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| Screening and Identification of Endophytic Fungus HU0460 for Biocontrol of Potato (Solanum tuberosum) Blackleg and the Mechanism of Its Metabolites Against Pectobacterium atroseptica |
| HUANG Yu-Han, MAO Lu-Tian*, LU Zhen-Zhang, JI Xia, XU Liang-Xiong, ZENG Xue-Yun, YE Yu-Zhen, HUANG Yan-Fen |
| College of Life Science, Huizhou University, Huizhou 516007, China |
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Abstract Potato blackleg disease (PBD) is a bacterial soil-borne disease caused by the pathogen Pectobacterium atroseptica (Pat), which seriously obstructs the development of the potato (Solanum tuberosum) industry. In this study, endophytic fungi were isolated from the stem and leaf tissues of Heritiera littoralis using the patato dextrose agar medium (PDA). Fungal metabolites were obtained by solid fermentation of corn medium and rice medium, and the highly active metabolites with significant resistance to Pat were screened using the filter paper method. High activity strain HU0460 was identified by combining morphological observation and ITS sequence comparison. The active components in the metabolites were obtained by ethanol extraction, concentration, and normal silica gel column chromatography. The antibacterial mechanism of the active components on Pat was explored using determination of bacteriostatic and growth curve, observation of cell morphology by scanning electron microscope, and detection of membrane potential by DiBAC4 (3) fluorescence probe. Furthermore, the inhibitory effect of the active metabolites on Pat was analyzed through tuber inoculation. The results showed that the corn fermentation product of the endophytic fungus HU0460 had good antibacterial activity against Pat. HU0460 was identified as Nigrospora sp. by morphology and ITS sequence.The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the bacteriostatic components isolated from the HU0460 corn fermentation medium by ethanol extraction and normal silica gel column chromatography on Pat were 2.44×10-3 and 3.91×10-2 μg/mL, respectively. Results of scanning electron microscope (SEM) showed that the degree of morphological deformation of Pat increased with the increase of the concentration of active metabolites. When the concentration reached the MBC, the Pat cells broke and the content of the cells was lost. The DiBAC4(3) fluorescence probe detection showed that the treatment of HU0460 active metabolites resulted in the up-regulation of the fluorescence intensity of the bacteria and depolarization of the membrane potential of Pat fluorescenceintensity which led to the infectivity to the tube was further confirmed by the tuber inoculation experiment. In this study, the endophytic fungus Nigrospora sp. HU0460 against Pat was isolated and analyzed. The isolated bacteriostatic metabolites have a good antibacterial effect on Pat, providing strain and reference data for the biological control of the PBD and the subsequent agricultural antibiotic research and development.
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Received: 01 November 2022
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Corresponding Authors:
*mlt@hzu.edu.cn
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