来源于湖北省梨轮纹病菌中真菌病毒多样性检测及其分析
|
摘要
一些真菌病毒严重影响寄主生长、产孢量、致病力。本研究对来源于湖北梨轮纹病菌携带真菌病毒情况进行分析,旨在挖掘具有生防潜能的真菌病毒资源。随机采集湖北省梨树发病枝干,分离、鉴定获得13个梨轮纹病菌株,其生长速率、致病性存在差异。提取dsRNA检测,发现12个菌株均携带不同大小的dsRNA片段。观察其dsRNA带型,HBWH-14 dsRNA含有1～7 kb 9条带,其他菌株携带的dsRNA条带位置均位于HBWH-14所含dsRNA带型中的某1条或多条对应的条带位置。RT-PCR检测发现部分菌株携带产黄青霉病毒BdCV1、双分体病毒BdPV1、单分体病毒BdTV1以及未分类的BdRV1。本研究未发现梨轮纹菌株所含病毒种类与致病力存在直接的相关性。HBWH-14原生质体脱毒获得后代100%携带的dsRNAs稳定遗传。
Some mycoviruses have effect on growth rate,sporulation ability,pathogenicity biological characteristics of fungi host. Aims at detection of dsRNA mycoviruses infecting Botryosphaeria dothidea strains collected from Hubei province,which are to be explored as bio-control resources. The obtained results are the followings.13 strains were isolated and cultured from pear branches with symptoms of ring spot disease to some extent in Wuhan city of Hubei province. The growth rate showed differences among 13 B. dothidiea strains,which exhibit different virulence. The patterns of dsRNAs were detected. The results show that 12 B. dothidiea strains have different sizes of molecular weight. The dsRNA profiles analysis indicate that the dsRNAs from HBWH-14 strain contain 9 bands with a molecular weight range of 1-7 kb,meanwhile the other strains exhibit one or more than dsRNA bands in accordance with that of HBWH-14 strain. In total,single or mixed infection with BdCV1,BdPV1,BdRV1 and a new member of Totiviridae( designated as BdTV1) are positive for the above one or more strains by RT-PCR detection; There was no correlation between mycoviruses and pathogenicity for 13 strains.dsRNAs from HBWH-14 regenerated strains are 100% positive by protoplast isolation.
Some mycoviruses have effect on growth rate,sporulation ability,pathogenicity biological characteristics of fungi host. Aims at detection of dsRNA mycoviruses infecting Botryosphaeria dothidea strains collected from Hubei province,which are to be explored as bio-control resources. The obtained results are the followings.13 strains were isolated and cultured from pear branches with symptoms of ring spot disease to some extent in Wuhan city of Hubei province. The growth rate showed differences among 13 B. dothidiea strains,which exhibit different virulence. The patterns of dsRNAs were detected. The results show that 12 B. dothidiea strains have different sizes of molecular weight. The dsRNA profiles analysis indicate that the dsRNAs from HBWH-14 strain contain 9 bands with a molecular weight range of 1-7 kb,meanwhile the other strains exhibit one or more than dsRNA bands in accordance with that of HBWH-14 strain. In total,single or mixed infection with BdCV1,BdPV1,BdRV1 and a new member of Totiviridae( designated as BdTV1) are positive for the above one or more strains by RT-PCR detection; There was no correlation between mycoviruses and pathogenicity for 13 strains.dsRNAs from HBWH-14 regenerated strains are 100% positive by protoplast isolation.
引文
[1]Zhai L F,Zhang M X,Lv G,et al.Biological and molecular characterization of four Botryosphaeria species isolated from pear plants showing ring rot and stem canker in China[J].Plant Disease,2014,98(6):716-726.
[2]Xu C,Wang C S,Sun X,et al.Multiple group I introns in the small-subunit rDNA of Botryosphaeria dothidea:implication for intra specific genetic diversity[J].PLoS ONE,2013,8(7):e67808.
[3]Xu C,Wang C S,Ju L L,et al.Multiple locus genealogies and phenotypic characters reappraise the causal agents of apple ring rot in China[J].Fungal Diversity,2015,71(1):215-231.
[4]He K P,Zhang J W,Wu C.Research progress review on pear ring rot(in Chinese)[J].China Plant Protection(中国植保导刊),2013,33(6):21-25.
[5]Zhao J,Zhao B M,Lu X L,et al.Biological characteristics of Macrophoma kuwatsukai Hara and foxicity of fungicides(in Chinese)[J].Journal of Shanghai Jiaotong University(上海交通大学学报),2016,34(3):79-84.
[6]Fan H,Ru J,Zhang Y,et al.Fengycin produced by Bacillus subtilis 9407 plays a major role in the biocontrol of apple ring rot disease[J].Microbiological Research,2017,199:89-97.
[7]Holgs M.Viruses associated with A Die-back disease of cultivated mushroom[J].Nature,1962,196(4858):962-965.
