柑橘叶点霉属(Phyllosticta spp.)真菌的遗传多样性分析
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摘要
利用筛选的11条ISSR引物对从我国柑橘主产区和国外收集的135株叶点霉属真菌菌株进行扩增,扩增产物进行凝胶电泳,扩增图谱用NTSYS-pc2.10软件进行群体聚类分析。结果显示,共扩增出116个DNA条带,其中多态性位点为112个,多态率为96.55%;平均每个引物可以扩增出10.55个条带,扩增产物大小在250~3 000 bp。聚类分析显示,中国柑橘叶点霉属真菌可以分为4个种,即P.citricarpa、P.citriasiana、P.capitalensis和P.citrichinaensis。以遗传相似性系数0.97为阈值时,柑橘黑斑病病原菌(P.citricarpa)种群可分为5个亚类,subclade-I的菌株来自中国的本地早、温州蜜柑、槾橘和南丰蜜橘,subclade-II的菌株均来自中国的砂糖橘,subclade-III的菌株来自非洲莫桑比克柠檬和葡萄柚、佛罗里达甜橙、南非甜橙和来自杭州市场上进口的澳橘,subclade-IV的菌株来自中国的甜橙,subclade-V的菌株来自中国的柠檬,表明我国柑橘黑斑病病原菌具有丰富的遗传变异,其遗传变异与寄主相关;我国与国外的柑橘黑斑病病原菌在起源上可能存在差异。P.citriasiana、P.capitalensis和P.citrichinaensis种群内也存在遗传变异,但其遗传变异与地理分布和寄主未发现相关性。该研究结果为研究柑橘叶点霉属真菌的遗传多样性奠定了基础。
One hundred and thirty-five Phyllosticta isolates collected from main citrus producing regions across China,and Africa,Australian,America were amplified using 11 ISSR primers. PCR products were electrophoresed at 95 V in a 1.5% agarose gel for 90 min,and then stained with ethidium bromide. The clustering analysis was performed using NTSYSpc version 2.10 software. The amplification results showed that 116 fragments were amplified,and 112 fragments displayed polymorphism that accounted for 96.55% in the total amplified fragments.The average number of bands amplified per primer was 10.55 and the length of the amplified fragments ranged from 250 to 3 000 bp. UPGMA cluster analysis based on ISSR data showed that Phyllosticta isolates collected from the citrus main producing regions in China were divided into four groups,that were associated with species of P. citricarpa,P. citriasiana,P. capitalensis and P. citrichinaensis,respectively. The group of P. citricarpa was divided into 5 subclades at a level of 0.97 similarity coefficient. Subclade I consisted of 28 isolates was collected from Citrus reticulata cv. Bendizao,Citrus unshiu,C. reticulata cv. Manju and C. reticulata cv.Nanfengmiju. Subclade II consisted of 5 isolates was collected from C. reticulata cv. Shatangju. Subclade III consisted of 7 isolates was collected from Mozambique lemon and grapefruit,American orange,South Africa orangeand Australian citrus. Subclade IV consisted of 13 isolates was collected from sweet orange in China. Subclade V consisted of 9 isolates was collected from lemon in China. These results showed that isolates of P. citricarpa collected from China had abundant genetic variation,and the genetic variation of P. citricarpa was relevant to its hosts. It may be different in origin between the pathogen of citrus black spot in China and the pathogen of citrus black spot in foreign countries.There was genetic variation within populations of P. citriasiana,P. capitalensis and P. citrichinaensis,but the genetic variation might be not relevant to its geographic distribution or hosts. The results would lay the foundation for the study of the genetic diversity of Phyllosticta species associated with citrus.
One hundred and thirty-five Phyllosticta isolates collected from main citrus producing regions across China,and Africa,Australian,America were amplified using 11 ISSR primers. PCR products were electrophoresed at 95 V in a 1.5% agarose gel for 90 min,and then stained with ethidium bromide. The clustering analysis was performed using NTSYSpc version 2.10 software. The amplification results showed that 116 fragments were amplified,and 112 fragments displayed polymorphism that accounted for 96.55% in the total amplified fragments.The average number of bands amplified per primer was 10.55 and the length of the amplified fragments ranged from 250 to 3 000 bp. UPGMA cluster analysis based on ISSR data showed that Phyllosticta isolates collected from the citrus main producing regions in China were divided into four groups,that were associated with species of P. citricarpa,P. citriasiana,P. capitalensis and P. citrichinaensis,respectively. The group of P. citricarpa was divided into 5 subclades at a level of 0.97 similarity coefficient. Subclade I consisted of 28 isolates was collected from Citrus reticulata cv. Bendizao,Citrus unshiu,C. reticulata cv. Manju and C. reticulata cv.Nanfengmiju. Subclade II consisted of 5 isolates was collected from C. reticulata cv. Shatangju. Subclade III consisted of 7 isolates was collected from Mozambique lemon and grapefruit,American orange,South Africa orangeand Australian citrus. Subclade IV consisted of 13 isolates was collected from sweet orange in China. Subclade V consisted of 9 isolates was collected from lemon in China. These results showed that isolates of P. citricarpa collected from China had abundant genetic variation,and the genetic variation of P. citricarpa was relevant to its hosts. It may be different in origin between the pathogen of citrus black spot in China and the pathogen of citrus black spot in foreign countries.There was genetic variation within populations of P. citriasiana,P. capitalensis and P. citrichinaensis,but the genetic variation might be not relevant to its geographic distribution or hosts. The results would lay the foundation for the study of the genetic diversity of Phyllosticta species associated with citrus.
