柿子炭疽病病原哈瑞炭疽菌(Colletotrichum horii)特异性PCR检测

doi:10.11924/j.issn.1000-6850.casb2026-0050

中国农学通报 ›› 2026, Vol. 42 ›› Issue (11): 140-150.doi: 10.11924/j.issn.1000-6850.casb2026-0050

柿子炭疽病病原哈瑞炭疽菌(Colletotrichum horii)特异性PCR检测

陈海铃¹^,²(), 李其利², 周浩¹, 宋锦康², 黄穗萍², 陈小林², 张禹², 唐利华²()

¹ 广西民族大学，南宁 530006
² 广西壮族自治区农业科学院植物保护研究所/农业农村部华南果蔬绿色防控重点实验室/广西作物病虫害生物学重点实验室，南宁 530007

收稿日期:2026-01-15 修回日期:2026-03-20 出版日期:2026-06-12 发布日期:2026-06-12
通讯作者:
唐利华，女，广西桂林人，副研究员，硕士，主要从事植物真菌病害及其防治研究工作。通信地址：530007 广西南宁市西乡塘区大学东路174号广西农业科学院，Tel：0771-3244445，E-mail：654123597@qq.com。
作者简介:
陈海铃，女，2001年出生，广西平南人，研究生，主要从事芒果、柿子炭疽病研究。通信地址：530007 广西南宁市西乡塘区大学东路174号广西农业科学院，Tel：0771-3244445，E-mail：2247922739@qq.com。
基金资助:
广西重点研发计划“芒果、柿子炭疽病监测预警与绿色防控技术研发示范”(桂科农AB241484041)

Specific PCR Detection of Causal Agent of Persimmon Anthracnose Colletotrichum horii

CHEN Hailing¹^,²(), LI Qili², ZHOU Hao¹, SONG Jinkang², HUANG Suiping², CHEN Xiaolin², ZHANG Yu², TANG Lihua²()

¹ Guangxi Minzu University, Nanning 530006
² Plant Protection Research Institute, Guangxi Academy of Agricultural Sciences/Key Laboratory of Green Prevention and Control on Fruits and Vegetables in South China, Ministry of Agriculture and Rural Affairs/Guangxi Key Laboratory of Biology for Crop Diseases and Insect Pests, Nanning 530007

Received:2026-01-15 Revised:2026-03-20 Published:2026-06-12 Online:2026-06-12

摘要/Abstract

摘要：

柿子炭疽病是柿树生产中最严重的真菌病害之一，哈瑞炭疽菌（Colletotrichum horii）是其优势致病菌，早期快速检测对病害防控至关重要。为开发一种能够快速、特异性检测哈瑞炭疽菌的PCR方法，并将其应用于柿子炭疽病的田间监测与快速诊断，本研究以多株炭疽菌、常见田间真菌和细菌为供试材料，比对TUB2(β-tubulin)、GAPDH(Glyceraldehyde-3-phosphate dehydrogenase)等基因序列，设计了4对引物，筛选出2对特异性引物(THF1/THR2、GHF1/GHR2)，并进行了PCR扩增、灵敏度测试及人工接种试验。结果显示：基于TUB2基因的引物THF1/THR2可扩增出211 bp的特异性条带，基于GAPDH基因的引物GHF1/GHR2可扩增出171 bp的特异性条带；两对引物的检测灵敏度均达100 pg/μL。在人工接种柿子叶片中，接种48 h即可检出目标条带，72 h信号显著增强。综上，本研究建立的双基因PCR检测方法特异性强、灵敏度高、结果可靠，可用于柿子炭疽病潜伏期、早期诊断及田间监测，为病害精准预警与绿色防控提供技术支撑。

关键词: 柿子, 炭疽病, 哈瑞炭疽菌, PCR检测, TUB2, GAPDH, 引物设计

Abstract:

Persimmon anthracnose is one of the most serious fungal diseases in persimmon production. Colletotrichum horii is the dominant pathogen. Early rapid detection is very important for disease prevention and control. To develop a rapid and specific PCR method for detecting Colletotrichum horii and apply it to field monitoring and rapid diagnosis of persimmon anthracnose, this study used multiple Colletotrichum species as test materials, compared the gene sequences of TUB2 (β-tubulin) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase), designed four primer pairs, and screened out two specific primer pairs (THF1/THR2, GHF1/GHR2) for PCR amplification, sensitivity testing, and artificial inoculation experiments. The results showed that the primer pair THF1/THR2 based on the TUB2 gene amplified a specific 211 bp band, and the primer pair GHF1/GHR2 based on the GAPDH gene amplified a specific 171 bp band; both primer pairs achieved a detection sensitivity of 100 pg/μL, and in artificially inoculated persimmon leaves, both primer pairs consistently detected the corresponding target bands. In conclusion, the PCR detection method established in this study is highly specific and sensitive, providing reliable technical support for field monitoring and rapid diagnosis of persimmon anthracnose.

