广东香葱炭疽病病原菌鉴定

doi:10.16420/j.issn.0513-353x.2020-1033

摘要/Abstract

摘要：

为明确广东香葱炭疽病的病原菌种类,在广东省香葱主要产区惠州市和韶关市采集香葱炭疽病疑似病株,分离、纯化病原菌;通过病原菌分生孢子悬液接种活体香葱植株和离体植株叶片,柯赫氏法则验证,明确病原菌的致病性;通过观察病原菌的形态特征,结合其核糖体内转录间隔区（ITS）、肌动蛋白（ACT）、几丁质合成酶1（CHS1）、甘油醛-3-磷酸脱氢酶（GAPDH）和β微管蛋白（TUB2）基因部分序列的多基因分子系统学分析等方法,确定病原菌的分类地位。从病样中分离获得23个形态特征相似的单分生孢子分离物。致病性人工接种结果表明,供试菌株分生孢子在20和25 ℃下均可侵染香葱产生与田间相似的炭疽病症状,老叶较嫩叶更易感病。20 ℃在PDA培养基上,供试菌株菌落呈灰色,菌丝生长速度较快;25和28 ℃菌丝生长较慢,菌落呈花瓣状;后期在菌落表面形成橘红色分生孢子堆。分生孢子梗透明,顶端簇生分生孢子;分生孢子透明,单细胞,微弯,大小13.1 ~ 19.6 µm × 3.2 ~ 3.9 µm。附着胞深棕色,形状不规则,大小11.7 ~ 25.5 µm × 5.0 ~ 14.3 µm;刚毛深棕色至浅棕色,基部膨大,顶端尖或圆滑,长为44.5 µm ~ 106.4 µm。这些形态特征与白蜡树刺盘孢（Colletotrichum spaethianum）基本一致。供试菌株的ITS、ACT、CHS1、GAPDH和TUB2基因序列与白蜡树刺盘孢模式菌株CBS167.49对应基因序列的一致性为99.46% ~ 100%。系统发育树显示,该菌株与白蜡树刺盘孢的3个菌株单独聚成一支。明确了广东香葱炭疽病的病原菌为白蜡树刺盘孢,这是白蜡树刺盘孢侵染香葱引起香葱炭疽病的首次报道。

关键词: 香葱, 炭疽病, 白蜡树刺盘孢, 病原鉴定

Abstract:

To clarify the taxonomy of the pathogen causing Allium ascalonicum anthracnose in Guangdong, diseased plant samples of A. ascalonicum showing anthracnose symptoms from Huizhou and Shaoguan in Guangdong were collected as test materials. Strains were isolated and purified by conidial suspension dilution method. The pathogenicity of these strains was determined by the Koch’s postulates with in vivo or in vitro inoculation of conidial suspension on plants of A. ascalonicum. The pathogen was identified based on morphological characteristics,combined with polygene sequence analysis and phylogenetic analysis method using partial sequences of internal ribosome transcribed spacer（ITS）,actin （ACT）,chitin synthase 1（CHS1）,glyceraldehyde-3-phosphate dehydrogenase（GAPDH）and β-tublin （TUB2）. Results showed that 23 single-conidium-isolates with similar morphological characteristics were obtained from diseased plant samples of A. ascalonicum showing anthracnose symptoms. Artificial in vivo and in vitro inoculation results showed that the tested isolate could infect A. ascalonicum and cause typical anthracnose symptoms both at 20 ℃ and 25 ℃. Old leaves were more susceptible than young leaves. On PDA medium,the colony was gray,and mycelia of the tested isolate grew evenly and fast at 20 ℃. But it grew abnormally and slowly,the colony showing petal-like shape at 25 ℃ and 28 ℃. Conidiomata in orange color were formed on the surface of the colony. Conidiophores were hyaline with conidia on the top. Conidia were hyaline,single-spore,slightly curved,13.1-19.6 µm × 3.2-3.9 µm. Appressoria were dark brown,irregular shapes,11.7-25.5 µm × 5.0-14.3 µm. Setae were light brown to dark brown,with enlarged base and pointy or rounded tip,44.5 to 106.4 µm in length. These morphological characteristics were similar with Colletotrichum spaethianum. Sequence identities of partial sequences of ITS,ACT,CHS1,GAPDH and TUB2 genes of representative isolates were 99.46% to 100% identical to the ex-holotype strain CBS 167.49. The phylogenetic tree showed that two representative isolates were grouped together with three C. spaethianum strains to form a single clade. This study clarified that the pathogen causing A. ascalonicum anthracnose in Guangdong was determined as C. spaethianum and reported for the first time that C. spaethianum could cause A. ascalonicum anthracnose.

