蜘蛛抱蛋(Aspidistra elatior)炭疽病病原菌的分离与鉴定

doi:10.3969/j.issn.1004-1524.2021.08.13

浙江农业学报 ›› 2021, Vol. 33 ›› Issue (8): 1461-1467.DOI: 10.3969/j.issn.1004-1524.2021.08.13

蜘蛛抱蛋(Aspidistra elatior)炭疽病病原菌的分离与鉴定

黄钰婷(), 孙洁, 张慧娟, 朱晏, 蒋明^*()

台州学院生命科学学院,浙江台州 318000

收稿日期:2020-10-18 出版日期:2021-08-25 发布日期:2021-08-27
作者简介:*蒋明,E-mail: jiangming1973@139.com
黄钰婷(1999—),女,浙江黄岩人,本科生,从事植物分子生物学研究。E-mail: 2278550422@qq.com
通讯作者: 蒋明
基金资助:
台州市大学生科技创新项目(2019年度)

Isolation and identification of pathogen causing anthracnose on Aspidistra elatior

HUANG Yuting(), SUN Jie, ZHANG Huijuan, ZHU Yan, JIANG Ming^*()

College of Life Sciences, Taizhou University, Taizhou 318000, China

Received:2020-10-18 Online:2021-08-25 Published:2021-08-27
Contact: JIANG Ming

摘要/Abstract

摘要：

为明确蜘蛛抱蛋(Aspidistra elatior)炭疽病病原菌的种类,本研究对采集的炭疽病样本进行分离纯化培养,得到1株蜘蛛抱蛋炭疽病病原真菌YYL,采用针刺回接实验测定其致病性,并在形态学鉴定基础上,克隆其ITS、ACT、GAPDH和CAL片段进行多基因联合鉴定。结果表明,该病原菌具有较强致病性,其菌落、菌丝和分生孢子形态符合百合炭疽菌(Colletotrichum lilii)的特征,且与百合炭疽菌相似度最高,其ITS和ACT序列与百合炭疽菌完全一致。综合形态学特征和分子生物学鉴定结果,确定引起蜘蛛抱蛋炭疽病的病原菌为百合炭疽菌。

关键词: 蜘蛛抱蛋, 炭疽病, 病原菌, 百合炭疽菌

Abstract:

To identify the pathogenic fungus causing anthracnose on Aspidistra elatior, tissue isolation method was applied on the samples collected and a fungus strain YYL was obtained. Acupuncture method was used to confirm its pathogenicity. Based on the morphological identification results, ITS, ACT, GAPDH, and CAL fragments were cloned for multigene analysis. The results showed that YYL had high pathogenicity, and the characteristics of colony, hyphae, and conidium were well matched to Colletotrichum lilii. The gene fragments from YYL shared the highest similarity with C. lilii, and the ITS and ACT sequences of both YYL and C. lilii were identical. By combining the morphological features and molecular biological identification results, C. lilii was identified as the pathogen causing anthracnose on Aspidistra elatior.

Key words: Aspidistra elatior, anthracnose, pathogen, Colletotrichum lilii

中图分类号:

S432.1

黄钰婷, 孙洁, 张慧娟, 朱晏, 蒋明. 蜘蛛抱蛋(Aspidistra elatior)炭疽病病原菌的分离与鉴定[J]. 浙江农业学报, 2021, 33(8): 1461-1467.

HUANG Yuting, SUN Jie, ZHANG Huijuan, ZHU Yan, JIANG Ming. Isolation and identification of pathogen causing anthracnose on Aspidistra elatior[J]. Acta Agriculturae Zhejiangensis, 2021, 33(8): 1461-1467.

图/表 7

表1 PCR引物和最佳退火温度

Table 1 PCR Primers and their optimum annealing temperatures

基因名称 Gene name	引物名称 Primer name	引物序列(5’→3’) Primer sequence (5’→3’)	退火温度 Annealing temperature/℃
ITS	ITS1	TCCGTAGGTGAACCTGCGG	56.3
	ITS4	TCCTCCGCTTATTGATATGC
ACT	512F	ATGTGCAAGGCCGGTTTCGC	62.1
	783R	TACGAGTCCTTCTGGCCCAT
GAPDH	GDF1	GCCGTCAACGACCCCTTCATTGA	56.3
	GDR1	GGGTGGAGTCGTACTTGAGCATGT
CAL	CL1	GARTWCAAGGAGGCCTTCTC	56.3
	CL2	TTTTTGCATCATGAGTTGGAC

图1 蜘蛛抱蛋炭疽病病叶特征 A,患病植株;B,病斑特写;C,菌丝通过气孔侵入叶片内部,2 500×。

Fig.1 Anthracnose symptoms on leaves of Aspidistra elatior A, Infected plants; B, Leaf lesion; C, Penetration by fungal hyphae through a stomatal pore, 2 500×.

图2 病原菌YYL的形态特征 A,菌落形态;B,菌丝形态;C,分生孢子形态;D,扫描电子显微镜下菌丝与孢子形态。

Fig.2 Morphological characteristics of pathogenic fungus YYL A, Colonial morphology; B, Hypha morphology; C, Conidia morphology; D, Morphology of hypha and spores under a scanning electron microscope.

图3 致病性检测 A,对照;B,接种病原菌的叶片。

Fig.3 Pathogenicity detection A, Control; B, Leaf inoculated with pathogenic fungus.

图4 不同基因片段PCR扩增产物凝胶电泳 M, DL 2000 DNA标准分子量;1~2,核糖体内转录间隔区;3~4,肌动蛋白基因;5~6,3-磷酸甘油醛脱氢酶基因;7~8,钙调蛋白基因。

Fig.4 Agarose gel electrophoresis of PCR products about different gene fragments M, DL 2000 DNA marker; 1-2, ITS; 3-4, ACT; 5-6, GAPDH; 7-8, CAL。

图5 ACT序列及其同源序列的多重比对 MH292787, Colletotrichum liriopes; MH292784, C. tofieldiae; KC843542, C. bletillum; MN752248, C. incanum; GU227908, C. lilii; MN433299, C. spaethianum; GQ856785, C. dematium; KC843538, C. guizhouensis; JQ005837, C. cereale.

Fig.5 Multiple alignment of ACT sequence and its homologous sequences

图6 基于基因片段及其同源序列构建的系统发育树 A,核糖体内转录间隔区;B,肌动蛋白基因;C,3-磷酸甘油醛脱氢酶基因;D,钙调蛋白基因。MH292787,Colletotrichum liriopes;MH292784,C. tofieldiae;KC843542,C. bletillum;MN752248,C. incanum;GU227908,百合炭疽菌;MN433299,白蜡树刺盘孢;GQ856785,黑线刺盘孢;KC843538,贵州刺盘孢;JQ005837,C. cereale。

Fig.6 Phylogenetic tree based on gene fragments and their homologous sequences A, ITS; B, ACT; C, GAPDH; D, CAL. MH292787, Colletotrichum liriopes; MH292784, C. tofieldiae; KC843542, C. bletillum; MN752248, C. incanum; GU227908, C. lilii; MN433299, C. spaethianum; GQ856785, C. dematium; KC843538, C. guizhouensis; JQ005837, C. cereale.

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蜘蛛抱蛋(Aspidistra elatior)炭疽病病原菌的分离与鉴定

Isolation and identification of pathogen causing anthracnose on Aspidistra elatior

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