Evaluation of the efficacy of Trichoderma and Pseudomonas species against bacterial wilt Ralstonia isolates of tomato ( Lycopersicum species)

Ralstonia solanacearum causes bacterial wilt of tomato and limits the crop production, and antagonistic microorganisms use to suppress the disease, of which Trichoderma and Pseudomonas species are the most effective agents to control bacterial wilt. In the present study, attempt was made to isolate these two microorganisms to evaluate their effectiveness to control R. solanacearum , the causal agent of bacterial wilt disease of tomato under greenhouse conditions. Thus R. solanacearum , Pseudomonas and Trichoderma spp. were isolated from wilted and healthy tomato plants grown from farmer's field in Ziway and Meki, Oromia Ethiopia. The virulence of the pathogen and the antagonistic effect of the bacteria and fungi were evaluated against R. solanacearum in vitro and in vivo condition. Based on the in vitro results the best two isolates were selected to show their antagonistic effect under greenhouse condition in single and combined designs. The result showed the pathogenicity test of the isolates were evaluated under greenhouse condition, and isolate AAURS1 showed highest virulence (75%) followed by isolate APPRCRS2 with pathogenicity of 50%. With regard to antagonism test, isolates AAURB20 and AAUTR23 showed the highest inhibition against R. solanacearum with inhibition zone of 16 and 15 mm, respectively. Among the treatments co-inoculation was more effective and reduced disease incidence by 13.33% and increased the bio-control efficacy by 72.22% when compared with individual treatment and negative control. The isolates significantly increased the plant height and dry weight by 72.33 cm and 12.18 g, respectively. Thus, the combined use of the biocontrol agents significantly reduced the incidence of tomato bacterial wilt disease. However, their performance should be evaluated using other yield parameters under field conditions to produce healthy tomato seedling to minimize the use of chemicals and reduce environmental pollution.


