两株贝莱斯芽胞杆菌鉴定及其对薄壳山核桃黑斑病病原菌的拮抗效果

doi:10.16409/j.cnki.2095-039x.2022.08.003

摘要/Abstract

摘要： 黑斑病为薄壳山核桃重要真菌性病害，为了实现薄壳山核桃黑斑病绿色防控，本研究旨在筛选优良的内生拮抗菌株，为黑斑病的生物防控提供生防资源。通过形态鉴定、生理生化特征测定和16S rDNA与GyrA基因分析，对薄壳山核桃内生拮抗菌进行鉴定；采用平板对峙法测定内生拮抗菌对黑斑病病原真菌（5属21种26株）菌丝生长的抑制能力，分离筛选到2株高效拮抗菌ZJJDZY和ZJJDYB，鉴定为贝莱斯芽胞杆菌Bacillus velezensis。平板对峙法测定结果表明，2株拮抗菌均对12种12株炭疽菌Colletotrichum spp.、1种4株葡萄座腔菌Botryosphaeria dothidea、2种2株链格孢菌Alternaria spp.、4种7株新拟盘多毛孢Neopestalotiopsis spp.和1种1株拟茎点霉菌Phomopsis sp.均具有拮抗效果。其中，对C. henanense PCZJJD18、C. sojae PCAHFY5、N. mesopotamica PCSCLZ2、A. alternata PCAHFY6、Phomopsis PCZJJD14 sp.的抑制率为最高，抑制率在57.1%～60.6%，且两株拮抗菌抑菌能力相似；不同浓度的拮抗菌对不同属病原真菌的抑制效果不同，浓度为10⁸ CFU/mL时，拮抗效果最好，随着浓度降低，拮抗效果显著减弱。该两个菌株对多种薄壳山核桃黑斑病病原真菌具有拮抗作用，具有较佳的生防潜力。

关键词: 薄壳山核桃, 黑斑病, 鉴定, 贝莱斯芽胞杆菌, 生物防治

Abstract: Black spot is an important pathogenic fungi disease infected pecan (Carya illinoensis). To develop green and efficient biological control method for pecan black spot disease, effective antagonistic endophytic strains were screened and identified as biocontrol resources against black spot disease. Based on morphological identification, physiological and biochemical characteristics, and phylogenetic analysis of 16S rDNA and GyrA gene, entophytic antagonistic strains isolated from pecan leaf were identified and their inhibition effects on mycelium growth of 26 isolates fungi caused black spot disease was detected by plate confrontation method. Two antagonistic entophytic strains screened from pecan leaf, named ZJJDZY and ZJJDYB, were identified as Bacillus velezensis. Both ZJJDZY and ZJJDYB showed significant antagonistic effects on 12 Colletotrichum species, 1 Botryosphaeria species, 2 Alternaria species, 4 Neopestalotiopsis species and 1 Phomopsis species. ZJJDZY and ZJJDYB demonstrated higher inhibition activity against PCZJJD18 C. henanense, PCAHFY5 C. sojae, PCSCLZ2 N. saprophytica, PCAHFY6 A. alternate and PCZJJD14 Phomopsis sp., with the inhibitory rates ranging from 57.1% to 60.6%, and the antibacterial ability of ZJJDZY and ZJJDYB was similar. Inhibition effects of ZJJDZY and ZJJDYB against different pathogenic fungi varied significantly at different concentrations, and the best antagonistic effect was shown at the concentration of 10⁸ CFU/mL. While, with the decrease of concentration, the antagonistic effect weakened significantly. Two entophytic antagonistic bacteria, ZJJDZY and ZJJDYB showed significant antagonistic effects against various pathogenic fungi causing black spot disease, which has the potential as biocontrol resource.

Key words: pecan, black spot disease, identification, Bacillus velezensis, biological control

中图分类号:

S476

王迪, 高岩, 吴小双, 张威, 舒金平, 张亚波, 翟凤艳. 两株贝莱斯芽胞杆菌鉴定及其对薄壳山核桃黑斑病病原菌的拮抗效果[J]. 中国生物防治学报, 2022, 38(6): 1572-1581.

