MANAGEMENT OF BACTERIAL WILT OF TOMATO BY

USE OF RESISTANT ROOTSTOCK

JARED NYAKUNDI ONDUSO

Bsc. Agric. (Hons), Nairobi

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT

FOR THE AWARD OF DEGREE OF

MASTERS OF SCIENCE IN CROP PROTECTION

DEPARTMENT OF PLANT SCIENCE AND CROP PROTECTION

FACULTY OF AGRICUTURE

UNIVERSITY OF NAIROBI

2014

i

DECLARATION

This is my original work and has not been presented for award of a degree or diploma in

any other university.

Jared Nyakundi Onduso …………..………………..…….. Date…………................

This thesis is submitted with our approval as the University supervisors

Prof. James W. Muthomi …………..…..…...………..… Date………………

Department of Plant Science and Crop Protection

University of Nairobi.

Prof. Eunice W. Mutitu ………..………...…………….... Date…………........

Department of Plant Science and Crop Protection

University of Nairobi.

ii

DEDICATION

This work is dedicated to my parents Mr. and Mrs. Alfayo Oute, my loving wife Rhoda,

sons Leo and Emmanuel, daughters Christine and Diana for their support, encouragement

and sacrifices during research work and thesis writing.

iii

ACKNOWLEDGEMENTS

I thank almighty God for his care and guidance throughout my life. This work might have

not succeeded without the immense support and guidance from my supervisors Prof.

James W. Muthomi and Prof. Eunice W. Mutitu who not only walked with me in every

step but offered training and mentorship during the entire work. I also wish to thank the

teaching and non-teaching staff of the University of Nairobi, Department of Plant Science

and Crop Protection for their immense support during the work. I thank the Technical

staff especially Godfrey Livasia who greatly put in many hours to work with me in the

laboratory as well as the field station. I also wish to recognize the support from Monsanto

Kenya for providing seeds for this work. I take the opportunity to thank Mr. Kinoti of

KARI NARL for the support in isolation of bacteria. I also thank Mr. Isaac Macharia and

Lucy Thungu and KEPHIS team at Muguga for assisting in serological for bacterial wilt

samples.

iv

TABLE OF CONTENTS

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

DEDICATION .................................................................................................................... II

ACKNOWLEDGEMENTS .............................................................................................. III

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

LIST OF FIGURES ........................................................................................................... ix

ACRONYMS AND ABBREVIATION ............................................................................. x

ABSTRACT ....................................................................................................................... xi

CHAPTER ONE: INTRODUCTION ............................................................................. 1

1.1 Background information ............................................................................................... 1

1.2 Problem Statement and Justification ............................................................................. 3

1.3 Objectives of the study.................................................................................................. 4

1.4 Hypothesis..................................................................................................................... 4

CHAPTER TWO: LITERATURE REVIEW ................................................................ 5

2.1 Tomato production in Kenya ........................................................................................ 5

2.2 Constraints to tomato production in Kenya .................................................................. 8

2.3 Bacterial wilt disease of tomato .................................................................................... 9

2.3:1 Causal agent of bacteria wilt ....................................................................................................... 9

2.3.2 Symptoms and signs of Ralstonia solanacearum ................................................................ 11

2.3.3 Epidemiology of bacterial wilt of tomato .............................................................................. 12

2.3.4 Disease distribution and host range of bacterial wilt of tomato............................... 14

2.4 Management of Bacterial wilt .................................................................................... 15

2.4.1 Cultural and phytosanitary options .......................................................................................... 15

2.4.2 Biological control options .......................................................................................................... 17

2.4.3 Chemical control options ............................................................................................................ 18

2.4.4 Use of resistance cultivars and rootstocks ............................................................................. 19

CHAPTER THREE: MATERIALS AND METHODS .............................................. 22

3.1 Determination of occurrence of bacterial wilt in open field

and greenhouse production ........................................................................................ 22

v

3.1.1 Description of study areas .......................................................................................................... 22

3.1.2 Determination of tomato production practices in key counties ....................................... 22

3.1.3 Assessment of bacterial wilt infection .................................................................................... 23

3.1.4 Isolation and identification of Ralstonia solanacearum .................................................... 24

3.2. Determinatin of effectiveness of reisistant tomato rootstock

in managing bacterial wilt under greenhouse production system ..................................... 25

3.2.1 Description of experimental sites ............................................................................................. 25

3.2.2 Experimental materials ................................................................................................................ 25

3.2.3 Experimental design and layout................................................................................................ 26

3.2.4 Crop management practices ....................................................................................................... 27

3.2.5 Assessments of bacterial wilt incidence and severity .............................................. 28

3.2.6 Assessment of fruit yield and quality ...................................................................................... 29

3.3 Evaluation of tomato rootstocks for bacterial wilt tolerance in the glasshouse .......... 29

3.3.1 Raising of seedlings and grafting ............................................................................................. 29

3.4.2 Experimental design and layout………………………………………….………..30

3.3.3 Inoculation and application of treatments……………………………….…………31

3.3:4 Assessments of bacterial wilt………………………………………………………31

3.4 Data analysis ............................................................................................................... 31

CHAPTER FOUR: RESULTS ...................................................................................... 32

4.1 Occurrence of bacterial wilt in open field and green house tomato production ...... 32

4.1.1 Tomato production practices ..................................................................................................... 32

4.1.2 Incidence and severity of bacterial wilt in farmers fields ................................................ 36

4.2. Tolerance of tomato rootstocks to R. solanacearum

under green house conditions………………………………………………………..39

4.2.1 Incidence and severity of rootstock to R.solanacearum………....…………………39

4.2.2 Yield for the varieties in selected farmers sites………………………………….…45

4.3 Tolerance of tomato rootstocks to bacterial wilt in glasshouse with R.

solanacearum inoculation ......................................................................................... 46

4.3:1 Plant mortality and bacterial wilt severity ............................................................. 46

4.3:2 Re-isolation of R. solanacearum from tomato rootstocks ..................................... 48

vi

CHAPTER FIVE: DISCUSSION .................................................................................. 50

5.1 Occurrence of bacterial wilt in key tomato producing counties ......................................... 50

5.2 Effectiveness of tolerant rootstocks in management of bacterial

wilt disease under field conditions ............................................................................................ 56

5.3 Tolerance of rootstock to bacterial disease under glasshouse condition .......................... 62

CHAPTER SIX: CONCLUSION AND RECOMMENDATIONS ............................ 64

6.1 Conclusions ........................................................................................................................................ 64

6.2 Recommendations ............................................................................................................................ 66

REFERENCES ................................................................................................................ 67

Appendix 1: Survey Questionnaire ............................................................................... 81

Appendix 2: Ingredient for R. solanacearum media .................................................... 81

vii

LIST OF TABLES

Table 2.1: Annual tomato Production (tons/hectare) in Kenya…………………...............5

Table 3.1 Descriptive features of rootstock varieties used in

both field and glass house experiments………………………………..……...26

Table 4.1 Percentage farmers grouping by gender and average farm

sizes in tomato growing counties in Kenya..……...............................................32

Table 4.2 Farmers percentage in irrigation water source and methods

of irrigation in tomato growing counties in Kenya ………............................... 33

Table 4.3 Percentage of farmers who cited different source of farmers

Extension service in tomato growing counties in Kenya ……………………...34

Table 4.4 Percentage of farmers who used selected seedling sources

in key tomato growing counties in Kenya……..............................................34

Table 4.5 Percentage of farmers adopting bacterial wilt management

practices in key tomato producing counties in Kenya ………………….........35

Table 4.6 Percentages of farmers who reported selected tomato diseases

in key tomato growing counties in Kenya…………...………………………...36

Table 4.7 Percentage samples testing positive to R. solanancearum on

selective media and serology test……………………………………. ……....38

Table 4.8 Bacterial wilt incidence, occurrence and percentage yield on

due to R. solancearum in key tomato growing counties in Kenya...…….…...39

Table 4.9 Percentage survival of tomato varieties from R. solanacearum

in farmers greenhouses……………………………………….………….........40

Table 4.10 Bacterial wilt browning scores from selected fields

in the experimental sites ………...……………….………...……..………….41

viii

Table 4.11 Bacterial oozing score of sampled plants in

selected experimental sites…………………………………………………..42

Table 4.12 Percentage colonies testing positive to R. solanacearum……………….…..44

Table 4.13 Harvested yield for each variety for selected experimental sites..………...…45

Table 4.14 Percentage survival of tomato varieties incubated with R. solanacearum

in glasshouse conditions at Kabete field station ………….…………………46

Table 4.15 Stem browning and oozing scores for R. solanacearum

for inoculated treatments in Kabete glass house experiment….…………...…48

Table 4.16 Percentage positive confirmatory tests for R. solanacearum

samples from the glass house experiment ………………...…………………49

ix

LIST OF FIGURES

Fig 4.1 Initial wilting of plants caused by R. solanacearum: In a farmer’s

field in Kiambu (B) in older crop in Isinya ……………………………………..37

Fig 4.2 Grafting union with plastic clip on (Figure C) and bacterial wilt

streaming (Figure D)…...………………………………………….………….…37

Fig 4.2 Colony positive to bacterial wilt pathogen on Kelman’s TC and SMSA………..37

x

ACRONYMS AND ABBREVIATION

AEZ Agro-ecological zones

AMF Arbuscular mycorrhizal fungi

ANOVA Analysis of Variance

AUDPC Area under disease progress curve

AVRDC Asian Vegetable Research and Development Center OEPP

ELISA Enzyme Linked Immunosorbent Assay

EPPO European and Mediterranean Plant Protection Organization

EU European Union

FAO Foo and Agricultural Organization of the United Nations

HCDA Horticultural Crops Development Authority

KARI Kenya Agricultural Research Institute

Kelman’s T.T.C. Kelman’s Triphenyl Tetrazolium Chloride

KHCP Kenya Horticultural Competitiveness Program

Masl Meters above sea level

NA Nutrient Agar

NARL National Agriculture Research Laboratories

PCR Polymerase Chain Reaction

PGPR Plant Growth Promoting Bacteria

pH Potential of Hydrogen

SMSA Semi-Selective Medium, South Africa

USAID United State Agency for International Development

xi

ABSTRACT

Bacterial wilt (Ralstonia solanacearum) is a devastating disease of tomato that is quickly

spreading and causing reduction in yield and income to farmers in Kenya. The objective

of the study was to determine the effectiveness of tomato rootstock in bacterial wilt

management. Effectiveness of Rootguard® and Nordox

® in controlling the diseases was

also evaluated. A survey was carried out in December 2011 in Kirinyaga, Kiambu,

Kajiado and Laikipia counties to collect tomato production information. Information on

production practices, diseases and pest present as well as control practices was gathered

by administering a questionnaire to the farmers in combination with farm evaluation

covering both open field and greenhouse production. The field experiment was carried

out in Kiambu, Ruiru, Karen and Isinya sites in farmers’ greenhouses where previous

crop was affected by bacterial wilt disease. A greenhouse variety Anna F1 was used as a

scion and was grafted on rootstock variety Cheong gang, Shin cheong gang and on a local

wild tomato variety. Efficacy of biological product Rootgard® and a copper fungicide

Nordox® were tested while non-grafted Anna F1 was used as a negative control. The

experiment was repeated under glasshoues conditions. In both field and glasshouse

experiment incidence of bacterial wilt, severity and yield data was collected.

From the survey it was found out that bacterial wilt is the most limitation factor to tomato

production in the counties with highest incidence recorded in Kiambu and lowest in

Kajiado. Higher wilt disease incidence was recorded in green houses as compared to open

field production. Poor agronomical practices were isolated as the important factor that

has contributed to the spread of the disease. Most farmers were found not to practice crop

xii

rotation and planted seedling from their own nurseries which were found to increase wilt

disease incidence. Poor field hygiene and flood irrigation use were also identified to aid

spreading of the disease.

Results strongly indicate that bacterial wilt disease severity and incidence was reduced

when wilt susceptible Anna F1 variety was grafted on Shin cheong gang rootstock.

Grafting susceptible Anna F1 tomato variety on Shin cheong gang variety reduced

bacterial wilt incidence by 95% and on rootstock variety Cheong gang by 92%. A wild

tomato variety also used as rootstock reduced disease incidence by 64%. Bacterial wilt

incidence for the varieties in the experimental sites differed with higher disease levels

being observed in Kiambu and Ruiru and least in Isinya and Karen. The tolerance levels

for each rootstock had a similar trend between the sites. Plants grafted onto Cheong gang

and Shin cheong gang had lowest disease incidence and produced high yield of tomato in

both quantity and quality when compared to non grafted Anna F1. This was true for both

field and glasshouse experiments. Tests on the grafted plants were negative to R.

solanacearum confirming that the pathogen was absent from the plants.

The study indicated that bacterial wilt can be effectively be managed by grafting

susceptible varieties on tolerant rootstock. With high level of tolerance grafted plants can

be planted in fields that are infected with R. solanacearum. Grafting also improved the

performance of the scion resulting in higher yield and better quality fruits than non-

grafted Anna F1.

xiii

1

CHAPTER ONE: INTRODUCTION

1.1 Background information

Tomato (Solanum lycopersicum) is native to South America and belongs to the family

Solanaceae (Jenkins, 1948). The crop is grown for its fruits which are used in salads or

cooked as a vegetable, in processed form as tomato paste (puree), tomato sauce, ketchup

and juice and the fruits are rich in vitamins A and C (AVRDC, 2003). Consumption of

tomatoes is gaining importance because it contains lycopene, a food component known to

reduce incidences of prostate cancer, heart and age related diseases as well as a source of

β-carotene (Fajinmi and Fajinmi, 2010). In Kenya the crop is mainly grown in open fields

under irrigation but also in plastic houses (greenhouses) to meet the increased demand for

tomato (Monsanto, 2013). Use of green houses guarantees continuous production

especially during rainy periods but also creates optimal conditions for proliferation of

many pathogens (Albajes et al., 1999). Poor methods of wilt disease management,

farmers are incurring big losses from the disease (Rivard et al., 2012). Key tomato

growing counties are Kajiado, Kirinyaga, Laikipia and Kiambu (KARI, 2005).

Top five tomato producers in the world accounts for 129 million tons with China

producing over a quarter of world production (FAO, 2013). It was estimated that in 2012

over 18,477 hectares were under tomato production in Kenya with an estimated

production of 539,151 tons (FAO, 2013). Poor practices such as continuous cropping of

the crop without rotation, furrow irrigation, poor field hygiene and use of infected

seedlings have lead to increased disease incidences (Monsanto, 2013).