INTRODUCTION
Tomato (Lycopersicon esculentum) is the second most important vegetable crop in the world next to potato (Gebisa et al., 2017). The center of origin of Solanum lycopersicum has been localized in the narrow band between the Andes mountain ranges and the Pacific coast of western South and extends from southern Ecuador to northern Chile, including the Galapagos Islands (Peralta et al., 2008). Tomatoes production accounts for about 4.8 million hectares of harvested landarea globally with an estimated production of 162 million tones. China leads world tomato production with about 50 million tones followed by India with 17.5 million tonnes (FAOSTAT, 2014). In Africa, the total tomato production for 2012 was 17.938 million tons with Egypt leading the continent with 8.625 million tones (FAOSTAT, 2014). It is an economically important vegetable in Ethiopia. According to the CSA (2019), the country produced 27,774.538 tons of tomato in 5235.19 hectares of land in 2018.
Ralstonia solanacearum is ranked as the second most important bacterial pathogen among the top ten economically important soil borne pathogens that cause severe yield losses on different solanaceous crops in different parts of the globe (Mansfied et al., 2012). Different studies showed the bacterial wilt pathogen inflict 50 to 100% loss on potato in Kenya (Muthoni et al., 2012), 88% on tomato in Uganda (Katafiire et al., 2005), 70% on potato in India (APS, 2005). It is one of the most destructive and widespread disease of tomato in Ethiopia and its prevalence is as high as 55% in major tomato producing areas of the country (Biratu et al., 2013). Different methods, mainly pesticides are employed to control bacterial wilt of tomatoes.
Chemical controls with Actigard (e.g., acibenzolar-Smethyl) and phosphorous acid effective to control bacterial wilt under at greenhouse and to a lesser extent field conditions (Pradhanang et al., 2005). The use of excessive agrochemicals is negatively perceived by consumers and supermarket chains due to residual chemicals in horticultural products. In addition use of chemical pesticides contaminate groundwater, enter food-chains, and pose hazards to animal health and to the user spraying the chemicals. Consequently, several members of the European Union (EU) (Sweden, Denmark, and Netherlands) decided in the mid-late 1980s to decrease the use of chemicals in agriculture by 50% and ban some of them through time within a 10-year period (Butt et al., 2001).
However, effective and long term control is possible by using a combination of diverse methods including the use of resistant/tolerance varieties, cultural practices, biological and chemical control as parts of an integrated pest management strategy to control bacterial wilt caused by R. solanacearum (Persley, 1986). The use of biological control agents alone and/or together with other control methods as part of integrated pest management (IPM) practices is widely employed to overcome these problems (Barbari, 2016).
Soil bacteria and fungi which flourish in the rhizosphere of plants and, stimulate plant growth are collectively known as plant growth promoting microorganisms Alelign 263 (PGPM). The most abundant and useful microorganisms in the rhizosphere are Pseudomonas, Bacillus, Burkholderia, Agrobacterium, Streptomyces, Trichoderma, Penicillium, and Gliocladium. These microorganisms are used with the aim of improving crop yield by augmenting nutrient availability, enhancing plant growth and protection of plants from diseases and pests (Vessey, 2003). They are capable of secreting hydrolytic enzymes and causing mycoparasitism on pathogens and narrow spectrum antagonistic activity compared to synthetic pesticides, and thus used singly or in combination with one another and chemicals in integrated pest management (IPM) to suppress plant-pathogens (Handima and Kalaivani, 2014).
Trichoderma and Pseudomonas species are the most frequently isolated fungi and bacteria in all the root ecosystems, respectively. Trichoderma spp. effective in controlling phytopathogens due to their ability to grow toward the hyphae of other fungi, coil around them and degrade the cell walls of the pathogen. Morsy et al. (2009) showed that, the dual application of Trichoderma viride and Bacillus subtilis decreased the percentage of pathogen infection and increased survival rate than single inoculation in tomato. The biocontrol potential of two Trichoderma spp. on sclerotia rot disease of tomato plants in Chile and Iceland was evaluated and the result showed that, Trichoderma harzianum and T. viride reduced the disease by 74.50 and 68.75%, respectively (Kator et al., 2015).
Several studies also showed that the application of these antagonists have a dramatic effect on bacterial wilt disease caused by R. solanacearum on tomato. Narasimha et al. (2013) showed that Trichoderma asperellum (T4 and T8) isolates delayed wilt development by R. solanacearum, effectively decreased the disease incidence (51%), improved plant growth promotion and increased fruit yield under field conditions.
Another study also showed that Trichoderma spp. AA2 and Pseudomonas fluorescens PFS were most potent inhibiting the growth of Ralstonia species and the field study indicated Trichoderma spp. and P, fluorescens alone were able to prevent 92 and 96% of the infection and combination of both were more effective, preventing 97% of infection compared to chemical control methods that prevented 94% of infection (Yendyo and Pandey, 2017). This shows the promising potential of native isolates of Trichoderma spp. and P, fluorescens as biocontrol agents against Ralstonia spp.
In order to identify successful microorganisms as biocontrol agents, continuous screening of new isolates is needed for effective formulation against specific E-mail: shashitu2020@gmail.com. Tel: +251919823556.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License pathogens. Therefore, this study was initiated with the objective of evaluating the efficacy of Trichoderma and Pseudomonas spp.
individually and in combination against bacterial wilt pathogen, R. solanacearum of tomato under in vitro and in vivo conditions.

Sample collection
Soil samples from the rhizosphere of healthy and bacterial wilt infected tomato plants were collected from different fields from Ziway and Meki along the Rift Valley, which is one of the most important vegetable producing areas in the country. Diseased plant samples were selected based on visible characteristic symptom of bacterial wilts (Yendyo and Pandey, 2017).

Isolation of R. solanacearum from wilted tomato plants
Isolation of the wilt pathogen was undertaken according to Kelman (1954). Diseased tomato stem samples were washed with tap water, and surface sterilized with 70% ethanol for 2 min and rinsed repeatedly in sterile water for 5 min. The samples were then suspended in the 5 ml sterile distilled water for 10 min to make them turbid due to oozing of bacterial cells from cut ends of diseased tissue. The bacterial suspensions were prepared to appropriate dilutions from which, 1 ml of the bacterial suspension was spread onto the surface of solidified Triphenyltetrazolium chloride agar (TZC) medium and incubated at 28±2°C for 48 h.

