南瓜白粉病拮抗内生细菌 Bacillus cereus B4-2 的分离鉴定及生防效果

Isolation and identification of endophytic Bacillus cereus B4- 2 against pumpkin powdery mildew and effect of biological control

摘要

从高抗白粉病的南瓜植株内分离筛选出对南瓜白粉病有较好防治效果的内生拮抗细菌菌株，为南瓜白粉病的生物防治提供新途径。采用孢子萌发抑制法、叶盘漂浮法和温室盆栽试验筛选出有较好防效的内生细菌，用形态观察、生理生化测定和分子生物学方法进行鉴定。结果表明，从高抗白粉病的南瓜自交系 22G3 植株内分离获得 29 株内生拮抗细菌菌株，5 个菌株对南瓜白粉菌的孢子萌发抑制率在 50%以上，其中菌株 B4-2 对白粉菌的孢子萌发抑制率最高，为 88.65%。叶盘和室内盆栽试验结果均表明，菌株 B4-2 对南瓜白粉病的防治效果最好，防治效果分别达 79.58%和 83.21%。不同稀释倍数的 B4-2 发酵液对南瓜白粉病均有一定的防治效果，随着稀释倍数的增大，防治效果降低，其中菌株 B4-2 发酵液原液对南瓜白粉病的防治效果最好，达 82.14%。通过形态特征、生理生化特征及基因序列分析，鉴定菌株 B4-2 为蜡样芽孢杆菌(Bacillus cereus)。综上所述，蜡样芽孢杆菌 B4-2 能较好地防治南瓜白粉病，有一定的开发潜力。

Abstract

In order to find the new biological control method against pumpkin powdery mildew, this experiment screened the endophytic antagonistic bacteria against pumpkin powdery mildew from high resistance plants. The spore germination inhibition method, leaf disk floating method and greenhouse pot experiment method were used to select endophytic bacteria with good control effect, which was identified by morphological observation, physiological and biochemical determination as well as molecular biology method. The results showed that 29 endophytic antagonistic bacterial strains were isolated from the high resistant pumpkin inbred line 22G3. Among them, 5 strains had an inhibition rate of over 50% on the spore germination of powdery mildew, and B4-2 had the highest inhibition rate of 88.65%. Both leaf disc and potting experiments showed that the strain B4-2 had the best control effect on pumpkin powdery mildew, with the control effects of 79.58% and 83.21%, respectively. All the different dilution ratios of B4-2 fermentation had certain control effects on pumpkin powdery mildew. As the dilution ratio increased, the prevention effect decreased. The prevention effect of original B4-2 solution was the best and was 82.14%. B4-2 was identified as Bacillus cereus by morphological, physiological, biochemical and gene sequence analysis methods. In summary, B. cereus B4-2 can effectively control pumpkin powdery mildew, which has certain potential for development.

关键词

南瓜 / 白粉病 / 蜡样芽孢杆菌 / 生物防治 / 防治效果

Key words

Pumpkin / Powdery mildew / Bacillus cereus / Biological control / Control efficiency

参考文献

[1] 黄子粤, 刘文君, 覃仁柳, 等. 不同品种南瓜内生细菌多样性及 PICRUSt 基因功能预测分析[J]. 中国农业科学, 2021, 54(18): 4018-4032.

[2] 魏照信, 李波, 荆爱霞. 南瓜白粉病的主要影响因素及防治技术[J]. 中国蔬菜, 2012(7): 28-29.

[3] REINHOLD-HUREK B, HUREK T. Living inside plants:Bacterial endophytes[J]. Current Opinion in Plant Biology, 2011, 14(4): 435-443.

[4] SINGH L P, GILL S S, TUTEJA N. Unraveling the role of fungal symbionts in plant abiotic stress tolerance[J]. Plant Signaling Behavior, 2011, 6(2): 175-191.

[5] 刘江苇, 刘颖, 徐婷, 等. 水稻内生菌研究进展及展望[J]. 生命科学研究, 2021, 25(3): 232-239.

[6] BONITO G, REYNOLDS H, ROBESON M S, et al. Plant host and soil origin influence fungal and bacterial assemblages in the roots of woody plants[J]. Molecular Ecology, 2014, 23(13): 3356-3370.

[7] KHARE E, MISHRA J, ARORA N K. Multifaceted interactions between endophytes and plant:Developments and prospects[J]. Frontiers Microbiology, 2018, 9: 2732.

[8] SANTOYO G, MORENO-HAGELSIEB G, OROZCO-MOSQUEDA M D C. Plant growth-promoting bacterial endophytes[J]. Microbiological Research, 2016, 183: 92-99.