Key words: Allium ascalonicum, anthracnose, Colletotrichum spaethianum, pathogen identification

中图分类号:

S633

于琳, 佘小漫, 汤亚飞, 蓝国兵, 李正刚, 何自福. 广东香葱炭疽病病原菌鉴定[J]. 园艺学报, 2022, 49(1): 129-140.

YU Lin, SHE Xiaoman, TANG Yafei, LAN Guobing, LI Zhenggang, HE Zifu. Identification of Pathogen Causing Allium ascalonicum Anthracnose in Guangdong[J]. Acta Horticulturae Sinica, 2022, 49(1): 129-140.

图/表 8

参考文献 42

[1]	Aveskamp M M, Verkley G J M, de Gruyter J, Murace M A, Perelló A, Woudenberg J H C, Groenewald J Z, Crous P W. 2009. DNA phylogeny reveals polyphyly of Phoma section Peyronellaea and multiple taxonomic novelties. Mycologia, 101:363-382. pmid: 19537209
[2]	Cai L, Hyde K D, Taylor P W J, Weir B S, Waller J M, Abang M M, Zhang J Z, Yang Y L, Phoulivong S, Liu Z Y, Prihastuti H, Shivas R G, McKenzie E H C, Johnston P R. 2009. A polyphasic approach for studying Colletotrichum. Fungal Diversity, 39:183-204.
[3]	Cannon P F, Damm U, Johnston P R, Weir B S. 2012. Colletotrichum - current status and future directions. Studies in Mycology, 73 (1):181-213. doi: 10.3114/sim0014 pmid: 23136460
[4]	Carbone I, Kohn L M. 1999. A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia, 91 (3),553-556. doi: 10.2307/3761358 URL
[5]	Chen Ai-chang, Wei Zhou-quan, Liu Xiao-juan. 2016. First report of anthracnose disease on Allium fistulosum in Gansu Province. Proceedings of the 2016 Annual Conference of the Chinese Society of Plant Pathology. Nanjing: China Agricultural Science and Technology Press: 6. (in Chinese)
	陈爱昌, 魏周全, 刘小娟. 2016. 大葱炭疽病在甘肃省首次发生报道. 中国植物病理学会2016 年学术年会论文集. 南京: 中国农业科学技术出版社: 6.
[6]	Chen Xin-qi, Xu Jie-mei, Liang Song-yun, Tsi Zhan-he, Lang Kai-yong, Mao Zu-mei, Xu Lang-ran. 1980. Flora reipublicae popularis sinicae:Tomus 14. Angiospermae Monocotyledoneae Liliaceae (1). Beijing: Science Press: 258. (in Chinese)
	陈心启, 许介眉, 梁松筠, 吉占和, 郎楷永, 毛祖美, 许朗然. 1980. 中国植物志(第十四卷)被子植物门单子叶植物纲百合科(一). 北京: 科学出版社: 258.
[7]	Damm U, Woudenberg J H C, Cannon P F, Crous P W. 2009. Colletotrichum species with curved conidia from herbaceous hosts. Fungal Diversity, 39:45-87.
[8]	Diao Y, Zhang C, Liu F, Wang W, Liu L, Cai L, Liu X. 2017. Colletotrichum species causing anthracnose disease of chili in China. Persoonia, 38:20-37. doi: 10.3767/003158517X692788 pmid: 29151625
[9]	Fan Ji-de, Lu Xin-juan, Liu Can-yu, Zhao Yong-qiang, Zhang Bi-wei, Yang Hai-feng, Yang Feng. 2020. Development status and countermeasures of onion and garlic vegetable industry in Jiangsu Province. Journal of Changjiang Vegetables,(8):24-26. (in Chinese)
	樊继德, 陆信娟, 刘灿玉, 赵永强, 张碧薇, 杨海峰, 杨峰. 2020. 江苏省葱蒜类蔬菜产业发展现状及对策建议. 长江蔬菜,(8):24-26.
[10]	Fang Zhong-da. 2007. Plant disease research methods.3rd ed. Beijing: China Agriculture Press:124-125. (in Chinese)
	方中达. 2007. 植病研究方法. 第3版. 北京: 中国农业出版社:124-125.
[11]	Galván G A, Wietsma W A, Putrasemedja S, Permadi A H, Kik C. 1997. Screening for resistance to anthracnose (Colletotrichum gloeosporioides Penz.) in Allium cepa and its wild relatives. Euphytica, 95:173-178. doi: 10.1023/A:1002914225154 URL
[12]	Gardes M, Bruns T D. 1993. ITS primers with enhanced specificity for basidiomycetes-application to the identification of mycorrhizae and rusts. Molecular Ecology, 2:113-118. pmid: 8180733
