我国苹果斑点落叶病菌携带dsRNA分析及一种dsRNA病毒的鉴定

doi:10.3864/j.issn.0578-1752.2021.22.007

摘要/Abstract

摘要：

【目的】苹果斑点落叶病在我国各苹果主产区均有发生,给苹果产业造成了严重的经济损失。苹果斑点落叶病是由链格孢苹果专化型（Alternaria alternata f. sp. mali）侵染引起的一种气传病害。本研究旨在探明我国苹果斑点落叶病菌携带dsRNA的情况,为田间防治斑点落叶病提供新的生防资源,并为病毒的多样性以及进化研究提供新的认识。【方法】从全国8个省份采集有典型症状的组织样本,通过组织分离和单孢分离法获得纯培养;通过dsRNA的提取及凝胶电泳明确我国苹果斑点落叶病菌携带dsRNA的情况;通过菌株培养特性、菌丝生长速率、在果实及叶片上的致病力测定揭示携带dsRNA病原菌的生物学性状;通过高通量测序技术、分子克隆技术对QY-2菌株携带的dsRNA病毒进行全基因组序列、基因组结构和系统进化分析。【结果】自我国苹果产区获得102株苹果斑点落叶病菌菌株的纯培养,通过dsRNA的提取及凝胶电泳明确了5个菌株带有明显的dsRNA条带,携带的dsRNA分为4种类型,类型I（菌株YT-3-7）dsRNA在8、2.5和1.5 kb左右;类型II（菌株SJZ-4）dsRNA在8 kb左右;类型III（菌株QY-2）dsRNA在3和0.8 kb左右;类型IV（菌株CL-2-6和SQ-1-1）dsRNA在2.5和1.5 kb左右。含有dsRNA的菌株培养性状多样,菌落特征、生长速率与dsRNA携带与否及类型没有明显的关系,含有dsRNA的菌株大多属于弱致病力菌株。QY-2在叶片和果实上的致病力均最弱,确定其携带的dsRNA病毒基因组包括5个dsRNA片段,分别为dsRNA1—dsRNA5,片段大小依次为3 665、3 054、2 824、2 819、831 nt,提交至GenBank,登录号分别为MK672910、MK672913、MK672912、MK672911、MK836314。编码蛋白的分子量依次为124、83、84、83、13 kD。经系统进化关系分析,与Alternaria alternata chrysovirus 1遗传关系最近,确定其为产黄青霉病毒科（Chrysoviridae）、β产黄青霉病毒属（Betachrysovirus）的Alternaria alternata chrysovirus,暂定命名为Alternaria alternata chrysovirus 2（AaCV2）。【结论】我国苹果斑点落叶病菌携带dsRNA群体多样,dsRNA的有无及类型与寄主病原菌培养性状没有明显相关性,携带dsRNA的菌株多为弱致病力菌株,致病力最低的QY-2菌株携带AaCV2。该菌株的弱致病力可能具有潜在的应用价值,可为苹果斑点落叶病提供新的生防资源。

关键词: 苹果斑点落叶病, 链格孢苹果专化型, dsRNA, 生物学性状, 致病力, 生物防治

Abstract:

【Objective】Apple Alternaria blotch occurs in all major apple producing areas in China, causing serious economic loss. Apple Alternaria blotch is a worldwide airborne disease caused by Alternaria alternata f. sp. mali. The objective of this study is to investigate the diversity of dsRNA of A. alternata f. sp. mali in China, and to provide new biological control resources for the disease and new understanding of virus diversity and evolutionary.【Method】Tissue samples with typical symptom were collected from eight provinces in China. Pure culture was obtained by tissue isolation and single spore separation. The diversity of dsRNA carried by A. alternata f. sp. mali in China was determined by dsRNA extraction and gel electrophoresis. The biological characteristics of dsRNA-carrying pathogens were determined by evaluation of culture characteristics, mycelial growth rate, and pathogenicity on fruits and leaves. By high throughput sequencing and molecular cloning techniques, the whole genome sequence, genome structure and phylogenetic for the mycovirus carried by QY-2 strain were analyzed.【Result】The pure culture of 102 strains of A. alternata f. sp. mali was obtained and five strains with obvious dsRNA bands were found. The dsRNA virus of A. alternata f. sp. mali in China could be divided into four types, there were about 8, 2.5 and 1.5 kb dsRNA in type I (strain YT-3-7), about 8 kb dsRNA in type II (strain SJZ-4), about 3 and 0.8 kb dsRNA in type III (strain QY-2), and about 2.5 and 1.5 kb dsRNA in type IV (strains CL-2-6 and SQ-1-1). The strains carrying dsRNA had diverse culture characteristics, and there was no significant relationship between the colony characteristics, growth rate and whether dsRNA carrying or not and its type. The strains carrying dsRNA were mostly of low pathogenicity type. It was found that QY-2 strain showed weak pathogenicity on leaves and fruits, so QY-2 was selected for virus identification. The genome of the dsRNA virus carried by the strain QY-2 consists of five dsRNA fragments, dsRNA1-dsRNA5, with the sequence sizes of 3 665, 3 054, 2 824, 2 819 and 831 nt, which were submitted to GenBank. The accession numbers are MK672910, MK672913, MK672912, MK672911, and MK836314. The molecular weights of coding proteins are 124, 83, 84, 83 and 13 kD, respectively. Phylogenetic analysis showed that it was closely related to AaCV1, so it was identified as Alternaria alternata chrysovirus belonging to Chrysoviridae and Betachrysovirus. The tentative designation is Alternaria alternata chrysovirus 2 (AaCV2). 【Conclusion】There are diverse dsRNAs in A. alternata f. sp. mali in China, and there is no significant correlation between dsRNA diversity and host pathogen culture traits. Most of the strains carrying dsRNA showed low pathogenicity. QY-2 strain with the least pathogenicity carries AaCV2. The low pathogenicity characteristic of this strain may have potential application value, which provides a new resource for biological control of apple Alternaria blotch.

