Abstract: The objective of this study was to evaluate the individual and combined efficacy of Pseudomonas fluorescens (EPI-5, KPI-7, ANR-2 and RTM-3) and Bacillus subtilis (KGI-4, PYR-3 and OCM-6) strains to promote the growth and yield parameters of tomato and to manage Fusarium wilt disease under in vitro and greenhouse conditions. The dominant pathogen which causes Fusarium wilt of tomato, was isolated and identified as Fusarium oxysporum f. sp. lycopersici (FOL). Twenty five native bacterial antagonists were isolated from healthy tomato rhizosphere soil in different geographical regions. Under in vitro conditions, the results revealed that the combined application of EPI (Pf-5)+KGI(Bs-4)+KPI (Pf-7) was found to effectively inhibit the mycelial growth of the pathogen (by 40%) when compared to application of individual strains of the bacterial antagonists. The above strains of P. fluorescens and B. subtilis were found compatible. Under greenhouse conditions, the combined application of EPI (Pf-5)+KGI (Bs-4)+KPI (Pf-7) exhibited the highest disease reduction. Also, tomato plants treated with EPI (Pf-5)+KGI (Bs-4)+KPI (Pf-7) strains showed a significant stimulatory effect on plant height and increased the dry weight of tomato plants up to 27% in comparison to the non-bacterized control. The combined strains also increased tomato fruit weight. It could be concluded that synergistic consortia of beneficial bacteria isolated from rhizosphere soil are perfectly able to promote plant growth and could be exploited for sustainable management of soil borne diseases especially, Fusarium wilt of tomato.
INTRODUCTION
Tomato (Solanum lycopersicum L.) is one of the most important vegetables grown and consumed worldwide. Its popularity is due to its high nutritive value, diversified use and nutritional significance as a source of vitamins A and C. It occupies number one position in its nutrient contribution to human diet. Tomato is cultivating in an area of 22,433 ha, with a production of 2, 82, 912 tonnes and a productivity of 12.61 tonnes ha-1 in Tamil Nadu (Anonymous, 2007). The crop is known to be affected by a number of diseases among which wilt caused by Fusarium oxysporum f. sp. lycopersici (Sacc.) Snyder and Hansen causes yield losses up to 50% (Lukyanenko, 1991). Wilt caused by species of Fusarium is one of the most serious disease problems of several agricultural, vegetable and fruit crops (Kloepper, 1991). The disease manifests as yellowing of the older leaves on one side of the stems close to fruit maturity. In the wilted plants, vascular tissue was dark brown and discoloration extended to the apex (Sheu and Wang, 2006). Presently, the management of tomato Fusarium wilt disease has been done with the application of chemical fungicides. However, it may not be sustainable in the longer run as chemical fungicides are known to cause residual toxicity, toxicity to non-target organisms and other environmental hazards and this has stimulated the search for biological options. Hence, recent attempts have been spotlighted on developing environmentally safe, long lasting and efficient management strategy for the management of plant diseases (Latha et al, 2009; Sundaramoorthy et al., 2012, 2013; Sundaramoorthy and Balabaskar, 2012).
Some priming strains of fluorescent Pseudomonads are also known as Plant Growth-promoting Rhizobacteria (PGPR) as they promote plant growth by secreting auxins, gibberellins and cytokinins (Dubeikovsky et al., 1993). Rhizobacterial strains of Pseudomonas and Bacillus spp. are also been used to reduce disease caused by a variety of soil borne pathogens (Larkin and Fravel, 1998). Several bacterial biocontrol agents including Pseudomonas fluorescens (Migula), Pseudomonas putida (Trevisan) and Bacillus subtilis (Ehrenberg) have been demonstrated to have promising biocontrol against F. oxysporum f. sp. lycopersici (Monda, 2002). Recently, Sundaramoorthy et al. (2012, 2013) and Sundaramoorthy and Balabaskar (2012) reported that combination of P. fluorescens strain (Pf1) and B. subtilis strain (EPCO16) effectively inhibited the growth of F. solani and Sarocladium oryzae in chilli and rice plants, respectively. Therefore, the present study was designed to evaluate the combined efficacy of Pseudomonas fluorescens and Bacillus subtilis strains against Fusarium wilt of tomato caused by Fusarium oxysporum f. sp. lycopersici.