[13]	Ghabrial S A, Suzuki N. 2009. Viruses of plant pathogenic fungi. Annual Review of Phytopathology, 47:353-384. doi: 10.1146/annurev-phyto-080508-081932 pmid: 19400634
[14]	Guan Y M, Liu Z B, Li M J, Wang Q X, Zhang Y Y. 2018. First report of Colletotrichum spaethianum causing anthracnose in Atractylodes japonica in China. Plant Disease, 102 (1):239.
[15]	Guerber J C, Liu B, Correll J C, Johnston P R. 2003. Characterization of diversity in Colletotrichum acutatum sensu lato by sequence analysis of two gene introns,mtDNA and intron RFLPs,and mating compatibility. Mycologia, 95 (5):872-895. pmid: 21148995
[16]	Guo M, Pan Y M, Dai Y L, Gao Z M. 2013. First report of leaf spot caused by Colletotrichum spaethianum on Peucedanum praeruptorum in China. Plant Disease, 97 (10):1380.
[17]	Hay F, Vaghefi N, Strickland D, Hadad R, Pethybridge S. 2018. First report of Colletotrichum fioriniae causing anthracnose of elephant garlic (Allium ampeloprasum var. ampeloprasum)in New York,USA. New Disease Reports, 38:1.
[18]	Jia Ju-sheng, Fang De-li. 2000. Anthracnose disease of onion and its control methods. Xinjiang Agricultural Sciences,(4):171-172. (in Chinese)
	贾菊生, 方德立. 2000. 洋葱炭疽病及其防治措施. 新疆农业科学,(4):171-172.
[19]	Kim W G, Hong S K, Kim J H. 2008. Occurrence of Anthracnose on Welsh Onion caused by Colletotrichum circinans. Mycobiology, 36 (4):274-276. doi: 10.4489/MYCO.2008.36.4.274 URL
[20]	Liu L, Zhang L, Qiu P, Wang Y, Liu Y, Li Y, Gao J, Hsiang T. 2020. Leaf spot of Polygonatum odoratum caused by Colletotrichum spaethianum. Journal of General Plant Pathology, 86:157-161. doi: 10.1007/s10327-019-00903-4 URL
[21]	Matos K S, Santana K F A, Catarino A M, Hanada R E, Silva G F. 2017. First report of anthracnose on welsh onion(Allium fistulosum)in Brazil caused by Colletotrichum theobromicola and C.truncatum. Plant Disease, 101 (6):1055.
[22]	Nischwitz C, Langston D, Sanders H F, Torrance R, Lewis K J, Gitaitis R D. 2008. First report of Colletotrichum gloeosporioides causing‘twister disease’of onion(Allium cepa)in Georgia. Plant Disease, 92 (6):974.
[23]	O’ Donnell K, Cigelnik E. 1997. Two divergent intragenomic rDNA ITS2 types within a monophyletic lineage of the fungus Fusarium are nonorthologous. Molecular Phylogenetics and Evolution 7:103-116. doi: 10.1006/mpev.1996.0376 URL
[24]	Okorley B A, Sossah F L, Dan D, Li Y, Fu Y. 2019. First report of Colletotrichum spaethianum causing anthracnose on Anemarrhena asphodeloides in China. Plant Disease, 103 (6):1414.
[25]	Pearson M N, Beever R E, Boine B, Arthur K. 2009. Mycoviruses of filamentous fungi and their relevance to plant pathology. Molecular Plant Pathology, 10:115-128. doi: 10.1111/mpp.2009.10.issue-1 URL
[26]	Rodriguez-Salamanca L M, Enzenbacher T B, Derie M L, du Toit L J, Feng C, Correll J C, Hausbeck M K. 2012. First report of Colletotrichum coccodes causing leaf and neck anthracnose on onions(Allium cepa)in Michigan and the United States. Plant Disease, 96 (5):769.
[27]	Russo V M, Pappelis A J. 1981. Observations of Colletotrichum dematium f.circinans on Allium cepa:halo formation and penetration of epidermal walls. Physiological Plant Pathology, 19 (1):127-136. doi: 10.1016/S0048-4059(81)80014-8 URL
