ABSTRACT
Tomato (Lycopersicon esculentum L.) is a key vegetable in Kenya, listed as second most economically important in the horticultural industry. The production of tomatoes has greatly been affected by bacterial wilt caused by Ralstonia solanacearum. Losses to 100% have been reported in both greenhouse and open fields growing conditions. Most of the bacterial wilt management strategies in place have not provided effective, safe and sustainable solution. Therefore, this study contributes, to sustainable tomato production by the use of Trichoderma species as an alternative method of managing bacterial wilt. The study determined antagonistic activity of Trichoderma species from different habitats against Ralstonia solanacearum in vitro and evaluated their efficacy in managing bacterial wilt of tomato at field level. Trichoderma species were isolated and identified from different soil habitats of Karura forest, compost, manure, coffee, and tomato fields. The dominant Trichoderma species were Trichoderma harzianum and Trichoderma asperellum and antagonistic check performed using dual plate technique against Ralstonia solanacearum. The antagonistic ability of the Trichoderma species was determined by measuring the growth radius as a percentage. The field experiments were further conducted in a randomized complete design (RCBD) replicated four times in three greenhouses at Naivasha, Mirera area. The treatments included; isolated Trichoderma asperellum, isolated Trichoderma harzianum, combination of isolated Trichoderma asperellum and Trichoderma harzianum, plots with no applications, commercial Trichoderma harzianum, commercial Trichoderma asperellum, combination of commercial Trichoderma harzianum and commercial Trichoderma asperellum. The isolated Trichoderma species were mass multiplied by growing in sterilized sorghum grains. The already infested greenhouse soil was re-inoculated with isolated Ralstonia solanacearum to ensure uniform pathogen levels. This was isolated from infected tomato plants and introduced one week earlier at 35 ml per pot and properly mixed to ensure uniformity. Trichoderma application was done at the transplanting stage of a greenhouse tomato variety Anna F1, and two more applications after every two weeks. The bacterial wilt incidence and severity assessment was then done weekly and yield data recorded based on physiological maturity of the tomato crops. The laboratory in vitro work indicated that the habitats with high organic matter and fewer disturbances in terms of cultivation had high Trichoderma presence. The habitats had a total of 42 Trichoderma harzianum isolates and nine Trichoderma asperellum. Trichoderma harzianum were 15 and four Trichoderma asperellum from forest habitat while three Trichoderma asperellum and 10 Trichoderma harzianum from compost habitat. The other habitats also had similar Trichoderma isolates with low frequency. Trichoderma asperellum and Trichoderma harzianum from the forest and compost habitats had the highest percentage inhibition in vitro. In greenhouse conditions, treatments with Trichoderma asperellum or Trichoderma harzianum at P ≤ 0.05 had significant reduction of bacterial wilt incidence and severity as compared to the plots with no applications done. The Trichoderma species combinations treatments had no significant difference from the single Trichoderma species applications at P ≤ 0.05. The incidence and severity of Ralstonia solanacearum were greatly reduced hence better yields in the Trichoderma treated plots. The results indicated that Trichoderma harzianum and Trichoderma asperellum were efficient in managing bacterial wilt in tomatoes an adoptable alternative management solution to bacterial wilt in tomatoes.
Keywords: Antagonism, Incidence, Habitats, Ralstonia solanacearum, Severity, Trichoderma spp.
