葡萄胶孢炭疽病菌复合种（Colletotrichum gloeosporioides species complexes）对嘧菌酯的抗药性研究

doi:10.16420/j.issn.0513-353x.2023-0494

摘要/Abstract

摘要：

研究了江苏丘陵地区葡萄胶孢炭疽病菌复合种（Colletotrichum gloeosporioides species complexes，CGSC）对嘧菌酯的敏感性及抗性分子机理。利用孢子萌发法和区分剂量法确定3个种（C. aenigma、C. viniferum和C. fructicola）51个单孢分离株对嘧菌酯的抑制中浓度（EC₅₀）和敏感性表型，采用田间人工接种防治方法评价其抗药性，通过嘧菌酯作用标靶基因（CYTB）序列分析确定抗性的分子机制。27个C. aenigma和16个C. viniferum中对嘧菌酯敏感的菌株分别有20和14个，EC₅₀分别为0.0388（0.0150 ~ 0.0770）和0.0614（0.0213 ~ 0.0906）mg · L^-1，而抗性菌株的EC₅₀均大于100 mg · L^-1；C. fructicola菌株均为抗性菌株，其EC₅₀大于100 mg · L^-1。田间接种防治试验证明嘧菌酯2倍推荐剂量（250 mg · L^-1）对抗性菌株C. fructicola MS206失去防效（防治效果7.11%）。所有抗性菌株的CYTB基因上都只含有G143A点突变；CYTB基因G143位氨基酸两侧内元呈现多样性。江苏丘陵地区葡萄炭疽病种群中的不同种对嘧菌酯的敏感性不同，抗药种群的流行是造成田间防效下降的主因子，所有抗性菌株的抗性分子机制均为CYTB基因G143A点突变，未发现其他点突变类型。

关键词: 葡萄, 炭疽病, 胶孢炭疽病菌复合种, 嘧菌酯, 抗药性, 分子机制

Abstract:

The sensitivity and resistance molecular mechanism of Colletotrichum gloeosporioides species complexes（CGSC）derived from the hilly area of Jiangsu Province to azoxystrobin were studied. Employing the methods of spore germination and discriminative dose（a concentration that fully inhibits mycelial growth of the sensitive strains），the effective inhibition medium concentration values（inhibits mycelia growth by 50% relative to the control，EC₅₀）to azoxystrobin and sensitivity types of 51 single spore isolates of CGSC（C. aenigma，C. viniferum and C. fructicola）were identified. The field control efficacy test inoculated with conidia suspension of sensitive，resistant and mixed strains（the resistant strain and sensitive strain with the same conidia concentration were mixed in equal volume）was used to evaluate the epidemic of fungicides resistance of CGSC. Furthermore，the molecular mechanisms of azoxystrobin were determined by the sequence analysis of the target gene（mitochondrial cytochrome b，CYTB）. Among the 27 strains of C. aenigma and 16 strains of C. viniferum，20 and 14 strains were sensitive to axoxystrobin，with EC₅₀ values of 0.0388（0.0150-0.0770）and 0.0614（0.0213-0.0906）mg · L^-1，respectively，while the EC₅₀ values of resistant strains were greater than 100 mg · L^-1. All the C. fructicola strains were resistant and their EC₅₀ values were greater than 100 mg · L^-1. Field inoculated test of azoxystrobin at twice recommended field dose（250 mg · L^-1）against resistant strains have lost the control efficacy（control efficacy 7.11%）. Sequence analysis of target gene（CYTB）showed that all resistant strains harbored G143A（Glycine substituted by Alanine）point mutation in CYTB. The CYTB can be divided into two types based on whether there are partial deletions in the sequence of the intro adjoining after the G143 amino acid position. The sensitivity of different species in CGSC to azoxystrobin exhibits heterogeneity. The prevalence of resistant populations was the main factor leading to a decrease in field control effectiveness. The genotype of resistance to azoxystrobin of CGSC belonged to the point mutation of single base of target gene，which resulted in the substitution of amino acids，i.e. G143A，and no other genotypes of point mutations were found in this research.

Key words: grape, grapevine anthracnose, Colletotrichum gloeosporioides species complexes, azoxystrobin, resistance, molecular mechanism

杨敬辉, 许媛, 肖婷, 褚姝频, 刘吉祥, 姚克兵. 葡萄胶孢炭疽病菌复合种（Colletotrichum gloeosporioides species complexes）对嘧菌酯的抗药性研究[J]. 园艺学报, 2024, 51(8): 1906-1912.

YANG Jinghui, XU Yuan, XIAO Ting, CHU Shupin, LIU Jixiang, YAO Kebing. Resistance of Colletotrichum gloeosporioides Species Complexes from Grape to Azoxystrobin[J]. Acta Horticulturae Sinica, 2024, 51(8): 1906-1912.

