捕食梨火疫病菌黏细菌菌株的筛选及其生防潜力

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

【目的】发掘梨火疫病生防资源,探究黏细菌在梨火疫病生物防治中的应用潜力。【方法】通过菌苔捕食和对峙共培养从实验室前期分离、保存的黏细菌菌株中筛选高效捕食梨火疫病菌的黏细菌菌株,并通过喷施梨离体花序和盆栽杜梨苗测定其防病效果。【结果】(1)46株供试黏细菌菌株对梨火疫病菌均具有捕食能力,其中菌株WCH05、FB02和WCH03对梨火疫病菌的平板捕食能力较强。(2)黏细菌菌株WCH05预处理(喷施)对预防梨花腐的效果显著,其7d平均防效为68.35%,与农用链霉素(68.20%)接近,其次是菌株FB02,防效达到63.24%,而菌株WCH03的防效相对较低,为50.36%;黏细菌菌株WCH05、FB02和WCH03预处理(喷施)能够显著降低盆栽杜梨苗嫩枝的枝枯率和病情指数(p<0.05),7~21 d的平均保护性防效分别为81.53%、76.38%和71.44%,治疗性防效分别为63.84%、51.13%和54.88%。(3)结合形态学特征和多基因序列分析,将WCH05和FB02鉴定为橙色黏球菌(Myxococcus fulvus),WCH03鉴定为黄色黏球菌(Myxococcusxanthus)。【结论】筛选获得3株对梨火疫病菌具有高捕食能力的黏细菌菌株,室内防效显著,具有较好的生物防治应用前景。

【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 con-trol 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 chemi-cal 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 100μL suspension of Erwinia amylovora was vertically inoculated on the surface of TPM nutrient-free medium,and it was allowed to dry naturally,3μL of myxobacteria suspen-sion was inoculated at the center of the moss for the moss predation test,which was periodically photo-graphed,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.Af-ter the 5th day,the pathogenic bacteria and moss were scraped,diluted and coated,and the number of vi-able bacteria was calculated.Two methods were used to screen out myxobacteria strains that could effi-ciently 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 0.22μm 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 inflores-cences and potted Pyrus seedlings as inoculation materials,and the disease prevention effect was deter-mined 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 spray-ing myxobacteria.【Results】(1)46 strains of the tested myxobacteria have predation ability on E.amy-lovora,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 de-creased 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 myxo-bacteria 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 myxobacte-ria strain WCH05 had a significant effect on the prevention of pear rot,and its average 7 d control effi-ciency 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.ful-vus 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.