Evaluation of Trichoderma spp., Pseudomonas fluorescence and Bacillus subtilis for biological control of Ralstonia wilt of tomato

Selection of the study plot

The sample collection and field trials were done at Agro Narayani Farm, Sukranagar, Chitwan District, Nepal (Latitude: 27.582016 and Longitude: 84.272259) where the bacterial wilt infection was recorded in the previous harvest. The tests in the field were conducted from 11 March 2017 to 8 July 2017 for 120 days in new transplants. At the time of transplant, compost fertilizer at the rate 2.94 kg/m², urea at the rate of 23.59 gm/m², potash at the rate of 29.49 gm/m², DAP 19.66 gm/m², borax at the rate 1.97 gm/m² and zinc at the rate 1.97 gm/m² were applied. At 45 days and 90 days of transplant NPK 20:20:20 was applied at the rate 9.83/m². Spacing of the plant was 50 cm in double row system of 50 × 50 cm. The plot size was 50 m² and total of 8 plots were used having 100 plants per plot. Weather data were also collected from online resources as reference.

Identification of bacterial wilt infection in the field

Physical symptoms, such as wilting of young leaves, discolored tissue at the dissected part of the stem base, and white, slimy ooze when the dissected part of the plant was kept in the glass of water, were used for the identification of infected plants²⁴.

Isolation and identification of R. solanacearum

Bacterial wilt infection in tomato plants (Variety: Manisha) grown in Chitwan, Nepal, was positively identified by S. Yendyo of Kishan Call Center (KCC). Six whole plants were brought to the Quality Control laboratory of Agricare Nepal Pvt. Ltd. in a sterile bag for the isolation of the pathogen.

R. solanacearum was isolated from dissected sections of the infected tomato plants on Casamino acid-Peptone-Glucose (CPG) Agar (casein hydrolysate 1 g/l, peptone 10 g/l, glucose 5g/l, agar 15g/l). Briefly, the stems of the infected plant were washed three times with autoclaved distilled water and then blot dried. After drying the stem were washed with 80% ethanol solution, then 1% sodium hypochlorite (NaOCl) solution was applied for 2 minutes. Final washing was done with autoclaved distilled water three times. The xylem of 2–3 cm from the stem was dissected and the sap was rubbed in CPG medium and inoculated for 48h at 28°C. Identification was done on the basis of the morphology of the colony on CPG medium, Gram staining and microscopic examination^25,26.

Isolation of antagonists

A total of 13 strains were used in this study (see Table 1). Six strains of Trichoderma spp., two strains of Pseudomonas fluorescence and one strain of Bacillus subtilis were isolated from soils and root soils collected from different sites in Nepal. ATCC 13525 strain of P. fluorescence from Microbiologics, St. Cloud, MN 56303, France was used as the reference for isolated Pseudomonas species. Trichoderma harzianum, Trichoderma virens and Trichoderma asperellum strains were provided by Tamil Nadu Agricultural University (TNAU), Coimbatore, Andhra Pradesh, India, and were used as reference species for isolated Trichoderma spp.

Trichoderma spp. were isolated by serial dilution method using TSM agar plate (K₂HPO₄: 0.9 g/l, MgSO₄: 0.2 g/l, KCl: 0.15 g/l, NH₄Cl: 1.05 g/l, Glucose 3 g/l, Rose Bengal: 0.15 g/l, agar 20 g/l, streptomycin: 100 mg/l, tetracycline: 50 mg/l)²⁷. 1 gm of each soil sample were suspended in 9 ml of sterile distilled water and vortexed (Accumax India, New Delhi-110058, India) for 5 min. The soil suspension was then serially diluted to 10^-3 and 10^-4. Pour plate technique was used by mixing 1 ml of the diluted soil suspension in 3 TSM agar plates for each sample and incubated at 25°C for 5 days. The strains were purified on TSM agar plates using sub-culture technique.

