognita in Umudike soil Mycorhizal fungus

Number of fruits/plant P. lilacinus

Mean

Control Single application

Double application

Control 1.00 1.67 2.33 1.67 G. etunicatum 2.00 2.67 3.67 2.78 G. mosseae 2.33 3.33 4.67 3.44 G. clarum 1.67 2.67 3.67 2.67 G. gigantea 2.67 4.00 4.67 3.78 G. deserticola 2.00 3.00 4.00 3.00 Mean 1.94 2.89 3.83 Total Fresh Weight of Fruit Control 10.38 28.68 37.00 25.36 G. etunicatum 37.09 44.89 67.86 49.95 G. mosseae 43.32 70.27 96.06 69.88 G. clarum 31.73 46.37 74.54 50.88 G. gigantea 56.36 73.38 105.93 78.55 G. deserticola 35.70 51.12 81.02 55.95 Mean 35.76 52.45 77.07

No of fruits Total fresh Wt. fruit LSD (0.05) for P. lilacinus (F) Means = 0.32 4.30 LSD (0.05) for Mycorrhizal fungus (M) = 0.45 6.09 LSD (0.05) for (FxM) Interaction Means = NS 10.54

100

These two species of AMF also combined with double application of the bionematicide to give

significantly (P ≤ 0.05) the highest fresh fruit yield.

The results of the effects of AMF inoculation and application of bioformulated P.lilacinus on

root gall and egg mass production on tomato infected with M. incognita in Uyo soil are presented

in Table 39. Eggmass production and root galling were significantly (P ≤ 0.05) impaired with

AMF inoculation and application of bionematicide compared with their respective control.

However, double application of the bionematicide was more effective in gall and egg production

inhibition than when it was single applied. For gall and eggmass inhibition, G. etunicatum and G.

deserticola were the most efficient species. The combination of G. etunicatum and G. mosseae

with double application of the bionematicide gave significantly the least number of galls per root

system and the least GI of 2.33. However, for egg production, the least number of eggmass was

obtained with G. etunicatum in combination with double application of the bioformulated

P.lilacinus. There was a reduction in EMI from 4.00 in the control plants to 2.00 in mycorrhizal

plants in combination with double application of the bionematicide excepting G. mosseae

inoculated plants with EMI of 3.00.

Fresh root weight of tomato plants were significantly (P ≤ 0.05) increased with AMF

inoculation and application of bionematicide in Uyo soil compared with their respective control

(Table 40). G. deserticola and G. mosseae were the most effective species. In most cases, double

application of the bionematicide in combination with AMF inoculation significantly increased

fresh root weight over single application with the exception of Gi. gigantea. Colonization of root

by AMF species differed significantly. The highest root colonizing species were G. deserticola

and G. mosseae. Application of P. lilacinus had no significant (P>0.05) effect on root

colonization by the AMF species. As in other soil types, there was a significant growth

enhancement with AMF inoculation and bionematicide application.

101

Table 39: Effects of arbuscular mycorrhizal fungus and P. lilacinus application on number of galls and Eggmasses per root system, gall index (GI)* and Eggmass Index* of tomato inoculated with M. incognita in Uyo soil

Mycorrhizal fungus

No. of Galls

P. lilacinus

Gall index

P. lilacinus

Control Single application

Double application

Mean Control Single application

Double application

Mean

Control 75.00 49.67 42.33 55.67 4.00 4.00 4.00 4.00

G. etunicatum 37.67 19.00 10.00 22.22 4.00 3.00 2.33 3.11

G. mosseae 47.33 25.00 11.33 27.89 4.00 3.00 2.33 3.11

G. clarum 45.00 27.67 22.33 31.67 4.00 3.00 3.00 3.33

Gi. gigantea 66.00 52.33 29.33 49.22 4.00 4.00 3.00 3.67

G. deserticola 32.33 21.67 17.00 23.67 3.67 3.00 3.00 3.22

Mean 50.56 32.56 22.55 3.95 3.33 2.94

No. of Eggmasses Eggmass Index

Control 56.67 25.00 16.00 32.56 4.00 3.00 3.00 3.33

G. etunicatum 19.00 9.00 5.67 11.22 3.00 2.00 2.00 2.33

G. mosseae 36.67 19.67 14.00 23.78 4.00 3.00 3.00 3.33

G. clarum 20.33 13.00 8.67 14.00 3.00 3.00 2.00 2.67

Gi. gigantea 25.67 14.00 7.33 15.67 3.00 3.00 2.00 2.67

G. deserticola 15.33 9.67 7.67 10.89 3.00 2.33 2.00 2.44

Mean 29.11 15.06 9.89 3.33 2.72 2.33 No. of Galls Gall Index No of Eggmasses Eggmass inde

LSD (0.05) for P. lilacinus (F) Means = 2.13 0.17 1.39 0.09 LSD (0.05) for Mycorrhizal (M) Means = 3.02 0.23 1.96 0.13 LSD (0.05) for (FXM) interaction Means= 5.22 0.41 3.40 0.23 *0 = Immune, 1 = Highly Susceptible, 2= Resistant, 3= Moderately susceptible 4 = Susceptible, 5 = Highly susceptible

102

Table 40: Effects of arbuscular mycorrhizal fungus and P. lilacinus application on fresh root weight (g)/plant, root colonization by AMF (%), shoot length (cm)/plant and dry shoot weight (g)/plant of tomato inoculated with M. incognita in Uyo soil

Mycorrhizal fungus

Fresh root weight

P. lilacinus

AMF root colonization

P. lilacinus

Control Single application

Double application

Mean Control Single application

Double application

Mean

Control 10.56 12.70 13.92 12.39 23.67 24.67 23.00 23.78

G. etunicatum 12.65 14.09 15.04 13.93 60.67 59.67 60.67 60.33

G. mosseae 13.26 14.69 15.62 14.52 67.67 69.67 68.67 68.67

G. clarum 13.72 13.78 14.45 13.98 61.67 59.33 61.67 60.89

Gi. gigantea 12.04 13.49 13.90 13.14 62.00 61.67 60.67 61.44

G. deserticola 14.84 15.99 16.47 15.77 70.33 71.67 70.67 70.89

Mean 12.85 14.12 14.90 57.67 57.78 57.56

Shoot length Dry shoot weight

Control 53.00 65.00 71.00 63.00 13.05 15.58 17.98 15.54

G. etunicatum 70.00 72.67 77.00 73.22 16.17 18.40 20.16 18.24(17.37)*

G. mosseae 65.67 72.67 78.00 72.11 17.70 20.02 22.58 20.10(29.34)

G. clarum 71.33 75.00 79.33 75.22 18.22 20.60 21.72 20.18(29.86)

Gi. gigantea 65.33 67.67 70.00 67.67 15.26 17.07 17.77 16.70(7.46)

G. deserticola 72.00 76.67 79.00 75.89 19.77 20.57 21.86 20.73(33.40)

Mean 66.22 71.61 75.72 16.70 18.71 20.35 Fresh Rt. Wt. Root Colonization Shoot length Dry shoot wt.

LSD (0.05) for P. lilacinus (F) Means = 0.35 NS 1.65 0.58 LSD (0.05) for Mycorrhizal (M) Means = 0.50 1.84 2.33 0.78 LSD (0.05) for (FXM) interaction Means= 0.86 NS 4.04 1.36 * Relative Mycorrhizal Effectiveness (RME)%

103

G. deserticola and G. clarum were the most efficient species. Double application of the

bionematicide significantly enhanced tomato growth more than single application. Shoot dry

matter accumulation was also significantly (P ≤ 0.05) enhanced with AMF inoculation and

bionematicide application. The highest dry shoot weight was obtained with G. mosseae

inoculated plants in combination with double application of the bionematicide. The most

efficient AMF species with reference to relative mycorrhizal effectiveness was G. deserticola

while the least was Gi. gigantea (Table 40).

Fruit-set and fresh fruit weight of tomato were significantly (P ≤ 0.05) enhanced with AMF

inoculation and application of bionematicide compared with their respective control in Uyo soil

(Table 41). Tomato seedlings inoculated with G. deserticola produced significantly the highest

fresh fruit yield followed by G. mosseae inoculated plants. Also, the combination of G.

deserticola or G. mosseae with double application of the bionematicide gave the highest fruit

yield relative to other treatment combinations.

104

Table 41: Effects of arbuscular mycorhizal fungus and P. lilacinus application on number of fruits per plant and total fresh fruit weight (g)/plant of tomato inoculated with M. incognita in Uyo soil Mycorhizal fungus

Number of fruits/plant P. lilacinus

Mean

Control Single application

Double application

Control 1.33 2.33 2.33 2.00 G. etunicatum 2.67 3.00 3.33 3.00 G. mosseae 3.33 4.00 4.67 4.00 G. clarum 1.33 3.67 3.33 2.78 G. gigantea 1.67 2.67 3.00 2.45 G. deserticola 4.00 4.33 4.67 4.33 Mean 2.39 3.33 3.56

Total Fresh Weight of Fruit

Control

11.20 36.88 45.52 30.20

G. etunicatum 34.84 49.05 55.71 46.53 G. mosseae 54.85 73.50 79.20 69.18 G. clarum 37.14 57.87 66.19 53.73 G. gigantea 33.96 54.28 62.02 50.09 G. deserticola 62.13 74.72 85.07 74.18 Mean 39.02 57.72 65.22 No of fruits Total fresh Wt. fruit LSD (0.05) for P. lilacinus (F) Means = 0.38 3.89 LSD (0.05) for Mycorrhizal fungus (M) = 0.53 5.23 LSD (0.05) for (FxM) Interaction Means = NS NS

105

Experiment VI. Evaluation of the Effects of P. lilacinus (PL GoldTM), Arbuscular

Mycorrhizal Fungi and Mucuna green manure on the pathogencity of M. incognita on

tomato

The results of the effects of inoculation of tomato seedlings with arbuscular mycorrhizal fungi

(AMF), inoculation with bioformulated nematicide and amendment with various Mucuna spp as

green manure on root galling by M. incognita are presented in Table 42. Soil amendment with all

the Mucuna spp significantly (P ≤ 0.05) reduced root galling compared with the unamended soil.

