INTERACTION OF Meloidogyne incognita AND Fusarium

INTERACTION OF Meloidogyne incognita AND Fusarium oxysporum f. sp.

vasinfectum Snyder and Hansen ON GROWTH, YIELD AND WILT SEVERITY ON

TWO OKRA (Abelmoschus esculentus) VARIETIES

BY

SAMUEL YAO AGBAGLO

(10508876)

THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN

PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF

MASTER OF PHILOSOPHY (MPHIL) CROP SCIENCE DEGREE

JULY, 2017

i

DECLARATION

I, Samuel Yao Agbaglo, do hereby declare that the work herein presented is the result of my

own investigation and that except other people‟s work, which have been duly acknowledged,

this thesis has never been presented to this university or elsewhere for any degree.

…………………………… …………………..

SAMUEL YAO AGBAGLO DATE

(STUDENT)

………………………………… …………………..

DR. S. T. NYAKU DATE

(MAIN SUPERVISOR)

………………………………. …………………..

DR. E. W. CORNELIUS DATE

(CO – SUPERVISOR)

ii

DEDICATION

To the memory of my late father, James Kwame Agbaglo

iii

ACKNOWLEDGEMENT

I wish to express my deepest gratitude to Almighty God for His protection, direction, favour

and compassion that paved the way for me through my course of study.

My heartfelt thanks go to my meticulous supervisors, Dr. S. T. Nyaku and Dr. E. W.

Cornelius for their endurance, guidance and direction towards the realization of this work.

Sirs, God richly bless you.

I am equally thankful and indebted to Mr. Samuel Osabutey, teaching assistant, Plant

Pathology laboratory, Crop Science Department–Legon and Mr Issac Bedu, a technician at

Plant Pathology Laboratory for their incalculable technical support during the laboratory and

field works. My sincere appreciation to you, my brothers.

My invaluable appreciation goes to Mr. E.W. Asante, the Principal Research Assistant at

Crop Science Department, University of Ghana and Mr. N. Agyekum of University of Ghana

farms for their technical assistance for the pot and field experiments.

I must again register my sincerest thanks to Ms. Elizabeth Annan for her support and prayers

throughout the course of study. Elizabeth, your chapter cannot be closed in my life.

I further record my heartfelt appreciation to my friends and course mates, Mr. Gyawu Bright,

Mr. Ablormeti Fred Kormla and Mr. Kingsley Ochar for their high sense of encouragement

and support. Thank you very much, my friends.

Finally, to all who in diverse ways contributed to the success of this research, may God never

forget you in times of predicaments.

iv

TABLE OF CONTENTS

Title Page No.

DECLARATION ........................................................................................................................ i

DEDICATION ........................................................................................................................... ii

ACKNOWLEDGEMENT ....................................................................................................... iii

TABLE OF CONTENTS ................................................................................................... iv - vi

LIST OF TABLES ................................................................................................................... vii

LIST OF FIGURES ........................................................................................................ viii–viii

LIST OF ABBREVIATIONS…………………………………………………………………x

LIST OF APPENDICES ........................................................................................................... xi

ABSTRACT ............................................................................................................................. xii

CHAPTER ONE ........................................................................................................................ 1

1.0 INTRODUCTION ....................................................................................................... 1

CHAPTER TWO ....................................................................................................................... 5

2.0 LITERATURE REVIEW ............................................................................................ 5

2.1 Origin and botany of okra ........................................................................................... 5

2.1.1 Origin ....................................................................................................................... 5

2.1.2 Botany ...................................................................................................................... 6

2.2 Importance of okra ...................................................................................................... 7

2.3 Varieties of Okra ......................................................................................................... 9

2.3.1 Tall green: .............................................................................................................. 11

2.3.1.1 Tall green, long pod: .............................................................................................. 11

2.3.1.2 Tall green, short pod: ............................................................................................. 11

2.3.2 Dwarf green: .......................................................................................................... 12

2.3.2.1 Dwarf green, long pod: .......................................................................................... 12

2.3.2.2 Dwarf green, short pod: ......................................................................................... 13

2.3.3 Lady‟s finger: ........................................................................................................ 13

2.3.3.1 Lady‟s finger, white pod: ....................................................................................... 13

2.3.3.2 Lady‟s finger, green pod: ....................................................................................... 14

2.4 Soilborne diseases of okra ......................................................................................... 15

2.4.1 Southern Blight (Sclerotium rolfsii) ...................................................................... 15

2.4.2 Damping off (Pythium spp., Macrophomina spp., Rhizoctonia spp.) ................... 15

v

2.4.3 Verticillium Wilt (Verticillium albo-atrum) .......................................................... 16

2.4.4 Southern Root knot nematode (Meloidogyne incognita) and its damage to crops 16

2.4.4.1 Identification of southern root-knot nematode ...................................................... 20

2.4.5 Fusarium wilt disease (Fusarium oxysporum f. sp. vasinfectum) and its damage to

crops……. ............................................................................................................................ 20

2.4.5.1 Identification of Fusarium oxysporum f. sp. vasinfectum ..................................... 21

2.6 Fungus–nematode interactions .................................................................................. 22

2.7 Diseases complexes and types of interactions (Synergistic, Antagonistic and Neutral

interactions). ......................................................................................................................... 25

2.7.1 Synergistic interaction ........................................................................................... 26

2.7.2 Antagonistic interaction/ Indirect effect: ............................................................... 27

2.7.3 Neutral interaction: ................................................................................................ 28

CHAPTER THREE ................................................................................................................. 29

3.0 MATERIALS AND METHODS .............................................................................. 29

3.1 Study Site .................................................................................................................. 29

3.2 Confirmation of pathogens for root knot disease and wilt of okra as M. incognita

and F. oxysporum f. sp. vasinfectum respectively. ............................................................... 30

3.2.1 Preparation of water agar (WA) ............................................................................ 30

3.2.2 Preparation of potato dextrose agar (PDA) .......................................................... 30

3.2.3 Isolation of Fusarium oxysporum f. sp. vasinfectum ............................................. 30

3.2.4 Identification of Fusarium oxysporum f. sp. vasinfectum ..................................... 31

3.2.5 Preparation of inoculum of Fusarium oxysporum f. sp. vasinfectum .................... 31

3.2.6 Inoculation of okra seedlings with F. oxysporum .................................................. 31

3.2.7 Re-isolation of the organism .................................................................................. 31

3.2.8 Extraction Meloidogyne incognita from diseased okra plants ............................... 32

3.2.9 Identification of M. incognita ................................................................................ 32

3.2.10 Preparation of inoculum of Meloidogyne incognita .............................................. 32

3.2.11 Inoculation of okra seedlings with inoculum ........................................................ 33

3.2.12 Re-extraction of the organism ............................................................................... 33

3.3 Evaluation of individual effect and combined interactions of M. incognita and F.

oxysporum f. sp. vasinfectum on the growth and yield of okra ............................................ 33

3.3.1 Pot experiment ....................................................................................................... 33

3.3.2 Field experiment .................................................................................................... 35

3.4 Data taken for pot and field experiments .................................................................. 36

vi

3.4.1 Plant height (cm) ................................................................................................... 37

3.4.2 Stem girth (cm) ...................................................................................................... 37

3.4.3 Chlorophyll content (CCI) ..................................................................................... 37

3.4.4 Fresh shoot weight (g) ........................................................................................... 37

3.4.5 Dry shoot weight (g) .............................................................................................. 37

3.4.6 Fresh root weight (g) ............................................................................................. 38

3.4.7 Dry root weight (g) ................................................................................................ 38

3.4.8 Determining okra fruit yield: ................................................................................. 38

3.4.9 Nematode Reproductive Index .............................................................................. 38

3.4.10 Number of nematode eggs/g root .......................................................................... 38

3.4.11 Root Knot (Gall) Index .......................................................................................... 39

3.4.12 Percentage wilt incidence ...................................................................................... 39

3.5 DATA ANALYSIS ................................................................................................... 39

CHAPTER FOUR .................................................................................................................... 40

4.0 RESULTS.................................................................................................................. 40

4.1 Causal organisms of root knot and wilt disease of okra ............................................ 40

4.1.1 Root knot disease of okra ...................................................................................... 40

4.1.2 Wilt of okra ............................................................................................................ 40

4.2 Individual, simultaneous and sequential interactions between Meloidogyne incognita

and Fusarium oxysporum f. sp. vasinfectum (FOV) on the growth and yield of okra ......... 42

4.2.1 Pot experiment ....................................................................................................... 42

4.2.2 Field Experiment ......................................................................................................... 52

4.3 Reproductive ability of Meloidogyne incognita on okra after Fusarium oxysporum f.

sp. vasinfectum infection under pot and field conditions ..................................................... 61

4.4 Effect of Meloidogyne incognita on the Fusarium wilt disease severity on two okra

varieties …………... ............................................................................................................ 65

CHAPTER FIVE ..................................................................................................................... 69

5.0 DISCUSSION ........................................................................................................... 69

CHAPTER SIX ........................................................................................................................ 73

6.0 CONCLUSIONS AND RECOMMENDATIONS ................................................... 73

6.1 Conclusion ................................................................................................................. 73

6.2 Recommendations ..................................................................................................... 74

REFERENCES ........................................................................................................................ 75

APPENDICES ......................................................................................................................... 95

vii

LIST OF TABLES

Table 1: Ten major okra producing countries in the world ....................................................... 8

Table 2: Production quantities of okra in Ghana ....................................................................... 9

Table 3: Some okra cultivars grown in Ghana......................................................................... 10

Table 4: Climatic data of experimental area ............................................................................ 29

Table 5: Description of treatments for pot experiment. ........................................................... 34

Table 6: Description of treatments for field experiment. ......................................................... 36

Table 7: Rating scale for wilt incidence……………………………………………………...39

Table 8: Fresh shoot weight and dry shoot weight for two okra varieties under various

inoculations in pot experiment (n = 3).............................................................................. 46

Table 9: Fresh root weight and dry root weight for two okra varieties under various

inoculations in pot experiment (n = 3).............................................................................. 48

Table 10: Number of pods for two okra varieties under various inoculations in pot experiment

(n = 3) ............................................................................................................................... 50

Table 11: Weight of pods and yield for two okra varieties under various inoculations in pot

experiment (n = 3) ............................................................................................................ 52


inoculations in field experiment (n = 3) ........................................................................... 57


inoculations in field experiment (n = 3) ........................................................................... 58

Table 14: Number of pods of „Essoumtem‟ and Clemson spineless inoculated serially with

Fusarium oxysporum f. sp. vasinfectum in field experiment (n = 3) ................................ 59

Table 15: Weight of pods and yield of „Essoumtem‟ and Clemson spineless inoculated

serially with Fusarium oxysporum f. sp. vasinfectum in field experiment (n = 3) ........... 61

Table 16: Number of eggs, gall index and reproductive factor for two okra varieties under

various inoculations in pot experiment (n = 3) ................................................................. 63

Table 18: Percentage wilt incidence for two okra varieties under various inoculations in pot

experiment (n = 3) ............................................................................................................ 66

Table 19: Percentage wilt incidence in „Essoumtem‟ and Clemson spineless inoculated

serially with Fusarium oxysporum f. sp. vasinfectum in field experiment (n = 3) .......... 68

viii

LIST OF FIGURES

Fig. 1: A. Okra plant and pod of tall green and B. Long pod okra .......................................... 11

Fig. 2: A. Okra plant and pod of tall green and B. Short pod okra .......................................... 12

Fig. 3: A. Okra plant and pod of dwarf green and B. Long pod okra ...................................... 12

Fig. 4: A. Okra plant and pod of dwarf green and B. Short pod okra ...................................... 13

Fig. 5: A. Okra plant and pod of lady‟s finger and B. White pod okra ................................... 14

Fig. 6: A. Okra plant and pod of lady‟s finger and B. Green pod okra ................................... 14

Fig. 7: Pots with seedlings laid for the pot experiment............................................................ 34

Fig. 9: Micrograph of female southern root-knot nematode, Meloidogyne incognita (A) and

nematode eggs (B). X 400 magnification for all the three figures. a – tail, b – ovaries, c –

neck, d – stylet knobs, e – stylet and f – eggs................................................................... 40

Fig. 10: Micrograph of a twelve-day old culture of Fusarium oxysporum f. sp. vasinfectum on

PDA (A) with Macroconidia (B), and Microconidia (C) (X 400) .................................... 41

Fig. 11: Height of okra plants inoculated individually and simultaneously with Meloidogyne

incognita and Fusarium oxysporum f. sp. vasinfectum in pot experiment (n = 3). F =

Fungus alone; N = Nematode alone; NF = Nematode and Fungus simultaneously; NF7 =

Fungus 7 days after nematode inoculation; NF14 = Fungus 14 days after nematode

inoculation; NF21 = Fungus 21 days after nematode inoculation and C = Control (un-

inoculated); A – Essoumtem, B – Clemson spineless. ..................................................... 42

Fig. 12: Girth of okra plants inoculated individually and simultaneously with Meloidogyne

incognita and Fusarium oxysporum f. sp. vasinfectum in pot experiment (n = 3). F =

Fungus alone; N = Nematode alone; NF = Nematode and Fungus simultaneously; NF7 =



inoculated); A – Essoumtem, B – Clemson spineless. ..................................................... 43

Fig. 13: Chlorophyll content of okra plants inoculated individually and simultaneously with

Meloidogyne incognita and Fusarium oxysporum f. sp. vasinfectum in pot experiment (n

= 3). F = Fungus alone; N = Nematode alone; NF = Nematode and Fungus

simultaneously; NF7 = Fungus 7 days after nematode inoculation; NF14 = Fungus 14

days after nematode inoculation; NF21 = Fungus 21 days after nematode inoculation and

C = Control (un-inoculated); A – Essoumtem, B – Clemson spineless. .......................... 44

Fig. 14: Height of okra plants inoculated serially with Fusarium oxysporum f. sp. vasinfectum

in field experiment (n = 3). F7 = fungus inoculated 7 days after seedling emergence, F14

ix

= fungus inoculated 14 days after seedling emergence, F21 = fungus inoculated 21 days

after seedling emergence, C = Control (un-inoculated); A = Essoumtem, B = Clemson

spineless. ........................................................................................................................... 53

Fig. 15: Girth of okra plants inoculated serially with Fusarium oxysporum f. sp. vasinfectum

in field experiment (n = 3). F7 = fungus inoculated 7 days after seedling emergence, F14

= fungus inoculated 14 days after seedling emergence, F21 = fungus inoculated 21 days

after seedling emergence, C = Control (un-inoculated); A = Essoumtem, B = Clemson

spineless. ........................................................................................................................... 54

Fig. 16: Chlorophyll content of okra plants inoculated serially with Fusarium oxysporum f.

sp. vasinfectum in field experiment (n = 3). F7 = fungus inoculated 7 days after seedling

emergence, F14 = fungus inoculated 14 days after seedling emergence, F21 = fungus

inoculated 21 days after seedling emergence, C = Control (un-inoculated); A =

Essoumtem, B = Clemson spineless. ................................................................................ 55

Fig 17: Effects of Meloidogyne incognita on the roots of okra. Root with galls in pot

experiment (A), root with galls in field experiment (B) and okra root without any gall

(C). .................................................................................................................................... 65

Fig. 19: Symptoms of Fusaiurm wilt on okra. The diseases showing symptoms from the top

of the plant (A) and disease showing symptoms from the soil line of the plant (B). ....... 68

x

LIST OF ABBREVIATIONS

ANOVA: Analysis of Variance

CRD: Completely Randomized Design

df: Degree of Freedom

FAO: Food and Agricultural Organization

FAOSTAT: Food and Agricultural Organization Statistics

FOV: Fusarium oxysporum f. sp. vasinfectum

GENSTAT: General Statistics

LSD: Least significant difference

Mi: Meloidogyne incognita

MOFA: Ministry of Food and Agriculture

m.s.: Means square

NARP: National Agriculture Research Project

N.P.K: Nitrogen, Phosphorus, Potassium

OELCV: Okra Enation Leaf Curl Virus

OYVMV: Okra Yellow Vein Mosaic Virus

PDA: Potato Dextrose Agar

PPN: Plant Parasitic Nematodes

RCBD: Randomized Completely Block Design

s.s.: Sum of squares

v.r.: Variance ratio

WA: Water Agar

xi

LIST OF APPENDICES

APPENDIX 1: Root–Knot Nematode Rating Chart – Bridge and Page ................................. 95

APPENDIX 2: Analysis of variance of plant height for pot experiment ................................. 96

APPENDIX 3: Analysis of variance of plant girth for pot experiment ................................... 97

APPENDIX 4: Analysis of variance of chlorophyll content for pot experiment .................... 98

APPENDIX 5: Analysis of variance of fresh shoot weight for pot experiment .................... 100

APPENDIX 6: Analysis of variance of dry shoot weight for pot experiment ....................... 100

APPENDIX 8: Analysis of variance of dry root for pot experiment ..................................... 101

APPENDIX 9: Analysis of variance of number of pods for pot experiment ......................... 101

APPENDIX 10: Analysis of variance of pod weight for pot experiment .............................. 101

APPENDIX 11: Analysis of variance of yield (kg/ha) for pot experiment ........................... 102

APPENDIX 12: Analysis of variance of gall index ............................................................... 102

APPENDIX 13: Analysis of variance of egg count ............................................................... 102

APPENDIX 14: Analysis of variance of reproductive factor for pot experiment ................. 103