Identification of virulent/avirulent isolates of R. solanacearum
The virulent and a virulent isolates of the pathogen were differentiated by Kelman method (1954) on TZC agar medium and compared with isolates obtained from Ambo Plant Protection Research Center. The virulent isolates were detected based on their pink or light red colored colonies with characteristic red center and whitish margin, whereas the avirulent isolates were differentiated on their colonies characterized by smaller, off-white and non-fluidal or dry texture on TZC medium after 24 h of incubation.

Pathogenicity test
Virulence of the isolates was carried out by inoculating them on the tomato seedlings according to Margaret et al. (2011). Tomato seeds were planted directly in 20 × 18 cm plastic pots containing sand and soil in the ratio of 2:1 (3 kg of soil and 1.5 kg of sand) soil and sand was obtained from AAU. Bacterial isolates were grown on nutrient broth medium for two days at 30°C, suspended in sterile distilled water and adjusted to OD 600 nm = 0.1 (approximately inoculum size of 10 8 CFU/ml) (Ran et al., 2005). Inoculation was made at the four true leaf stages by injecting into the stem with a needle. Plants inoculated with sterile water served as control and pots were regularly watered. Tomato plants were observed for development of typical wilt symptoms, and the severity of bacterial wilt was recorded based on the severity scale as follows: (% of shoot wilted, using a scale of 0-5 where 0=No symptoms, 1=one leaf wilted (1%-25%), 2=2 or 3 leaves wilted (26%-49%), 3=half plant wilted (50%-74%), 4=all leaves wilted (75%-100%), 5=Plant dead) (Tans- Kersten et al., 2001).

Biochemical characterization of isolates
The selected virulent isolates were also inoculated on nutrient agar plates and incubated at 28°C for 24 h for biochemical characteristics including Gram reaction, catalase test, oxidase test, motility and indole production test.

Isolation of antagonists from tomato rhizosphere soil
Isolation of the bacterial and fungal antagonists was carried out using soil dilution method according to Johnsen and Nielsen (1999). Ten grams of rhizosphere soil sample collected from healthy tomato plants was prepared to appropriate dilutions (10 -1 to 10 -5 ) and 10 -3 to 10 -5 plated on to KB (King's B medium) for rhizobacteria and PDA for Trichoderma spp. (fungal antagonists). The Petri plates were incubated at 25°C for 7 days for fungal antagonists and at 28°C for two days for rhizobacteria.

In vitro antagonism test against the pathogen
The antagonism tests were carried out on the fungal and bacterial isolates against the bacterial wilt pathogen in vitro used disk diffusion method (Nguyen and Ranamukhaarachchi, 2010). The bacterial wilt pathogen was grown on nutrient broth for 48 h from which, 100 µml was swapped onto Petri plates with nutrient agar. And the bacterial antagonist grow on nutrient broth for 48 h and Trichoderma were grown in Potato Dextrose Broth (PDB) (20 g/l dextrose, 4 g/l potato extract and 15 g/l agar) for 7 days and sterilized Paper disc (5 mm) was immersed in each test antagonist solution and was spotted at the center of the pathogen-inoculatedplate. Paper disc immersed in sterile distilled water and spotted at the center of the plates with the pathogen was used as control. Plates were incubated at 28°C for 48 h to measure inhibition zone of diameter.

Morphological characterization of antagonist fungi
Morphological characterizations of the fungal antagonists were performed by growing them on PDA at 25°C for 7 days. They were characterized by observing their cultural characteristics (colony color on the front and reverse side of the plate, growth rate, conidiophore branching, conidial shape and compared with the culture collection from Addis Ababa University (AAU).

Biochemical characterization of antagonist bacteria
The selected bacterial antagonistic isolates were characterized by the biochemical tests including Gram differentiation and Gram reaction, growth at 41°C, catalase test, oxidase test, pigment production, gelatin liquefaction, hydrogen cyanide production, ammonia production, phosphate solubilization, and carbohydrate fermentation test by using standard methods (Bhargavi and Tallapragada, 2016).

Compatibility test
In vitro compatibility test between the selected bacterial and fungus isolate was conducted using dual culture method in order to determine whether they can be used in combination. Thus, an overnight culture of the bacterium grown in King's B broth was streaked on one side of a Petri-dish containing NA of 2% sucrose. The other side of the Petri-dish was inoculated with 1 cm disc of 7 days old Trichoderma spp. The plates were then incubated at 25°C to test the presence of inhibition diameter between the two isolates.