图/表 3

参考文献 22

[1]	Bardas G A, Veloukas T, Koutita O, Karaoglanidis G S. 2010. Multiple resistance of Botrytis cinerea from kiwifruit to SDHIs,QoIs and fungicides of other chemical groups. Pest Manag Science,66:967-973.
[2]	Bartlett D W, Clough J M, Godwin J R, Hall A A, Hamer M, Parr-Dobrzanski B. 2002. The strobilurin fungicides. Pest Manag. Science,58:649-662.
[3]	Baumler S, Felsenstein F G, Schwarz G. 2003. CAPS and DHPLC analysis of a single nucleotide polymorphism in the cytochrome b gene conferring resistance to strobilurins in field isolates of Blumeria graminis f. sp. hordei. Journal Phytopathology,151:149-152.
[4]	Bolton M D, Rivera V, Secor G. 2013. Identification of the G143A mutation associated with QoIs resistance in Cercospora beticola field isolates from Michigan,United States. Pest Management Science,69:35-39.
[5]	Deng Weiping, Yang Min, Du Fei, Yang Jizhong, Zhu Shusheng. 2013. Identification of the pathogen causing grape anthracnose in Yunnan and its pathogenicity. Journal of Plant Protection, 40 (1):61-67. (in Chinese)
	邓维萍, 杨敏, 杜飞, 杨积忠, 朱书生. 2013. 云南葡萄产区葡萄炭疽病病原鉴定及致病力分析. 植物保护学报, 40 (1):61-67.
[6]	Gisi U, Sierotzki H, Cook A, McCaffery A. 2002. Mechanisms influencing the evolution of resistance to Qo inhibitor fungicides. Pest Management Science,58:859-867.
[7]	Hu M J, Grabke A, Dowling M E, Holstein H J, Schnabel G. 2015. Resistance in Colletotrichum siamense from peach and blueberry to Thiophanato-Methyl and Azoxystrobin. Plant Disease, 99 (6) :806-814.
[8]	Inada M, Ishii H, Chung W H, Yamada T, Yamaguchi J, Furuta A. 2008. Occurrence of strobilurin-resistant strains of Colletotrichum gloeosporioides (Glomerella cingulate),the causal fungus of strawberry anthracnose. Japan Journal Phytopathology,74:114-117.
[9]	Kim Y S, Dixon E W, Vincessi P, Farman M L. 2003. Field resistance to strobilurin(QoI)fungicides in Pyricularia grisea caused by mutations in the mitochondrial cytochrome b gene. Phytopathology,93:891-900.
[10]	Lei Yan, Lin Xiongjie, Chen Ting, Liu Xinming, Cai Shenghua, Fan Guocheng. 2014. Pathogen identification and pathogenicity analysis of grape ripe rot in Fujian. Journal of Fruit Science, 31 (6):1123-1127. (in Chinese)
	雷龑, 林雄杰, 陈婷, 刘鑫铭, 蔡盛华, 范国成. 2014. 福建葡萄炭疽病病原鉴定及致病性分析. 果树学报, 31 (6):1123-1127.
[11]	Li Hongxia, Liu Zhaoyun, Wang Jianxin, Zhou Mingguo. 2005. Baseline sensitivity of Colletotrichum gloeosporioides and C. capsica from capsicum to azoxystrobin. Acta Phytopathologica Sinica, 35 (1):73-77. (in Chinese)
	李红霞, 刘照云, 王建新, 周明国. 2005. 辣椒炭疽病菌对嘧菌酯的敏感性测定. 植物病理学报, 35 (1):73-77.
[12]	Li Yang, Liu Changyuan, Chen Xiurong, Zhao Kuihua, Miao Zeyan, Liang Chunhao, Wang Hui. 2009. Identification of the grape anthracnose and its sensitivity to carbendazim in Liaoning. Plant Protection, 35 (4):74-77. (in Chinese)
	李洋, 刘长远, 陈秀蓉, 赵奎华, 苗则彦, 梁春浩, 王辉. 2009. 辽宁省葡萄炭疽菌鉴定及对多菌灵敏感性研究. 植物保护, 35 (4):74-77.
[13]	Ma Z H, Felts D, Michailides T J. 2003. Resistance to azoxystrobin in Alternaria isolates from pistachio in California. Pesticide Biochemistry and Physiology,77:66-74.
[14]	Melksham K J, Weckert M A, Steel C C. 2002. An unusual bunch rot of grapes in sub-tropical regions of Australia caused by Colletotrichum acutatum Australas. Plant Pathology,31:193-194.
[15]	Peng L J, Sun T, Yang Y L, Cai L, Hyde K D, Bahkali A H, Liu Z Y. 2013. Colletotrichum species on grape in Guizhou and Yunnan Provinces,China. Mycoscience,54:29-41.
[16]	Peres N A R, Souza N L, Peever T L, Timmer L W. 2004. Benomyl sensitivity of isolates of Colletotrichum acutatum and C. gloeosporioides from Citrus. Plant Disease,88:125-130.
[17]	Sierotzki H, Frey R, Wullschleger J. 2007. Cytochrome b gene sequence and structure of Pyrenophora teres and P. tritici-repentis and implications for QoI resistance. Pest Management Science,63:225-233.
[18]	Southworth E A. 1891. Ripe rot of grapes and apples. Mycology,80:692-696.
[19]	Sung K H, Wan G K, Hae K Y, Kyung J C. 2008. Morphological variations,genetic diversity and pathogenicity of Colletotrichum species causing grape ripe rot in Korea. The Plant Pathology Journal,24:269-278.
[20]	Wei L L, Zheng H H, Zhen P C, Chen W C, Zheng J Q, Chen C J, Cao A B. 2021. Molecular and biochemical characterization of Colletotrichum gloeosporioides isolates resistant to azoxystrobin from grape in China. Plant Pathology, 70 (6):1300-1309.
[21]	Xu Yuan, Xiao Ting, Chu Shupin, Liu Jixiang, Rui Dongming, Yao Kebing, Yang Jinghui. 2022. Diversity analysis of the Colletotrichum spp. causing grape anthracnose in Jurong City of Jiangsu Province. Journal of Nangjing Agricultural University, 45 (1):78-85. (in Chinese)
	许媛, 肖婷, 褚姝频, 刘吉祥, 芮东明, 姚克兵, 杨敬辉. 2022. 江苏省句容市葡萄炭疽病菌多样性及对苯并咪唑类杀菌剂的抗药性分析. 南京农业大学学报, 45 (1):78-85.
[22]	Yamamoto J, Sato T, Tomioka K. 1990. Occurrence of ripe rot of grapes(Vitis vinifera L.) caused by Colletotrichum acutatum Simmonds ex Simmonds. Annual Phytopathology Social Japan,65:83-86.