MATERIALS AND METHODS
Isolation and purification of pathogens: Infected vascular tissues from stem and root regions of tomato cultivar (PKM 1) showing wilt symptoms were collected separately from farmer’s field. Tissue bits were surface sterilized with 10% sodium hypochlorite for 5-10 min. and subsequently three washings with sterile distilled water. Then, they were placed on Potato Dextrose Agar (PDA) medium separately and incubated at the laboratory conditions at 25±3°C for five days (Fig. 1a). The fungi were purified separately by transferring the tip of the mycelia into PDA slants and maintained as stock cultures for further studies (Fig. 1b).
Isolation and maintenance of bacterial native antagonists from tomato rhizosphere soil: Rhizosphere soil from healthy tomato plants were collected from different locations. The identified bacterial antagonists, viz., Pseudomonas spp. and Bacillus spp. were isolated by serial dilution technique using King’s B medium (King et al., 1954) and nutrient agar media (Allen, 1953), respectively. The bacterial antagonists were further purified on their respective media and compared with the isolates maintained in laboratory.
Compatibility among bacterial strains: The isolates of Pseudomonas and Bacillus were tested for their compatibility among each other following the method of Fukui et al. (1994). The compatibility was determined for P. fluorescens and B. subtilis strains using NA medium. The bacterial strains were streaked horizontally and vertically to each other. The plates were incubated at room temperature (28±2°C) for 72 h and observed for the inhibition zone. Absence of inhibition zone indicates the compatibility with respective bacterial strains and the presence of inhibition zone indicated the incompatibility.
In vitro effect of individual and combined bacterial native antagonists against FOL: Dual culture technique described by Nandakumar et al. (2001, 2002) was adopted to study the effect of antagonists Pseudomonas spp. and Bacillus spp. against F. oxysporum f. sp. lycopersici.
| Fig. 1(a-b): | Isolation and purification of Fusarium oxysporum f.sp. lycopersici (FOL) from wilt infected tomato tissue bits, (a) Isolation of FOL and (b) Axenic culture of FOL |
Nine mmillimeter disc of fifteen days old fungal cultures were placed on PDA medium one cm away from the edge of the plate, separately. Bacterial antagonists were streaked separately at opposite side of the Petri plate (Vidhyasekaran et al., 1997). Plates were incubated at 25±3°C for seven days. For each fungus and each antagonist three replicated plates were maintained. Per cent inhibition over control was calculated (Vincent, 1927) as per the equation:
| PI | = | Per cent inhibition over control |
| C | = | Growth of test pathogen with absence of antagonist (mm) |
| T | = | Growth of test pathogen with antagonist (mm) |
Preparation of individual and mixture of PGPE and PGPR bioformulations: A loopful of P. fluorescens and B. subtilis were inoculated into the sterilized KB and nutrient broth, respectively and incubated in a rotary shaker at 150 rpm for 48 h at room temperature (28±2°C). After 48 h of incubation, the broth containing 9x108 cfu mL-1 was used for the preparation of talc-based formulation. To 400 mL of bacterial suspension, 1 kg of talc powder (sterilized at 105°C for 12 h), calcium carbonate 15 g (to adjust the pH to neutral) and Carboxy Methyl Cellulose (CMC) 10 g (adhesive) were mixed under sterile conditions, following the method described by Nandakumar et al. (2001). After shade drying overnight, it was packed in polypropylene bag and sealed. At the time of application the population of bacteria in talc formulation was not less than 2.5-3x108 cfu g-1. For bacterial strain mixture, the bacterial strains were grown separately and the strains that are going to make up the mixture were added equally (v/v) and finally mixed with talc powder, CaCO3 and CMC (Nandakumar et al., 2001).