Antagonistic test of the isolates against the test pathogen on tomato under greenhouse condition
Tomato seeds from local Gelelima and Galilea varieties that were obtained from Melkasa Agricultural Research Center were sown in seedling bed. After 25 days, the seedlings were transplanted in pots filled with potting mixture (soil: sand at 2:1 w/w/) at the rate of three seedlings per pot. Inoculum of the pathogen and the selected biocontrol agents; Pseudomonas and Trichoderma were prepared at 10 8 cfu ml -1 and conidial suspension of (10 8 spores ml -1 ), respectively as described by Sivan et al. (1984). 50 ml of the mixed inoculum of the pathogen and antagonists were inoculated into the pots at the same time using soil drench method (Algam et al., 2010). Each treatment was replicated thrice in completed randomized design (CRD). The treatments were: where BE = Biocntrol efficacy, DIC = Disease incidence of control, GPE=growth promotion efficacy. Plant growth was measured in terms of shoot height and shoot dry weight 2 months after sowing. For dry weight measurement, plants were dried in an oven at 70°C for 3 days to constant weights.

Data analysis
All statistical analysis was performed with SAS software. Measured variables were submitted to one-way ANOVA. Duncan's multiple range test was used for mean values discrimination at 5% level.

Cultural and biochemical tests for identification of R. solanacearum
A total of fifteen bacterial isolates were collected from infected tomato plants with bacteria wilt, of which four isolates that showed the typical cultural characteristics of virulent R. solanacearum were selected for in vivo pathogenicity studies (Table 1). These isolates exhibited pink or light red colonies or red center with whitish margin. All of them were rod shaped, Gram negative, non-spore forming, motile, and catalase and oxidase positive and indole negative bacteria (data not shown). These results conformed to the characteristics of virulent strains of R. solanacearum on TZC medium after 24 h of incubation reported elsewhere (Kelman, 1954;Narasimha et al., 2013).

Pathogenicity tests
The result showed that bacterial wilt of tomato occurred within 15 to 21 days after inoculation. All isolates were pathogenic on tomato plants and produced typical symptoms of wilt. Isolate AAURS1 exhibited the highest disease incidence (75% wilting) followed by 50% of wilting with APPRCRS2, whereas isolates AAURS3 and AAURS4 induced weak infection on the host (Table 1). Other reports also showed 50 to 71% wilting on different tomato varieties (Abo-Elyousr and Asran, 2009). El-Ariqi et al. (2005) also reported that different isolates of R. solanacearum caused 52 to 97% of wilting. Selim et al. (2011) have also reported that different isolates of R. solanacearum showed different wilt incidence ranging from 40 to 96%.

Isolation and screening of plant growth promoting antagonist
A total of twenty rhizobacterial and six fungal isolates were collected and preliminarily screened for their antagonistic property on the test pathogen. They were evaluated against two isolates of R. solanacearum using paper disc diffusion method under in vitro conditions. The data showed that the bacterial isolate, AAURB20 had the highest mean inhibition diameter of 15 and 16 mm followed by the fungus, AAUTR23, isolate with inhibition diameters of 14 mm against the two test pathogens AAURS1 and APPRCRS2, respectively (Table  2). This implies that the antagonists have potential to be used in the greenhouse for in vivo bio protection of tomato plant. The in vitro antagonistic activity of P. fluorescens was also reported by Alyie et al. (2008) where P. fluorescens isolates (PF20) had the greatest inhibition zone in vitro against R. solanacearum with the inhibition diameter of 14.15 mm and other two isolates (PR-3-I-x, PR-4-I-x) showed 3.2 and 3.5 mm, respectively. This suggests that the mode of action or the type of antibacterial metabolite production may vary among the isolates tested (Williams and Asher, 1996). The inhibitory activity of P. fluorescens against the pathogen in the study is in line with that of Henok et al. (2007), Aliye et al. (2008) and Yendyo and Pandey (2017) where they reported that isolates of P. fluorescens had significantly inhibited under the bacterial growth of R. solanacearum under in vitro conditions. The in vitro antagonistic activity of T. asperellum was reported by Narasimha et al. (2013) that inhibit the growth of R. solanacearum with inhibition zone ranging from 11 to 27 mm diameter.