APPENDIX 15: Analysis of variance of wilt incidence for pot experiment ......................... 103

APPENDIX 16: Analysis of variance of plant height for field experiment .......................... 103

APPENDIX 17: Analysis of variance of plant girth for field experiment ............................. 105

APPENDIX 18: Analysis of variance of plant girth for field experiment ............................. 106

APPENDIX 19: Analysis of variance of fresh shoot weight for field experiment ................ 107

APPENDIX 20: Analysis of variance of dry shoot weight for field experiment ................... 108

APPENDIX 21: Analysis of variance of fresh root weight for field experiment .................. 108

APPENDIX 22: Analysis of variance of dry root weight for field experiment ..................... 108

APPENDIX 23: Analysis of variance for number of pods for field experiment ................... 109

APPENDIX 24: Analysis of variance for weight of pod for field experiment ...................... 109

APPENDIX 25: Analysis of variance of yield for field experiment ..................................... 109

APPENDIX 26: Analysis of variance of gall index for field experiment .............................. 110

APPENDIX 27: Analysis of variance of egg count for field experiment .............................. 110

APPENDIX 28: Analysis of variance of reproductive factor for field experiment ............... 110

APPENDIX 29: Analysis of variance of wilt incidence for field experiment ....................... 111

xii

ABSTRACT

The interaction of the fungus, Fusarium oxysporum f. sp. vasinfectum and the nematode,

Meloidogyne incognita on wilt disease on „Essoumtem‟ and Clemson spineless was examined

in pot and field experiments from September, 2016 to June, 2017 in the University of Ghana

farms. The fungus used for inoculation of four weeks old „Essoumtem‟ and Clemson

spineless seedlings for pot experiment and one week old „Essoumtem‟ and Clemson spineless

seedlings for field experiment was confirmed as Fusarium oxysporum and the nematode was

confirmed as Meloidogyne incognita. The individual, simultaneous and sequential inoculation

of second stage juveniles (at 1000 J2/kg soil) of Meloidogyne incognita and Fusarium

oxysporum (1.1 x 106 cells/kg soil) resulted in significant suppressed plant growth parameters

viz., plant height, plant girth, chlorophyll content, fresh and dry shoot weight and fresh and

dry root weight. Maximum suppression in plant growth parameters were observed on plants

that received NF21 and F21 treatments for pot and field experiments respectively. The least

suppression of plant growth parameters was observed in separate inoculations of the two

pathogens for both pot and field experiments. Maximum suppression in yield parameters was

observed in plants that received NF21 and F21 treatments for pot and field experiments

respectively. Minimal yield suppression was observed for individual inoculations (N and F

treatments) in both pot and field experiments. M. incognita reproduction, as reflected by

number of galls/root and eggs in root system, was least in plants inoculated with NF21

treatment and highest for plants inoculated with NF7 on both „Essoumtem‟ and Clemson

spineless for pot experiment. Again, M. incognita reproduction was least in plants inoculated

with F21 treatment and highest for plants inoculated with F7 on „Essoumtem‟ and Clemson

spineless for field experiments. The highest severity of wilt disease was observed on plants

that received NF21 and F21 treatment in both pot and field experiment.

xiii

Key words: root-knot Meloidogyne incognita; root-rot Fusarium oxysporum f. sp.

vasinfectum; Essoumtem; Clemson Spineless; interaction; suppression

1

CHAPTER ONE

1.0 INTRODUCTION

Okra (Abelmoschus esculentus) is regarded amongst other species of plants from the

Malvaceae family as extensively known and consumed specie of crop (Naveed et al., 2009)

and equally known to be economically significant vegetable crop cultivated in many parts of

the world (Oyelade et al., 2003; Andras et al., 2005; Saifullah and Rabbani, 2009). Hibiscus

was the genus into which okra was categorized later classifications reassigned it under

Abelmoschus and differentiated with copious features from the genus Hibiscus (Aladele et al.,

2008).

In Northern Ghana, the crop is grown for its high commercial value for deprived growers and

significance as a component in the diet of the people. Within developing countries such as

Ghana, okra is cultivated with ease and it represents an essential cash crop for families in

underprivileged areas (Filgueira, 2003). Okra is basically a tropical and subtropical crop

responsive to frost, temperature, drought and water logging. Fruits of the crop are found in a

fresh state in almost all markets in Ghana during the rainy season and in a dehydrated form

during the dry season. Some of the okra varieties commonly grown in Ghana are Lady‟s

finger, Quim Bombo, Asutem, Clemson Spineless and Labadi Dwarf. (MOFA, 2013).

The characteristics of okra adapts it to various uses such as its fresh leaves, buds, flowers,

succulent pods, soft stems and seeds (Mihretu et al., 2014). Its economic importance cannot

be overemphasized as it contains food nutrients such as proteins, calcium, vitamins C and

carbohydrates in larger quantities (Owolarafe and Shotonde, 2004; Gopalan et al., 2007;

Arapitsas, 2008; Dilruba et al., 2009; Naveed et al., 2009; Benchasri, 2012). Okra seeds are

also good source of protein and oil and a substitute for coffee (Calisir and Yildiz, 2005). Okra

2

is mainly cultivated as a vegetable crop and has the potential of being cultivated for the

production of oil since it contains as high as 20 to 40% of oil (Sorapong, 2012; MEF, 2013).

The young immature fruits are essentially fresh fruits that can be eaten in different forms

(Benchasri, 2013) and could also be boiled, fried or cooked for meals (Akintoye et al., 2011).

The highest concentration of nutrients realized in okra is obtained when the fruit is allowed

about seven days on the plant (Ndunguru and Rajabu, 2004; Agbo et al., 2008).The mucilage

from the fruit is able to bind bile and cholesterol carrying toxins released by the liver. Several

dishes are made out of the entire plant since the plant is versatile (Madison, 2008; Maramag,

2013).

Root-knot nematodes (Meloidogyne spp.) are regarded among the five most dangerous plant

pathogens of crops worldwide (Kayani et al., 2013; Mukhtar et al., 2013). There are four

commonly occurring species of nematodes out of 100 described species. These are the

southern root-knot nematode (Meloidogyne incognita), sugarcane eelworm (M. javanica),

peanut root-knot nemadoe (M. arenaria) and root-knot nematode (M. hapla). Among these

four species of nematodes, M. incognita is the most destructive and therefore of economic

importance (Hussain et al., 2012; Kayani et al., 2012a, 2012b, 2013; Barros et al., 2014).

Meloidogyne incognita has been established to infect a wide range of crops causing root-knot

disease (Khan et al., 2005; Anwar et al., 2007; Hussain et al., 2012, 2016; Kayani et al.,

2013; Ntidi et al., 2016; Shigueoka et al., 2016). Root-knot infections have been reported to

cause a surge in incidence and severity of wilt disease caused by bacteria and fungi (Shahbaz

et al., 2015; Tariq-Khan et al., 2016).

Fusarium wilt (Vascular wilt), caused by the fungus Fusarium oxysporum f. sp. vasinfectum

(Atk) Snyder and Hansen, is one of the utmost significant diseases on Malvaceae species. The

pathogen, Fusarium oxysporum f. sp. vasinfectum causes a disease called vascular wilt in

okra and cotton (Cia and Salgado, 1997). Some other diseases that infects okra are

3

Fusariumwilt (Fusarium oxysporum f. sp. vasinfectum), Damping-off (Pythium sp.), Powdery

mildew (Erysiphe cichoracearum), Southern blight (Sclerotium rolfsii), Verticillium wilt

(Verticillium albo-atrum), Alternaria leaf spot (Alternaria alternata) and Okra leaf curl (Okra

leaf curl virus)(Atia and Tohamy, 2004; Raid and Palmateer, 2006).

Nematode feeding on plants predisposes or breaks down their resistance of these plants to

infection by other plant pathogens which use wounds created by nematode feeding as

infection courts (France and Abawi, 1994). Plant parasitic nematodes found in the

rhizosphere interact with a wide range of plant pathogens such as bacteria, viruses and fungi.

Plant parasitic nematode and fungi interact on the same crop to cause wilting of the crop

(Powell, 1971; Francl and Wheeler, 1993).

The first record of a study on nematode-fungus interaction on plants was in 1892 (Atkinson

1892) which stimulated interest in the interaction between nematodes and fungi in nematode-

fungus disease complex. Some studies have been conducted on the interaction between

Fusarium oxysporum and Meloidogyne incognita on host crops (Khpalwak, 2012) but not

much work has been done on the interaction between Fusarium oxysporum and Meloidogyne

incognita on okra in Ghana. The concept of nematode-fungi relationship in plant disease has

been researched into but the mechanism of interaction is not fully understood and further

research need to be conducted on it (Abawi and Barker, 1984; Back et al., 2002; Castillo et

al., 2003; Mokbel et al., 2007).

The objectives of the present study are therefore to:

1. confirm the pathogens for root knot disease and wilt of okra as M. incognita and F.

oxysporum f. sp. vasinfectum respectively.

4

2. evaluate individual, simultaneous and sequential inoculations between Meloidogyne

incognita and Fusarium oxysporum f. sp. vasinfectum on the growth and yield of

„Essoumtem‟ and Clemson spineless.

3. determine the reproductive ability of Meloidogyne incognita on „Essoumtem‟ and

Clemson spineless after Fusarium oxysporum f. sp. vasinfectum infection.

4. evaluate the effect of Meloidogyne incognita on the severity of Fusarium wilt disease

on „Essoumtem‟ and Clemson spineless.

5

CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 Origin and botany of okra

2.1.1 Origin

Okra has two accepted names the world over; Abelmoschus esculentus and Hibiscus

esculentus (Kumar et al., 2010) and it is known to be native of countries in West Africa

(Joshi et al., 1974; Kochhar, 1986). Among the old cultivated crops in the world is okra and

its cultivation cannot be underestimated. Egyptians in 1216 A.D. gave the first record of okra

as being originated from Egypt but other agronomists such as Vavilov also documented the

origin of okra to be from Ethiopia (Lamont, 1999). Okra is believed to have originated from

Ethiopia (Getachew, 2001; Simmone et al., 2004; Dandena, 2010; Sathish and Eswar, 2013)

and was then disseminated to Northern part of Africa, the Mediterranean, Arabia and in India

by 1300 BC (Nzikou et al., 2006). Little information is readily available about the history and

distribution of the crop due to fastness of the spread of the crop from Ethiopia to the rest of

the world. The movement of the okra crop from Ethiopia down to India is really not

documented (Tindall, 1983).

Various scientists have proposed that okra, Abelmuschus esculentus, originated from

countries such as India, West Africa, Ethiopia, Tropical Asia, Pakistan and Burma

(Benchasri, 2012). Through Brazil and Dutch Guinea, okra reached the new Worlds and

slaves migrating from Africa to North America also sent the crop to other parts of the world

(Hamon et al., 1990; Bish et al., 1995). Wild and cultivated varieties of the okra crop exist

and some few examples are Abelmoschus esculentus, A. caillei, A. moschatus, A. manihot, A.

ficulneus and A. tetraphyllus. Edible species amongst those cultivated include Abelmoschus

manihot L. and A. moschatus L. (Stevel, 1988; Siemonsma, 1991).

6

2.1.2 Botany

Each parent okra plant has a complete diploid set of chromosomes (Siemonsma, 1982) with

various varieties of okra exhibiting incredible dissimilarity in plant shape, fruit type, plant

size and colour. Morphological studies show that okra plant is semi woody, fibrous

herbaceous annual with growth habit described as imprecise (Nonnecke, 1989). The plant has

a far reaching taproot with numerous fibrous roots moving towards all directions in the

rhizosphere of the plant. The seeds are round, kidney shaped, dicotyledonous and with

epigeal germination (Hamon et al., 1991; Ariyo, 1993). Okra has both the male and female

reproductive part of the same flower and is self-attuned (Martin, 1983; Hamon et al., 1990).

Within 24 hours, the flowers remain opened and are mostly self-pollinating but honeybees

and humble bees may equally hasten its pollination. Pollen grains of the crop are usually very

large and so pollination (self and cross pollination) is mostly successful by insects aid

(Hamon and Koechlin, 1991; Al-Ghzawi et al., 2003). The flower fully opens (anthesis) at

dawn; throughout the morning the flower remains open and closes later in the day when the

sun is of high intensity (Mitidieri and Vencovsky, 1974). The season, insect population and

cultivar affect the level of cross pollination that takes place. The number of flowers formed is

not different in terms of whether the crop was pollinated by insects or wind (Al-Ghzawi et

al., 2003).

Okra is reproduced usually using seeds (sexual) and has 90–100 days from planting till the

plant is unable to bear fruit. It is an annual plant with its stem very robust, erect and with

variable branches with height ranging from 0.5 to 4.0 metres high (Tripathi et al., 2011).

Between 4th

and 6th

day, okra fruits, fruit length, and diameter increases tremendously but it is

at this stage that okra is normally harvested for use (Tripathi et al., 2011). Usually, okra

begins to produce much fibre starting from the 9th

day and depending upon the variety, okra

7

plants produce fruit indefinitely (Nath, 1976). To obtain a good yield, good quality fruit and

huge size of fruit, harvesting is carried out every other day (Ramu, 1976).

Varieties and ecology of an area affects the rate of cross-fertilization on okra. Long humid

and warm periods are conducive for okra cultivation and it prefers temperatures ranging

between 24°C and 28°C. Though fruiting is delayed with too much higher temperatures, the

plant grows faster with high temperatures but temperature ranging from 40°C to 42°C causes

flower abortion (Tripathi et al., 2011). Loose, well-drained, relatively light loamy soil with

pH of 6.0 to 6.8 is ideal for the cultivation of okra (Tripathi et al., 2011).

2.2 Importance of okra

The various parts such as fresh leaves, buds, flowers, succulent pods, soft stems and seeds of

okra makes it have many benefits (Mihretu et al., 2014). The immature parts of okra can

equally be utilized as salads, soup and stews, prepared in fresh, dry, boil or fried forms

(Ndunguru and Rajabu, 2004). Extracts can be obtained from okra fruits that would be used

in preparing stews, soups and sauces. Cholesterol and bile acids are mostly bound by the

mucilage obtained from okra fruits. Okra plants have several uses and can therefore be used

in preparing several dishes (Madison, 2008; Maramag, 2013). The seeds of okra are good

source of protein and oil. Many small scale manufacturers have used okra to produce oil. The

seeds of okra can equally be roasted and ground to be used as a substitute for coffee (Calisir

and Yildiz, 2005). Though okra is used by small scale manufacturers, it can equally be used

by large scale industries (Adetuyi et al., 2011). Okra is used to reduce insecurity in the food

sector and also minimize malnutrition in countries of the world. Due to the increasing growth

in human population and expanding oil sector, there is a corresponding increasing in the

demand for vegetable oils (Schalau, 2002). Due to the high oil content in okra, its cultivation

on a large scale is induced greatly to produce large quantities of oilseeds. Okra contains about

20–40% of oil (Sorapong, 2012; MEF, 2013).

8

2.2 World okra production

Okra can survive in subtropical and tropical arrears the world over (Aladele et al., 2008;

Alam and Hossain, 2008; Kumar et al., 2010; Wammanda et al., 2010).It can be cultivated

both as a garden crop and equally as a commercial crop (Rubatzky and Yamaguchi,

1997).Commercially okra is believed to be cultivated in several countries in the world such as

in India, Iran, Ethiopia, Turkey, Western Africa, Ghana, Japan, Yugoslavia, Bangladesh,

Afghanistan, Pakistan, Myanmar, Malaysia, Thailand, Brazil, Cyprus and the U.S.A.

(Qhureshi, 2007;Benjawan et al. 2007; Benchasri, 2012). In Table 1 is major okra producing

countries in the world.

Table 1: Ten major okra producing countries in the world

Producing country Production quantity (tons)

India 9,623,718

Nigeria 2,039,500

Sudan 284,000

Cote d‟Ivoire 139,187

Iraq 125,583

Pakistan 112,983

Cameroon 80,689

Ghana 66,360

Egypt 55,166

Benin 48,907

Source: FAOSTAT (2016).

2.3 Okra production in Ghana

African countries such as Sudan, Egypt, Ghana and Nigeria are noted for production of the

crop in commercial quantities (Joshi et al., 1974; FAOSTAT, 2008).Okra production, sale

and consumption are carried out in all the ten regions of Ghana. In Ghana, okra is the fourth

most popular vegetable after garden eggs, pepper and tomatoes (Sinnadurai, 1973; Lamont

Jr., 1999). Okra, hot pepper and eggplant are tolerant to high climatic conditions thereby

making the crops easy to cultivate (Sinnadurai, 1973). As an early maturing crop, it is mainly

9

cultivated for its succulent fruits. It is also grown for its‟ succulent leaves and stem (Bamire

and Oke, 2003; Aladele et al., 2008; Alam and Hossain, 2008; Kumar et al., 2010;

Wammanda et al., 2010). In Ghana, this traditional crop is largely cultivated in Brong-Ahafo,

Ashanti, Northern, Volta, Greater Accra and Central regions (NARP, 1993). The quantity of

okra produced in Ghana annually is presented in Table 2.

Table 2: Production quantities of okra in Ghana

Year Production quantity (tons)

2014 66,360

2013 63,860

2012 60,000

2011 55,000

2010 50,000

2009 47,000

2008 80,453

2007 108,000

2006 105,000

2005 104,389

2004 99,486

2003 99,224

2002 99,945

2001 101,917

2000 100,000

Source: FAOSTAT (2016).