[28]	Salunkhe V N, Anandhan S, Gawande S J, Ikkar R B, Bhagat Y S, Mahajan V. 2018a. First report of Colletotrichum truncatum causing anthracnose of mouse garlic(Allium angulosum)in India. Plant Disease, 102 (1):240.
[29]	Salunkhe V N, Anandhan S, Gawande S J, Ikkar R B, Bhagat Y S, Mahajan V, Singh M. 2018b. First report of anthracnose caused by Colletotrichum spaethianum on Allium ledebourianum in India. Plant Disease, 102 (10):2031.
[30]	Santana K F A, Garcia C B, Matos K S, Hanada R E, Silva G F, Sousa N R. 2016. First report of anthracnose caused by Colletotrichum spaethianum on Allium fistulosum in Brazil. Plant Disease, 100 (1):224.
[31]	Sikirou R, Beed F, Hotègni J, Winter S, Assogba-Komlan F, Reeder R, Miller S A. 2011. First report of anthracnose caused by Colletotrichum gloeosporioides on onion(Allium cepa)in Benin. New Disease Reports, 23:7.
[32]	Sydow P. 1897. Beiträge zur Kenntnis der Pilzflora der Mark Brandenburg. I. Hedwigia Beiblatt, 36:157-164.
[33]	Tong Xian-ming, Wang Zheng-yi, Shi Zhi-long. 1998. Studies on the pathogen and the biological characteristics of scallion anthracnose. Acta Phytophylacica Sinica, 25 (3):249-252. (in Chinese)
	童贤明, 王政逸, 施志龙. 1998. 浙江省舟山市矗头炭疽病病原及其生物学特性研究. 植物保护学报, 25 (3):249-252.
[34]	Vieira W A S, Michereff S J, Oliveira A C, Santos A, Câmara M P S. 2014. First report of anthracnose caused by Colletotrichum spaethianum on Hemerocallis flava in Brazil. Plant Disease, 98 (7):997.
[35]	White T J, Bruns T, Lee S, Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics//Innis M A,Gelfand D H,Sninsky J J,White T J. PCR Protocols:a guide to methods and applications. San Diego,California,U.S.A: Academic Press:315-322.
[36]	Wu Yahong, Zhao Qing, Wang Haiping. 2021. Research progress on the classification of Allium plants. Acta Horticulturae Sinica, 48 (7):1418-1428. (in Chinese)
	武亚红, 赵青, 王海平. 2021. 葱属植物分类研究进展. 园艺学报, 47 (7):1418-1428.
[37]	Yang L, Lu X H, Jing Y L, Li S D, Wu B M. 2019. First report of common bean(Phaseolus vulgaris)stem rot caused by Colletotrichum spaethianum in China. Plant Disease, 103 (1):151.
[38]	Yang Y, Liu Z, Cai L, Hyde K D. 2012. New species and notes of Colletotrichum on daylilies(Hemerocallis spp.). Tropical Plant Pathology, 37 (3):165-174. doi: 10.1590/S1982-56762012000300001 URL
[39]	Yu L, Sang W, Wu M, Zhang J, Yang L, Zhou Y, Chen W, Li G. 2015. Novel hypovirulence-associated RNAmycovirus in the plant-pathogenic fungus Botrytis cinerea:molecular and biological characterization. Applied and Environment Microbiology, 81 (7):2299-2310. doi: 10.1128/AEM.03992-14 URL
[40]	Yu Lin, She Xiao-man, Lan Guo-bing, Tang Ya-fei, Li Zheng-gang, Deng Ming-guang, He Zi-fu. 2020. Identification of pathogen causing sweet potato scab in Guangdong. Journal of Southern Agriculture, 51 (3):579-585. (in Chinese)
	于琳, 佘小漫, 蓝国兵, 汤亚飞, 李正刚, 邓铭光, 何自福. 2020. 广东甘薯疮痂病病原菌鉴定. 南方农业学报, 51 (3):579-585.
[41]	Zeng Da-xing, Qi Pei-kun, Jiang Zi-de. 2002. Identification of the falcate Colletotrichum on vegetable using RAPD analysis. Acta Phytopathologica Sinica, 32 (2):170-174. (in Chinese)
	曾大兴, 戚佩坤, 姜子德. 2002. 应用RAPD技术快速鉴定蔬菜上的弯孢类炭疽菌. 植物病理学报, 32 (2):170-174.
[42]	Zhao W, Wang T, Chen Q Q, Chi Y K, Swe T M, Qi R D. 2016. First report of leaf spot caused by Colletotrichum spaethianum on Lilium lancifolium in China. Plant disease, 100 (11):2328.