Efficacy of bacterial native antagonistic mixture on the incidence of Fusarium wilts disease under greenhouse conditions: A pot culture study was conducted to test the antagonistic potential of native antagonists against F. oxysporum f. sp. lycopersici. Potting mixture (red soil: sand: decomposed FYM at 1:1:1 w/w/w) was prepared and autoclaved 1 hr for two consecutive days and filled in earthern pots of 5 kg capacity. Tomato seeds (cv. PKM1) were sown in autoclaved pot mixture in earthern pots. After 25 days, the seedlings were pulled out from the pots and dipped in their respective formulation for 2 h ensuring that the roots alone were immersed in the inoculum and then transplanted in pots at the rate of four seedlings per pot (5 kg capacity). Bacterial native strains EPI (Pf-5), KPI (Pf-7), KGI (Bs-4), EPI (Pf-5)+KGI (Bs-4), KGI (Bs-4) +KPI (Pf-7) and EPI (Pf-5)+KGI (Bs-4)+KPI (Pf-7) were effective against F. oxysporum f. sp. lycopersici in vitro were selected. Soil application with the formulation was done 15 days and 30 days after transplantation. The F. oxysporum f.sp. lycopersici mass multiplied on sand maize medium was incorporated in to the pots at 5% cent (w/w). The observation on the per cent disease incidence was recorded at the time of harvest. Each treatment was replicated thrice in Randomized Block Design (RBD). The scheduled treatment details were as given below:
Statistical analysis: The data were statistically analyzed (Gomez and Gomez, 1984) and the treatment means were compared by Duncan’s Multiple Range Test (DMRT). The package used for a nalysis was IRRI-Stat version 92-a developed by International Rice Research Institute Biometrics Units, The Philippines.
RESULTS
Compatibility among bacterial strains: Twenty five native bacterial antagonists were isolated from healthy tomato rhizosphere soil in different geographical regions (Table 1). P. fluorescens (EPI-5, KPI-7, ANR-2 and RTM-3) and B. subtilis (KGI-4, PYR-3 and OCM-6) strains were tested for their compatibility in vitro. None of the antagonistic bacteria inhibited each other, suggesting that these bacterial native antagonists were compatible with each other.
In vitro screening of bacterial native antagonists against the radial mycelial growth F. oxysporum f. sp. lycopersici: Among the twenty five bacterial native antagonists, seven promising bacterial antagonistic P. fluorescens and B. subtilis strains (Table 2) were tested individually and in combination to assess the radial growth of FOL.
| Table 1: | Bacterial native antagonists isolated from rhizosphere soil of tomato |
| *PGPR-Plant growth promoting rhizobacteria | |
| Table 2: | Selected priming bacterial native antagonists isolates from tomato rhizosphere soil |
| *PGPR- Plant growth promoting rhizobacteria | |
| Table 3: | Effect of bacterial native antagonists on the mycelia growth of F. oxysporum f. sp. lycopersici |
| *Mean of three replications; In a column, mean followed by a common letter are not significantly different at the 5% level by DMRT | |
All the treatments were effective in reducing the mycelia growth of the pathogen. However, the combined application of EPI (Pf-5)+KGI (Bs-4)+ KPI (Pf-7) resulted in the least mycelia growth with 54.00 mm (Fig. 2). The control plates recorded the highest mycelia growth of 90.00 mm (Table 3).
Effectiveness of bacterial native antagonists on wilt incidence and yield parameters under glasshouse conditions: The application of bacterial native antagonists through seedling dip and soil application was found effective in suppressing wilt incidence (by 12.52-25.50%). Conspicuously, a combined application of EPI (Pf-5)+ KGI (Bs-4)+KPI (Pf-7) antagonistic bacterial formulation was recorded least wilt incidence (by 12.52%) followed by KGI (Bs-4)+KPI (Pf-7) by (15.33%) and EPI (Pf-5)+ KGI (Bs-4) by (17.45%) than any of the strains treated individually (Fig. 3b, Table 4). Among the treatments, Carbendazim (0.1%) was found to be the most effective and recorded the least wilt incidence of 10.26% compared to control (57.75%).
