Stem canker disease caused by Rhizoctonia solani is one of the destructive diseases of potatoes that leads to the loss of the annual yield of the product all over the world. Disease control methods include planting disease-free seed tubers and using chemical compounds that inhibit the fungus. The continuous use of fungicides causes the emergence of resistant strains and environmental pollution. Bacillus species have a unique ability to multiply rapidly, compete for space and nutrients with the Rhizoctonia pathogen, and survive under adverse environmental conditions. They secrete different types of antimicrobial compounds such as lipopeptides, antibiotics, enzymes, and volatile organic substances. They also strengthen plant growth and induce systemic resistance. The current study was conducted to investigate the biocontrol and growth of Bacillus strains in reducing potato stem canker disease and improving the growth characteristics of the plant.
Materials and Methods
To evaluate the inhibitory mechanisms of Bacillus strains on Rhizoctonia pathogen, three methods including dual culture assay, production of antifungal volatile substances, and antibiotic production were used. All experiments were performed in two separate experiments based on a completely randomized design with 20 treatments and three repetitions. The biocontrol characteristics of the antagonists consisting of the production of hydrolyzing enzymes, siderophore, biosurfactant, and biofilm development were evaluated. Five selected strains were identified using 16S rRNA sequencing. Also, their effect on the germination of sclerotia of the pathogenic fungus and the morphology of its mycelium was checked. In pot experiments, in addition to candidate strains (Ba1, Ba5, Ba8, Ba11, Ba12, Ba5 + Ba11), other defined treatments including Carbendazim, Iprodione-Carbendazim, potassium phosphite, negative control (healthy plant), and positive control (fungus infected plant) were determined and disease scale calculated. The results of biological tests were analyzed by SPSS software version 24.
Results and Discussion
In dual culture screening, strains Ba12 and in the production tests of volatile and antibiotic compounds, strains Ba11 and Ba8 respectively showed the highest inhibition against the studied fungal pathogen. Apart from the 3 mentioned strains, Ba1 and Ba5 candidates are in the next degrees of inhibiting the growth of the fungus. In evaluating the production of extracellular enzymes, Ba8 had the highest amount of chitinase and Ba12 had the highest production of cellulase, protease, and pectinase. Also, strains Ba1 and Ba5 secreted all the examined enzymes except pectinase. Five candidate strains also belong to moderate and strong biofilm-producing groups. All of them led to mycelium swelling and fragmentation and cytoplasmic coagulation in R. solani AG3 mycelia. According to the sequencing data, selected isolates including Ba1, Ba5, Ba8, Ba11, and Ba12 belonged to B. mojavensis, Bacillus sp., B. pumilus, Bacillus sp., and B. velezensis, respectively. Among the selected Bacillus strains, Ba5 and Ba8 showed zero disease index and controlled the disease, just like the Iprodione-Carbendazim fungicide. Another strain (Ba8) which controlled the pathogen 100% in greenhouse experiments, also produced high chitinase, which is effective in destroying the fungal wall and also completely prevented the germination of Rhizoctonia sclerotia. The combined treatment did not show an additive effect compared to the single treatment.
Due to their genetic and metabolic diversity, Bacillus species are well-adapted to a wide range of environmental conditions. Based on the results of the present research, it seems that the usage of Ba8 (B. pumilus) and Ba5 (Bacillus sp.) strains in combination with other available control methods should be appropriate for stem canker reduction. However, achieving the optimal formulation and validating the results requires field experiments on a larger scale.