EVALUATION OF BACILLUS AND TRICHODERMA SPECIES FOR BIOLOGICAL CONTROL OF BACTERIAL WILT CAUSED BY RALSTONIA SOLANACEARUM IN TOMATO

Tomato (Lycopersicum esculentum) has been rated second most important vegetable crop in Kenya. Bacterial wilt caused by Ralstonia solanacearum, is a major biotic constraint to tomato productivity with yield losses of up to 64%. The available management strategies such as cultural practices and use of chemicals are limited in effectiveness. This study therefore focused on biological control agents (BCAs) in the management of Ralstonia solanacearum. Bacillus and Trichoderma isolates that are antagonistic to Ralstonia solanacearum and other important bacteria pathogens were screened and identified in vitro. The study also evaluated the effect of the Bacillus and Trichoderma isolates in management of bacterial wilt disease under field conditions.

Twenty-eight Trichoderma species isolated from local soils at Kabete and 19 Bacillus isolates retrieved from earlier screened isolates maintained at the Plant Science and Crop Protection Department, University of Nairobi were used as the antagonists against three pathogens. Paper disc method was used to test the antagonistic activity of the isolates against Ralstonia solanacearum, Xanthomonas campestris pv campestris and Pseudomonas spp in vitro. The experiment was conducted in a complete randomized design, with three replicates. Antagonistic activity was assessed by measuring the radius of zone of inhibition (ZOI) of the pathogen due to the antagonist. Field experiments were conducted in a randomized complete block design at Kabete and Mwea sites in Kenya. The treatments included; 3 Trichoderma isolates (T1, T2 and T4), 2 Bacillus isolates (CB64 and CA7), a mixture of T1, T2 and T4, chemical standard and distilled water as control. Trichoderma and Bacillus isolates were grown on sterilized sorghum grain and cow manure carriers, respectively. Antagonist’s inoculation was carried out by dipping tomato plants for 30 minutes in each treatment suspension. Each treatment was applied at a rate of 150ml/plant hole and this was repeated after 35 days. Soils were sampled prior to transplanting, 60 days and 112 days after transplanting for quantification of R. solanacearum population and at 126 days for determining the total microbial count in the soil. Bacterial wilt incidence was assessed every week by counting the number of wilted plants in each plot. Yield parameters were assessed at physiological maturity.

In vitro studies showed that 10 Bacillus and 11 Trichoderma isolates had varied antagonistic activity against all the pathogens tested. Trichoderma isolate T1 was the most effective in inhibiting the growth of Ralstonia solanacearum with a mean ZOI measuring 13.5mm while Bacillus isolates CB64 was the best antagonist with a mean ZOI measuring 4.3mm. 

Trichoderma isolate T28 and Bacillus isolate CA5 showed the highest ZOI of 15.2mm and 6.6mm, respectively against Xanthomonas campestris. The antagonists screened gave lower activity against Pseudomonas sp. compared to other pathogens. Trichoderma isolate T28 showed the highest ZOI of 9.3mm and Bacillus isolate CB14 and CB22 gave similar ZOI of 5.3mm. Isolates of Trichoderma showed better activity by more than 56.67% compared to isolates of Bacillus. All the treatments evaluated under field conditions significantly reduced bacterial wilt incidence and severity at P≤ 0.05 than the control at Kabete and Mwea sites. Trichoderma isolate T1 followed by Bacillus isolate CB64 were the best in reducing the disease incidence by more than 61.66% and 53%, respectively at both sites. Treatment CB64 and T1 had the highest reduction of R. solanacearum population in the soil by 93.17% and 92.07%, respectively. However, control had a pathogen increase of 20.40%. The total microbial count was highest in Bacillus treated plots in both sites. Isolate CB64 had the highest count of 1.32×105 CFU/ml at Kabete and 1.21×105 CFU/ml at Mwea site. CB64 and T1 performed significantly better compared to the standard, while the mixture of isolates T1, T2 and T4 performed poorest in all parameters. The treatments also increased the yield of tomato. Results from this study showed that Trichoderma and Bacillus isolates are effective biological control agents for use in management of bacterial wilt.

Keywords: Tomato, bacterial wilt, Ralstonia solanacearum, Trichoderma, Bacillus

 

 

 

 

Table 3. 6: Radius of zone of inhibition (mm) induced by Trichoderma isolates against

Table 4.4: Severity index (%) of bacterial wilt disease, 126 days after treatment application 59 Table 4. 5: Quantification of Ralstonia solanacearum population in the soil after application

Table 4. 7: Bacteria and fungi diversity after 126 days of soil treatment based on colony groups (with similar characteristics) 65

 

Figure 3.6: ZOI of Bacillus isolates against (a) Pseudomonas sp, (b) Ralstonia solanacearum, (c) Xanthomonas campestris pv campestris and (d) control (filter paper disc dipped in sterile water). 30

Figure 3.7: Mean radius of zone of inhibition (ZOI) in mm (Millimeters) induced by Bacillus isolates against three plant pathogens after 48 hours of incubation 35