Key words: persimmon, anthracnose, Colletotrichum horii, PCR detection, TUB2, GAPDH, primer design

中图分类号:

S436.63

陈海铃, 李其利, 周浩, 宋锦康, 黄穗萍, 陈小林, 张禹, 唐利华. 柿子炭疽病病原哈瑞炭疽菌(Colletotrichum horii)特异性PCR检测[J]. 中国农学通报, 2026, 42(11): 140-150.

CHEN Hailing, LI Qili, ZHOU Hao, SONG Jinkang, HUANG Suiping, CHEN Xiaolin, ZHANG Yu, TANG Lihua. Specific PCR Detection of Causal Agent of Persimmon Anthracnose Colletotrichum horii[J]. Chinese Agricultural Science Bulletin, 2026, 42(11): 140-150.

图/表 10

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No.	Strain no.	Species	Host	Accession no.
1	FJ11-1	Colletotrichum asianum	Mangifera indica	MH636523^a
2	GD10-1	Colletotrichum siamense	Mangifera indica	MH636415^a
3	HN47-2	Colletotrichum fructicola	Mangifera indica	MH636458^a
4	HN23-5	Colletotrichum cordylinicola	Mangifera indica	MH636464^a
5	HN32-1	Colletotrichum tropicale	Mangifera indica	MH636466^a
6	HN42-2	Colletotrichum gigasporum	Mangifera indica	MH636467^a
7	JH7-2	Colletotrichum plurivorum	Mangifera indica	MW507147^a
8	YN31-4	Colletotrichum cliviicola	Mangifera indica	MH636508^a
9	YN32-2	Colletotrichum endophytica	Mangifera indica	MH636509^a
10	YN33-1	Colletotrichum liaoningense	Mangifera indica	MH636504^a
11	YN51-1	Colletotrichum scovillei	Mangifera indica	MH636512^a
12	GZ15-1	Colletotrichum gloeosporioides	Mangifera indica	MH636440^a
13	GZ23-3	Colletotrichum musae	Mangifera indica	MH636444^a
14	YN34-1	Colletotrichum karstii	Mangifera indica	MH636506^a
15	PLG3-1	Colletotrichum horii	Persimmon	MT982860^a
16	YB53-2	Epicoccum sorghinum	Mangifera indica	/
17	EGY16A-1	Pseudofusicoccum violaceum	Violet	/
18	YB43-2	Alternaria brassicae	Mangifera indica	/
19	HF	Neoscytalidium dimidiatum	Hylocereus undulatus	N
20	TY6-1	Phyllosticta capitalensis	Mangifera indica	PV259393^a
21	TY9-2	Botryosphaeria	Vitis vinifera	/
22	YB33-1	Exserohilum	Mangifera indica	/
23	YMWK	Rhizoctonia solani	Zea mays	N
24	FY27-1	Phanerochaete sordida	/	/
25	HZ40-3	Neopestalotiopsis sonneratae	Prunus salicina	N
26	TEY15-1	Fusarium sulawesiense	Prunus salicina	OM432482^a
27	STJ	Penicillium digitatum	Citrusreticulata	N
28	G2-1	Pantoea anthophila	Mangifera indica	/
29	Y5-1	Pantoea vagans	Mangifera indica	OL413424.1^b
30	G10-1	Pantoea cypripedii	Mangifera indica	/
31	Y11-2	Pantoea dispersa	Mangifera indica	/
32	Y12-1	Xanthomonas	Mangifera indica	/
33	PLY8-1	Colletotrichum fructicola	Persimmon	MT982874^a
34	PLY4-2	Colletotrichum karstii	Persimmon	MT982871^a
35	YSY2-2	Colletotrichum cliviae	Persimmon	MT982887^a
36	PLY16-2	Colletotrichum siamense	Persimmon	MT982879^a
37	GCG7-2	Colletotrichum horii	Persimmon	MT982820^a
38	GCG-6-1	Colletotrichum horii	Persimmon	MT982818^a
39	PLG-5-1A	Colletotrichum horii	Persimmon	MT982862^a

