山茶花腐病病原菌的分离鉴定及生物学特性

普梅英; 武自强; 张诗文; 李艳杰; 牛锦华; 付芳丽; 耿芳; 陈龙清; 王超

doi:10.19303/j.issn.1008-0384.2022.010.011

山茶花腐病病原菌的分离鉴定及生物学特性

doi: 10.19303/j.issn.1008-0384.2022.010.011

西南林业大学/国家林业和草原局西南风景园林工程技术研究中心/云南省功能性花卉资源及产业化技术工程研究中心/云南省森林灾害预警与控制实验室，云南　昆明　650224

基金项目: 国家重点研发计划项目（2019YFD100100005）；云南省教育厅科学研究基金项目（2021Y259、2021J0172）；西南山地森林保育与利用教育部重点实验室开放基金项目（KLESWFU-202005）；国家基础科学公共科学数据中心项目（ NBSDC-DB-03）

详细信息

作者简介:
普梅英（1997−），女，硕士研究生，研究方向：植物保护（E-mail：670868861@qq.com）

通讯作者:
王超（1980−），男，博士，研究方向：植物资源及利用（E-mail：47188127@qq.com）

中图分类号: S 432
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出版历程
- 收稿日期: 2022-06-23
- 修回日期: 2022-07-18
- 网络出版日期: 2022-10-21
- 刊出日期: 2022-10-30

Pathogen Identification and Biological Characterization of Camellia Petal Blight Disease

Southwest Research Center for Engineering Technology of Landscape Architecture, State Forestry and Grassland Administration/Yunnan Engineering Research Center for Functional Flower Resources and Industrialization/College of Landscape Architecture and Horticulture Sciences/Key Laboratory of Forest Disaster Warning and Control in Universities of Yunnan Province/Southwest Forestry University, Kunming, Yunnan　650224, China

摘要

摘要: 目的明确山茶花腐病的致病菌及其生物学特性，提升山茶花期品质和观赏价值。方法采用组织分离法对云南省昆明市昆明植物园山茶的花腐病进行病原菌分离，通过致病性测定、形态学和分子生物学鉴定确定病原菌，并进行生物学特性的研究。结果从病组织中分离得到1株菌株，将其回接于健康花瓣上，接种后划破的花瓣病症较为明显，花朵失色变黄褐，严重时能使花瓣干腐或湿腐，并且长菌丝体，与田间的症状一致，符合柯赫氏法则的致病性测定。PDA培养基上菌丝偏黄，培养后期长黑色的孢子，子囊孢子柠檬状，经合并多基因（ITS、LSU、EF1）系统发育分析，其与假螺卷毛壳菌（Chaetomium pseudocochliodes）聚集在同一分支。对假螺卷毛壳菌（C. pseudocochliodes）的生物学特性研究结果显示，该病原菌的最适培养温度为25 ℃，对光照条件没有特殊要求，最适培养基为LB和OMA培养基，以牛肉膏作为氮源和以果糖作为碳源的利用率最高，致死温度为55 ℃/10 min。结论假螺卷毛壳菌（C. pseudocochliodes）是山茶花腐病的病原菌，该病原菌的适宜温度为25 ℃，不受光照影响，培养基为LB和OMA培养基，以牛肉膏作为氮源和以果糖作为碳源的利用率最高，致死温度为55 ℃/10 min。
- 山茶 /
- 花腐病 /
- 假螺卷毛壳菌 /
- 生物学特性
Abstract: Objective The pathogen and its biological characteristics of camellia petal blight disease were investigated for improving the quality and protecting the ornamental value of the floral plant. Method A pathogen was isolated from of diseased Camellia japonica at the Botanical Garden in Kunming City by tissue separation method and confirmed by pathogenicity assay as well as morphological and molecular biological identifications. Biological characteristics of the isolate were studied. Result A suspected microbe was isolated from the diseased tissue and inoculated onto healthy camellia flower petals. With the appearance of yellowing and browning petals with dry or wet rots and fungal mycelial growth, the similar symptoms shown on the infected plants in the field, the isolate was studied further for a confirmed identification. On a PDA medium, the fungal colonies displayed yellow mycelia and produced black spores and lemon-like ascospores at late stage. The phylogenetic analysis of multiple genes of ITS, LSU, and EF1 clustered the isolate with Chaetomium pseudocochliodes clade. Subsequently, the optimal conditions for the growth of the isolated fungus were found to be at 25 ℃ on LB and OMA culture medium,without special requirement of light,beef paste as a nitrogen source and fructose as a carbon source have the highest utilization rates, and the lethality at 55 ℃ in 10 min. Conclusion C. pseudocochliodes was identified as the pathogen that caused floral petal blight disease on C. japonica. The pathogen grew optimally at 25 ℃ on LB and OMA culture medium ,without a specific light requirement, beef paste as a nitrogen source and fructose as a carbon source have the highest utilization rates, and it was killed by exposure to 55 ℃ for 10 min.
- Camellia japonica /
- petal blight disease /
- Chaetomium pseudocochliodes /
- biological characteristics

HTML全文

图 1 山茶花腐病田间和接种后症状及病原菌在PDA培养基上的形态学特征

a. 田间感病病花症状；b. 田间健康花；c、d. 室内离体接种后花腐症状；e. 菌落形态；f. 子囊孢子。

Figure 1. Symptoms of petal blight disease on field and inoculated C. japonica and morphology of pathogen on PDA

a: symptoms on field flower; b: healthy flower; c and d: symptoms on inoculated camellia flower;e: colony morphology; f: ascospores.