Significantly, the least number of galls was obtained with M. jaspaeda amendment followed by

M. ghana. Mycorrhizal inoculation significantly (P ≤ 0.05) inhibited gall formation compared

with the uninoculated control. The most efficient species in gall inhibition was G. mosseae.

Inoculation of the tomato plants with the bionematicide significantly (P≤ 0.05) reduced root

galling relative to the uninoculated plants. Interaction between AMF and Mucuna amendment

was significant. Among all the Mucuna spp, and with the unamended soil, AMF inoculation

significantly reduced root galling compared with the non-mycorrhizal plant excepting G.

deserticola in combination with M. pruriens utilis. Interaction between mycorrhizal inoculation

and bionematicide application was significant. In both mycorrhizal and non-mycorrhizal plants,

application of P. lilacinus significantly inhibited root galling. The interaction among the three

factors was significant. Generally, there was a significant inhibition in root galling when plants

were inoculated with the two bicontrol agents and the soil amended with Mucuna compared with

the control. Tomato plants inoculated with G. mosseae and P. lilacinus and the soil amended

with the various Mucuna species in most cases had significantly the fewest number of galls per

root system compared with other treatment combinations, as shown in Plate 6. Inoculation of G.

deserticola in combination with the other two factors followed G. mosseae in gall reduction.

106

Table 42: Effects of arbuscular mycorrhizal fungi, P. lilacinus and Mucuna spp soil amendment on number of galls/root system of tomato inoculated with M. incognita Mucuna spp Mycorrhizal fungus

P. lilacinus

Vo** V 0xF V1 V1xF V2 V2xF V3 V3xF V4 V4xF V5 V5xF MF F M

*M 0 F0*** F1

105.67 22.00

65.78 13.56

38.33 18.67

30.44 10.89

22.33 10.67

15.33 8.06

55.67 19.67

39.78 13.00

19.67 9.67

13.11 7.67

65.00 21.33

46.89 15.22

51.11 17.00

35.22 11.40

34.06

(M x V) M1

F0 F1

63.83 75.67 11.67

28.50 29.67 10.67

16.50 15.67 8.33

37.67 40.00 15.67

14.67 13.67 9.00

43.17 49.67 17.67

37.39 12.17

24.78

(M x V) M2

F0 F1

43.67 44.33 9.33

20.17 27.67 6.67

12.00 11.33 4.33

27.83 29.67 9.67

11.33 9.67 4.67

33.67 35.00 11.00

26.28 7.61

16.94

(M x V) M3

F0 F1

26.83 60.00 14.67

17.17 32.33 10.00

7.83 15.67 9.67

19.67 42.33 12.33

7.17 13.00 8.67

23.00 48.33 18.67

35.28 12.33

23.81

(M x V) M4

F0 F1

37.33 44.33 12.33

17.00 25.00 9.00

12.67 11.67 10.00

27.33 40.00 11.00

10.83 10.33 8.67

33.50 45.67 12.33

29.50 10.56

20.03

(M x V) M5

F0 F1

28.33 64.67 11.33

20.00 29.67 10.33

10.83 15.33 5.33

25.50 31.00 9.67

9.50 12.33 9.67

29.00 37.67 10.33

31.78 8.72

20.25

(M x V) 38.00 28.50 10.33 20.33 8.83 24.00 V-mean

39.67

20.67

11.69

26.39

10.39

31.05

*Mo = Control **Vo = Control ***Fo = Control LSD(0.05) Mycorrhiza means(M) = 1.01 M1 = G. etunicatum V1 = M. pruriens utilis F1 = P. lilacinus applied LSD(0.05) for Mucuna means(V) = 1.01 M2 = G. mosseae V2 = M. ghana LSD(0.05) for P. lilacinus means(F) = 0.59 M3 = G. clarum V3 = M. cochichinensis LSD(0.05) (M x V) interaction means = 2.28 M4 = Gi.. gigantea V4 = M. jaspaeda LSD(0.05) (M x F) interaction means = 1.43 M5 = G. deserticola V5 = M. pruriens IR2 LSD(0.05) (V x F) interaction means = 1.43 LSD(0.05) (M x F x V) interaction means = 3.51

107

M0V0F0: Control

M2V4F1: G. mosseae + M. jaspaeda + Bionematicide application

Plate 6: Lightly galled and heavily galled roots of Tomato plants due to treatment effects.

108

Soil amendment with Mucuna significantly (P ≤ 0.05) reduced gall index (GI) compared with the

unamended soil with the exception of M. pruriens IR2 and M. cochichinensis (Table 43). The lowest

gall index of 2.36 was obtained with M. jaspaeda. Similarly, mycorrhizal inoculation significantly

(P≤0.05) reduced gall index relative to the uninoculated plant excepting G. clarum. The most

efficient species in gall index reduction was G. mosseae followed by G. deserticola. Inoculation of

tomato plants with the bionematicide significantly (P≤0.05) reduced gall index relative to the

uninoculated plant. Interactions between mycorrhizal and Mucuna amendment was significant. In

the nonmycorrhizal plants, only soil amended with M. jaspaeda and M. ghana significantly reduced

gall index compared with the unamended soil. However, among the AMF species, there was a

significant decrease in gall rating when the soil was amended with the Mucuna spp compared with

the unamended soil excepting M. cochichinensis and M. pruriens IR2. Although, there was no

significant (P>0.05) interaction among the three factors, the gall rating of tomato plants infected by

M. incognita was lower in soils amended with Mucuna and plants inoculated with the two biocontrol

agents compared with the control. The gall index (GI) of 4.67 was recorded for the control plants

and were rated highly susceptible, while those soil amended with M. jaspaeda and M. ghana and

inoculated with the two biocontrol agents had in most cases plants with GI = 2.0, rated resistant.

Eggmass production by M. incognita followed the trend of root galling (Table 44). Soil

amendment with Mucuna, inoculation of plants with AMF and the bionematicide significantly (P ≤

0.05) inhibited egg production compared with their respective controls. However, G. mosseae and

M. ghana were the most efficient AMF and Mucuna species, respectively. The interaction among the

three factors was significant (P ≤ 0.05) in eggmass production inhibition. In most cases, eggmass

production was reduced more when the three factors where combined relative to single or double

application.

109

Table 43: Effects of arbuscular mycorrhizal fungi; P. lilacinus and Mucuna spp soil amendment on root gall index (GI) of tomato inoculated with M. incognita

Mucuna spp Mycorrhizal Fungus

p. lilacinus

Vo** V

0xF V

1 V

1xF V

2 V

2xF V

3 V

3xF V

4 V

4xF V

s V

5xF MF F M

M0* F

0

***

F1

4.67 3.00

4.11 2.67

4.00 3.00

3.44 2.33

3.00 2.33

2.94 2.11

4.00 3.00

3.83 2.72

3.00 2.00

2.72 2.00

4.00 3.00

3.94 2.83

3.78 2.72

3.50 2.44

3.25

(M0xv)

M1

F

0

F1

3.83 4.00 2.67

3.50 3.33 2.33

2.67 3.00 2.00

3.50 4.00 3.00

2.50 3.00 2.00

3.50 4.00 3.00

3.56 2.50

3.03

(M2xv)

M2

F

0

F1

3.33 4.00 2.00

2.83 3.00 2.00

2.50 2.67 2.00

3.50 3.33 2.33

2.50 2.00 2.00

3.50 3.67 2.67

3.11 2.17

2.64

(M3xv)

M3

F

0

F1

3.00 4.00 3.00

2.50 4.00 2.44

2.33 3.00 2.00

2.83 4.00 3.00

2.00 3.00 2.00

3.17 4.00 3.00

3.67 2.56

3.11

(M4xv)

M4

F

0

F1

3.50 4.00 2.67

3.17 3.00 2.00

2.50 3.00 2.33

3.50 4.00 2.67

2.50 2.33 2.00

3.50 4.00 3.00

3.89 2.44

2.92

(M5xv)

M5

F

0

F1

3.33 4.00 2.67

2.50 3.33 2.33

2.67 3.00 2.00

3.33 3.67 2.33

2.17 3.00 2.00

3.50 4.00 2.33

3.50 2.28

2.89

(M5xv)

3.83 3.50 2.67 3.50 2.50 3.50

v-mean 3.39 2.89 2.53 3.28 2.36 3.39

M0 = Control **Vo = Control ***F0 = Control LSD(0.05) Mycorrihza means(M) = 0.15

M1 = G.etunicatum V1 = M. pruriens utilis F1 = P. lilacinus applied LSD(0.05) for Mucuna means (V) = 0.15

M2 = G. mosseae V2 = M. ghana LSD(0.05) for P. lilacinus means (F) = 0.08

M3 = G. clarum V3 = M. cochichinensis LSD(0.05) (MXV) Interaction means = 0.36

M4 = G. gigantea V4 = M. jaspaeda LSD(0.05) (MXF) Interaction means = NS

M5 = G. deserticola V5 = M. pruriens IR2 LSD(0.05) (VXF) Interaction means = 0.21

LSD(0.05) (MXFXV) Interaction means = NS

110

Table 44: Effects of arbuscular mycorrhizal fungus, P. lilacinus and Mucuna spp soil amendment on number of egg masses/ root system of tomato inoculated with M. incognita

*M0 = Control **Vo = Control ***F0\ = Control LSD(0.05) Mycorrihza means(M) = 1.04

M1 = G.etunicatum V1 = M. pruriens utilis F1 = P. lilacinus applied LSD(0.05) for Mucuna means (V) = 1.04

M2 = G. mosseae V2 = M. ghana LSD(0.05) for P. lilacinus means (F) = 0.60

M3 = G. clarum V3 = M. cochichinensis LSD(0.05) (MXV) Interaction means = 2.54

M4 = G. gigantean V4 = M. jaspaeda LSD(0.05) (MXF) Interaction means = 1.47

M5 = G. deserticola V5 = M. pruriens IR2 LSD(0.05) (VXF) Interaction means = 1.47