TABLE OF CONTENTS
DECLARATION i
DECLARATION OF ORIGINALITY FORM ii
DEDICATION iii
ACKNOWLEDGMENT iv
TABLE OF CONTENTS v
LIST OF ABBREVIATIONS viii
LIST OF FIGURE S ix
LIST OF TABLES x
GENERAL ABSTRACT xi
CHAPTER 1:
INTRODUCTION
1.1. Background information 13
1.2. Problem statement 15
1.3. Justification of the study 16
1.4. Objectives 17
1.5. Hypothesis 17
CHAPTER 2:
LITERATURE REVIEW
2.1. History of tomato production in Kenya 19
2.2. Biotic challenges to tomato production in Kenya 20
2.3. Requirements for tomato production in Kenya 20
2.4. Bacterial wilt in tomatoes 21
2.4.1. The occurrence of bacterial wilt in Kenya 21
2.4.2. The causal agent of bacterial wilt of tomatoes 22
2.4.3. Epidemiology of bacterial wilt of tomatoes 23
2.4.4. Management of bacterial wilt in tomatoes 24
2.5. Biological control agents used in crop protection 26
2.5.1. The biological control agents of pests and diseases 26
2.5.2. Trichoderma species in management of plant diseases 27
CHAPTER 3:
ABUNDANCE OF ANTAGONISTIC TRICHODERMA SPECIES IN SOILS FROM DIFFERENT HABITATS
3.1. Introduction 31
3.2. Materials and methods 32
3.2.1. Characteristics of habitats sources of the Trichoderma isolates 32
3.2.2. Sampling and collection of soil 33
3.2.3. Isolation and identification of Trichoderma species from the soil 33
3.2.4. Isolation of Ralstonia solanacearum and pathogenicity test 34
3.2.5. Screening of Trichoderma isolates for antagonism against Ralstonia solanacearum 34
3.2.6. Data analysis 35
3.3. Results 35
3.3.1. The Trichoderma species isolated from the different habitats 35
3.3.2. The isolated Ralstonia solanacearum 37
3.3.3. Antagonism of Trichoderma species against Ralstonia solanacearum 39
3.4. Discussion 41
3.5. Conclusion 42
CHAPTER 4:
EFFICACY OF TRICHODERMA SPECIES IN MANAGING RALSTONIA SOLANACEARUM IN TOMATOES
4.1. Introduction 45
4.2. Materials and Methods 46
4.2.1. Description of the experimental site 46
4.2.2. Culture and multiplication of Trichoderma spp for field application 46
4.2.3. Isolation and multiplication of Ralstonia solanacearum 47
4.2.4. Experimental design and layout 48
4.2.5. Assessment of bacterial wilt incidence and severity 49
4.2.6. Assessment of growth and yield 49
4.2.7. Data analysis 50
4.3. Results 50
4.3.1. Multiplied Trichoderma species and Ralstonia solanacearum 50
4.3.2. Bacterial wilt incidence and severity 50
4.3.3. Growth of the tomato plants in the treatments 54
4.3.4. Yield of the tomato plants in the treatments 54
4.4. Discussion 56
4.5. Conclusion 58
CHAPTER 5:
GENERAL DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS
5.1. General Discussion 59
5.2. Conclusion 60
5.3. Recommendations 60
REFERENCES 61
L I S T OF A B B R E V I A T I O N S
FAO Food Agricultural Organization
CABI Centre of Agricultural Biodiversity
KARI Kenya Agricultural Research Institute
EPPO European Plant Protection Organization
RCBD Randomized Complete Block Design
CAN Calcium Ammonium Nitrate
NPK Nitrogen Phosphorous Potassium
FAME Fatty Acid and Methyl esters
GLM General Linear model
LSD Least Significant Difference
PDA Potato Dextrose Agar
CFU Colony Forming Units
PCR Polymerase Chain Reaction
TZC Triphenyl Tetrazolium Chloride
L I S T OF F I G U R E S
Figure 3.1: Colony growth of Trichoderma spp from different habitats at day 4 36
Figure 3.2: Isolated plant pathogen Ralstonia solanacearum with virulent colonies pink color plated on TZC media 37
Figure 3.3: Trichoderma harzianum labelled A and T asperellum as B with different conidiophores shapes viewed on a light microscopic at ×100 37
Figure 3.4: The populations of Trichoderma species in different soil habitats 39
Figure 3.5: Growth activity of A - Trichoderma harzianum and B -Trichoderma asperellum
antagonism against Ralstonia solanacearum by dual plate technique on PDA 41
Figure 4.1: Pure cultures of Ralstonia solanacearum isolated from the infested greenhouse.50 Figure 4.2: Mass multiplication of Trichoderma spp on sorghum grains 51
Figure 4.3: Bacterial streaming test using wilted tomato plant stem section suspended in plain water 53
Figure 4.4: The harvested tomato grades: grade 1, 2 and 3 55
L I S T OF T A B L E S
Table 3.1: Morphological characteristics of identified Trichoderma species 36
Table 3.2: Percentage Trichoderma frequency from the different habitats 38
Table 3.3: Trichoderma harzianum from different habitats antagonism on Ralstonia solanacearum at day 3, 6 and 9 40