[8]Anagnostakis S L.Biological control of chestnut blight[J].Science,1982,215(4532):466-471.
[9]Nuss D L.Biological control of chestnut blight:an example of virus-mediated attenuation of fungal pathogenesis[J].Microbiological Reviews,1992,56(4):561-576.
[10]Eusebio-CopeA,Sun L Y,Tanaka T,et al.The chestnut blight fungus for studies on virus/host and virus/virus interactions:From a natural to a model host[J].Virology,2015,477:164-175.
[11]Wang L P,Jiang J J,Wang Y F,et al.Hypovirulence of the phytopathogenic fungus Botryosphaeria dothidea:association with a coinfecting chrysovirus and a partitivirus[J].Journal of Virology,2014,88(13):7517-7527.
[12]Zhai L F,Xiang J,Zhang M X,et al.Characterization of a novel double-stranded RNA mycovirus conferring hypovirulence from the phytopathogenic fungus Botryosphaeria dothidea[J].Virology,2016,493:75-85.
[13]Zhai L F,Hong N,Zhang M X,et al.Complete dsRNAsequence of a novel victorivirus isolated from the pear stem wart fungus Botryosphaeria dothidea[J].Archives of Virology,2015,160(2):613-616.
[14]Wang L H,Luo H,Wang G P,et al.The study for the effect of Botryosphaeria dothidea Chrysovirus 1(designated as BdCV1)isolate belonging to the member of the Chrysoviridae family,on growth and pathogenicity of Botryosphaeria dothidea strain infection with pear(in Chinese)[J].Journal of Fruit Science(果树学报),2017,(10):1330-1339.
[15]White T J,Bruns T,Lee S,et al.Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics[A].PCR Protocols[C].Holland:Elsevier,1990:315-322.
[16]Ignazio C,Linda M K.A method for designing primer sets for speciation studies in filamentous ascomycetes[J].Mycologia,1999,91(3):553-556.
[17]Glass N L,Donaldson G C.Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes[J].Applied and Environmental Microbiology,1995,61(4):1323-1330.
[18]Freeman S,Katan T,Shabi E.Characterization of Colletotrichum gloeosporioides isolates from avocado and almond fruits with molecular and pathogenicity tests[J].Applied And Environmental Microbiology,1996,62(3):1014-1020.
[19]Yang F,Hong N,Wang G P.Mycovirus dsRNA extraction kit and its application:ZL201310072994.3[P].2015-06-03.
[20]Wu M D.Study of mycovirus from Botrytis spp.(in Chinese)[D].Wuhan:Huazhong Agricultural University(武汉:华中农业大学),2012.
[21]Cheng L,Lun Y Y,Sun G W,et al.Optimization of protoplast preparation and regeneration conditions of Valsa mali var.mali(in Chinese)[J].Shandong Agricultural Sciences(山东农业科学),2014,46(8):109-112.
[22]Phillips A J L,Alves A,Abdollahzadeh J,et al.The Botryosphaeriaceae:genera and species known from culture[J].Studies in Mycology,2013,76(1):51-167.
[23]Li X G,Yang Q S,Lin J,et al.Study on resistances of pear varieties to branch ring-rot disease and their inheritance(in Chinese)[J].Acta Agriculturae Jiangxi(江西农业学报),2009,21(11):59-60.
[24]Li M T,Occurrencelaws and prevention and control technology of pear ring rot(in Chinese)[J].Journal of Agricultural Catastrophology(农业灾害研究),2013,3(4):25-28.
[25]Chiba S,Salaipeth L,Lin Y H,et al.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[J].Journal of Virology,2009,83(24):12801-12812.
[26]Salaipeth L,Chiba S,Eusebio-Cope A,et al.Biological properties and expression strategy of Rosellinia necatrix megabirnavirus 1 analysed in an experimental host,Cryphonectria parasitica[J].Journal of General Virology,2014,95(3):740-750.
[27]Liu S,Xie J T,Cheng J S,et al.Fungal DNA virus infects a mycophagous insect and utilizes it as a transmission vector[J].Proceedings of the National Academy of Sciences of the United States of America,2016,113(45):12803-12808.
[28]Xie J,Jiang D.New insights into mycoviruses and exploration for the biological control of crop fingal disese[J].Annual Review Phytopathology,2014,52:45-68.责任编辑:于金枝
[2]Xu C,Wang C S,Sun X,et al.Multiple group I introns in the small-subunit rDNA of Botryosphaeria dothidea:implication for intra specific genetic diversity[J].PLoS ONE,2013,8(7):e67808.
[3]Xu C,Wang C S,Ju L L,et al.Multiple locus genealogies and phenotypic characters reappraise the causal agents of apple ring rot in China[J].Fungal Diversity,2015,71(1):215-231.
[4]He K P,Zhang J W,Wu C.Research progress review on pear ring rot(in Chinese)[J].China Plant Protection(中国植保导刊),2013,33(6):21-25.