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图 2 山茶花腐病病原菌的基因电泳图

a、b、c分别为ITS、LSU、EF1区间电泳图，M为DL2 000的DNA Marker，1为SX-3，2为SX Ⅰ，3为YH Ⅰ。

Figure 2. Gene electrophoresis of camellia petal blight disease pathogen

a, b, and c: electrophoretic map of primer ITS, LSU and EF1 respectively; M: DNA marker of DL2 000; 1: SX-3; 2: SX Ⅰ; 3: YH Ⅰ.

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图 3 基于ITS、LSU和EF1的多基因系统发育进化树

Figure 3. Multigene phylogenetic tree based on ITS, LSU and EF1 gene sequences

下载: 全尺寸图片幻灯片

表 1 引物序列表

Table 1. Primer sequences

基因 Gene	引物 Primer	序列 Sequence	文献来源 Reference
ITS	ITS1	5′-TCCGTAGGTGAACCTGCGG-3′	Henson等^[17]
ITS	ITS4	5′-TCCTCCGCTTATTGATATG-3′	Henson等^[17]
LSU	NL1	5′-GCATATCAATAAGCGGAGGAAAAG -3′	刘彬等^[18]
LSU	NL4	5′-GGTCCGTGTTTCAAGACGG-3′	刘彬等^[18]
EF1	EF1-983F	5′-GCYCCYGGHCAYCGTGAYTTYAT-3′	谭秀梅等^[19]
EF1	EF1-2218R	5′-ATGACACCRACRGCRACRGTYTG-3′	谭秀梅等^[19]

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表 2 温度对菌株SX-3菌丝生长的影响

Table 2. Effect of temperature on mycelial growth of SX-3

温度 Temperature/℃	菌落直径 Colony diameter/cm	温度 Temperature/℃	菌落直径 Colony diameter/cm
10	3.967±0.0577 d	30	7.833±0.2887 b
15	5.500±0.5000 c	35	6.267±0.0577 c
20	7.733±1.1676 b	40	1.033±0.0577 e
25	8.833±0.2887 a
不同小写字母表示各处理菌落直径差异显著（P＜0.05），下同。
The different lowercase alphabet shows that the factor to the diameter of the colony has significantly difference (P＜0.05), The same applies below.

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表 3 光照对菌株SX-3菌丝生长的影响

Table 3. Effect of illumination on mycelial growth of SX-3

光照情况 Lighting condition	菌落直径 Colony diameter/ cm	光照情况 Lighting condition	菌落直径 Colony diameter/ cm
24 h光照 24 h lighting	8.800±0.1732 a	24 h黑暗 24 h darkness	8.600±0.6928 a
12 h交替 12 h alternation	8.500±0.0000 a	正常光照 Normal lighting	8.667±0.0577 a

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表 4 培养基对菌株SX-3菌丝生长的影响

Table 4. Effect of culture medium on mycelial growth of SX-3

培养基 Culture medium	菌落直径 colony diameter/ cm	培养基 Culture medium	菌落直径 Colony diameter/ cm
OMA	8.500±0.0000 b	PDA	8.167±0.2887 c
CMA	8.500±0.0000 b	LB	9.000±0.0000 a

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表 5 氮源对菌株SX-3菌丝生长的影响

Table 5. Effect of nitrogen sources on mycelial growth of SX-3

氮源 Nitrogen source	菌落直径 Colony diameter/cm	氮源 Nitrogen source	菌落直径 Colony diameter/cm
硝酸钠 Sodium nitrate	8.500±0.0000 a	硝酸钾 Potassium nitrate	8.333±0.2887 a
蛋白胨 Peptone	8.500±0.0000 a	甘氨酸 Glycine	8.667±0.2887 a
牛肉膏 Beef extract	8.500±0.0000 a	缺氮 Nitrogen deficiency	7.200±0.1000 c
酵母膏 Yeast cream	7.967±0.2517 b

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表 6 碳源对菌株SX-3菌丝生长的影响

Table 6. Effect of carbon sources on mycelial growth of SX-3

碳源 Carbon source	菌落直径 Colony diameter/cm	碳源 Carbon source	菌落直径 Colony diameter/cm
蔗糖 Sucrose	7.967±0.4509 b	阿拉伯树胶糖 Gum Arabic sugar	3.567±0.3055 c
葡萄糖 Glucose	8.433±0.0577 a	果糖 Fructose	8.500±0.0000 a
木糖 Xylose	8.467±0.0577 a	缺碳 Carbon deficiency	7.500±0.0000 b
麦芽糖 Maltose	8.333±0.1155 ab

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表 7 菌株SX-3致死温度测定

Table 7. Lethal temperature of SX-3

致死温度 Lethal temperature/ ℃	菌落直径 Colony diameter/cm	致死温度 Lethal temperature/ ℃	菌落直径 Colony diameter/cm
35	7.633±0.3215 b	55	0.000±0.0000 d
40	8.367±0.1528 a	60	0.000±0.0000 d
45	7.133±0.9074 b	65	0.000±0.0000 d
50	2.467±0.1528 c

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