Morphological and biochemical characterization P. fluorescens
Based on the antagonistic potential characteristics, twelve isolates of P. fluorescens were studied in detail for colony, colour, growth type, cell shape, and fluorescens of the isolates. All the isolates showed similar results with regard to round yellow colony texture on King's B medium with production of fluorescent pigment gelatin liquefaction positive, catalase, oxidase, Gram stain negative, positive KOH and lack of growth at 41°C. This, together with rod shape cell morphology and fast growth further confirmed the isolates to be P. fluorescens as reported by earlier workers (Meera and Balabaskar, 2012).

Carbohydrate fermentation test for bacterial isolates
The isolates utilized the tested carbohydrates and produced yellow color on the medium, which was an indication of the utilization of each carbohydrate. All isolates were capable of utilizing glucose followed by maltose, fructose and lactose (Table 3). The utilization of different carbohydrate sources by the isolates was similar with P. fluorescens reported by Henok et al. (2007).

Morphological characterization of fungi
The fungal isolates were characterized by fast growth with dark green mycelia colony on PDA. Microscopic study revealed that it produced globes to ellipsoidal conidial shape, which was much branched.

Plant growth promoting rhizobacteria (PGPR) characterization of rhizobacteria
Among isolates that were screened for their plant growth promoting activities viz., HCN production, ammonia production, and phosphate solubilization. Isolate AAURB20 and AAURB19 exhibited strong HCN production followed by isolates AAURB8 and AAURB16 (Table 4). Among test isolates, AAURB 7 and AAURB 20 displayed three PGP characters; whereas most of the isolates exhibited only one of the PGP characters (Table  5). The strains of P. fluorescence isolated from rice fields are found to produce HCN against S. oryzae (Meera and Balabaskar, 2012). Another important trait of PGPR, that may indirectly influence the plant growth, is the production of ammonia. In this study, isolate AAURB7 and AAURB20 produced ammonia. Another study showed that 95% of the isolates from the rhizosphere of rice crops produced ammonia (Joseph et al., 2007).
Phosphorous is a major essential macronutrient for biological growth and development. With regard to solubilization of inorganic phosphate four isolates (33%) (AAURB7, AAURB16, AAURB19, and AAURB20) were able to solubilize phosphate in the plate-based assay, by showing a clear halo zone around the colony. Several species of Pseudomonas such as P. fluorescens, P. aeruginosa and Bacillus species have been reported as good phosphate solubilizers in agricultural soils (Jha et al., 2013).

Compatibility test
The compatibility test between the selected isolate, AAURB20 and selected fungal isolate AAUTR23 indicated that, the colonies of the fungus and the bacterium met on the 7th day without showing inhibitory activity with one another. This observation was the basis for testing a combination of the two antagonists as "mixed culture" in the greenhouse trial. Similarly, under in vitro compatibility between T. viride and P. fluorescens was reported by Ephrem et al. (2011) with no inhibition between them. Isolate characters AAUTR21 AAUTR22 AAUTr23 AAUTR24 AAUTR25 AAUTR26 Colony growth rate (cm) 8-9 cm in 6 days 8-9 cm in 6 days 8-9 cm in 3 days 8-9 cm in 5 days 8-9 cm in 4 days 8-9 cm in 4 days Colony colour Green