2

Pests and diseases not only cause reduction of produce and quality but also increase cost

of production (Monsanto, 2013). Among the most devastating diseases attacking tomato

is bacterial wilt caused by R. solanacearum (Smith). This disease cause massive deaths of

plants resulting in reduced yields and incomes to farmers (Taylor et al., 2011). The

disease affects a wide range of crops especially members of solanaceae family such as

pepper, brinjals, and potatoes as well as their weed relatives and ornamental crops

(Champoiseau and Momol, 2009; Elphinstone, 2005).

R. solanacearum is a soil borne disease and once it infects the soil it easily spreads within

the field as well as to adjacent fields not only affecting the crop but also rendering the

farm unusable to production of any solanaceous crops (Sequeira, 1993). The disease is

known to spread very quickly through furrow irrigation as well as rain (Taylor et al.,

2011). Control of the disease is challenging as use of chemical products including

antibiotics, fertilizers and fungicides are not effective in managing the disease (Hartman

and Elphinstone, 1994). Use of cultural practices such as crop rotation has faced

challenges due to unpredictable survival of the pathogen (Sequeira, 1993), the pathogen

has the ability to survive in the soil for a period of up to 10 years even in the absence of

any vegetation (Sequeira, 1993; Smith et al., 1995).

Management of the disease is challenging as current practices and chemical products

have not been effective in managing the disease. Use of tolerant rootstock tomato

varieties has been widely used in the management of many soil borne diseases including

bacterial wilt (McAvoy et al., 2012). The use of tolerant rootstock has been in use since

early 20th

century in Asia with estimated grafted crops at 81% in Japan and 54% in South

3

Korea (McAvoy et al., 2012). This technology has been tested in other parts of the world

with impressive results but because the pathogen is composed of a number of distinct

strains, varieties tolerant to the disease in one region may not stand the disease in another

region (Freeman et al., 2011). Field studies show that grafting is one of the most

promising options in stemming the impact of bacterial wilt on tomato production as well

as increasing the overall productivity of tomato cultivars (Rivard et al., 2012, Taylor et

al., 2011).

1.2 Problem Statement and Justification

R. solanacearum being soil borne poses serious challenges in its management especially

in already infected fields leading to reduced incomes to small scale growers (Taylor et

al., 2011). A sustainable, affordable and effective control method needs to be introduced

to prevent further crop loss. The management strategy must guarantee continuous and

increased production of tomato. Tomato production being a key income earner to some

families it will ensure increased incomes for the farmer and fair prices to the consumers

(Taylor et al., 2011). Among the many methods available for R. solanacearum

management is use tolerant rootstock. Rootstocks a well as resistant varieties will provide

sustainable and environmentally sound management option (Freeman et al., 2011). To

test the tolerance of the varieties, an experiment was set where as susceptible variety was

grafted on two rootstock varieties and a local wild tomato. High tolerant rootstock variety

tested in key tomato producing regions in the experiment will be recommended for

commercialization with the farmers to manage the disease.

4

1.3 Objectives of the study

The main objective of the study was to develop effective option of managing R.

solanacearum by use of resistant rootstock.

The specific objectives were:

i. To determine the occurrence of R. solanacearum in major tomato producing

counties in Kenya.

ii. To evaluate the effectiveness of tomato rootstocks, Nordox® and Rootgard

® in

managing R. solanacearum

1.4 Hypothesis

i. Bacterial wilt is the most limiting disease for tomato farmers in key tomato

producing areas in Kenya

ii. Root stock variety Shin cheong gang F1 variety, and Nordox®

and Rootgard

® are

effective in controlling R. solanacearum

5

CHAPTER TWO: LITERATURE REVIEW

2.1 Tomato production in Kenya

Horticultural industry is one of the key income generating sectors of the Kenyan

economy with over one million tons of vegetables produced annually. Of the production

90% is consumed locally and 10% destined for the export market (Wiersinga et al.,

2008). Tomato is one of the most important vegetable crops ranking second to Brassica

mainly cabbage and kales in quantities produced as well as value and is ranked first in a

prioritization of vegetable crop value chains (KARI, 2005). Tomato is a financially

attractive horticultural crop with a potential for high incomes for small scale farmers and

provides a potential source of employment to many rural and peri-urban residents (Mbaka

et al., 2013). Tomato is mainly grown in low rainfall areas with increased production in

semi arid areas with the use of irrigation technology (KARI, 2005). Production of the

crop is mainly carried out by small scale growers with land sizes between 0.5-2.5 ha

(Mbaka et al., 2013). It is estimated that production of the crop is carried out on 18,477

ha as indicated in Table 2.1 with an annual production of 539,151 metric tons (FAO,

2013) with an estimated value of KES 14.2 billion (HCDA, 2011).

Table 2.1: Annual tomato production (tons/hectare) in Kenya

Year

2007 2008 2009 2010 2011

Production (tones) 559,680 400,270 526,922 539,151 407,374

Total hectares 18,656 13,500 17,500 18,477 18,178

Yield (t/ha) per season 15.0 14.8 15.1 14.6 11.2

(Source FAO, 2013)

6

Production of tomato over the years has been carried in open field conditions but in the

recent past adoption of plastic tunnels popularly referred to as ‘greenhouses’ is on the

rise (Mbaka et al., 2013). The innovation has attracted more educated youths to

horticultural farming since it is perceived as smart, modern and cutting edge technology

(Mbaka et al., 2013). Tomato seeds supply has been achieved by support from seed

companies through the supply of a range of varieties to meet farmer’s demand. The main

tomato varieties grown in Kenya can be categorized into those grown in greenhouses and

those grown in the open field. Varieties grown in the green house include Anna F1,

Nemoneta F1, Corazzon F1, Tylka F1, Claudia F1, Chonto F1 and Prostar F1 (Monsanto,

2013). In open field production system the open pollinated (OPV) varieties commonly

grown include Riogrande and Cal-J supplied by a number of seed companies (Monsanto,

2013) This varieties have replaced earlier varieties such as Money Maker, Fortune,

Kentom, Neema 1400, Neema 1200, Caltana, Manset, Rotade (Wiersinga et al,. 2008)

which are no longer popular with farmers. Hybrids varieties such as Eden F1, Assila F1,

Kilele F1, Valoria F1, Shanty F1, Nuru F1 and Tropicana F1 have been introduced to

offer increased yield and disease tolerance (Monsanto, 2013).

Tomatoes are fairly adaptable plants, but grow well in warm conditions with optimum

temperatures of 15°C -25 °C (Waiganjo et al., 2006). Very low temperature can delay

colour formation as well as ripening while temperatures above 300C inhibit fruit set,

flavor and Lycopene development (Waiganjo et al., 2006). Tomatoes grow well in a wide

range of soil types, which are high in organic matter, well-drained and with a pH range of

5 to 7.5 and moderate rains (Wiersinga et al., 2008). Wet conditions increase disease

7

incidences and affect fruit ripening. With the greenhouse technology, farmers are now

able to utilize small pieces of land to produce high quality tomato for specialized markets

(Mbaka et al., 2013). Production of tomato is carried out in two main seasons based on

availability of rains as well as anticipated pest and disease pressure. The main production

areas for open field tomato types include Kirinyaga, Nyandarua, Kajiado and Narok

counties with Mwea, Loitoktok and Rumuruti being the main production regions

(Monsanto, 2013). Raised plastic tunnel production system is mainly practiced in the

outskirts of Nairobi, Kajiado, Kiambu and Nakuru Counties (Mbaka et al., 2013).

The main marketing channels for tomatoes include supply to open air markets through

middle men, direct orders to supermarket and groceries chains as well as local hotels

(Wiersinga et al., 2008). In the recent past, well established market linkages between the

farmer and the market in sub-contract supply arrangements with leading groceries chains

such as Fruit and juice, Zucchini and specialized vegetable markets in Westland and

Parklands in Nairobi has emerged (Monsanto, 2013). This requires established farms and

farmers who are able to consistently supply the product. A large percentage of the

farmers still market their tomatoes through markets such as Wakulima market, Githurai,

Kangemi and City Park. The biggest challenge hindering small scale farmers from

accessing specialized markets include quality issues, consistent supply, presence of

pesticide residues and poor product hygiene (Wiersinga et al., 2008). Tomato retailers

cite perishability, price fluctuations, poor transport networks from the farms and lack of

storage facilities as challenges. Transport of produce from the field is usually done by use

of wooden boxes loaded in trucks resulting in damages fruits (KHCP, 2011).

8

2.2 Constraints to tomato production in Kenya

Key production challenges for tomato crop include pests and disease (Singh et al., 2014a,

Black et al., 2003) as well as marketing (KHCP, 2011). The major insect pests attacking

the crop include; whiteflies, nematodes, spider mites, thrips, Leaf miners, African

bollworm and aphids (KARI, 2005; Waiganjo et al., 2006). Whiteflies are key pest

especially in dry seasons as they suck plant sap reducing plant productivity and also

cause sooty mould on fruits which reduce fruit quality. Whiteflies also spread viral

diseases such as tomato yellow leaf curl virus that can wipe out fields (Monsanto, 2013).

During dry periods especially those associated with dust, red spider mites (Tetranychus

spp) can infest the tomato plant sucking sap which can resulting in leaf curling, webbing

and loss of leaves directly affecting yield (Knapp, 1999). Physiological disorders such as

nutrient deficiencies, water logging and drought can also affect the crop causing

significant losses in quality and quantity. The crop is also affected by calcium deficiency

that causes blossom end rot disease on fruits (KARI, 2005).

Diseases remain the biggest challenge in tomato production. It is estimated that there are

more than 200 known diseases affecting tomatoes (Jones, 2008). Tomato diseases are

rampant in lowlands, highlands, tropics and can cause 15-95% crop loss (Abdullah, 1988;

Hayward, 1991; Tahat et al., 2010). Some of the major diseases affecting this crop in

Kenya include; early and late blight, Fusarium wilt, yellow leaf curl virus, tobacco

mosaic virus, septoria leaf spot, powdery mildew, bacterial canker and bacterial spot

(KARI, 2005; Singh et al., 2014b). In their effort to control pests and diseases farmers

use pesticide products excessively with over 40 applications per season recorded in some

9

tomato fields (Waiganjo et al., 2006). The uncontrolled and unregulated application of

the pesticides continue to be an occupational health hazard to the farmer, cause food

poisoning to the consumer and more importantly degrade the environment. Some farmers

have reported health issues which have been linked to the effects of pesticide and poor

use of pesticide products (Waiganjo et al., 2006).

Bacterial wilt has been reported by growers in the country with over 64% crop loss

reported for open field production and up to 100% in green houses production systems

threatening production of the crop in the country (Mbaka et al., 2013). This disease is

challenging due to its destructive nature, wide host range and geographical distribution,

and it is believed to be the most important bacterial disease of plants in tropical,

subtropical and warm temperate zones of the world (Hayward, 1991; Kelman, 1954). In

addition to the loss of plants and yield, the disease is blamed for abandonment of

cultivation of tomatoes on previously productive farms due to serious infestation of the

disease as the pathogen persists in a wide range of crop and weed hosts (Kelman, 1963).

Bacterial wilt is a threat to tomato production because of limited control strategies the

disease that cannot be effectively managed with chemicals.

2.3 Bacterial wilt disease of tomato

2.3:1 Causal agent of bacteria wilt

Ralstonia solanacearum is an important pathogen of many crops (Tahat and

Kumaruzaman, 2010) and was reported for the first time at the end of the 19th

Century on

potato, tobacco, tomato and groundnut in Asia, southern USA and South America (EPPO,

10

2004). The disease is highly infectious both in soil and in soilless culture causing wilt of

plants especially in solanaceae family. Affected crops include: tomato, eggplant, and

sweet pepper (Jenkins and Averre, 1983). The pathogen is a gram-negative, rod shaped,

largely aerobic bacterium that is 0.5-0.7x1.5-2.0 μm in size capable of surviving for long

at -80°C in liquid culture broth containing 40% glycerol (Denny, 2006).

The pathogen is a complex of bacterial species grouped into races. Five biovars on the

ability produce acid from panel carbohydrates (Denny, 2006) and recently genomovars,

phylotypes and sequevars (Prior, 1990b). Race 1 is a poorly-defined group with a very

wide host range and is endemic to the southern United States as well as Africa, Asia and

South America. Race 2 is known to attack bananas and is mainly found in Central

America and Southeast Asia. Race 3 is distributed worldwide and has primarily been

associated with potato. Race 4 affects ginger mainly in Asia and Hawaii and race 5

affects mulberries in China (Denny, 2006; Kelman, 1997). Use of genetic printing

techniques such as RFLP has greatly helped to classify the pathogen into divisions

(Hayward, 2000) with division I (biovars 3, 4 and 5 originating in Asia) and II (biovars

1,2A and 2T, originating in South America). Further taxonomical division mainly based

on nucleic sequences analysis into phyllovars and sequevars has been proposed (Poussier

et al., 2000; Taghavi et al., 1996).

The pathogen is a quarantine pest worldwide and has been listed as a select agent plant

pathogen under the Agricultural bioterrorism Act of 2002 in the USA (Champoiseau and

Momol, 2009). The origin of the pathogen is not clear, but Hayward (1991) suggested

11

that it predates the geological separation of the continents as the bacterium has been

found in virgin jungle in South America and Indonesia. However, race 3 biovar 2 strains

are believed to originate in the Andean highlands and this near-clonal subgroup is widely

distributed in tropical ones throughout the world and some temperate regions including

Europe and northern Asia (Tahat and Kumaruzaman, 2010).

The pathogen can be isolated from any parts of the plant and in most cases the stems are

used (Mwangi et al., 2008). The pathogen can also be isolated from the soil, waste, or

surface using a number of available media. SMSA medium as modified by Elphinstone et

al., (1996) has been used successfully in Europe (Elphinstone et al., 1998). Isolation from

symptomatic material can easily be performed using YPGA non-selective medium or

Kelman’s tetrazolium medium (EPPO, 2004). Detection of latent infection can be done

by performing an immuno-fluorescence test and/or selective plating on SMSA medium

which are eventually combined with optional PCR assays, ELISA or fluorescent in situ

hybridization tests can be formed after carrying out procedures to increase sensitivity

(OEPP, 2004; Lelliott and Stead, 1987). The bacterium is usually re-isolated from plants

by taking a stem section above the ground and placing it in a small volume of sterile

distilled water or 50 mm phosphate buffer, plating on YPGA and/or SMSA medium, and

observing for typical colonies (EU, 1998).

