甘肃部分地区苹果褐腐病病原分离鉴定及拮抗细菌筛选

doi:10.13560/j.cnki.biotech.bull.1985.2023-0234

生物技术通报 ›› 2023, Vol. 39 ›› Issue (10): 209-218.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0234

甘肃部分地区苹果褐腐病病原分离鉴定及拮抗细菌筛选

蒋晶晶¹^,³(), 周昭旭¹^,³, 杜蕙¹^,³, 吕昭龙², 王春明¹^,³, 郭建国¹^,³, 张新瑞¹^,³, 李继平¹^,²^,³()

1.甘肃省农业科学院植物保护研究所，兰州 730070
2.甘肃农业大学植物保护学院，兰州 730070
3.农业农村部天水作物有害生物科学观测试验站，天水 741299

收稿日期:2023-03-16 出版日期:2023-10-26 发布日期:2023-11-28
通讯作者: 李继平，男，研究员，研究方向：经济作物病害及微生物资源；E-mail: lijiping@gsagr.ac.cn
作者简介:蒋晶晶，女，助理研究员，研究方向：经济作物病害病理学及生物防治；E-mail: jingjingziyu@163.com
基金资助:
甘肃省农业科学院科技支撑计划(2021GAAS07);甘肃省农业科学院科技支撑计划(2021GAAS17);甘肃省农业科学院科技支撑计划(2023GAAS18);国家重点研发计划(2018YFD0201404-4);国家绿肥产业技术体系旱地绿肥病虫草害综合防控(CARS-22-G-20);甘肃省林业和草原科技创新项目(GYCX[2020]03-1)

Isolation and Identification of Apple Brown Rot Pathogen in Parts of Gansu and Screening of Antagonistic Bacteria

JIANG Jing-jing¹^,³(), ZHOU Zhao-xu¹^,³, DU Hui¹^,³, LYU Zhao-long², WANG Chun-ming¹^,³, GUO Jian-guo¹^,³, ZHANG Xin-rui¹^,³, LI Ji-ping¹^,²^,³()

1. Institute of Plant Protection, Gansu Academy of Agricultural Sciences, Lanzhou 730070
2. College of Plant Protection, Gansu Agricultural University, Lanzhou 730070
3. Scientific Observing and Experimental Station of Crop Pests in Tianshui, Ministry of Agriculture and Rural Affairs, Tianshui 741299

Received:2023-03-16 Published:2023-10-26 Online:2023-11-28

摘要/Abstract

摘要：

明确甘肃省静宁县及榆中县苹果褐腐病的病原种类，并筛选优质病原拮抗菌，为苹果采后病害的生物防治提供资源。采用常规组织分离法对苹果褐腐病病果进行病原菌分离纯化、柯赫氏法则验证、分子生物学方法结合形态学鉴定；采用平板对峙法筛选拮抗细菌，利用形态学、生理生化和分子生物学方法对拮抗菌进行鉴定，用离体果实测定拮抗菌防病效果。结果表明，引起甘肃静宁县及榆中县苹果褐腐病的病原菌为云南链核盘菌（Monilia yunnanensis），筛选获得的最优拮抗细菌X2-2为解淀粉芽孢杆菌（Bacillus amyloliquefaciens），对M. yunnanensis的菌丝抑制率达（81.29±0.57）%，在离体果实上拮抗菌的防治效果达98.8%，能够显著抑制苹果褐腐病病斑在果实上的扩展速度。云南链核盘菌为甘肃部分地区苹果褐腐病主要病原菌，该研究筛选出的拮抗细菌X2-2抑菌效果显著，具有作为优质生防菌株的潜力。

关键词: 苹果褐腐病, 云南链核盘菌, 解淀粉芽孢杆菌

Abstract:

This work aims to clarify the pathogenic species of apple brown rot in Jingning county and Yuzhong county of Gansu province and to screen high-quality pathogen antagonistic bacteria for providing resources for biological control of apple postharvest diseases. The pathogens were isolated and purified by conventional tissue isolation method, verified by Koch's postulates, and identified by molecular biology method combined with morphology. The antagonistic bacteria were screened by plate confrontation, identified by morphological, physiological, biochemical and molecular biological methods, and the disease control effects of the antagonistic bacteria were determined by in vitro fruits. The results showed that the pathogens causing apple brown rot in Jingning county and Yuzhong county of Gansu province were Monilia yunnanensis. The optimal antagonistic bacterium X2-2 was Bacillus amyloliquefaciens, which inhibited（81.29±0.57）% of the mycelium of M. yunnanensis and 98.8% of the control effect of the antagonistic bacteria on the isolated fruits. It significantly inhibited the expansion rate of M. yunnanensis lesion spots of apple fruits. M. yunnanensis was the main pathogen of apple brown rot in some areas of Gansu province. The endogenous antagonistic bacterium X2-2 screened in this study had significant antibacterial effect and had the potential of being a high-quality biocontrol strain.

Key words: apple brown rot, Monilia yunnanensis, Bacillus amyloliquefaciens

蒋晶晶, 周昭旭, 杜蕙, 吕昭龙, 王春明, 郭建国, 张新瑞, 李继平. 甘肃部分地区苹果褐腐病病原分离鉴定及拮抗细菌筛选[J]. 生物技术通报, 2023, 39(10): 209-218.

