Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (22): 4787-4799.doi: 10.3864/j.issn.0578-1752.2021.22.007

• PLANT PROTECTION • Previous Articles     Next Articles

Analysis of dsRNA Carried by Alternaria alternata f. sp. mali in China and Identification of a dsRNA Virus

CAO YuHan(),LI ZiTeng(),ZHANG JingYi,ZHANG JingNa,HU TongLe,WANG ShuTong,WANG YaNan(),CAO KeQiang()   

  1. College of Plant Protection, Hebei Agricultural University, Baoding 071001, Hebei
  • Received:2021-04-06 Accepted:2021-05-10 Online:2021-11-16 Published:2021-11-19
  • Contact: YaNan WANG,KeQiang CAO E-mail:c897836047@163.com;lzt304838566@163.com;wyn3215347@163.com;cao_keqiang@163.com

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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

Table 1

The information of apple Alternaria blotch samples in this study"

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

Table 2

Primers used in this study"

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
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

Fig. 1

DsRNA types of A. alternata f. sp. mali in China M: DL8000; 1: YT-3-7; 2: SJZ-4; 3: QY-2; 4: CL-2-6; 5: SQ-1-1"

Fig. 2

Cultivation type I of A. alternata f. sp. mali strains"

Fig. 3

Cultivation type II of A. alternata f. sp. mali strains"

Fig. 4

The mycelial growth rate of different A. alternata f. sp. mali strains"

Fig. 5

Spots size of different strains of A. alternata f. sp. mali on cherry-apple leaves and apple fruits"

Fig. 6

Pathogenicity of different strains of A. alternata f. sp. mali on cherry-apple leaves"

Fig. 7

Pathogenicity of different strains of A. alternata f. sp. mali on apple fruits"

Table 3

Virus information in A. alternata f. sp. mali strain QY-2 by high-throughput sequencing"

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

Fig. 8

A schematic diagram of the genomic organization of A. alternata f. sp. mali QY-2 dsRNA virus"

Table 4

Similarity of nucleotide and amino acid sequences between A. alternata f. sp. mali strain QY-2 and other mycoviruses"

名称
Name		 ORF1 ORF2 ORF3 ORF4 ORF5
nt (%) aa (%) nt (%) aa (%) nt (%) aa (%) nt (%) aa (%) nt (%) aa (%)
AaCV1 77 93 75 91 70 80 76 90 95 87
BdCV1 59 56 54 47 40 35 51 47
AtCV1 57 55 50 44 42 32 50 43
PjCV2 54 52 49 40 42 32 56 43

Fig. 9

Phylogenetic tree based on RNA-dependent RNA polymerase amino acid sequence of mycovirus"

Fig. 10

Identification of AaCV2 in A. alternata f. sp. mali strain QY-2 by RT-PCR"

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