我国苹果轮纹病弱毒菌株中病毒的鉴定及其携带情况的检测

doi:10.3864/j.issn.0578-1752.2025.03.006

Abstract

Abstract:

【Objective】Apple ring rot is one of the serious fungal diseases in apple production caused by Botryosphaeria dothidea. The aim of this study is to obtain strains of B. dothidea with weak pathogenicity carrying dsRNA viruses, identify the types of viruses, and clarify the distribution of these viruses in China, so as to provide new biological control resources for the prevention and control of apple ring rot and new insights into the diversity and systematic evolution of fungal viruses. 【Method】Samples of branches with typical symptoms of apple ring rot were collected from across China, and pure cultures were obtained through tissue isolation and single-spore isolation. Virus-carrying strains were identified through dsRNA band analysis, and the types of dsRNA viruses carried by the virus-carrying strain WH-2L were identified using high-throughput sequencing and molecular cloning techniques. The presence of two types of dsRNA viruses in B. dothidea from six provinces (autonomous region) in China was determined by RT-PCR. Pathogenicity differences among representative strains carrying different viruses were clarified through pathogenicity tests. Finally, the transmission characteristics of the two viruses were revealed through analysis of vertical and horizontal transmission properties. 【Result】For the first time in apple-producing regions of China, strains of B. dothidea causing apple ring rot were found to be co-infected with two viruses: Botryosphaeria dothidea chrysovirus 1 (BdCV1), belonging to the family Chrysoviridae and genus Chrysovirus, and Botryosphaeria dothidea victorivirus 2 (BdVV2), belonging to the family Totiviridae and genus Victorivirus. It was clarified that BdCV1 and BdVV2 are widely distributed in B. dothidea causing apple ring rot in China. BdCV1 was detected in strains from Liaoning, Shandong, Henan, Hebei, Shaanxi, and Xinjiang, except for Yan’an in Shaanxi and Shijiazhuang in Hebei, with an average detection rate of 53.6%. BdVV2 occurred in Liaoning, Shandong, Henan, Hebei, and Shaanxi, but was not detected in Yan’an in Shaanxi, Shijiazhuang and Handan in Hebei, Aksu in Xinjiang, Taian and Qingdao in Shandong, with an average detection rate of 28.6%. It was clarified that the pathogenicity of strains with co-infection of the two viruses and single infection of BdCV1 on branches, apple fruits, and pear fruits was significantly reduced. The vertical transmission efficiency of BdCV1 and BdVV2 was 100%, and the horizontal transmission efficiency was 9% and 3%, respectively. 【Conclusion】The strain WH-2L of B. dothidea with weak pathogenicity carries two viruses, BdCV1 and BdVV2. The detection rates of BdCV1 and BdVV2 in B. dothidea causing apple ring rot in six apple-producing regions in China were 53.6% and 28.6%, respectively. Both viruses can cause reduced pathogenicity in their hosts, with high vertical transmission efficiency and certain horizontal transmission efficiency, and have potential for development as biological control resources for apple ring rot.

Key words: Botryosphaeria dothidea, apple ring rot, dsRNA, fungal virus, pathogenicity, biological control

CONG QiQi, ZHANG JingYi, MENG XiangLong, DAI PengBo, LI Bo, HU TongLe, WANG ShuTong, CAO KeQiang, WANG YaNan. Identification of Hypovirus in Apple Ring Rot Fungus Botryosphaeria dothidea and Detection of Virus-Carrying Status in China[J].Scientia Agricultura Sinica, 2025, 58(3): 478-492.

