抗稻瘟病的生防细菌筛选与鉴定
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摘要
为获取优良的水稻抗稻瘟病的生防菌株,以稻瘟病菌(Pyricularia oryzae)为指示菌,针对2016年和2017年采自辽宁省7个水稻主产区的46份水稻田土样,采用土壤稀释平板法从土样中分离纯化得到541株菌株,其中真菌137株,细菌288株,放线菌116株;再经菌株发酵液处理水稻种子,清水和氨基寡糖素处理为空白对照,待水稻长至3叶1心期时采用活体苗期接种的方法对已分离出的菌株进行筛选,得到对稻瘟病有较好诱导抗性的生防菌株5株,标记为DdN5-6、DdN6-1、PJN13-7、PJN13-5和YKN8-7,防治效果为76.13%～58.03%,其中DdN6-1防治效果最好,达到76.13%。本试验研究菌株发酵液浸种对水稻生长的促进作用,室内种子萌发试验结果表明,经菌株DdN6-1和PJN13-5发酵液浸种处理后的水稻种子发芽率分别达到88.89%和90.00%,明显高于对照;在相同的生长时间和生长条件下,经菌株PJN13-5发酵液处理的水稻根长最长,达到9.57 cm,高于其他菌株处理;经5株菌株发酵液处理的水稻种子,长至3叶1心期时与对照相比根系较粗壮,其中DdN6-1发酵液处理的水稻幼苗抗病效果最好,菌株PJN13-5发酵液处理的水稻幼苗促生效果最好。结合形态特征、生理生化特性和16S rDNA基因序列测定对筛选出的菌株进行鉴定,鉴定结果表明:菌株DdN5-6、PJN13-7和YKN8-7为巨大芽孢杆菌(Bacillus megaterium);菌株DdN6-1为蜡样芽孢杆菌(Bacillus cereus);菌株PJN13-5为纺锤形赖氨酸芽孢杆菌(Lysinibacillus fuormis)。
In order to obtain a good biocontrol strain of rice resistant to rice blast, 46 paddy soil samples were collected in2016 and 2017 from 7 main rice production areas in Liaoning Province, using Pyricularia oryzae as indicator. 541 strains were isolated and purified from soil samples by soil dilution plate method, including 137 fungi, 288 bacteria and 116 actinomycetes;When rice seeds were treated with strain fermentation broth, clear water and amino oligosaccharides were used as blank control,and the isolated strains were screened by the method of seeding in vivo at the first heart stage of 3 leaves.Five biocontrol strains with good resistance to rice blast were obtained, which were labeled as DdN5-6, DdN6-1, PJN13-7, PJN13-5 and YKN8-7, in the range of 76.13%-58.03%, among which the best control effect of DdN6-1 was 76.13%.In this experiment, the effect of soaking with fermentation broth on the growth of rice was studied. The results of indoor seed germination test showed that the germination rate of rice seeds after soaking with strain DdN6-1 and PJN13-5 was 88.89% and 90.00% respectively higher than that of the control.Under the same growth time and condition, the root length of rice treated with strain PJN13-5 fermentation broth was the longest, which reached 9.57 cm higher than that of other strains, and the rice seeds treated with five strains fermentation broth. Compared with the control, the root system of rice seedlings treated with DdN6-1 fermentation liquid was the best, and that of rice seedlings treated with strain PJN13-5 fermentation liquid was the best.Combined with morphological characteristics, physiological and biochemical characteristics and 16 s rDNA gene sequencing, the selected strains were identified.The results showed that DdN5-6, PJN13-7 and YKN8-7 were Bacillus megaterium; DdN6-1 was Bacillus cereus; Strain PJN13-5 as Lysinibacillus fuormis.
In order to obtain a good biocontrol strain of rice resistant to rice blast, 46 paddy soil samples were collected in2016 and 2017 from 7 main rice production areas in Liaoning Province, using Pyricularia oryzae as indicator. 541 strains were isolated and purified from soil samples by soil dilution plate method, including 137 fungi, 288 bacteria and 116 actinomycetes;When rice seeds were treated with strain fermentation broth, clear water and amino oligosaccharides were used as blank control,and the isolated strains were screened by the method of seeding in vivo at the first heart stage of 3 leaves.Five biocontrol strains with good resistance to rice blast were obtained, which were labeled as DdN5-6, DdN6-1, PJN13-7, PJN13-5 and YKN8-7, in the range of 76.13%-58.03%, among which the best control effect of DdN6-1 was 76.13%.In this experiment, the effect of soaking with fermentation broth on the growth of rice was studied. The results of indoor seed germination test showed that the germination rate of rice seeds after soaking with strain DdN6-1 and PJN13-5 was 88.89% and 90.00% respectively higher than that of the control.Under the same growth time and condition, the root length of rice treated with strain PJN13-5 fermentation broth was the longest, which reached 9.57 cm higher than that of other strains, and the rice seeds treated with five strains fermentation broth. Compared with the control, the root system of rice seedlings treated with DdN6-1 fermentation liquid was the best, and that of rice seedlings treated with strain PJN13-5 fermentation liquid was the best.Combined with morphological characteristics, physiological and biochemical characteristics and 16 s rDNA gene sequencing, the selected strains were identified.The results showed that DdN5-6, PJN13-7 and YKN8-7 were Bacillus megaterium; DdN6-1 was Bacillus cereus; Strain PJN13-5 as Lysinibacillus fuormis.