2.3.2 Symptoms and signs of Ralstonia solanacearum

Plants affected by the disease show wilted leaves and stems usually visible at the warmest

time of the day, the wilting persist until the plant dies where youngest leaves are the first

12

to be affected and have a flaccid appearance (EPPO, 2004). In most cases the stem near

the root produces many adventitious root buds and roots indicating infection to the

vascular bundle (EPPO, 2004). Plants may exhibit discoloration of the vascular system by

showing a streaky brown to yellow cream discoloration and the plant leaves may have a

bronze stint and epinasty of the petiole (Agrios, 2005). The permanent wilting usually

occurs due to a massive invasion of the cortex which may result to water-soaked lesions

on the external surface of the stem; if an infected stem is cut crosswise tiny drops of dirty

white or yellowish viscous ooze exude which indicates presence of bacterial cell from

several vascular bundles (Champoiseau and Momol, 2009; OEPP, 2004). In some cases

the plant may have latent infection where none of these symptoms, even under typical

environmental conditions that are ideal for the pathogen are expressed (Adebayo and

Ekbo, 2005; Ariboud et al., 2014).

2.3.3 Epidemiology of bacterial wilt of tomato

Ralstonia solanacearum is both a soil borne and waterborne pathogen, the bacterium is

known to survive and disperse in infected soil or water which can form a reservoir source

of inoculum from season to season (Fajinmi and Fajinmi, 2010). The ability of the

pathogen to survive in soils and water is responsible for continuous spread in surface

irrigated fields. The pathogen infects host plants primarily through roots and the vascular

bundle entering the plant through wounds formed by lateral root emergence, those caused

by soilborne organisms especially insects and nematodes (Adebayo and Ekpo, 2006).

Disease transmission can also occur through wounds from mechanical damage when

carrying out cultural activities (Champoiseau and Momol, 2009). McCarter (1991)

13

reported that in special cases, plant-to plant spread can occur when the bacteria move

from roots of infected plants to roots of nearby healthy plants but also long distance

movement of the pathogen can occur with transportation of latently infected seedlings.

The bacteria has the ability to survive for years in infected water, wet soils or in soil

layers greater than 75cm from where it can be dispersed and only antagonistic

microorganisms and environmental factors, mainly temperature, soil type and soil

moisture can affect its survival (Champoiseau and Momol, 2009; Denny, 2006). Spread

of bacteria by aerial means and subsequent plant contamination through foliage is not

known to occur, thus making R. solanacearum a non airborne pathogen (Agrios, 2005).

Survival of the pathogen from season to season occurs in host plants, volunteer crops,

solanaceous weeds and in crop residues.

High temperatures (29-35ºC) play a major role in pathogen growth and disease

development as temperatures below 18ºC inhibit disease development (Hayward, 1991)

but the pathogen can survive in a physiological latent state (Champoiseau and Momol,

2009). Several other factors that affect pathogen survival in soil as well as in water may

also favor disease development including; soil type and structure, soil moisture content,

organic matter in soil, water pH and salt content, and the presence of antagonist

microorganisms (Fajinmi and Fajinmi, 2010). The infection rate of the disease is

positively influenced by the type of soil. Abdullah et al. (1983) found that soil type and

moisture levels individually as well as in combination had a significant effect on the

severity of the bacterial wilt of groundnut. It has been reported that low disease

incidences occur in organic soils with organic matter more than 65% (Abdullah, 1988).

14

Jatala et al. (1988) reported that nematode injury to the plant enhance disease spread as

well as severity and by controlling the nematodes will reduce disease spread as well as

severity. The synergistic interaction between root-knot nematode (Meloidogyne spp.) and

R. solanacearum on a variety of hosts is widely recognized. Root infection by nematodes

has a positive correlation with bacterial wilt as the wounds created by the nematodes on

the root system provide points of pathogen entry (Agrios, 2005).

2.3.4 Disease distribution and host range of bacterial wilt of tomato

The first record of Ralstonia solanacearum was reported by Burrill (1890) in Japan.

Across the world there are differences between R. solanacearum races and biovars

depending on the geographical distribution (Hayward, 1991). Biovar 1 is predominant in

USA and biovar 3 in Asia, whereas biovar 2 and 5 occur in Australia (Pitkethley, 1981).

Strains of R. solanacearum affects over 200 plant species in over 50 families and has

been reported in many parts of the world (Champoiseau and Momol, 2009) race 1 being

the most common (Denny, 2006; Mwangi et al., 2008). The pathogen is also reported to

cause Moko diseases in banana (Peeter et al., 2013). One plant species that is seriously

affected by bacterial wilt is tomato and the efforts to grow tomato widely in the tropics

have generally been hampered by this disease (Hayward, 2000).

It has also been found that biovar 4 occurs in India and Indonesia. In Africa the bacterial

wilt disease has been recorded in Egypt, Kenya, Libya, South Africa, Zambia and

Burundi (Gitaitis and McCarter, 1992). In the Philippines, biovars 1-4 have been found

and in Asia biovar 3 is a predominant biovar in lowland regions. Strawberry is a host in

15

Japan and Taiwan but not in the southeastern USA (Hayward, 1991). Around 43 plant

species were found to be hosts of the bacterial wilt disease in Malaysia especially biovar

3 race 2 (Abdullah, 1982).

Bacterial wilt disease was also observed in Cameron Highland of Malaysia at about 1,545

Masl. The survival of the pathogen in host or weed plants has been documented as the

disease was isolated from the infected crop plants and weeds at the farm of University

Putra Malaysia, Selangor (Gitaitis and McCarter, 1992). Two new hosts for R.

solanacearum: davana (Artemisia pallens) and coleus (Coleus forskohlii) were recorded

by Chandrashekara and Prasannakumar (2010). Both are important crops in medicinal

and aromatic industries in India.

2. 4 Management of Bacterial wilt

2.4.1 Cultural and phytosanitary options

The control of R. solanacearum is challenging once the pathogen has infested the soil

(Jones, 2008). Even with the effort of the farmers to adopt integrated disease

management strategies such as cultural practices, crop rotation and use of resistant

cultivars, limited success has been recorded (Mbaka et al., 2013). Among the cultural

practices, crop rotation, intercropping or incorporation of green manure and planting a

susceptible crop such as mung bean before the cultivation are among the many

documented cultural practices that can be used to manage the disease (Hartman et al.,

1994). The infection by this disease can be significantly reduced by using non-susceptible

16

crops and crop rotation for 5-7 years (Smith et al., 1995). Use of crop rotation has been

shown to reduce disease incidence, it is limited in management as the pathogen

population continues to proliferate because the pathogen is able to survive in the soil over

a long time but also complicated with existence of weeds and volunteer crops of

solanaceous family (Fajinmi and Fajinmi, 2010). Crop rotation with a non-susceptible

crop provides some control, but this can be limited as the diseases has a very wide host

range (Saddler, 2005). In the United States, Southern tomato transplant growers

sometimes manage the disease by avoiding infected fields (Hayward, 1964).

There is some significant reduction of the disease when organic manure is used as

demonstrated by Islam and Toyota (2004) where bacterial wilt of tomato was suppressed

when poultry and farmyard manure was added to the soils increasing microbial activity.

The application of the organic amendment and compost released biologically active

substances from crop residues and soil microorganisms such as allelochemicals have

been reported to reduce the disease (Chellemi et al., 1997)

In locations where the pathogen is not present, it is critical to prevent introduction and if

inadvertently introduced, subsequent movement of the pathogen should be prevented.

Planting certified disease free seedling from registered plant raisers, disinfecting

equipments after working in a field, controlled use of flood irrigation and avoiding

overhead irrigation can reduce spread of the disease (McCarter, 1991). Growers should

monitor potentially infected sites for early detection and subsequent eradication of

pathogen (Fajinmi and Fajinmi, 2010). Weeds can also host the pathogen and their

17

control will contribute to the disease management especially those around the tomato

field and irrigation reservoirs (Champoiseau and Momol, 2009; Momol, 2005)

2.4.2 Biological control options

Use of biological controls products for soil borne pathogen has gained popularity in

recent years due to environmental concerns raised on the use of chemical products in

disease control (Haas and De’fago, 2005). Biological control methods have been widely

accepted and advocated for as key practice in sustainable agriculture with the biggest

potential of the biological control being microorganisms, arbuscular mycorrhizal fungi

(AMF) (Sharma and John, 2002; Tahat et al., 2010) and some naturally occurring

antagonistic rhizobacteria such as Bacillus sp., Pseudomonas sp. (Guo et al., 2004). Use

of AMF in agricultural crops can provide protection against soil-borne pathogens by

reducing the root diseases caused by a number of soil pathogens (Sharma et al., 2004;

Trotta et al., 1996). A number of mechanisms are involved in controlling and suppression

of the pathogen by mycorrhizal fungi roots among them exclusion of pathogen, changed

nutrition, lignifications of cell wall, and exudation of low molecular weight compounds

(Chellemi et al., 1997; Tahat et al., 2011).

Other biological agents that have been used for the disease management include;

Fluorescent pseudomonads such as Pseudomonas fluorescens which are antagonistic to

soil-borne pathogens by production of antimicrobial substances, competition for space,

nutrients and indirectly through induction of systemic resistance (Kavitha and Umesha,

2007). It was reported by Wall and Sanchez (1992) that bacteriophages, which are

18

capable of attacking the R. solanacearum, have been used as a bio-control. Plant growth

promoting bacteria (PGPR) strains are reported to be a promising bio-control agent to

control R. solanacearum. A fungi, Pythium oligandrum has been reported by Akira et al.

(2009) to suppress bacterial wilt caused by R. solanacearum but yet to produced and

formulated for use on a commercial scale.

2.4.3 Chemical control options

Bacterial wilt control using chemicals is a challenge because of the localization of the

pathogen inside the xylem and its ability to survival in the soil. There are no known

eradication bactericides available for chemical control of the bacterial wilt disease

(Hartman et al., 1994), while others reported that it is difficult to control bacterial with

chemicals (Grimault et al., 1994). Earlier use of the banned soil fumigant such as methyl

bromide in controlling bacterial wilt disease did not succeed (Chellemi et al., 1997). Ji et

al. (2005) reported some control by use of phosphoric acid. Soil treatments, including

modification of soil pH, solarization and application of stable bleaching powder reduced

bacterial populations and disease severity on a small scale (Saddler, 2005). There is a

report of significance control of R. solanacearum with application of urea fertilizer as

well as potassium Nitrate fertilizer but their use on commercial scale has not yet been

tested. Use of chemical product to manage the disease ultimately contributes to

environmental degradation apart from being labor intensive and expensive (Fajinmi and

Fajinmi, 2010).

19

The bactericides Terlai has been tested in Taiwan under both greenhouse and field

conditions (Hartman et al., 1994) and it was found that chemical control through soil

fumigation and antibiotics (Penicillin, Ampicillin, Tetracycline and Streptomycin) has

shown little suppression of the pathogen. Application of a resistance inducer such as

acibenzolar-S-methyl (Actigard -Syngenta) or in combination with moderately resistant

varieties can give increased control against the disease. Similarly, application of another

product Thymol, a plant -derived volatile chemical has also shown to give positive results

in managing the disease (Champoiseau and Momol, 2009).

2.4.4 Use of resistance cultivars and rootstocks

Host resistance is an efficient and effective component in integrated management of

bacterial diseases and some tomato cultivars provide moderate resistance against bacterial

wilt disease (Peregrine, 1982). This is has been made possible by genetic improvement

on varieties to increase tolerance against R. solanacearum (Liao et al., 1998). The use of

resistant varieties has been reported to be the most effective and practical method to

control bacterial wilt (Black et al., 2003; Grimault et al., 1994). R. solanacearum is a

complex and heterogeneous species group with a wide host range (Kelman et al., 1961)

high variability in its biochemical properties (Cuppels, 1978; Hayward, 1964) serological

reactions (Schaad et al., 1978) membrane proteins (Dristig and Dianese, 1990) and phase

susceptibility (Okabe and Goto, 1963) confirming the existence distinct strains posing a

challenge in breeding for resistance. Although the species occur worldwide, distribution

of individual strains is not uniform leading to resistance breakage of a highly tolerant

variety from a different geographical area (Black et al., 2003).

20

Resistance to R. solanacearum has been reported in some tomato genotypes but

incorporation of resistance into materials with good horticultural characteristics has been

difficult (Peregrine, 1982; Peterson et al., 1983). R. solanacearum strain type, genetic

variability of the plant and reproducibility of the inoculation technique may affect the

selection of resistant material (Prior et al., 1990a). Some R.solanacearum resistant

cultivars have been developed from the Asian Vegetable Research and Development

Center (AVRDC). However, their resistance is restricted to locations, climate, and strains

of the pathogen and soil characteristics (AVRDC, 2003). A number of tomato varieties

have been developed with significant levels of resistance for certain environments

(Gomes et al., 1998); in a number of cases the stability in regions with high temperatures

and humidity especially in lowland tropics is difficulty to achieve as resistance breaks

when variety is transferred to a different region (Hayward, 1991; Hanson et al., 1996).

Use of resistance varieties and rootstock provide a more stable strategy in management

but performance will vary with temperature and location (Wang et al., 1998). Abdullah

(1998) found that the degree of susceptibility to bacterial wilt is significantly different

among six tomato cultivars which were tested and this indicated that the additive genes

were more important than the non-additive genes. Grafting susceptible tomato cultivars

onto resistant tomato or other solanaceous rootstock has recorded high control against

Asian strains of R. solanacearum and this technology has been used in many parts of the

world (Black et al., 2003; Saddler, 2005). Grafting experiments has indicated that the

percentage of wilting of Ponderosa scions was less on Hawaii 7996 rootstocks than that

on the most resistant rootstock (LS-89) used in Japan. Hawaii 7996 could be an

21

alternative genetic source for breeding for resistance to bacterial wilt (Nakaho et al.,

2004). Use of grafting techniques provides new possibilities as resistant rootstocks can be

grafted with preferred commercial tomato varieties (Taylor et al., 2011). In future R

.solanacearum management with resistant varieties and resistant rootstocks will offer the

most economic and more sustainable solution to the problem of R. solanacearum (Mbaka

et al., 2013; Wang, 2005).

22

CHAPTER THREE: MATERIALS AND METHODS

3.1 Determination of occurrence of bacterial wilt in open field and greenhouse

production

3.1.1 Description of study areas

A survey was carried out in selected tomato growing counties in Kenya including Kirinya,

Kajiado, Kiambu and Laikipia counties. Kirinyaga county is one of the counties in

Central Kenya and is of high agricultural potential (Waiganjo et al., 2006). The region has

an annual rainfall of 800-2200 mm, temperature range of 9.7 - 21.60C with deep and

moderately fertile soils. With two main rivers Thiba and Nyamidi, irrigation water is

readily available to farmers enabling them to utilize 85% of the 112,700 ha for agriculture.

