猕猴桃溃疡病生防菌群的构建及其生防效应

doi:10.11707/j.1001-7488.LYKX20230653

摘要/Abstract

摘要：

目的: 从猕猴桃本体分离获得生防细菌构建生防菌群，明确浙江临安猕猴桃溃疡病病原菌的类群，探究生防菌群的生防效应，为猕猴桃溃疡病的防治提供新思路。方法: 从浙江临安猕猴桃种植区分离溃疡病菌并鉴定；基于健康、发病猕猴桃枝条内生菌的宏基因组测序比较和R语言分析，以该病菌为靶标分离生防细菌，构建生防菌群；通过离体共培养和活体接种试验，验证菌群的生防效应。结果: 1）浙江临安的猕猴桃溃疡病由丁香假单胞菌猕猴桃致病变种HY-05引起。枝条内生菌的宏基因组测序结果表明，与发病的猕猴桃植株相比，健康的猕猴桃枝条内部细菌群落之间更易形成积极的互作关系。2）基于R语言分析，从猕猴桃健康植株分离获得病原菌HY-05的拮抗菌株红树林动球菌ZC-3和蒙氏肠球菌ZC-5，以及ZC-3的益生菌嗜根寡养单胞菌ZC-6，构建成生防菌群。3）离体共培养试验结果表明，各供试菌株均能抑制菌株HY-05的生长，其中以ZC-3、ZC-5和ZC-6构建的生防菌群效果最佳，抑制率达100%；植株活体试验中，添加各生防菌组合的处理均可不同程度抑制猕猴桃溃疡病的发生，其中以ZC-3、ZC-5和ZC-6构建的生防菌群处理效果最佳，防治效果达56.00%，与发病对照相比差异极显著（P<0.01）。结论: 发生自浙江临安的猕猴桃溃疡病由丁香假单胞菌猕猴桃致病变种引起，从猕猴桃植株内分离获得的生防菌株构建的生防菌群经离体和活体实验证实具有实际生防效应。

关键词: 猕猴桃溃疡病, 宏基因组测序, R语言, 生防菌, 菌群构建, 生防效应

Abstract:

Objective: In this study, biocontrol bacteria were isolated from kiwifruit and used for construction of biocontrol bacterial community. The pathogen of kiwifruit bacterial canker was identified in Lin'an, Zhejiang Province. This study aims to explore the biocontrol effect of the bacterial community on the pathogen of kiwifruit bacterial canker, so as to provide new insights for the prevention and control of kiwifruit bacterial canker. Method: The pathogenic bacteria causing canker disease of kiwifruit were isolated from kiwifruit planting areas in Lin'an, Zhejiang Province and identified. Based on the comparison and analysis of the metagenomes of endophytic bacteria in healthy and diseased kiwifruit branches using R language, antagonistic bacteria against the pathogenic bacteria were isolated, and a biocontrol bacterial community was constructed. The biocontrol effect of the bacterial community on the pathogenic bacteria was validated through in vitro co-cultivation and in vivo inoculation experiments. Result: 1) Kiwifruit bacterial canker in Lin'an, Zhejiang Province was caused by Pseudomonas syringae pv. actinidiae HY-05. The metagenomic sequencing results of endophytic bacteria in kiwifruit branches showed that positive interactions were more likely to form among the bacterial communities in healthy kiwifruit branches compared to diseased ones. 2) Based on R language analysis, antagonistic strains, Kineococcus mangrovi ZC-3 and Enterococcus mundtii ZC-5, against the pathogenic strain HY-05, as well as a probiotic strain, Stenotrophomonas rhizophila ZC-6, were isolated from healthy kiwifruit plants, and a biocontrol bacterial community was constructed. 3) In the in vitro co-cultivation experiment, all tested strains inhibited the growth of HY-05, with the biocontrol bacterial community constructed by ZC-3, ZC-5, and ZC-6 showing the best inhibitory effect, with a suppression rate of 100%. In the in vivo plant experiment, treatments with different biocontrol bacterial combinations were able to inhibit the occurrence of kiwifruit bacterial canker to varying degrees, with the biocontrol bacterial community constructed by ZC-3, ZC-5, and ZC-6 showing the best preventive effect, with a control efficiency of 56.00%, which was significantly different from the diseased control (P<0.01). Conclusion: Kiwifruit bacterial canker in Lin'an, Zhejiang Province is caused by P. syringae pv. actinidiae. The biocontrol bacterial community constructed from beneficial bacteria isolated from kiwifruit plants is proven to have practical biocontrol effects through in vitro and in vivo experiments. This study enriches the research system of biological control of bacterial canker in kiwifruit from a theoretical perspective, and the biocontrol bacterial community obtained can be used for precise prevention and control of kiwifruit bacterial canker, which is of practical significance for the development of the kiwifruit industry.