2.3 Varieties of Okra

Plant breeders have improved genetically the characteristics of okra plants in terms of plant

height, branching, leaves, maturity, pods and fibre development (Corley, 1985; Scott et al.,

1990). Some of the hybrids of okra plants have also allowed for close spacing and increased

yields than the former varieties. Three types of okra are available generally, namely; tall

green, dwarf green and lady‟s finger. Each of the varieties is sub-divided in terms of length

and colour of the pods resulting in many more varieties i.e., tall green, long pod; dwarf green,

10

Table 3: Some okra cultivars grown in Ghana

Name of variety Cultivated regions Specific locality

Nkuruma hene Brong Ahafo Berekum

Asontem Brong Ahafo Kintampo

Nkuruma tia Brong Ahafo Berekum

Nsafitaa Brong Ahafo Jema

Debo Brong Ahafo Nsokaw

Atuogya Brong Ahafo Kintampo

Muomi Greater Accra Accra

Asontem-Gar Greater Accra Accra

Awoale Nkuruma Greater Accra Prampram

Labadi Greater Accra Accra

Legon fingers Greater Accra Accra

Spineless Greater Accra Dowenya

Volta Greater Accra Accra

Awoale Nkuruma Greater Accra Accra

Agbodro Greater Accra Dodowa

Agbodrofe Greater Accra Dodowa

Agbodroga Greater Accra Dodowa

Ngruma Greater Accra Ayikuma

New york Volta Dzodze

Fitiri Volta Agbozume

Nkuruma hwam Ashanti Kumasi

KNUST Ashanti Kumasi

Nkuruma tenten Ashanti Kumasi

Tech Nkuruma Ashanti Kumasi

Nsapan Ashanti Mankranso

Asante abe‟ Ashanti Mankranso

Bekwaso Ashanti Bekwai

Gyeabatan Ashanti Bekwai

Mamolega Upper East Bolgatanga

Wune mana Upper East Navrongo

Source: Ahiakpa et al. (2013).

11

long pod; tall green, short pod; lady‟s finger, green pod; dwarf green and short pods and

lady‟s finger, white pod (Beattie, 1905). The cultivars of okra cultivated in Ghana are shown

in the Table 3 above.

2.3.1 Tall green: The height of this type of okra plant is 10 cm to 40 cm; it grows upright

and is not able to spread its leaves but all the stems are erect; has large leaves borne out of

petioles; pods appear in the axil of the plant and on short stem, colour of the pod is green.

2.3.1.1 Tall green, long pod: Long pods of 8 cm to 13 cm as at the time it gets ready for the

market. When allowed to mature fully, it reaches 18cm to 28 cm (Fig 1).

Fig. 1: A. Okra plant and pod of tall green and B. Long pod okra

2.3.1.2 Tall green, short pod: Short pods of 4 cm to 5 cm when succulent for marketing but

can grow as long as 8 cm to 13 cm when allowed to mature fully (Beattie, 1905) (Fig. 2).

A B

12

Fig. 2: A. Okra plant and pod of tall green and B. Short pod okra

2.3.2 Dwarf green: The height of this type of okra is 51cm to 107 cm; it spreads near the

ground, it is bushy and its leaves appear rather small on slender or weak petioles; and pods

appear green in colour.

2.3.2.1 Dwarf green, long pod: Long pods of 5 cm to 10 cm when ready for harvesting to

the market. If allowed to fully mature with fibre, it can grow up to 15 cm to 25 cm. This

variety of okra is earlier and high yielding than others. In diameter, this variety can grow up

to 4 cm; its leaves are usually divided (Fig. 3).

Fig. 3: A. Okra plant and pod of dwarf green and B. Long pod okra

A B

A B

13

2.3.2.2 Dwarf green, short pod: Short pods of 4 cm to 8 cm when market ready; can grow

up to 8cm to 15cm when allowed to mature fully. Can equally grow up to 4 cm to 6 cm in

diameter and its leaves are large (Beattie, 1905) (Fig. 4)

Fig. 4: A. Okra plant and pod of dwarf green and B. Short pod okra

2.3.3 Lady’s finger: The height of this variety of okra is about 90 cm. It appears heavily

branched and bushy; leaves are large and borne on a long petiole and the lower leaves appear

to be as long as 60 cm. The plant is light in colour. The colour of the pod of this variety

differentiates it from others. Pods grow as long as 10cm to 13 cm when ready for the market;

grows to 15 cm to 25 cm when allowed to fully mature. The fruit is covered with numerous

soft hairs.

2.3.3.1 Lady’s finger, white pod: The pods of this variety appear nearly white or greenish

(Fig. 5).

A B

14

Fig. 5: A. Okra plant and pod of lady’s finger and B. White pod okra

2.3.3.2 Lady’s finger, green pod: pods from this variety appear pale green and in some few

cases some appear nearly pure green (Beattie, 1905) (Fig 6).

Fig. 6: A. Okra plant and pod of lady’s finger and B. Green pod okra

A B

A B

15

2.4 Soilborne diseases of okra

2.4.1 Southern Blight (Sclerotium rolfsii)

Southern blight, caused by the soil borne fungus Sclerotium rolfsii, is a severe disease that

attack crop plants such as; vegetables, fruits, field crops, ornamentals, and turf grasses. Crop

type, soil conditions and environmental conditions are some few factors that aid in losses of

crop yield. Favourable conditions such as; wet conditions, acidic soils and warm weather

(24–35°C) augments disease development. Sclerotium rolfsii can attack any part of the plant

especially those that touches the soil or on plant parts just close to the ground (Ferrin, 2015).

Warm and humid environment is conducive for the development of the disease on okra. The

fungus, Sclerotium rolfsii, infects the roots and lower stems thereby making the okra plant

exhibit a progressive wilt symptom. The mycelium of the fungus could be seen around the

point where the stem touches the soil surface when conditions are favourable. A large amount

of white sclerotia develops in some few days. With time these structures, sclerotia, turn

brown in colour and develops into a size similar to mustard seed. Since the fungus survives

saprophytically, it is able to survive and serve as a survival structure for this fungus over a

number of years. Movement of the soil infested with the pathogen, Sclerotium rolfsii, infests

other soils as well (Raid and Palmateer, 2006).

2.4.2 Damping off (Pythium spp., Macrophomina spp., Rhizoctonia spp.)

Damping-off disease is highly favoured by cool cloudy weather, high humidity, wet soils,

overcrowding and compacted soils (Tripathi, 2011). This disease reduces plant population as

the disease affects the emergence of seedlings after germination. Seedlings are mostly killed

by damping-off either before or just after the emergence of the okra seedlings (Raid and

Palmateer, 2006).

16

When there is infection of damping-off before emergence of seedling, poor germination

occurs due to seed decay underneath the soil. Seedlings fall on the ground and die if there is

an attack of the okra seeds before emergence of the seedling. Cool soil is a condition that

predisposes the okra plant to pre-emergence damping-off.

Factors such as the amount of pathogen and the environmental conditions affect the severity

and intensity of the disease. Tissues beneath the water-soaked lesion become soft when the

attack affects the okra seedling around the collar region. Young seedlings of okra can topple

over and die when the pathogen infect the crop at or below the soil line (Raid and Palmateer,

2006).

2.4.3 Verticillium Wilt (Verticillium albo-atrum)

Verticillium wilt is a fungal disease caused by Verticillium albo-atrum on okra. Symptom of

slight leaf yellowing is observed on lower and older leaves. Around midday when the

temperature is very high, wilting of plants increases. Before the plant finally dies, the

progression of the wilt from the lower leaves to upper leaves occurs. When the lower portion

of the stem is cut longitudinally, discolouration of the vascular bundles is observed. Soils

with high pH are conducive for the rapid growth and development of Verticillium wilt (Raid

and Palmateer, 2006).

2.4.4 Southern Root knot nematode (Meloidogyne incognita) and its damage to crops

Several factors have devastating effects on okra but the most damaging ones are pests and

diseases. Nematodes are known to be the most common animal group on earth and are un-

segmented, worm-like, in nature (Decraemer and Hunt, 2013). Many insects and plant

pathogens such as viruses, nematodes, fungi and mycoplasms heavily attack okra (Hussain et

al., 2011; Ahmad et al., 2012; Arain et al., 2012; Iqbal et al., 2012; Srivastava et al., 2012).

17

Nematodes are believed to be organisms that create infection courts for other pathogens such

as fungi, to penetrate (Storey and Evans, 1987).

The spread of root-knot nematode – Meloidogyne spp. is a limiting factor in agricultural

productivity the world over (Sasser et al., 1982; Taylor et al., 1982). Over 100 species of the

genus, Meloidogyne have been discovered (Onkendi et al., 2014) but considerable damage is

attributed to only 6 of the species under the genus (Adam et al., 2007). Root-knot nematodes

are regarded as the most economically important and diverse form of all the nematodes

(Ferraz and Brown, 2002) and are found worldwide (Karssen et al., 2013). Within 25 days

and at temperature of 27°C, nematodes complete their life cycle but when temperatures are in

the extreme low or high, nematodes are able to survive longer periods. Several biotic causes

of plant stress and crop losses exists but the single major one that is difficult to control is

Meloidogyne spp. (Bird and Kaloshian, 2003; Hussain et al., 2015).

Root-knot nematodes (Meloidogyne spp.) are regarded among the foremost five plant

pathogens and the first among the ten most significant genera of plant parasitic nematodes the

world over (Kayani et al., 2013; Mukhtar et al., 2013). There is formation of typical galls on

roots of affected plants and that this causes serious retardation in growth of vegetables. In

collaboration with other organisms such as Ralstonia solanacearum, the nematode causes

upsurge in wilt diseases of vegetables (Iqbal and Mukhtar, 2014; Iqbal etal., 2014; Aslam et

al., 2015; Shahbaz et al., 2015).

There are four commonly occurring species of nematodes out of 100 described species. These

include M. incognita (Kofoid and White) Chitwood; M. javanica (Treub) Chitwood; M.

arenaria (Neal) Chitwood; and M. hapla (Chitwood) (Skantar et al., 2008; Hunt and Handoo,

2009; Perry et al., 2009; Lunt et al., 2014). Among the four commonly occurring nematodes,

18

M. incognita (Kofoid and White) Chitwood is regarded as the most destructive and hence is

regarded as nematode of economic importance (Hussain et al., 2012; Kayani et al., 2012,

2012, 2013; Barros et al., 2014). There are about 2000 known species of crop plants that are

attacked by the southern root-knot nematodes (Meloidogyne incognita), the most widely

distributed nematode (Sikora and Fernandez, 2005). Some losses caused annually by root-

knot nematodes in the tropical zones are 22% in okra, 29% in tomato, 24% in potato, 23% in

eggplant, 28% in beans and 25% in pepper (Sasser, 1979).

The nematode enters the host root using the stylet and enzymes that soften the tissues of the

plant (Wieczorek et al., 2014). It moves to adjacent cell within the cell wall towards the end

of the root (Goto et al., 2013) where it looks for cells to establish and develop (Bartlem et al.,

2014). The female nematode begins laying eggs after the male has left the tissue of the host.

Root-knot nematodes cause severe retardation in growth of crop plants especially vegetables

due to the formation of distinctive galls. The southern root-knot nematode, Meloidogyne

incognita, infects a wide variety of crops the world over (Khan et al., 2005; Anwar et al.,

2007; Hussain et al., 2012, 2016; Kayani et al., 2013; Ntidi et al., 2016; Shigueoka et al.,

2016).

Vegetables are the most susceptible and commonly attacked crops by nematodes (Osman et

al., 2012; Youssef et al., 2012; Naz et al., 2016: Podestá et al., 2016; Zhou et al., 2016).

Meloidogyne spp. that causes root-knot disease on okra reduces yield drastically and could

reach up to 27% loss (Sikora and Fernandez, 2005). Root-knot infections have been reported

to cause a surge in incidence and severity of wilt disease that are caused by bacteria and fungi

(Shahbaz et al., 2015; Tariq-Khan et al., 2016).

19

Due to the build-up of substantial amount of inocula of root-knot nematodes (M. spp.), there

is significant increase in yield losses (Kayani et al., 2013). Growing similar okra varieties

increases the amount of inoculum in the soil when it is cultivated year after year without

rotation (Hussain et al., 2011, 2014). Photosynthesis is seriously affected when nematodes

are allowed to feed around root tissues thereby reducing the ability of plant roots to absorb

water for growth. The inability of the root tissues to absorb water for photosynthesis create

room for soil-borne fungi such as Fusarium oxysporum to invade the plants through the

incisions created by the nematode (Anwar and Van Gundy, 1993; Stirling et al., 2004). The

initial population density of M. incognita and yield loss relationship was rigorously studied

on soya bean (Fourie et al., 2010), tomato (Jaiteh and Akromah, 2012) and cotton (Davis and

May, 2005) and was concluded that the higher the initial population of nematodes, the higher

the loss in yield. Okra plants develop chlorosis, stunting and unthriftness anytime they are

heavily infested with root-knot nematodes (Archana and Saxena, 2012). But crop damage and

yield losses may not necessarily occur just because of the presence of plant parasitic

nematode since for a specific field, damage threshold level of the nematode population may

be so inadequate to cause significant damage (Schomaker and Been, 2006; Khan, 2008).

Yield is reduced severely in susceptible varieties than resistant crops when female nematodes

reproduce at a faster rate. This may even occur when the two varieties of moringa are

subjected to the nematode infection at the same time (Anwar et al., 2007). Vegetable crops

are most susceptible hosts for nematode attack though other plants are equally preferred by

nematodes (Sasser, 1980). The yield of okra, tomato, and brinjal suffered 90.9, 46.2 and 2.3%

losses, respectively, due to Meloidogyne incognita infestation at the rate of 3–4 larvae/g soil

under field conditions (Bhati and Jain, 1977).

20

2.4.4.1 Identification of southern root-knot nematode

Nematologists face major hindrance in diagnosing the attack on crops from Meloidogyne spp.

since there are various species of Meloidogyne with similar morphological characteristics. In

the past, plant pathology laboratories (specializing in nematology) were able to identify

nematodes using certain features; major amongst them is the use of a perennial pattern

identified on the female nematode. In recent times however, nematologists are able to

develop new tools for identifying species of organisms. These were even achieved in short

times, involving less financing but with high level of accuracy (Oliveira et al., 2011).

Meloidogyne spp. are regarded as group of obligate plant pathogens which are also endo-

pathogenic in nature. The females of the species are sedentary and stay within root tissues,

develop and lay eggs inside gelatinous sacs (Karssen et al., 2013).

2.4.5 Fusarium wilt disease (Fusarium oxysporum f. sp. vasinfectum) and its damage to

crops

Fusarium represents one of the most important genus of the Ascomycetes. This genus is

responsible for massive economic losses to crop plants due to decreases in harvest yields and

the quality of foods (Leslie and Summerell, 2006). Eighty percent of crops are infected with

at least one disease initiated by a Fusarium species (Leslie and Summerell, 2006).

In most horticultural crops, fungi cause major crop diseases and massive yield loss of about

70% which presupposes that fungi are of grave concern in crop production (Agrios, 2005).

Nematode presence on plants predisposes or breaks down the resistance of the plants to the

infection of Fusarium which finds its way into plants when there is an incision (France and

Abawi, 1994). Genetic resistance of plants is broken when nematode populations are high in

the rhizosphere thereby causing soil-pathogenic fungi to infect the crop. The presence of both

pathogens, Meloidogyne incognita and Fusarium oxysporum, concomitantly reduces the

resistance of plants to diseases.

21

When the two pathogens appear to attack plants concomitantly, the severity of the damage is

greater than when the organisms attack separately (Jonathan and Gajendran, 1998; Jeffers and

Roberts, 2003). In all soil types the world over, Fusarium oxysporum is well represented as a

soil borne fungus (Burgess, 1981). The rhizosphere of plants is populated with this species of

fungus (Gordon & Martyn, 1997). Garrett (1970) reported that this fungus is able to survive

in the rhizosphere of plants for longer periods due to its saprophytic nature.

Fusarium, members of the genus are saprophytic and are distributed widely throughout the

tropical and subtropical zones the world over (Burgess, 1981). Fusarium wilt, caused by the

fungus Fusarium oxysporum f. sp. vasinfectum (Atk) Snyder and Hansen, is a prevalent and

prominent disease on Malvaceae species. The pathogen, Fusarium oxysporum f.

sp.vasinfectum, causes a dangerous disease called vascular wilt on cotton and okra (Cia and

Salgado, 1997). Fusarium wilt is known to be responsible for significant yield losses in many

areas where okra crop is cultivated (Silva et al., 2007).

2.4.5.1 Identification of Fusarium oxysporum f. sp. vasinfectum

Fusarium oxysporum f. sp. vasinfectum (FOV) is a wide range wilt causing pathogen that

attacks several species of crops from the genus Gossypium as well as species of Malvaceae,

Leguminosae and Solanaceae. Different wilt causing agents are regularly found together in

the same crop field and it appears that groups of Fusarium spp. are difficult to distinguish

morphologically from FOV. The similarities in symptoms associated with the species of

Fusarium makes it difficult to identify FOV which is regarded by pathologists as the most

serious wilt agent. Symptoms of FOV infection do not appear until a considerable time after

the infection has establishes itself especially in moderately resistant plants (Holliday, 1980).

To be able to identify FOV, isolations are done and the culture observed under the

22

microscope and morphological characteristics based on descriptions made by Barnett and

Hunter (2006), Leslie and Summerell (2006) and other workers.