Key words: apple Alternaria blotch, Alternaria alternata f. sp. mali, dsRNA, biological characteristic, pathogenicity, biological control

曹钰晗,李紫腾,张静怡,张静娜,胡同乐,王树桐,王亚南,曹克强. 我国苹果斑点落叶病菌携带dsRNA分析及一种dsRNA病毒的鉴定[J]. 中国农业科学, 2021, 54(22): 4787-4799.

CAO YuHan,LI ZiTeng,ZHANG JingYi,ZHANG JingNa,HU TongLe,WANG ShuTong,WANG YaNan,CAO KeQiang. Analysis of dsRNA Carried by Alternaria alternata f. sp. mali in China and Identification of a dsRNA Virus[J]. Scientia Agricultura Sinica, 2021, 54(22): 4787-4799.

图/表 14

表1

本研究中苹果斑点落叶病样本信息"

序号 Number	菌株编号 Strain number	采集地点 Collection location	采集时间 Collection time	品种 Cultivar	树龄 Tree age (a)
1	ND	河北保定Baoding, Hebei	05-10	中秋王Mid-autumn King	10
2-7	WA-3, WA-8, WA-17, WA-20, WA-25, WA-28	河北武安Wuan, Hebei	05-12	富士Fuji	10
8-13	QY-2, QY-3, QY-4, QY-5, QY-6, QY-8	河北保定Baoding, Hebei	05-22	藤木一号Vine Wood One	10
14-16	SMX-1-1, SMX-1-3, SMX-1-9	河南三门峡Sanmeixia, Henan	06-22	金冠Golden Delicious	6
17	SMX-2-1	河南三门峡Sanmeixia, Henan	06-22	嘎啦Gala	6
18	SMX-3-1	河南三门峡Sanmeixia, Henan	06-22	红尾Red Tail	6
19-24	WH-5, WH-8, WH-12, WH-15, WH-17, WH-3-1	山东威海Weihai, Shandong	06-21	威海金Weihai Gold	3
25-31	SQ-1-1, SQ-1-2, SQ-1-5, SQ-1-6, SQ-1-7, SQ- 1-8, SQ-1-9	河南商丘Shangqiu, Henan	06-21	礼泉Liquan	9
32-33	SQ-2-2, SQ-2-3	河南商丘Shangqiu, Henan	06-21	新红星New Red Star	24
34-40	SQ-3-1, SQ-3-6, SQ-3-7, SQ-3-8, SQ-3-11, SQ- 3-13, SQ-3-14	河南商丘Shangqiu, Henan	06-21	长富2号Changfu 2	24
41-44	CL-2-6, CL-2-3, CL-2-9, CL-2-12	河北昌黎Changli, Hebei	06-21	太平洋玫瑰Pacific Rose	4
45-49	CL-3-1-1, CL-3-2-1, CL-3-2-2, CL-3-2-3, CL-3-2-4	河北昌黎Changli, Hebei	06-21	秋香Qiuxiang	3
50-55	CL-4-1, CL-4-2, CL-4-9, CL-4-7, CL-4-6, CL-4-13	河北昌黎Changli, Hebei	06-21	北斗Beidou	4
56	YA	陕西延安Yan’an, Shaanxi	06-23	延丰Yanfeng	8
57-62	LN-2, LN-6, LN-7, LN-8, LN-9, LN-10	辽宁Liaoning	06-29	绿帅Lvshuai	13
63	YT-2-1	山东烟台Yantai, Shandong	07-03	富士Fuji	5
64-66	YT-3-1, YT-3-7, YT-3-9	山东烟台Yantai, Shandong	07-03	岳红Yuehong	13
67-69	YT-4-1, YT-4-2, YT-4-3	山东烟台Yantai, Shandong	07-03	粉红女士Pink Lady	13
70-77	HLJ-1-3, HLJ-1-4, HLJ-1-6, HLJ-1-7, HLJ-1-8, HLJ-1-9, HLJ-1-10, HLJ-1-11	黑龙江牡丹江Mudanjiang, Heilongjiang	07-14	QRZ-1	6
78-85	HLJ-2-1, HLJ-2-2, HLJ-2-3, HLJ2-5, HLJ-2-6, HLJ2-7, HLJ2-8, HLJ-2-9	黑龙江牡丹江Mudanjiang, Heilongjiang	07-14	QRZ-2	5
86-90	GS-1-1, GS-1-2, GS-1-8, GS-1-14, GS-1-17	甘肃天水Tianshui, Gansu	07-17	富士Fuji	未知Unknown
91-95	GS-3-2, GS-3-3, GS-3-7, GS-3-11, GS-3-12	甘肃天水Tianshui, Gansu	07-17	金冠Golden Delicious	未知Unknown
96-100	SJZ-1, SJZ-2, SJZ-3, SJZ-4, SJZ-8	河北石家庄Shijiazhuang, Hebei	07-20	嘎啦Gala	8
101	YN-1-2	云南石林Shilin, Yunnan	08-20	红露Honglu	4
102	YN-2-3	云南弥勒Mile, Yunnan	08-20	富士Fuji	3