[9] WAZNY R, ROZPADEK P, DOMKA A. The effect of endophytic fungi on growth and nickel accumulation in Noccaea hyperaccumulators[J]. Science of the Total Environment, 2021, 768: 144666.

[10] VARDHARAJULA S, SHAIK Z A, KRISHAN PRASAD VURUKONDA S S. Plant growth promoting endophytes and their interaction with plants to alleviate abiotic stress[J]. Current Biotechnology, 2017, 6(3): 252-263.

[11] 何珊, 田志宏. 植物内生细菌生物学效应的研究进展[J]. 安徽农学通报, 2020, 26(4): 20-23.

[12] 崔文艳, 罗喜燕, 黄高, 等. 金银花白粉病生防内生菌株的筛选、鉴定及其对白粉病的防效[J]. 西南农业学报, 2023, 36(5): 958-964.

[13] 龚双军, 向礼波, 薛敏峰, 等. 小麦根部内生细菌 ZY-1 对小麦白粉菌的抑菌活性及拮抗机理[J]. 中国生物防治学报, 2016, 32(5): 635-641.

[14] 马英元, 栾非时, 马鸿艳, 等. 甜瓜白粉病内生拮抗细菌的筛选鉴定及其防治效果的研究[J]. 植物保护, 2011, 37(2): 25-30.

[15] 中华人民共和国农业部. 农药室内生物测定试验准则杀菌剂第 11 部分:防治瓜类白粉病试验盆栽法:NY/T 1156.11-2008[S]. 北京: 中国农业出版社, 2008.

[16] 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.

[17] 张琦, 刘应敏, 杨东燕, 等. 贝莱斯芽孢杆菌 SM2 对番茄灰霉病的生防效果[J]. 中国瓜菜, 2024, 37(2): 66-73.

[18] SENTHILKUMAR M, ANANDHAM R, MADHAIYAN M. Endophytic bacteria:Perspectives and applications in agricultural crop production[M]//DINESH K M. Bacteria in Agrobiology:Crop Ecosystems.Berlin:Springer, 2011: 61-96.

[19] 赵杰, 田琼金, 聂蔓茹, 等. 红芸豆根腐病拮抗内生细菌的筛选和鉴定[J]. 山西农业科学, 2023, 51(10): 1226-1232.

[20] 张庆琛, 刘应敏, 朱晓琴, 等. 薄荷内生细菌 X7 的分离鉴定及其对番茄黄萎病的防效[J]. 中国瓜菜, 2023, 36(2): 29-34.

[21] PRIHATININGSIH N, DJATMIKO H A, LESTARI P. Endophytic bacteria:An emerging tool for biological control bacterial leaf blight of paddy[J]. IOP Conference Series-Earth and Environmental Science, 2021, 653: 012080.

[22] 高沙尔·卡依尔哈力, 热子亚·麦麦吐逊, 祖丽皮亚·玉努斯. 地锦草内生细菌多样性、拮抗及促生特性测定[J]. 微生物学通报, 2021, 48(2): 392-406.

[23] YUAN X M, LYU S J, ZHANG H Q, et al. Complete genome sequence of novel isolate SYJ15 of Bacillus cereus group, a highly lethal pathogen isolated from Chinese soft shell turtle (Pelodiscus sinensis)[J]. Archives of Microbiology, 2020, 202(1): 85-92.

[24] FAYAD N, AWAD M K, MAHILLON J. Diversity of Bacillus cereus sensu lato mobilome[J]. BMC Genomics, 2019, 20: 436.

[25] 朱海云, 马瑜, 柯杨, 等. 猕猴桃溃疡病菌拮抗菌的筛选、鉴定及其对植物病原真菌的抗性[J]. 生物技术通报, 2021, 37(6): 66-72.

[26] 王刚, 刘凤英, 王淼, 等. 内生细菌 B3-7 的运动性参与其在小麦根系的内生定殖和对小麦全蚀病的生物防治[J]. 植物病理学报, 2011, 41(5): 526-533.

[27] 彭双, 闫淑珍, 陈双林. 具杀线虫活性植物内生细菌的筛选和活性产物[J]. 微生物学报, 2011, 51(3): 368-376.

[28] 王聪. 蜡样芽孢杆菌 BCM2 生防南方根结线虫的影响因素研究[D]. 南京: 南京师范大学, 2018.

[29] 周瑚, 刘剑波, 谭陈勇, 等. 蜡样芽孢杆菌 YN917 对水稻稻瘟病菌的拮抗机理及应用[J]. 农药, 2023, 62(3): 213-216.

基金资助

甘肃省农业科学院科研条件建设及成果转化项目(2022GAAS33)

国家重点研发计划项目(2022YFD1602110)