Figure 4. 2: Disease progress curve for percentage disease incidence due to Trichoderma isolates at Kabete site (K) and Mwea site (M) 56

Figure 4. 4: R. solanacearum population in the soil (x 106 cfu/ml) at Kabete (K) and Mwea

 

 

Tomato (Solanum lycopersicum) has been documented as one of the most important vegetable crop belonging to the nightshade family of Solanaceae (Adams et al., 2006). The crop is reported to have originated from South America (Jenkins, 1948). Taylor, (1986) confirmed tomato originated from South America by documenting geographical dispersal of the native wild relatives location at 0º to 20º S and 64º to 81º W where the species breed freely in the wild. Tomato was first domesticated in Mexico through pre-Hispanic culture (Jenkins, 1948). According to Champoiseau and Momol, (2008), during 1870, an American botanist Alexander W. Livingston devoted his entire life on this crop where he conducted selective breeding on improving and uplifting tomato into the customary form, we are aware of today. According to Athertonand, (1986), tomato was introduced in Africa by early Europeans during the pre- colonial period in the 16th century.

Food and Agriculture Organization (FAO) reported that in 2017, the area under tomato cultivation was 4.8 million hectares which produced 188 million tons with a market revenue of more than 190.4 billion USD globally (FAOSTAT, 2019). China is the leading tomato producers in the world accounting for 33% of the global production followed by India, United States of America, Turkey and Egypt (FAOSTAT, 2017 and 2019). In Africa, tomato is cultivated on 1.3 million ha which produces an average of 37.8 million tons annually (FAOSTAT, 2017). Egypt is the biggest tomato producer in Africa recording an average production of 7.3 million tons in 2017 followed by Nigeria, Tunisia and Morocco (FAOSTAT, 2019). Kenya is among the African’s top ten leading producers of tomato, cultivated on 0.4 million hectares which produces about 280, 000 tons annually (FAOSTAT, 2017). Therefore, Kenya accounts for 0.2% of the world production. The crop in Kenya is mainly grown in open fields under irrigation and in greenhouses to meet the increasing demand for tomato (Monsanto, 2013). In Kenya, the key counties where tomato is grown includes; Kirinyaga, Laikipia, Kiambu and Kajiando (KARI, 2005).

Tomato has a wide range of uses and contribute to a healthy, well balanced diet. Tomato fruits are consumed fresh in salads or cooked in sauces, soup and meat or fish dishes. In addition, they can be processed into puree, jam, paste, powder, juices, tomato sauce and canned or dried into economically important processed products (Ochilo et al., 2019). Tomato are excellent source of important micronutrients like iron, potassium, ascorbic acid and antioxidants like vitamins C, B, amino acids. These nutrients are what makes tomato highly recommended by dieticians and nutritionists for controlling cholesterol and for weight reduction (Basco et al., 2017).

Poor agricultural practices like continuous cropping of tomato crop without rotation, furrow irrigation, poor field hygiene and use of infected seedlings have led to increased disease incidences (Monsanto, 2013). Pests that include; leaf miner, American bollworm, nematodes, mites and diseases such as tomato blights, tomato wilts, blossom end rot not only cause reduction of yield and quality but also increase the cost of production (Monsanto, 2013). Among the serious diseases that attack tomato, bacterial wilt is one of the most devastating. Bacterial wilt caused by Ralstonia (formally Pseudomonas) solanacearum (Smith) causes massive death of plant resulting in yield and income reduction for farmers (Yabuuchi et al., 1995).

Ralstonia solanacearum is a soil borne pathogen that once it infects the soil, it is easily spread within the adjacent fields (Virendra, 2017). This is mainly through contaminated irrigation/flowing water, transplants and this not only affect the crop but also renders the farm unusable to production of any Solanaceae crops (Champoiseau and Momol, 2008). The pathogen invades the roots and colonizes the xylem vessels causing wilt especially in tropical and sub-tropical regions (Champoiseau and Momol, 2008). The bacteria produces several phytotoxins such as extracellular polysaccharide and combination of plant cell wall degrading enzymes such as endoglucanase and polygalacturonase that enables it to invade plant’s natural mechanism (Yendyo et al., 2017). This pathogen infects a wide host range causing disease to approximately 450 crop species that include; tomato, potato, eggplant, tobacco, pepper, among others (Maji and Chakrabartty, 2014).

Management of bacterial wilt disease is challenging because the available methods are limited in their effectiveness. The long-term use of chemical products such as bactericides and fungicides induce resistance in the pathogen making it tolerant to chemical application (Maji and Chakrabartty, 2014). There are hardly available chemicals to manage bacterial diseases except the copper based and antibiotics that are used for human and animal medicine and are therefore highly restricted (Yendyo et al., 2017). Use of cultural practices such as crop rotation has faced challenges due to unpredictable survival of the pathogen (Sequeira, 1993). R. solanacearum has ability to survive in the soil for long period of time even in the absence of vegetation (Champoiseau, and Momol, 2008). Use of resistant tomato variety is not effective since the pathogen has a number of distinct strains that makes a variety resistant to the disease in one region become susceptible in another (Onduso, 2014). It is also difficult to get good resistant tomato varieties at reasonable price with qualities preferred by consumers (Onduso, 2014). The limited effectiveness of these management strategies calls for a focus on alternative methods to manage bacterial wilt disease. Biological methods that can be used to manage diseases have been an attractive area of study by many researchers.