Key words: kiwifruit bacterial canker, metagenomic sequencing, R language, biocontrol bacteria, bacterial community construction, biocontrol effect

中图分类号:

S718.43

尹志诚,顾佳颖,李南羿,张昕. 猕猴桃溃疡病生防菌群的构建及其生防效应[J]. 林业科学, 2024, 60(12): 101-110.

Zhicheng Yin,Jiaying Gu,Nanyi Li,Xin Zhang. Construction and Control Effect of Biocontrol Flora of Kiwifruit Bacterial Canker[J]. Scientia Silvae Sinicae, 2024, 60(12): 101-110.

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级别 Degree	代表值 Representative value	分级标准 Classified standard
Ⅰ	0	接种部位未出现病斑或伤口完全愈合 No lesions appear at the vaccination site or the wound is completely healed
Ⅱ	1	接种部位出现较浅褐色病斑，病斑面积<0.1 cm² Light brown lesions appear at the inoculation site, lesion area<0.1 cm²
Ⅲ	2	接种部位出现褐色病斑， 0.1 cm²≤ 病斑面积<0.2 cm² Brown lesions appear at the inoculation site, 0.1 cm²≤lesion area<0.2 cm²
Ⅳ	3	接种部位出现深褐色病斑， 0.2 cm²≤ 病斑面积<0.3 cm² Dark brown lesions appear at the inoculation site, 0.2 cm²≤lesion area<0.3 cm²
Ⅴ	4	接种部位出现锈红色病斑，病部凹陷， 0.3 cm²≤病斑面积 Rust-red lesions appear at the inoculation site, lesion area is sunken, 0.3 cm²≤lesion area

菌株编号 Strain code	菌落形态 Colony morphology	革兰氏染色反应 Gram stain reaction	氧化酶反应 Oxidase reaction	过氧化氢酶反应 Catalase reaction
ZC-3	菌落扁平，边缘不规则，中间白，外圈呈半透明雪花状，透明圈大，菌落整体呈淡粉色。 Colonies flattened, irregular edge, white in the middle, translucent snowflake-like outer ring, large transparent circle, overall pale pink color of the colony.	+	?	+
ZC-5	菌落扁平，边缘规则，圆形，中间白色，边缘呈半透明状。 Colonies flattened, with regular, rounded edges, white in the center, with translucent edges.	+	?	?
ZC-6	菌落凸起，边缘规则，圆形，质地黏稠，中间黄色，边缘呈乳黄色半透明状，透明圈较大。 Colonies are convex, with regular, rounded edges, thick texture, yellow in the middle, milky yellow translucent edges, and large transparent circles.	?	?	+

处理方法 Treatment	病情指数 Disease index (%)	防治效果 Prevention and control effect (%)	显著水平 Significance level
仅接种病原菌 Only pathogenic bacteria	78.13	—	A
接种病原菌与菌株ZC-5 Inoculation pathogen and strain ZC-5	62.50	20.01	A
接种病原菌与生防菌群 Inoculation pathogenic bacteria and biocontrol flora	34.38	56.00	B

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