花椒根腐病拮抗菌W-5的鉴定及发酵条件的优化

Identification of Antagonistic Strain W-5 to Zanthoxylum bungeanum Root Rot and Optimization of Fermentation Conditions

腐皮镰刀菌(Fusarium solani)是引起花椒根腐病的主要致病菌,该病严重威胁着花椒产业的发展。为筛选出花椒根腐病的生防菌株,本研究采用土壤平板梯度稀释法和平板对峙法从花椒根系土壤中筛选出对腐皮镰刀菌具有拮抗作用的菌株,通过形态特征观察结合16S rDNA基因序列分析对菌株进行分类地位确定,并在单因素试验基础上结合Box-Behnken中心组合设计的响应面法优化拮抗菌株的发酵条件。结果表明,分离获得的菌株W-5为枯草芽孢杆菌(Bacillus subtilis),且该菌对腐皮镰刀菌的抑制率达到82.7%,对其他植物病原菌(禾谷镰刀菌、梅毒镰刀菌、藤仓赤霉菌、松针刺盘孢菌和层生镰刀菌)均有较高的抑菌活性,说明其抑菌谱的广泛性;能够分泌蛋白酶和纤维素酶且产嗜铁素能力极强,能够减轻离体培养下花椒根片的发病症状;响应面法优化后的菌株W-5的最佳发酵条件为:温度31.3℃、pH值7.5、接种量3.1%、转速180.5 r/min,此条件下OD_(600)值达到1.832。综之,拮抗菌W-5对花椒根腐病防效优良,具有生防潜力,建立并优化的拮抗菌W-5的发酵条件体系可为花椒根腐病生物药剂的研制提供技术参考。

Fusarium solani is the main pathogen causing the root rot disease of Zanthoxylum bungea-num,which seriously threatens the development of Z.bungeanum industry.In order to screen out biocontrol strains of Z.bungeanum root rot disease,the soil plate gradient dilution method and plate confrontation method were used to screen strains with antagonistic effects against F.solani in Z.bungeanum root soil.The classifica-tion status of the strains was determined through morphological observation and 16S rDNA gene sequence anal-ysis.The fermentation conditions for the antagonistic strains were optimized by single factor tests and response surface methodology with Box-Benhnken central combination design.The results showed that the isolated strain W-5 belonged to Bacillus subtilis,and its inhibition rate against F.solani reached 82.7%.It also had higher antibacterial activity against other plant pathogens such as F.graminearum,F.syphilis,Gibberella fujikuroi,Colletotrichum fioriniae and F.proliferatum,indicating it had broad antibacterial spectrum.It could secrete proteases and cellulases,and had strong ability to produce ferritin,which could alleviate the disease occur-rence of Z.bungeanum root slices under in vitro culture.The optimal fermentation conditions for the strain W-5 optimized by response surface methodology included temperature of 31.3℃,pH 7.5,inoculation amount of 3.1%and rotational speed at 180.5 r/min.Under these conditions,the OD_(600) value reached 1.832.In summa-ry,the antagonistic strain W-5 had excellent control effects on Z.bungeanum root rot disease,so it had bio-control potential.The established and optimized fermentation system for the antagonistic strain W-5 could pro-vide technical references for the development of biological agents for Z.bungeanum root rot disease.