Biological control agents (BCAs) are naturally occurring soil microorganisms that aggressively attack plant pathogen, suppress the diseases and help in control of pests and weeds (Singh et al., 2014). The mechanism involved in biological control includes; parasitism, antibiosis, competition for nutrient and space, cell wall degradation by lytic enzymes and induced disease resistance (Singh, 2013). The idea of using microbes as a method of pests and disease management dates backs in the 19th Century. Since then, several studies have been done to control bacterial wilt of tomato with application of BCAs such as Trichoderma spp, Bacillus spp, Pseudomonas spp among others (Kumur and Ganesan, 2006; Singh, 2013; Maji and Chakrabartty, 2014; Yendyo et al., 2017). It is therefore important to evaluate the antagonistic ability of BCAs against the pathogen and incorporate them into successful disease management strategy.

Tomato production in the country has been exposed to multiple risks that results in large losses both qualitative and quantitative hence decreasing tomato profitability. This decrease is caused by both pest and diseases. Insect pests that include leaf miner, flea beetles, fruit borers, aphids and whiteflies affect the crop from the time of emergence to harvest. Diseases that affect tomato include blights, mildews, cankers, and wilts with collective losses of up to 100%. Among the diseases affecting tomato production, bacterial wilt is one of the most destructive disease and is caused by Ralstonia solanacearum. Being a soil borne pathogen, it is difficult to manage R. solanacearum especially in the already infected fields and this has led to reduced income for small scale growers (Taylor et al., 2011).

The farmers involved in horticultural export find it difficult to manage bacterial wilt disease because there are hardly known chemicals used to manage bacteria diseases. The available chemicals are normally copper based fungicides that are limited in effectiveness and antibiotics that are used for human and animal medicine hence highly restricted. Use of chemicals has led to more problems as they are non-biodegradable and they pollute the environment which has greatly contributed to the current climate change. In addition, chemical residues on the produce cause health problems to human beings. Furthermore, chemical pesticides are harmful to farmers applying them and continuous application has led to build up of resistance among pathogens and pests (Maji and Chakrabartty, 2014). Target markets for tomato produce such as EU market have set strict quality requirement for food produce that includes: safe, clean produce free of chemical residues and safe for human consumption. This has greatly affected farmers who use chemicals intensively as their produce are rejected in the market.

Cultural practices like crop rotation used for management of R. solanacearum have challenges due to unpredictable nature of the pathogen. The pathogen has ability to survive in the soil for long period of time even in the absence of vegetation (Champoiseau and Momol, 2008). The pathogen is composed of a number of distinct strains hence varieties tolerant/resistant to the disease in one region may be susceptible in another (Onduso, 2014). All this factor has adverse effects on livelihood of everyone in the society due to aggravating issues of food insecurity which consequently leads to hunger. Most people especially the farmers whose livelihoods depend on agriculture end up in poverty and this may lead to decline in a country’s economy.

Tomato is one of the most important vegetable crops in Kenya and the devastating effect of R. solanacearum on tomato production has increased loss of yields. In an effort to meet the demand of tomato, farmers result to use of chemicals. However, concerns on the toxicity of the chemical products used and their retention potential in the food chain has led to a shift from exclusive use of chemicals to use of biological control methods or the judicial use of chemicals in an integrated package.

Satisfying consumer needs, taking care of human health and the environmental safety can be achieved through sustainable, affordable and effective disease management strategies. The method used should guarantee continuous and increased production of quality/quantity tomato produce. This will ensure increased income for tomato growers and fair prices for the consumers (Taylor et al., 2011).

BCAs are plant growth promoting bacteria and fungi that have antagonistic activity against R. solanacearum. Hence, they are able to reduce R. solanacearum inoculum intensity, disease incidence and severity with no residue effect on tomato produce. Moreover, they do not cause environmental pollution or other adverse effects that are associated with chemicals. In the long run they slow down or reduce the rate of climate change that has caused numerous problems. Use of BCAs is an effective method as some antagonists have broad spectrum of activity hence can manage different pathogens in one application and once, they establish, the strategy reduces the cost of production. Most microbial antagonists are found naturally in soils and their efficacy can be enhanced through addition of BCAs. The present study involves screening, identifying and evaluating Bacillus and Trichoderma spp that have antagonistic effect against R. solanacearum in vitro and under field conditions. This will assist researchers in coming up with effective BCAs to recommended for commercial use in management of bacterial wilt of tomato.

 

The general objective is to contribute to sustainable horticultural production through the use of BCAs in the management of bacterial wilt of tomato.

Specific objectives: