沙福芽孢杆菌GX-H6的分离鉴定及对水稻细菌性条斑病的防病效果

doi:10.13560/j.cnki.biotech.bull.1985.2022-1153

生物技术通报 ›› 2023, Vol. 39 ›› Issue (5): 243-253.doi: 10.13560/j.cnki.biotech.bull.1985.2022-1153

沙福芽孢杆菌GX-H6的分离鉴定及对水稻细菌性条斑病的防病效果

任沛东¹^,²(), 彭健玲¹^,², 刘圣航¹^,², 姚姿婷¹^,³, 朱桂宁¹^,³^,⁴, 陆光涛², 李瑞芳¹^,³^,⁴()

1.广西农业科学院植物保护研究所, 南宁 530017
2.广西大学生命科学与技术学院, 南宁 530005
3.农业农村部华南果蔬绿色防控重点实验室，南宁 530017
4.广西作物病虫害生物学重点实验室, 南宁 530017

收稿日期:2022-09-20 出版日期:2023-05-26 发布日期:2023-06-08
通讯作者: 李瑞芳，女，副研究员，研究方向：微生物学、分子植物病理学；E-mail: ruifangli@gxaas.net
作者简介:任沛东，男，博士研究生，研究方向：分子植物病理学；E-mail: nmgpeytonren@126.com
第一联系人：
彭健玲为共同第一作者
基金资助:
国家自然科学基金项目(32160617);广西农业科学院科技发展基金(桂农科2021JM13);广西农业科学院科技发展基金(桂农科2021ZX23);广西农业科学院科技发展基金(桂农科2021YT063)

Isolation and Identification of a Bacillus safensis Strain GX-H6 and Its Biocontrol Effect on Bacterial Leaf Streak of Rice

REN Pei-dong¹^,²(), PENG Jian-ling¹^,², LIU Sheng-hang¹^,², YAO Zi-ting¹^,³, ZHU Gui-ning¹^,³^,⁴, LU Guang-tao², LI Rui-fang¹^,³^,⁴()

1. Plant Protection Research Institute, Guangxi Academy of Agricultural Sciences, Nanning 530017
2. College of Life Science and Technology, Guangxi University, Nanning 530005
3. Key Laboratory of Green Prevention and Control on Fruits and Vegetables in South China Ministry of Agriculture and Rural Affairs, Nanning 530017
4. Guangxi Key Laboratory of Biology for Crop Diseases and Insect Pests, Nanning 530017

Received:2022-09-20 Published:2023-05-26 Online:2023-06-08

摘要/Abstract

摘要：

水稻细菌性条斑病（bacterial leaf streak, BLS）由稻黄单胞菌稻生致病变种（Xanthomonas oryzae pv. oryzicola, Xoc）侵染引起，已成为我国南方水稻种植区的一个重要病害。为了筛选防治BLS的生防菌，以Xoc 野生型菌株GX01为指示菌，采用含菌平板稀释法和牛津杯法，从花生根际土壤中筛选到一株对Xoc具有拮抗作用的细菌，编号为GX-H6。根据形态学观察、生理生化特征以及16S rDNA和进化树分析，鉴定该菌株属于沙福芽孢杆菌（Bacillus safensis）。拮抗实验表明，B. safensis GX-H6菌株能够对多种黄单胞菌以及植物病原真菌具有较好的拮抗活性，尤其对引起水稻发生白叶枯病（bacterial blight, BB）的稻黄单胞菌稻致病变种（Xanthomonas oryzae pv. oryzae，Xoo）的拮抗效果最显著。温室和田间水稻植株试验表明，GX-H6菌株能较好地防治BLS和BB。对GX-H6菌株的基因组进行分析，发现该菌株的基因组中拥有与抗真菌、环境竞争相关的基因，同时也拥有地衣杆菌素（lichenysin）、植物阿唑霉素（plantazolicin）和溶杆菌素（bacilysin）合成相关基因。这为生产应用提供了新的微生物资源，以及为后续的抑菌机理研究提供了新材料。

关键词: 水稻条斑病菌, 黄单胞菌, 沙福芽孢杆菌, 拮抗, 生物防治

Abstract:

Bacterial leaf streak（BLS）, caused by Xanthomonas oryzae pv. oryzicola（Xoc）, is emerging as a serious disease in rice fields of southern China. In order to screen biocontrol bacteria for controlling BLS, we had Xoc wild-type strain GX01 as indicator, a bacterium with antagonistic effect to Xoc was isolated from the samples of peanut rhizosphere soil by using bacterial plate dilution and Oxford cup methods, and named as GX-H6. Based on the morphological, physiological and biochemical data, 16S rDNA and evolutionary tree analysis, this strain was identified to be Bacillus safensis. Antagonistic experiments revealed that the B. safensis GX-H6 strain effectively repressed the growths of several xanthomonads and plant pathogenic fungi, particularly the bacterium Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight（BB）of rice. Further greenhouse and field rice plant experiments showed that this strain effectively controlled BLS and BB of rice. Genome analysis indicated that the GX-H6 strain contained a variety of genes related to antifungal, environmental competition, and the production of lichenysin, plantazolicin and bacilysin. Overall, the B. safensis strain GX-H6 is a potential novel biocontrol agent for developing pesticides and exploring novel bacteriostatic mechanism in future.

Key words: Xanthomonas oryzae pv. oryzicola, xanthomonads, Bacillus safensis, antagonistic ability, biocontrol

任沛东, 彭健玲, 刘圣航, 姚姿婷, 朱桂宁, 陆光涛, 李瑞芳. 沙福芽孢杆菌GX-H6的分离鉴定及对水稻细菌性条斑病的防病效果[J]. 生物技术通报, 2023, 39(5): 243-253.

REN Pei-dong, PENG Jian-ling, LIU Sheng-hang, YAO Zi-ting, ZHU Gui-ning, LU Guang-tao, LI Rui-fang. Isolation and Identification of a Bacillus safensis Strain GX-H6 and Its Biocontrol Effect on Bacterial Leaf Streak of Rice[J]. Biotechnology Bulletin, 2023, 39(5): 243-253.

图/表 10

图1 GX-H6的形态特征与拮抗效果 A: GX-H6对Xoc GX01的拮抗效果；B&C: 菌落形态和显微镜观察结果

Fig. 1 Morphological characteristics and antagonistic effect of GX-H6 A: Antibacterial effect of GX-H6 on Xoc GX01. B: Colonies morphology of strain GX-H6. C: Microscopic observation of strain GX-H6

图2 基于16S rDNA基因和gyrB基因序列的试验菌株与其他芽孢杆菌的系统发育树刻度尺代表0.1%的序列差异

Fig. 2 Phylogenetic tree of tested strains and other Bacillus based on 16S rDNA gene and gyrB gene sequence The scale indicates 0.1% sequence difference

图3 GX-H6对细菌的拮抗谱 A：GX-H6对不同Xoc分离株的拮抗（1：3-1分离株；2：4-1分离株；3：5-1分离株；4：6分离株；5：6-2分离株；6：6-3分离株；7：7-1分离株；8：8-1分离株；9：8-2分离株；10：2015分离株）；B：GX-H6对不同Xoo分离株和其他植物病原菌的拮抗（1: Xoo PXO99A；2: Xoo K74；3: Xoo 1-1分离株；4: Xoo 1-2分离株；5: Xoo 1-3分离株；6: Xcc 8004；7: Xac 9 分离株；8: Xac 12 分离株；9: 燕麦噬酸菌燕麦亚种 Pas-1；10: 丁香假单胞菌番茄致病变种DC3000；11：栖木槿假单胞菌Y2分离株；12：栖木槿假单胞菌Y3分离株）。图中数据为平均直径±标准差（mm）, 不同字母表示存在显著性差异 P<0.05

