基于根表真菌群落与病原菌鉴定探究‘宁杞5号’枸杞根腐病的发生机制

doi:10.13560/j.cnki.biotech.bull.1985.2024-0187

摘要/Abstract

摘要：

【目的】比较两种植区易感病品种‘宁杞5号’枸杞健康株和患病株的根表真菌群落组成和分离到的腐根真菌，明确‘宁杞5号’根腐病的病原菌，探究两种植区‘宁杞5号’根腐病的发生原因。【方法】采用高通量测序技术研究枸杞根表真菌群落的组成特征，采用组织分离法从两种植区枸杞腐根中分离腐根真菌，基于形态学和ITS、EF-1α基因序列对腐根真菌进行分类学鉴定，依据科赫法则进行致病性研究。【结果】两种植区‘宁杞5号’枸杞健康株和患病株之间的根表真菌群落组成均表现出明显差异，主要体现在患病后镰刀菌属（Fusarium）相对丰度升高，被孢霉属（Mortierella）相对丰度降低，伴随着Fusicolla和Pseudogymnoascus等未知类群丰度增加，而两种植区健康株根表真菌群落结构趋于一致。自QXBZ腐根分离到36株真菌，分别鉴定为尖孢镰刀菌（Fusarium oxysporum）、腐皮镰刀菌（F. solani）、红贝俄式孔菌（Earliella scabrosa）、立枯丝核菌（Rhizoctonia solani）和Penicillium pimiteouiense，自QTBZ腐根分离29株真菌，分别鉴定为尖孢镰刀菌、腐皮镰刀菌、红贝俄式孔菌、粉红黏帚菌（Clonostachys rosea）、P. pimiteouiense、新知镰刀菌（F. andiyazi）、桃色顶孢霉（Acremonium persicinum），其中腐皮镰刀菌的分离频率在两种植区均为最高，尖孢镰刀菌次之。回接实验确定腐皮镰刀菌、尖孢镰刀菌、新知镰刀菌和立枯丝核菌是‘宁杞5号’枸杞根腐病的病原菌，其中新知镰刀菌为新发现的枸杞根腐病病原菌。【结论】‘宁杞5号’根腐病发生主要与根表真菌群落结构改变以及病原菌种类密切相关，与种植区关系不大，F. solani、F. oxysporum、F. andiyazi和R. solani是‘宁杞5号’根腐病的病原菌。

关键词: 枸杞, 根表真菌群落, 根腐病, 病原菌, 分离鉴定

Abstract:

【Objective】 This paper is to compare the composition of rhizoplane fungal communities in susceptible strain ‘Ningqi-5’ between the healthy and diseased plants in the two planting areas（QXBZ and QTBZ）, as well as the isolated rot root fungi. It is also to clarify the pathogens causing root rot disease in ‘Ningqi-5’, and to explore the reasons for the root rot disease in ‘Ningqi-5’.【Method】High-throughput technology was applied to investigate the compositional characteristics of the rhizoplane fungal community. Tissue isolation method was to isolate the rot-root fungi the decayed roots of Lycium barbarum. They were taxonomically identified based on the morphological characteristics and the sequences of ITS and EF-1α gene fragments. The pathogenicity bythe rotten-root fungi was verified based on Koch's law.【Result】 The root rhizoplane fungal community composition differed significantly between the healthy and diseased plants of ‘Ningqi-5’ in both planting areas, showing the increase in relative abundance of Fusarium and decrease in relative abundance of Mortierella in the diseased plants, accompanying with the increase of unknown taxa like Fusicolla and Pseudogymnoascus. While the fungal community structure on the rhizoplane of the healthy plants in both planting areas tended to be consistent. The 36 strains isolated from the rotten rot of QXBZ were classified as Fusarium oxysporum, F. solani, Earliella scabrosa, Rhizoctonia solani, and Penicillium pimiteouiense. The 29 strains isolated from the rotten rot of QTBZ were classified as F. oxysporum, F. solani, E. scabrosa, Clonostachys rosea, P. pimiteouiense, F. andiyazi and Acremonium persicinum. Isolation frequency of F. solani was the highest in both planting areas, followed by F. oxysporum. Based on the artificial infection test, F. solani, F. oxysporum, R. solani, and F. andiyazi were identified as the pathogens of ‘Ningqi-5’ root rot disease, among which F. andiyazi was a newly discovered pathogen of L. barbarum root rot.【Conclusion】In this study, it is clarified that the occurrence of ‘Ningqi-5’ root rot disease is mainly closely related to the changes in the structure of the root rhizoplane fungal community and pathogens. F. solani, F. oxysporum, F. andiyazi, and R. Solani are the pathogens of ‘Ningqi-5’ root rot disease.