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序号Number	菌株编号 Strain number	采集地点 Collection site	分离部位 Separation site	采集时间 <BOLD>C</BOLD>ollection time	品种 Cultivar	树龄 Tree age
1-10	LY-1L, LY-2L, LY-3L, LY-4L, LY-5L, LY-6L, LY-7L, LY-8L, LY-9L, LY-10L	辽宁省营口市 Yingkou, Liaoning	枝干Branch	2023-04	富士Fuji	11
11-14	3L-10, 7L-10, 9L-10, 10L-10	山东省威海市 Weihai, Shandong	枝干Branch	2023-04	烟富8 Fuji 8	10
15-20	WH-1L, WH-2L, WH-3L, WH-6L, WH-7L, WH-9L	山东省威海市 Weihai, Shandong	枝干Branch	2023-04	威海金Harlikar	7
21-27	SMX-3L, SMX-4L, SMX-5L, SMX-8L, SMX-9L, SMX-20L, SMX-80L	河南省三门峡市 Sanmenxia, Henan	枝干Branch	2023-03	-	-
28-36	CL-1L, CL-2L, CL-3L, CL-4L, CL-5L, CL-6L, CL-7L, CL-8L, CL-10L	河北省秦皇岛市 Qinhuangdao, Hebei	枝干Branch	2023-03	富士Fuji	8
37-40	BS-2L, BS-5L, BS-9L, BS-10L	陕西省渭南市Weinan, Shaanxi	枝干Branch	2023-03	富士Fuji	15
41	76	陕西省延安市Yan’an, Shaanxi	树皮Bark	2011-05	-	-
42-50	52, 68, 70, 72, 74, 99, 978, 1109, 99-3	河北省保定市Baoding, Hebei	枝干、果实、树皮Branch, fruit, bark	2016-04	-	-
51	77	河北省石家庄市Shijiazhuang, Hebei	树皮Bark	2024-05	-	-
52	53	河北省邯郸市Handan, Hebei	枝干Branch	2024-05	-	-
53	AKS	新疆阿克苏市Akesu, Xinjiang	枝干Branch	2016-04	-	-
54-55	TA-1, TA-2	山东省泰安市Taian, Shandong	枝干Branch	2024-05	-	-
56	QD	山东省青岛市Qingdao, Shandong	枝干Branch	2024-05	-	-
57-64	529, 530, 531, 532, 534, 536, 537, 539	日本未知地区 Unknown region in Japan	枝干、果实 Branch, fruit	2018-09	-	-

引物名称 Primer name	序列 Sequence (5′-3′)	片段大小 Product size (bp)	用途 Purpose	退火温度 Tm (℃)	来源 Source
BdCV1-F	TGCAATGCTGGATGTGTACG	516	BdCV1鉴定 BdCV1 identification	59.4	自行设计 Designed by authors
BdCV1-R	AGCGGTTTCTGCCAATTCG	516	BdCV1鉴定 BdCV1 identification	59.4	自行设计 Designed by authors
StPV-F	GGAGAACTAGACGACGGGTT	511	StPV鉴定 StPV identification	53.0	自行设计 Designed by authors
StPV-R	ACTTCGGTTCAGCGCTATCT	511	StPV鉴定 StPV identification	53.0	自行设计 Designed by authors
BfPV1-F	CTACACCAAAACCCCAACCG	531	BfPV1鉴定 BfPV1 identification	53.0	自行设计 Designed by authors
BfPV1-R	GGTCGGCAATCACACCTTTT	531	BfPV1鉴定 BfPV1 identification	53.0	自行设计 Designed by authors
PdPV-pa-F	CATCTGTGACGGACCGGTAT	565	PdPV-pa鉴定 PdPV-pa identification	53.0	自行设计 Designed by authors
PdPV-pa-R	CGCCTATCAACTTGTCCTGC	565	PdPV-pa鉴定 PdPV-pa identification	53.0	自行设计 Designed by authors
CfCV1-F	TTACTTGGAGCAGGAGGGTG	519	CfCV1鉴定 CfCV1 identification	53.0	自行设计 Designed by authors
CfCV1-R	ACACGAGCCCATTGATACCT	519	CfCV1鉴定 CfCV1 identification	53.0	自行设计 Designed by authors
CpMV1-F	ATGATCAGGTCCTTTGGGCA	525	CpMV1鉴定 CpMV1 identification	56.0	自行设计 Designed by authors
CpMV1-R	AAAACGCGGTCTGGATTCAC	525	CpMV1鉴定 CpMV1 identification	56.0	自行设计 Designed by authors
AOV-F	CGCAAGGTACGGTGGTTATG	541	AOV鉴定 AOV identification	56.0	自行设计 Designed by authors
AOV-R	CACCAACATTTCTGCGGGAT	541	AOV鉴定 AOV identification	56.0	自行设计 Designed by authors
BdVV2-F	CCGGTAACTGGGTCAACCTC	500	BdVV2鉴定 BdVV2 identification	59.4	[22]
BdVV2-R	CCGTGCGCTTCAATAAGTCG	500	BdVV2鉴定 BdVV2 identification	59.4	[22]