2.6 Fungus–nematode interactions

There are numerous interrelationships between pathogens where one, or both species, support

or surge the deterioration caused by the other pathogen (Powell, 1971; Bergeson, 1972; Mai

and Abawi, 1987; Taylor, 1990; Evans and Haydock, 1993; Back et al., 2002).Further, it is

essential to comprehend and appreciate the relevance of each such interrelationship between

pathogens so as to develop an appropriate management practice for the control of the

pathogen.

Fusarium oxysporum f. sp. vasinfectum and Meloidogyne incognita was reported in the

rhizosphere for the first time on cotton plants that were severely affected by Fusarium wilt

(Atkinson, 1892). In recent years, the nematode–fungus complexes have been receiving

rigorous attention from various pathologists (Sasser, 1989; Khan and Reddy, 1993).

Diseases caused by Fusarium spp. and plant parasitic nematodes have certain interaction on

cultivated crops and much attention was on interrelationships in the rhizosphere of host

plants. Fusarium wilt intensity in the presence of nematodes or to the derailment of the

barrier put up by the nematode causes more damage to the crops (Powell, 1971).

Meloidogyne incognita, M. arenaria and M. javanica were confirmed to be the cause in

severity of root-rot fungi on beans crop (Al-Hazmi and Al-Nadary, 2015).

A minimum level of nematode infection predisposes the host plant to plant pathogenic fungi

invasion. Low population density of either Fusarium oxysporum f. sp. vasinfectum or

23

Meloidogyne incognita combined with high population density of either of the two results in

high incidence of wilt symptoms on cotton (Garber et al., 1979).

The simultaneous application of the fungus, Fusarium oxysporum f. sp. pisi and the root-knot

nematode, Meloidogyne incognita caused death of plants just after 45 days (Padilla et al.,

1980).The resistance of tomato to Fusarium wilt decreased when root-knot nematodes attack

the crop (Young, 1939). Single gene resistance (I gene) of tomato to the fungus F. oxysporum

f. sp. lycopersici was broken by the infection of the plant with root-knot nematode. Fusarium

infection is rigorously enhanced when there is the presence of nematode in the rhizosphere of

the plant (France and Abawi, 1994).

Concomitant inoculation of the two pathogens or inoculation of the nematode, Meloidogyne

incognita, 10 days prior to fungus inoculation enormously affected the growth in plant height

and shoot weight with equally high incidence of wilt attack (Singh et al., 1981). There was

minimal incidence of the wilt disease when soil was inoculated with fungus only on French

bean.Sharma and Cerauskas (1985) reported that inoculation of M. javanica and F.

oxysporum f. sp. ciceri in chickpea concomitantly, reduces shoot and root weights than when

either of the pathogen is inoculated alone.

In the presence of root-knot nematode, Rhizoctonia-root roton green beans was more severe

including the disease, root rot disease complex, caused by R. solani and M. incognita.(Batten

and Powell, 1971; Reddy et al., 1979; Sharma and Gill, 1979; Chahal and Chhabra, 1984; Ali

and Venugopal, 1992; France and Abawi, 1994; Shahzad and Ghaffar, 1995; Anwar and

Khan, 2002; Bhagwati et al., 2007; Mokbel et al., 2007; Abuzar, 2013).

24

A study on the interaction between M. incognita and Machophomina phaseolina on Coleus

forskohlii was done in control environment (glass house) by Senthamarai et al., (2006). When

inoculation of fungus was done prior to nematode inoculation, multiplication of the nematode

was slow. The study also showed that when there is concomitant application of the two

pathogens (fungus and nematode) there was 100% root-knot disease. Within 15 days, when

fungus is inoculated followed by nematode, there is severe reduction in the growth of the

plant as compared to if the nematode is inoculated first before the fungus or inoculation of

fungus alone (Senthamarai et al., 2006)..

The combined and individual effect of M. incognita and F. oxysporum f. sp. pisi infection

was identified as the cause of great reduction in growth parameters of pea crop as measured

against un-inoculated plants (Haseeb et al., 2007). There was a significant reduction in the

growth of plant when nematode was inoculated before inoculating the fungus and also when

the two pathogens were inoculated concomitantly. Pre-inoculation of the field with fungus

prior to the nematode decreases the reproduction and galling on the roots of the plant.

Similarly, the presence of nematodes increases the infection rate of fungus.

The inter-relationship between F. oxysporum f. sp. lycopersici and southern root knot

nematode (M. incognita) on tomato under controlled conditions in the green house has been

investigated (Samuthiravalli and Sivakumar, 2008). When the plant was with the fungus

inoculated alone little harm was caused compared to the simultaneous inoculation of the two

pathogens. There was synergy between the two pathogens on the level of harm caused to the

plant. When the two pathogens were inoculated at the same time, there was a reduction in

plant height.

25

Ganaie and Khan (2011) reported that isolates of M. incognita and F. solani solely caused a

severe reduction in the height of plants, fresh weight, dry weight, number of fruits and fruits

weight as compared to un-inoculated plots which served as control. But the nematode, M.

incognita caused more reduction in the growth parameters as compared to the fungus, F.

solani. When tomato plants were inoculated first with M. incognita before F. solani (N+F),

serious damages were recorded. When the inoculation was done concomitantly and

sequentially, the pathogens‟ multiplication, number of galls and number of females were

adversely affected in all the treatments applied to the crops.

2.7 Diseases complexes and types of interactions (Synergistic, Antagonistic and

Neutral interactions).

For a disease to develop on farms, conditions such as host plant, virulent pathogen and

favourable environment condition are required. The important role that nematodes play in the

development and spread of disease that are caused by soilborne organisms has been reported

worldwide. Atkinson (1892) reported that the presence of M. incognita along with F.

oxysporum in the rhizosphere of cotton plants increases the severity of wilt disease caused by

plant pathogenic fungi. Increased attention has been given to the nematode-fungus complexes

in recent times. Several workers have developed the interest in studying the interaction or

relationship between the two important plant pathogens (Sasser, 1989b; Khan and Reddy,

1993). Such nematode–fungus disease complexes in many instances involve root-knot

nematodes (Meloidogyne spp.), though numerous other endoparasitic and ectoparasitic

nematodes have played a major role in causing diseases related to those caused by soilborne

organisms. Previously, nematode-fungus complexes have be studied by many scientists

(Powell, 1971; Bergeson, 1972; Evans and Haydock, 1993 and Back et al., 2002), but this

review looks at the mechanism underpinning synergistic, antagonistic, symbiotic and/or

neutral interactions between two or more pathogens.

26

Interactions between two or more organisms can either be synergistic, antagonism or neutral

(Khan, 2008). For the interaction between these organisms to be successful there should be

vectors of fungal pathogen, mechanical wound agents, host modifiers, rhizosphere modifiers

and resistance breakers (Ravichandra, 2014).

2.7.1 Synergistic interaction

Plant Parasitic Nematodes–pathogen complexes show how soilborne microbes affect the

development of plant diseases. Root-rot disease increases when M. arenaria and root-rot

disease concomitantly appear on a host plant. Several host plants that are attacked by root-

knot nematodes increase the severity of root-rot disease (Anwar and Khan, 2006; Poornima

and Subramanian, 2006; Bhagawati et al., 2007; Mokbel et al., 2007). Synergistic interaction

gives rise to disease complexes (Back et al., 2002). An interaction is synergistic if the

association between two organisms results in plant damage greater than the sum of individual

damage (1 + 1 > 2). There is positive synergy where there is an association between two

pathogens and the resultant damage to plant exceeds the sum of individual damage by the two

organisms (1 + 1 > 2). The timing of the inoculation of fungus and nematode seems to play a

major role in the interaction of the two pathogens (Back et al., 2006; Bhattarai et al., 2009).

Young plants were seen to be more susceptible to disease complexes than older plants

(Polychronopoulos et al., 1969; Bhattarai et al., 2010).

There can be reduction in the resistance of the host but it does not necessarily mean it is from

the interaction between the nematode and fungus. It is scientifically proven that where there

is synergistic interaction between Heterodera schachtii and Rhizoctonia solani, the host

plants‟ resistance was not broken (Hillnhütter et al., 2011). Development of disease

27

complexes can also be tremendously affected by some abiotic factors such as temperature,

soil type and meteorological conditions (Back et al., 2002). There are variances in the

synergistic interaction of Meloidogyne javanica and R. solani on soybean (Agu, 2002).

2.7.2 Antagonistic interaction/ Indirect effect:

Both nematodes and fungi have certain benefits during their interaction (Back et al., 2002;

Evans and Haydock, 1993). When a fungus and a nematode are found on the same host, one

of the organisms indirectly affects the other. Some of the possible indirect effects on the

organisms are root space competition, metabolite production to nematode from fungi,

nematode preying on fungus and/or fungus invading nematode (Al-Hazmi and Al-Nadary,

2015).

Antagonistic interactions result where an association between nematode and fungus leads to

plant damage less than that expected from the sum of the individual organisms, (1 + 1 < 2).

Major experiments carried out in nematological research considered the ability of the fungus,

Fusarium spp. to emit toxic compounds that have tremendous effect on the behaviour and life

stages of saprophytic and plant parasitic nematodes (Krizkova et al., 1979; Mani and Sethi,

1984). Several species and strains of Fusarium spp. show the ability and capacity of yielding

mycotoxins (Marasas et al., 1984) that play major role in interfering with the life stages and

normal behavior of the nematodes. Plant-microbe interactions is beneficial to plants,

including suppression of diseases, production of more nutrients and their absorption and

improved resistance to abiotic and biotic pressures which leads to high plant productivity

(Lugtenberg et al., 2002; Weller et al., 2002; Morrissey et al., 2004; Haas and Défago 2005;

Berg, 2009; Mendes et al., 2011; Selvakumar et al., 2012; Zamioudis and Pieterse 2012;

Badri et al., 2013; Zolla et al., 2013).

28

2.7.3 Neutral interaction:

Neutral interaction exists between two pathogens (nematode and fungus) when their

interaction can cause plant damage that adds up to the sum of individual damage by the

pathogens (1 + 1 = 2). Although synergistic and antagonistic interactions can be

demonstrated experimentally, neutral interaction can be difficult to observe. This is because

neutral associations can result in similar plant damage to that seen in additive associations,

where nematode and fungus are known not to interact with one another (Back et al., 2002).

29

CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1 Study Site

The experiment was carried out in the University of Ghana farm, Legon. The experimental

site had a mean rainfall of 55 mm a mean temperature of 32.3°C and relative humidity of

78% from September, 2016 to January, 2017 when the pot experiment was conducted (Table

4). The mean rainfall, temperature of and relative humidity from March, to June, 2017 were

56 mm, 32.5 °C and 69% respectively for the field experiment (Table 4).

Table 4: Climatic data of experimental area

Month Rainfall

(mm)

Relative Humidity (%) Temperature (°C)

At 06:00 h

GMT

At 15:00 h

GMT

Minimum Maximum

Oct., 2016 118.2 96 81 31.1 24.0

Nov., 2016 36.4 96 78 32.8 24.9

Dec., 2016 25.0 95 74 32.6 24.3

Jan., 2017 40.6 93 63 32.7 24.6

Feb., 2017 73.5 95 70 32.9 25.1

Mar., 2017 23.5 95 68 33.6 25.5

Apr., 2017 64.3 94 68 32.1 25.2

May, 2017 80.5 95 70 32.6 25.1

June, 2017 55.7 95 68 31.6 25.5

Source: Ghana Meteorological Service Agency, Mempeasem, Legon, 2017.

The soils at the site are classified under the Adentan series and are relatively light clayey soils

with low fertility. Isolation of the Fusarium oxysporum f. sp. vasinfectum and extraction of

the Meloidogyne incognita was carried out at the Plant Pathology Laboratory, Department of

Crop Science, University of Ghana, Legon.

30

3.2 Confirmation of pathogens for root knot disease and wilt of okra as M. incognita

and F. oxysporum f. sp. vasinfectum respectively.

3.2.1 Preparation of water agar (WA)

Three grams of dehydrated agar (Oxoid Ltd., Basingstoke, Hampshire, England) was

weighed into 100 ml distilled water in a 250 ml conical flask. The conical flask was then

plugged with nonabsorbent cotton and covered with aluminium foil to prevent wetting of

cotton wool during condensation. The mixture was autoclaved at 121°C for 15 minutes after

which the cotton wool plug was removed; the neck of the conical flask was flamed. About 10

ml water agar was poured into sterilized 9 cm petri dishes and allowed to solidify.

3.2.2 Preparation of potato dextrose agar (PDA)

Potato Dextrose Agar (Oxoid Ltd., Basingstoke, Hampshire, England) was prepared by

dissolving 3.9 g of the agar powder in 100 ml of distilled water in a 250 ml conical flask. The

conical flask was plugged with cotton wool and covered with aluminium foil. The content

was shaken vigorously to obtain a thoroughly mixed substance. The mixture was autoclaved

at 121°C for 15 minutes. The PDA was then poured in 9 cm Petri dishes and allowed to cool.

3.2.3 Isolation of Fusarium oxysporum f. sp. vasinfectum

Suspected diseased okra plants with symptoms of Fusarium wilt were collected from atomic

site, Accraand brought to the Plant Pathology Laboratory. The isolation of the pathogen was

done on Water Agar (WA) (20 g L−1

; Oxoid, Basingstoke, UK). Pieces of the infected tissues

were first surface sterilized with 1 % sodium hypochlorite (NaClO) for about 60 s. It is

blotted dry and plated singly on WA. The plates were incubated for 7 days, after which the

fungus that grew was further sub-cultured on Potato Dextrose Agar (PDA) (39 g L−1

; Oxoid,

Basingstoke, UK) to obtain a pure culture. The culture produced macroconidia and

31

microconidia after eight days and twelve days of incubation, chlamydospores were produced.

Slides of isolated fungus were prepared and its morphological features studied under

compound microscope to confirm its identity.

3.2.4 Identification of Fusarium oxysporum f. sp. vasinfectum

The colony and spore characteristics were studied using twelve days old the pure culture of

the fungus. Identification of the fungus was based on growth rate and colour of culture and

morphology of mycelia, conidia and sporulating structures as described by Barnett and

Hunter (2006) and Leslie and Summerell (2006).

3.2.5 Preparation of inoculum of Fusarium oxysporum f. sp. vasinfectum

The fungus was inoculated into the potato dextrose agar (PDA) aseptically under a laminar

flow and incubated at room temperature (26ºC) for two weeks. The two week old culture was

scraped into a beaker with sterile distilled water and blended. Sterile distilled water was

added to obtain 1000 cm3

of the suspension. The fungal spore suspension (approximately 1.1

x 105 cells per 5 ml) was calculated using haemocytometer (Booth, 1971).

3.2.6 Inoculation of okra seedlings with F. oxysporum

Four weeks old seedlings (10 seedlings for pot experiment and 30 seedlings for field

experiment) of „Essoumtem‟ and Clemson Spineless were inoculated with 50 ml (1.1 x 106

cells) of Fusarium oxysporum f. sp. vasinfectum inoculum suspensions. Rhizosphere of the

okra seedlings was dug to expose the roots and the required quantity (1.1 x 106

cells) of the

inoculum was poured around the exposed roots and covered.

3.2.7 Re-isolation of the organism

Five plants per plot infected (showing the symptoms of Fusarium wilt) after sixty days

following the final inoculation were sent to the Plant Pathology laboratory, Department of

32

Crop Science, University of Ghana, Legon and the suspected causal organism was re-isolated

on water agar and later sub-cultured on PDA and observed under a compound microscope.

3.2.8 Extraction Meloidogyne incognita from diseased okra plants

Five plants per plot infected with root-knot disease were obtained from a field, placed into

polythene bags in an ice chest with ice and transported to the Plant Pathology laboratory and

kept at 4˚C. The roots were washed gently under running tap, cut into approximately 1–2 cm

pieces and vigorously shaken in a bottle containing 0.5% NaClO for 5 minutes. Eggs from

the roots were collected on a 38 µm sieve and washed in a beaker (Hussey and Barker, 1973).

3.2.9 Identification of M. incognita

A compound microscope was used to determine the Meloidogyne incognita juveniles in the

aliquot suspension. Two milliliters of the nematode suspension was pipetted after bubbling

air through the suspension for homogeneity and dispensed into a counting dish for nematode

identification. Nematode identification was done with the aid of pictorial key of Mai and

Lyon (1975).

3.2.10 Preparation of inoculum of Meloidogyne incognita

The eggs were placed on a 1% water agar for 10 days to hatch into second-stage juvenile (J2).

The nematode suspension was poured into a measuring cylinder to estimate inoculum

density. The numbers of juveniles were estimated in ten aliquots of 1 mL in a counting dish

under a dissecting microscope at a magnification of X 40 and their mean calculated. The total

number of juveniles was extrapolated based on the total volume of the suspension. To

concentrate the juveniles‟ suspension, it was left to settle down for several hours and the extra

water was decanted leaving the bottom undisturbed.

http://scialert.net/fulltext/?doi=ajbs.2015.42.50&org=12#129254_b

http://scialert.net/fulltext/?doi=ajbs.2015.42.50&org=12#129254_b

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3.2.11 Inoculation of okra seedlings with inoculum

Four weeks old seedling of the two varieties of okra were inoculated with 1000 second

juvenile (J2) of Meloidogyne incognita. Just before inoculations, feeder root of seedlings were

exposed by carefully removing the top layer of soil and a required quantity of nematode

suspension was poured uniformly around the exposed roots using a sterilized pipette.