Fig. 3 Antagonistic spectrum of GX-H6 against bacteria A: Antagonistic spectrum of GX-H6 against different Xoc strains（1: 3-1 isolate; 2: 4-1 isolate; 3: 5-1 isolate; 4: 6 isolate; 5: 6-2 isolate; 6: 6-3 isolate; 7: 7-1 isolate; 8: 8-1 isolate; 9: 8-2 isolate; 10: 2015 isolate）. B: Antagonistic spectrum of GX-H6 against different Xoo strains and other plant pathogens（1: Xoo PXO99A strain; 2: Xoo K74 strain; 3: Xoo 1-1 isolate; 4: Xoo 1-2 isolate; 5: Xoo 1-3 isolate; 6: Xcc 8004 strain; 7: Xac 9 isolate; 8: Xac 12 isolate; 9: A.avenae subsp. avenae Pas-1 strain; 10: P.syringae pv. tomato DC3000 strain; 11：P.hibiscicola Y2 isolate; 12：P.hibiscicola Y3 isolate）. The data is average diameter± standard deviation in the Figure, and different letters indicate significant differences at the P < 0.05

图4 GX-H6对不同真菌分离株的拮抗试验 A:平板对峙定性分析；B: 介质含菌法定量分析。图中数据为平均直径±标准差（cm）,不同字母分别表示差异显著性P < 0.05。1：新暗色柱节孢；2：果生刺盘孢；3：灰葡萄孢；4：尖孢镰刀菌古巴专化型4号小种；5：胶孢炭疽菌；6：稻瘟病菌

Fig. 4 Antagonistic spectrum of GX-H6 against six plant pathogenic fungi strains A: Qualitative analysis via plate confrontation method. B: Quantitative analysis via bacterial plate method. The data in the Figure are average diameter± standard deviation, and different letters indicate significant differences at P < 0.05. 1: N. novaehollandiae. 2: C. plurivorum. 3: B. cinere. 4: F. oxysporum f. sp. cubense, Foc. 5: C. gloeosporioides. 6: M. oryzae

图5 温室植株检测GX-H6对Xoc的生物防治效果 *表示差异显著（P<0.05）

Fig. 5 Biocontrol effect of GX-H6 on Xoc in greenhouse plants * indicates significant（P<0.05）

图6 温室植株检测GX-H6对 Xoo的生物防治效果 *表示差异显著（P<0.05），**表示差异极显著（P<0.01）

Fig. 6 Biocontrol effect of GX-H6 strain on Xoo in green-house plants * indicates significant（P<0.05）, and ** indicates significant（P<0.01）

表1 GX-H6生防菌剂防治水稻细菌性条斑病试验结果

Table 1 Experimental results of GX-H6 biocontrol agent against bacterial leaf streak of rice

药剂处理 Treatment	用量 Dose/（L·hm^-2）	药前病情指数 Pre-treament disease index	第3次药后10 d病情指数 Post-treatment disease index	防效 Relative control effect/%	差异显著性 Significance
GX-H6生防菌剂	7.5	0.6	6.8	65.0	bC
	11.25	0.6	6.1	68.6	bBC
	22.5	0.7	5.2	72.8	aAB
20%噻菌铜悬浮剂	1.875	0.7	4.6	76.0	aA
清水对照（CK）		0.7	19.2	─	─

表2 B. safensis GX-H6的基因组特征

Table 2 Genomic characteristics of B. safensis GX-H6

特征 Characteristics	值 Value
Toal length	3 763 758 bp
GC content	41.51%
ORF number	3 754
ORF total length	3 301 671 bp
Longest ORF length	10 707 bp
Shortest ORF length	90 bp
Average ORF length	879 bp
ORF/Genome（coding percentage）	87.72%

图7 B. safensis GX-H6的基因组特征 A：B. safensis GX-H6的基因组圈图；B：B. safensis GX-H6的GO富集；C：B. safensis GX-H6的KEGG富集

Fig. 7 Genomic characteristics of B. safensis GX-H6 A：The genomic circle map of B. safensis GX-H6. B：GOSlim illustration of B. safensis GX-H6；C：KEGG chart of B. safensis GX-H6