LSD(0.05) (MXFXV) Interaction means = 3.59

Mucuna spp Mycorrhizal Fungus

p. lilacinus

Vo** V

0xF V

1 V

1xF V

2 V

2xF V

3 V

3xF V

4 V

4xF V

s V

5xF MF F M

M0* F

0

***

F1

85.33 12.00

39.22 7.28

17.33 8.00

11.83 4.89

10.67 3.67

6.44 2.72

19.00 11.67

16.78 7.17

11.33 5.00

7.94 3.39

25.00 10.00

19.0 7.28

28.11 8.39

16.87 5.45

18.25

(M0xv)

M1

F

0

F1

48.67 42.33 8.00

12.67 13.33 4.67

7.17 6.33 2.33

15.33 19.33 7.33

8.17 9.00 3.67

17.50 20.00 8.33

18.39 5.72

12.06

(M1xv)

M2

F

0

F1

25.17 22.33 3.33

9.00 8.67 2.00

4.33 4.67 1.33

13.33 11.33 4.67

6.33 5.33 1.67

14.17 14.33 5.33

11.11 3.06

7.08

(M2xv)

M3

F

0

F1

12.83 30.33 9.00

5.33 12.67 5.00

3.00 7.33 3.33

8.00 17.00 6.67

3.50 10.00 4.33

9.83 19.33 9.00

16.11 6.22

11.17

(M3xv)

M4

F

0

F1

19.67 20.67 4.67

8.83 9.00 6.00

5.33 4.33 3.33

11.83 13.67 8.33

7.17 6.33 3.33

14.17 19.67 6.33

12.28 5.33

8.81

(M4xv)

M5

F

0

F1

12.67 34.33 6.67

7.50 10.00 3.67

3.83 5.33 2.33

11.00 20.33 4.33

4.85 5.67 2.33

13.00 15.67 4.67

15.22 4.00

9.61

(M5xv)

20.50 6.83

2.83 12.33 4.00 10.17

v-mean 23.25 8.36 4.58 11.97 5.67 13.14

111

The least number of eggmass per root system was obtained when the soil was amended with M.

ghana or M. jaspaeda and inoculated with the two biocontrol agents. Eggmass index (EMI)

followed the trend of eggmass production (Table 45). The interaction among the three factors

was significant (P ≤ 0.05). The eggmass index (EMI) of the control plant was reduced from 4.00

to 2.00 when the two biocontrol agents where combined with soil amendment with Mucuna. The

least EMI of 1.00 was obtained in soils amended with M.ghana or M. jaspaeda and inoculated

with G. mosseae and the bionematicide. G. mosseae was closely followed by G. deserticola with

EMI = 1.33.

The rhizophere soil nematode population was significantly (P ≤ 0.05) reduced with the

inoculation of AMF, bioformulated P.lilacinus and Mucuna amendment compared with their

respective controls (Table 46). G. mosseae and M. jaspaeda were the most efficient in nematode

larval population reduction among the AMF and Mucuna species, respectively. The combined

application of the three factors significantly (P ≤ 0.05) reduced nematode population more than

single or double application. However, the least number of nematode larvae was obtained in pots

where the soil was amended with M. ghana or M. jaspaeda and inoculated with G. mosseae and

bionematicide.

The amendment of soil with Mucuna and inoculation with AMF as well as bioformulated

P. lilacinus significantly (P ≤ 0.05) increased fresh root weight compared with their respective

control (Table 47). Gi. gigantea and M. ghana were the most efficient AMF and Mucuna

species, respectively. The interaction between mycorrhiza and Mucuna amendment was

significant in their effects on fresh root of tomato plants. Mycorrhizal inoculation in combination

with the various Mucuna spp excepting M. jaspaeda significantly (P ≤ 0.05) enhanced fresh root

weight of tomato plants compared with the non-mycorrhizal plants. Interaction between

mycorrhiza and P.lilacinus was singnificant.

112

Table 45: Effects of arbuscular mycorrhizal fungi, P. lilacinus and Mucuna spp soil amendment on Eggmass index (EMI) of tomato inoculated with M. incognita Mucuna spp Mycorrhizal fungus

P. lilacinus

Vo** V 0xF V1 V1xF V2 V2xF V3 V3xF V4 V4xF Vs V5xF MF F M

*M 0 F0*** F1

4.00 2.67

3.61 2.11

3.00 2.00

2.56 1.89

2.33 2.00

2.06 1.61

3.00 2.67

2.94 2.11

2.67 2.00

2.17 1.72

3.00 2.33

3.00 2.06

3.00 2.28

35.22 11.40

2.64

(M0 x V) M1

F0 F1

3.33 4.00 2.00

2.50 3.00 2.00

2.17 2.00 1.33

2.83 3.00 2.00

2.33 2.00 2.00

2.67 3.00 2.00

2.83 1.89

2.36

(M1 x V) M2

F0 F1

3.00 3.00 2.00

2.50 2.00 1.33

1.67 2.00 1.00

2.50 2.67 2.00

2.00 2.00 1.00

2.50 3.00 2.00

2.44 1.56

2.00

(M2 x V) M3

F0 F1

2.50 3.67 2.00

1.67 3.00 2.00

1.50 2.00 2.00

2.33 3.00 2.00

1.50 2.33 2.00

2.50 3.00 2.00

2.83 2.00

2.42

(M3 x V) M4

F0 F1

2.83 3.00 2.00

2.50 2.00 2.00

2.00 2.00 2.00

2.50 3.00 2.00

2.17 2.00 2.00

2.50 3.00 2.00

2.50 2.00

2.25

(M4 x V) M5

F0 F1

2.50 4.00 2.00

2.00 2.33 2.00

2.00 2.00 1.33

2.50 3.00 2.00

2.00 2.00 1.33

2.50 3.00 2.00

2.72 1.78

2.25

(M5 x V) 3.00 2.17 1.67 2.50 1.67 2.50 V-mean

2.86

2.22

1.83

2.53

1.94

2.53

*Mo = Control **Vo = Control ***Fo = Control LSD(0.05) Mycorrhiza means(M) = 0.15 M1 = G. etunicatum V1 = M. pruriens utilis F1 = P. lilacinus applied LSD(0.05) for Mucuna means(V) = 0.15 M2 = G. mosseae V2 = M. Ghana LSD(0.05) for P. lilacinus means(F) = 0.08 M3 = G. clarum V3 = M. cocochinensis LSD(0.05) (M x V) interaction means = 0.36 M4 = G. gigaspora V4 = M. jaspaeda LSD(0.05) (M x F) interaction means = 0.21 M5 = G. deserticola V5 = M. pruriens IR2 LSD(0.05) (M x V) interaction means = 0.50

113

Table 46: Effects of arbuscular mycorrhizal fungi; P. lilacinus and Mucuna spp soil amendment on number of nematode larvae /200 g soil of tomato inoculated with M. incognita

Mucuna spp

Mycorrhizal Fungus

P. lilacinus

V0** V 0xF V1 V1xF V2 V2xF V3 V3xF V4 V4xF V5 V5xF MF F M

M0** F0***

F1

4.00

3.20

3.78

2.98

3.58

3.12

3.42

2.89

3.28

2.98

3.10

2.73

3.77

3.21

3.58

3.05

3.01

2.68

2.83

2.56

3.86

3.25

3.65

3.02

3.59

3.07

3.39

2.87

3.33

(M0xV) 3.60 3.35 3.13 3.49 2.85 3.56

M1 F0

F1

3.85

2.97

3.45

2.92

3.03

2.66

3.67

3.08

2.90

2.62

3.66

3.14

3.43

2.90

3.16

(M1xV) 3.41 3.18 2.85 3.37 2.76 3.40

M2 F0

F1

3.64

2.79

3.31

2.48

2.96

2.35

3.36

3.00

2.67

2.36

3.51

2.79

3.24

2.63

2.94

(M2xV) 3.22 2.90 2.66 3.18 2.52 3.14

M3 F0

F1

3.77

3.02

3.59

2.95

3.17

2.84

3.73

3.04

2.85

2.63

3.67

3.19

3.46

2.95

3.20

(M3xV) 3.40 3.27 3.00 3.38 2.73 3.43

M4 F0

F1

3.50

3.01

3.14

2.90

2.98

2.80

3.55

3.00

2.72

2.59

3.67

2.91

3.26

2.87

3.07

(M4xV) 3.28 3.02 2.89 3.27 2.65 3.29

M5 F0

F1

3.84

2.92

3.47

2.99

3.18

2.76

3.42

2.97

2.83

2.47

3.54

2.81

3.38

2.82

3.10

(M5Xv 3.38 3.22 2.97 3.20 2.65 3.17

V-mean 3.38 3.16 2.92 3.32 2.69 3.33

M0=Control ** Vo=Control *** F0= Control LSD(0.05) Mycorrihza means(M) = 0.02 M1=G.etunicatum V1=M. pruriensutilis F1= P. lilacinus applied LSD(0.05) for Mucuna means (V) = 0.02 M2=G. mosseae V2=M. ghana LSD(0.05) for P. lilacinusmeans (F) = 0.01 M3=G. clarum V3=M. cochichinensis LSD(0.05)(MXV) Interaction means = 0.04 M4=G. gigantea V4=M. jaspaeda LSD(0.05) (MXF) Interaction means = 0.02 M5=G. deserticola V5=M. pruriens IR2 LSD(0.05) (VXF) Interaction means = 0.02 LSD(0.05) (MXFXV) Interaction means = 0.06

114

Table 47: Effects of arbuscular mycorrhizal fungus, P. lilacinus and Mucuna spp soil amendment on fresh root weight (g) /plant of tomato inoculated with M. incognita Mycorrhizal fungus

