香梨黑斑病病原菌分离及拮抗菌筛选鉴定

doi:10.6048/j.issn.1001-4330.2022.10.023

新疆农业科学 ›› 2022, Vol. 59 ›› Issue (10): 2538-2545.DOI: 10.6048/j.issn.1001-4330.2022.10.023

• 植物保护·微生物·农业生态环境·畜牧兽医 • 上一篇下一篇

香梨黑斑病病原菌分离及拮抗菌筛选鉴定

鲁晏宏¹^,²(), 郝金辉¹^,², 詹发强², 王宁², 侯新强², 杨蓉², 包慧芳²(), 龙宣杞²()

1.新疆大学生命科学与技术学院,乌鲁木齐 830046
2..新疆农业科学院微生物应用研究所,乌鲁木齐 830091

收稿日期:2021-12-23 出版日期:2022-10-20 发布日期:2022-12-21
通信作者: 包慧芳,龙宣杞
作者简介:鲁晏宏(1996-),女,黑龙江人,硕士研究生,研究方向为微生物资源与利用,(E-mail)luyanhong@stu.xju.edu.cn
基金资助:
新疆维吾尔自治区天山优秀青年"两种生防菌对链格孢菌抑制机理及协同作用研究"(2020Q027);新疆农业科学院科技创新重点培育专项“库尔勒香梨果园营建关键技术优化集成”(xjkcpy-2020006);新疆人社厅自治区高层次人才引进工程

Isolation of Pathogenic Bacteria and Screening and Identification of Antagonistic Bacteria of Black Spot of Fragrant pear

LU Yanhong¹^,²(), HAO Jinhui¹^,², ZHAN Faqiang², WANG Ning², HOU Xinqiang², YANG Rong², BAO Huifang²(), LONG Xuanqi²()

1. College of Life Science and Technology, Xinjiang University, Urumqi 830046, China
2. Research Institute of Applied Microbiology, Xinjiang Academy of Agricultural Sciences, Urumqi 830091, China

Received:2021-12-23 Online:2022-10-20 Published:2022-12-21
Correspondence author: BAO Huifang, LONG Xuanqi
Supported by:
Outstanding Youth Fund of Xinjiang"Study on Inhibition Mechanism and Synergic Effect of Two Biocontrol Bacteria on Alternatia"(2020Q027);Key Cultivation of Science and Technology Innovation of Xinjiang Academy of Agricultural Sciences " Key Technology Optimization Integration of Korla Pear Orchard Construction"(xjkcpy-2020006);High-level Talent Introduction Project of Xinjiang

摘要/Abstract

摘要：

【目的】从香梨树体中筛选出对香梨采后黑斑病病原菌具有拮抗作用的内生菌,为香梨采后生物保鲜奠定基础。【方法】采用组织分离法,对香梨采后黑斑病病原菌及梨树内生菌进行分离,采用ITS基因与16S rRNA基因引物分别对分离出的病原菌和拮抗菌进行测序,采用平板对峙法测定内生菌的拮抗效果,并对拮抗效果优良的拮抗菌采用离体果实进行防效测定。【结果】分离得到香梨采后黑斑病病害病原菌1株,鉴定为Alternatia alternata XL₂。从梨树叶片及枝条中分离出内生菌16株,其中8株对Alternatia alternata XL₂有抑制作用,8株拮抗菌均为芽孢杆菌属,分别为Bacillus velezensis 5株、Bacillus paralicheniformis 、Bacillus siamensis 和Bacillus tequilensis 各1株,牛津杯琼脂扩散实验中Bacillus velezensis NY₂、NY₇抑菌效果最佳,其次为NY₁₅,抑菌圈直径均可达20 mm以上。离体果实抑菌实验中短期效果最佳的为NY₂,使用第4 d时抑菌率可达80%以上,中长期效果较佳的为NY₁₁,使用第7 d天时仍可达20%以上。【结论】筛选获得2株优良的香梨采后黑斑病拮抗菌。

关键词: 黑斑病; 内生菌; 拮抗菌; 应用效果

Abstract:

【Objective】 The purpose of this study is to screen the endophytic bacteria with antibacterial effect from the Korla pear tree with a view to using the endophytic biocontrol agent to keep the pear fresh after harvesting.【Method】 The pathogen of postharvest black spot of fragrant pear and endophytic bacteria were isolated by tissue separation method and then the isolated strains were sequenced by ITS gene and 16S rRNA gene.Also, the plate confrontation method was used to screen the antagonistic bacteria against Alternatia alternata XL₂ from the screened endophytic bacteria, and bacteriostasis experiments of the antagonistic bacteria were conducted in vitro fruits.【Result】 The pathogen of postharvest diseases of Korla pear Alternatia alternata XL₂ was obtained in this experiment, and 16 strains of endophytic bacteria were isolated from the leaves and branches of pear trees, among which 8 strains had inhibitory effect on Alternatia alternata XL₂.They were 1 Bacillus paralicheniformis, 5 Bacillus velezensis, 1 Bacillus siamensis and 1 Bacillus tequilensis.Among them, Bacillus velezensis NY₂, NY₇ and NY₅ had the best antibacterial effect in the Oxford cup experiment, and the diameter of antibacterial zone was more than 20 mm.In the bacteriostatic experiment of Korla pear, NY₂ and NY₁₅ had the best bacteriostatic effect, and the bacteriostatic rate was more than 30%.In the bacteriostatic test in vitro fruits, the bacteriostatic rate of NY₂ reached more than 80% in the fourth day after inoculation, and the bacteriostatic effect of NY₁₁ was more stable than others.【Conclusion】 In this experiment, 2 strains of endophytic bacteria with antibacterial effect are screened.