Main crops grown in Kirinyaga county include tomato, french beans and onion. Laikipia

County is in the Rift Valley region and was selected to represent medium altitude low

rainfall (400- 750 mm), with average temperature of 16.6 to 260C. Kiambu county is

situated in Central part of Kenya with annual rainfall of about 1500 mm annually with

well drained high fertility soil suitable for farming (KARI, 2005). Kajiado county is dry

with less than 500 mm annually rainfall with predominantly black cotton soil (KARI,

2005). The survey was carried out from December 2011.

3.1.2 Determination of tomato production practices in key counties

A structured questionnaire (Appendix I) was administered to each respondent farmer. The

survey covered both greenhouse as well as open field production systems. To assist in

identification of the disease by farmers, pictures of plants affected by R. solanacearum to

distinguish it from other wilting diseases. Purpose random sampling was used to select

23

households where a farm on either side of the road but interviews only granted to farmers

who have been active in tomato growing in the last six months. An average of twenty

farmers was visited and interviewed per county. The information gathered included social

economic indicators such as land size, gender, period the farmers has been active in

farming, and the main crops grown. Data on whether irrigation was carried out in the farm

and water source, disease and pest levels, practices such as crop rotation, use of fertilizer

and manure, and management practices used by the farmer in management of diseases

especially bacterial wilt of tomato was collected. Data on bacterial wilt incidence as well

as estimated yield losses incurred by the farmer in the previous crop was also collected.

3.1.3 Assessment of bacterial wilt infection

Disease incidence in the field was determined by visual assessment using signs and

symptoms. Diagonal sampling method was used where 10 plants were systematically

picked from the farmers field for assessment. Sampled plants were checked for bacterial

wilt symptoms including wilting and stem discoloration and findings recorded. Percentage

disease incidence per farm was calculated by dividing the affected plants by the total

plants assessed multiplied by hundred. Disease incidence for the county was calculated

using the average from the sampled farms. Field diagnostic tests for bacterial wilt were

carried out by cutting a tomato stem of about eight centimeter in length picked from the

root base of a wilted plant and the stem portion placed in a clear glass beaker filled with

clear water. The presence of oozing milky exudates from the cut stem section was proof

that the pathogen was R. solanacearum (Goszczynska et al., 2000). In addition, five

sample plants were collected per farmer for further laboratory tests and pathogen isolation.

24

3.1.4 Isolation and identification of Ralstonia solanacearum

Stem samples cut of about 15 cm of the stem above the ground collected from the field

were cut into small pieces and surface sterilized with a solution of 1% sodium

hypochloride and triple rinsed in sterile water. The pieces were macerated in sterile

distilled water and allowed to stand for two minutes. Using a wire loop, the extract was

streaked on nutrient agar (23g nutrient agar dissolved in a liter of distilled water), SMSA

(1g casamino acids, 10g bacto-peptone, 5g dextrose, 5ml glycerol and 15g bacto-agar

dissolved in 1 liter distilled water) and Kelmans’ triphenyl tetrazolium chloride media (1g

casamino acids, 10g bacto-peptone, 5g dextrose and 15g bacto-agar dissolved in a liter of

distilled water). The inoculated petri dishes were incubated up side down for a period of

30 - 48 hours. R. solanacearum colonies were large, elevated, fluidal, and either entirely

white or with a pale red center as given by Kelman et al. (1961). Bacterial culture of R.

solanacearum isolated were purified and preserved in sterilized distilled water and stored

at 4°C in screw cap bottle for experimental use.

Nitrocellulose membrane enzyme linked immunosorbent assays (NCM-ELIZA)

procedure was carried out on the samples. Using a sterilized knife samples were cut into

small pieces of the stem and placed in small polybags. Extraction buffer (3 ml/g) was

then added and placed in iced containers. Crashing of the samples using wooden rollers

was done gently to increase surface. 500 μl of extraction buffer and 500 μl of SMSA

media (1/2 ml) were added prior to crashing. The samples were then incubated for 48

hours and serology test done by loading into ELIZA plates. Samples with colour changes

conforming to positive check were recorded.

25

3.2 Determination of effectiveness of resistant tomato rootstock in managing

bacterial wilt under greenhouse production system

3.2.1 Description of experimental sites

Experiments were carried out in greenhouses in Kiambu and Karen representing a higher

rainfall, cooler region and Ruiru and Isinya sites representing lower rainfall warmer

region. Karen area has an elevation of 1,661 masl with an average annual temperature of

17.70C, average annual rainfall of 925 mm with predominant black cotton soil. Kiambu

area has an elevation of 1,720 masl with an annual rainfall average of 989 mm, average

temperature of 18.70C with red clay to loamy soils. Ruiru site was located in a drier

region with annual average rainfall of 798 mm with average temperatures of 19.50C and

Black cotton to red loamy soils. Isinya site which was located dry region 1,707 masl with

annual average rainfall of 550 mm, average temperature of 23.50C and black cotton to

loamy soil.

3.2.2 Experimental materials

All the experiments were conducted in farmers owned greenhouses with R. solanacearum

incidence levels of 25% in Isinya, 30% in Karen, 45% and Kiambu 50% from the survey

data. During land preparation thorough mixing of the soil was done to ensure uniform

distribution of the pathogen. Farm yard manure used was from the same farm and land

preparation methods used was similar for all the sites. Selected rootstock varieties

Cheong gang and Shin Cheong gang documented to have high resistance to bacterial wilt

were used as indicated in table 3.1.

26

Table 3.1 Descriptive features of rootstock varieties used in both field and glass house

experiments

Rootstock Cheong Gang Shin Cheong Gang

Vigour Very high Very high

Bacterial wilt resistance High tolerance High tolerance

Nematodes tolerance Intermediate tolerance (IR) Intermediate tolerance (IR)

Source Asian breeding program Asian breeding program

Supplier Seminis Seminis

Source: Monsanto (2013)

Tomato seedlings were raised in the nursery before transplanting. The seedlings were

grafted using top grafting 10 days after emergence when they were 1.5 mm thick.

Grafting was done in moist chambers where same thickness scion and root stock were cut

at 45 degrees and joined by plastic clips. After grafting the seedling were transferred to a

humidity house for four days before being taken to the healing chamber. Transplanting

was done after 10 days. Non grafted seedlings remained in the greenhouse before they

were transplanted.

3.2.3 Experimental design and layout

Farmers with existing greenhouses were selected and each green house had six beds of

1.2 m in diameter. Each bed was subdivided into four plots measuring 4 m in length.

Two beds formed an experimental block. The treatments used were:

1. Anna F1

2. Anna F1 grafted on Wild tomato

27

3. Shin Cheong Gang rootstock

4. Cheong Gang rootstock

5. Anna F1 grafted on Shin Cheong Gang rootstock

6. Anna F1 grafted on Cheong Gang rootstock

7. Anna F1 treated with Rootgard SP® (Biomicrobial biopesticide)

8. Anna F1 treated with Nordox ®

(Copper oxychloride)

Randomized complete block design (RCBD) was used to allocate the treatments in each

for the experiment. A spacing of 90 cm between the plants and 45 cm between rows was

used. Rootgard® supplied by Juanco SPS which is a cocktail of plant useful

microorganisms specially formulated with nutrients and enzymes was used. The

microorganisms include Trichoderma spp., Bacillus spp., Pseudomonas spp., Aspergillus

spp., Chaetomium spp., Escherichia spp. and Azorobacter spp. This micro-organisms

range makes Rootgard® to function as insecticide as well as fungicide (Waiganjo et al.,

2006). Nordox® supplied by Bayer is a copper based fungicide which has been reported

to have appreciable control on R. solanacearum but also has been recorded to have some

activity on soil bacterial pathogens when drenched. Application of Nordox® and

Rootgard® treatments was applied two weeks before transplanting at the rate of 50g/m

2.

3.2.4 Crop management practices

Transplanting was done in the evening when the weather was cool to increase chances of

survival for the seedlings. During transplanting 150 kg/Ha of Di-ammonium phosphate

(47% P205) was used. Two weeks after transplanting, 200Kg/ ha Urea (46% N) was

applied followed by Calcium Ammonium Nitrate (CAN) in the fourth week at the rate of

28

200Kg/ha (27%N) and in weeks seven, nine and twelve. Nitogen, Phosphate, potassium

compound fertilizer (N=17%, P205=17%, K=17%) was applied at the rate of 150Kg/ha.

Foliar feed Wuxal® was applied weekly at 50ml/20l water. The crop was kept free of

weeds by manual weeding and uprooting of weeds as per normal farmers practice.

Irrigation was carried out once every two. Crop support (trellised) was carried as per

farmers practice. Pruning to removal of side shoots, laterals, old leaves, diseased leaves

and branches done to reduce fungal diseases. Standard pest and disease management

program used by farmers was used except for the management of R. solanacearum.

3.2.5 Assessments of bacterial wilt incidence and severity

The numbers of wilted as well as dead plants were counted recorded every week.

Assessment of disease severity was carried out by cutting stems of wilted and dead plants

and scoring for stem browning of the stems using a score of 0 - 3 where 0 - no browning,

1 - light brown colour restricted to 2 cm from the stem base, 2 - light brown colour spread

more than 2 cm from the base and 3 - Dark brown colour that is wide spread browning of

the vascular tissue (Ephinstone et al., 1998). Bacterial streaming from the stems was also

tested by suspending the stems in clear water in a beaker and the ooze rate score of 0 - 3

given where 0 - no ooze, 1 - thin strands of bacteria oozing stops in 3 minutes, 2 -

continuous thin flow that is unrestricted and 3 - heavy ooze turning the water turbid in 2

minutes (Ephinstone et al., 1998). Confirmatory tests on the presence of bacterial wilt

pathogen were done by isolation on nutrient agar and Kelmans’ TTC solid agar; where

unique R. solanacearum colony characteristics, colony colour and tendency of the colony

to flow were checked.

29

3.2.6 Assessment of fruit yield and quality

Ripe fruits were harvested 75 days after transplanting, un-marketable fruits especially

very small (fruits weighing less than 50 g) and continued to 100 days after transplanting,

deformed or those damaged by pests and diseases were removed before weighing. The

weight per harvest per plant was recorded per plant for each harvest. A total of 10

harvests were carried out with an average of two harvests per week being carried out.

Using number of plants per square area for the corresponding plant spacing, the yield per

plot was extrapolated to give yield in tons per hectare.

3.3 Evaluation of tomato rootstocks for bacterial wilt tolerance in the glasshouse

3.3.1 Raising of seedlings and grafting

Seeds were raised in the nursery in spindling trays with cocoa peat being used as the

media. Normal nursery practices were adopted where limited fertilizer use in the media

and increased foliar feeds were used. About 10 days after emergence, the seedling

attained a thickness of 1.5mm required for grafting. Grafting was carried out in moist

chambers where same thickness scion and root stock were cut at 450 and joined by plastic

clips. After grafting the varieties were placed in labeled trays before being taken to

moisture chambers. This process was carried out to ensure high grafting success is

achieved. The grafted seedlings were transferred to humidified chambers with a relative

humidity of 80% for five days to allow the graft union to heal. The seedlings were

transferred to the normal nursery where healing process was allowed for two weeks

before they were transplanted.

30

3.4.2 Experimental design and layout

Pots with capacity of 5 litres were filled with media composed of yard manure: soil: sand

in the ratio of 1:3:2 volume to volume. The pots were uniformly filled to the three quarter

level to allow easy transplanting and irrigation to avoid overflow. A sauce to collect

excess water was placed beneath each of the pots. The treatments in the experiment were

as follows:

1. Anna F1

2. Anna F1 grafted on wild tomato

3. Cheong Gang

4. Shin Cheong gang

5. Anna F1 grafted on Cheong gang

6. Anna F1 grafted on Shin Cheng gang

7. Anna F1 treated with Rootgard®

8. Anna F1 treated with Nordox ®

One set of the treatment was inoculated with R. solanacearum at 1x109 colony forming

units per ml and the control set inoculated with sterile water. Treatment with Nordox®

and Rootgard® was carried out 10 days before transplanting. Five plants were

transplanted into each of the pots with each treatment having three replicates.

Randomized complete block design was used in the laying of the plots in the glass house.

One plot was made of three pots with a total of 15 plants. Standard fertilizer program

was adopted for the experiment 20g of NPK fertilizer (N=17: P205=17: K=17) was

applied per week and was repeated once every two weeks. Standard Agronomical

practices were carried including; irrigation, weed control, pest and disease management.

31

3.3.3 Inoculation and application of treatments

Inoculation was carried out one week after transplanting by passing a sterile scalpel close

to the stem on one side of the pot to a depth of 5-6 cm to cause injury to the secondary

roots according to Winstead and Kelman (1952) and Mwangi et al, (2008). Four mls of

the standardized bacterial suspension (1x109 colony forming units per ml) was poured

over the roots and washed with 100 ml of water. The inoculation was repeated 24 hours

later on the opposite side of the stem and a high soil moisture level maintained afterwards

by frequent watering.

3.3:4 Assessments of bacterial wilt

A weekly record of the number of wilted and dead plants was recorded in each pot. Stem

browning and bacterial oozing score was collected at 60 days after transplanting. This

was done by selecting and evaluating one plant per pot to make three plants per

treatment.

3.4 Data analysis

Survey data was analyzed using SPSS program where frequency of various parameters

from the questionnaire was analyzed to generate trends for the counties. Analysis of

Variance (ANOVA) of data from both the green house and glass house experiments was

carried out using GENSTAT® statistical program, 13

th edition and means were separated

by Fischer Protected LSD at 5% significance level.

32

CHAPTER FOUR: RESULTS

4.1 Occurrence of bacterial wilt in open field and green house tomato production

4.1.1 Tomato production practices

In all the counties farming operations in the farms were mainly owned or managed by

males (Table 4.1). Higher female farm ownership was recorded in Kiambu county and

lower in Laikipia, Kajiado and Kirinyaga counties. Total farm sizes in all the counties

ranged from 0.5 acres to 12 acres with Laikipia county having a minimum of one acre to

a maximum of 8 acres. Kirinyaga county had farm sizes ranging from 1 acre to 4.5 acres.

The county of Kajiado had the highest percentage of farmers adopting irrigation (Table

4.2). In Laikipia, Kiambu and Kajiado counties most of the farmers use borehole as a

source of water for irrigation with drip system adopted as a method of irrigation. In

contrast most farmers in Kirinyaga county mainly used rivers as a source of water and

have adopted furrow irrigation (Table 4.2) irrigation method. In this county investment in

plastic pipes and motorized pumps used to pump water from the water to the fields were

very common. This is aimed at improving water use efficiency.