P. lilacinus

Vo** V0xF V

1 V

1xF V

2 V

2xF V

3 V

3xF V

4 V

4xF V

5 V

5xF MF F M

*M0 F

0

*** 12.83 17.25 15.99 19.81 18.79 21.31 17.54 20.47 20.61 21.45 15.68 19.91 16.91 20.03

F1 15.75 19.27 19.18 21.22 21.18 22.64 19.48 21.41 21.52 21.77 18.41 21.09 19.26 21.23 18.08

(M0 x V) 14.29 17.59 19.99 18.51 21.07 17.05 M

1 F

0 14.75 18.26 20.29 19.39 21.46 19.17 18.99

F1 19.17 20.86 22.62 21.34 20.79 20.81 20.84 19.91

(M1 x V) 19.96 19.56 21.45 20.37 21.13 19.99 M

2 F

0 16.81 19.94 21.42 21.23 21.35 20.75 20.25

F1 20.90 21.54 20.32 21.98 22.06 22.45 21.54 20.89

(M2 x V) 18.86 20.74 20.87 21.61 21.70 21.59 M

3 F

0 18.67 19.06 20.46 21.50 20.78 21.08 20.26

F1 19.27 19.99 23.87 20.75 21.12 22.31 21.29 20.74

(M3 x V) 18.97 19.52 22.17 21.12 20.95 21.69 M

4 F

0 21.42 22.72 24.63 22.61 24.18 21.01 22.76

F1 22.20 24.45 25.53 24.31 23.66 21.69 23.64 23.20

(M4 x V) 21.81 23.59 25.08 23.46 23.92 21.35 M

5 F

0 19.00 22.88 22.24 20.58 20.32 21.78 21.13

F1 18.33 21.27 22.32 20.59 21.46 20.85 20.80 20.97

(M5 x V) 18.67 22.07 22.28 20.58 20.89 21.32 V-mean 18.26 20.51 21.97 20.94 21.61 20.50 *M0 = Control **Vo = Control ***F0 = Control LSD(0.05) Mycorrihza means(M) = 0.61

M1 = G.etunicatum V1 = M. pruriens utilis F1 = P. lilacinus applied LSD(0.05) for Mucuna means (V) = 0.61

M2 = G. mosseae V2 = M. ghana LSD(0.05) for P. lilacinus means (F) = 0.35

M3 = G. clarum V3 = M. cochichinensis LSD(0.05) (MXV) Interaction means = 1.49

M4 = G. gigantea V4 = M. jaspaeda LSD(0.05) (MXF) Interaction means = 0.86

M5 = G. deserticola V5 = M. pruriens IR2 LSD(0.05) (VXF) Interaction means = NS

LSD(0.05) (MXFXV) Interaction means = NS

115

In both mycorrhizal and non-mycorrhizal plant excepting G. deserticola, bionematicide

application significantly enhanced fresh root biomass of tomato relative to no application.

Although, the interaction among the factors was not significant (P>0.05) , combined application

of all the factors produced higher fresh root weight than single or double application.

Root colonization by the AMF was significantly (P≤0.05) enhanced by Mucuna

amendment and bionematicide application compared with their respective controls (Table 48).

The colonization rate was significantly higher in the mycorrhizal plants than the non mycorrhizal

plants. The uninoculated plants were lightly colonized by the indigenous AMF species. Gi.

gigantea and M. cochichinensis treated plants had significantly (P≤ 0.05) the highest rate of

mycorrhizal root colonization. Interaction between mycorrhiza and Mucuna was significant. Gi.

gigantea inoculated plants in combination with all the Mucuna species had the highest root

colonization. Also, the interaction between Mucuna and bionematicide had a significant effect on

root colonization by AMF. In soil amended with M. pruriens utilis and M. ghana, as well as

unamended soil, there was a significant increase in AMF root colonization due to bionematicide

application. With the exception of G. deserticola and Gi. gigantea, bionematicide inoculation

significantly increased AMF root colonization in both mycorhizal and non mycorrhizal plants.

The results of the effects of AMF inoculation, Mucuna amendment and bionematicide

application on shoot length of tomato infected with M. incognita are presented in Table 49. Soil

amendment with Mucuna, inoculation with mycorrhiza and application of bioformulated P.

lilacinus significantly (P ≤ 0.05) enhanced tomato growth compared with their respective

control. M. ghana and Gi. gigantea were significantly the most efficient Mucuna and AMF

species, respectively in this regard. The interaction of the three factors was significant in growth

improvement. In most cases, inoculation of AMF when combined with Mucuna soil amendment

and bionematicide application resulted in significant higher plant height than when the factors

where applied singly or double.

116

Table 48: Effects of P. lilacinus inoculation and Mucuna spp soil amendment on percentage root colonization by arbuscular mycorrhizal fungus of tomato inoculated with M. incognita

Mucuna spp Mycorrhizal

Fungus P.

lilacinus V0** V 0xF V1 V1xF V2 V2xF V3 V3xF V4 V4xF V5 V5xF MF F M

M0** F 0***

F1

18.00

24.33

57.50

62.44

20.00

26.33

62.06

65.72

22.33

26.33

64.94

66.61

24.00

27.00

66.83

67.78

22.33

22.33

65.50

65.61

22.67

22.00

65.67

64.78

21.56

24.72

63.75

65.49

23.14

(M0xV) 21.17 23.17 24.33 25.50 22.33 22.33

M1 F0

F1

50.33

57.67

57.33

60.00

61.00

63.00

63.67

66.00

62.00

63.33

63.67

63.00

59.67

62.17

60.92

(M1xV) 54.00 58.67 62.00 64.83 62.67 63.33

M2 F0

F1

71.00

75.00

74.00

80.00

81.00

83.33

83.00

84.00

82.00

82.67

81.67

81.00

78.78

81.00

79.89

(M2xV) 73.00 77.00 82.17 83.50 82.33 81.33

M3 F0

F1

62.33

67.67

68.67

72.33

70.67

72.00

74.67

73.33

72.33

72.00

72.00

71.00

70.11

71.39

70.75

(M3xV) 65.00 70.50 71.33 74.00 72.17 71.50

M4 F0

F1

77.67

80.00

82.33

82.67

81.67

84.00

82.00

82.67

82.00

82.33

82.33

81.33

81.33

82.17

81.75

(M4xV) 78.33 82.50 82.83 82.33 82.17 81.83

M5 F0

F1

65.67

70.00

70.00

73.00

73.00

71.00

73.67

73.67

72.33

71.00

71.67

70.33

71.06

71.50

71.28

(M5xV 67.83 71.50 72.00 73.67 71.67 71.00

V-mean 59.97 63.87 65.78 67.31 65.56 65.22

M0=Control ** Vo=Control *** F0= Control LSD(0.05) Mycorrihza means(M) = 0.83 M1=G.etunicatum V1=M. pruriensutilis F1= P. lilacinus applied LSD(0.05) for Mucuna means (V) = 0.83 M2=G. mosseae V2=M. ghana LSD(0.05) for P. lilacinusmeans (F) = 0.48 M3=G. clarum V3=M. cochichinensis LSD(0.05)(MXV) Interaction means = 2.03 M4=Gi.. gigantea V4=M. jaspaeda LSD(0.05) (MXF) Interaction means = 1.17 M5=G. deserticola V5=M. pruriens IR2 LSD(0.05) (VXF) Interaction means = 1.17 LSD(0.05) (MXFXV) Interaction means = NS

117

Table 49: Effects of arbuscular mycorrhizal, P. lilacinus and Mucuna spp soil amendment on shoot length (cm/plant) of tomato inoculated with M. incognita

Mucuna spp Mycorrhizal Fungus

p. lilacinus

Vo** V0xF V1 V1xF V2 V2xF V3 V3xF V4 V4xF Vs V5xF MF F M

*M0 F0***

F1

52.33 67.67

68.61 71.78

62.00 67.67

69.22 74.61

69.00 73.33

75.17 78.50

60.33 63.33

69.83 71.11

69.67 72.00

74.22 76.72

58.67 63.67

69.56 70.89

62.00 6794

71.10 73.94

64.97

(M0xv) M1

F0

F1

60.00 68.33 70.67

64.83 66.67 71.67

71.17 71.00 78.00

61.83 66.67 65.00

70.83 73.00 77.67

61.17 66.67 69.67

68.72 72.11

70.42

(M1xv) M2

F0

F1

69.50 72.33 76.33

69.17 67.67 76.33

74.50 77.00 77.33

65.83 70.00 71.67

75.33 74.67 83.67

68.17 70.33 71.33

72.00 76.11

74.06

(M2xv) M3

F0

F1

74.33 70.00 71.33

72.00 70.67 77.67

77.17 80.00 80.67

70.83 74.67 75.67

79.17 74.67 73.33

70.83 72.67 79.00

73.78 76.28

75.03

(M3xv) M4

F0

F1

70.67 76.00 71.67

74.17 76.33 79.67

80.33 81.00 79.67

75.17 77.33 79.33

74.00 81.33 80.33

75.83 74.67 72.00

77.78 77.11

77.44

(M4xv) M5

F0

F1

73.83 72.87 73.00

78.00 72.00 74.67

80.33 73.00 82.00

78.33 70.00 71.67

80.83 72.00 73.33

73.33 74.33 69.67

72.33 74.06

73.19

(M5xv)

72.83

73.33 77.50 70.83 72.67 72.00

v-mean 70.19 71.92 76.83 70.47 75.47 70.22

*M0 = Control ** Vo = Control *** F0 = Control LSD(0.05) Mycorrihza means(M) = 1.07

M1 = G.etunicatum V1 = M. pruriens utilis F1 = P. lilacinus applied LSD(0.05) for Mucuna means (V) = 1.07

M2 = G. mosseae V2 = M. ghana LSD(0.05) for P. lilacinus means (F) = 0.62

M3 = G. clarum V3 = M. cochichinensis LSD(0.05) (MXV) Interaction means = 2.62

M4 = Gi. gigantea V4 = M. jaspaeda LSD(0.05) (MXF) Interaction means = 1.51

M5 = G. deserticola V5 = M. pruriens IR2 LSD(0.05) (VXF) Interaction means = 1.51

LSD(0.05) (MXFXV) Interaction means = 3.70

118

The greatest growth enhancement was obtained with either Gi gigantea or G. clarum inoculation in

combination with the various Mucuna spp and with bionematicide application.