WANG Di, GAO Yan, WU Xiaoshuang, ZHANG Wei, SHU Jinping, ZHANG Yabo, ZHAI Fengyan. Identification of Two Strains of Bacillus velezensis Isolated from Carya illinoensis Leaf and Their Antagonistic Effects on Pecan Black Spot Pathogen[J]. Chinese Journal of Biological Control, 2022, 38(6): 1572-1581.

参考文献

[1] 何旭东, 郑纪伟, 田雪瑶,等.薄壳山核桃品种亲缘关系分析与指纹图谱构建[J]. 林业科学研究, 2021, 34(4): 95-102.
[2] 周梦钰. 薄壳山核桃复合林分土壤理化性质及微生物多样性的研究[D]. 合肥: 安徽农业大学, 2021.
[3] Fabrizio G C, Elmarie V W, Gesine M C. Propagation of pecan (Carya illinoensis): A review[J]. African Journal of Biotechnology, 2018, 17(18): 586-605.
[4] Tres M V. Potential applications of pecan residual biomasses: A review[J]. Biointerface Research in Applied Chemistry, 2020, 10: 5524-5531.
[5] 张潇丹. 薄壳山核桃矿质养分及果实性状研究[D]. 重庆: 西南大学, 2021.
[6] 孟珂. 薄壳山核桃黑斑病病原菌鉴定及室内药剂筛选[D]. 新乡: 河南科技学院, 2020.
[7] 常君, 姚小华, 张亚波, 等. 松针刺盘孢菌侵染对薄壳山核桃不同抗性品种酶活性和酚类物质的影响[J]. 西南大学学报(自然科学版), 2022, 44(3): 52-58.
[8] 陈于, 王敏, 朱灿灿, 等. 常州金坛地区不同薄壳山核桃品种黑斑病抗性田间调查[J]. 江苏林业科技, 2018, 45(6): 26-29.
[9] Zhang Y B, Meng K, Shu J P, et al. First report of anthracnose on pecan (Carya illinoensis) caused by in China[J]. Plant Disease, 2019, 103(6): 1432.
[10] 孟珂, 张亚波, 常君, 等. 8种杀菌剂对9种薄壳山核桃炭疽病病原菌的毒力测定[J]. 林业科学研究, 2021, 34(1): 153-164.
[11] 王德浩. 马铃薯黑痣病的生物防治及其机理研究[D]. 泰安: 山东农业大学, 2020.
[12] 罗云艳. 烟草根黑腐病根际拮抗菌的筛选、鉴定及其生物防治研究[J]. 杨凌: 西北农林科技大学, 2021.
[13] Jaffuel G, Imperiali N, Shelby K, et al. Protecting maize from rootworm damage with the combined application of arbuscular mycorrhizal fungi, Pseudomonas bacteria and entomopathogenic nematodes[J]. Scientific Reports, 2019, 9(1): 1–12.
[14] Liang L Q, Fu Y J, Deng S S, et al. Genomic, antimicrobial, and aphicidal traits of Bacillus velezensis ATR2, and its biocontrol potential against ginger rhizome rot disease caused by Bacillus pumilus[J]. Microorganisms, 2022, 10(1): 63.
[15] 陈泽斌, 靳松, 张永福, 等. 植物内生菌生物防治研究进展及存在的问题[J]. 昆明学院学报, 2014, 36(3): 40-42.
[16] Kotb E. Purification and partial characterization of serine fibrinolytic enzyme from Bacillus megaterium KSK-07 isolated from kishk, a traditional egyptian fermented food[J]. Applied Biochemistry and Microbiology, 2015, 51(1): 34-43.
[17] Lim S B Y, Junqueira A C M, Uchida A, et a1. Genome sequence of Bacillus velezensis SGAir0473, isolated from tropical air collected in Singapore[J]. Genome Announcements, 2018, 6(27): 00642-18.
[18] 张妙宜, 云天艳, 周登博, 等. 甲基营养型芽胞杆菌的研究进展[J]. 热带农业科学, 2017, 37(9): 66-71.
[19] Huang L, Li Q C, Hou Y, et al. Bacillus velezensis strain HYEB5-6 as a potential biocontrol agent against anthracnose on Euonymus japonicus[J]. Biocontrol Science and Technology, 2017, 27(5): 636-653.