Key words: black spot; endophytic; antagonistic bacteria; apply effect

中图分类号:

S436.412

鲁晏宏, 郝金辉, 詹发强, 王宁, 侯新强, 杨蓉, 包慧芳, 龙宣杞. 香梨黑斑病病原菌分离及拮抗菌筛选鉴定[J]. 新疆农业科学, 2022, 59(10): 2538-2545.

LU Yanhong, HAO Jinhui, ZHAN Faqiang, WANG Ning, HOU Xinqiang, YANG Rong, BAO Huifang, LONG Xuanqi. Isolation of Pathogenic Bacteria and Screening and Identification of Antagonistic Bacteria of Black Spot of Fragrant pear[J]. Xinjiang Agricultural Sciences, 2022, 59(10): 2538-2545.

图/表 9

图1 病原菌分离及回接注:(a)XL1病原菌菌株平板内形态为(b)XL2病原菌菌株平板内形态(c)XL2回接发病图(d)XL2感染病原菌香梨纵切图(e)XL3病原菌菌株平板内形态为(f)XL3回接发病图 (g)XL3感染病原菌香梨纵切图

Fig.1 Isolation and reinoculated symptoms of pathogenic bacteria Note:(a) the morphology of XL1 pathogen strain in the plate (b) the morphology of XL2 pathogen strain in the plate (c) the incidence diagram of XL2 back-graft (d) the slitting diagram of XL2 infected pathogen fragrant pear(e) The morphology of XL3 pathogen strains in the plate (F) the incidence diagram of XL3 tie-back (G) the slitting diagram of XL3 infection pathogen fragrant pear

图2 XL2基于rDNA-ITS基因序列的PCR扩增

Fig.2 PCR amplification of strain XL2 based on ITS gene sequences

图3 基于16S rRNA基因序列采用Neighbor-Joining法构建病原菌系统发育树注:分支上的数字为Bootstrap值,表示构建系统进化树时计算1000次时形成该节点的百分比,只显示大于50%的的值;括号内数值为GenBank登录号;标尺0.1代表10%的16S r RNA基因序列的进化差异。

Fig.3 Phylogenetic tree based on Neighbor-Joining of isolated strain XL2 Note:The bootstrap values(%) presented at the branches were calculated from 1000 replications,only showing bootstrap values higher than 50%.Numbers in parentheses are GenBank accession numbers;The scale bar 0.1 represents 100 nucleotide substitutions per 1000 nucleotides.

表1 拮抗菌发酵液对链格孢抑菌效果

Table1 Oxford cup AGAR diffusion inhibition results

菌株编号 Strain No.	抑菌圈直径 Diameter of inhibition zone(mm)
NY₁	10.33±1.53^d
NY₂	23.43±0.51^a
NY₅	14.67±0.58^c
NY₇	23.33±0.58^a
NY₉	13.33±1.15^c
NY₁₀	9.33±0.58^d
NY₁₁	14.33±1.41^c
NY₁₅	21.73±0.25^b

图4 拮抗菌对XL2抑菌作用

Fig.4 Antagonistic activity of antagonist against XL2

图5 拮抗菌基于16s rRNA基因序列的PCR扩增

Fig.5 PCR amplification of antagonistic bacteria based on 16S rRNA gene sequences

图6 基于16S rRNA基因序列采用Neighbor-joining法构建的拮抗菌系统发育树注:分支上的数字为Bootstrap值,表示构建系统进化树时计算1000次时形成该节点的百分比,只显示大于50%的的值;括号内数值为GenBank登录号;标尺0.1代表10%的16S r RNA基因序列的进化差异。

Fig.6 Phylogenetic tree based on Neighbor-joining of isolated antagonistic bacteria Note:The bootstrap values(%) presented at the branches were calculated from 1000 replications,only showing bootstrap values higher than 50%.Numbers in parentheses are GenBank accession numbers;The scale bar 0.1 represents 100 nucleotide substitutions per 1000 nucleotides.

表2 发酵液在库尔勒香梨对病原菌抑制率

Table 2 Inhibitory rate of pathogenic bacteria in Korla fragrant pear

菌株编号 Strain No.	防效Control effect(%)
NY₁	11.71±0.55^c
NY₂	31.30±1.19^a
NY₅	15.24±2.37^c
NY₇	12.95±2.67^c
NY₉	4.30±0.21^d
NY₁₀	13.52±1.15^c
NY₁₁	21.72±2.98^b
NY₁₅	24.49±2.48^b

图7 香梨抑菌测定复测注:标有*表示与其余组差异显著(P<0.05)

Fig.7 The bacteriostatic rate of fragrant pear was determined Note:Marked with * indicates significant difference with other groups (P<0.05)

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香梨黑斑病病原菌分离及拮抗菌筛选鉴定

Isolation of Pathogenic Bacteria and Screening and Identification of Antagonistic Bacteria of Black Spot of Fragrant pear

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