Mikrobiol. Z. 2020; 82(4):41-52.
Endophytes from Medicinal Plants as Biocontrol Agents against Fusarium Caused Diseases
V. Shurigin1, D. Egamberdieva1, S. Samadiy1, G. Mardonova1, K. Davranov2
1National University of Uzbekistan
4 University Str., Tashkent, 100174, Uzbekistan
2Institute of Microbiology of AS RUz
7B Kadyriy Str., Tashkent, 700128, Uzbekistan
Objective. The objective of this research was to reveal bacterial endophytes isolated from some medicinal plants, which have biocontrol activity against phytopathogenic fungi Fusarium solani and Fusarium oxysporum. Methods. The bacterial endophytes were isolated from shoots and roots of medicinal sage (Salvia officinalis L.), fennel (Foeniculum vulgare Mill.), tansy (Tanacetum vulgare L.), medicinal calendula (Calendula officinalis L.), pale yellow iris (Iris pseudacorus L.), horseradish (Armoracia rusticana G. Gaertn., B. Mey. & Scherb.). The endophytes were isolated after grinding of the surface sterilized roots or shoots, dilution of the cell juice in phosphate buffered saline and spreading the suspension on Tryptic Soy Agar. The grown colonies were picked up in 4 days. The endophytes were checked for in vitro antagonistic activity against phytopathogenic fungi Fusarium solani and F. oxysporum on Petri dishes using Chapek medium. The endophytes were examined for the ability to inhibit the same phytopathogenic fungi in pot experiment. The soil was infected with fungi and the seeds of cotton and cucumber were inoculated with endophytes. 4 weeks after sowing the number of survived plants was defined. The bacterial isolates were identified using 16S rRNA gene analysis. For DNA extraction the heat treatment method was used. Extracted DNA was used as template for 16S rRNA gene analysis. Sequencing was performed using ABI PRISM BigDye 3.1 Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems). The 16S rRNA sequences were identified using BLAST and comparisons with the GenBank nucleotide data bank from the NCBI. The strains were checked for production of HCN, hydrolytic enzymes (chitinase, lipase and protease) and siderophores. All experiments were conducted in 3 replications. Results. In total 151 isolates were obtained from 6 medicinal plants. Only 20 isolates inhibited fungi Fusarium oxysporum, Fusarium solani or both of them. HRT18, AST3, BST3 and FST7 bacterial isolates were the most effective in biocontrol activity against disease caused by Fusarium oxysporum on a cotton plants in pot experiments. The most effective bacterial isolates having biocontrol activity against disease caused by Fusarium solani on a cucumber were KRT5, FRT13, FRT12, CST6 and BST10. In total 20 bacterial isolates were identified using 16S rRNA gene analysis as follows: Serratia ficaria KRT5, Raoultella ornithinolytica KRT12, Agrobacterium tumefaciens HRT9, Klebsiella oxytoca BST12, Pseudomonas putida FRT13, Pseudomonas kilonensis FRT12, Pseudomonas lini FRN1, Agrobacterium vitis HRT14, Bacillus toyonensis HRT5, Pseudomonas azotoformans HRT18, Pseudomonas syringae AST3, Klebsiella pneumoniae BST3, Xanthomonas translucens HRT10, Enterobacter ludwigii KRT16, Paenibacillus typhae KRN1, Pseudomonas extremaustralis CST6, Pseudomonas oryzihabitans FST7, Pseudomonas chlororaphis BST10, Agrobacterium vitis KRN2, and Pseudomonas jessenii BRT3. It was revealed that all strains having biocontrol properties could produce at least one of the tested substances (HCN, siderophores, chitinase, protease and lipase), which are considered as antifungal and by means of which the biological control can be realized. Conclusions. The strains possessing high biocontrol activity towards phytopathogenic fungi can be proposed for usage as a part of biofungicides after field experiments.
Keywords: medicinal plants, endophytes, bacteria, Fusarium oxysporum, Fusarium solani, growth inhibition.
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