Pseudomonas fluorescence were isolated by serial dilution method using King’s B agar (Peptone: 20g/l, K₂HPO₄: 1.5 g/l, MgSO₄: 1.5 g/l, glycerol: 10 ml/l, agar: 20g/l)²⁸. 1 gm of each soil sample were suspended in 9 ml of sterile distilled water and vortexed (Accumax India, New Delhi-110058, India) for 5 min. The soil suspension was then serially diluted to 10^-6. Pour plate technique was used by mixing 1 ml of the diluted soil suspension in 3 King’s B agar plates for each sample and incubated at 27°C for 48 h. The strains were purified on King’s B agar plates using sub-culture technique.

Bacillus subtilis was isolated by serial dilution method using Nutrient Agar (Peptic digest of animal tissue: 5 g/l, NaCl: 5 g/l, beef extract: 1.5 g/l, yeast extract: 1.5 g/l, agar: 20g/l)²⁹. 1 gm of each soil sample were suspended in 9 ml of sterile distilled water and vortexed (Accumax India, New Delhi-110058, India) for 5 min. The soil suspension was then serially diluted to 10^-6. Pour plate technique was used by mixing 1 ml of the diluted soil suspension in 3 NA plates for each sample and incubated at 27°C for 48 h. The strains were purified on NA agar plates by using sub-culture technique.

Evaluating the efficacy of antagonists against Ralstonia solanacearum

All thirteen isolates were screened against R. solanacearum by agar well diffusion technique³⁰. R. solanacearum on CPG agar plates were transferred to nutrient broth and shaken in a rotary shaker (Talboys, Henry Troemner, LLC, USA) at 100 rpm at 27°C for 24 h. Similarly, the TSM, King’ B and NB were prepared for all Trichoderma spp., P. fluorescence and B. subtilis, respectively, and incubated for 7 days, 48 h and 48h, respectively. After incubation of the antagonists, 5 ml of broth suspension were centrifuged at 5000 rpm for 5 min and the supernatant was stored at 4°C for further procedure. Then, R. solanacearum suspension of 10⁸ cfu/ml was prepared as per McFarland 0.5 turbidity method³¹ and was swabbed on NA plates. Holes of 5 mm were punched into the agar plate and 40 µl of the supernatant prepared were added separately and the plates were incubated at 27°C for 48 h. Inhibition of R. solanacearum growth was assessed by measuring the radius in mm of the zone of inhibition (ZOI) after incubation.

For microscopic visualization of the inhibition, CPG agar plates were prepared to provide the most favorable growth to R. solanacearum, and the respective 5 mm mycelial discs of Trichoderma species were added in the center of the plate after cotton swabbing from the CPG broth of R. solanacearum. For B. subtilis and P. fluorescence, the line was streaked parallel to the streak of R. solanacearum in two different CPG agar plates using dual culture technique. After 72 hr of incubation, live microscopic examination on the culture plate was done using a digital microscope (Olympus CX-43, Tokyo, Japan). Images were captured to visualize the interaction of the individual strains of biocontrol agents with R. solanacearum.

Effect of sucrose on the population of biocontrol agents

There is common practice in Nepal of keeping bio-based products in 5–10% (w/v) sucrose solution for 2–4 h before application to the plant. Hence, to evaluate the effect of 5% (w/v) sucrose solution on the cell number (growth) of the biocontrol agents, 1 ml of concentrate containing 1 × 10⁹ cells ml^-1 was kept in 5% sucrose solution, made using autoclaved distilled water. Cell count was taken using a Hemocytometer (Reichert, Buffallo, NY, USA) with trypan blue at 1 h, 2 h, 3 h and 24 h to observe the effect on microbial population.

Evaluating the effects of biocontrol agents in the field

For the field study, the concentrates containing 10⁹ cells/ml of the respective biocontrol agent were used. The densities of the cells were determined using Hemocytometer (Reichert, Buffallo, NY, USA)³². The TSM broth of all six isolated native Trichoderma species viz. AA2, AG3, AKD, A5, A9 and A10 were mixed in equal proportion to prepare 1 liter of concentrate containing 10⁹ cells/ml. Similarly, two native P. fluorescence species viz. PFB and PFS were mixed in an equal proportion to prepare 1 liter of concentrate containing 10⁹ cells/ml. Concentrate of native B. subtilis viz. BS containing 10⁹ cells/ml was used to analyze the effect of B. subtilis as a possible biocontrol agent.