表3 B. safensis GX-H6基因组中次生代谢产物预测

Table 3 Prediction of secondary metabolites in B. safensis GX-H6 genome

区域Region	起始Start	终止End	最似产物Most similar	类型Type	相似度Similarity
1	693 587	724 627	N/A	Betalactone	N/A
2	1 190 336	1 231 436	N/A	T3PKS	N/A
3	1 271 177	1 291 720	N/A	Terpene	N/A
4	1 357 616	1 386 025	Fengycin	Betalactone	53%
5	1 575 524	1 597 704	Plantazolicin	LAP,RRE-containing	91%
6	2 112 286	2 140 932	Carotenoid	Terpene, siderophore	50%
7	2 287 110	2 308 015	N/A	RRE-containing	N/A
8	2 781 557	2 863 632	Lichenysin	NRPS	92%
9	3 002 623	3 012 922	N/A	RiPP-like	N/A
10	3 231 420	3 278 568	Bacillibactin	NRPS	53%
11	3 545 166	3 586 587	Bacilysin	Other	85%

参考文献 23

[1]	Niño-Liu DO, Ronald PC, Bogdanove AJ. Xanthomonas oryzae pathovars: model pathogens of a model crop[J]. Mol Plant Pathol, 2006, 7(5): 303-324. doi: 10.1111/j.1364-3703.2006.00344.x pmid: 20507449
[2]	Ou SH, Commonwealth Mycological Institute(Great Britain). Rice diseases[M]. 2nd ed. Surrey: UK: Commonwealth Mycological Institute; 1985.
[3]	Suh JP, Jeung JU, Noh TH, et al. Development of breeding lines with three pyramided resistance genes that confer broad-spectrum bacterial blight resistance and their molecular analysis in rice[J]. Rice(NY), 2013, 6(1): 5.
[4]	Chen XF, Liu PC, Mei L, et al. Xa7, a new executor R gene that confers durable and broad-spectrum resistance to bacterial blight disease in rice[J]. Plant Commun, 2021, 2(3): 100143. doi: 10.1016/j.xplc.2021.100143 URL
[5]	Wang CL, Fan YL, Zheng CK, et al. High-resolution genetic mapping of rice bacterial blight resistance gene Xa23[J]. Mol Genet Genomics, 2014, 289(5): 745-753. doi: 10.1007/s00438-014-0848-y pmid: 24715026
[6]	岑贞陆, 黄思良, 李容柏, 等. 稻种材料抗细菌性条斑病性鉴定[J]. 安徽农业科学, 2007, 35(22): 6850-6851, 6853.
	Cen ZL, Huang SL, Li RB, et al. Identification of resistance of rice materials to bacterial leaf streak[J]. J Anhui Agric Sci, 2007, 35(22): 6850-6851, 6853.
[7]	Xu XM, Xu ZY, Li ZY, et al. Increasing resistance to bacterial leaf streak in rice by editing the promoter of susceptibility gene OsSULRT3;6[J]. Plant Biotechnol J, 2021, 19(6): 1101-1103. doi: 10.1111/pbi.13602 pmid: 33942463
[8]	Zhao XY, Zhao XM, Wei YM, et al. Isolation and identification of a novel antifungal protein from a rhizobacterium Bacillus subtilis strain F3[J]. J Phytopathol, 2013, 161(1): 43-48. doi: 10.1111/jph.12015 URL
[9]	Wang XL, Wang L, Wang J, et al. Bacillus cereus AR156-induced resistance to Colletotrichum acutatum is associated with priming of defense responses in loquat fruit[J]. PLoS One, 2014, 9(11): e112494. doi: 10.1371/journal.pone.0112494 URL
[10]	王刚, 沈永红, 王俊芳, 程希. 蜡样芽孢杆菌B3-7菌株对小麦全蚀病菌的抑制作用[J]. 河南大学学报: 自然科学版, 2005, 35(1): 62-64.
	Wang G, Shen YH, Wang JF, et al. Inhibitory action of bacilis Cereus B3-7 strain on Gaeumannomyces graminis var tritici[J]. J Henan Univ Nat Sci, 2005, 35(1): 62-64.
[11]	李生樟, 陈颖, 杨瑞环, 等. 一株拮抗黄单胞菌的贝莱斯芽孢杆菌的分离和鉴定[J]. 微生物学报, 2019, 59(10): 1969-1983.