目的序列 Target sequence	引物名称 Primer	引物序列（5′-3′） Primer sequence	参考文献 Reference
ITS	ITS1F	CTTGGTCATTTAGAGGAAGTAA	Gardes & Bruns,1993
	ITS4	TCCTCCGCTTATTGATATGC	White et al.,1990
ACT	ACT-512F	ATGTGCAAGGCCGGTTTCGC	Carbone & Kohn,1999
	ACT-783R	TACGAGTCCTTCTGGCCCAT	Carbone & Kohn,1999
CHS1	CHS-79F	TGGGGCAAGGATGCTTGGAAGAAG	Carbone & Kohn,1999
	CHS-354R	TGGAAGAACCATCTGTGAGAGTTG	Carbone & Kohn,1999
GAPDH	GDF1	GCCGTCAACGACCCCTTCATT	Guerber et al.,2003
GAPDH	GDR1	GGGTGGAGTCGTACTTGAGCATGT	Guerber et al.,2003
TUB2	T1	AACATGCGTGAGATTGTAAGT	O’ Donnell & Cigelnik,1997
	Btub4R	CCRGAYTGRCCRAARACRAAGTTGTC	Aveskamp et al.,2009

目的序列 Target sequence	引物名称 Primer	引物序列（5′-3′） Primer sequence	参考文献 Reference
ITS	ITS1F	CTTGGTCATTTAGAGGAAGTAA	Gardes & Bruns,1993
	ITS4	TCCTCCGCTTATTGATATGC	White et al.,1990
ACT	ACT-512F	ATGTGCAAGGCCGGTTTCGC	Carbone & Kohn,1999
	ACT-783R	TACGAGTCCTTCTGGCCCAT	Carbone & Kohn,1999
CHS1	CHS-79F	TGGGGCAAGGATGCTTGGAAGAAG	Carbone & Kohn,1999
	CHS-354R	TGGAAGAACCATCTGTGAGAGTTG	Carbone & Kohn,1999
GAPDH	GDF1	GCCGTCAACGACCCCTTCATT	Guerber et al.,2003
GAPDH	GDR1	GGGTGGAGTCGTACTTGAGCATGT	Guerber et al.,2003
TUB2	T1	AACATGCGTGAGATTGTAAGT	O’ Donnell & Cigelnik,1997
	Btub4R	CCRGAYTGRCCRAARACRAAGTTGTC	Aveskamp et al.,2009