JIANG Jing-jing, ZHOU Zhao-xu, DU Hui, LYU Zhao-long, WANG Chun-ming, GUO Jian-guo, ZHANG Xin-rui, LI Ji-ping. Isolation and Identification of Apple Brown Rot Pathogen in Parts of Gansu and Screening of Antagonistic Bacteria[J]. Biotechnology Bulletin, 2023, 39(10): 209-218.

图/表 10

图1 苹果褐腐病果实自然发病症状及病原菌接种症状 A-B：病害症状（A：‘秦冠’，B：‘红富士’）；C，E：菌饼接种5 d（C：‘秦冠’，E：‘红富士’）；D，F：孢子悬浮液接种5 d（D：‘秦冠’，F：‘红富士’）

Fig. 1 Disease characteristics of brown rot of apple and symptoms of pathogenic inoculation A-B: Disease symptom（A: ‘Qinguan apple’. B: ‘Red Fuji’ apple）. C, E: Fungus cake inoculation for 5 d（C: ‘Qinguan’ apple. E: ‘Red Fuji’ apple）. D, F: Spore suspension inoculation for 5 d（D: ‘Qinguan’ apple. F: ‘Red Fuji’ apple）

图2 苹果褐腐病菌的菌落形态及分生孢子形态 A：菌落正面；B：菌落背面；C：分生孢子梗（10×40）；D：分生孢子（10×40）；标尺=20 μm

Fig. 2 Colony and conidial morphology of apple brown rot bacteria A: The front of the colony. B: The back of the colony. C: Conidiophores（10×40）. D: The conidium（10×40）. Scale bar=20 μm

图3 基于rDNA-ITS序列采用邻接法构建的系统发育树

Fig. 3 Phylogenetic tree constructed using the neighbour-joining based on rDNA-ITS sequences

表1 拮抗菌对苹果褐腐病菌的抑制作用

Table 1 Inhibition effect of antagonistic bacterium on M. yunnanensis

菌株编号 Strain No.	抑菌率 Inhibition rate/%	抑菌带 Inhibition bands/mm
X2-2	81.29±0.57a	7.42±0.17a
Zyx-3	80.08±0.66b	5.42±0.51a
X1-5	74.64±0.97c	4.25±0.38a
X1-6	71.29±0.62d	4.11±0.42a
P-sy-3	70.88±0.52d	3.20±0.55a

图4 拮抗菌X2-2对苹果褐腐病菌的拮抗效果

Fig. 4 Effect of antagonistic bacteria X2-2 against M. yun-nanensis

图5 接种时间对病斑面积和防治效果的影响 A：先接菌饼，24 h后接X2-2菌悬液；B：菌饼+菌悬液；C：先接X2-2菌悬液，24 h后接菌饼。图中小写字母表示在P<0.05水平差异显著

Fig. 5 Effect of inoculation time on lesion area and control effect A: Inoculated with the M. yunnanensis first, and then inoculated with the X2-2 bacterial suspension after 24 h. B: M. yunnanensis + bacterial suspension. C: First inoculated with X2-2 bacterial suspension, then inoculated with M. yunnanensis after 24 h. Lowercase letters indicate significant difference at P<0.05 level

图6 X2-2菌悬液对苹果褐腐病病斑扩展的抑制效果 A：先接菌饼，24 h后接X2-2菌悬液；B：菌饼+菌悬液；C：先接X2-2菌悬液，24 h后接菌饼

Fig. 6 Inhibition effect of X2-2 bacterial suspension on the lesion expansion of M. yunnanensis A: Inoculated with the M. yunnanensis first, and then inoculated with the X2-2 bacterial suspension after 24 h. B: M. yunnanensis + bacterial suspension. C: First inoculated with X2-2 bacterial suspension, then inoculated with M. yunnanensis after 24 h

图7 拮抗菌X2-2的单菌落（A）和革兰氏染色（B）

Fig. 7 Colony（A）and Gram stain（B）of antagonistic bacterium X2-2

表2 拮抗菌X2-2的生理生化鉴定结果

Table 2 Physiological and biochemical identification results of antagonistic bacterium X2-2

指标 Index	结果 Result	指标 Index	结果 Result
革兰氏染色 Gram staining	+	V.P试验 V.P test	+
淀粉水解 Amylolysis	+	甲基红试验 Methyl red test	+
明胶液化 Gelatin liquefaction	+	柠檬酸盐 Citrate utilization	+
酪蛋白水解 Caseinhydrolyzation	-	硫化氢试验 Hydrogen sulfide test	-
7% NaCl	+	接触酶 Catalase	+

图8 基于16S rRNA（A）和gyrA（B）基因序列构建的拮抗菌X2-2的系统发育树

Fig. 8 Phylogenetic tree of antagonistic bacterium X2-2 based on 16S rRNA (A) and gyrA (B) sequences

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甘肃部分地区苹果褐腐病病原分离鉴定及拮抗细菌筛选

Isolation and Identification of Apple Brown Rot Pathogen in Parts of Gansu and Screening of Antagonistic Bacteria

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