Accumulation of dry matter in the shoot was significantly (P ≤ 0.05) enhanced with the

application of biformulated P.lilacinus, Mucuna soil amendment and AMF inoculation compared

with their respective control (Table 50). Gi. gigantea inoculated plants significantly had the highest

dry shoot matter. The relative mycorrhizal effectiveness (RME) values were 7.82, 11.70, 11.75, 13.30

and 17.58% for G. etunicatum, G. mosseae, G. deserticola, G. clarum and Gi. gigantea, respectively.

M. ghana and M. jaspaeda were the most efficient species in the enhancement of shoot dry matter

accumulation in tomato. The interaction of the three factors was significant in shoot dry matter

accumulation in tomato. Generally, Gi. gigantea inoculation in combination with most of the

Mucuna species amendment and with bionematicide application produced plants with significantly

higher dry shoot weight compared with other treatments.

There was a significant (P ≤ 0.05) enhancement in the number of fruit set by tomato plantss

with Mucuna soil amendment and AMF inoculation compared with their respective control (Table

51). M. jaspaeda and M. ghana amended soil significantly produced plants with the highest number

of fruits. For AMF, Gi. gigantea inoculated plants produced the highest number of fruits. The

interaction among the three factors was significant. Generally, the combination of the three factors in

most cases significantly induced the formation of higher number of fruits compared with where one

or two factors was used. The combination of Gi. gigantea with M. ghana and G. mosseae with M.

jaspaeda produced the highest number of fruits per plant.

119

Table 50: Effects of arbuscular mycorrhizal, P. lilacinus and Mucuna spp soil amendment on dry shoot weight (g)/plant of

tomato inoculated with M. incognita

Mucuna spp Mycorrhizal fungus

P. lilacinus

Vo** V 0xF V1 V1xF V2 V2xF V3 V3xF V4 V4xF Vs V5xF MF F M

*M 0 F0*** F1

11.59 16.44

16.07 18.14

15.66 18.35

17.51 19.74

19.69 20.77

21.69 22.90

16.37 17.48

19.46 19.40

20.42 21.45

21.88 22.18

17.31 19.04

20.57 20.56

16.84 18.92

19.47 20.49

17.80

(M0 x V) M1

F0 F1

14.02 15.25 17.18

17.01 16.47 19.78

20.23 21.59 22.31

16.93 17.69 17.43

20.94 21.33 21.37

18.17 20.03 21.37

18.73 19.89

19.31

(M1 x V) M2

F0 F1

16.21 17.51 18.51

18.12 17.45 19.70

21.95 21.24 20.65

17.56 19.37 20.00

21.31 20.85 24.55

20.70 20.58 21.51

19.50 20.82

20.16

(M2 x V) M3

F0 F1

18.01 18.18 19.14

18.58 17.13 20.56

20.95 22.65 24.23

19.68 21.27 20.57

22.70 22.52 21.14

21.05 20.19 20.79

19.99 21.07

20.53

(M3 x V) M4

F0 F1

17.66 18.28 19.39

18.84 19.72 21.08

23.44 24.11 24.71

20.92 21.92 21.37

21.83 24.29 23.97

20.49 22.64 20.58

21.83 21.85

21.84

(M4 x V) M5

F0 F1

18.84 17.63 18.20

20.40 18.60 19.00

24.41 20.65 24.74

21.64 20.17 19.57

24.13 21.87 20.67

21.61 20.79 20.08

19.95 20.38

20.17

(M5 x V) 17.92 18.80 22.69 19.87 21.27 20.43 V-mean

17.11

18.63

22.28

19.43

22.03

20.41

*Mo = Control **Vo = Control ***Fo = Control LSD(0.05) Mycorrhiza means(M) = 0.41 M1 = G. etunicatum V1 = M. pruriens utilis F1 = P. lilacinus applied LSD(0.05) for Mucuna means(V) = 0.41 M2 = G. mosseae V2 = M. ghana LSD(0.05) for P. lilacinus means(F) = 0.24 M3 = G. clarum V3 = M. cochichinensis LSD(0.05) (M x V) interaction means = 0.01 M4 = Gi.gigantea V4 = M. jaspaeda LSD(0.05) (M x F) interaction means = 0.58 M5 = G. deserticola V5 = M. pruriens IR2 LSD(0.05) (V x F) interaction means = 0.58 LSD(0.05) (M x F x V) interaction means =1.42

120

Table 51: Effects of arbuscular mycorrhizal, P. lilacinus and Mucuna spp soil amendment on number of fruits per plant of tomato inoculated with M. incognita Mucuna spp Mycorrhizal fungus

P. lilacinus Vo** V0xF V1 V1xF V2 V2Xf V 3 V3xF V4 V4xF Vs V5xF MF F M

*M 0 F0*** F1

2.33 3.33

3.83 3.72

3.00 3.68

4.17 4.89

4.00 4.68

4.83 5.56

3.00 3.33

3.94 4.22

5.33 6.00

5.61 5.33

4.00 4.33

4.50 4.17

3.61 4.22

4.48 4.65

3.92

(M0 x V) M1

F0 F1

2.83 3.67 3.33

3.33 3.33 4.33

4.33 6.00 5.00

3.17 3.33 4.33

5.68 5.33 4.67

4.17 4.00 4.00

4.28 4.28

4.28

(M1 x V) M2

F0 F1

3.50 4.33 4.33

3.83 3.67 6.00

5.50 5.00 4.00

3.83 3.33 4.33

5.00 5.67 7.00

4.00 4.67 4.00

4.44 4.94

4.69

(M2 x V) M3

F0 F1

4.33 3.67 3.67

4.83 4.67 4.00

4.50 4.67 6.00

3.83 5.00 4.67

6.33 5.67 4.33

4.33 4.33 4.00

4.67 4.44

4.56

(M3 x V) M4

F0 F1

3.67 5.33 4.00

4.33 5.67 5.33

5.33 5.67 7.33

4.83 4.67 5.33

5.00 6.67 5.33

4.17 4.33 4.33

5.39 5.28

5.33

(M4 x V) M5

F0 F1

4.67 3.67 3.67

5.50 4.67 6.00

6.50 3.67 6.33

5.00 4.33 3.33

6.00 5.00 4.67

4.33 5.67 4.33

4.50 4.72

4.61

(M5 x V) 3.67 5.33 5.00 3.83 4.83 5.00 V-mean

3.78

4.53

5.19

4.08

5.47

4.33

*Mo = Control **Vo = Control ***Fo = Control LSD(0.05) Mycorrhiza means(M) = 0.40

M1 = G. etunicatum V1 = M. pruriens utilis F1 = P. lilacinus applied LSD(0.05) for Mucuna means(V) = 0.40

M2 = G. mosseae V2 = M. ghana LSD(0.05) for P. lilacinus means(F) = NS

M3 = G. clarum V3 = M. cochichinensis LSD(0.05) (M x V) interaction means = 0.98

M4 = Gi. gigantea V4 = M. jaspaeda LSD(0.05) (M x F) interaction means = NS

M5 = G. deserticola V5 = M. pruriens IR2 LSD(0.05) (V x F) interaction means = 0.57

LSD(0.05) (M x F x V) interaction means = 1.39

121

Tomato fresh fruit weight was significantly (P≤ 0.05) enhanced when the soil was amended with

Mucuna and inoculated with the two bicontrol agents (Table 52) compared with their various

controls .Gi. gigantea and M. jaspaeda were the most efficient AMF species and Mucuna

species, respectively. Interaction among the three factors was significant (P ≤ 0.05). M. jaspaeda

and M. ghana were the most efficient species in increasing the total fresh fruit weight of tomato

when combined with most of the AMF species and inoculated with the bionematicide. The

highest fresh fruit yield of tomato was obtained with G. mosseae inoculated plants

(139.46g/plant) and Gi. gigantea inoculated plants (136.06 g/plant) grown in M. jaspaeda

amended soil and inoculated with P.lilacinus. Generally, the combination of the three factors

resulted in significantly higher fresh fruit weight compared with single or double application of

the treatments. Plate 7 shows potted tomato plants with fruits.

122

Table 52: Effects of arbuscular mycorrhizal fungi, P.lilacinus and Mucuna spp soil amendment on total fresh fruit of weight (g/plant) of tomato inoculated with M. incognita

*Mo = Control **Vo = Control ***Fo = Control LSD(0.05) Mycorrhiza means(M) = 3.92 M1 = G. etunicatum V1 = M. pruriens utilis F1 = P. lilacinus applied LSD(0.5) for Mucuna means (V) = 3.92 M2 = G. mosseae V2 = M.ghana LSD(0.05) for P. lilacinus means (F) = 2.92 M3 = G. clarum V3 = M. cochichinensis LSD(0.05) (M x V) Interaction means =9.73 M4 = G. gigantea V4 = M. jaspaeda LSD(0.05) (M x F) Interaction means =5.62 M5 = G. deserticola V5 = M. pruriens IR2 LSD(0.05) (V x F) Interaction means = NS LSD(0.05) (M x F X V) Interaction means = 13.76

Mucuna spp . Mycorrhizal fungus

P. lilacinus V0 VoXF V

1 V,xF V

2 V

2xF V

3 V

3xF V

4 V

4xF V

5 VBXF MF F M

•Mo Fo*** 39.02 62.74 60.70 75.52 88.89 96.85 61.72 81.76 107.99 112.54 70.21 88.28 71.42 86.28 Fi 53.00 74.47 70.56 97.30 94.57 109.59 85.02 96.21 119.31 126.34 88.03 98.42 85.07 100.39 78.2 (M0xV) 46.01 65.63 91.70 73.37 113.65 79.12 M

1 FO 56.00 72.00 100.31 74.22 100.60 81.81 80.82

F1 68.61 84.92 110.58 95.26 116.95 93.97 95.01 87.92 (M1xV) 62.30 78.46 105.45 84.74 108.67 87.89 M