Before the application in the tomato plots at Agro Narayani Farm, the prepared concentrate of biocontrol agents were taken to the field and were further diluted at the rate of 2ml/l of tap water containing 5% (w/v) sucrose. After 2 h of incubation in 5% sucrose water, the diluted solutions were applied in the root of tomato plants at the rate of 100 ml per plant. The processes of applications were repeated every 7 days for 8 weeks (total of 8 applications) by preparing fresh dilutions in 5% sucrose solutions 2 h prior to applications. Effects after the 8 weeks of continuous application were measured in the field by identifying the number of plants that underwent recovery after treatment. 6 plots were treated with the biocontrol agent and 2 plots were used as controls. Chemical treatment was done in one plot (positive control plot) using the combination of Agricin (9% Streptomycin Sulphate and 1% Tetracycline Hydrochloride) at the rate of 100 ml of 0.1% (w/v) solution per plant from Agricare Nepal Pvt. Ltd., Nepal and Bavistin (50% carbendazim) at the rate of 0.2 % (w/v) solution per plant from Crystal Crop Protection Pvt. Ltd., India. For negative control no treatment methods were selected in one plot.

The treatment plots were designed such that the effects of the individual biocontrol agent and effects of combination treatment can be studied (Table 2).

Statistical analysis

Statistical analysis of the data was done using IBM SPSS Statistics (ver 23) and figures and data were made through Microsoft Excel 2007 and Microsoft Word 2007.

Weather data

Weather data of Chitwan District for temperature, humidity and rainfall were collected from www.worldweatheronline.com from March 2017 to July 2017 (see Table 3).

The bacterial wilt outbreak was reported in the late May, 2017, when the temperature and humidity level increased. Higher temperature and moisture favors the growth of R. solanacearum³³

Identification of bacterial wilt in the field

From the field examination of the tomato plants, observation revealed that the leaves were flaccid, adventitious roots started to appear on the stem and ooze appeared after dipping the stem in water. Also, field experts from KCC confirmed the presence of bacterial wilt infection, due to their years of experience in plant disease diagnosis.

Isolation and identification of R. solanacearum

Infected plant saps from six xylems showed similar bacterial colonies on CPG medium. All the colonies were similar to avirulent type, as the appearance was white or cream-colored, irregularly-round, fluidal, and opaque on CPG medium^34,35. Gram staining and observation using a microscope showed that the bacteria were gram negative, rod shaped and non-spore forming, which further confirmed that the bacteria was R. solanacearum.

Evaluating the efficacy of antagonists against Ralstonia solanacearum

All 13 strains tested showed antagonistic effect against R. solanacearum, with inhibition zone radii ranging from 13 to 21.33 mm (Table 4). P. fluorescence PFS isolated from Parthenium rhizoplane soil was most potent compared to other P. fluorescence strains. Trichoderma virens ATV and Trichoderma harzianum ATH provided by TNAU were the least and most potent species. Among six natively isolated Trichoderma sp., AA2 isolated from Parthenium rhizoplane soil was most potent and AKD and A9 were least potent. However, the activities of native Trichoderma spp. were satisfactory in the term of inhibition zone shown. Bacillus subtilis BS isolated from Bhaktapur top soil did not show satisfactory inhibition activity.

The effect of biocontrol agents against R. solanacearum was analyzed at a microscopic level in dark phase using image analyzer (Olympus CX-43, Tokyo, Japan). Figure 1–Figure 3 show the distinct inhibitory effect on the growth and survival of R. solanacearum caused by different biocontrol agents. From the figures it can be seen that the most of the pathogenic cells (R. solanacearum) were either killed or growth was retarded or limited in or towards the region of growth of antagonists as compared to region away from the growth of antagonists.