Exposed roots were covered immediately by leveling the soil to avoid drying of the

nematodes introduced. Watering was done as and when necessary.

3.2.12 Re-extraction of the organism

Five plants per plot infected (showing the symptoms of root knot) at sixty days after final

inoculation were sent to the Plant Pathology laboratory, Department of Crop Science,

University of Ghana, Legon and the suspected causal organism was re-extracted. The roots

were washed gently under running tap, cut into approximately 1–2 cm pieces and vigorously

shaken in a bottle containing 0.5% NaClO for 5 minutes. Eggs from the roots were collected

on a 38 µm sieve and washed in a beaker (Hussey and Barker, 1973).

3.3 Evaluation of individual effect and combined interactions of M. incognita and F.

oxysporum f. sp. vasinfectum on the growth and yield of okra

3.3.1 Pot experiment

Two (2) varieties of okra, 'Essoumtem' and 'Clemson spineless', were used in this experiment.

The soil (light loamy soil) for the pot experiments was sterilized at 105˚C for three days and

left overnight to cool. It was then used to fill plastic pots (5 kg of the sterilized soil per pot)

with drainage holes at the base (Fig. 7).

34

Fig. 7: Pots with seedlings laid for the pot experiment

The treatment used in the pot experiment has been explained (Table 5). The treatments used

in the experiment were seven.

Table 5: Description of treatments for pot experiment.

Treatment Description

F Okra plants inoculated with Fusarium oxysporum only.

N Okra plants inoculated with Meloidogyne incognita only.

NF Meloidogyne incognita and Fusarium oxysporum inoculated

simultaneously on okra.

NF7 Fusarium oxysporum inoculated 7 days after Meloidogyne incognita on

okra.

NF14 Fusarium oxysporum inoculated 14 days after Meloidogyne incognita

on okra.

NF21 Fusarium oxysporum inoculated 21 days after Meloidogyne incognita

on okra.

C Okra plants with neither Meloidogyne incognita nor Fusarium

oxysporum inoculation (control).

The conidial suspension of the fungal pathogen, approximately 1.1 x 106 cells of F.

oxysporum f. sp. vasinfectum in 50 ml water were applied to F and NF (Table 5) only in three

35

(3) the replications. The spore suspension was thoroughly mixed with the soil of each pot. At

the same time, the suspension of 1000 M. incognita eggs in 5 ml water was also mixed

thoroughly with only the NF (Table 3.2) and the other treatments such as NF7, NF14 and

NF21 (Table 3.2). The fungal spore suspension was later added to the NF7, NF14 and NF21

(Table 3.2) in 7 days, 14 days and 21 days respectively after the nematode inoculation.

Control treatments included those pots with neither the fungus nor the nematode pathogen

application.

The experiment has a 2 x 7 factorial laid out in a Completely Randomized Design (CRD)

with three replications. An experimental plot measured 2 m by 1.2 m (2.4 m2). Alleys

between the blocks were 1 m while that between the plots was 50 cm. A total of 42 plots were

used and each plot had 15 okra plants with planting distance of 60 cm x 70 cm. five plants

were tagged as record plants. Each block or replication is made up of 14 plots.

Three seeds were planted and thinned to one plant per hill. Watering was done daily and

weeds were controlled fortnightly by hand picking prior to canopy closure. NPK (15-15-15)

was applied three weeks after planting (3 g/plant) to replenish the lost nutrients.

3.3.2 Field experiment

The field experiment was conducted on the University farm, Legon from March, 2017 to

June, 2017. The land was ploughed and harrowed to obtain a fine tilth and plots demarcated.

Soil samples were collected from each plot for nematode estimations before okra seeds were

planted. The experiment had a 2 x 4 factorial treatment combinations arranged in

Randomized Complete Block Design (RCBD) with three replications. Each experimental plot

measured 2.8 m by 2.5 m (7 m2). Alleys between the blocks were 1 m while that between the

plots of the same replication was 50 cm. A total of 24 plots were used and each plot consisted

36

of 30 plants spaced in 50 cm x 70 cm apart. Five plants were used within the plot as record

plants. Each block or replication is made up of 8 plots.

The two varieties of okra, „Essoumtem‟ and Clemson Spineless, were obtained from

Agriseed, Accra. At planting, three seeds were sown and thinned to one plant per hill.

Watering was done daily and intermittently, weed control was done by hoeing and

handpicking fortnightly. Insecticide (Taurus) was also applied at a rate of 5 ml/15 L

Knapsack sprayer to control termites that attacked the pots.

Description of treatments used on the field experiment is laid out in Table 6 below. The field

on which the experiment was done had already infected light loamy soil.

Table 6: Description of treatments for field experiment.

Treatment Description

F7 Fusarium oxysporum inoculated 7 days after okra seedling

emergence in Meloidogyne incognita infested field.





C Un-inoculated field – Control.

The conidial suspension of the fungal pathogen, approximately 1.1 x 106

cells of F.

oxysporum f. sp. vasinfectum in 50 ml water were applied to F7, F14 and F21 in 7 days, 14

days and 21 days respectively. Control treatments included those plots with only nematodes.

3.4 Data taken for pot and field experiments

The plant growth parameters taken were fresh and dry shoot and root weights, plant height,

plant girth and chlorophyll content. Yield parameters taken were number of fruits per plant

37

and weight of fruits per plant). Nematode Reproductive Index (initial nematode population in

soil/ final nematode population in soil), number of nematode eggs/g root, and gall (root knot)

index were also determined using a scale developed by Bridge and Page, (1980). Also, the

incidence of wilt caused by Fusarium oxysporum f. sp. vasinfectum was determined using a

scale developed by Nene et al., (1981).

3.4.1 Plant height (cm)

Plant height was taken four weeks after planting with two weeks intervals for four times and

repeated fortnightly until the last harvest. The heights of the plants were taken from the soil

level to the highest tip of the plant with a meter rule.

3.4.2 Stem girth (cm)

The girth of the plants after the first leaf was measured four weeks after planting with two

weeks intervals for four times using a string and meter rule.

3.4.3 Chlorophyll content (CCI)

The chlorophyll content of the okra plants at four weeks after planting were taken with a

SPAD meter at two weeks intervals for four times. The forceps sensor was clamped to the

leaf and the chlorophyll content taken on three leaves per plant and averaged.

3.4.4 Fresh shoot weight (g)

Selected plants were carefully uprooted after harvest and the upper portion (shoot) of the okra

plant was chopped up and weighed with an electronic balance and recorded.

3.4.5 Dry shoot weight (g)

This was determined by chopping up the upper portion of the plant above the soil level and

drying in an electronic oven at 70 °C for 96 hours and weighed with an electronic balance

and recorded.

38

3.4.6 Fresh root weight (g)

The plant was carefully uprooted and the lower portion (root) of the record plant of okra was

chopped up and weighed on electronic balance and recorded.

3.4.7 Dry root weight (g)

The root of the record plants was dried in an electronic oven at 80 °C for 72 hours when

constant weight was obtained. This was recorded as dry root weight.

3.4.8 Determining okra fruit yield:

The plants of each plot were harvested at edible maturity stage and weighed for pod yields

per plot which was converted into pod yield per hectare (kg/ha) using the formula:

Pod yield (kg/ha) = Pod weight (kg) x 10000 m2

Harvested area (m2)

3.4.9 Nematode Reproductive Index

The nematodes extracted from the soil at the beginning of the study represent the initial

nematode population (Pi). The nematodes extracted from the soil at the end of the study

formed the final nematode population (Pf). The reproduction factor (Rf) was calculated as

follows:

Reproductive factor = Final nematode population

Initial nematode population

3.4.10 Number of nematode eggs/g root

Root systems of the record plants were weighed and placed in a closeable container. 10%

Clorox solution (50 ml Clorox and 450 mL water) was poured on the root system to cover it.

The container was shaken continually for 5 minutes and the solution was poured through a 71

µm-mesh sieve nested on a 36 µm--mesh sieve. The eggs were rinsed under tap water to

reduce the concentration of the Clorox. The 36 µm -mesh sieve were finally collected into 50

ml falcon tubes. Counting of the eggs was done in 1 ml of a well-mixed sample multiply by

100 to get the number of eggs per root system.

39

3.4.11 Root Knot (Gall) Index

Roots from record plants were uprooted and washed under tap. The specimen is blot dried

and the level of root galling scored using root knot nematode rating chart by Bridge and Page,

(1980) (Appendix 1).

3.4.12 Percentage wilt incidence

A rating scale for wilt incidence was utilized for the determination of Fusarium disease

incidence (Nene, et al., 1981) (Table 7).

Percentage (%) wilt = Number of plants wilted× 100

Total number of plants

3.5 DATA ANALYSIS

Data collected for both pot and field experiments were subjected to analysis of variance

(ANOVA) using GenStat (12th

) edition and where there was significant difference, the least

significant difference (LSD p ≤ 0.05) was used for mean separation.

Table 7: Rating scale for wilt incidence

Scale Rating scale

No wilt 1

10% or less wilted 3

11 – 20% wilted 5

21 – 50% wilted 7

51% and more wilted 9

Source: Nene et al., 1981

40

CHAPTER FOUR

4.0 RESULTS

4.1 Causal organisms of root knot and wilt disease of okra

4.1.1 Root knot disease of okra

The nematode extracted from the soil and roots appeared pear-shaped (b) with long and

projecting neck (c), a long and robust stylet (e) and a prominent rounded stylet knobs (d)

(Fig. 9 B). The tail (hyaline) which is bluntly round is terminus (a) and largely un-striated

(Fig. 9 A). It also showed a slightly curved spicules and a crescentic gubernaculum. The egg

of M. incognita is elongated, ellipsoid and stunted in shape (Fig. 9 C). The eggs were laid by

the female in an enclosed gelatinous sac. Based on the characteristics observed, the nematode

species used in the study was identified as Meloidogyne incognita.

Fig. 9: Micrograph of female southern root-knot nematode, Meloidogyne incognita (A) and

nematode eggs (B). X 400 magnification for all the three figures. a – tail, b – ovaries, c –

neck, d – stylet knobs, e – stylet and f – eggs.

4.1.2 Wilt of okra

After eight days of incubation on PDA, macroconidia and microconidia (Fig. 10 B and C)

were observed. Twelve days later, chlamydospores were also observed. The pathogen

A C B a

b

c

d

e

f

41

produced concentric, cottony, fluffy pinkish pigment in the medium (Fig. 10 A). As the

culture aged small, single and bi-celled conidia were observed under the microscope. The

hyaline and multicelled macroconidia were sickled-shaped and 3-septated. Based on the

cultural and morphological characteristics of the pathogen, it was identified as Fusarium

oxysporum Booth (1971).

Fig. 10: Micrograph of a twelve-day old culture of Fusarium oxysporum f. sp.

vasinfectum on PDA (A) with Macroconidia (B), and Microconidia (C) (X 400)

A B C

D

42

4.2 Individual, simultaneous and sequential interactions between Meloidogyne

incognita and Fusarium oxysporum f. sp. vasinfectum (FOV) on the growth and yield of

okra

4.2.1 Pot experiment

Plant height

There was a steady increase in plant height on Essoumtem okra plants up to 8 WAP.

Inoculation of the treatments, NF14 and NF21, resulted in the least plant height (43.0 cm).

The highest plant height (54.0 cm) was observed in control plants (Fig. 11).

Plant height increased steadily from week 4, 6, and 8, afterward growth was retarded until to

week 10, this was however not observed in the un-inoculated Clemson spineless plants (62

cm) which had the highest plant height. Inoculation of the treatment, NF21, resulted in the

least plant height (44 cm) followed by simultaneous inoculation of the fungus and nematode

(47 cm) on plants (Fig. 11).

Fig. 11: Height of okra plants inoculated individually and simultaneously with Meloidogyne

incognita and Fusarium oxysporum f. sp. vasinfectum in pot experiment (n = 3). F = Fungus

alone; N = Nematode alone; NF = Nematode and Fungus simultaneously; NF7 = Fungus 7

days after nematode inoculation; NF14 = Fungus 14 days after nematode inoculation; NF21 =

Fungus 21 days after nematode inoculation and C = Control (un-inoculated); A – Essoumtem,

B – Clemson spineless.

0

10

20

30

40

50

60

0 4 6 8 10

He

igh

t (c

m)

Duration (weeks)

A

F

N

NF

NF7

NF14

NF21

C 0

10

20

30

40

50

60

70

0 4 6 8 10

He

igh

t (c

m)

Duration (weeks)

B

F

N

NF

NF7

NF14

NF21

C

43

Plant girth

There were significant differences (p ≤ 0.05) in plant girth among the treatments for week 10

in the Essoumtem okra plants. Nematode (M. incognita) and fungus (FOV) decreased plant

girth except in un-inoculated plants. Plants that received NF14 has the least plant girth

(7.7 cm) and the highest plant girth (11.3 cm) was observed in the un-inoculated plants

(Fig. 12).

There was significant increase in plant girth on Clemson spineless okra plants in all the

treatments from week 4, 6 and 8. These increases reduced towards the 10th

week except in

plants that were not inoculated (11.9 cm). The least plant girth (6.8 cm) was observed on

plants inoculated with fungus 21 days after nematode inoculation (Fig. 12).

Fig. 12: Girth of okra plants inoculated individually and simultaneously with Meloidogyne

incognita and Fusarium oxysporum f. sp. vasinfectum in pot experiment (n = 3). F = Fungus

alone; N = Nematode alone; NF = Nematode and Fungus simultaneously; NF7 = Fungus 7


Fungus 21 days after nematode inoculation and C = Control (un-inoculated); A – Essoumtem,

B – Clemson spineless.

0

2

4

6

8

10

12

0 4 6 8 10

Pla

nt

girt

h (

cm)

Duration (weeks)

A

F

N

NF

NF7

NF14

NF21

C0

2

4

6

8

10

12

14

0 4 6 8 10

Pla

nt

girt

h (

cm)

Duration (weeks)

B

F

N

NF

NF7

NF14

NF21

C

44

Chlorophyll content

There were significant differences in chlorophyll content in Essoumtem okra plants (p ≤ 0.05)

among the treatments. M. incognita and F. oxysporum infection decreased chlorophyll

content of the plants. Plants that received NF7 treatments showed the least chlorophyll

content (35.3 CCI). Highest chlorophyll content (50.3 CCI) was observed on control

(Fig. 13).

There were significant differences in the various treatments applied to the Clemson spineless

okra plants. The least chlorophyll content (31.7 CCI) was observed on plants inoculated with

NF21 treatment. Un-inoculated plants were observed to show the highest chlorophyll content

(49.4 CCI) (Fig. 13).

Fig. 13: Chlorophyll content of okra plants inoculated individually and simultaneously with

Meloidogyne incognita and Fusarium oxysporum f. sp. vasinfectum in pot experiment (n = 3).

F = Fungus alone; N = Nematode alone; NF = Nematode and Fungus simultaneously; NF7 =



inoculated); A – Essoumtem, B – Clemson spineless.

0

10

20

30

40

50

60

0 4 6 8 10

Ch

loro

ph

yll c

on

ten

t

Duration (weeks)

A

F

N

NF

NF7

NF14

NF21

C 0

10

20

30

40

50

60

0 4 6 8 10

Ch

loro

ph

yll c

on

ten

t

Duration (weeks)

B

F

N

NF

NF7

NF14

NF21

C

45

Fresh and dry shoot weight

There were significant differences (p ≤ 0.05) with respect to fresh shoot weight of okra

among treatments in Essoumtem okra plants. The total fresh shoot weights of plants

inoculated with the two pathogens were lower compared to un-inoculated plants. Plants that

received the NF21 treatment, were significantly different (p ≤ 0.05) from the other

treatments, for fresh shoot weight (767 g). Plants that received F, N, NF and NF7 were not

significantly different from each other. The highest fresh shoot weight (1,554 g) were on the

control plants (Table 8).

There were significant differences (p ≤ 0.05) in fresh shoot weight of Clemson spineless okra

among the treatments. Fresh shoot weight was significantly lower (593 g) (p ≤ 0.05) in plants

that received NF21. Plants that received F, N, NF and NF7 were not significantly different

from each other. The highest fresh shoot weight (1,273 g), however, was recorded in the


The highest percentage reductions (50.6% and 53.4%) in fresh shoot weight of okra were

observed on plants that received NF21 treatment for Essoumtem and Clemson spineless

respectively. The least percentage reductions (12.9% and 17.7%) for plants receiving N

treatment were observed on Essoumtem and Clemson spineless okra plants respectively

(Table 8).

Significant differences (p ≤ 0.05) existed for dry shoot weight within Essoumtem variety.

Plants that received NF21 treatments showed the least dry shoot weight (471 g). Plants that

46

received N, NF and NF7 were not significantly different from each other. The highest dry

shoot weight (1,210 g) was observed on control plants (Table 8).

Significant differences (p ≤ 0.05) existed for dry shoot weight within the Clemson spineless

okra variety. Plants that received NF21 showed the least dry shoot weight (329 g). Plants that

received N, NF and NF7 were not significantly different. The highest dry shoot weight

(973 g) was observed on control plant (Table 8).