Effects of isolates on disease incidence and biocontrol efficacy
The biocontrol efficacy and antagonistic effect of the treatments on disease incidence was highly significant (p≤0.05) when compared with the control treatments. The highest disease incidence of 80 and 60% was recorded from the control (Pathogen infection only) on Galilea and Gelelima varieties, respectively. All treatments reduced disease incidence ranging from 13 to 35% and biocontrol efficacy of 48 to 72% (Table 6). Similar results were also reported by Selim et al. (2011) plants treated with PGPR isolates significantly disease reduced ranging from 15 to 57% compared to infected control, as well as greater amount of biomass compared to the control. The combined treatments exhibited the lowest value (13.33%) of disease incidence as well as the highest value (72.22%) of biocontrol efficacy against R. solanacearum, on Gelelima variety. While isolate AAURB20 exhibited the highest (31.11%) disease incidence and lowest value (61.11%) of biocontrol efficacy on Galilea variety, and 35 and 48% on Gelelima variety, respectively ( Table 6). The results could be attributed to the synergistic effect between the combinations of the two microorganisms in this treatment. These results were in harmony with those reported by Yendyo and Pandey (2017) that Trichoderma spp. and P. fluorescence seem to be more effective than treatment using each individual biocontrol agent that has been achieved 97% of biocontrol efficacy. The dual application of T. viride and B. subtilis decreased the percentage of pathogen infection and increased survival rate than single inoculation in tomato (Morsy et al., 2009). Another study showed that the number of wilted chickpea infected with Fusarium oxysporium plants was reduced by 67.93% due to inoculation/suppression by T. harzianum (Subhani et al., 2013). The highest percentage of disease incidence was found on galilee variety, which may be due to variety resistance. These results were in harmony with those reported by Chatterjee et al. (1997) which stated that differences of wilt incidence and severity were due to diversity of host plants, the virulence of the pathogen, and other environmental factors.

Plant growth promotion efficacy of antagonists in greenhouse condition
Results of this experiment showed that antagonists (bioagents) stimulated plant growth promotion under greenhouse conditions and indicated that tomato plants treated with rhizobacteria and Trichoderma strains significantly grew better than control biomass increase of tomato plants treated with rhizobacteria and Trichoderma strains are shown in Table 7.
Significant differences (P≤0.05) among treatments regarding plant height and biomass were observed. Plants treated with combined isolates of AAURB20+AAUTR23 showed the highest values of plant height, and dry weight (72.33 cm and 12.73 g), respectively, when compared with the control (AAURS1) and plants treated by individual isolates AAURB20 and AAUTR23 in variety two (Table 7). Likewise plants treated with isolates AAURB20+AAUTR23 showed high GPE (%) (62% and 81.5%) for height and dry weight, respectively in variety two (Table 7).
Significant differences (P≤0.05) among treatments regarding plant height and biomass were also noted on variety one (Table 7). Plants treated with combined isolates of AAURB20+AAUTR23 presented the highest values of plant height and dry weight (67 cm and 11.25 g), respectively, when compared with the control (AAURS1) and plants treated by individual isolates AAURB20 and AAUTR23 in variety one (Table 7). Generally, combined treatments showed the best performance compared to individual treatments. Significant differences were observed in the vegetative growth parameters due to the inoculation of isolated bioinoculants. This result was in harmony with that of Nguyen and Ranamukhaarachchi (2010) on tomato. The use of beneficial microorganisms as biocontrol agents led to enhance plant growth parameters (70.4 cm plant height and 19.5 g of dry weight). Such enhancement may be due to induce plant resistance (De Meyer et al., 1998), production of extracellular enzymes and antifungal or antibiotics, which reduce the negative effect of biotic stress on plant and produce growth promoting substances (Szczech and Shoda, 2004). Similar results also reported by Selim et al. (2011) plants treated with PGPR isolates significantly reduced disease compared to infected control, as well as caused greater amount of biomass compared to the control.

Conclusion
A total of twenty rhizobacterial and six fungal isolates were preliminarily screened for their antagonistic property on the test pathogen using paper disc diffusion method under in vitro conditions. Isolate, AAURB20 gave the highest mean inhibition diameter of 15 and 16 mm followed by the fungus, AAUTR23, isolate with inhibition diameters of 14 mm against the two test pathogens AAURS1and APPRCRS2, respectively.
Based on the in vitro inhibition test and PGB properties, one isolate of Pseudomonas spp. and one isolates of Trichoderma had shown better inhibitory effects on the test pathogen strains selected for green house study. Combined treatments of Trichoderma and Pseudomonas spp. (AAURB20+AAUTR23) showed best performance compared to individual treatments, although individual isolates reduce bacterial wilt incidence and increase biomass of tomato significantly compared to the pathogen inoculated control. Therefore, the use of this bioagent would be important to manage bacterial wilt at greenhouse conditions. However, further study will be required to use the bio-agent in the field condition for biocontrol development program.