| Fig. 2 (a-d): | Combined efficacy of bacterial native antagonistic activity against wilt pathogen (FOL) under in vitro condition, (a) EPI (Pf-5)+KGI (Bs-4), (b) KGI (Bs-4)+KPI (Pf-7), (c) EPI (Pf-5)+KGI (Bs-4)+KPI (Pf-7) and (d) Control |
| Table 4: | Efficacy of bacterial native antagonistics against fusarial wilts of tomato (cv. PKM1) under greenhouse conditions |
| 1Fusarium oxysporum f.sp. lycopersici (‘+’ Presence; ‘-‘ Absence),*Mean of three replications, In a column, mean followed by a common letter are not significantly different at the 5% level by DMRT | |
Also, the results of this experiment revealed that the combined application of EPI (Pf-5)+KGI (Bs-4)+KPI (Pf-7) antagonistic bacterial formulation significantly increased the plant height (by 73.62 cm), Dry weight (by 127 mg) and fruit yield (by 288.389 g) when compared to individual strains and untreated control (Fig. 3a, Table 4).
DISCUSSION
Accumulating evidence from literature has shown that compatible multiple strains appear to be an important pre-requisite for the desired effectiveness of strains and more consistent disease suppression (Ganeshamoorthi et al., 2008; Latha et al., 2009; Sundaramoorthy et al., 2012, 2013, Sundaramoorthy and Balabaskar, 2012). In the present study, about twenty five isolates of PGPR were isolated from the rhizosphere of tomato. Similarly, most of the research workers have isolated PGPR from varying ecosystems. P. fluorescens was isolated predominantly from suppressive soils for the management of soil borne diseases (Ongena et al., 1999; Zehnder et al., 2000; Weller et al., 2002). The present in vitro study clearly showed that the bacterial antagonists P. fluorescens (EPI-5, KPI-7, ANR-2 and RTM-3) and B. Subtilis strains (KGI-4, PYR-3 and OCM-6) were compatible and effectively inhibited the growth of FOL. The inhibitory effect of Pseudomonas fluorescens and Bacillus subtilis against F. oxysporum f. sp. lycopersici under in vitro condition has been reported by several workers (Podile and Dube, 1985; Sarathchandra et al., 1993).
| Fig. 3(a-b): | Effect of combined application bacterial antagonistic in the management of tomato wilt disease under greenhouse conditions, (A) Control, (B) EPI (Pf-5)+KGI (Bs-4), (C) KGI (Bs-4)+KPI (Pf-7) and (D) EPI (Pf-5)+KGI (Bs-4)+KPI (Pf-7) |
Several strains of Pseudomonas and Bacillus spp. have been reported to produce wide array of antibiotics viz., 2,4, diacetylphloroglucinol, oligomycin, phenazine, pyoluteorin, pyrolnitrin, pyocyanin, iturin, bacillomycin, zwittermycin A and surfactin which are responsible for their antifungal action (Yu et al., 2002). Results from the present study clearly indicated maximum reduction in mycelia growth due to the combination of biocontrol strains than individual strains suggesting the synergism among bacterial native antagonistics in reducing the mycelia growth of the pathogen.
The treatment with combination of EPI (Pf-5)+ KGI (Bs-4)+KPI (Pf-7) increased the plant growth tomato more than did individual biocontrol strains. Similar results on increased plant growth due to combined application of Pf1+Py15+Bs16+Zimmu in tomato (Latha et al, 2009) and EPCO16+EPC5+Pf1 in chilli and tomato (Sundaramoorthy et al., 2012; Sundaramoorthy and Balabaskar, 2012) were also reported. In the greenhouse studies also the treatment with combination of EPI (Pf-5)+KGI (Bs-4)+KPI (Pf-7) resulted in a significantly least wilt disease incidence than any of the strains treated individually. Although, the chemical treatment with Carbendazim (0.1%) recorded the least disease incidence it is noteworthy to observe that the treatment with combination with EPI (Pf-5)+KGI (Bs-4)+KPI (Pf-7) also produced almost comparable results in reducing the disease incidence.
CONCLUSION
Obviously, the results of the present study demonstrated that combined application of bacterial native antagonistic EPI (Pf-5)+KGI (Bs-4)+KPI (Pf-7) is a promising approach for the eco friendly management of Fusarium wilt disease caused by F. oxysporum f. sp. lycopersici and enhancing the growth of the tomato plants.