No.	Strain no.	Species	Host	Accession no.
1	FJ11-1	Colletotrichum asianum	Mangifera indica	MH636523^a
2	GD10-1	Colletotrichum siamense	Mangifera indica	MH636415^a
3	HN47-2	Colletotrichum fructicola	Mangifera indica	MH636458^a
4	HN23-5	Colletotrichum cordylinicola	Mangifera indica	MH636464^a
5	HN32-1	Colletotrichum tropicale	Mangifera indica	MH636466^a
6	HN42-2	Colletotrichum gigasporum	Mangifera indica	MH636467^a
7	JH7-2	Colletotrichum plurivorum	Mangifera indica	MW507147^a
8	YN31-4	Colletotrichum cliviicola	Mangifera indica	MH636508^a
9	YN32-2	Colletotrichum endophytica	Mangifera indica	MH636509^a
10	YN33-1	Colletotrichum liaoningense	Mangifera indica	MH636504^a
11	YN51-1	Colletotrichum scovillei	Mangifera indica	MH636512^a
12	GZ15-1	Colletotrichum gloeosporioides	Mangifera indica	MH636440^a
13	GZ23-3	Colletotrichum musae	Mangifera indica	MH636444^a
14	YN34-1	Colletotrichum karstii	Mangifera indica	MH636506^a
15	PLG3-1	Colletotrichum horii	Persimmon	MT982860^a
16	YB53-2	Epicoccum sorghinum	Mangifera indica	/
17	EGY16A-1	Pseudofusicoccum violaceum	Violet	/
18	YB43-2	Alternaria brassicae	Mangifera indica	/
19	HF	Neoscytalidium dimidiatum	Hylocereus undulatus	N
20	TY6-1	Phyllosticta capitalensis	Mangifera indica	PV259393^a
21	TY9-2	Botryosphaeria	Vitis vinifera	/
22	YB33-1	Exserohilum	Mangifera indica	/
23	YMWK	Rhizoctonia solani	Zea mays	N
24	FY27-1	Phanerochaete sordida	/	/
25	HZ40-3	Neopestalotiopsis sonneratae	Prunus salicina	N
26	TEY15-1	Fusarium sulawesiense	Prunus salicina	OM432482^a
27	STJ	Penicillium digitatum	Citrusreticulata	N
28	G2-1	Pantoea anthophila	Mangifera indica	/
29	Y5-1	Pantoea vagans	Mangifera indica	OL413424.1^b
30	G10-1	Pantoea cypripedii	Mangifera indica	/
31	Y11-2	Pantoea dispersa	Mangifera indica	/
32	Y12-1	Xanthomonas	Mangifera indica	/
33	PLY8-1	Colletotrichum fructicola	Persimmon	MT982874^a
34	PLY4-2	Colletotrichum karstii	Persimmon	MT982871^a
35	YSY2-2	Colletotrichum cliviae	Persimmon	MT982887^a
36	PLY16-2	Colletotrichum siamense	Persimmon	MT982879^a
37	GCG7-2	Colletotrichum horii	Persimmon	MT982820^a
38	GCG-6-1	Colletotrichum horii	Persimmon	MT982818^a
39	PLG-5-1A	Colletotrichum horii	Persimmon	MT982862^a