	Li SZ, Chen Y, Yang RH, et al. Isolation and identification of a Bacillus velezensis strain against plant pathogenic Xanthomonas spp[J]. Acta Microbiol Sin, 2019, 59(10): 1969-1983.
[12]	李生樟, 刘昭, 杨瑞环, 等. 一株拮抗水稻条斑病菌的蜡样芽孢杆菌的分离和鉴定[J]. 江苏农业科学, 2020, 48(7): 127-136.
	Li SZ, Liu Z, Yang RH, et al. Isolation and identification of a Bacillus cereus strain against plant pathogenic Xanthomonas oryzae[J]. Jiangsu Agric Sci, 2020, 48(7): 127-136.
[13]	黄梦桑, 杨瑞环, 阎依超, 等. 一株具有防治水稻条斑病潜力的高地芽孢杆菌181-7[J]. 植物病理学报, 2021, 51(6): 962-974.
	Huang MS, Yang RH, Yan YC, et al. Bacillus altitudinis 181-7, a potential biocontrol agent against bacterial leaf streak of rice[J]. Acta Phytopathol Sin, 2021, 51(6): 962-974.
[14]	Yang RH, Li SZ, Li YL, et al. Bactericidal effect of Pseudomonas oryziphila sp. nov., a novel Pseudomonas species against Xanthomonas oryzae reduces disease severity of bacterial leaf streak of rice[J]. Front Microbiol, 2021, 12: 759536. doi: 10.3389/fmicb.2021.759536 URL
[15]	Yamamoto S, Harayama S. PCR amplification and direct sequencing of gyrB genes with universal primers and their application to the detection and taxonomic analysis of Pseudomonas putida strains[J]. Appl Environ Microbiol, 1995, 61(3): 1104-1109. doi: 10.1128/aem.61.3.1104-1109.1995 URL
[16]	Singh R, Paul D, Jain RK. Biofilms: implications in bioremediation[J]. Trends Microbiol, 2006, 14(9): 389-397. pmid: 16857359
[17]	袁智鹏. 解淀粉酶芽孢杆菌产抗真菌物质的提取、分离、初步鉴定及生防活性的探究[D]. 烟台: 烟台大学, 2022.
	Yuan ZP. Study on extraction, isolation, preliminary identification and biocontrol activity of antifungal substances produced by Bacillus amylase[D]. Yantai: Yantai University, 2022.
[18]	Kajimura Y, Sugiyama M, Kaneda M. Bacillopeptins, new cyclic lipopeptide antibiotics from Bacillus subtilis FR-2[J]. J Antibiot(Tokyo), 1995, 48(10): 1095-1103.
[19]	Besson F, Michel G. Mycosubtilins B and C: minor antibiotics from mycosubtilin-producer Bacillus subtilis[J]. Microbios, 1990, 62(251): 93-99. pmid: 2115097
[20]	Malanicheva IA, Kozlov DG, Efimenko TA, et al. New antibiotics produced by Bacillus subtilis strains[J]. Mikrobiologiia, 2014, 83(4): 445-450. pmid: 25844455
[21]	Chen H, Wang L, Su CX, et al. Isolation and characterization of lipopeptide antibiotics produced by Bacillus subtilis[J]. Lett Appl Microbiol, 2008, 47(3): 180-186. doi: 10.1111/j.1472-765X.2008.02412.x pmid: 19552782
[22]	杨杰. 枯草杆菌fmb60的NRPS和Ⅰ型PKS基因簇代谢产物发掘及其生物活性研究[D]. 南京: 南京农业大学, 2017.
	Yang J. Genomics-driven discovery of NRPS and type I PKS metabolites from Bacillus subtilis fmb60 and their bioactivity[D]. Nanjing: Nanjing Agricultural University, 2017.
[23]	申琪瑶. 利用Red/ET重组技术克隆和异源表达芽孢杆菌天然产物基因簇及海洋拮抗细菌分离[D]. 济南: 山东大学, 2016.
	Shen QY. Cloning and heterologous expression of natural product gene clusters from Bacillus using red/ET recombineering and the separation of antagonistic bacterium from marine environment[D]. Jinan: Shandong University, 2016.