Figure 1. Digital images (400X) of microscopic analysis in dark phase representing the interactions of different Trichoderma species against Ralstonia solanacearum.

A1–J1 shows strains AA2, AG3, AKD, A5, A9, A10, ATA, ATH and ATV, respectively (see Table 1), growing on R. solanacearum, which was cotton swabbed onto CPG agar plates. A2–J2 represents growth of R. solanacearum 4 cm away from the growth of Trichoderma spp., viz., A2, AG3, AKD, A5, A9, A10, ATA, ATH and ATV, respectively, on the plate. Negative control (i.e., blank plate without any swabbing) and positive controls (i.e. R. solanacearum without Trichoderma species) are also included. The images reveal that the population of R. solanacearum is significantly less and most of the cells are dead in the region of growth of samples treated with Trichoderma spp., compared to the region 4 cm far from the growth of Trichoderma spp.

Figure 2. Digital images (400X) of microscopic analysis in bright field representing the interactions of Pseudomonas fluorescence species against Ralstonia solanacearum.

PFA, PFB and PFS represent P. fluorescence (PF) species (see Table 1), and RS represents R. solanacearum. Both PF and RS were streaked near to each other to see the interaction between the two species. PF tended to grow on the side of RS, whereas RS tended to restrict the growth towards the PF species. RS positive control (without PF streak nearby) tended to spread, which confirms the spreading pattern of RS.

Figure 3. Digital image (400X) of microscopic analysis in bright field representing the interaction of Bacillus subtilis against Ralstonia solanacearum.

BS represents Bacillus subtilis species (see Table 1), and RS represents Ralstonia solanacearum. Both BS and RS were streaked near to each other to see the interaction between two species.BS has completely overgrown the RS streak on the CPG agar plate, which suggests that there has been an interaction between RS and BS.

Effect of sucrose on the population of biocontrol agents

The effect of sucrose (5% w/v) in the concentrate mixture of individual biocontrol agents was analyzed (Figure 4), which showed that there was profound increase in the number of cells of biocontrol agents after 2h of incubation compared with the initial population. The cell count between 2h and 3 h of incubation was not significant as compared with 2 h and 24 h of incubation. Thus, 2 h of incubation in sucrose solution can be considered as optimal time, as lengthier time can result in growth of contaminant in the solution whilst using tap water in the field.

Figure 4. Effect of sucrose on the growth of Pseudomonas fluorescence, Trichoderma spp. and Bacillus subtilis.

An increase in a number of cells of biocontrol agents was seen over time when these agents were kept in 5 % (w/v) sucrose solution (n=3).

Evaluating the effects of biocontrol agents in the field

Before applications, 2ml of respective biocontrol agents of (10⁹ cells/ml) were incubated in 5% (w/v) of sucrose solution and incubated for 2 hr. The prepared dilution was thus applied at the rate of 100 ml per plant in the root region every week. 8 applications were done over 8 weeks. 8 plots (100 plant/plot) were selected, out of which one plot was used as positive control/chemical treatment plot (Agricin+Bavistin) and one plot as negative control/zero treatment plot (no treatment given).

The results are displayed in Table 5 and show that the application inhibited the bacterial wilt infection (R. solanacearum) in tomato plants by and the highest rate of plant recovered was 97% from treatment using antagonists, which was comparable with the plant recovery of 94% using the chemical treatment (Agricin and Bavistin). Only 37% of plants were recovered in the plot where no treatment methods were applied. Field Images (Figure 5) show a clear visualization of growth of plants and severity of infection in treated and untreated plot.

Figure 5. Difference in the field between treated and untreated plots.

Field images clearly reveal that the treatment with biocontrol agents has helped to eliminate the bacterial wilt disease in the field after 8 weeks of application. (A) shows the growth and vigor of plants treated with biocontrol agents; (B) shows growth and severity of infection occurred in untreated plot.