Journal of Plant Pathology & Microbiology

Evaluation of the Efficacy of Trichoderma and Pseudomonas Species against Bacterial Wilt (Ralstonia Isolates) of Tomato (Lycopersicum Spp.)

Shashitu Aleling^* Ethiopian Institute of Agricultural Research, Debrezeit Research Center, Addis Ababa, Ethiopia
^*Correspondence: Shashitu Aleling, Ethiopian Institute of Agricultural Research, Debrezeit Research Center, Addis Ababa, Ethiopia, Tel: +251919823556, Email:

Received: 08-Mar-2021 Published: 19-Jul-2021, DOI: 10.35248/2157-7471.21.12.564

Introduction

Tomato (Lycopersicon esculentum) is the second most important vegetable crop in the world next to potato [1]. The center of origin of Solanum lycopersicum, (S. section Lycopersicon) 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 [2]. Tomatoes production accounts for about 4.8 million hectares of harvested land area 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 [3]. In Africa, the total tomato production for 2012 was 17.938 million tons with Egypt leading the continent with 8.625 million tones [3]. It is an economically important vegetable in Ethiopia. According to the Central Statistics Authority of Ethiopia, the country produced 27,774.538 tons of tomato in 5235.19 hectares of land in 2018 [4].

R. 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 [5]. Different studies showed the bacterial wilt pathogen inflict 50-100% loss on potato in Kenya [6], 88% on tomato in Uganda [7], 70% on potato in India [8]. 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. Different methods, mainly pesticides are employed to control bacterial wilt of tomatoes. Chemical controls with Actigard (e.g., Acibenzolar-S-methyl) and phosphorous acid effective to control bacterial wilt under at greenhouse and to a lesser extent field conditions [9]. 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 [10].

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 [11]. 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 [12].

Soil bacteria and fungi which flourish in the rhizosphere of plants and, stimulate plant growth are collectively known as plant growth promoting microorganisms (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 [13]. 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 [14].

Trichodermaand Pseudomonas spp.  are the most frequently isolated fungi and bacteria in all the root ecosystems respectively. Trichoderma species 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. [15] showed that, the dual application of T. viride and B. subtilis decreased the percentage of pathogen infection and increased survival rate than single inoculation in tomato. The biocontrol potential of two Trichoderma species on sclerotia rot disease of tomato plants in Chile and Iceland was evaluated and the result showed that, T. harzianum and T. viride reduced the disease by74.50%, and 68.75%, respectively [16].

Several studies also showed that the application of these antagonists have a dramatic effect on bacterial wilt disease (Ralstonia solanacearum) of tomato. Narasimha, et al. [17] 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 P. fluorescens PFS were most potent inhibiting the growth of Ralstonia spp. ,and the field study indicated Trichoderma spp.  and Pseudomonas 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 [18]. This shows the promising potential of native isolates of Trichoderma spp. and Pseudomonas 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 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.

Materials and Methods

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 [18].

Isolation of Ralstonia solanacearum from wilted tomato plants

Isolation of the wilt pathogen was undertaken according to Kelman A [19]. Diseased tomato stem samples were washed with tap water, and surface sterilized with 70% ethanol for 2 minutes and rinsed repeatedly in sterile water for 5 minutes. The samples were then suspended in the five-milliliter sterile distilled water for ten minutes 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 Triphenyl Tetrazolium Chloride Agar (TZC) medium and incubated at 28 ± 2°C for 48 hrs.

Identification of virulent/avirulent isolates of Ralstonia solanacearum

The virulent and a virulent isolates of the pathogen were differentiated by Kelman method [19] on Tetrazolium Chloride (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 hours of incubation.

Pathogenicity test

Virulence of the isolates was carried out by inoculating them on the tomato seedlings according to Margare et al. [20]. 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 108 CFU/ml) [21]. 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% to 25%), 2= 2 or 3 leaves wilted (26% to 49%), 3=half plant wilted (50% to 74%), 4= all leaves wilted (75% to 100%), 5=Plant dead).

Biochemical characterization of isolates

The selected virulent isolates were also inoculated on nutrient agar plates and incubated at 28ºC for 24 hrs 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 [22]. Ten gram 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 [23]. The bacterial wilt pathogen was grown on nutrient broth for 48 hr from which, 100 µml was swapped onto Petri plates with nutrient agar. And the bacterial antagonist grow on nutrient broth for 48 hrs and Trichoderma were grown in Potato Dextrose Broth (PDB) (20 g/l dextrose, 4 g/l potato extract and 15g/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-inoculated-plate. 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 48hrs to measure inhibition zone.

Morphological characterization of fungi antagonists

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 AAU.

Biochemical characterization of bacterial antagonists

The selected bacterial antagonistic isolates were characterized by the following biochemical tests including Gram differentiation and gram reaction, growth at 41oc, catalase test, oxidase test, pigment production, gelatin liquefaction, hydrogen cyanide production, ammonia production, phosphate solubilization, and carbohydrate fermentation test by using standard methods.

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 containing 2% sucrose. The other side of the petri-dish was inoculated with 1 cm disc of 7 days old Trichoderma sp. The plates were then incubated at 25°C to test the presence of inhibition between the two isolates.