[20] Cui L X, Yang C D, Wei L J, et a1. Isolation and identification of an endophytic bacteria Bacillus velezensis 8-4 exhibiting biocontrol activity against potato scab[J]. Biological Control, 2020, 141: 104156.
[21] 李苗苗, 张家韬, 崔传斌, 等. 三株芽胞杆菌的生防特性研究[J]. 中国烟草科学, 2020, 41(4): 80-89.
[22] 孙旺旺, 闫丽, 陈昌龙, 等. 生菜软腐和菌核病拮抗菌贝莱斯芽胞杆菌BPC6鉴定与防效[J], 中国生物防治学报, 2020, 36(2): 231-240.
[23] Buchanan R E, Gibbons N E. Bergey's manual of systematic bacteriology[M]. Beijing: Science Press, 1999.
[24] 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, 62-65.
[25] Ruiz-Garcia C, Bejar V, Martinez-Checa F, et al. Bacillus velezensis sp. nov., a surfactant-producing bacterium isolated from the river Velez in Malaga, southern Spain[J]. International Journal of Systematic and Evolutionary Microbiology, 2005, 55(1): 191-195.
[26] 郭鹤宝, 王星, 何山文, 等. 表型特征结合基因组分析鉴定不同菌落形态 Bacillus velezensis ACCC19742[J]. 生物技术通报, 2020, 36(2): 142-148.
[27] Liu G Q, Kong Y Y, Fan Y J, et a1. Whole-genome sequencing of Bacillus velezens LS69, a strain with a broad inhibitory spectrum against pathogenic bacteria[J]. Journal of Biotechnology, 2017, 249: 20-24.
[28] Lim S M, Yoon M Y, Choi G J, et a1. Diffusible and volatile antifungal compounds produced by an antagonistic Bacillus velezens G341 against various phytopathogenic fungi[J]. The Plant Pathology Journal, 2017, 33(5): 488-498.
[29] Nannan C, Gillis A, Caulier S, et a1. Complete genome sequence of Bacillus velezens CN026 exhibiting antagonistic activity against gram-negative foodborne pathogens[J]. Genome Announcements, 2018, 6(4): e01543-17.
[30] Cao Y, Pi H, Chandrangsu P, et al. Antagonism of two plant-growth promoting Bacillus velezensis isolates against Ralstonia solanacearum and Fusarium oxysporum[J]. Scientific Reports, 2018, 8(1): 1-14.
[31] 孙力. 贝莱斯芽胞杆菌Can L-30挥发性有机物活性研究及菌剂研制[D]. 武汉: 华中农业大学, 2020.
[32] 沙月霞, 王琦, 李燕. 稻瘟病生防芽胞杆菌的筛选及防治效果[J]. 中国生物防治学报, 2016, 32(4): 474-484.
[33] 谢林艳, 狄义宁, 何丽莲, 等. 甘蔗赤腐病内生拮抗菌yc89的筛选及鉴定[J]. 微生物学报, 2020, 60(4): 739-748.
[34] Cui L X, Yang C D, Wei L J, et a1. Isolation and identification of an entophytic bacteria Bacillus velezensis 8-4 exhibiting biocontrol activity against potato scab[J]. Biological Control, 2020, 141: 104156.
[35] Toral L, Rodríguez M, Béjar V, et al. Crop protection against Botrytis cinerea by rhizhosphere biological control agent Bacillus velezensis XT1[J]. Microorganisms, 2020, 8(7): 992.
[36] 李万芹, 何鹏飞, 吴毅歆, 等. 香蕉内生贝莱斯芽胞杆菌yx-11的分离鉴定与功能研究[J]. 江西农业学报, 2021, 33(12): 8-13.
[37] 胡党振, 徐嫒媛, 于梦怡, 等. 芽胞杆菌处理对柑桔苗木黄龙病抑制效果[J]. 中国农业大学学报, 2021, 26(8): 72-83.
[38] 欧阳晓伦. 贝莱斯芽胞杆菌PZ-3 (Bacillus velezensis PZ-3) 分离、鉴定及其对植物病原真菌的抑菌效果[D]. 大庆: 黑龙江八一农垦大学, 2021.
[39] 万青, 包晓东, 李文彬, 等. 一株贝莱斯芽胞杆菌B18的分离纯化与鉴定及其对香蕉枯萎病的防效[J]. 基因组学与应用生物学, 2021, 40(z3): 3209-3215.