Introduction
Potato is one of the most important food crops after wheat, rice, and corn. Plant pathogens caused quantitative and qualitative damages to this crop in all stages of growth. Ralstonia solanacearum causing the widespread disease known as bacterial wilt, Pectobacterium atrosepticum causing blackleg of potato, Pectobacterium carotovorum (Pc) causing soft rot disease of various plant hosts and blackleg in potato, and pathogenic Streptomyces spp. which causes common scab, are important problems in the cultivation of this plant all over the world. The conventional techniques to manage these diseases, i.e., applying chemical pesticides have adverse effects on human health and the environment. Furthermore, these methods caused the emergence of resistance among phytopathogens. Therefore, biocontrol agents are essential for reducing the need to dangerous pesticides and protecting plants from diseases. The Streptomyces genus of gram-positive bacteria has a capacity to produce diverse antimicrobial compounds, such as metabolites and organic compounds. This study aimed to select and identify non pathogenic Streptomyces spp. with efficient antagonistic activity against the mentioned pathogens in vitro conditions. Also, the effect of selected antagonist Streptomyces spp. was evaluated on the soft rot incidence and the fresh and dry weight of potato plants grown in sterile soil and in soil infested with Pectobacterium carotovorum in greenhouse conditions.
Materials and Methods
In the present study, P. carotovorum strain 8, P. atrosepticum strain SCR, R. solanasearum strain 4, pathogenic Streptomyces sp. strain 12 and 200 isolates belonging to the Streptomyces genus were obtained from department of plant protection ,University of Zanjan. The pathogenicity test was performed on potato tubers for 200 isolates of Streptomyces spp. and the isolates without pathogenicity were selected for antagonistic activity against four mentioned pathogenic bacteria according to cross streak assay and chloroform test on nutrient agar medium. Also, the antibacterial efficacy of culture filtrates from selected antagonist isolates was evaluated in laboratory conditions, and then gas chromatography analysis was performed for the metabolites. Phenotypic and molecular identification of the selected antagonist isolates were conducted using conventional phenotypic tests and 16S rRNA gene sequencing. Also, phylogenic relationships of the selected antagonist isolates with other sequences available in the GenBank database were determined and a phylogenetic tree based on partial 16S rRNA gene sequences was constructed using the Maximum Likelihood method with 1000 bootstrap replicates. In the greenhouse conditions, antagonistic activity of selected strains was evaluated as the pretreatment of potato tubers (cv. Marfona) with selected antagonist Streptomyces spp. isolates, either individually or in combination, and planted tubers in the sterile soil and in soil infested with P. carotovorum strain 8. Then 40 days after inoculation, the disease incidence and fresh and dry weight in different treatments were assessed.
Results and Discussion
Among 200 isolates of Streptomyces spp., 30 non pathogenic isolates were selected according to pathogenicity test on potato tubers and their antagonistic activity was assessed by cross streak and chloroform assays towards the mentioned pathogenic species. The isolates AEFB88, 7 and AEB133 exhibited inhibitory activity against pathogenic bacteria. The inhibition zone diameter produced by bacterial antagonists ranged from 10 to 50mm. Also, the culture filtrates of three antagonist isolates showed growth inhibition of P. atrosepticum and P. carotovorum seven to 14 days after extraction. In the gas-chromatography of culture filtrates of selected isolates, compounds such as tetramethoxystyrene and trifluoromethylbenzylamine were detected in the isolate 7, benzene dicarboxylic was identified in the isolate AEFB88, and benzothiophene acetamide and isothiourea were detected in the isolate AEB133. Phylogenetic tree based on partial 16S rRNA gene sequences showed the taxonomic position of selected antagonists. Isolates AEFB88, 7 and AEB133 formed a clade with the type strains of Streptomyces deccanensis, Streptomyces albogriseolus and Streptomyces griseus, respectively. In the greenhouse conditions, the effect of selected isolates on the disease incidence showed that although none of the three isolates protected the potato plants completely against Pc, but the Streptomyces sp. AEB133 isolate was effective against Pc and reduced the disease incidence by 60% compared to the positive control. Therefore, biological control of soil-borne pathogens is facilitated by microorganisms that are active in the root zone. Moreover, the effect of the selected antagonist isolates on growth parameters of potato plants revealed that the Streptomyces sp. AEB133 isolate increased the fresh and dry weight of the inoculated plants significantly in comparison with the negative and positive controls. This isolate increased the fresh and dry weight of potato plant, 51g and 8g respectively, in the steril soil. Also, in the presence of P. carotovorum, the highest fresh and dry weight, with averages of 36g and 7g, was related to the Streptomyces sp. AEB133 isolate.
Various studies have determined that Streptomyces species are sustainable alternatives to chemical fertilizers that stimulate plant growth by producing substances such as indole-3-acetic acid, cytokinin, and siderophores. Moreover, they are bioinoculants against bacteria, fungi, nematodes, and viruses, and protect plants by producing antimicrobial metabolites, enzymes, nutrient competition, mycoparasitism, and plant defense regulation.
Conclusion
This study demonstrates that Streptomyces sp. strain AEB133 has significant antagonistic capacity against important bacterial pathogens of potato in vitro and in vivo conditions. In addition to disease control, this strain promotes plant growth factors compared to the controls. This allows us to affirm that it is possible candidate for use in biological control programs in the production of this crop as part of sustainable agriculture practices.