表1

表2

图1

图2

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图5

图6

图7

表3

图8

表4

图9

图10

参考文献 30

[1]	JUNG K H. Growth inhibition effect of pyroligneous acid on pathogenic fungus, Alternaria mali, the agent of Alternaria blotch of apple. Biotechnology and Bioprocess Engineering, 2007, 12: 318-322. doi: 10.1007/BF02931111
[2]	胡同乐, 曹克强, 王树桐, 甄文超. 生长季苹果斑点落叶病流行主导因素的确定. 植物病理学报, 2005, 35(4): 374-377.
	HU T L, CAO K Q, WANG S T, ZHEN W C. Study on the main factor for the epidemic of Alternaria blotch on apple. Acta Phytopathologica Sinica, 2005, 35(4): 374-377. (in Chinese)
[3]	MIYAMOTO Y, ISHII Y, HONDA A, MASUNAKA A, TSUGE T, YAMAMOTO M, OHTANI K, FUKUMOTO T, GOMI K, PEEVER T L, AKIMITSU K. Function of genes encoding Acyl-CoA synthetase and enoyl-CoA hydratase for host-selective ACT-toxin biosynthesis in the tangerine pathotype of Alternaria alternata. Phytopathology, 2009, 99(4): 369-377. doi: 10.1094/PHYTO-99-4-0369
[4]	LI Y, ZHANG L Y, ZHANG Z, CONG P H, CHENG Z M. A simple sequence repeat marker linked to the susceptibility of apple to Alternaria blotch caused by Alternaria alternata apple pathotype. Journal of the American Society for Horticultural Science, 2011, 136(2): 109-115. doi: 10.21273/JASHS.136.2.109
[5]	吴玉星, 李美娜, 周宗山, 仇贵生, 金纪艳, 乔壮. 8种杀菌剂防治苹果斑点落叶病试验. 中国果树, 2007(2): 28-30.
	WU Y X, LI M N, ZHOU Z S, QIU G S, JIN J Y, QIAO Z. Experiment of eight fungicides against apple Alternaria blotch. China Fruits, 2007(2): 28-30. (in Chinese)
[6]	任璐, 史晓晶, 姚众, 韩巨才, 赵晓军. 苹果斑点落叶病菌对戊唑醇敏感基线建立及抗性突变体适合度. 植物病理学报, 2017, 47(3): 380-388.
	REN L, SHI X J, YAO Z, HAN J C, ZHAO X J. Baseline sensitivity of Alternaria alternata f. sp. mali to tebuconazole and fitness of resistant mutants. Acta Phytopathologica Sinica, 2017, 47(3): 380-388. (in Chinese)
[7]	赵国康, 李焰, 张树武, 徐秉良, 刘佳. 5种植物源农药对苹果斑点落叶病的防效评价. 中国果树, 2020(5): 46-49.
	ZHAO G K, LI Y, ZHANG S W, XU B L, LIU J. Evaluation the control effects of five botanical fungicides on apple Alternaria leaf spot(Alternaria mali). China Fruits, 2020(5): 46-49. (in Chinese)
[8]	FERNANDO W G D, NAKKEERAN S, ZHANG Y, SAVCHUK S. Biological control of Sclerotinia sclerotiorum (Lib.) de Bary by Pseudomonas and Bacillus species on canola petals. Crop Protection, 2007, 26(2): 100-107. doi: 10.1016/j.cropro.2006.04.007
[9]	ANAGNOSTAKIS S L. Biological control of chestnut blight. Science, 1982, 215(4532): 466-471. doi: 10.1126/science.215.4532.466
[10]	NUSS D L. Biological control of chestnut blight: An example of virus-mediated attenuation of fungal pathogenesis. Microbiological Reviews, 1992, 56(4): 561-576. doi: 10.1128/mr.56.4.561-576.1992
[11]	HOLLINGS M. Viruses associated with a die-back disease of cultivated mushroom. Nature, 1962, 196(4858): 962-965. doi: 10.1038/196962a0
[12]	GHABRIAL S A, NIBERT M L. Victorivirus, a new genus of fungal viruses in the family Totiviridae. Archives of Virology, 2009, 154(2): 373-379. doi: 10.1007/s00705-008-0272-x
[13]	YU J, KWON S J, LEE K M, SON M, KIM K H. Complete nucleotide sequence of double-stranded RNA viruses from Fusarium graminearum strain DK3. Archives of Virology, 2009, 154(11): 1855-1858. doi: 10.1007/s00705-009-0507-5
[14]	SHEPHERD H S. Viruslike particles in tentoxin-producing strains of Alternaria alternata. Journal of Virology, 1988, 62(10): 3888-3891. doi: 10.1128/jvi.62.10.3888-3891.1988
[15]	ZABALGOGEAZCOA I, PETRUNAK D, CHRIST B J, GILDOW F E. Unencapsidated double-stranded RNA associated with membrane vesicles in isolates of Alternaria solani. Mycological Research, 1997, 101(5): 604-608. doi: 10.1017/S0953756296003097
[16]	HAYASHI N, TSUGE T, KOBAYASHI H, NISHIMURA S. The presence of double-stranded RNAs in Alternaria alternata Japanese pear pathotype and their participation in AK-toxin productivity. Japanese Journal of Phytopathology, 1988, 54(2): 250-252. doi: 10.3186/jjphytopath.54.250