Table 3.4: Trichoderma asperellum from different habitats antagonism on Ralstonia 40
Table 4.1: Bacterial wilt incidence across the treatments 52
Table 4.2: Bacterial wilt severity across the treatments 52
Table 4.3: Stem browning across the treatments 53
Table 4.4: Ralstonia solanacearum ooze from the wilted tomatoes in the treatments 53
Table 4.5: Tomato height up to 1st fruit set in the treatments 55
Table 4.6: Tomato yield in kilograms per treatment 55
CHAPTER 1
INTRODUCTION
1.1. Background information
Tomato (Lycopersicon esculentum) is the second relevant vegetable crop after potatoes, at 4.85 million ha per year globally (FAOSTAT, 2019). Tomato is an annual plant originating from the South American Andes (Bedassa, Fufa, & Aga 2020; Saleem et al., 2013), with reports of the Netherlands and Mexico being the world’s leading producers (Costa & Heuvelink, 2018). In sub-Saharan Africa, Kenya is amongest the leading countries in tomato production with 410,033 tones (Ochilo et al., 2019), constituting 7% of the total horticultural produce and second-leading vegetable in Kenya (Momanyi et al., 2019). In Africa, tomatoes are important food and economic source (Mansour et al., 2019; Wafula, Waceke, & Macharia 2018). In Kenya, tomato ranks as the second widely cultivated crop in value and production after potato (Mitra & Yunus, 2018). Lycopersicon esculentum is a vegetable grown world wide due to its numerous health benefits (Liu et al., 2018) such as its high lycopene content an antioxidant, additionally reduces the chances of Type two diabetes which is related to chronic (Hassan & Barde, 2020) and cardio-vascular diseases (Banihani, 2018). Tomato production has risen over the years in Kenya. However, it suffers losses from biotic and abiotic elements (Nakhungu et al., 2021). Biotic factors involve several fungal and bacterial diseases where annual tomato production is restricted by bacterial wilt (Boyaci et al., 2021). The disease is known to occur in tomatoes (Kago et al., 2019) and other solanaceous crops (Manda et al., 2020). Bacterial wilt causes severe economic impact even to the world's big solanaceous vegetables producers like India, Italy, Portugal, Spain, Brazil, Indonesia, USA, Israel, Colombia, China, Kenya, with many more vegetables cultivating countries (Costa & Heuvelink, 2018).
Ralstonia solanacearum, which is a soil-borne pathogen is the causal agent for bacterial wilt (Kumar, & Sood 2021; Siregar et al., 2021), which occurs worldwide but is more severe in temperate, equatorial, and subequatorial areas (Yabuuchi et al., 1995). It affects beyond 450 species of plants, with most susceptible crops from the solanaceous family (Kurabachew & Ayana, 2017; Lebeau et al., 2011; Lee et al., 2018). Additionally, the bacterium can manifest itself in over 200 various species of plants, including tomato, eggplant, and tobacco (Hayward, 2006; Tsuchiya, 2014). The pathogen is phytopathogenic bacteria monitored globally owing to its persistence, destructiveness, extensive geographic distribution, and wide host range (Huet, 2014). Increased soil dampness (−0.5 to −1 bar) (Jiang et al., 2021) and high temperature (24 °C to 35 °C) supports the survival, pathogen dispersal, and easier multiplication (Nesmith & Jenkins, 1985).
The soil is the principal origin of inoculum for the pathogen where it could persist to 40 years (Chiranjeevi & Raghavendra, 2021) with temperatures of 20 °C - 25 °C (Denny, 2007). Not with standing Ralstonia solanacearum fully loses its viability at 0 % soil wetness after six months. This situation does not crop up in temperate, tropical, and subtropical regions (Singh et al., 2015). The dispersal mode is irrigation, infected soil, latently infected weeds, use of contaminated tools, seed materials, and insect vectors (Deberdt et al., 2014). The pathogen easily enters plants through wounds and natural openings (Wijayanti et al., 2021). Invading the xylem vessels spreading to the other plant parts (Genin, 2010). It multiplies (1010 cells cm-1 of the stem) by developing high exopolysaccharides leading to obstruction of vessels and killing the host. Bacterial wilt invasion is also noted to occur at the root level, preceded by the occupation of the roots (Ingel et al., 2021). Through the intercellular spaces they access the xylem vessels where high multiplication happens, causing the wilting symptoms and finally, death (Hikichi et al., 2017).