沙福芽孢杆菌GX-H6的分离鉴定及对水稻细菌性条斑病的防病效果

Isolation and Identification of a Bacillus safensis Strain GX-H6 and Its Biocontrol Effect on Bacterial Leaf Streak of Rice

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 10

参考文献 23

相关文章 15

编辑推荐

Metrics

本文评价

[1]	褚睿, 李昭轩, 张学青, 杨东亚, 曹行行, 张雪艳. 黄瓜枯萎病拮抗芽孢杆菌的筛选、鉴定及其生防潜力[J]. 生物技术通报, 2023, 39(8): 262-271.
[2]	马俊秀, 吴皓琼, 姜威, 闫更轩, 胡基华, 张淑梅. 蔬菜软腐病菌广谱拮抗细菌菌株筛选鉴定及防效研究[J]. 生物技术通报, 2023, 39(7): 228-240.
[3]	章乐乐, 王冠, 柳凤, 胡汉桥, 任磊. 芒果炭疽病拮抗菌分离、鉴定及生防机制研究[J]. 生物技术通报, 2023, 39(4): 277-287.
[4]	易希, 廖红东, 郑井元. 植物内生真菌防治根结线虫研究进展[J]. 生物技术通报, 2023, 39(3): 43-51.
[5]	王伟宸, 赵进, 黄薇颐, 郭芯竹, 李婉颖, 张卓. 芽胞杆菌代谢产物防治三种常见植物病原真菌的研究进展[J]. 生物技术通报, 2023, 39(3): 59-68.
[6]	杨东亚, 祁瑞雪, 李昭轩, 林薇, 马慧, 张雪艳. 黄瓜茄病镰刀菌拮抗芽孢杆菌的筛选、鉴定及促生效果[J]. 生物技术通报, 2023, 39(2): 211-220.
[7]	罗宁, 焦阳, 茆振川, 李惠霞, 谢丙炎. 木霉菌对根结线虫和孢囊线虫防治机理研究进展[J]. 生物技术通报, 2023, 39(2): 35-50.
[8]	马赛买, 李同源, 马燕军, 韩富军, 彭海, 孔维宝. 几丁质酶在农作物病虫害生物防治中的研究进展[J]. 生物技术通报, 2023, 39(10): 29-40.
[9]	张玲, 张荣意, 刘盛科, 谭志琼. 瓜类细菌性果斑病菌拮抗细菌的筛选及其抑菌作用[J]. 生物技术通报, 2023, 39(1): 253-263.
[10]	孙卓, 王妍, 韩忠明, 王云贺, 赵淑杰, 杨利民. 防风根际真菌的分离鉴定及其生防潜力评价[J]. 生物技术通报, 2023, 39(1): 264-273.
[11]	祖雪, 周瑚, 朱华珺, 任佐华, 刘二明. 枯草芽孢杆菌K-268的分离鉴定及对水稻稻瘟病的防病效果[J]. 生物技术通报, 2022, 38(6): 136-146.
[12]	胡珊, 梁卫驱, 黄皓, 徐匆, 罗华建, 胡楚维, 黄晓彦, 陈仕丽. 中药渣堆肥中解磷细菌的筛选、鉴定及其拮抗作用[J]. 生物技术通报, 2022, 38(3): 92-102.
[13]	严聪文, 苏代发, 代庆忠, 张振荣, 田云霞, 董琼娥, 周文星, 陈杉艳, 童江云, 崔晓龙. 草莓病害的生物防治研究进展[J]. 生物技术通报, 2022, 38(12): 73-87.
[14]	施兆荣, 孙述俊, 张广荣, 梅大海, 刘彦超, 杨成德. 甜瓜黑斑病菌的拮抗细菌筛选、鉴定及发酵条件优化[J]. 生物技术通报, 2022, 38(1): 115-124.
[15]	舒洁, 张仁军, 梁应冲, 陈雅琼, 张娟, 郭建, 陈穗云. 植物源与微生物源生物制剂复配防治根结线虫病[J]. 生物技术通报, 2021, 37(7): 164-174.