Table 4.1 Percentage farmers grouping by gender and average farm sizes in tomato

growing counties in Kenya.

Gender

County

Male

Female

Average farm size (acres)

Kirinyaga 90.0 10.0 2.1

Kiambu 78.9 21.1 2.8

Laikipia 90.0 10.0 3.5

Kajiado 89.5 10.5 2.8

33

Table 4.2 Farmers source of irrigation water and methods of irrigation used in key tomato

growing counties in Kenya.

Irrigation County

Water source Method Kirinyaga Kiambu Laikipia Kajiado

Bore hole Drip 5.0 47.4 32.5 70.1

Overhead 0.0 0.0 2.5 3.6

Furrow 0.0 0.0 0.0 0.0

Dam Drip 0.0 5.3 18.8 0.0

Overhead 0.0 0.0 5.2 0.0

Furrow 0.0 0.0 0.0 0.0

River Drip 0.0 10.2 9.5 0.0

Overhead 15.0 5.6 5.5 0.0

Furrow 30.0 0.0 0.0 0.0

No irrigation - 50.0 31.6 30.0 26.3

Sample size 25 22 20 16

Advice from farm input shops (Agrovets) ranked top as a key source of information for

farmers on crop production and protection with Kirinyaga recording the highest

percentage at 45% (Fig 4.3). A high number of farmers in all the counties had attended

agronomy training especially from seed companies. Farmers were also found to rely on

their experience to make crop protection decisions. Services by professional consultants

individually or from advisory and consulting private firms were found in Kiambu and

Kajiado counties (Fig 4.3). Extension service especially from the ministry of Agriculture

was reported in all counties but only low percentages of farmers were found to be

utilizing their advice. Reports of increased training and field work offered by the seeds

company was evident, but was more towards hybrid seeds. It was observed that farmers

who depended on same source of advice practiced same agronomic practices.

34

Table 4.3 Percentage of farmers who cited different sources of extension service in key

tomato growing counties in Kenya.

Source Laikipia Kirinyaga Kiambu Kajiado

Farm input shops 20.0 45.0 31.6 15.8

Professional consultants 0.0 0.0 10.4 26.3

Extension officers 10.0 5.0 5.3 10.5

Others farmers 25.0 10.0 21.1 10.5

Farmer knowledge 30.0 40.0 26.4 36.8

Company trainings 15.0 0.0 5.3 0.0

Sample size 25 22 20 16

Table 4.4 Percentage farmers in key tomato producing counties in Kenya who sourced

tomato seedlings from indicted seedling sources.

Source of seedlings Kirinyaga Kiambu Laikipia Kajiado

Registered nurseries 0.0 52.6 20.0 47.4

Soil nursery 100.0 42.1 80.0 52.6

Direct planting 0.0 5.3 0.0 0.0

Sample size 25 22 20 16

Soil nursery was found to be the key method of raising the seedlings in the counties

(Table 4.4). Use of artificial media (coco-peat) and trays to raise the seedling was

reported in Kiambu and Laikipia. Use of seedlings from plant raisers and nurseries was

reported to be gaining importance due to high cost of seeds. Nurseries in Naivasha and

Kitengela were among those identified to sell disease free seedlings. Direct seeding was

reported in Kiambu but was limited to green house farmers with very good irrigation

system. In Kirinyaga county farmers were found to produce soil raised seedling for sale.

35

Farm hygiene as a disease management strategy was highly adopted in Laikipia (Table

4.5). Crop rotation as a practice strategy to manage the disease was not well adopted in

all the counties with less 50% compliance. It was noted that there was lower disease

incidence with farmers who practiced good farming practices. Highest usages of chemical

control products were recorded in Kirinyaga and Kiambu counties. Uprooting infected

plants was mostly carried out in Kirinyaga and Laikipia. Use of foot paths with water

containing a disinfectant (Sodium Hypochloride - Jik) was found to be higher in green

house production with Kiambu farmers as shown in Table 4.5. Foot paths were less

common in open field production systems especially in Kajiado and Kirinyaga increasing

the risk of disease spread from one field to the other. Farmers who practiced

intercropping reported lower bacterial wilt incidence levels.

Table 4.5 Percentage of farmers adopting bacterial wilt management practices in key

tomato producing counties in Kenya.

Laikipia Kirinyaga Kiambu Kajiado

Good farming practice

Farm hygiene 63.2 55.0 52.6 57.9

Crop rotation 38.0 45.5 25.0 45.5

Weeding 100.0 100.0 100.0 100.0

Intercropping

21.1 35.0 36.8 15.8

Management options for R. Solanacearum

Chemical products 46.7 72.7 65.0 57.0

Uprooting infected plants 70.0 85.0 60.0 15.5

Foot baths and hygiene 50.0 10.1 75.0 14.0

Tolerant varieties 16.7 35.5 28.8 12.5

Sample size 25 22 20 16

36

4.1.2 Incidence and severity of bacterial wilt in farmers fields

Bacterial wilt of tomato was reported in all the counties with the county of Kiambu

reporting the highest incidence at 37.4%; followed closely by Kirinyaga county and

lowest in Kajiado county (Fig 4.6). In Kiambu high disease incidences were recorded

mostly in green houses in Ruiru and Kamiti with poor rotation programs. In Kajiado

county the disease reported were mainly powdery mildew, early blight with cases of

bacterial wilt now being reported in greenhouses (Fig 4.6). Downey mildew and early

blight diseases were mainly observed in harvesting stage in green houses but in all other

counties the incidences was below 25% except Kiambu county where incidence was 53%

for early blight. Bacterial spot was recorded in the counties but the incidence was low

except in open field crops in Laikipia county. Fusarium wilt incidence was highest in

Kiambu with no reports of the disease in laikipia county. In all counties viral diseases

were reported but the effects was minimal.

Table 4.6 Percentage of farmers who reported tomato the indicated diseases in key

tomato growing counties in Kenya.

Diseases Laikipia Kirinyaga Kiambu Kajiado

Bacterial spot 16.7 0.0 5.3 0.0

Downy mildew 23.3 20.0 6.3 0.0

Late blight 27.8 60.0 52.6 12.1

Powdery mildew 2.2 0.0 10.5 21.1

Early blight 18.9 25.0 53.7 16.3

Fusarium wilt 0.0 11.1 15.0 10.8

Bacterial wilt 23.3 35.0 37.4 15.8

Sample size 25 22 20 16

37

Figure 4.1 Initial wilting of plants caused by R.solanacearum: A) in a farmer’s field in

Kiambu (B) in older crops in Kajiado

Figure 4.2 Grafting union (Figure C) and bacterial wilt streaming (Figure D)

Figure 4.3 Colonies of to R. solanacearum on Kelman’s TTC (E) and SMSA media(D).

A B

D C

F E

38

The common symptom of R. solanacearum observed was general plant wilting crops as

affected (Figure 4.1). The wilting was observed to be unique from other wilts as plant

remained green unlike other soil pathogens where wilting occurred with yellowing of

leaves. Wilted plants were observed to completely dry after a few days. Growth of R.

solanacearum on growth media is shown in Figure 4.2. It was observed that the disease

was damaging during flowering and worsens towards fruit stage and harvesting.

In Kiambu county losses up to 100% reported, in general lower losses were reported in

open field crops as shown in Table 4.8. Farmers reported that they had been affected by

the disease for a period of more than 4 years. About 10% of farmers in Laikipia and

Kiambu reported new disease incidence in 2012 (Table 4.8). Laboratory tests for samples

from field on NA, NCM-ELISA, Kelman’s TTC and SMSA had a similar disease level as

was recorded in the field (Table 4.7).

Table 4.7 Percentage samples testing positive to R. solancearum on selective solid media

and serology test.

Test Laikipia Kirinyaga Kiambu Kajiado

Bacterial stream 24 32 55 19

Colony on NA 32 55 70 44

Colony on SMSA 32 45 60 31

NCM-ELISA 32 45 60 31

Kelman’s TC 32 45 60 31

RE-Inoculation 32 41 60 31

Means 28 45 60 31

Sample size 25 22 20 16

NA-Nutrient Agar Media, NCM-ELISA- Enzyme Linked Immunosorbent Assay on Nitrocellulose Membrane,

Kelman’s TTC (Kelman’s Triphenyl Tetrazolium Chloride), SMSA.

39

Table 4.8 Bacterial wilt incidence, occurrence and percentage yield losses due to

R. solanacearum in key tomato growing counties in Kenya.

Region

Wilt %

Incidence

% crop loss

Disease occurrence period

<1 year 1-2years >2years

Kirinyaga (n=25) 35.0 11.0 5.0 20.0 25.0

Laikipia (n=20) 23.3 25.0 10.0 15.8 10.0

Kiambu (n=22) 37.4 45.0 10.5 26.3 10.5

Kajiado (n=18) 15.8 6.3 5.3 8.5 11.1

4.2 Tolerance of tomato rootstocks to R.solanacearum under greenhouse conditions

4.2.1 Incidence and severity of rootstock to R.solanacearum

Grafting of Anna F1 on both Cheong Gang and Shin Cheong Gang resulted in very high

survival percentages in all the sites (Table 4.9). In all the sites, survival percentage was

over 92% indicating that the tolerance of the rootstock varieties was very high. In Ruiru

and Kiambu site where over 80 % of the non grafted Anna F1 deaths from bacterial wilt

were recorded. The grafted variety showed very high tolerance. Grafting of the Anna F1

variety onto wild tomato improved tolerance of the susceptible of variety to bacterial wilt.

There was no significant difference between survival rates for non grafted Anna F1 and

one treated with Rootgard® or Nordox

®. Survival rates for the Rootgard

® treatments were

significantly higher than Nordox®

treatments in Isinya and Ruiru in the second season.

Lower survival rates were recorded in Ruiru and Kiambu counties as the infection

occurred in early stages of crop development and lower in Karen and Isinya site where

infection occurred at a later stage in crop development. Higher percentage survival was

recorded in the first season than in the second seasons in all the treatments (Table 4. 9).

40

Table 4.9 Percentage survival of tomato varieties from R.solanacearum in experimental

greenhouses in Kenya.

Site

Variety Isinya Karen Kiambu Ruiru Mean

Season 1

Anna F1 65.3 b 64.0 b 1.3 d 21.3 c 38.0 d

Anna F1 + Wild tomato 94.7 a 94.7 a 85.3 b 78.7 b 88.3 b

Cheong Gang 98.7 a 98.7 a 96.0 a 98.7 a 98.0 a

Shin Cheong gang 100.0 a 100.0 a 98.7 a 98.7 a 99.3 a

Anna F1 + Cheong Gang 94.7 a 94.7 a 90.7 ab 97.3 a 94.3 a

Anna F1 + S. Cheong Gang 98.7 a 98.7 a 93.3 ab 98.7 a 97.3 a

Anna F1 + Rootgard®

59.3 b 64.7 b 18.7 c 24.0 c 41.7 cd

Anna F1 + Nordox ® 63.3 b 74.7 b 14.7 c 25.3 c 44.5 c

L.S.D (P=0.05) 14.2 10.7 10.4 13.5 11.46

C.V. % 2.8 2.8 3.8 2.8 1.2

Season 2

Anna F1 53.3 c 46.0 b 20.0 b 15.3 c 33.7 d

Anna F1+ Wild tomato 96.7 a 85.3 a 85.3 a 83.3 b 87.7 b

Cheong Gang 98.7 a 96.0 a 90.7 a 97.3 ab 95.7 a

Shin Cheong gang 98.0 a 98.0 a 93.3 a 100.0 a 97.3 a

Anna F1 + Cheong gang 96.0 a 94.0 a 94.7 a 97.3 ab 95.5 a

Anna F1 + S.Cheong Gang 97.3 a 98.0 a 93.3 a 97.3 ab 96.5 a

Anna F1 + Rootgard®

72.0 b 54.7 b 20.0 b 18.7 c 41.3 c

Anna F1 + Nordox ® 64.0 bc 58.0 b 21.3 b 8.7 c 38.0 cd

L.S.D. (P=0.05) 15.5 15.2 16.1 16.2 5.05

C.V. % 1.2 3.1 3.1 4.7 1.7

Note:

Means with similar letters are not significantly difference P=0.05, means separated by Fischer

Protected LSD

41

Bacterial wilt severity scores were significantly lower in Anna F1 grafted on rootstock

Cheong Gang and Shin Cheong Gang, rootstock reduced disease severity up to 94 %. No

significant deference between severity for Anna F1, Anna F1 treated with Rootgard® and

Anna F1 treated with Nordox® which had the highest browning score. Severities of the

varieties for R. solanacearum were similar across the sites and between the two seasons.

Table 4.10 Bacterial wilt browning scores from field experimental sites

Site

Variety Isinya Karen Kiambu Ruiru Mean

Season 1

Anna F1 0.8 bc 2.4 d 2.3 c 2.8 d 2.1 d

Anna F1 + wild tomato 0.2 ab 0.6 b 0.6 a 0.8 b 0.5 b

Cheong Gang 0.1 a 0.1 a 0.1 a 0.1 a 0.1 a

Shin Cheong gang 0.0 a 0.1 a 0.0 a 0.0 a 0.0 a

Anna F1 + Cheong gang 0.4 ab 0.3 ab 0.2 a 0.9 b 0.5 b

Anna F1 + S. Cheong Gang 0.1 a 0.0 a 0.1 a 0.1 a 0.1 a

Anna F1 + Rootgard®

1.1 c 1.2 c 1.7 b 1.9 c 1.5 c

Anna F1+ Nordox® 1.8 d 2.2 d 2.1 bc 2.7 d 2.2 d

L.S.D. (P=0.05) 0.6 0.4 0.6 0.6 0.3

C.V. % 40.3 23.8 14.5 5.4 5.7

Season 2

Anna F1 1.1 b 2.2 c 2.3 b 2.9 c 2.1 d

Anna F1+wild tomato 0.5 ab 0.4 a 0.2 a 0.8 a 0.5 b

Cheong Gang 0.0 a 0.1 a 0.0 a 0.0 a 0.0 a

Shin Cheong gang 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a

Anna F1 + Cheong gang 0.2 a 0.2 a 0.1 a 0.6 a 0.3 ab

Anna F1 + S. Cheong gang 0.1 a 0.1 a 0.2 a 0.2 a 0.1 ab

Anna F1 + Rootgard®

1.3 bc 1.1 b 2.2 b 2.3 bc 1. 8 c

Anna F1+ Nordox® 2.0 c 0.3 a 2.2 b 1.7 b 1.6 c

L.S.D. (P=0.05) 0.9 0.7 0.8 0.8 0.4

C.V. % 18.9 27.7 25.4 9.2 6.6

Means with same letters are not significantly different P=0.05

Score 0 - no browning, 1 - light browning restricted to 2 cm, 2 - light brown colour spread more than 2 cm

and 3 - Dark brown colour wide spread browning (Ephinstone et al., 1998).