In the field conditions, symptoms of the disease occur in the mature tomato plants. The leaves frequently wilt during the day and recover at night or in the early morning hours. If the weather is favorable enough, with high soil humidity and high temperatures, the disease can lead to wilting of the entire plant (Wang et al., 2021) and eventually death (Hong et al., 2011). Bacterial wilt majorly affects plants starting from vegetative to their fruiting stages. The leaves maintain their green colour, but eventually, the whole plant wilts abruptly in hot and humid climate, conducive for pathogen growth (Singh et al., 2015). In the progressive stages, the green nature of the leaves of the wilted plants persists (Zohoungbogbo et al., 2021) and the vascular tissues turn to brownish-yellow. In the field, the disease is rampant in the more damp sections nevertheless, plants indicating symptoms of the disease can be found randomly. The plants inffected by Ralstonia solanacearum additionally dwarf as a result of insufficient water and inadequate nutrient take-up (Hong et al., 2011).
The present integrated management strategies involve; resistant cultivars and germplasm (Ravishankar et al., 2021; Pandey et al., 2020), soil sterilization (Enfinger et al., 1979; Ganiyu et al., 2020), crop rotation (Michel et al., 1996), grafting techniques (Kaushal et al., 2020). Use of coco-peat as growing media (Black et al., 2003; Singh et al., 2015), irrigation using seawater (Elsas et al., 2001) and screening of antagonists (Lwin & Ranamukhaarachchi, 2006). Planting of pathogen-free transplants (Pradhanang et al., 2005), with other crop protection methods. Although, these strategies have demonstrated to be insubstantial because of the complicated nature of soil-borne pathogens, expansive host range, extensive distribution of Ralstonia Solanacearum (Hayward, 2006). The development of resistant cultivars has been limited to averagely tolerant cultivars which are defined by location, climate, and resistance to strains of the pathogen (Adhikari et al., 2020; Chaudhary et al., 2021). Transplants can reduce the dispersal of the bacterium, but because it is a soil- borne pathogen, majority of the crops in the field can still be infected. Use of crop rotation can be complicated owing to the diverse host range of Ralstonia solanacearum strains, and that the pathogen can live or colonize various types of weeds (Hayward, 2006).
The management of bacterial wilt is hence challenging from the methods suggested (Mamphogoro et al., 2020) and that are widely used to manage the disease. The insubstantial effectiveness of the current management strategies warrants alternative methods to manage the disease (Aguk et al., 2018). Vast studies have therefore commenced on the use of biological control agents in managing plant disease. Biological control agents are soil microorganisms that occur naturally whose mode of action include initiation of host resistance through the release of plant growth stimulating hormones (Haidar et al., 2016). Additionally, they use competition of nutrients, parasitism, antibiosis and cell wall degrading enzymes. Numerous studies have been carried on the use of BCAs in management of plant diseases these include the use of Bacillus spp, Trichoderma spp and many more (Al-Ani, 2017; Konappa et al., 2018; Wang et al., 2021). Therefore the need to evaluate the performance of Trichoderma spp from different native habitats against Ralstonia solanacearum.
1.2. Problem statement
The production of tomatoes is challenged by various factors worldwide, including living and non-living factors (Gharbi et al., 2017; Zhou et al., 2019). In Kenya, biotic factors have a major economic impact on tomato production, consisting of pests, fungal, bacterial, and viral diseases (Ochilo et al., 2019) where bacterial wilt is of concern. Bacterial wilt has been observed to be endemic in different areas in Kenya, including Kirinyaga, Kiambu, Bomet, Kajiado areas (Kago et al., 2019; Kones et al., 2020) Nakuru, Muranga, and Nyandarua counties. The disease can lead to up to 100% loss of the whole crop (Kamuyu, 2017; Mbaka et al., 2013; Rivard & Louws, 2008). These resulting in low income from the growing of tomatoes due to reduced productivity of the crop (Onduso, 2014).