The highest percentage reductions in dry shoot weight of okra (61.1% and 66.2%) were

observed on plants that received NF21 treatment for Essoumtem and Clemson spineless

respectively. The least percentage reductions (7.5 % and 32.1 %) were observed for

Essoumtem plants that received N treatments and Clemson spineless plants that received NF7

treatment respectively. Control plants did not show any reduction in dry shoot weight

(Table 8).


inoculations in pot experiment (n = 3)

Type of

inoculation

Fresh shoot weight (g) % Reduction Dry shoot weight (g) % Reduction

V1 V2 V1 V2 V1 V2 V1 V2

F 940.0 b* 1047.0 b 39.5 17.8 597.0 ab 705.0 ab 50.7 27.5

N 1354.0 b 858.0 b 12.9 32.6 1119.0 b 574.0 b 7.5 41.0

NF 1259.0 b 857.0 b 19.0 32.7 1036.0 b 452.0 b 14.4 53.5

NF7 1079.0 b 964.0 b 30.5 24.3 855.0 b 661.0 b 29.3 32.1

NF14 994.0 ab 748.0 ab 36.0 41.2 716.0 ab 518.0 ab 40.8 46.8

NF21 767.0 a 593.0 a 50.6 53.4 471.0 a 329.0 a 61.1 66.2

C 1554.0 c 1273.0 c – – 1210.0 c 973.0 c – –

*Means followed by the same letter in a column are not significantly different at LSD (p ≤ 0.05). F =

Fungus alone; N = Nematode alone; NF = Nematode and Fungus simultaneously; NF7 = Fungus 7


Fungus21 days after nematode inoculation and C = Control (un-inoculated). V1 = Essoumtem, V2 =

Clemson spineless

47

Fresh and dry root weight

There were significant differences (p ≤ 0.05) in fresh root weight among the different

treatments within the Essoumtem okra plants. Appreciable levels of reductions were observed

in plants inoculated individually and sequentially with both nematode and fungal pathogens.

However, plants that received the NF21 treatment showed the least (221 g) fresh shoot

weight. Plants that received F, N, NF, NF7 and NF14 did not significant differ from each

other for fresh root weight. The highest fresh root weight (727 g) was observed on control

plants (Table 9).

There were also significant differences (p ≤ 0.05) in the fresh root weight of plants among the

treatments for Clemson spineless okra plants. Plants that received the NF21 treatment had the

least fresh root weight (300 g). Plants that received F, N, NF, NF7 and NF14 did not

significantly differ from each other for fresh root weight. The highest fresh root weight

(628 g) was observed on control plants (Table 9).

The highest percentage reductions (69.6% and 52.2%) in fresh root weight of okra were

observed on Essoumtem and Clemson spineless plants that received NF21 treatment

respectively. The least percentage reductions (15.4 % and 20.0 %) were observed for

Essoumtem plants that received NF treatment and Clemson spineless plants that received F

treatment respectively (Table 9).

Significant differences (p ≤ 0.05) existed among dry root weight treatments for Essoumtem

plants. Essoumtem plants that received NF21 treatment showed the least dry root weight

(85 g). Plants that received F, N, NF, NF7 and NF14 treatments did not significantly differ

from each other, in relation to dry root weights. The highest dry root weight (573 g) was

observed on control plants (Table 9).

48

There were significant differences (p ≤ 0.05) among dry root weight treatments for Clemson

spineless okra plants. Plants inoculated with NF21 treatment showed the least dry root weight

(116 g). Plants that received F, N, NF, NF7 and NF14 treatments did not significantly differ

from each other. The highest dry root weight (267 g) was observed on control plants

(Table 9).

The highest percentage reductions (85.2 % and 56.6 %) in dry root weight of okra were

observed on both Essoumtem and Clemson spineless plants that received NF21 treatments

respectively. The least percentage reductions (31.2 % and 22.5 %) were observed on

Essoumtem plants that received NF treatment and Clemson spineless plants that received

NF7 treatments respectively (Table 9).


inoculations in pot experiment (n = 3)

Type of

inoculation

Fresh root weight (g) % Reduction Dry root weight (g) % Reduction

V1 V2 V1 V2 V1 V2 V1 V2

F 506.0 ab* 503.0 ab 30.4 20.0 277.0 ab 145.0 ab 51.7 34.4

N 531.0 ab 398.0 ab 27.0 36.6 276.0 ab 147.0 ab 51.8 44.9

NF 615.0 ab 349.0 ab 15.4 44.4 394.0 ab 141.0 ab 31.2 47.2

NF7 597.0 ab 390.0 ab 17.9 37.9 312.0 ab 207.0 ab 45.5 22.5

NF14 532.0 ab 311.0 ab 26.8 50.5 337.0 ab 150.0 ab 41.2 43.8

NF21 221.0 a 300.0 a 69.6 52.2 85.0 a 116.0 a 85.2 56.6

C 727.0 b 628.0 b – – 573.0 b 267.0 b – –



days after nematode inoculation; NF14 = Fungus 14 days after nematode inoculation; NF21 = Fungus

21 days after nematode inoculation and C = Control (un-inoculated); V1 = Essoumtem, V2 = Clemson

spineless

49

Yield parameters

Yield parameters showed significant differences (p ≤ 0.05) among the various treatments in

the Essoumtem okra plants. The number of pods (21) were significantly reduced in plants

inoculated with NF21 treatment. The maximum number of pods (42) were observed on


Yield parameters showed significant differences among the different inoculation treatments

on Clemson spineless okra plants. The number of pods (30) were significantly reduced in

plants that received NF21 treatment. The maximum number of pods (53) were observed on

the control plants (Table 10).

The highest percentage reductions (50.0 % and 43.4 %) in number of pods of okra were

observed on Essoumtem and Clemson spineless plants that received the NF21 treatments

respectively. The least percentage reductions (19.0 % and 22.6 %) were in Essoumtem plants

that received NF treatment, and Clemson spineless plants that received the F treatment

(Table 10).

50

Table 10: Number of pods for two okra varieties under various inoculations in pot

experiment (n = 3)

Type of

inoculation

No. of pods % Reduction

Essoumtem Clemson

spineless

Essoumtem Clemson

spineless

F 33.0 c* 41.0 c 21.4 22.6

N 32.0 bc 38.0 bc 23.8 28.3

NF 34.0 bc 39.0 bc 19.0 26.4

NF7 29.0 abc 35.0 abc 31.0 34.0

NF14 25.0 ab 34.0 ab 40.5 35.8

NF21 21.0 a 30.0 a 50.0 43.4

C 42.0 d 53.0 d – –




21 days after nematode inoculation and C = Control (un-inoculated).

Weight of pods was significantly affected by the pathogens (fungus and nematode) on

Essoumtem okra plants. The lowest weight of pod (521 g) was observed on plants inoculated

with NF21 treatment. The highest weight of pods (945 g) were observed on control plants

(Table 11).

Weight of pods was significantly affected by the pathogens on Clemson spineless okra plants.

The lowest weight of pod (564 g) was observed on plants that received NF21 treatment. The

highest weight of pods (1,090 g) was observed on control plants (Table 11).

The highest percentage reductions (44.9 % and 48.3 %) in weight of pods of okra were

observed on Essoumtem plants and Clemson spineless plants both inoculated with NF21

treatments. The least percentage reductions (32.5 % and 28.2 %) were observed on

51

Essoumtem plants that received NF treatment and Clemson spineless plants that received F

treatment respectively with no percentage reduction on control plants (Table 11).

Yield was affected significantly on Essoumtem okra plants due to the interaction of the two

pathogens. The highest yield was (1,575.0 kg/ha) observed on control plants. The least of

yields (868.3 kg/ha) were observed on plants treated with NF21 (Table 11).

Yield was affected significantly on Clemson spineless okra plants due to the interaction of the

two pathogens. The highest yield (1,816.7 kg/ha) was recorded by control plants. Plants that

received NF21 treatments showed the least yield (940 kg/ha) (Table 11).

The highest percentage reductions (44.9 % and 48.3 %) in yield of okra were observed on

plants that received NF21 treatment, for Essoumtem and Clemson spineless respectively. The

least percentage reductions (19.0 % and 22.6 %) were recorded on Essoumtem plants that

received NF treatment and Clemson spineless plants that received F treatment respectively

with no percentage reduction on control plants (Table 11).

52

Table 11: Weight of pods and yield for two okra varieties under various inoculations in

pot experiment (n = 3)

Type of

inoculation

Weight of pods (g) % Reduction Yield (kg/ha) % Reduction

V1 V2 V1 V2 V1 V2 V1 V2

F 628.0 a* 783.0 a 33.5 28.2 1,046.7 a 1,305.0 a 33.5 28.2

N 604.0 a 676.0 a 36.1 38.0 1,006.7 a 1,126.7 a 36.1 38.0

NF 638.0 a 744.0 a 32.5 31.7 1,063.3 a 1,240.0 a 32.5 31.7

NF7 605.0 a 638.0 a 36.0 41.5 1,008.3 a 1,063.3 a 36.0 41.5

NF14 558.0 a 627.0 a 41.0 42.5 930.0 a 1,045.0 a 41.0 42.5

NF21 521.0 a 564.0 a 44.9 48.3 868.3 a 940.0 a 44.9 48.3

C 945.0 b 1,090.0 b – – 1,575.0 b 1,816.7 b – –




21 days after nematode inoculation and C = Control (un-inoculated); V1 = Essoumtem, V2 = Clemson

spineless

4.2.2 Field Experiment

Plant height

There were significant differences in Essoumtem plant height (p ≤ 0.05) among the various

treatments. Plant height showed significant increase up to 8 WAP but the rate of growth

slowed at 10 WAP for plants that received F21, F14 and F7 treatment. When the plants

received F21 treatment minimum height (14.8 cm) was observed. The maximum height (17.7

cm) was observed on plants inoculated with F21 treatment (14.8 cm) (Fig. 14).

There were significant differences in plant height among the treatments inoculated on the

Clemson spineless okra plants. Maximum plant height (23.1 cm) was observed on control

plants. Minimum plant height (16.8 cm) was observed on plants that received F21 treatment

(Fig. 14).

53

Fig. 14: Height of okra plants inoculated serially with Fusarium oxysporum f. sp. vasinfectum

in field experiment (n = 3). F7 = fungus inoculated 7 days after seedling emergence, F14 =

fungus inoculated 14 days after seedling emergence, F21 = fungus inoculated 21 days after

seedling emergence, C = Control (un-inoculated); A = Essoumtem, B = Clemson spineless.

Plant girth

There was significant difference (p ≤ 0.05) in plant girth among treatments on Essoumtem

okra plants. The least plant girths (2.8 cm) at 10 weeks after planting were observed on plants

that received F7 and F21 treatment. The control showed the highest plant girth (3.5 cm)

(Fig. 15).

There was significant difference (p ≤ 0.05) in plant girth among treatments on Clemson

spineless. When the plants received F21 treatment, the least plant girth (1.9 cm) was

observed. The control showed the highest plant girth (2.9 cm) (Fig. 15).

0

5

10

15

20

25

0 4 6 8 10

Pla

nt

he

igh

t (c

m)

Duration (weeks)

A

F7

F14

F21

C

0

5

10

15

20

25

0 4 6 8 10

Pla

ant

he

igh

t (c

m)

Duration (weeks)

B

F7

F14

F21

C

54

Fig. 15: Girth of okra plants inoculated serially with Fusarium oxysporum f. sp. vasinfectum

in field experiment (n = 3). F7 = fungus inoculated 7 days after seedling emergence, F14 =

fungus inoculated 14 days after seedling emergence, F21 = fungus inoculated 21 days after

seedling emergence, C = Control (un-inoculated); A = Essoumtem, B = Clemson spineless.

Chlorophyll content

There were significant differences in chlorophyll content (p ≤ 0.05) among the different

treatments on Essoumtem okra plants. The lowest chlorophyll content (43 CCI) was observed

on plants inoculated with F21 treatment. The highest chlorophyll content (56.8 CCI) was

recorded by un-inoculated plants (Fig. 16).

There were significant differences in chlorophyll content (p ≤ 0.05) among the different

treatments on Clemson spineless. The lowest chlorophyll content (32.5 CCI) was observed on

plants inoculated with F21 treatment. The highest chlorophyll content (45.6 CCI) was

recorded by control plants (Fig. 16).

0

0.5

1

1.5

2

2.5

3

3.5

4

0 4 6 8 10

Pla

nt

girt

h (

cm)

Duration (weeks)

A

F7

F14

F21

C0

0.5

1

1.5

2

2.5

3

3.5

0 4 6 8 10

Pla

nt

girt

h (

cm)

Duration (weeks)

B

F7

F14

F21

C

55

Fig. 16: Chlorophyll content of okra plants inoculated serially with Fusarium oxysporum f.

sp. vasinfectum in field experiment (n = 3). F7 = fungus inoculated 7 days after seedling

emergence, F14 = fungus inoculated 14 days after seedling emergence, F21 = fungus

inoculated 21 days after seedling emergence, C = Control (un-inoculated); A = Essoumtem, B

= Clemson spineless.

Fresh and dry shoot weight

There was significant difference in fresh shoot weight (p ≤ 0.05) among the different

inoculations of the two pathogens on Essoumtem okra plants. Fresh shoot weight was

significantly reduced by all the inoculations. Plants inoculated with F21 treatment showed the

least fresh shoot weight (98.6 g). The highest fresh shoot weight (140.7 g) was recorded by


There was significant difference in fresh shoot weight (p ≤ 0.05) among the different

inoculations of the two pathogens on Clemson spineless okra plants. Fresh shoot weight was

significantly reduced by all the inoculations. Plants inoculated with F21 treatments showed

the least fresh shoot weight (21 g). The highest fresh shoot weight (49.1 g) was observed on


0

10

20

30

40

50

60

0 4 6 8 10

Ch

loro

ph

yll c

on

ten

t

Duration (weeks)

A

F7

F14

F21

C0

10

20

30

40

50

0 4 6 8 10

Ch

loro

ph

yll c

on

ten

t

Duration (weeks)

B

F7

F14

F21

C

56

The highest percentage reductions (57.2 % and 5.8 %) in fresh shoot weight of okra were

observed on Clemson spineless plants and Essoumtem plants that received F21 treatments

respectively. The minimum percentage reductions (1.1 % and 32.2 %) were observed on

Essoumtem and Clemson spineless plants both inoculated with F7 treatments (Table 12).

There was significant difference in dry shoot weight showed (p ≤ 0.05) among the different

inoculation on Essoumtem okra plants. When fungus was inoculated with F21 treatment,

minimum dry shoot weight (9.5 g) was observed. The highest dry shoot weight (22.3 g) was

observed by control plants (Table 12).

There was significant difference in dry shoot weight showed (p ≤ 0.05) among the different

inoculations on Clemson spineless okra plants. When F21 treatment was inoculated to the

plants the minimum dry shoot weight (4.9 g) was observed. The highest dry shoot weight (9.3

g) was recorded by control plants (Table 12).

The highest percentage reductions (57.4 % and 47.3 %) in dry shoot weight of okra were

observed on plants inoculated with F21 treatments for Essoumtem showing and Clemson

spineless plants respectively. The least percentage reductions (22.9 % and 18.3 %) were

observed on Essoumtem and Clemson spineless plants that received F7 treatments

respectively (Table 12).

57


inoculations in field experiment (n = 3)

Type of

inoculation

Fresh shoot weight (g) % Reduction Dry shoot weight (g) % Reduction

V1 V2 V1 V2 V1 V2 V1 V2

F7 103.5 a* 33.3 ab 1.1 32.2 17.2 bc 7.6 b 22.9 18.3

F14 96.2 a 29.7 a 8.1 39.5 11.8 ab 6.5 a 47.1 30.1

F21 98.6 a 21.0 a 5.8 57.2 9.5 a 4.9 a 57.4 47.3

C 104.7 a 49.1 b – – 22.3 c 9.3 c – –

*Means followed by the same letter in a column are not significantly different at LSD (p ≤

0.05). F7 = fungus inoculated 7 days after seedling emergence, F14 = fungus inoculated 14

days after seedling emergence, F21 = fungus inoculated 21 days after seedling emergence, C

= Control (un-inoculated); V1 = Essoumtem, V2 = Clemson spineless.

Fresh and dry root weight

Fresh root weight showed significant difference (p ≤ 0.05) in fresh root weight among

treatments on Essoumtem okra plants. Sequential inoculation of both pathogens caused

significant reduction in the weight of fresh root. The lowest fresh root weight (23.6 g) was

shown by plants inoculated with F21 treatment. The highest root weight (39.3 g) was

observed on control plants (Table 13).

There was significant difference (p ≤ 0.05) in fresh root weight with the various treatments on

Clemson spineless okra plants. The lowest fresh root weight (7.2 g) was observed on plants

inoculated with F21 treatments. The highest root weight (16.5 g) was observed on control

plants (Table 13).

The highest percentage reductions (39.9 % and 56.4 %) in fresh root weight of okra were

observed on plants inoculated with F21 treatments for Essoumtem and Clemson spineless

plants respectively. The least percentage reductions (23.6 % and 7.2 %) were observed on

58

Essoumtem and Clemson spineless plants that received F21 treatments respectively

(Table 13).

Dry root weight on Essoumtem okra plant was significantly reduced by the inoculations of

the fungus to already nematode infested soil. Plant inoculation with F21 treatment showed the

minimum dry root weight (2.6 g). The highest dry root weight (6.3 g) was recorded on


Dry root weight was significantly reduced on Clemson spineless okra plants. Plant

inoculation with F21 treatment showed the minimum dry rot weight (1.1 g). The highest dry

root weight (3.1 g) was recorded on control plants (Table 13).