[5]Zhao J,Zhao B M,Lu X L,et al.Biological characteristics of Macrophoma kuwatsukai Hara and foxicity of fungicides(in Chinese)[J].Journal of Shanghai Jiaotong University(上海交通大学学报),2016,34(3):79-84.
[6]Fan H,Ru J,Zhang Y,et al.Fengycin produced by Bacillus subtilis 9407 plays a major role in the biocontrol of apple ring rot disease[J].Microbiological Research,2017,199:89-97.
[7]Holgs M.Viruses associated with A Die-back disease of cultivated mushroom[J].Nature,1962,196(4858):962-965.
[8]Anagnostakis S L.Biological control of chestnut blight[J].Science,1982,215(4532):466-471.
[9]Nuss D L.Biological control of chestnut blight:an example of virus-mediated attenuation of fungal pathogenesis[J].Microbiological Reviews,1992,56(4):561-576.
[10]Eusebio-CopeA,Sun L Y,Tanaka T,et al.The chestnut blight fungus for studies on virus/host and virus/virus interactions:From a natural to a model host[J].Virology,2015,477:164-175.
[11]Wang L P,Jiang J J,Wang Y F,et al.Hypovirulence of the phytopathogenic fungus Botryosphaeria dothidea:association with a coinfecting chrysovirus and a partitivirus[J].Journal of Virology,2014,88(13):7517-7527.
[12]Zhai L F,Xiang J,Zhang M X,et al.Characterization of a novel double-stranded RNA mycovirus conferring hypovirulence from the phytopathogenic fungus Botryosphaeria dothidea[J].Virology,2016,493:75-85.
[13]Zhai L F,Hong N,Zhang M X,et al.Complete dsRNAsequence of a novel victorivirus isolated from the pear stem wart fungus Botryosphaeria dothidea[J].Archives of Virology,2015,160(2):613-616.
[14]Wang L H,Luo H,Wang G P,et al.The study for the effect of Botryosphaeria dothidea Chrysovirus 1(designated as BdCV1)isolate belonging to the member of the Chrysoviridae family,on growth and pathogenicity of Botryosphaeria dothidea strain infection with pear(in Chinese)[J].Journal of Fruit Science(果树学报),2017,(10):1330-1339.
[15]White T J,Bruns T,Lee S,et al.Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics[A].PCR Protocols[C].Holland:Elsevier,1990:315-322.
[16]Ignazio C,Linda M K.A method for designing primer sets for speciation studies in filamentous ascomycetes[J].Mycologia,1999,91(3):553-556.
[17]Glass N L,Donaldson G C.Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes[J].Applied and Environmental Microbiology,1995,61(4):1323-1330.
[18]Freeman S,Katan T,Shabi E.Characterization of Colletotrichum gloeosporioides isolates from avocado and almond fruits with molecular and pathogenicity tests[J].Applied And Environmental Microbiology,1996,62(3):1014-1020.
[19]Yang F,Hong N,Wang G P.Mycovirus dsRNA extraction kit and its application:ZL201310072994.3[P].2015-06-03.
[20]Wu M D.Study of mycovirus from Botrytis spp.(in Chinese)[D].Wuhan:Huazhong Agricultural University(武汉:华中农业大学),2012.
[21]Cheng L,Lun Y Y,Sun G W,et al.Optimization of protoplast preparation and regeneration conditions of Valsa mali var.mali(in Chinese)[J].Shandong Agricultural Sciences(山东农业科学),2014,46(8):109-112.
[22]Phillips A J L,Alves A,Abdollahzadeh J,et al.The Botryosphaeriaceae:genera and species known from culture[J].Studies in Mycology,2013,76(1):51-167.
[23]Li X G,Yang Q S,Lin J,et al.Study on resistances of pear varieties to branch ring-rot disease and their inheritance(in Chinese)[J].Acta Agriculturae Jiangxi(江西农业学报),2009,21(11):59-60.
[24]Li M T,Occurrencelaws and prevention and control technology of pear ring rot(in Chinese)[J].Journal of Agricultural Catastrophology(农业灾害研究),2013,3(4):25-28.
[25]Chiba S,Salaipeth L,Lin Y H,et al.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[J].Journal of Virology,2009,83(24):12801-12812.
[26]Salaipeth L,Chiba S,Eusebio-Cope A,et al.Biological properties and expression strategy of Rosellinia necatrix megabirnavirus 1 analysed in an experimental host,Cryphonectria parasitica[J].Journal of General Virology,2014,95(3):740-750.
[27]Liu S,Xie J T,Cheng J S,et al.Fungal DNA virus infects a mycophagous insect and utilizes it as a transmission vector[J].Proceedings of the National Academy of Sciences of the United States of America,2016,113(45):12803-12808.
[28]Xie J,Jiang D.New insights into mycoviruses and exploration for the biological control of crop fingal disese[J].Annual Review Phytopathology,2014,52:45-68.责任编辑:于金枝