Most of the growers of tomatoes have resorted to abandoning their greenhouses and fields stopping farming activities of growing tomatoes and crops susceptible to the disease for a long time (Kamuyu, 2017; Mbaka et al., 2013). There are limited conventional solutions for management of bacterial wilt as they have been banned due to their non-biodegradability in the environment (Aguk et al., 2018) such as Methyl bromide. The other methods used in managing bacterial wilts such as grafting, crop rotation and other cultural practices have additionally not given satisfactory results. This therefore warrants the need for sustainable, effective, and safe methods to be utilized in the management of bacterial wilt of tomatoes. These issues have greatly affected all those involved within the production and consumption chain of tomatoes. Additionally, this impacts negatively on the economical aspect of the society at large and Kenya’s food security.
1.3. Justification of the study
In tomato production, bacterial wilt is the commonest disease for both open field and greenhouse setup (Kago et al., 2019). The management of Ralstonia solanacearum has been difficult as the pathogen has proven to persist for duration in the soils and wide geographical distribution (Mihovilovich et al., 2017). The strategies in managing bacterial wilt over the years have included the use of disease-free planting materials or tolerant varieties; crop rotation, and chemical use. Disease-free planting materials involving grafting to more tolerant tomato varieties have been reported to manage bacterial wilt (Alividza, 2019), although a costly method to a small-scale farmer. Tolerant tomato varieties that have been tried still show different levels of susceptibility to the pathogen (Michael et al., 2020), not giving the farmer proper tolerance against the disease. Crop rotation has been observed to be ineffective as the pathogen can endure and survive in the soils for a long time (Jiang et al., 2017; Mihovilovich et al., 2017; Yang-Xian et al., 2015). Chemicals are known to affect bacterial wilt however, they are very few (Aguk et al., 2018) and have become ineffective due to overuse (Shiva et al., 2018). Therefore, chemicals as a management strategy are insufficient (Namisy et al., 2019) and not sustainable. Increased use of chemical techniques involving bactericides have been reported (Marian et al., 2018) but have been seen to cause negative consequences on the surroundings and human health (Satapute et al., 2019). Reports have also shown that certain chemical molecules pose a high risk to the environment once they start undergoing the degradation process (Kumar et al., 2020; Sharma et al., 2020), whose results are detrimental. They are greatly being placed in reduced microbial life due to microbial degradation (Tudi et al., 2021) and pollution in the environment (Warra & Prasad, 2020). The handlers and users who are the farmers are also at risk as some of the pesticide formulations have heavy metals that are lethal to human health (Dhananjayan et al., 2020).
Therefore the need for alternative safe, sustainable, and effective methods in controlling bacterial wilt of tomatoes (Morais et al., 2019). According to Kumar (2017), (BCAs) have been used as antagonistic plant pathogenic agents. These BCAs exhibit characteristics that involve self-sustaining ability, reduced input of non-replenishable resources, scattered across after the first establishment, and provision of continuous disease suppression (Whipps, 2007). Research has shown that combining BCAs like Trichoderma species and Bacillus in the management of Ralstonia solanacearum gives promising results (Kariuki et al., 2020; Konappa et al., 2018). Therefore, the need for use microbial-based pesticides, which are deemed more sustainable and safer, as an alternative solution in managing the disease (Todorović, 2017). This ensures the farmers' safety during handling and application of the biological control agents in regards to their health (Abd-Elgawad, 2020), is cost-effective (Bhusal & Mmbaga 2020; Messmer et al., 2021), and safe to environmental microbial life (Kumari et al., 2020) hence this study.
1.4. Objectives
The general objective of the study was to contribute to effective management of bacterial wilt of tomato by the using Trichoderma species.
The specific objectives were
i. To determine the abundance of Trichoderma species from different habitats and their antagonistic activity against Ralstonia solanacearum in vitro.
ii. To evaluate efficacy of Trichoderma species in managing bacterial wilt of tomatoes.
1.5. Hypothesis
The following hypotheses were to be investigated in this study.
i. Native Trichoderma species are not abundant in soils and have no antagonistic activity against Ralstonia solanacearum in vitro.
ii. Native Trichoderma species have no effects on bacterial wilt incidence and severity in field conditions.
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