2 Fo 66.73 69.13 93.29 76.08 123.14 82.01 85.06

F1 86.15 118.88 95.86 100.19 139.46 93.78 105.72 95.39

(M2xV) 76.44 94.60 94.57 88.14 131.30 87.89 M

3 Fo 68.33 78.65 92.60 100.58 102.88 87.59 88.44

F1 71.87 85.79 120.96 102.88 123.96 103.51 105.45 94.34

(M3xV) 70.10 82.22 106.78 101.58 113.42 95.55 M

4 Fo 80.91 103.10 115.34 101.92 135.05 105.19 106.92

F1 94.38 105.76 133.72 107.60 136.06 106.01 113.92 110.42

(M4xV) 87.64 104.43 124.53 104.76 135.55 105.60

M5 Fo 65.46 69.55 90.69 76.02 105.58 102.89 85.03

F1 72.81 117.91 101.93 86.60 122.49 105.24 101.16 93.10 (M5xV) 69.13 93.73 96.31 81.31 114.03 104.06 V-mean 68.61 86.41 103.22 88.98 119.44 93.35

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Plate 7: Potted tomato plants with fruits

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DISCUSSION

The high mycorrhizal spore density found in Nsukka and Obubra soils may be

attributed in part to the soil type as both were sandy loam in texture and also to low available P

contents. Generally, soils from locations with high P-content had relatively lower mycorrhizal

spore density .High availability of P in soils has been implicated for low spore density and

colonization of plants by AMF (Carling et al.,1989;Smith,1988). Higher nematode density in

Calabar ,Obubra and Ogoja soils could be attributed to higher sand contents of those soils as

reported by Agu(2002) and Olowe(2005).

In experiment I, none of the Mucuna species tested was galled and no eggmass was found in

their roots. However, the check plant (tomato cv. Roma VF) was heavily galled with more than

100 eggmasses per root system. The Mucuna species were rated immune to M. incoginta

infection while the check tomato plant was rated highly susceptible. The susceptibility of the

tomato cultivar to M. incogita validates the virulence of the root- knot nematode species used

in this trial. Many authors have emphasized that, for the cultural control of nematode pests with

rotational crops and/or green manure crops, the host status of the crop intended for this purpose

must be ascertained (Ritzenger and McSorley, 1998 Queneherve et al. 1998; Stirling and

Stirling, 2003; Marla et al., 2008). The non- host status of the five Mucuna species to M.

incognita in this trial validates the report of Rodriguez- kabana et al. (1992) Queneherve et al.

(1998) who evaluated Florida and Mozambique accessions of M. pruriens utilis (Syn. M.

deeringiana) against three species of Meloidogyne and established that they were non- hosts. In

Nigeria, Caveness (1988) had reported the effectiveness of Mucuna pruriens utilis in

suppressing the population of various plant parasitic nematodes when used as a cover crop. The

exudates from the roots of Mucuna have long been implicated in the suppression of population

of Meloidogyne spp in the soil (Vincente and Acosta, 1987). The trial also showed a significant

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reduction in the number of root- knot nematode juveniles recovered from the rhizosphere soil

of the different Mucuna species compared with the susceptible tomato plant. It is possible that

some nematicidal/nematostatic constituents such as aliphatic ester triacontyl tetracosanate and

aliphatic alcohol 1-triacontanol, β- sitosterol + stigmasterol, fatty acids, allantoin, etc. could

have been released to the rhizosphere, thus disrupting the coordination and normal activities of

nematodes, as observed by earlier researchers (Vargas et al., 1996; Nogueira et al., 1996;

Barbosa et al., 1999). However, the concentration of the nematicidal /nematostatic compounds

present in the various species of Mucuna may differ as reflected in the difference in nematode

larval population among the Mucuna species. This, however, calls for further research.

Successive increase in the rate of Mucuna amendment as green manure significantly inhibited

root galling and eggmass production by M. incognita on tomato. The response followed a

negative linear regression model. This finding confirms the report by Ritzinger and McSorley

(1998) who concluded that the best rate of velvet bean or castor amendment for nematode

suppression and plant growth enhancement could be predicted through curvilinear or linear

regression equations. Mucuna jaspaeda and M. ghana amendment at 10 t/ha had the highest

nematode suppressing effect. In literature, experiments involving Mucuna used as cover or

green manure crop in nematode management had always utilized Mucuna pruriens utilis

(McSorley and Galler, 1992; Weaver et al., 1993; McSorley and Dickson, 1995; Queneherve et

al., 1998; Nogueira et al, 1996; Barbosa et al., 1999). Thus, it is apparaent from this trial that

M. jaspaeda and M. ghana may have a higher profile of the nematicidal constituents than the

popular M. pruriens utilis. Such species and varietal differences have been observed in

marigold (Tagetes spp) and sunn hemp (Crotalaria juncea) by Ploeg (1999) and Marla et al.

(2008), respectively. Various mechanisms have been advocated for the possible effects of

organic amendments on the tripartite interaction of nematode- plant- soil system (Oka, 2010,

McSorley, 2011, Thoden et al., 2011). The release of pre- existing nematicidal constituent,

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generation of nematicidal compounds such as organic acids, ammonia, nitrogenous compounds,

etc. during degradation, enhancement or stimulation of nematode antagonistic organisms,

increase in plant resistance/tolerance by microorganisms or natural compounds and changes in

the physicochemical properties of the soil are some of the mechanisms listed by these authors.

However, Akhtar and Malik (2000) had cautioned that it may be difficult to distinguish which

mechanisms are the most important as they may operate simultaneously. It is likely that, some

of these mechanisms could have been involved in the suppression of M. incognita population in

this trial.

The Mucuna spp differed in their mineral contents. M. jaspaeda and M.ghana with high

N content and narrow C: N ratios showed higher nematicidal/nematotoxic activity against M.

incoginta. Ritzinger and McSorley (1998) had earlier reported a high macro and micro

elements composition of M. deerigiana with a low C:N ratio of 8.68:1. Rodriguez-kabana et al.

(1987) and Agu (2007) had observed that organic materials with low C:N ratios and high

protein or amine type of N content were more potent in nematode suppression. This could

justify the higher efficacy of M. jaspaeda and M. ghana in nematode suppression observed in

this trial compared with the other Mucuna species. In this trial, nematodes may have been

killed by the release of nitrous acid which is reported to be more effective in acidic soils than

ammonia, as a product of decomposition (Oka, 2010). Ammonia is easily converted to

ammonium ion in acidic soils and ammonium ion is less nematicidal. Generally, the Mucuna

spp had narrow C: N ratios with the exception of M. pruriens 1R2 which had 21.28:1.

McSorley and Frederick (1999) had reported a C: N ratio of 18.6:1 for M. pruriens utilis, while

Blum and Rodriguez- Kabana (2006) had reported 17.32:1 for the same species. However, our

result shows that M. pruriens utilis had C:N ratio of 11.40:1. This variation could be attributed

to the differences in environmental factors. M. pruriens IR2 also showed the least nematicidal

property, validating the claim by Rodriguez- kabana et al. (1987) that organic materials with C:

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N ratio > 20 may not possess nematicidal properties due to slow decomposition and release of

the active constituents.

Increase in the rate of Mucuna amendment resulted in an increase in growth, dry mater

yield and total fresh fruit yield of tomato. This was depicted as a positive linear regression

response model. However, there was a decrease in these variables as the rate was increased

from 8 to 10 t/ha, especially in soils amended with M.jaspaeda and M. ghana. This could have

signaled phytotoxicity. Rodriguez- kabana et al. (1987) had cautioned that organic materials

with very low C:N ratios when applied at higher rates may be phytotoxic to crops and this

could be remedied by boosting the C contents of such material. The increase in growth and

yield of tomato plants with increase in amendment rate of Mucuna could be attributed in part to

various mechanisms of nematode suppression and induced tolerance/ resistance which may

have operated simultaneously (Akhtar and Malik, 2000). The growth and yield of tomato plants

grown in amended soil could have been increased relative to unamended soil as result of

nutrients released during the decomposition process, improvement in soil structure, water

retention capacity , chemical properties,etc, (Adigbo et al., 2003). For instance, analysis of the

Mucuna spp showed high contents of macro and micro elements needed for the growth of

tomato. In theory, application of 8 t/ha of M.ghana on dry weight basis could release up to

380kg N/ha.Van Noordwijk et al. (1995) estimated that 83% of Mucuna N was available to a

subsequent crop and very little for the second crop. Adigbo et al. (2003) estimated the fertilizer

equivalent of Mucuna pruriens utilis applied at 6.67 DM t/ha to be 30 kg N/ha. Reduction in

root galling in amended soil could have resulted to better nutrients and water absorption,

translocation and photosynthetic efficiency of the tomato plants relative to heavily galled roots

in the unammended soil (Onkendi et al., 2014). This could possibly justify the higher fruit yield

obtained in soils amended with Mcuna spp.

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In experiment III, results obtained indicated that the test plant was highly susceptible to

M. incognita. This could be attributed in part to both the condusive soil condition and the

ambient temperature during the period of the experiment. Soils with high proportion of sand

permit increased penetration, reproduction and damage by root-knot neumatodes (Agu, 2002;

Olowe, 2005 Windham and Barker, 1986). The average temperature of 310C during the trial

period could have boosted the activity of M. incognita with attendant higher damage potential

(Trudgill, 1995). The efficacy of the Mucuna spp in suppressing nematode population and its

infectivity on tomato was consistent with that obtained in experiment II. Mucuna jaspaeda was

the most effective followed by M. ghana. Arbuscular mycorrhizal fungus (AMF) inoculation

significantly inhibited galling and eggmass production by M. incognita. The effect differed

among the AMF species. This corroborates the findings of earlier researchers (Diederichs,

1987; Jothi and Sundarababu, 2000; Zhang et al., 2008). Diederichs (1987) Observed that

Glomus manihotis and Gigaspora margarita were more efficient in reducing nematode damage

and growth enhancement of chick pea than the other endophytes. Also, Zhang et al. (2008)

reported G. mosseae as the most efficient AMF species in root- knot disease suppression and

growth improvement on cucumber. In this trial, gall and eggmass production were more

efficiently inhibited by Gi. gigantea and G. mosseae. The mechanisms involved in nematode

suppression and growth improvement of mycorrhizal plants are still controversial. Induced

systemic resistance due to improved host’s nutrition has been advocated (Gosling et al., 2006).