引文
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[17]陈刘军,俞仪阳,王超,等.蜡样芽孢杆菌AR156防治水稻纹枯病机理初探[J].中国生物防治学报,2014,30(1):107-112.
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[19] SHA Y,WANG Q,YAN L.Suppression of Magnaporthe oryzae and interaction between Bacillus subtilis and rice plants in the control of rice blast[J].Springer Plus,2016,5(1):1238.
[20]陈志谊.芽孢杆菌类生物杀菌剂的研发与应用[J].中国生物防治学报,2015,31(5):723-732.
[21]吕黎,王蕾,周佳敏,等.巨大芽孢杆菌的研究现状及应用[J].农业科学研究,2014,35(3):48-52.
[22]管珺,胡永红,杨文革,等.蜡样芽孢杆菌防治植物病虫害的研究进展[J].现代农药,2007,6(4):7-10.
[23]刘露,李丽,闫洪雪.巨大芽孢杆菌的应用研究进展[J].北方农业学报,2016,44(4):117-120.
[24]李姝江,朱天辉,谯天敏.花椒根腐病生防芽孢杆菌的筛选鉴定及定殖和防治效果[J].西北农林科技大学学报(自然科学版),2016,44(4):114-122.
[2]沙月霞,王琦,李燕.稻瘟病生防芽孢杆菌的筛选及防治效果[J].中国生物防治学报,2016,32(4)474-484.
[3]王巧兰,郭刚.水稻稻瘟病生物防治研究进展[J].河南农业科学,2005(10):10-13.
[4] DAGDAS YF,YOSHINO K,DAGDAS G.Septin-mediated plant cell invasion by the rice blast fungus,Magnaporthe oryzae[J].Science,2012,336(6088):1590-1595.
[5]张传清,周明国,朱国念.稻瘟病化学防治药剂的历史沿革与研究现状[J].农药学学报,2009,11(1):72-80.
[6] SHAN H,ZHAO M,CHEN D,et al.Biocontrol of rice blast by the phenaminomethylacetic acid producer of Bacillus methylotrophicus strain BC79[J].Crop Protection,2013,44:29-37.
[7]韩长志.植物病害生防菌的研究现状及发展趋势[J].中国森林病虫,2015,34(1):33-37.
[8]马桂珍.植物真菌性病害诱导抗性研究进展[J].河北农业技术师范学院学报,1997,11(3):55-59.
[9] RVAN P,NIEMANN GJ,SCHIPPERS B.Induced resistance and phytoalexin accumulation in biologica control of Fusarium wilt of carnation by Pseudomonas sp. strain WCS 417r[J].Phytopathology,1991,81:728-734.
[10]魏国汶.水稻诱导抗病性研究进展[J].江西农业学报,2007,19(8):46-49.
[11]罗文芳,魏松红,王海宁,等.水稻纹枯病生防菌的筛选与鉴定[J].沈阳农业大学学报,2017,48(6):660-665.
[12]李思博,魏松红,王海宁,等2015~2016年辽宁省稻瘟病菌种群动态分析[J].沈阳农业大学学报,2017,48(3):284-289.
[13] SEBASTIEN.Standard Evaluation System for Rice[S].1996:17-18.
[14]回虹燕,王媛媛,朱晓峰,等.拮抗线虫的花生根瘤内生细菌筛选及鉴定[J].中国油料作物学报,2016,38(2):223-229.
[15]东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001:353-370.
[16]布坎南R E,吉本斯N E.伯杰细菌鉴定手册[M].8版.中国科学院微生物研究所《伯杰细菌鉴定手册》翻译组,译.北京:科学出版社,1984:729-758.
[17]陈刘军,俞仪阳,王超,等.蜡样芽孢杆菌AR156防治水稻纹枯病机理初探[J].中国生物防治学报,2014,30(1):107-112.
[18] ZHANG C,ZHANG X,SHEN S.Proteome analysis for antifungal effects of Bacillus subtilis KB-1122 on Magnaporthe grisea P131[J].World Journal of Microbiology and Biotechnology,2014,30(6):1763-1774.
[19] SHA Y,WANG Q,YAN L.Suppression of Magnaporthe oryzae and interaction between Bacillus subtilis and rice plants in the control of rice blast[J].Springer Plus,2016,5(1):1238.
[20]陈志谊.芽孢杆菌类生物杀菌剂的研发与应用[J].中国生物防治学报,2015,31(5):723-732.
[21]吕黎,王蕾,周佳敏,等.巨大芽孢杆菌的研究现状及应用[J].农业科学研究,2014,35(3):48-52.
[22]管珺,胡永红,杨文革,等.蜡样芽孢杆菌防治植物病虫害的研究进展[J].现代农药,2007,6(4):7-10.
[23]刘露,李丽,闫洪雪.巨大芽孢杆菌的应用研究进展[J].北方农业学报,2016,44(4):117-120.
[24]李姝江,朱天辉,谯天敏.花椒根腐病生防芽孢杆菌的筛选鉴定及定殖和防治效果[J].西北农林科技大学学报(自然科学版),2016,44(4):114-122.