菌种 Strains	菌株总数 Number of isolate	敏感表型 Sensitivity phenotype	菌株数 Number of isolate	EC₅₀/（mg · L^-1）
C. aenigma	27	敏感Sensitivity	20	0.0388（0.0150 ~ 0.0770）
C. aenigma		抗性Resistance	7	> 100
C. viniferum	16	敏感Sensitivity	14	0.0614（0.0213 ~ 0.0906）
C. viniferum		抗性Resistanc	2	> 100
C. fructicola	8	抗性Resistanc	8	> 100

菌种 Strains	菌株总数 Number of isolate	敏感表型 Sensitivity phenotype	菌株数 Number of isolate	EC₅₀/（mg · L^-1）
C. aenigma	27	敏感Sensitivity	20	0.0388（0.0150 ~ 0.0770）
C. aenigma		抗性Resistance	7	> 100
C. viniferum	16	敏感Sensitivity	14	0.0614（0.0213 ~ 0.0906）
C. viniferum		抗性Resistanc	2	> 100
C. fructicola	8	抗性Resistanc	8	> 100

菌种 Strains	表型 Phenotype	菌株 Isolate	突变位点 Mutation site	内元类型 Intro type
Colletotrichum aenigma	敏感 Sensitivity	BT75、BT77、BT85、BT167、BT172、BT175、BT232、HB94、HY140、HY141、HY143、HY144、HY145、HY147、HY148、HY149、HY151、MS189、MS190、MS192	—	b
	抗性 Resistance	BT174、HY142、HY150、MS132、MS136、MS200、MS207	G143A	b
Colletotrichum viniferum	敏感 Sensitivity	HB93、HY45、BT28、BT39、BT165、BT125、HY105、HY154、HY161、MS8、MS58、MS68、MS134、MS195	—	a
	抗性 Resistance	MS211、HY155	G143A	a
Colletotrichum fructicola	抗性 Resistance	HY197、HY198、MS130、MS135、MS201、MS206、MS210、MS212	G143A	b

菌种 Strains	表型 Phenotype	菌株 Isolate	突变位点 Mutation site	内元类型 Intro type
Colletotrichum aenigma	敏感 Sensitivity	BT75、BT77、BT85、BT167、BT172、BT175、BT232、HB94、HY140、HY141、HY143、HY144、HY145、HY147、HY148、HY149、HY151、MS189、MS190、MS192	—	b
	抗性 Resistance	BT174、HY142、HY150、MS132、MS136、MS200、MS207	G143A	b
Colletotrichum viniferum	敏感 Sensitivity	HB93、HY45、BT28、BT39、BT165、BT125、HY105、HY154、HY161、MS8、MS58、MS68、MS134、MS195	—	a
	抗性 Resistance	MS211、HY155	G143A	a
Colletotrichum fructicola	抗性 Resistance	HY197、HY198、MS130、MS135、MS201、MS206、MS210、MS212	G143A	b

接种菌株Inoculated strain	病果率/% Disease fruit ratio	防效/% Control efficacy
MS190（C. aenigma，敏感Sensitivity）	9.40	89.89
MS206（ C. fructicola，抗性Resistance）	77.78	7.11
MS190 + MS206	53.29	45.08