The highest percentage reductions (58.7 % and 64.5 %) in dry root weight of okra were

observed on plants inoculated with F21 treatments for Essoumtem and Clemson spineless

plants respectively. Less percentage reductions (19.0 % and 19.4 %) in dry root weight were

observed on Essoumtem and Clemson spineless plants respectively (Table 13).


inoculations in field experiment (n = 3)

Type of

inoculation

Fresh root weight (g) % Reduction Dry root weight (g) % Reduction

V1 V2 V1 V2 V1 V2 V1 V2

F7 31.5 a* 10.0 a 19.8 39.4 5.1 bc 2.5 a 19.0 19.4

F14 28.9 a 8.9 a 26.5 46.1 3.9 ab 1.7 a 38.1 45.2

F21 23.6 a 7.2 a 39.9 56.4 2.6 a 1.1 a 58.7 64.5

C 39.3 a 16.5 b – – 6.3 c 3.1 a – –




= Control (uninoculated); V1 = Essoumtem, V2 = Clemson spineless

59

Yield parameters

There was significant difference (p ≤ 0.05) in the number of pods among treatments on

Essoumtem okra plants. Plants inoculated with F14 and F21 treatments showed the least

number of pods (12). The highest number of pods (16) were observed on control plants.

There was no significant difference in number of pods between plants that received F7

treatment and control (Table 14).

There were significant differences (p ≤ 0.05) in the number of pods among treatments on

Clemson spineless okra plants. Plants inoculated with F21 treatment showed the least number

of pods (7.0). The highest number of pods (18) was recorded on control plants (Table 14).

The highest percentage reductions (25.0 % and 63.4 %) in number of pods of okra were

observed on Essoumtem and Clemson spineless plants inoculated with F21 treatments

respectively. The least percentage reductions (12.5 % and 27 %) were observed on

Essoumtem and Clemson spineless plants that received F7 treatments respectively (Table 14).

Table 14: Number of pods of ‘Essoumtem’ and Clemson spineless inoculated serially

with Fusarium oxysporum f. sp. vasinfectum in field experiment (n = 3)

Type of

inoculation

No. of pods % Reduction

Essoumtem Clemson

spineless

Essoumtem Clemson

spineless

F7 14.0 a* 13.0 a 12.5 27.3

F14 12.0 a 15.0 a 25.0 19.7

F21 12.0 a 7.0 a 25.0 63.4

C 16.0 a 18.0 a – –



days after seedling emergence, F21 = fungus inoculated 21 days after seedling emergence.

60

The weight of pods was significantly lower (77.8 g) in Essoumtem okra plants received F21

treatment. There was no significant difference in plants that received F7, F14 and F21

treatments. The highest weight of pods (161.4) were observed on control plants (Table 15).

The weight of pods were significantly lower (30.4 g) in Clemson spineless okra plants that

were inoculated withF21 treatment. There was no significant difference in plants that

received F7 and F14 treatments. The highest weight of pods (111.1 g) was observed on


The highest percentage reductions (51.8 % and 72.6 %) in weight of pods of okra were

observed on Essoumtem and Clemson spineless plants inoculated with F21 treatments

respectively. The least percentage reductions (40.5 % and 31.7 %) in pod weight were

observed on Essoumtem and Clemson spineless plants which received F7 treatments

respectively (Table 15).

Yield significantly reduced the in plants serially inoculated with the two pathogens on

Essoumtem okra plants. The plants inoculated with F21 treatments showed the least yield

(130.0 kg/ha). The highest yield was recorded on control plants (269.0 kg/ha) (Table 15).

Yield significantly reduced the in Clemson spineless okra plants serially inoculated with the

two pathogens. The plants inoculated with F21 treatment showed the least yield (51.0 kg/ha).

The highest yield (185.0 kg/ha) was observed on control plants (Table 15).

The highest percentage reductions (51.7 % and 72.4 %) in yield of okra were observed on

Essoumtem and Clemson spineless plants inoculated with F21 treatments respectively. .The

least percentage reductions (40.5 % and 31.4 %) were observed in yield of both Essoumtem

and Clemson spineless that received F21 treatments respectively (Table 15).

61

Table 15: Weight of pods and yield of ‘Essoumtem’ and Clemson spineless inoculated

serially with Fusarium oxysporum f. sp. vasinfectum in field experiment (n = 3)

Type of

inoculation

Weight of pods (g) % Reduction Yield (kg/ha) % Reduction

V1 V2 V1 V2 V1 V2 V1 V2

F7 96.1 a* 75.9 ab 40.5 31.7 160.0 a 127.0 ab 40.5 31.4

F14 88.3 a 71.4 ab 45.3 35.7 147.0 a 119.0 ab 45.4 35.7

F21 77.8 a 30.4 a 51.8 72.6 130.0 a 51.0 a 51.7 72.4

C 161.4 b 111.1 b – – 269.0 b 185.0 b – –

*Means followed by the same letter in a column are not significantly different at LSD (p ≤ 0.05).F7 =

fungus inoculated 7 days after seedling emergence, F14 = fungus inoculated 14 days after seedling

emergence, F21 = fungus inoculated 21 days after seedling emergence, V1 = Essoumtem, V2 =

Clemson spineless.

4.3 Reproductive ability of Meloidogyne incognita on okra after Fusarium oxysporum

f. sp. vasinfectum infection under pot and field conditions

Number of eggs, root galls and reproductive factor on two okra varieties in Pot

experiment

The number of eggs, root galls and reproductive factor of M. incognita and F. oxysporum

inoculated on Essoumtem okra plants in pots showed significant difference among

treatments. The highest (261.0) egg numbers were observed on plants that were inoculated

with N treatment while the lowest (0.0) were observed in plants that were inoculated with F

treatment and control plants (Table 16).

The number of eggs, root galls and reproductive factor of M. incognita and F. oxysporum

inoculated on Clemson spineless okra plants in pots showed significant differences. There

was significant difference in number of eggs among treatments. The highest (257.0) was

observed on plants that received N treatment. The lowest (0.0) was observed in plants that

that received with F treatment and control plants. There was no significant difference

between plant that received NF7 and NF14 treatment (Table 16).

62

There was significant difference in galling on Essoumtem okra plants. The highest (2.9) was

observed on plants that received N treatments. The lowest (0.7) was observed on plants that

received F treatments and control plants. There was no significant difference in galling

among all inoculations that involved the nematode (N, NF, NF7, NF14 and NF21) (Table 16).

There was significant difference in galling on Clemson spineless okra plants. The highest

(3.1) was observed on plants that received N treatments. The lowest (0.7) was observed on

plants inoculated with F treatment and control plants (Table 16).

There was significant difference in reproductive factor (RF) among treatments on Essoumtem

okra plants. The highest (1.3) was observed on plants that received N treatments. The lowest

(0.7) was recorded on plants that received F treatments and control plants. There was no

significant difference among plant that was inoculated with nematodes (N, NF, NF7, NF14

and NF21) (Table 16).

There was significant difference in reproductive factor among treatments on Clemson

spineless okra plants. The highest reproductive factor (1.3) was observed on plant that

received N treatments. The lowest (0.7) was observed on plants inoculated with F treatment

and control plants (Table 16).

63

Table 16: Number of eggs, gall index and reproductive factor for two okra varieties

under various inoculations in pot experiment (n = 3)

Type of

inoculation

Nematode reproduction parameters

Number of eggs/ root Gall index Reproductive factor

V1 V2 V1 V2 V1 V2

F 0.0 0.0 0.0 0.0 0.00 0.00

N 261.0 257.0 7.7 9.0 1.3 1.3

NF 241.0 240.0 4.3 5.0 1.0 1.0

NF7 200.0 199.0 2.3 3.0 0.9 0.9

NF14 87.0 85.0 3.0 3.0 0.6 0.7

NF21 20.0 13.0 1.0 1.0 0.6 0.5

C 0.0 0.0 0.0 0.0 0.00 0.00

LSD 22.4 20.5 1.2 1.2 0.06 0.07

*Figures in parenthesis are square root transformed. F = Fungus alone; N = Nematode alone; NF =

Nematode and Fungus simultaneously; NF7 = Fungus 7 days after nematode inoculation; NF14 =

Fungus 14 days after nematode inoculation; NF21 = Fungus 21 days after nematode inoculation and C

= Control. V1 = Essoumtem, V2 = Clemson spineless

Number of eggs, root galls, and reproductive factor on two okra varieties in field

experiments

There were significant differences (p ≤ 0.05) in number of egg among treatment on

Essoumtem okra plants. The highest number of eggs (47.0) was observed on control plants.

Plants that received F21 treatments had the least (20.0 g) egg numbers (Table 17).

There were significant differences among the number of eggs for the various treatments. The

highest number of eggs (39.0) was observed on control plants. Plants that received F21

treatment had the least egg numbers (16.0) (Table 17).

Within the Essoumtem okra plants, the highest gall index (6.0) was observed on the control

plants. The lowest gall index (1.5) was observed on plants that received the F21 treatment

(Table 17).

64

The highest gall index (5.0) for Clemson spineless okra plants was on the control plants. The

lowest gall index (1.5) was observed on plants that received the F21 treatment (Table 17).

The highest reproductive factor (1.2) among treatment for Essoumten okra plants was

observed on control plants. The least reproductive factor (0.9) was however, observed on

plants that received the F21 treatment (Table 17).

Within the treatments for Clemson spineless plants, the highest reproductive factor (1.2) was

observed on control plants. The least reproductive factor (0.7) was observed on plants that

received the F21 treatment (Table 17).

The galls (A and B) on the roots of okra with the control (C) showing no sign of the

nematodes attack (Fig. 17).

Table 17: Number of eggs, gall index and reproductive factor of ‘Essoumtem’ and

Clemson spineless inoculated serially with Fusarium oxysporum f. sp. vasinfectum in

field experiment (n= 3)

Type of

inoculation

Nematode reproduction parameters

No. of eggs/ root Gall index Reproductive factor

Essoumtem Clemson

spineless

Essoumtem Clemson

spineless

Essoumtem Clemson

spineless

F7 25.0 23.0 1.7 2.0 1.1 1.1

F14 23.0 20.0 3.0 2.5 1.0 0.9

F21 20.0 16.0 1.5 1.5 0.9 0.7

C 47.0 39.0 6.0 5.0 1.2 1.2


0.05); F7 = fungus inoculated 7 days after seedling emergence, F14 = fungus inoculated 14


= control (un-inoculated)

65

Fig 17: Effects of Meloidogyne incognita on the roots of okra. Root with galls in pot

experiment (A), root with galls in field experiment (B) and okra root without any gall

(C).

4.4 Effect of Meloidogyne incognita on the Fusarium wilt disease severity on two

okra varieties

Percentage wilt incidence in pot experiment

There was significant increase (p ≤ 0.05) in wilt incidence among treatments on Essoumtem

okra plants. The highest disease incidence (73 %) was observed on plants that received NF21

treatment. The lowest (0 %) was observed on plants that received N treatment and on control

plants (Table 18).

There was significant increase (p ≤ 0.05) in wilt incidence among treatments on Clemson

spineless okra plants. The highest disease incidence (82 %) was observed on plants that

received NF21 treatment. The lowest (0 %) was recorded on plants that received N treatment

and control plants. There were no wilt symptoms on control plants and plants that received N

treatment (Table 18).

A B C

a b c

66

Table 18: Percentage wilt incidence for two okra varieties under various inoculations in

pot experiment (n = 3)

Type of inoculation % Wilt incidence

Essoumtem Clemson

spineless

F: Fungus alone 37.0 32.0

N: Nematode alone 0.0 0.0

NF: Nematode and Fungus inoculated simultaneously 41.0 43.0

NF7: Fungus inoculated 7 days after nematode inoculation 45.0 42.0



C: Control (un-inoculated) 0.0 0.00

*Figures in the parenthesis are square root transformed.

Some of the symptoms of the Fusarium wilt on okra (A, B and C) with the control (D)

showing no symptoms of the wilt of okra in the pot experiment (Fig. 18).

67

Fig. 18: Reaction of two okra varieties to the two pathogens (M. incognita and FOV) inoculations in

pot experiment. A – shows typical symptom of Fusarium oxysporum on okra in pot, B – a plot

showing the infection of Fusarium oxysporum on okra in pot, C – advance stage of infection of

Fusarium oxysporum and D – a plot showing disease-free plot

Percentage wilt incidence in field experiment

Wilt increased significantly in all the treatments applied to „Essoumtem‟ variety of okra.

Incidence of wilt was severe in all the three treatments of F7 (12 %), F14 (423 %) and F21

(59 %).The control (0 %) showed no wilt incidence since no fungus was inoculated. The

treatment which showed the highest percentage wilt was the F21 treatment (Table 19).

There was significant increase in wilt incidence in all the treatments. Incidence of wilt was

severe in all the three treatments of serial fungus inoculation. Treatment where plants

received F21 treatments showed the highest incidence of wilt (65 %) on Clemson spineless.

The least wilt incidence (0 %) was observed on control plants where no fungus was

A B

C D

68

inoculated. The treatment which recorded the highest percentage wilt was plants treated with

F21 (Table 19).

Table 19: Percentage wilt incidence in ‘Essoumtem’ and Clemson spineless inoculated

serially with Fusarium oxysporum f. sp. vasinfectum in field experiment (n = 3)

Type of inoculation % Wilt incidence

Essoumtem Clemson

spineless

F7: Fungus inoculated 7 days after seedling emergence 12.0 12.0



C: Un-inoculated (control) 0.0 0.0

Some of the symptoms of the Fusarium wilt on okra (A and B) in the field experiment

(Fig. 19).

Fig. 19: Symptoms of Fusaiurm wilt on okra. The diseases showing symptoms from the

top of the plant (A) and disease showing symptoms from the soil line of the plant (B).

A B a

b

69

CHAPTER FIVE

5.0 DISCUSSION

In this study, the shorter plant height (43 cm and 44 cm), smaller plant girth (9 cm and 8.1

cm), and lower chlorophyll contents (37 CCI and 32 CCI) in inoculated plants in pot

experiments for „Essoumtem‟ and Clemson spineless okra plants respectively due to root

damage in the pot experiment reducing the ability plants to take up water and minerals from

the soil via the roots to aid in photosynthesis. The shorter plant height (15 cm and 17.1 cm),

smaller plant girth (2.8 cm and 1.9 cm), and lower chlorophyll contents (43 CCI and 32 CCI)

in inoculated plants in field experiments for „Essoumtem‟ and Clemson spineless okra plants

respectively were due to root damage. Growth and development of leaf tissue, chlorophyll

contents, are affected when there is inadequate supply of water, minerals, energy and

photosynthates (Khan and Khan, 2007). As reported by Khan and Khan (2007), damage of

roots of okra plants does not allow absorption of water and mineral from the soil thereby

causing stunting. Wilting and stunting of plants occurs when nematode infestation is

prevalent (William and Robert, 2007).

In the current study, plants that received sequential treatments (NF21, NF14 and NF7) had

higher reductions in their plant growth parameters e.g., fresh shoot weight (50.6 % and 53.4

%), dry shoot weight (32.1 % and 7.5 %), fresh root weight (69.6 % and 52.2 %) and dry root

weight (15.4 % and 20.0 %) for „Essoumtem‟ and Clemson spineless okra varieties

respectively in pot experiment. Within the field experiment, plants that received sequential

treatments (F21, F14 and F7) had a fairly high reductions in their plant growth parameters

e.g., fresh shoot weight (57.2 % and 5.8 %), dry shoot weight (57.4 % and 47.3 %), fresh root

weight (39.9 % and 56.4 %) and dry root weight (58.7 % and 64.5 %) for „Essoumtem‟ and

Clemson spineless okra varieties respectively. The isolates of M. incognita and F. solani

70

caused a severe reduction in the height of plants (43.83 %), fresh shoot (52.81 %) and root

weight (60.99 %) and dry shoot (57.75 %) and root weight (67.96) as compared to the plots

that were not inoculated (Ganaie and Khan, 2011). Comparably, there is higher percentage of

reductions in plant growth parameters in the current study as reported by Ganaie and Khan,

2011.

In the present study, there was significant reduction in number of pods, weight of pods and

yield was observed for interaction between nematode and fungus on „Essoumtem‟ and

Clemson spineless in both pot and field experiments. In the pot experiments, number of pods

(21 and 30), weight of pods (521 g and 567 g) and yield (868 kg/ha and 940 kg/ha) was very

low for plants that received F21 treatments in both „Essoumtem‟ and Clemson spineless

varieties respectively. In the field experiments, number of pods (12 and 7), weight of pods

(77.8 g and 30.4 g) and yield (130 kg/ha and 51 kg/ha) was very low for plants that received

F21 treatments in both „Essoumtem‟ and Clemson spineless varieties respectively.

Nematodes are found to be serious pest of okra plant damaging plant stands thereby delaying

the production of okra pods by almost 80 % (Bolles and Johnson, 2012). Reports show that

infection of crops by Meloidogyne spp. causes severe growth impairment and yield losses

(Hussain et al., 2011; Kayani et al., 2013; Mukhtar et al., 2013; Barros et al., 2014). The

figures observed in the current study gave credence to the fact that nematodes reduce the

production of pods in okra plants especially of the field where nematode population was high.

The poor growth of leaves of plant consequently leads to decrease in yield (Hussain et al.,

2016; Kayani et al., 2017).