Also, changes in the root morphology and histopathology of the host, increased production of

phytoalexins, phenols, lignin, phenylalanine, chitinase, etc. to the detriment of the nematode

partner have been suggested (Morandi, 1996; Masadeh et al., 2004).

The combined inoculation of tomato plants with AMF and amendment of soil with

Mucuna spp resulted in the greatest gall and eggmass production inhibition as well as growth

and yield enhancement relative to sole application. This finding is in line with the report of

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earlier workers (Ehte shamul-Hague et al., 1996, Rao et al., 1996; Rao et al., 1995; Goswami et

al., 2007; Siddiqui and Akhtar, 2008b). Soil amendments with organic matter have been

reported to simulate soil food webs, thus increasing the population of free-living nematode,

plant growth promoting rhizobacteria, fungi and nematode antagonists (Oka, 2010, McSorley,

2011). Also, plant growth promotion could be linked to nutrient release from the mineralization

process of the Mucuna green manure amendement as earlier discussed. The greatest root- knot

nematode suppression, growth and yield enhancement in tomato were obtained from soils

amended with M. jaspaeda or M. ghana in combination with Gi. giagantea or G. mosseae.

Experiment IV was a confirmation of experiment III in the field. The results of the field

experiment followed the trend of experiment III in the greenhouse. However, the fruit yield in

the field experiment was higher as expected. The fruit yield in the Screenhouse experiment was

very low, perhaps due to the high temperature experienced during the growth period. Air and

night temperatures of more than 30 and 22oC, respectively, are responsible. for reduced fruit

development and enhanced vegetative growth in tomato (Shankara et al., 2005). In the field

experiments, none of the Mucuna species planted and ploughed in as green manure was

infected by M. incognita confirming the non- host status of these Mucuna spp as illustrated in

the Screenhouse experiment. Comparatively, tomato plants were galled more in the greenhouse

trial (experiment III) than the field trial (experiment IV). This could be explained from two

perspectives. The Screenhouse trial had plants artificially inoculated with 5,000 eggs of M.

incognita. In the field, inoculum density may vary due to spotting factor as nematodes are not

evenly distributed in the field. Of course, this spotting factor accounted for higher root- knot

incidence recorded in some mycorrhizal plants relative to non- mycorrhizal plants. Secondly, in

the field, Mucuna were planted and the foliage incorporated into the soil in situ as green

manure. Since from this trial, it has been established that the Mucuna species are non- hosts of

M. incoginta, then it follows that for the three months of growth, root- knot nematodes could

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have been starved as there was no good host for them to survive on. Also, the roots of these

Mucuna species must have released some nematicidal/nematostatic compounds which could

have seriously reduced the pre- plant nematode density. This trial typically illustrates the

combined action of crop rotation and green manuring in nematode management. Although,

Mucuna seeds are yet to be utilized as livestock feed or human food due to anti-nutritional

factors, the farmers could benefit by planting it for just three months and then plough in. The

nematode suppression and biological nitrogen fixation as well as the nutrients released during

its decomposition may as well off set the cost of nematicide application and synthetic fertilizer

(Gosling et al, 2006). Thus, combination of M. jaspaeda and M. ghana with Gi. gigantea or G.

mosseae gave the best nematode control and yield of tomato in the field.

The results of experiment V showed wide variation in root galling of the tomato plants

by M. incoginta among the soils from the different locations. The highest number of galls was

found in Ogoja soil with sandy texture. Galling was also severe in Calabar with loamy sand

texture as well as Obubra and Umudike with sandy loam texture. However, galling was not

severe in Ikom soil with sandy clay loam texture as well as Nsukka and Uyo soils with sandy

loam texture. Many interacting factors of the environment may account for this variation in

galling. However, soils with high proportion of sand as opposed to a relatively high amount of

fine particles do enhance nematode activities (Agu, 2002; Olowe, 2005). This could have

accounted for higher infection of tomato roots by M. incognita in soils from locations with high

sand content. In almost all the locations, double application of the bionematicde (P. lilacinus)

significantly inhibited galling and eggmass production than single application. This observation

is in line with the report of earlier workers (Cabanillas and Barker, 1989; Bruckner, 2004;

Nasresfahani and Ansari Pour, 2006) who recommended split application of P. lilacinus for

effective management of root-knot nematodes. Since, P. lilacinus is an egg parasitic fungus,

timing of treatment application to coincide with the susceptible stage of the nematode pest is

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important. Across the soils from the different locations, there was a synergistic interaction

between AMF and P. lilacinus in nematode suppression as well as growth and yield

enhancement of tomato infected with M. incoginta. Application of the bionematicide at

transplanting and two weeks later in combination with the various AMF species resulted in the

greatest gall inhibition and yield improvement. These findings corroborate the report of earlier

researchers (Akhtar and Siddiqui, 2008; Al-Raddad, 1995; Mahmood, 1995). However, the

efficacy of the AMF species varied among the soil types. In combination with P. lilacinus

application, the most effective AMF species were: G. etunicatum and G. deserticola (Nsukka

and Obubra soils), G. etunicatum and G. mosseae (Calabar and Uyo soils), Gi. gigantea and G.

etunicatum (Ikom and Ogoja soils) and Gi. gigantea and G. mosseae (Umudike soil). The

difference in AMF species effectiveness could partly be attributed to the differences in soil

properties and their adaptability to variation in climatic factors such as temperature. For

instance, it has been reported that Gigaspora spp do not proliferate in clayey Vertisols in

tropical soils but adapt well to sandy soils (Lekberg et al., 2007). In Nigeria, it has been

reported recently that G. clarum and G. deserticola are more abundant in the savanna

agroecology, G. etunicatum and Gi. gigantea adapt better to the humid forest zone, while

Glomus mosseae occurred in large population in all the agroecological zones (Dare et al.,

2013). It could be that, G. mosseae and G. gigantea are more adaptable to the highly available

P content of Calabar, Uyo and Umudike soils. High available phosphorus levels have been

reported to retard AMF activity (Carling et al., 1989; Smith, 1988). The interaction of some

other soil microbes with AMF may be injurious to the latter. Recently, Singh et al. (2014)

observed that Fusarium oxysporum f.sp. lycopersici and Trichoderma harzianum inhibited

tomato root colonization by some Glomus species thus reducing their growth enhancing ability.

Also, Flor-Peregrin et al. (2014) had reported that AMF (Funneliformis mosseae) was more

effective than Rhizophagus irregularis in combination with Pasteura Penetrans in root knot

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disease control in tomato. The explanation of how biocontrol agents differ in their control

activities appears complex, as the multifaceted interactions taking place in the rhizosphere are

influenced by so many factors. However, this trial showed a compatible interaction between

AMF and bioformulated P. lilacinus in the management of M. incognita on tomato. This

compatible interaction could have resulted from early inoculation of the tomato plants with the

AMF. Inoculation of tomato seedlings at the nursery stage with AMF could have placed the

fungus at a competitive advantage over indigenous micro-organisms and arbuscular

mycourhizal fungi. The AMF may have established in the root epidermis before the

inducement of giant cells by the root-knot nematode which was also under attack by P.

lilacinus. Again, it is possible that AMF may have colonized the feeding sites before the root-

knot nematode, thereby starving them to death. This could have led to the reduction in

nematode population and subsequent reduction in galling. As few juveniles were able to

penetrate as they escaped antagonism by AMF, P. lilacinus must have colonized the eggs laid

by the matured females, killed the first larval stage and thus prevented egg hatch. NasrEstahni

and Ansari Pour (2006) observed that P. lilacinus penetrates the eggs of root-knot nematodes

and develops profusely inside and over (being filled with the fungus mycelium), completely

inhibiting juvenile development of the nematode inside the egg. It is apparent from this trial

that, the deployment of various antagonistic strategies by AMF species against M. incognita in

combination with egg parasitism by the bioformulated P. lilacinus could possibly account for

the synergistic interaction observed between the two biocontrol agents in managing the pest in

tomato.

The most effective AMF species in gall suppression and fruit yield enhancement across the

soil types were G. etunicatum and G. deserticola, respectively. Thus, it appears that G.

etunicatum is very efficient in nematode antagonism but not very effective in plant growth and

yield enhancement. This finding calls for the evaluation of mixtures of AMF in combination

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with P. lilacinus in the management of root-knot disease in subsequent studies. However, some

commercial bioformulations containing mixtures of AMF are available. An example is Stanes

symbion vam plus(R) (Glomus fasciculatum and Gigaspora spp) which has been tested against

fungal root-rot and root-knot diseases of potato (Abd-El-Khair and El- Nagdi, 2014).

Experiment VI evaluated the combination of AMF, amendment of soil with

Mucuna and application of the bioformulated P. lilacinus in the management of M. incoginta.

The results obtained indicated that combined application of the three control agents

significantly reduced root galling and nematode reproduction with a corresponding significant

increase in growth and fruit yield of tomato relative to sole or double application. M. jaspaeda

in combination with G. mosseae or Gi gigantea and bionematicide application produced the

best result. The compatibility of P. lilacinus with AMF in root-knot nematode management has

been discussed already in the previous section. However, worthy of note is that soil amendment

with the Mucuna spp did not adversely affect the growth and establishment of the two

biocontrol agents. Root colonization by AMF was however increased with Mucuna soil

amendment. Also, in most cases, gall inhibition, growth and yield enhancement were increased

by P. lilacinus application in soils amended with Mucuna as green manure relative to

unamended soil. These findings are in conformity with the report of earlier investigators

(Rodriguez-kabana et al., 1987; Goswami et al., 2007; Siddiqui and Akhtar, 2008b; El-sherif

and Ismail, 2009; Rao et al., 1996; Serfoji et al., 2010).

