Screening and biocontrol potential of myxobacteria preying on pathogenic bacteria causing pear fire blight

Abstract: 【Objective】Pear fire blight is one of the destructive diseases of Rosaceae plants, which was first discovered successively in Yili and Bazhou in Xinjiang in 2016, and if effective preventive and control measures can’t be taken to curb the spread and proliferation of the disease, not only will it cause heavy damage to Xinjiang’s characteristic advantageous pear industry and face the risk of destruction, but also will bring a major threat to Xinjiang and even the national fruit industry. At present, pear fire blight control methods mainly include phytosanitary, pruning, eradication of diseased plants and chemical control and other ways, but these methods have some disadvantages. Research and practice have shown that biological control can achieve good preventive effect, compared with chemical pesticides, and has the outstanding advantages of strong selectivity, not easy to produce resistance, safety and high efficiency. Therefore, the aim of this study was to explore the biological resources for controlling pear fire blight and determine the potential of myxobacteria in the biological control for pear fire blight. 【Methods】In this study, a 100 μL suspension of Erwinia amylovora was vertically inoculated on thesurface of TPM nutrient-free medium, and it was allowed to dry naturally, 3 μL of myxobacteria suspension was inoculated at the center of the moss for the moss predation test, which was periodically photographed, and the predation was completed by scraping the plate of the pathogen moss after the 5th day, and the number of residual viable bacteria was counted by diluting and coating the moss. Myxobacteria were inoculated near the edge of the dried pathogen moss, and the face-off culture was carried out. After the 5th day, the pathogenic bacteria and moss were scraped, diluted and coated, and the number of viable bacteria was calculated. Two methods were used to screen out myxobacteria strains that could efficiently prey on pear fire blight bacteria from pre- laboratory isolates and preserved mucoid bacterial strains. After shaking cultivation of myxobacteria to the stable stage by LBS medium, the fermentation filtrate was obtained and filtered with 0.22 μm microporous filter membrane to remove bacteria, and the decontaminated fermentation filtrate was obtained. Take 1 mL of decontaminated fermentation filtrate and 0.1 mL of pathogenic bacterial suspension to be co-cultured for 24 h. Dilute the coating and count the number of residual viable bacteria to explore the effect of its metabolites on pathogenic bacteria. In order to clarify the effectiveness of myxobacteria against pear fire blight, we used pear isolated inflorescences and potted Pyrus seedlings as inoculation materials, and the disease prevention effect was determined with two methods, that is, protective and therapeutic tests, by spraying myxobacteria first for 24 hours and then spraying pathogenic bacteria, and by spraying pathogenic bacteria first and then spraying myxobacteria.【Results】(1) 46 strains of the tested myxobacteria have predation ability on E. amylovora, and compared with the control group, the residual viable bacterial number of 9.7×108 cfu ·mL-1 decreased to (3.7×103 ) - (2.4×107 ) cfu·mL-1 , of which strains WCH05, FB02 and WCH03 had stronger plate predation ability on E. amylovora. In the bacterial moss predation test, myxobacteria strain WCH05 had the strongest ability to prey on E. amylovora, and the residual viable bacterial count decreased to 3.7×103 cfu ·mL-1 , followed by strains FB02 and WCH03. In the standoff culture test, strain WCH05 had the strongest ability to expand outward to prey on E. amylovora, and the residual viable bacterial count of E. amylovora decreased to 2.6×103 cfu·mL-1 compared with the control group of 2.8× 108 cfu ·mL-1 , followed by strains WCH03 and FB02. (2) The residual viable bacterial counts of myxobacteria WCH05, FB02 and WCH03 after co-cultivation with E. amylovora were all in the same order of magnitude compared with the control, with no significant difference, suggesting that the three strains preyed on pear fire blight pathogens mainly by direct contact. (3) Pretreatment (spraying) of myxobacteria strain WCH05 had a significant effect on the prevention of pear rot, and its average 7 d control efficiency was 68.35%, which was close to that of agricultural streptomycin (68.20%), followed by FB02 (63.24%), but WCH03 (50.36%) was relatively low. Pretreatment (spraying) of myxobacterium strains WCH05, FB02 and WCH03 could significantly reduce the dead shoot rate and disease index of potted pear seedlings (p＜0.05), and the average protective efficacy from 7 to 21 days was 81.53%, 76.38% and 71.44%, respectively. The therapeutic efficacy was 63.84%, 51.13% and 54.88%, respectively. (4) WCH05 and FB02 were identified as Myxococcus fulvus, while WCH03 was identified as M. xanthus based on the morphological characteristics and multi-gene sequence analysis.【Conclusion】The M. fulvus WCH05 in the strains WCH05, FB02 and WCH03 has the best biocontrol effect on pear fire blight bacteria, and is expected to be developed as a biocontrol agent for pear fire blight, laying a foundation for the biological control for pear fire blight.