Number of eggs, gall index and reproductive factor were highly reduced due to the

interaction between nematode and fungus pathogen on both „Essoumtem‟ and Clemson

spineless in pot and field experiments. Severe reduction was observed in the sequential

inoculation of nematodes compared to fungus and simultaneous inoculation of both

71

pathogens on „Essoumtem‟ and Clemson spineless in both pot and field experiment. The

fungus forms film of mat over the roots thereby reducing the population of the nematodes in

the root. The final population of nematodes in the roots is thereby reduced drastically. The

combined effect of M. incognita and other fungal pathogens such as, F. oxysporum reduced

gall index in tomato (Nagesh et al., 2006). Root-knot nematode and root-rot fungus has also

been reported to have synergistic effects on various crops (Golden and Van Gundy, 1975;

Chahal and Chabra, 1984; Ali and Venugopal, 1992; Walker, 1994; Prasad, 1995; Bhagwati

et al., 2007). These reports therefore give credence to the findings in this research work.

The reproducibility of eggs and adults of M. incognita was slowed by the presence of F.

oxysporum, especially in the two sequential treatments (NF21 and F21; NF14 and F14

treatment) on both „Essoumtem‟ and Clemson spineless in pot and field experiment. Several

soil-borne fungi affect the reproduction of Meloidogyne spp. on numerous vegetable crops

(Mokbel et al., 2007). Nematode reproduction was tremendously reduced greatly due to the

severe damage caused by F. oxysporum. Synergistic effect of M. incognita and R. solani has

also been reported okra where they cause reduction in growth and yield of the crop (Bhagwati

et al., 2007).

The severity of root rot caused by F. oxysporum increased in the presence of M. incognita.

Severity of the root rot disease was more prominent when the inoculation by M. incognita

preceded the F. oxysporum by three weeks on „Essoumtem‟ and Clemson spineless in both

pot and field experiment. Severe plant damage (1 + 1 ˃ 2) was realized when both pathogens

were concomitantly or simultaneously inoculated. Specific fungi and other plant pathogens

interact to form disease complexes (Begum et al., 2012). In the presence of root-knot

nematode, Rhizoctonia root rot was more severe. Root-rot disease complex, caused by R.

solani and M. incognita has been reported when root-knot nematode is present on green

beans (Bhagwati et al., 2007; Mokbel et al., 2007 and Abuzar, 2013). In this study,

72

concomitant and sequential infection of Meloidogyne incognita and F. oxysporum resulted in

more damages than the individual infection of the two pathogens on both „Essoumtem‟ and

Clemson spineless. Wilt severity caused by R. solani in a root-knot nematode infested field is

higher on green beans (Bhagwati et al., 2007; Mokbel et al., 2007; Abuzar, 2013).

73

CHAPTER SIX

6.0 CONCLUSIONS AND RECOMMENDATIONS

6.1 Conclusion

The southern root knot nematode, Meloidogyne incognita and Fusarium oxysporum f.

sp. vasinfectum were confirmed as the pathogens responsible for root knot disease and

Fusarium wilt respectively on okra.

Plant height, plant girth, chlorophyll content, fresh shoot weight, dry shoot weight,

fresh root weight and dry root weight were drastically reduced when nematode (M.

incognita) and fungus (F. oxysporum f. sp. vasinfectum) were simultaneously

inoculated on both „Essoumtem‟ and Clemson spineless, than when they were

inoculated on the plants individually.

The reproductive ability of M. incognita was reduced by the introduction of F.

oxysporum f. sp. vasinfectumon the two okra varieties, „Essoumtem‟ and Clemson

spineless.

Serial and simultaneous inoculation of M. incognita andF. oxysporum f. sp.

vasinfectumincreased significantly wilt incidence on „Essoumtem‟ and Clemson

spineless in both pot and field trials compared to when they were inoculated

individually. The inoculation of M. incognita 21 days prior to F. oxysporum f. sp.

vasinfectum, however, resulted in the most severe wilt incidence in the two okra

varieties („Essoumtem‟ and Clemson spineless) used in the experiment.

74

6.2 Recommendations

It is therefore recommended that:

The okra varieties used in this experiment, „Essoumtem‟ and Clemson spineless have

reacted differently to the two plant pathogens. Other okra varieties should be used to

study the interaction between M. incognita and F. oxysporum in wilt incidence.

The two pathogens, M. incognita and F. oxysporum should be controlled as early as

possible as the two can cause devastating damage on okra plants if allowed to fester

for at least 3 weeks (21 days).

In future studies, where nematode infestation is high, inoculation of fungus should be

done within 21 days to obtain a high incidence of wilt severity.

Further studies should be carried out on the interaction of the two pathogens (M.

incognita and F. oxysporum) looking at the correlation and regression analysis on the

two varieties of okra.

75

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APPENDICES

APPENDIX 1: Root–Knot Nematode Rating Chart – Bridge and Page

Source: Bridge and Page, 1980

96

APPENDIX 2: Analysis of variance of plant height for Pot experiment

Variate: Plant Height 4 WAP

Source of variation d.f. s.s. m.s. v.r. F pr.

TREATMENT 6 15.740 2.623 1.36 0.265

VARIETY 1 195.006 195.006 101.09 <.001

TREATMENT.VARIETY 6 2.959 0.493 0.26 0.953

Residual 28 54.013 1.929

Total 41 267.718



TREATMENT 6 60.469 10.078 1.94 0.110

VARIETY 1 311.604 311.604 59.86 <.001


Residual 28 145.747 5.205

Total 41 546.999



TREATMENT 6 340.52 56.75 3.71 0.008

VARIETY 1 354.38 354.38 23.19 <.001


Residual 28 427.80 15.28

Total 41 1252.66

97



TREATMENT 6 873.40 145.57 9.69 <.001

VARIETY 1 309.43 309.43 20.60 <.001


Residual 28 420.51 15.02

Total 41 1700.36

APPENDIX 3: Analysis of variance of plant girth for pot experiment

Variate: Plant Girth 4 WAP


TREATMENT 6 0.56000 0.09333 2.47 0.048

VARIETY 1 0.01167 0.01167 0.31 0.583


Residual 28 1.06000 0.03786

Total 41 2.12500



TREATMENT 6 2.8781 0.4797 2.50 0.046

VARIETY 1 0.2288 0.2288 1.19 0.284


Residual 28 5.3800 0.1921

Total 41 10.3564

98



TREATMENT 6 5.856 0.976 0.86 0.538

VARIETY 1 0.040 0.040 0.04 0.852


Residual 28 31.900 1.139

Total 41 40.781



TREATMENT 6 34.866 5.811 4.40 0.003

VARIETY 1 0.400 0.400 0.30 0.586


Residual 28 36.973 1.320

Total 41 76.131

APPENDIX 4: Analysis of variance of chlorophyll content for pot experiment

Variate: Chlorophyll Content 4 WAP


TREATMENT 6 62.761 10.460 1.15 0.360

VARIETY 1 61.492 61.492 6.77 0.015


Residual 28 254.420 9.086

Total 41 407.820

99



TREATMENT 6 52.951 8.825 1.25 0.311

VARIETY 1 1.760 1.760 0.25 0.621


Residual 28 197.563 7.056

Total 41 283.080



TREATMENT 6 264.255 44.042 5.46 <.001

VARIETY 1 28.274 28.274 3.50 0.072


Residual 28 225.919 8.069

Total 41 566.866



TREATMENT 6 1036.54 172.76 16.77 <.001

VARIETY 1 39.75 39.75 3.86 0.059


Residual 28 288.37 10.30

Total 41 1393.82

100

APPENDIX 5: Analysis of variance of fresh shoot weight for pot experiment

Variate: Fresh shoot weight


TREATMENT 6 1812059. 302010. 6.00 <.001

VARIETY 1 552918. 552918. 10.99 0.003

TREATMENT.VARIETY 6 350669. 58445. 1.16 0.355

Residual 28 1409306. 50332.

Total 41 4124952.

APPENDIX 6: Analysis of variance of dry shoot weight for pot experiment

Variate: Dry Shoot weight


TREATMENT 6 1639801. 273300. 6.41 <.001

VARIETY 1 687974. 687974. 16.13 <.001


Residual 28 1194167. 42649.

Total 41 4038645.

APPENDIX 7: Analysis of variance of fresh root for pot experiment

Variate: Fresh root


TREATMENT 6 546616. 91103. 1.77 0.141

VARIETY 1 154631. 154631. 3.01 0.094


Residual 28 1438087. 51360.

Total 41 2278599.

101

APPENDIX 8: Analysis of variance of dry root for pot experiment

Variate: Dry Root


TREATMENT 6 319604. 53267. 1.93 0.110

VARIETY 1 201409. 201409. 7.31 0.012


Residual 28 771548. 27555.

Total 41 1424442.

APPENDIX 9: Analysis of variance of number of pods for pot experiment

Variate: Number of pods


TREATMENT 6 1796.33 299.39 9.26 <.001

VARIETY 1 632.60 632.60 19.56 <.001


Residual 28 905.33 32.33

Total 41 3383.83

APPENDIX 10: Analysis of variance of pod weight for pot experiment

Variate: Pod weight


TREATMENT 6 874461. 145744. 9.25 <.001

VARIETY 1 82606. 82606. 5.24 0.030


Residual 28 441118. 15754.

Total 41 1418872.

102

APPENDIX 11: Analysis of variance of yield (kg/ha) for pot experiment

Variate: YIELD (kg/ha)


TREATMENT 6 15180958. 2530160. 9.25 <.001

VARIETY 1 1434344. 1434344. 5.24 0.030


Residual 28 7657722. 273490.

Total 41 24632022.

APPENDIX 12: Analysis of variance of gall index

Variate: Gall Index for pot experiment


TREATMENT 6 318.4762 53.0794 106.16 <.001

VARIETY 1 1.5238 1.5238 3.05 0.092


Residual 28 14.0000 0.5000

Total 41 336.4762

APPENDIX 13: Analysis of variance of egg count

Variate: Egg count for pot experiment


TREATMENT 6 490025.9 81671.0 439.77 <.001

VARIETY 1 5.4 5.4 0.03 0.866


Residual 28 5200.0 185.7

Total 41 495287.1

103

APPENDIX 14: Analysis of variance of reproductive factor for pot experiment

Variate: Reproductive factor


TREATMENT 6 8.911046 1.485174 678.83 <.001

VARIETY 1 0.001360 0.001360 0.62 0.437


Residual 28 0.061259 0.002188

Total 41 8.982853

APPENDIX 15: Analysis of variance of wilt incidence for pot experiment

Variate: Wilt Incidence


TREATMENT 6 30785.90 5130.98 398.34 <.001

VARIETY 1 0.02 0.02 0.00 0.966


Residual 28 360.67 12.88

Total 41 31355.07

APPENDIX 16: Analysis of variance of plant height for Field experiment

Variate: Plant Height at 4 WAP


REP stratum 2 3.123 1.562 0.56

TREATMENT 3 9.605 3.202 1.16 0.361

VARIETY 1 28.602 28.602 10.32 0.006


Residual 14 38.797 2.771

Total 23 84.398

104



REP stratum 2 19.240 9.620 2.28

TREATMENT 3 12.338 4.113 0.97 0.433

VARIETY 1 44.827 44.827 10.62 0.006


Residual 14 59.107 4.222

Total 23 144.565

Variate: Plant Height at 8WAP


REP stratum 2 16.843 8.422 3.13

TREATMENT 3 19.808 6.603 2.45 0.106

VARIETY 1 9.004 9.004 3.35 0.089


Residual 14 37.670 2.691

Total 23 85.680



REP stratum 2 18.481 9.240 5.03

TREATMENT 3 149.102 49.701 27.07 <.001

VARIETY 1 14.107 14.107 7.68 0.015


Residual 14 25.706 1.836

Total 23 207.838

105

APPENDIX 17: Analysis of variance of plant girth for field experiment

Variate: Plant Girth at 4 WAP


REP stratum 2 0.08563 0.04282 0.90

TREATMENT 3 0.15498 0.05166 1.09 0.385

VARIETY 1 0.02535 0.02535 0.54 0.477


Residual 14 0.66317 0.04737

Total 23 1.05438



REP stratum 2 0.12243 0.06122 0.98

TREATMENT 3 0.15793 0.05264 0.84 0.492

VARIETY 1 0.04167 0.04167 0.67 0.427


Residual 14 0.87277 0.06234

Total 23 1.36953



REP stratum 2 0.1633 0.0817 0.50

TREATMENT 3 0.6046 0.2015 1.24 0.332

VARIETY 1 3.3004 3.3004 20.35 <.001


Residual 14 2.2700 0.1621

Total 23 6.8596

106



REP stratum 2 0.2925 0.1463 1.00

TREATMENT 3 2.2746 0.7582 5.18 0.013

VARIETY 1 3.0104 3.0104 20.58 <.001


Residual 14 2.0475 0.1463

Total 23 7.9262

APPENDIX 18: Analysis of variance of plant girth for field experiment

Variate: Chlorophyll Content at 4 WAP


REP stratum 2 45.890 22.945 3.50

TREATMENT 3 8.649 2.883 0.44 0.728

VARIETY 1 230.144 230.144 35.09 <.001


Residual 14 91.826 6.559

Total 23 386.278



REP stratum 2 11.50 5.75 0.30

TREATMENT 3 18.09 6.03 0.31 0.814

VARIETY 1 809.84 809.84 42.27 <.001


Residual 14 268.24 19.16

Total 23 1129.02

107



REP stratum 2 36.94 18.47 1.49

TREATMENT 3 22.30 7.43 0.60 0.625

VARIETY 1 726.40 726.40 58.71 <.001


Residual 14 173.23 12.37

Total 23 979.70



REP stratum 2 28.599 14.299 2.76

TREATMENT 3 745.844 248.615 47.99 <.001

VARIETY 1 773.389 773.389 149.29 <.001


Residual 14 72.526 5.180

Total 23 1629.168

APPENDIX 19: Analysis of variance of fresh shoot weight for field experiment

Variate: Fresh Shoot Weight


REP stratum 2 1593.2 796.6 2.85

TREATMENT 3 4600.5 1533.5 5.49 0.011

VARIETY 1 35105.9 35105.9 125.58 <.001


Residual 14 3913.6 279.5

Total 23 45763.9

108

APPENDIX 20: Analysis of variance of dry shoot weight for field experiment

Variate: Dry Shoot Weight (g)


REP stratum 2 62.106 31.053 4.96

TREATMENT 3 255.720 85.240 13.62 <.001

VARIETY 1 395.606 395.606 63.20 <.001


Residual 14 87.628 6.259

Total 23 872.017

APPENDIX 21: Analysis of variance of fresh root weight for field experiment

Variate: Fresh Root Weight (g)


REP stratum 2 121.18 60.59 0.98

TREATMENT 3 503.24 167.75 2.72 0.084

VARIETY 1 2447.63 2447.63 39.65 <.001


Residual 14 864.32 61.74

Total 23 3969.89

APPENDIX 22: Analysis of variance of dry root weight for field experiment

Variate: Dry Root Weight (g)


REP stratum 2 7.107 3.554 2.24

TREATMENT 3 27.715 9.238 5.81 0.009

VARIETY 1 33.654 33.654 21.18 <.001


Residual 14 22.250 1.589

Total 23 92.816

109

APPENDIX 23: Analysis of variance for number of pods for field experiment

Variate: Number of pods


REP stratum 2 180.08 90.04 3.90

TREATMENT 3 184.79 61.60 2.67 0.088

VARIETY 1 2.04 2.04 0.09 0.771


Residual 14 323.25 23.09

Total 23 751.96

APPENDIX 24: Analysis of variance for weight of pod for field experiment

Variate: Weight of pod (g)


REP stratum 2 9316. 4658. 4.26

TREATMENT 3 21280. 7093. 6.49 0.006

VARIETY 1 6809. 6809. 6.23 0.026


Residual 14 15313. 1094.

Total 23 54115.

APPENDIX 25: Analysis of variance of yield for field experiment

Variate: Yield (kg)


REP stratum 2 19013. 9506. 4.26

TREATMENT 3 43430. 14477. 6.49 0.006

VARIETY 1 13896. 13896. 6.23 0.026


Residual 14 31250. 2232.

Total 23 110438.

110

APPENDIX 26: Analysis of variance of gall index for field experiment

Variate: Gall Index


REP stratum 2 16.750 8.375 2.20

TREATMENT 3 85.000 28.333 7.45 0.003

VARIETY 1 32.667 32.667 8.59 0.011


Residual 14 53.250 3.804

Total 23 208.000

APPENDIX 27: Analysis of variance of egg count for field experiment

Variate: Egg count


REP stratum 2 56.6 28.3 0.13

TREATMENT 3 2692.8 897.6 4.27 0.025

VARIETY 1 121.5 121.5 0.58 0.460


Residual 14 2946.1 210.4

Total 23 6167.8

APPENDIX 28: Analysis of variance of reproductive factor for field experiment

Variate: Reproductive factor


REP stratum 2 0.16127 0.08064 1.49

TREATMENT 3 0.31905 0.10635 1.96 0.167

VARIETY 1 0.10010 0.10010 1.84 0.196


Residual 14 0.75999 0.05429

Total 23 1.35956

111

APPENDIX 29: Analysis of variance of wilt incidence for field experiment

Variate: Wilt Incidence


REP stratum 2 61.083 30.542 3.06

TREATMENT 3 12928.125 4309.375 432.22 <.001

VARIETY 1 51.042 51.042 5.12 0.040


Residual 14 139.583 9.970

Total 23 13230.958

Documents

INTERACTION OF Meloidogyne incognita AND Fusarium