42

Highest bacterial oozing score was observed on Anna F1, Anna F1 treated with

Rootgard® and Anna F1 treated with Nordox

®. Bacterial ooze was reduced by up to 90 %

for grafted varieties when compared to Anna F1. There was significantly higher oozing

score in the second season. There was no significance difference between oozing score

for the control Anna F1, and when treated with Rootgard® or Nordox

® (Table 4.11).

Table 4.11Bacterial oozing score of sampled plants from experimental sites.

Site Mean

Variety Isinya Karen Kiambu Ruiru

Season 1

Anna F1 1.2 c 0.9 b 1.0 cd 1.8 e 1.2 c

Anna F1 + wild tomato 0.3 ab 0.2 a 0.6 bc 0.7 bc 0.4 b

Cheong Gang 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a

Shin Cheong gang 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a

Anna F1 + Cheong gang 0.3 ab 0.1 a 0.2 ab 0.3 ab 0.3 ab

Anna F1 + S. Cheong gang 0.0 a 0.0 a 0.1 ab 0.1 a 0.1 a

Anna F1 + Rootgard®

0.8 bc 1.0 b 1.4 d 1.1 cd 1.1 c

Anna F1 + Nordox® 0.8 bc 1.1 b 1.3 d 1.3 de 1.1 c

L.S.D. (P=0.05) 0.6 0.6 0.5 0.5 0.3

C.V. % 33.9 10.2 14.3 22.6 12.2

Season 2

Anna F1 0.9 b 1.2 c 1.3 b 1.3 bc 1.2 b

Anna F1+ Wild variety 0.2 a 0.3 ab 0.3 a 0.4 a 0.3 a

Cheong Gang 0.0 a 0.0 a 0.1 a 0.0 a 0.0 a

Shin Cheong gang 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a

Anna F1 + Cheong gang 0.1 a 0.6 abc 0.1 a 0.2 a 0.3 a

Anna F1 + S.Cheong gang 0.2 a 0.1 a 0.1 a 0.1 a 0.1 a

Anna F1 + Rootgard®

1.2 b 1.3 c 1.1 a 1.1 b 1.2 b

Anna F1+ Nordox® 1.4 b 1.1 bc 1.6 b 1.7 c 1.4 b

L.S.D. (P=0.05) 0.7 0.9 0.6 0.5 0.3

C.V. % 32.7 43.5 24.8 30.9 9.6

Means with same letters are not significantly different P=0.05.

Score: 0 - no ooze, 1 - thin strands of bacteria oozing stops in 3 minutes, 2 - continuous thin flow that is

unrestricted and 3 - heavy ooze turning the water turbid in 2 minutes (Ephinstone et al., 1998).

43

Anna F1 had the highest oozing score scores of 1.2 in all the sites and seasons. Oozing

score for Anna F1 was higher but not significantly different in Anna F1 treated with

Nordox® in and Anna F1 treated with Rootgard

®. Higher than average score was

recorded in season two in Karen site for Anna F1 grafted on Cheong Gang (Table 4.11).

Confirmatory tests on bacterial wilt for the rootstock variety Cheong Gang and Shin

Cheong Gang were negative (Table 4.12). Wild tomato had less positive samples than

Anna F1, Anna F1 treated with Nordox® and Anna F1 treated with Rootgard

®. Some of

the samples that did not test positive for bacterial streaming tested positive for

R.solancearum with other confirmatory tests (Table 4.12). The colony characteristic was

positive for R. solanacearum on Kelmans’TTC media where fluidy pink colonies were

produced. The pathogen was positive on SMSA and NCM-ELISA tests. From the tests

Kelman’s TC and ELISA tests were found to be the most accurate and more reliable in

detection of R. solanacearum.

44

Table 4. 12 Percentage colonies testing positive for R. Solanacearum from the sites

Season 1

Season 2

Streaming Isinya Karen Kiambu Ruiru Isinya Karen Kiambu Ruiru

Anna F1 60 40 20 40 20 0 60 60

Ann + Wild tomato 20 0 0 0 20 0 20 0

Anna + Cheong Gang 20 0 20 0 0 0 0 0

Anna + S. Cheong Gang 0 0 0 0 0 0 0 0

Anna + Rootgard® 40 40 20 40 20 20 40 40

Anna + Nordox ® 20 60 40 40 40 40 20 60

Colony in on NA

Anna F1 80 60 60 80 60 60 60 100

Ann + Wild tomato 40 20 40 40 20 40 20 40

Anna + Cheong Gang 20 0 40 0 0 20 0 0

Anna + S. Cheong Gang 0 0 0 20 0 0 20 0

Anna + Rootgard® 60 60 60 60 60 40 60 60

Anna + Nordox ® 80 80 60 60 40 80 80 100

Colony in SMSA

Anna F1 80 60 60 60 60 60 60 80

Ann + Wild tomato 20 20 20 40 20 20 20 40

Anna + Cheong Gang 20 0 40 0 0 20 0 0

Anna + S. Cheong Gang 0 0 0 20 0 0 20 0

Anna + Rootgard® 40 40 60 60 60 40 40 60

Anna + Nordox ® 60 80 40 40 40 80 80 80

Kelmans' TTC

Anna F1 80 60 60 60 60 60 60 80

Ann + Wild tomato 20 20 20 20 20 20 20 40

Anna + Cheong Gang 20 0 40 0 0 40 0 0

Anna + S. Cheong Gang 0 0 0 40 0 0 20 0

Anna + Rootgard® 40 40 60 60 60 40 40 60

Anna + Nordox ® 60 80 40 40 40 80 80 80

NCM-ELISA

Anna F1 80 60 60 60 60 60 60 80

Ann + Wild tomato 20 20 20 40 20 20 20 40

Anna + Cheong Gang 20 0 40 0 0 20 0 0

Anna + S. Cheong Gang 0 0 0 40 0 0 20 0

Anna + Rootgard® 40 40 60 60 60 40 40 60

Anna + Nordox ® 60 80 40 40 40 80 80 80

NA-Nutrient Agar Media, NCM-ELISA- Enzyme Linked Immunosorbent Assay on Nitrocellulose Membrane,

Kelman’s TTC (Kelman’s Triphenyl Tetrazolium Chloride), SMSA media

45

4.2.2 Yield for the varieties in selected sites

Anna F1 grafted on Shin cheong gang and Cheong gang had the highest yield in both

season one and two (4.13). On average the yield was higher in grafted variety Shin

cheong gang by 65% in the first season and by 46 % in the second season. Higher yield

for all the varieties were recorded in Isinya and Karen than for Ruiru and Kiambu sites.

(Table 4.13). There was no significant yield difference between non grafted Anna F1 and

where treated with Rootgard® and Nordox

®.

Table 4.13 Harvested yield for each variety for selected experimental sites.

Variety yield in T/ha Isinya Karen Kiambu Ruiru Mean

Season 1

Anna F1 46.0 b 40.2 b 29.2 bc 30.2 b 38.4 c

Anna F1+wild tomato 28.2 c 32.4 c 26.6cd 40.4 bc 24.8d

Cheong Gang 20.4 c 16.8 c 10.0 d 17.0 d 18.0 d

Shin Cheong gang 20.2 c 14.4 c 22.6 d 18.4 cd 18.2d

Anna F1 + Cheong gang 8.8 b 54.2 a 40. 8 b 54.2 a 48.6 b

Anna F1 +S. Cheong gang 72.6 a 65.0 a 56.4 a 65.6 a 63.6 a

Anna F1 + Rootgard®

54.2 ab 36.4 b 49.2 ab 23.4 bcd 40.0 c

Anna F1 + Nordox®

22.4 b 36.8 b 50.4 ab 27.0 bc 38.8 c

L.S.D. (P=0.05) 24.2 20.0 10. 8 10.6 5.0

C.V. % 8.9 7.8 7.8 6.5 4.2

Season 2

Anna F1 40.4 a 49.2 a 48.2 a 29.6 c 40.6 b

Anna F1+wild tomato 36.6 b 30.4 cd 36.8b 33.2 cd 20.0 d

Cheong Gang 21.0 b 17.4 cd 25.4 b 19.0 cd 19.6d

Shin Cheong gang 18.6 b 10.2 d 24.6 b 14.6 d 16.8 d

Anna F1 + Cheong gang 38.4 a 27.2 ab 50. 8 a 40.4 b 40.8 a

Anna F1 +S. Cheong gang 51.4 a 39.0 ab 52.2 a 58.8 a 59.2 a

Anna F1 + Rootgard®

40.2 a 26.4 bcd 25.0 b 26.4 c 29.2 c

Anna F1 + Nordox®

42. 8a 28.2 bc 26.6 b 23.6 cd 29.0 c

L.S.D. (P=0.05) 15.4 17.0 20.4 13. 8 5.2

C.V. % 6.9 12.9 2.6 1.9 3.1

Figures with same letter are not significantly different. Means separated by Fischers protected LSD P=0.05

46

Higher average yield of an average of 12 % was recorded in the first season as compared

to the second season. Treatment of Anna F1 with Rootgard® and Nordox

® products did

not significantly improve yield.

4.3 Tolerance of tomato rootstocks to bacterial wilt in glasshouse with R.

solanacearum inoculation

4.3:1 Plant mortality and bacterial wilt severity

Highest survival was recorded with Anna F1 grafted on Cheong gang and Shin cheong

gang (Table 4.14). Non grafted Anna F1 was most susceptible in both season one and

two. Grafting reduced R.solanacearum incidence by 82% for Cheong gang rootstock and

over 93% for shin cheong gang. Local wild tomato reduced disease incidence by 73%.

There was reduced variety survival in season two compared to season one.

Table 4.14 Percentage survival of tomato varieties incubated with R. solanacearum in

glasshouse conditions at Kabete field station.

Variety and treatments Season 1 season 2

Anna F1 20.0 c 13.3 c

Anna F1 grafted on wild tomato 66.7 b 80.0 b

Cheong Gang 93.3 a 86.7 ab

Shin Cheong gang 100.0 a 93.3 ab

Anna grafted on Cheong gang 80.0 ab 86.7 ab

Anna grafted on Shin Cheng gang 93.3 a 93.3 ab

Anna treated with Rootgard®

26.7 c 20.0 c

Anna treated with Nordox® 40.0 c 20.0 c

L.S.D (P=0.05) 20.9 13.6

C.V. % 15.3 10.1

Figures with same letter are not significantly different. Means separated by Fischers protected LSD P=0.05

47

There was no significant difference between the survival percentages of Anna F1 and

Anna F1 treated with Rootgard® and Nordox

®. Survival percentages of non grafted

rootstock were similar with grafted treatments (Table 4.14).

Disease severity was lowest in grafted Anna F1 on Shin Cheng gang and Cheong gang.

Grafting reduced disease severity by 94% with lowest disease severity with Shin cheong

gang. Anna grafted on the wild tomato had significantly lower severity than Anna F1

having reduced the severity by 23% in the first season and 31% in the second season.

There was no significance difference between Anna F1, Anna treated with Rootgard® and

Anna F1 treated with Nordox® (Table 4.15). There was no significant difference in

browning scores between the two seasons.

Oozing score was lowest in Anna F1 grafted on rootstocks (Table 4.15), oozing rate was

reduced by 93% in the first season and 84%. Anna grafted on wild tomato showed

significantly lower oozing score than Anna F1 in the two seasons. Oozing score for Anna

grafted on the wild tomato was not significantly different from the Anna F1 grafted on

rootstocks in season one but significantly higher than the rootstocks in season two. Anna

F1 treated with Nordox® and Anna F1 treated with Rootgard

® had lower oozing score

than the control in the first season but showed no significant difference in the second

season (Table 4.15).

48

Table 4.15 Stem browning and oozing scores for R. solanacearum from inoculated

treatments in glasshouse experiment.

Stem discoloration score Oozing score

Variety Season 1 Season 2 Season 1 Season 2

Anna F1 2.6 e 2.6 d 1.3 c 1.2 d

Anna F1 grafted on wild tomato 0.6 c 0.8 c 0.2 a 0.7 bc

Cheong Gang 0.2 ab 0.2 ab 0.0 a 0.0 a

Shin Cheong gang 0.1 a 0.1 a 0.0 a 0.0 a

Anna F1 grafted on Cheong gang 0.4 bc 0.4 b 0.2 a 0.3 ab

Anna F1 grafted on Shin Cheng gang 0.1 a 0.1 a 0.1 a 0.2 ab

Anna treated with Rootgard®

1.9 d 2.3 d 1.1 bc 1.1 cd

Anna treated with Nordox® 2.0 d 2.4 d 1.0 b 1.1 cd

L.S.D. (P=0.05) 0.3 0.2 0.3 0.5

C.V. % 27.2 12.9 69.1 112.2

Browning scale

0 - no browning, 1 - light brown colour restricted to 2 cm, 2 - light brown colour spread more than 2 cm

and 3 - Dark brown colour wide spread browning of the vascular tissue (Ephinstone et al., 1998).

Oozing scale

0 - no ooze, 1 - thin strands of bacteria oozing stops in 3 minutes, 2 - continuous thin flow that is

unrestricted and 3 - heavy ooze turning the water turbid in 2 minutes (Ephinstone et al., 1998).

4.3:2 Re-isolation of R. solanacearum from tomato rootstocks

Anna F1 grafted on Cheong gang and Shin cheong gang plant samples tested negative for

the R. solanacearum with bacterial streaming, SMSA, Kelmans’ Nutrient Agar and

ELISA indicating absence of the pathogen. R. solanacearum was isolated from Anna F1,

Anna F1 treated with Nordox®

and Rootgard® (Table 4.16) indicating presence of the

pathogen. This confirmed that the pathogen did not thrive in the grafted treatments but

multiplied in susceptible varieties.

49

Table 4.16 Percentage positive confirmatory tests for R. solanacearum samples

from the glass house experiment.