序列编号 Sequence number	病毒分类 Virus classification	同源序列登录号 Accession number	序列相似性 Sequence similarity (%)
2L-1	Chrysoviridae, Betachrysovirus	YP_010839425	96.09
2L-4	Chrysoviridae, Betachrysovirus	AJD14833.1	92.35
2L-5	Chrysoviridae, Betachrysovirus	AJD14833.1	92.26
2L-8	Chrysoviridae, Betachrysovirus	YP_009353026.1	97.70
2L-10	Chrysoviridae, Betachrysovirus	YP_009353028.1	91.04
2L-11	Chrysoviridae, unclassified Chrysoviridae	UVZ34692.1	87.50
2L-12	Chrysoviridae, unclassified Chrysoviridae	UVZ34692.1	91.16
2L-14	Chrysoviridae, Betachrysovirus	YP_009353026.1	96.20
2L-19	Chrysoviridae, Betachrysovirus	AJD14833.1	92.41
2L-20	Chrysoviridae, Betachrysovirus	AJD14833.1	92.26

编号 Number	种名 Species name	病毒分类 Virus classification	寄主 Host	病毒来源reads数目Virus reads number
1	产黄青霉病毒 Botryosphaeria dothidea chrysovirus 1 (BdCV1)	Chrysoviridae, Betachrysovirus	Botryosphaeria dothidea	13381204
2	葡萄座腔菌整体病毒2 Botryosphaeria dothidea victorivirus 2 (BdVV2)	Totiviridae, Victorivirus	Botryosphaeria dothidea	11509973
3	葡萄球菌病毒Staphylococcus virus 29 (StPV29)	Siphoviridae, Phietavirus	Staphylococcus aureus	24898
4	灰葡萄藻1型 Botryotinia fuckeliana partitivirus 1 (BfPV1)	Partitiviridae, unclassified Partitiviridae	Botrytis cinerea	6025
5	分枝杆菌病毒 Pseudogymnoascus destructans partitivirus-pa (PdPV-pa)	Partitiviridae, unclassified Partitiviridae	Pseudogymnoascus destructans	4010
6	刺盘孢菌病毒 Colletotrichum fructicola chrysovirus 1 (CfCV1)	Chrysoviridae, Betachrysovirus	Colletotrichum fructicola	848
7	有丝分裂病毒1 Cryphonectria mitovirus 1 (CpMV1)	Mitoviridae, Mitovirus	Cryphonectria parasitica	568
8	赭色曲霉病毒Aspergillus ochraceous virus (AOV)	Partitiviridae, Gammapartitivirus	Aspergillus ochraceous	222

序号 Number	采集地点 Collection site	BdCV1		BdVV2
序号 Number	采集地点 Collection site	阳性菌株数/总菌株数 Positive number/total number	检出率 Detection rate (%)	阳性菌株数/总菌株数 Positive number/total number	检出率 Detection rate (%)
1-10	辽宁地区Liaoning region	6/10	60	5/10	50
11-23	山东地区Shandong region	9/13	69	4/13	30
24-30	河南三门峡Sanmenxia, Henan	3/7	43	1/7	14
31-50	河北地区Hebei region	8/20	40	3/20	15
51-55	陕西地区Shaanxi region	3/5	60	3/5	60
56	新疆阿克苏Akesu, Xinjiang	1/1	100	0/1	0
总计Total		30/56	53.6	16/56	28.6

Identification of Hypovirus in Apple Ring Rot Fungus Botryosphaeria dothidea and Detection of Virus-Carrying Status in China

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