[17]	AOKI N, MORIYAMA H, KODAMA M, ARIE T, TERAOKA T, FUKUHARA T. A novel mycovirus associated with four double- stranded RNAs affects host fungal growth in Alternaria alternata. Virus Research, 2009, 140(1/2): 185-187.
[18]	LIN Y H, ZHANG H L, ZHAO C J, LIU S X, GUO L H. The complete genome sequence of a novel mycovirus from Alternaria longipes strain HN28. Archives of Virology, 2015, 160(2): 577-580. doi: 10.1007/s00705-014-2218-9
[19]	KOMATSU K, KATAYAMA Y, OMATSU T, MIZUTANI T, FUKUHARA T, KODAMA M, ARIE T, TERAOKA T, MORIYAMA H. Genome sequence of a novel victorivirus identified in the phytopathogenic fungus Alternaria arborescens. Archives of Virology, 2016, 161(6): 1701-1704. doi: 10.1007/s00705-016-2796-9
[20]	ZHONG J, SHANG H H, ZHU C X, ZHU J Z, ZHU H J, HU Y, GAO B D. Characterization of a novel single-stranded RNA virus, closely related to fusariviruses, infecting the plant pathogenic fungus Alternaria brassicicola. Virus Research, 2016, 217: 1-7. doi: 10.1016/j.virusres.2015.11.012
[21]	向均. 我国梨黑斑病病原菌种类及一种dsRNA病毒的鉴定[D]. 武汉: 华中农业大学, 2017.
	XIANG J. Identification of the pathogenic fungi of pear black spot disease from China and a dsRNA virus in them[D]. Wuhan: Huazhong Agricultural University, 2017. (in Chinese)
[22]	OKADA R, ICHINOSE S, TAKESHITA K, URAYAMA S I, FUKUHARA T, KOMATSU K, ARIE T, ISHIHARA A, EGUSA M, KODAMA M, MORIYAMA H. Molecular characterization of a novel mycovirus in Alternaria alternata manifesting two-sided effects: Down-regulation of host growth and up-regulation of host plant pathogenicity. Virology, 2018, 519: 23-32. doi: 10.1016/j.virol.2018.03.027
[23]	李朋华. 部分链格孢属真菌的形态学及多基因鉴定[D]. 郑州: 河南农业大学, 2016.
	LI P H. Identification of partial Alternaria by morphology and multiple-gene analysis[D]. Zhengzhou: Henan Agricultural University, 2016. (in Chinese)
[24]	李波. 核盘菌弱毒菌株SX276生物学特性及其携带RNA病毒的研究[D]. 武汉: 华中农业大学, 2016.
	LI B. Research on biological characteristics and RNA viruses from the hypovirulent strain SX276 of Sclerotinia sclerotiorum[D]. Wuhan: Huazhong Agricultural University, 2016. (in Chinese)
[25]	CHIBA S, SALAIPETH L, LIN Y H, SASAKI A, KANEMASTU S, SUZUKI N. A novel bipartite double-stranded RNA mycovirus from the white root rot fungus Rosellinia necatrix: Molecular and biological characterization, taxonomic considerations, and potential for biological control. Journal of Virology, 2009, 83(24): 12801-12812. doi: 10.1128/JVI.01830-09
[26]	YU X, LI B, FU Y P, XIE J T, CHENG J S, GHABRIAL S A, LI G, YI X H, JIANG D H. Extracellular transmission of a DNA mycovirus and its use as a natural fungicide. Proceedings of the National Academy of Sciences of the United States of America, 2013, 110(4): 1452-1457.
[27]	MELZER M S, DUNN M, ZHOU T, BOLAND G J. Assessment of hypovirulent isolates of Cryphonectria parasitica for potential in biological control of chestnut blight. Canadian Journal of Plant Pathology, 1997, 19(1): 69-77. doi: 10.1080/07060669709500576
[28]	赵迎彤. Botryosphaeria dothidea病毒dsRNA的多样性研究[D]. 泰安: 山东农业大学, 2014.
	ZHAO Y T. Diversity of dsRNA from Botryosphaeria dothidea fungal viruses[D]. Taian: Shandong Agricultural University, 2014. (in Chinese)
[29]	王利华, 罗慧, 王国平, 王利平. 来源于产黄青霉病毒科成员的分离物对梨轮纹病菌生长及其致病力的影响. 果树学报, 2017, 34(10): 1330-1339.
	WANG L H, LUO H, WANG G P, WANG L P. Effect of Botryosphaeria dothidea chrysovirus 1 isolate belonging to the Chrysoviridae family on growth and pathogenicity of the B. dothidea strain infection in pears. Journal of Fruit Science, 2017, 34(10): 1330-1339. (in Chinese)
[30]	MA G P, ZHANG X F, HUA H H, ZHOU T, WU X H. Molecular and biological characterization of a novel strain of Alternaria alternata chrysovirus 1 identified from the pathogen Alternaria tenuissima causing watermelon leaf blight. Virus Research, 2020, 280: 197904. doi: 10.1016/j.virusres.2020.197904

