疮痂链霉菌拮抗菌HD9-1的筛选及功能评价

Screening and functional evaluation of strain HD9-1 against Streptomyces scabies

【背景】近年来,由土传致病链霉菌引起的疮痂病在我国马铃薯产区大面积暴发,严重影响块茎的商品价值,由于缺乏有效防控措施,其危害程度呈逐年加重趋势。然而拮抗菌可有效抑制病原菌的繁殖,其防控效果优异,对环境友好,逐渐引起了人们的关注。【目的】分离筛选对致病链霉菌具有拮抗功能的细菌,鉴定其分类学名,评价其主要生物学特性,为研发高效防病菌剂提供菌种资源。【方法】以疮痂链霉菌为靶向微生物,从马铃薯疮痂病高发土样中筛选拮抗菌株,通过形态学、生理生化和分子生物学试验确定其分类地位;用盆栽试验初步判定其对马铃薯疮痂病的防控效果,研究其广谱抗病性和耐盐碱特性,结合生产中常用化学农药的敏感性测定,综合评价其应用前景。【结果】筛选出一株对致病疮痂链霉菌(Streptomyces scabies)有显著拮抗效果的菌株HD9-1,抑菌圈直径约31mm,菌落呈圆形、淡黄色,边缘较为整齐,革兰氏染色阳性,菌体杆状,宽度约为0.75μm,长度为1.0-2.5μm。以蔗糖作为唯一碳源,能产生β-半乳糖苷酶、精氨酸双水解酶、赖氨酸脱羧酶和鸟氨酸脱羧酶;培养24 h后代谢产物中含有3.44 mg/L的吲哚乙酸;16S rRNA基因序列与枯草芽孢杆菌(Bacillus subtilis)RUB聚类到同一分支,确定该菌株为Bacillus subtilis;盆栽试验结果表明,HD9-1对马铃薯微型薯疮痂病的防效达59.15%;菌体可耐受pH 3.0-11.0、NaCl含量1%-11%的盐碱环境和100℃水浴30 min;对中生菌素、苯醚甲环唑、氟菌·霜霉威、春雷霉素、氟硅唑、吡唑醚、嘧菌酯和甲基硫菌灵等常用化学农药不敏感,对大丽轮枝菌(Verticillium dahliae)、尖孢镰刀菌(Fusarium oxysporum)、立枯丝核菌(Rhizoctonia solani)等农作物病原菌均具有一定的抑制效果。【结论】菌株HD9-1鉴定为枯草芽孢杆菌,对马铃薯疮痂病防控效果较好,且具有良好的环境适应性,可作为防治马铃薯土传病害的高效复合菌剂的备选菌株。

[Background]The soil-borne potato common scab(CS),caused by Streptomyces spp.,is rampant in China,resulting in tremendous economic loss.With not efficient control measure available,the pathogen become more aggressive.Biocontrol bacteria which are environmentally friendly and show ideal effect in controlling the reproduction of the pathogen have attracted the interest of scholars.[Objective]To isolate and identify antagonistic bacterial isolate against Streptomyces scabies,determine the taxonomic status,evaluate the major biological traits,and thus to provide resources for the development of biocontrol agents.[Methods]Antagonistic isolates were screened from soil with high incidence of potato CS.The taxonomic status was confirmed with morphological,physiological,biochemical,and molecular methods.The control effect was analyzed by pot trials.Then,the potential application of the isolate was evaluated by broad-spectrum resistance(BSR)test,salt-alkali tolerance assay,and sensitivity tests to chemical fungicides.[Results]HD9-1 with significant antagonistic effect on S.scabies was screened out,which exhibited an inhibition zone diameter of 31 mm.The bacterial cells were rod-shaped and Gram-positive,with width of 0.75μm and length of 1-2.5μm.Colony of the isolate was yellowish,with smooth round edge.HD9-1 used sucrose as the only carbon source and producedβ-galactosidase,arginine dihydrolase,lysine decarboxylase,and ornithine decarboxylase.IAA(3.44 mg/L)was detected in liquid medium after 24 h of culture.With the 16S rRNA sequence highly homologous to that of Bacillus subtilis RUB,HD9-1 was determined to be B.subtilis.The pot trial results showed that the scab-control efficiency was 59.15%.Under medium culture conditions,HD9-1 showed tolerance at pH 3.0-11.0,NaCl concentration of 1%-11%,and temperature of up to 100℃(water bath for 30 min).This isolate was insensitive to zhongshengmycin,difenoconazole,fluconazole propamocarb,kasugamycin,flusilazole,pyrazole ether,azoxystrobin and thiophanate methyl.Meanwhile,it showed certain control effect on other crop pathogens,such as Verticillium dahliae,Fusarium oxysporum,and Rhizoctonia solani.[Conclusion]HD9-1,a B.subtilis strain,displays significant control effect on potato CS and strong adaptability,which can be a potential candidate for developing compound microbial agent against some potato soil-borne diseases.