Variety Streaming NA SMSA TTC ELISA

Season 1

Anna F1 60 80 60 60 60

Anna F1 grafted on wild tomato 20 40 40 40 40

Cheong Gang 0 0 0 0 0

Shin Cheong gang 0 0 0 0 0

Anna grafted on Cheong gang 0 0 0 0 0

Anna grafted on Shin Cheng gang 0 0 0 0 0

Anna treated with Rootgard®

40 40 40 40 40

Anna treated with Nordox® 60 60 40 40 40

Season 2

Anna F1 20 80 60 60 60

Anna F1 grafted on wild tomato 40 60 40 40 40

Cheong Gang 0 0 0 0 0

Shin Cheong gang 0 0 0 0 0

Anna grafted on Cheong gang 20 20 0 0 0

Anna grafted on Shin Cheng gang 0 0 0 0 0

Anna treated with Rootgard®

40 80 60 60 60

Anna treated with Nordox® 60 60 60 60 60

NA-Nutrient Agar Media, NCM-ELIZA- Enzyme Linked Immunosorbent Assay on Nitrocellulose Membrane,

TTC (Kelman’s Triphenyl Tetrazolium Chloride), SMSA media

50

CHAPTER FIVE: DISCUSSION

5.1 Occurrence of bacterial wilt in key tomato producing counties

Ralsotonia solanacearum incidence varied greatly between the four counties surveyed as

well as within the counties with Kiambu county leading with a disease incidence of

37.4%, with as higher as 65% incidence recorded in a number of greenhouses. This could

be attributed to conditions in the green house that favoured pathogen multiplication.

Singh et al. (2014a) showed that the micro-climate inside the green house (Temperatures

of 280C-30

0C, 80%-90% RH and wet soil) favors rapid pathogen multiplication and

disease. Level of the pathogen in the soil was high in Kiambu and Ruiru compared to

Karen and Isinya in tandem with disease incidence recorded in the previous crops.

Disease incidence was evaluated using disease signs and symptoms, it was assumed that

all infected plants produced wilt symptoms, however some may not have shown wilting

symptoms in spite of pathogen presence (Aribaud et al., 2014). Confirmatory tests for R.

solananearum showed higher positive samples in Kiambu and Ruiru and fewer in Isinya

and Karen. Wilted samples collected from the field tested positive with the various

diagnostic media including SMSA, NA, Kelman’s TTC and NCM-ELISA. This not only

confirmed the causal organism as was reported by Zheng et al. (2009) and Peeters et al.

(2013) but separated the diseases symptoms from the other diseases.

Higher crop losses were recorded in Kiambu county with an average of 45% recorded as

the disease occurred in the early stages of plant growth leading to high plant deaths.

Intensive farming and lack of crop rotation could was identified as the key reason for

51

high disease, this has also been reported by Mbaka et al. (2013). Crop loss in the

greenhouse was much higher than in open field as more crop rotation was practiced in

open fields. Similar trend was observed in India where crop losses of up to 90% was

reported in the greenhouse compared to losses of 25-60% reported for open field tomato

(Singh et al., 2014a) and 29% losses recorded in a study in Taiwan (Hartman et al.,

1994).

Bacterial wilt was reported to have affected farmers since 2008 with more farmers

reporting the disease for the first time in 2011. About 10% of the farmers in Kajiado and

Laikipia reported new infections in the year 2011 comparing to 20% in Kirinyaga and

26.3% Kiambu counties. This confirmed that the high rate of disease spread was due to

practices that favours disease development in the counties. Practices such as use of flood

irrigation in Kirinyaga county accounted for over 40 % disease spread. It was found that

fields that had higher nematodes infestation were more affected by bacterial wilt. This

relationship has also been reported by Jatala et al. (1988). Areas with high nematode

population showed higher R. solanacearum levels. Root injuries from nematodes

providing entry avenues for R. solanacearum leading to higher infection (Singh et al.

2014b). Due to the relationship management of bacterial wilt must also go hand in hand

with that of nematodes. A crop rotation program designed to manage bacterial wilt must

also be effective against root-knot nematodes (Swarnam and Vekmurugan, 2013).

Farmers who practiced crop rotation were found to have less bacterial wilt incidences as

pathogen survival and multiplication reduces with a non-host in a rotation program.

52

Reduction of bacterial wilt with crop rotation has also been reported by Autrique and

Potts (1978) and Melton and Powell (1991). Although crop rotation reduces bacterial wilt

incidence, its usage is limited by choice of crops with high returns. It was found to be a

challenge especially where land used for farming is leased. Irrespective of the previous

crop farmers in the four counties showed preference to tomato due to its higher income

potential. The findings are also in line with reports by Jackson and Gonzalez (1981) and

Wimer et al. (2009) who reported that long term crop rotation for smallholder farmers is

challenging since land is a constraint. Some farmers included a solanaceous crop in the

rotation program before or after tomato nullifying the primary benefits of a crop rotation

program (Ateka et al., 2001). Planting a tomato crop near infected banana plantation

should also be avoided as the bananas can be a source of pathogen (Peeters et al., 2013).

Maize has been reported to reduce the occurrence of bacterial wilt when intercropped

with a tomato crop. This is because maize favor growth of P. cepacia which is

antagonistic to R. solanacearum (Autrique and Potts 1987).

Farmers who used farm yard manure as well as those who had adopted intercropping

system reported lower disease incidence this is because manure is known to increase soil

micro-organisms which can out-compete R. solanacearum reducing disease incidence

(Vinatzer et al., 2014). Intercropping can reduce plant diseases by 73% (Boudreau, 2013)

as there is direct pathogen inhibition due to non-preference to a non host. From the study

there was no relationship between those farmers who used fertilizer and those who dint

contrary to Chang and Hsu (1988) and Hsu and Chang (1989) who reported that use of

urea and Calcium oxide fertilizers reduced survival of R. solanacearum in the soil.

53

Bacterial wilt spread through irrigation water was found to contribute to increased disease

incidences in Kirinyaga county where flood irrigation was commonly used. Over 75% of

farmers in this county use flood irrigation with water sourced from furrows and rivers.

Irrigation water from furrows is likely to be infected and can potentially carry with it the

bacterial pathogen to the next farm. It was also found out that farmers dispose infected

plants into canals aiding in spread of the disease as dead or wilted plants acts as source of

disease inoculum to the next farm. Disease spread through contaminated irrigation water

was also reported by Waiganjo et al. (2006). In Kajiado county farmers use water from

boreholes and have adopted drip irrigation method which was found to help to reduce

disease incidence and severity. Drip irrigation ensures adequate water is applied at the

rhizosphere reducing surface runoff which can potentially help to spread diseases. This

has also supported reported by Vinatzer et al. (2014) and Ramsubhag et al. (2012) that

drip irrigation is used, chances of disease spreading through water are reduced.

It was noted that movement of soil embedded on shoes and farm implements has

contributed to increased rate of disease spread. High incidence of bacterial wilt was also

noted in farms where poor field hygiene was practiced. Use of disinfectants such as

sodium hypochloride in foot baths was observed in Kiambu and significant reduction of

the disease noted. Greenhouse that had a shoe disinfecting basin before entry into the

green house reported reduced disease incidence. This has also been reported by Mbaka et

al. (2013) who did similar work in region. Observing field hygiene practices including

cleaning shoes using disinfectants in foot baths has also been reported to reduce disease

infestation (Dudek 2008; Meng, 2013).

54

Establishing nurseries on soil especially where the soils are already infected by R.

solanacearum was identified to contribute significantly to the spread of the disease in the

counties. Infected seedlings from such nurseries continue to infect clean fields spreading

the disease. This was mainly noted in Kirinyaga county and has also been reported by

Nyangeri et al. (1984) and Ajanga (1993) who observed that the use of infected seedlings

was among the most important means by which the disease spread. With high cost of

hybrid seeds about 5% of farmers select fruits which they extract seeds from fruits for

subsequent planting. Fruits from infected plant may produce infected seeds leading to

disease spread when infected seeds are planted in the nursery. R. solanacearum spread

through infected seeds has also been reported by Moffet et al. (1981) and Ramsubhag et

al. (2012) who showed that bacterial wilt can be transmitted to cotyledons and leaves of

tomato and capsicum plants from contaminated seeds. Machmud and Middleton (1991)

isolated R. solanacearum from ground nut seed confirming transmission of disease by

seeds. On the contrary farmers in Kiambu and Kajiado counties had fully adopted the

usage of certified seeds hence reducing disease spread through seed.

To avoid disease risks associated with soil nurseries farmers in Kiambu and Kajiado

counties have adopted the use of trays seedlings where artificial media is used. Use

artificially media guarantees disease free seedling as reported by Mbaka et al. (2013).

The trays and media were found to be available from farm input shops. With this method

farmers can reduce seed wastage but also achieve excellent crop stand as seedlings from

trays have a high rate of survival (Weirsinga et al. 2008). There exist a huge opportunity

for nurseries and plant raiser to supply seedlings with biggest limitation being cost of

55

transport from the nurseries to farmers field. Importing of forest soil and use of pots is

gaining importance in bacterial wilt management in green houses. Plastic bags and pots

used with forest soil was also reported but challenges of compaction of such soil limiting

roots movement reported. Importation of soils from forests is costly due to high transport

costs of soil in and out of the green house but also there are not may forests remaining.

Information available showed that there was success in the first year of production but

this practice was not sustainable due to poor phytosanitary practices carried out by

farmers (Mbaka et al., 2013, Masinde et al., 2011).

From the study farmers lacked adequate information on sound crop and disease

management. Poor disease identification was also noted with most growers, this can lead

to wrong disease management practices. Field disease identification plates available to

farmers can help in identification of diseases. Solke et al. (2012) recommended the use of

field disease testing immunoprinting kits and lateral devices which can help the farmers

quickly test and identify disease causing organism. Poor advice result in wrong disease

management practices leading to increased cost of production and yield loss. Mbaka et al.

(2013) and Wirsinga et al. (2008) also reported farmers’ dependence in farm inputs shops

for information. Farm input shops usually will sell pesticide products with disregard to

efficacy on bacterial wilt (Waiganjo et al., 2006). Some of the products that farmers have

been advised to use include copper based fungicides such as Nordox, Kocide and

Compracaffaro, hydrogen peroxide, Rovral (Iprodione) and even fumigants such as

Basamid®. These products if excessively used could lead to environmental degradation

and pollution (Huat, 2014) as well as higher residue levels in fresh produce.

56

5.2 Effectiveness of tolerant rootstocks in management of bacterial wilt disease

under field conditions

Grafted Anna F1 on Shin cheong gang recorded high tolerance to bacterial wilt disease

across all the sites. The percentage survival was followed closely by grafted Anna F1 on

rootstock Cheong gang. From the experiment the rootstock varieties Shin cheong gang

and Cheong gang reduced disease incidence by over 94%. High tolerance of these

varieties has also been reported by other scientists. In Painter, Virginia a trial carried out

where a susceptible variety was grafted on Cheong Gang a disease incidence of less than

6.5% was recorded and in Quincy Florida disease incidence for rootstock Cheong Gang

was highly reduced (McAvoy et al., 2012 and Paret et al., 2011). Chaudhari et al. (2012)

reported that Cheong gang and Shin cheong gang recorded very low disease. In an

experiment a variety grafted on Cheong gang and Shin cheong gang showed no incidence

of bacterial wilt even under elevated disease pressures (Chaudhari et al., 2012).

Other rootstock varieties such as Dai Honmei have also showed low bacterial wilt

incidence when compared with a non-grafted susceptible tomato as reported by Rivard et

al. (2012). In another study Cardoso et al. (2012) used rootstock Hawaii 7996 to achieved

high control of bacterial wilt. In Taiwan a tolerant rootstock variety reduced bacterial wilt

incidence by up to 100% (Lin, 2008). Grafting experiments indicated that the percentage

of wilting of Ponderosa scions was less on Hawaii 7996 rootstocks than that on the most

resistant rootstock (LS-89) used in Japan (Nakaho et al., 2004). In their review King et

al. (2010) and Black et al. (2003) confirmed that rootstocks can enhance disease

tolerance. Use of grafting tomatoes has greatly increased in the recent past (Rivard and

Louws, 2008) and is showing potential in the management of wilt disease.

57

Tolerance levels of rootstock varieties to bacterial wilt were consistent across the sites

with higher R. solanacearum level recorded in the second season. Increase in disease

incidence in the second year could be attributed to increased pathogen level in the soil in

the second season as no rotation was done after the first planting. Having mixed the soil

well pathogen level was homogenous within the green has as recommended by Genin et

al. (2012). It has been reported that bacterial wilt strains and pathotypes, pathotypes of R.

solanacearum which coexisted in a competitive growth system in the field, and their

distribution determine the severity of R. solanacearum (Zheng et al., 2014; Ramsubhag et

al., 2012). The sites had different soil types and disease level did not correlate to a

certain soil type. This was in contrast to Abdullah et al. (1983) who found out that soil

type and moisture levels individually and in combination had a significant effect on the

severity of bacterial wilt of ground nuts.

Use of tolerant varieties to the disease has been reported (Techawongstien, 2009) in

experiments carried out in Eastern shore agricultural research and extension centre in

Virginia by Wimer et al. (2009) varieties BHN 669 with greater yield potential showed

high tolerance to the disease. In India no variety was resistant among the 70 genotypes

tested by Singh et al. (2014b). However some of the varieties tested showed high

tolerance. There are reports that new varieties Kilele from Syngenta, Rambo from Kenya

Highland seeds and VL-642 from Seminis have high tolerance to bacterial wilt and are

under trial in the country (Monsanto, 2013). Breeding work utilizing germplasm Hawaii

7996 (Black et al., 2003) and NC 1953-64N (Yamakawa, 1982) is in course in Japan in

the effort to get a resistant tomato variety.

58

Grafting susceptible Anna F1 on to wild cherry tomato reduced bacterial wilt up to 87%

indicating that the wild cherry tomato has bacterial wilt tolerance and can used in

breeding programs to improve the preferred tomato varieties. High tolerance to bacterial

wilt by the wild variety is possible because the variety has tolerance gene that have been

passed on from generation to generation. High bacterial wilt tolerance was also reported

by Lu et al. (2003) when wild Chinese tomato cultivars were used the varieties were

reported to have reduced disease incidence up to 100% as well as delaying the symptoms

when compared with non grafted susceptible varieties.

This study showed biological product Rootgard®

containing Trichoderma spp., Bacillus

spp., Pseudomonas spp., Aspergillus spp., Chaetomium spp., Escherichia spp. and

Azorobacter spp. was not effective in reducing bacterial wilt disease as percentage

survival was not significantly different from non-grafted Anna F1. This is in contrast to

reports of bio-control reported. Pathogen P. fluorescens, controlled bacterial wilt on

potato in both field and laboratory trials (Ciampi-Panno et al., 1989). Pseudomonas

fluorescens was reported to be effective in managing bacterial wilt of tobacco (Liao et al.