Key words: Lycium barbarum, rhizoplane fungal community, root rot disease, pathogen, isolation and identification

张亚亚, 李盼盼, 高惠惠, 贾晨波, 徐春燕. 基于根表真菌群落与病原菌鉴定探究‘宁杞5号’枸杞根腐病的发生机制[J]. 生物技术通报, 2024, 40(9): 238-248.

ZHANG Ya-ya, LI Pan-pan, GAO Hui-hui, JIA Chen-bo, XU Chun-yan. Exploring on the Pathogenesis of Root Rot of Lycium barbarum cv. ‘Ningqi-5' Based on the Rhizoplane Fungal Community and Pathogens Identification[J]. Biotechnology Bulletin, 2024, 40(9): 238-248.

图/表 5

图1 ‘宁杞5号’枸杞健康株和患病株的根表真菌群落结构 A：门水平；B：属水平；C：属水平的PCoA分析；QXJK：杞鑫健康株；QXBZ：杞鑫患病株；QTJK：杞泰健康株；QTBZ：杞泰患病株

Fig. 1 Fungal community structure on the rhizoplane of the healthy and diseased plants of ‘Ningqi-5’ A: Phyla level. B: Genus level. C：PCoA on genus level. QXJK and QXBZ denote the healthy and the diseased plants of Qixin plantation, respectively. QTJK and QTBZ denote the healthy and the diseased plants of Qitai plantation, respectively

图2 代表性腐根真菌的菌落形态与菌丝、孢子形态

Fig. 2 Morphologies of colony, hyphae and spore of representative strains

图3 分离自QXBZ和QTBZ腐根的65株腐根真菌的系统发育树 A：基于QXBZ腐根真菌的ITS序列；B：基于QXBZ镰刀菌的EF-1α序列；C：基于QTBZ腐根真菌的ITS序列；D：基于QTBZ镰刀菌的EF-1α序列

Fig. 3 Phylogenetic trees of 65 strains of fungi isolated from the rotten root of QXBZ and QTBZ A: Based on ITS sequences of fungi isolated from the rotten root of QXBZ. B: Based on EF-1α sequences of Fusarium isolated from the rotten root of QXBZ. C: Based on ITS sequences of fungi isolated from the rotten root of QTBZ. D: Based on EF-1α sequences of Fusarium isolated from the rotten root of QTBZ

表1 患病株（QXBZ和QTBZ）腐根中分离的真菌数量与相应的分离频率

Table 1 Number and isolation frequency of fungi isolated from the rotten root of diseased plants（QXBZ and QTBZ）

样品 Sample	分类 Classification	腐皮镰刀菌 F. solani	尖孢镰刀菌 F. oxysporum	粉红黏帚菌 C. rosea	立枯丝核菌 R. solani	红贝俄式孔菌 E. scabrosa	桃色顶孢霉 A. persicinum	青霉菌 P. pimiteouiense	新知镰刀菌 F. andiyazi	总计 Total
QXBZ	数量 Number/Strains	17	11	—	1	6	—	1	—	36
	分离频率 Frequency/%	47.2	30.6	—	2.8	16.7	—	2.8	—	100
QTBZ	数量 Number/Strains	15	7	1	—	3	1	1	1	29
	分离频率 Frequency/%	51.7	24.1	3.4	—	10.3	3.4	3.4	3.4	100

图4 接种不同菌株前后枸杞的植株表型与根部表型

Fig. 4 Phenotypes of L. barbarum before and after inoculation with different strains

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