物种名称	菌株名称	寄主	地理来源	GenBank登录号
物种名称	菌株名称	寄主	地理来源	ITS	TUB2	GAPDH
C. cliviae	GCY1-2A	Persimmon	China	MT982835	MW046811	MW032377
C. cliviae	GCY4-2	Persimmon	China	MT982837	MW046813	MW032379
C. cliviae	PLY14-2	Persimmon	China	MT982878	MW046854	MW032420.1
C. cliviae	YSY2-2	Persimmon	China	MT982887	MW046863	MW032429
C. fructicola	PLY8-1	Persimmon	China	MT982874	MW046850	MW032416
C. fructicola	YSY1-2	Persimmon	China	MT982886	MW046862	MW032428
C. fructicola	YSY3-3	Persimmon	China	MT982888	MW046864	MW032430
C. fructicola	YSY5-2	Persimmon	China	MT982889	MW046865	MW032431
C. fructicola	YSY8-3	Persimmon	China	MT982890	MW046866	MW032432
C. horii	PLG5-1A	Persimmon	Pingle, Guilin	MT982862	MW046838	MW032404
C. horii	PLG3-1	Persimmon	Pingle, Guilin	MT982860	MW046836	MW032402
C. horii	PLG3-2	Persimmon	China	MT982861	MW046837	MW032403
C. horii	PLG6-1	Persimmon	China	MT982864	MW046840	MW032406
C. karstii	LPY4-2B	Persimmon	China	MT982858	MW046834	MW032400
C. karstii	PLY7-2	Persimmon	China	MT982873	MW046849	MW032415
C. karstii	GCY21-3	Persimmon	China	MT982850	MW046826	MW032392
C. karstii	PLY2-2	Persimmon	China	MT982869	MW046845	MW032411
C. karstii	PLY4-2	Persimmon	China	MT982871	MW046847	MW032413
C. siamense	GCG17-1	Persimmon	China	MT982829	MW046805	MW032371
C. siamense	PLY16-2	Persimmon	China	MT982879	MW046855	MW032421
C.asianum	FJ11-1	Mangifera indica	China	MH636523	MH622733	MH681402
C.asianum	FJ18-3	Mangifera indica	China	MH636528	MH622738	MH681407
C.asianum	FJ36-6	Mangifera indica	China	MH636539	MH622749	MH681418
C.asianum	QZ-3	Mangifera indica	China	MF039834	MF039805	MF040765
C.asianum	YN55-1	Mangifera indica	China	MH636503	MH622713	MH681382
C.cliviicola	YN8-2	Mangifera indica	China	MH636487	MH622697	MH681366
C.cliviicola	YN31-4	Mangifera indica	China	MH636508	MH622718	MH681387
C.cordylinicola	HN23-5	Mangifera indica	China	MH636464	MH622674	MH681343
C.endophyticum	YN32-2	Mangifera indica	China	MH636509	MH622719	MH681388
C.endophyticum	HN37-2	Mangifera indica	China	MH636453	MH622663	MH681331
C.endophyticum	HN37-6	Mangifera indica	China	MH636453	MH622663	MH681332
C.endophyticum	YN32-6	Mangifera indica	China	MH681390	MH636511	MH622721
C.fructicola	GZ15-1	Mangifera indica	China	MH636440	MH622650	MH681319
C.fructicola	HN47-2	Mangifera indica	China	MH636458	MH622668	MH681337
C.fructicola	FJ32-6	Mangifera indica	China	MH636534	MH622744	MH681413
C.fructicola	FJ35-5	Mangifera indica	China	MH636538	MH622748	MH681417
C.fructicola	GZ1-2	Mangifera indica	China	MH636427	MH622637	MH681306
C.gigasporum	HN42-2	Mangifera indica	China	MH636467	MH622677	MH681346
C.gloeosporioides	GZ14-G-1	Mangifera indica	China	MH636439	MH622649	MH681318
C.liaoningense	YN33-1	Mangifera indica	China	MH636504	MH622714	MH681383
C.musae	GZ23-3	Mangifera indica	China	MH636444	MH622654	MH681323
C.plurivorum	JH7-2	Mangifera indica	China	JF898133	MW507372	MW507371
C.scovillei	HN44-3	Mangifera indica	China	MH636457	MH622667	MH681336
C.scovillei	YN51-1	Mangifera indica	China	MH636512	MH622722	MH681391
C.scovillei	HN44-2	Mangifera indica	China	MH636456	MH622666	MH681335
C.siamense	YN40-1-2	Mangifera indica	China	MH636499	MH622709	MH681378
C.siamense	GD8-4	Mangifera indica	China	MH636416	MH622626	MH681293
C.siamense	GD10-1	Mangifera indica	China	MH636415	MH622625	MH681294
C.siamense	YN56-1-1	Mangifera indica	China	MH636497	MH622707	MH681376
C.siamense	YN56-2-1	Mangifera indica	China	MH636498	MH622708	MH681377
C.tropicale	HN32-1	Mangifera indica	China	MH636466	MH622676	MH681345