1998), and reduced bacterial spot disease in field conditions (Kavitha and Umesha,

2007). Aspiras and cruz (1986) recorded high survival of tomato from 0% in infected

soil to 60% when the soil was treated with microbial antagonistic B. polymyxa and 90%

when the soil was treated with P. fluorescens. Recent work done in Ethiopia by Biratu et

al. (2013) done to screen indigenous actinobacteria for their antibacterial activity against

R. solanacearum proved that they have antagonistic activity against the pathogen and can

be used as a component of integrated programs for the disease management. It was not

59

clear why Rootgard® use was not effective in managing the disease as earlier reported but

moisture levels in the soil and soil-micro-organism dymanics could have inhibited its

activity.

In both field and glass house experiments there was no significant reduction of bacterial

wilt disease with Nordox® treatment. This confirms reports that bactericides have no

significant control activity on the disease (Hartman et al., 1991), as it is difficult to

control bacterial with chemicals (Grimault et al., 1993). In an experiment carried out by

Wimer et al. (2009) chemical product Actigard-Syngenta containing acibenzolar-S-

Methyl dint not reduce bacterial wilt diseases although it is believed to stimulate systemic

acquired resistance to bacterial wilt. There are reports that plant extracts can reduce wilt

disease, in Ethiopia leaf extract of some plants were reported by Alemu et al. (2014) to

exhibit inhibitory characteristics against R. solanacearum. Use of leaf extract has also

been reported by Wagura (2011) and use Lantana camara plant extract by Mary (2011).

Soil amendments such as sodium nitrates suppressed R. solanacearum population and

incorporation of Sun hemp as green manure was effective in controlling bacterial wilt in

the green house in the experiments carried out by Hartman et al. (1994). Amendments

containing urea and calcium oxide affect other soil micro-organisms affecting the

survival of R. solanacearum (Chang and Hsu, 1988; Hsu and Chang, 1989). In the French

West Indies Prior (1990b), found out that urea increased host resistance to bacterial wilt.

Use of chemical products is greatly discouraged because it reduces soil and water quality

(Swanrnam and Velmurugan, 2013). Soil solarization has also been reported to reduce

pathogen levels in the soil (Ioannou et al., 2001).

60

High vascular browning index was observed in the Anna F1, Anna F1 treated with

Nordox® and Rootgard

®. This indicated high severity of the varieties to R. solanacearum

as the pathogen was able to establish and colonize the stem producing the browning

colour. Anna F1 grafted onto tolerant varieties Cheong gang and Shin cheong gang

showed lower vascular browning indicating limited pathogen activity. Similar trends

showing less disease severity with the rootstocks were observed with tolerant rootstock in

Florida (Paret et al., 2012). Pathogen confirmatory test carried out from samples from

the grafted varieties showed no pathogen presence on solid media and ELISA tests while

samples from non–grafted showed high level of pathogen presence.

There was a strong relationship between the level of disease infestation and oozing score,

when infected stems were cut across the stem tiny drops of dirty white or yellowish

viscous ooze exudate from several vascular bundles confirming presence of the bacteria

(Champoiseau et al., 2009). The oozing score was directly proportional with level of

disease. The bacterial ooze score was highest in the un-grafted susceptible Anna F1,

Anna F1 treated with Nordox® and Rootgard

® confirming high disease level. Anna F1

grafted on wild tomato had significantly lower oozing score than Anna F1. Heavily

wilted plants recorded higher bacterial oozing but in some cases there was no oozing

especially where the plants were dry after being killed by the disease. Best time to isolate

pathogen was when the plant has completely wilted and not when dry.

More samples from the susceptible Anna F1 tested positive when compared to grafted

Ann F1 on the two rootstocks. There was no difference in the percentage of the samples

61

that tested positive between the ELISA, Kelmans’ TTC, Nutrient Agar and SMSA

indicating that the presence of the pathogen in the samples was indicative of the

symptoms observed. This was consistent with similar studies carried out by Aribaud et al.

(2014) showing that wilted plants tested positive with common used media.

Higher yield were recorded in Anna F1 grafted on Shin cheong gang rootstock and on

Cheong gang. Higher yield was as a result of all the plants surviving till end of harvest

period but also the rootstock varieties had enhanced root system which increased nutrient

uptake (McAvoy et al., 2012), water and minerals leading to increased yield (Freeman et

al., 2011). Production from grafted Anna F1 was highest with excellent fruit quality.

Similar studies in USA where rootstock variety Cheong gang was used a higher yield was

achieved compared to non grafted varieties that had low production (McAvoy et al.,

2012). Anna F1 grafted on Shin cheong gang rootstocks produced better fruit quality and

fruits shape, also reported by Taylor et al. (2011) and Paret et al. (2012).

Yield was lowest in the susceptible Anna F1 variety as well as Anna F1 treated with

Nordox® and Anna F1 treated with Rootgard

® as most plants died before full harvest. In

Similar experiments in Andaman and Nicobar Islands India grafting negatively affected

yield (Singh et al., 2014b). In tests carried out by Wimer et al. (2009). The yield of

treatments where Actigard was applied did not show any significance difference with

those non treated varieties. Therefore, grafting procedure with locally available materials

and techniques is desirable for rapid adoption of grafting benefits to farmers (Huat et al.,

2014).

62

5.3 Tolerance of rootstock to bacterial disease under glasshouse condition

In the glasshouse experiment where inoculation with R. solanacearum was carried out

disease incidence level observed was consistent with field experiment. The survival

percentage was highest in Anna F1 grafted on rootstock variety Shin cheong gang and

Cheong gang results. Survival of susceptible Anna F1 grafted on the wild tomato variety

recorded was higher than non grafted Anna F1. This indicates that the wild variety has

tolerance bacterial wilt and can be used as a grafting variety but also as a source of

resistance genes.

Treatment with Nordox® and Rootgard

® on Anna F1 did not improve tolerance to wilt,

this was in line with greenhouse experiments. This confirmed that the products Nordox®

and Rootgard® were not effective in controlling the disease. Browning was highest in

susceptible Anna F1, Anna F1 treated with Nordox® and Anna F1 treated with

Rootgard®. This indicated that these varieties had the highest disease incidence a fact that

is line with lower survival rates recorded. Lowest disease severity was recorded in

susceptible Anna F1 grafted on rootstocks Cheong gang and Shin cheong gang varieties.

Bacterial oozing was lowest in Anna F1 grafted on Cheong gang and Shin Cheong gang.

Confirmatory test with ELISA, SMSA, Kelmans’ TTC, Nutrient Agar confirmed the

disease level where higher percentage of the samples tested positive with non grafted

Anna F1 and those samples treated with Nordox® and Rootgard

®. Similar experiment

carried out by Zheng et al. (2014) where pathogens isolated from infected plants in

various disease levels produced unique pathogenic characteristics and populations

densities when plated in media.

63

Use of artificial media can be used as a management option as all treatments as non

inoculated pots had no disease. This was also reported by Masinde (2011). Because some

plants don’t show symptoms even with stem colonization with bacterial wilt causing

pathogen (Aribaud et al., 2014, Meng, 2013) farmers should test their plant periodically

to ensure that early infection is detected and control strategies put in place.

64

CHAPTER SIX: CONCLUSION AND RECOMMENDATIONS

6.1 Conclusion

Bacterial wilt of tomato was found to be present in all four counties and is one of the

biggest challenges affecting farmers in Kenya. The study showed that poor farming

practices have contributed to the quick spread of the disease within field as well as into

new areas. The study also indicated that farmers have limited information on the disease

and are using less effective methods hence continue to loose crops to the disease as well

as incur increased cost of production.

Practicing crop rotation was found to positively contribute to reduction of bacterial wilt

disease. The choice of crop and period of rotation was found have an effect on the level

of disease in a crop. Managing weeds especially those of solanaceous family can ensure

that the pathogen does not continue to multiply in absence of a susceptible crop. Control

of nematodes can help to reduce disease levels hence managing the severity of the disease

in infected fields. Farmers should practice field hygiene and use seedlings from certified

nurseries had less wilt incidences than those who used soil nurseries. Use of furrow

irrigation is discouraged as it was identified as a major contributing factor in the spread

of the pathogen and farmers should use alternative irrigation methods that have reduce

disease spreading risk.

65

Use of second generation seeds from infected plants a practice observed in Kirinyaga

county should be avoided as this contribute transmission of pathogen from the seeds to

the seedling in new fields, use of certified seeds is recommended. Grafting a susceptible

variety on rootstock Shin cheong gang can greatly increase the survival of a susceptible

variety from bacterial wilt disease and as it was found to be effective in managing R.

solanacearum. From the experiment grafted varieties recorded higher survival

percentages and reducing disease severity

Use of rootstock Shin cheong gang can help to reclaim already abandoned land parcels

that were unsuitable for production of tomato. These rootstock varieties if used in

combination with good agricultural practices and field hygiene can greatly reduce effect

of the disease and reduce spread. Use of copper product Nordox® and Rootgard

® was

found not to be effective in managing bacterial wilt.

High tolerance to the disease exhibited by the wild tomato indicates that indigenous wild

tomato has inherent tolerance characteristics for the disease. This variety can be used as

rootstock varieties or used for breeding programs.

66

6.2 Recommendations

1. Shin cheong gang F1 is recommended for use as a rootstock variety for grafting

for bacterial wilt management. There is need for partnership with plant raisers in

to enhance technology adoption and make supply of grafted seedlings possible.

2. Studies on costs benefit analysis of using rootstock varieties should be done to

compare it with the use of resistant varieties.

3. Studies to map distribution and occurrence of bacterial wilt strains in the country

should be carried to predict tolerance of varieties when their strain tolerance range

is known.

4. In addition to grafting technology farmers should adopt other disease management

practices such as restricted entry into growing areas, supervise all visitors and use

of foot baths for disinfection purposes. Use of artificial media and trays seedling

production adoption is recommended.

5. Further study on the use of wild tomato as a rootstock but also a source of

resistance genes for bacterial wilt as the variety is more adapted to local is

recommended.

6. More work to test resistant varieties which do not require grafting should be done.

67

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APPENDICES

Appendix 1: Survey Questionnaire

Details of respondent

Name of farmer: …………………………………………………………………………

Date: ……………………….…… Sex: Male Female

Responded owner/manager/employee/ other: ………………… Household size: ……

Particulars of the area

County: ……………………. ….. Location: ………………………………...………….

Tomato in greenhouse Open field Altitude: .………………….….………

Farm size (Acres): …………….. Main soil type: ………..…..……….……..……..……

Agro-Ecological Zone…………………………………………………………………….

Please read and fill as appropriate

1. How long have you been farming on the farm? …………………..………

2. What crops are you currently growing please give approximate size in acres

i. ……………………………………………………. …………….

ii. ………………………………….………………… …………….

iii. ………………………………….………………… …………….

3. For how long have you been growing tomato…………………………..

4. Which tomato varieties you are growing? Variety and reason (advantages)

i. …………….. ………………..………………...................................

ii. ……………… …………………………………………..……………

iii. ……………… …………………………………………..……………

5. Do you carry irrigation farming yes No

i. What percentage of the farm is under irrigation…………. Which type of

irrigation adopted; Drip Furrow overhead sprinklers Pivot

ii. Source of irrigation water; River Lake borehole Rain dam

6. Do you practice crop rotation yes No . Use organic manure yes No

i. Use of fertilizers; DAP CAN/UREA NPK Others………….

Method of application; Broadcasting Drip (Fertigation) others..…

ii. What is the average yield per acre in crates (1 crate 60kg) ………….

where do you sell the tomato harvest? ……………………………...........

82

iii. Who do you consult on farming issues……………………………………

7. What are the main challenges of growing tomato; start with the most challenging

Pests: Control method used:

i. …………………………………… ………………………………………

ii. …………………………………… ………………………………………

iii. …………………………………… ………………………………………

Diseases:

i. …………………………………… ……………………………………..

ii. …………………………………… ……………………………………..

iii. …………………………………… ……………………………………..

Other challenges:

i. …………………………………… ……………………………………..

ii. …………………………………… ……………………………………..

iii. …………………………………… ……………………………………..

8. Have you seen bacterial wilt disease (picture) problem on your crop? Yes No

If yes, for how long has this been a problem………………what is the average

loss in % that you have per crop………………………………..……………

9. How have you been managing the disease to achieve production?

a) Use of chemicals sprays/fumigation….………..……………….…………….

b) Use of tolerant variety(s) …….….…………………………………………...

c) Uprooting affected plants……………………………………………………..

d) Crop rotation and manures …...…………………………………..………….

e) Use disinfectant footbath……………………………………………………..

f) Irrigation/fertilizer management……………………………………………...

g) Any other (specify)…………….……………………………………………..

10. Are you willing to plant tolerant variety; Yes No Why?………………….

Additional information……………………………………………………………..

………………………………………………………………………………………

……………………………………………………………………………………………

………………….

Thank you very much for your participation

83

Appendix 2: Ingredients for R. solanacearum isolation media

A. Nutrient Agar (NA)

Nutrient Agar (Difco) 23.0 g

Distilled water 1.0 L

Dissolve ingredients and sterilise by autoclaving at 121 °C for 15 min.

B. Kelman’s Tetrazolium Medium (Kelmans TTC)

Casamino Acids (Difco) 1.0 g

Bacto-Peptone (Difco) 10.0 g

Dextrose 5.0 g

Bacto-Agar (Difco) 15.0 g

Distilled water 1.0 L

Dissolve ingredients and sterilise by autoclaving at 121 °C for 15 min.

Cool to 50 °C and add a filter-sterilised solution of 2,3,5-triphenyl tetrazolium chloride

(Sigma) to obtain a final concentration of 50 mg per L.

C. SMSA medium (Englebrecht, 1994 as modified by Elphinstone et al., 1996)

Basal medium

Casamino acids (Difco) 1.0 g

Bacto-Peptone (Difco) 10.0 g

Glycerol 5.0 ml

Bacto-Agar (Difco) 15.0 g

Distilled Water 1.0 L

Dissolve ingredients and sterilise by autoclaving at 121 °C for 15 min.

84

Cool to 50 °C and add filter-sterilised aqueous stock solutions of the following

ingredients to obtain the specified final concentrations:

Crystal Violet (Sigma) 5 mg per L

Polymixin-B-Sulphate(Sigma P-1004) 600 000 U (appro.100 mg) per L

Bacitracin (Sigma B-0125) 1250 U (approximately 25 mg) per L

Chloramphenicol (Sigma C-3175) 5 mg per L

Penicillin-G (Sigma P-3032) 825 U (approximately 0.5 mg) per L

2,3,5-triphenyl tetrazolium chloride (Sigma) 50 mg per L