引物名称 Primer name	引物序列 Primer sequence (5′-3′)	条带大小 Product size (bp)	用途 Purpose	来源 Source
CRV1-5F	GCAAAAAAGAACTAAAGGAC	800	AaCV2 ORF5的扩增 Amplification of AaCV2 ORF5	自行设计 Designed in this study
CRV1-5R	ACACACAAATGGGATACC	800	AaCV2 ORF5的扩增 Amplification of AaCV2 ORF5	自行设计 Designed in this study
CRV1-1F	ATCTCAAGGTACCGGAGTCA	450	AaCV2 RT-PCR验证 Identification of AaCV2 by RT-PCR	自行设计 Designed in this study
CRV1-1R	GCTGCTTAAACATCACCTGCA	450	AaCV2 RT-PCR验证 Identification of AaCV2 by RT-PCR	自行设计 Designed in this study

基因ID Unigene ID	基因长度 Unigene length (nt)	蛋白描述 Protein description	登录号 Accession number	E-value	Reads数量 Number of Reads	RPKM
Contig 10	3665	RNA-dependent RNA polymerase [AaCV1]	BBC27878.1	0.00E+00	1451570	4820
Contig 41	3054	Putative coat protein [AaCV1]	BBC27881.1	3.5E-176	2331542	9292
Contig 38	2824	Hypothetical protein [AaCV1]	BBC27880.1	2.9E-108	1167609	5032
Contig 24	2819	Hypothetical protein [AaCV1]	BBC27879.1	2.3E-137	1268863	5478