物种名称	菌株名称	寄主	地理来源	GenBank登录号
物种名称	菌株名称	寄主	地理来源	ITS	TUB2	GAPDH
C. cliviae	GCY1-2A	Persimmon	China	MT982835	MW046811	MW032377
C. cliviae	GCY4-2	Persimmon	China	MT982837	MW046813	MW032379
C. cliviae	PLY14-2	Persimmon	China	MT982878	MW046854	MW032420.1
C. cliviae	YSY2-2	Persimmon	China	MT982887	MW046863	MW032429
C. fructicola	PLY8-1	Persimmon	China	MT982874	MW046850	MW032416
C. fructicola	YSY1-2	Persimmon	China	MT982886	MW046862	MW032428
C. fructicola	YSY3-3	Persimmon	China	MT982888	MW046864	MW032430
C. fructicola	YSY5-2	Persimmon	China	MT982889	MW046865	MW032431
C. fructicola	YSY8-3	Persimmon	China	MT982890	MW046866	MW032432
C. horii	PLG5-1A	Persimmon	Pingle, Guilin	MT982862	MW046838	MW032404
C. horii	PLG3-1	Persimmon	Pingle, Guilin	MT982860	MW046836	MW032402
C. horii	PLG3-2	Persimmon	China	MT982861	MW046837	MW032403
C. horii	PLG6-1	Persimmon	China	MT982864	MW046840	MW032406
C. karstii	LPY4-2B	Persimmon	China	MT982858	MW046834	MW032400
C. karstii	PLY7-2	Persimmon	China	MT982873	MW046849	MW032415
C. karstii	GCY21-3	Persimmon	China	MT982850	MW046826	MW032392
C. karstii	PLY2-2	Persimmon	China	MT982869	MW046845	MW032411
C. karstii	PLY4-2	Persimmon	China	MT982871	MW046847	MW032413
C. siamense	GCG17-1	Persimmon	China	MT982829	MW046805	MW032371
C. siamense	PLY16-2	Persimmon	China	MT982879	MW046855	MW032421
C.asianum	FJ11-1	Mangifera indica	China	MH636523	MH622733	MH681402
C.asianum	FJ18-3	Mangifera indica	China	MH636528	MH622738	MH681407
C.asianum	FJ36-6	Mangifera indica	China	MH636539	MH622749	MH681418
C.asianum	QZ-3	Mangifera indica	China	MF039834	MF039805	MF040765
C.asianum	YN55-1	Mangifera indica	China	MH636503	MH622713	MH681382
C.cliviicola	YN8-2	Mangifera indica	China	MH636487	MH622697	MH681366
C.cliviicola	YN31-4	Mangifera indica	China	MH636508	MH622718	MH681387
C.cordylinicola	HN23-5	Mangifera indica	China	MH636464	MH622674	MH681343
C.endophyticum	YN32-2	Mangifera indica	China	MH636509	MH622719	MH681388
C.endophyticum	HN37-2	Mangifera indica	China	MH636453	MH622663	MH681331
C.endophyticum	HN37-6	Mangifera indica	China	MH636453	MH622663	MH681332
C.endophyticum	YN32-6	Mangifera indica	China	MH681390	MH636511	MH622721
C.fructicola	GZ15-1	Mangifera indica	China	MH636440	MH622650	MH681319
C.fructicola	HN47-2	Mangifera indica	China	MH636458	MH622668	MH681337
C.fructicola	FJ32-6	Mangifera indica	China	MH636534	MH622744	MH681413
C.fructicola	FJ35-5	Mangifera indica	China	MH636538	MH622748	MH681417
C.fructicola	GZ1-2	Mangifera indica	China	MH636427	MH622637	MH681306
C.gigasporum	HN42-2	Mangifera indica	China	MH636467	MH622677	MH681346
C.gloeosporioides	GZ14-G-1	Mangifera indica	China	MH636439	MH622649	MH681318
C.liaoningense	YN33-1	Mangifera indica	China	MH636504	MH622714	MH681383
C.musae	GZ23-3	Mangifera indica	China	MH636444	MH622654	MH681323
C.plurivorum	JH7-2	Mangifera indica	China	JF898133	MW507372	MW507371
C.scovillei	HN44-3	Mangifera indica	China	MH636457	MH622667	MH681336
C.scovillei	YN51-1	Mangifera indica	China	MH636512	MH622722	MH681391
C.scovillei	HN44-2	Mangifera indica	China	MH636456	MH622666	MH681335
C.siamense	YN40-1-2	Mangifera indica	China	MH636499	MH622709	MH681378
C.siamense	GD8-4	Mangifera indica	China	MH636416	MH622626	MH681293
C.siamense	GD10-1	Mangifera indica	China	MH636415	MH622625	MH681294
C.siamense	YN56-1-1	Mangifera indica	China	MH636497	MH622707	MH681376
C.siamense	YN56-2-1	Mangifera indica	China	MH636498	MH622708	MH681377
C.tropicale	HN32-1	Mangifera indica	China	MH636466	MH622676	MH681345

设计区	引物		序列(5'-3')
TUB2	上游引物	THF1	GGGGCTAACCAGAGTTCTTTCA
	下游引物	THR2	TTAGCGGTCAACCACCAATGT
	上游引物	THF3	GTTACATTTGCAAGAACAACGG
GAPDH	上游引物	GHF1	CCTCCAAACTTGTCATGACTTCACTTC
	下游引物	GHR2	TGAATGTGACAGACGGGGCTTATT
	上游引物	GHF3	TTATCCGCACCCCGCCG

柿子炭疽病病原哈瑞炭疽菌(Colletotrichum horii)特异性PCR检测

Specific PCR Detection of Causal Agent of Persimmon Anthracnose Colletotrichum horii

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