种类	菌株	GenBank登录号GenBank accession number
Species	Isolate	ITS	ACT	CHS1	GAPDH	TUB2
Colletotrichum lilii	CBS 186.30	GU227811	GU227909	GU228301	GU228203	GU228105
	CBS 109214	GU227810	GU227908	GU228300	GU228202	GU228104
C. liriopes	CBS 119444	GU227804	GU227902	GU228294	GU228196	GU228098
C. spaethianum	BLC1	MW295524	MW298835	MW298836	MW298837	MW298838
	CBS 167.49	GU227807	GU227905	GU228297	GU228199	GU228101
	CBS 100063	GU227808	GU227906	GU228298	GU228200	GU228102
	CBS 101631	GU227809	GU227907	GU228299	GU228201	GU228103
	WYC1	MW295525	MW298839	MW298840	MW298841	MW298842
C. tofieldiae	CBS 495.85	GU227801	GU227899	GU228291	GU228193	GU228095
C. trichellum	CBS 217.64	GU227812	GU227910	GU228302	GU228204	GU228106
C. verruculosum	IMI 45525	GU227806	GU227904	GU228296	GU228198	GU228100
C. gloeosporioides	CBS 112999	JX010152	JX009531	JX009818	JX010056	JX010445
C. theobromicola	ICMP 18649	JX010294	JX009444	JX009869	JX010006	JX010447
C. truncatum	CBS 151.35	GU227862	GU227960	GU228352	GU228254	GU228156
Monilochaetes infuscans	CBS 869.96	JQ005780	JQ005843	JQ005801	JX546612	JQ005864

种类	菌株	GenBank登录号GenBank accession number
Species	Isolate	ITS	ACT	CHS1	GAPDH	TUB2
Colletotrichum lilii	CBS 186.30	GU227811	GU227909	GU228301	GU228203	GU228105
	CBS 109214	GU227810	GU227908	GU228300	GU228202	GU228104
C. liriopes	CBS 119444	GU227804	GU227902	GU228294	GU228196	GU228098
C. spaethianum	BLC1	MW295524	MW298835	MW298836	MW298837	MW298838
	CBS 167.49	GU227807	GU227905	GU228297	GU228199	GU228101
	CBS 100063	GU227808	GU227906	GU228298	GU228200	GU228102
	CBS 101631	GU227809	GU227907	GU228299	GU228201	GU228103
	WYC1	MW295525	MW298839	MW298840	MW298841	MW298842
C. tofieldiae	CBS 495.85	GU227801	GU227899	GU228291	GU228193	GU228095
C. trichellum	CBS 217.64	GU227812	GU227910	GU228302	GU228204	GU228106
C. verruculosum	IMI 45525	GU227806	GU227904	GU228296	GU228198	GU228100
C. gloeosporioides	CBS 112999	JX010152	JX009531	JX009818	JX010056	JX010445
C. theobromicola	ICMP 18649	JX010294	JX009444	JX009869	JX010006	JX010447
C. truncatum	CBS 151.35	GU227862	GU227960	GU228352	GU228254	GU228156
Monilochaetes infuscans	CBS 869.96	JQ005780	JQ005843	JQ005801	JX546612	JQ005864