Antagonistic test of the isolates against the test pathogen on tomato under greenhouse condition

Tomato seeds from local Gelelima variety and Galilea variety 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 108cfu ml–1and conidial suspension of (108 spores ml–1) respectively as described by Sivan, et al. [24]. Fifty ml of the mixed inoculum of the pathogen and antagonists were inoculated into the pots at the same time using soil drench method [25]. Each treatment was replicated thrice in completed randomized design (CRD). The treatments were;

T1 Ralstonia solanacearum+ Trichoderma. (AAURS+AAURB20)

T2 Ralstonia solanacearum+ pseudomonas (AAURS+AAUTR23)

T3Ralstoniasolanacearum+ Trichoderma spp.  + pseudomonas (AAURS+AAURB20+AAUTR23)

T4 Inoculated control with Ralstonia solanacearum (diseased control) (AAURS) and

T5 un-inoculated control (healthy control) (DW)

According to Song, et al. [26], wilt incidence was calculated by the following formula:

Where

BE = Biocontrol 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 variables measured were subjected to one-way ANOVA. Duncan’s multiple range tests was applied when one-way ANOVA revealed significant differences (P < 0.05). All statistical analysis was performed with SAS software.

Results and Discussion

Cultural and biochemical tests for identification of Ralstonia 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. solanacerum 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 hours of incubation reported elsewhere [19].

Table 1: Variations in pathogenicity of R. solanacearum isolates on the host tomato variety (Gelilema).

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 50-71% wilting on different tomato varieties [27]. El-Ariqi, et al. [28] also reported that different isolates of R. solanacearum caused 52%-97% of wilting. Selim, et al. [29] have also reported that different isolates of R. solanacearum showed different wilt incidence ranging from 40%-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 Ralstonia solanacerum using paper disc diffusion method under in vitro conditions.

The data showed that the bacterial isolate, AAURB20 showed the highest mean inhibition diameter of 15mm and 16 mm followed by the fungus, AAUTR23, isolate with inhibition diameters of 14 mm against the two test pathogens AAURS1and 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 Aliye, et al. [30] 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 ) 3.2 showed 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 [31]. The inhibitory activity of P. fluorescens against the pathogen in the study is in line with that of Henok, et al. [32], Aliye, et al. [30] and Yendyo, et al. [18] where they reported that isolates of P. fluorescens had significantly inhibited under the bacterial growth of R. solanacearum under in vitro condition.

Table 2: Antagonistic activity of antagonists against R. solanacerum under in vitro condition grown on NA medium and incubated at 28°C for 2 days.

The in vitro antagonistic activity of Trichoderma sperellum was reported by Narasimha, et al. [17] that inhibit the growth of Ralstonia solanacerum with inhibition zone ranging from 11mm-27mm 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. Those 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 Pseudomonas fluorescens as reported by earlier workers Meera and Balabaskar [33].

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., [32].

Isolates	Fructose	Glucose	Lactose	Maltose
AAURB 1	+	+	±	+
AAURB 3	+	+	+	+
AAURB6	+	+	+	+
AAURB 7	+	+	+	+
AAURB 8	+	+	+	+
AAURB 11	±	+	±	±
AAURB 12	±	+	+	+
AAURB16	±	+	±	±
AAURB 17	+	+	+	+
AAURB 18	±	+	±	±
AAURB19	+	+	+	+
+	+	+	+

Table 3: Carbohydrate fermentation test results of different indigenous bio-control agents.

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 (Table 4).

Table 4: Morphological characterization of fungi.

PGPR characterization of rhizbacteria

Among isolates that were screened for their plant growth promoting activities viz., HCN production, ammonia production, phosphate solubilization. Isolate AAURB20 and AAURB19 exhibited strong HCN production followed by isolates AAURB8 and AAURB16. 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 [33].

Table 5: Characterization of rhizobacteria for their PGPR characters.

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 [34].

Phosphorous is a major essential macronutrient for biological growth and development. With regard to solubilization of inorganic phosphate four isolates 4 (33%) (AAURB7, AAURB16, AAURB19, and AAURB20) of 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 [35].

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. Similary, under in vitro compatibility between T. viride and P. fluorescens was reported by Ephrem, et al. [36] there was no inhibition between them.

Effects of isolates on disease incidence and biocontrol efficacy

The bio control efficacy and antagonistic effect of the treatments on disease incidence was highly significant (p ≤ 0.05) when compared to 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%-35%; and bio control efficacy of 48%-72% (Table 6). Similar results also reported by Selim et al. [29] plants treated with PGPR isolates significantly disease reduced ranging from (15%-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 bio control efficacy against R. solanacearum, on Gelelima variety. While isolate AAURB20 exhibited the highest (31.11%) disease incidence and lowest value (61.11%) of bio control 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, et al. [18] that Trichoderma spp.  and P. fluorescence seems to be more effective than treatment using each individual biocontrol agent that has been achieved 97% of bio control efficacy.

Table 6: Effect of AAURB20, AAUTR23, and their combination (AAURB20+AAUTR23) on disease incidence.

The dual application of T. viride and B. subtilis decreased the percentage of pathogen infection and increased survival rate than single inoculation in tomato [15]. 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 [37]. 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.[38] 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).

Table 7: Effect of plant growth promotion of antagonists on tomato.

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 best performance compared to individual treatments. Significant differences were observed in the vegetative growth parameters due to the inoculation of isolated bio-inoculants. This result was in harmony with that of Nguyen and Ranamukhaarachchi [23] 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 [39], production of extracellular enzymes and antifungal or antibiotics, which reduce the negative effect of biotic stress on plant and produce growth promoting substances [40]. Similar results also reported by Selim, et al. [29] 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

The combined use of the biocontrol agents significantly reduced the incidence of tomato bacterial wilt disease. Therefore the use of this bioagent would be important for to manage bacterial wilt at greenhouse conditions.

Acknowledgements

I would like to acknowledge Ethiopian institute of agricultural research and Healthy Seedling Project supported by the Ethiopian Biotechnology Institute (EBTI) for financial support; and the Department of Microbial, Cellular and Molecular Biology, College of Natural and Computational Sciences, Addis Ababa University, for providing me laboratory space and equipment, and technical support to undertake the research work.

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