梨灰霉病菌拮抗细菌Pseudomonas extremorientalis的鉴定、发酵条件优化及防效评价

Identification, Optimization of Fermentation Conditions of Antagonistic Bacterium Pseudomonas extremorientalis against Botrytis cinerea on Pear and Evaluation of Its Biocontrol Efficacy

灰霉病是梨贮藏期的主要病害,发病率高,为害严重。为了有效控制该病害发生,利用平皿稀释法从梨根际土壤中分离内生细菌,并采用琼脂平板扩散法进行抑菌活性初筛,筛选出1株拮抗细菌L21,基于形态特征、生理生化特性及16S rRNA和gyrB序列分析,鉴定为Pseudomonas extremorientalis。采用生长速率法测定菌株L21的无菌发酵液对12种病原真菌均有抑制作用,采用Salkowski比色法测定菌株L21能够分泌吲哚-3-乙酸(IAA),采用酪蛋白培养基平板检测菌株L21可以产生胞外蛋白酶。在单因素试验的基础上,通过Plackett-Burman试验设计筛选出pH、硫酸锰和转速3个影响发酵的重要因素,利用最陡爬坡试验和Box-Behnken试验设计,获得菌株L21的最佳发酵条件为蔗糖20 g/L、牛肉膏20 g/L、硫酸锰3.00 g/L、甘油1 g/L、K2HPO4 1.50 g/L、接种量8%、温度34℃、pH 7.31、转速200.66 r/min和培养时间15 h。处理96 h后,菌株L21对梨灰霉病的防效为74.23%,与多菌灵的防效差异不显著。本研究表明菌株L21具有广谱抑菌活性,为植物真菌病害的生物防治提供新的生防资源。

Pear gray mold,an important postharvest disease with high incidence,causes serious damage during the storage period.To control this disease efficiently,endophytic bacteria strains were isolated from pear rhizosphere soil by plating dilution method,and screened based on antifungal activity by using agar plate diffusions method.Antagonistic bacterium strain L21 was obtained,and identified as Pseudomonas extremorientalis based on the morphological,physiological and biochemical characteristics,and 16 S rRNA and gyrB sequence analysis.The antifungal effects of cell-free fermentation broth of strain L21 on twelve pathogenic fungi were obvious by using mycelial growth rate method.Strain L21 could produce indoleacetic acid(IAA)by Salkowski colorimetric method.Strain L21 could produce extracellular by using casein medium plate.Based on single factor tests,three important factors of pH,temperature and yeast powder were screened by Plackett-Burman experimental design.The optimal fermentation conditions for sucrose 20 g/L,beef extract 20 g/L,manganese sulfate 3.00 g/L,glycerol 1 g/L,K2 HPO41.50 g/L,inoculation amount 8%,cultural temperature 34℃,pH 7.31,rotational speed 200.66 r/min and cultural time 15 hours were determined by steepest ascent experiment and Box-Behnken experimental design.At 96 hours after application,the control efficacy of strain L21 was 74.23%,and no significant difference compared with that of carbendazim.Strain L21 showed strong inhibition effect and wide antagonistic spectrum,which has the potential to be the novel biocontrol agent for plant fungal diseases control.