The bulk of evidence from the findings of these authors supports the hypothesis that

biological control agents colonize and proliferate on the host plant better with soil organic

amendments compared with unamended soils. Oka (2010) has hypothesized that soil organic

amendments simulate soil food web thus providing source of carbon and other nutrients needed

by microbes, biocontrol agents inclusive. Thus, from our results it is possible that the Mucuna

used as a green manure may be playing multifunctional role in nematode management. The

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release of nematicidal constituents as it decomposes could be very beneficial in nematode

population suppression (Nogueira et al., 1996; Vincente and Acosta, 1987; Vargas et al., 1996).

Nutrients released during its decomposition (Adigbo et al., 2003) could benefit the first crop

and thus may lead to induced tolerance/resistance to pests and diseases (Casky et al., 1998). In

the real farm situation, using Mucuna as a short-term cover/green manure crop could benefit

the farmer immensely as it is a non-host to M. incognita, the population of this nematode

species could be reduced drastically during the three months of growth as illustrated in

experiment IV. Moreover, there is increase in the N-Pool of the soil due to biological nitrogen

fixation by Rhizobium spp. Thus, although the three months period of Mucuna cover could be

considered by farmers as inefficient land utilization, the benefits derived may outweigh the

cost. It appears from the results of these experiments, early inoculation of tomato seedlings at

the nursery stage could have helped the plants in the procurement of less mobile elements

(P,Ca,Cu, Zn, etc.) through increased root absorptive surface and suppression of nematode

population through various mechanisms of antagonism. Also, with the combined action of egg

parasitism by P. lilacinus and the beneficial effects soil amendment with Mucuna, the tomato

plants were at a competitive advantage with M. incognita compared with the control plants.

135

SUMMARY, CONCLUSION AND RECOMMENDATIONS

SUMMARY

Five Screenhouse experiments and one field experiment were conducted at the

Teaching and Research Farm of the Department of Crop Science, Faculty of Agriculture,

University of Calabar, Cross River State. Experiment 1 was carried out in the Screenhouse to

ascertain the host status of five Mucuna species to Meloidogyne incognita. It was laid out in a

completely randomized design (CRD) having six treatments represented by five Mucuna

species (M. pruriens utilis, M. ghana, M. cochichinensis, M. jaspaeda and M. pruriens IR2)

plus a check (susceptible tomato cv. Roma VF) with five replications. The Mucuna spp and the

check plant were inoculated with 5,000 eggs of M. incognita/plant. Experiment 11 evaluated

the effects of five Mucuna species used as green manures against M. incognita. The treatments

were five rates (2, 4, 6, 8 and 10 t/ha on dry matter basis) of each Mucuna spp applied as green

manure and soil without amendment served as control (0 t/ha). The 26 treatments were laid out

in a completely randomized design with three replications. Tomato (cv. Roma VF) seedlings

were inoculated with 5,000 eggs of M. incognita and grown to full maturity. Experiments III

and IV evaluated the effects of Mucuna spp green manure in combination with arbuscular

mycorrhizal fungi (AMF) against M. incognita in the Screehouse and field, respectively. The

Screenhouse experiment was laid out as a 6 x 6 factorial in CRD with three replications. The

treatments were combinations of five species of Mucuna applied at 8 t/ha each and five species

of AMF: Glomus etunicatum, G. mosseae, G. clarum, G. deserticola and Gigaspora gigantea)

plus their respective controls. The tomato seedlings were inoculated with AMF at the nursery

stage. During transplanting, each seedling was inoculated with 5,000 eggs of M. incognita. For

the field experiment, it was a split-plot laid out in randomized complete block design with three

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replicates. The main-plot was planted with the respective Mucuna species and ploughed-in after

three months. Natural fallow plot served as control. The main–plot was split to contain the

AMF species as the sub-plots. Tomato plants grown in the field were naturally infected with M.

incognita. In experiment V, the effects of bioformulated P. lilacinus and AMF against M.

incognita were evaluated in different soil types. Top soils were collected from Calabar, Ikom,

Obubra and Ogoja (Cross River State), Nsukka (Enugu State), Umudike (Abia State) and Uyo

(Akwa Ibom State) and the experimental design was a 3 x 6 factorial in CRD with three

replications. Three levels of P. lilacinus application were combined with six levels of AMF

species. The tomato seedlings were inoculated with 5, 000 eggs of M. incognita and grown to

full maturity. Experiment VI was a 6x6x2 factorial laid out in CRD with three replications. The

treatments included six levels each of Mucuna species and AMF species and two levels of P.

lilacinus application. The tomato seedlings were inoculated with 5,000 eggs of M. incognita /

plant. Data were collected on number of galls and egg masses/root system, gall index (0-5

scale), nematode larvae/ 200 g soil, mycorrhizal root colonization (%), weight (g) of fresh root,

dry shoot, shoot length(cm)/plant, number and total fresh fruit weight(g)/plant and analysed by

Analysis of Variance (ANOVA). Significant means were separated using fishers’ least

significant difference (F-LSD) at 5% probability level. Tomato responses to rates of Mucuna

were tested with a linear or curvilinear regression analysis at 1% probability level. Roots of all

the Mucuna spp in both the Screenhouse and field trials were neither galled nor had egg masses

and were rated immune to M. incognita infection, with a gall index (GI) of 0.00. The tomato

plant (control) was highly susceptible, with GI rating of 5.00. The number of nematode larvae

on tomato rhizosphere was significantly (P < 0.05) higher than that of Mucuna species. Among

the Mucuna species, M. jaspaeda haboured significantly (p< 0.05) the lowest nematode

larvae. In all the Mucuna species, successive increase in the rate of amendment resulted in a

significant (p<0.05) decrease in the number of galls, eggs masses, nematode larvae but with a

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significant (p<0.05) enhancement in growth, plant dry matter and fresh fruit yield. M. jaspaeda

and M. ghana amendment produced plants that had significantly ( p < 0.05) the fewest galls

and egg masses. These two Mucuna species had the lowest C:N ratio. The highest fruit yield of

50.26 g/plant was obtained with 8 t/ha of M. jaspaeda amendment. There was a highly

significant (p < 0.01) inverse (r > - 0.80) linear relationship between number of galls and rates

of amendment in all the Mucuna spp but a positive (r > 0.70) linear relationship with respect to

total fresh fruit weight. In both Screenhouse and field experiments, mycorrhizal inoculation and

soil amendment with Mucuna significantly (P < 0.05) suppressed root galling and nematode

reproduction but enhanced growth and fruit yield of tomato compared with their respective

controls. Mucuna amendment significantly (P < 0.05) enhanced root colonization by AMF.

Application of both control agents was more effective than sole application. The highest total

fresh fruit yield (409.00g / plant) was obtained in plots whose plants were inoculated with Gi

gigantea and soil amended with M. jaspaeda. Application of bioformulated P. lilacinus or

AMF inoculation significantly ( p < 0.05) inhibited root galling and eggmass production by

M. incognita in all the soils from all locations while there was a significant ( p <0.05)

enhancement in growth, dry matter content and fresh fruit yield of tomato. Double application

of the bio-nematicide was significantly (p < 0.05) more effective than single application. In

combination with P. lilacinus application , the most effective AMF species were: G.

etunicatum and G. deserticola (Nsukka and Obubra soils ), G. etunicatum and G. mosseae

(Calabar and Uyo soils), Gi gigantea and G. etunicatum (Ikom and Ogoja soils) and Gi.

gigantea and G. mosseae (Umudike soil). Combined application of the three control agents

significantly ( P < 0.05) inhibited galling and nematode reproduction with a corresponding

significant ( p <0.05) increase in growth and fruit yield of tomato relative to sole application.

The highest fresh fruit yield of 139.46g and 136.06 g/plant were obtained from G. mosseae and

138

Gi. gigantea inoculated plants, respectively grown in M. jaspaeda amended soils with P.

lilacinus applied.

.

CONCLUSION

In conclusion, the trials have shown that Mucuna could be used as a short-term

rotation/green manure crop in combination with early inoculation of tomato seedlings with

arbuscular mycorrhizal fungi in the management of M. incognita. The best Mucuna species

were: M. jaspaeda and M. ghana which could perfectly substitute the popular M. pruriens

utilis. The bioformualted P. lilacinus was effective in reducing infectivity of M. incognita in all

the soils obtained from the different locations in southeastern states, of Nigeria. Among the

AMF species, G. etunicatum was the most effective in nematode control while G. deserticola

was the best in growth and yield enhancement. The three control agents acted synergistically in

root-knot disease control and growth enhancement of tomato. Field evaluation of the

bionematicide in combination with Mucuna as cover/green manure crop and arbuscular

mycorrhizal fungus inoculation of tomato seedlings may offer a sound environmentally friendly

means of managing this pest.

RECOMMENDATIONS

(1) Farmers should be encouraged to plant Mucuna preferably (M. jaspaeda and M. ghana) as a

short-term rotation crop and incorporate the foliage for the control of M. incognita.

(2) Tomato seedlings should be inoculated with effective species of AMF like Glomus

etunicatum, G. deserticola, G. gigantea and G. mosseae, for the control of M. incognita.

139

(3) The bioformulated P. lilacinus could be used at the manufacturer’s recommended rate in

combination with effective species of AMF and Mucuna after thorough field trials in various

agroecological zones of Nigeria.

(4) The nematicidal compounds present in M. jaspaeda and M. ghana as well as other species

of Mucuna should be evaluated.

(5) Research should be geared towards isolating indigenous (Nigerian) P. lilacinus for possible

formulation for nematode control in Nigeria.

(6) Isolation of pure cultures of indigenous AMF species should be carried out and

experiments involving mixtures of species for root-knot nematode control should be

considered.

(7) The efficacy of the two biocontrol agents and Mucuna species used in this work should be

tested on other root-knot nematodes species and even against other nematode genera prevalent

in Nigeria.

140

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