引文
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[9]Baayen R P,Bonants P J M,Verkley G,et al.Nonpathogenic isolates of the citrus black spot fungus,Guignardia citricarpa,identified as a cosmopolitan endophyte of woody plants,G.mangiferae(Phyllosticta capitalensis)[J].Phytopathology,2002,92(5):464-477.
[10]Zavala M G M,Er H L,Goss E M,et al.Genetic variation among Phyllosticta strains isolated from citrus in Florida that are pathogenic or nonpathogenic to citrus[J].Tropical Plant Pathology,2014,39(2):119-128.
[11]Zietkiewicz E,Rafalski A,Labuda D.Genome fingerprinting by simple sequence repeat(SSR)-anchored polymerase chain reaction amplification[J].Genomics,1994,20(2):176-183.
[12]Xu Y M,Liu X M,Wang J M.Applications of ISSRin the researches of the fungal plant pathogens(in Chinese)[J].Chinese Agricultural Science Bulletin(中国农学通报),2011,27(9):358-361.
[13]Zhou S G,Smith D R,Stanosz G R.Differentiation of Botryosphaeria species and related anamorphic fungi using inter simple or short sequence repeat(ISSR)fingerprinting[J].Mycological research,2001,105(8):919-926.
[14]Wang X H,Li H Y.Optimization of inter-simple sequence repeat PCR reaction system of Phyllosticta citricarpa(in Chinese)[J].Acta agriculturae boreali-sinica(华北农学报),2016,31(1):123-127.
[15]Rohlf F J.Ntsys-pc:Numerical taxonomy and multivariate analysis system version 2.1[Z].Exeter Software,Setauket,New York.2000.
[16]Slippers B,Crous P W,Denman S,et al.Combined multiple gene genealogies and phenotypic characters differentiate several species previously identified as Botryosphaeria dothidea[J].Mycologia,2004,96(1):83-101.
[17]Phillips A,Alves A,Correia A,et al.Two new species of Botryosphaeria with brown,1-septate ascospores and Dothiorella anamorphs[J].Mycologia,2005,97(2):513-529.
[18]Prihastuti H,Cai L,Chen H,et al.Characterization of Colletotrichum species associated with coffee berries in northern Thailand[J].Fungal Diversity,2009,39:89-109.
[19]Motohashi K,Inaba S,Anzai K,et al.Phylogenetic analyses of Japanese species of Phyllosticta sensu stricto[J].Mycoscience,2009,50(4):291-302.
[2]Junior G J S,da Silva Scapin M,Silva F P,et al.Spray volume and fungicide rates for citrus black spot control based on tree canopy volume[J].Crop Protection,2016,85:38-45.
[3]Schubert T S,Dewdney M M,Peres N A,et al.First report of Guignardia citricarpa associated with citrus black spot on sweet orange(Citrus sinensis)in North America[J].Plant Disease,2012,96(8):1225.
[4]EPPO.PM 7/17(2):Guignardia citricarpa[J].EPPOBulletin,2009,39(3):318-327.
[5]Baker R,Bragard C,Candresse T,et al.Scientific opinion on the risk of Phyllosticta citricarpa(Guignardia citricarpa)for the EU territory with identification and evaluation of risk reduction options[J].EFSAJournal,2014,12(2):3557.
[6]Glienke C,Pereira O L,Stringari D,et al.Endophytic and pathogenic Phyllosticta species,with reference to those associated with Citrus Black Spot[J].Persoonia-Molecular Phylogeny and Evolution of Fungi,2011,26:47-56.
[7]Wulandari N F,To-anun C,Hyde K D,et al.Phyllosticta citriasiana sp.nov.,the cause of Citrus tan spot of Citrus maxima in Asia[J].Fungal Diversity,2009,34(1):23-39.
[8]Wang X H,Chen G Q,Huang F,et al.Phyllosticta species associated with citrus diseases in China[J].Fungal Diversity,2012,52(1):209-224.
[9]Baayen R P,Bonants P J M,Verkley G,et al.Nonpathogenic isolates of the citrus black spot fungus,Guignardia citricarpa,identified as a cosmopolitan endophyte of woody plants,G.mangiferae(Phyllosticta capitalensis)[J].Phytopathology,2002,92(5):464-477.
[10]Zavala M G M,Er H L,Goss E M,et al.Genetic variation among Phyllosticta strains isolated from citrus in Florida that are pathogenic or nonpathogenic to citrus[J].Tropical Plant Pathology,2014,39(2):119-128.
[11]Zietkiewicz E,Rafalski A,Labuda D.Genome fingerprinting by simple sequence repeat(SSR)-anchored polymerase chain reaction amplification[J].Genomics,1994,20(2):176-183.
[12]Xu Y M,Liu X M,Wang J M.Applications of ISSRin the researches of the fungal plant pathogens(in Chinese)[J].Chinese Agricultural Science Bulletin(中国农学通报),2011,27(9):358-361.
[13]Zhou S G,Smith D R,Stanosz G R.Differentiation of Botryosphaeria species and related anamorphic fungi using inter simple or short sequence repeat(ISSR)fingerprinting[J].Mycological research,2001,105(8):919-926.
[14]Wang X H,Li H Y.Optimization of inter-simple sequence repeat PCR reaction system of Phyllosticta citricarpa(in Chinese)[J].Acta agriculturae boreali-sinica(华北农学报),2016,31(1):123-127.
[15]Rohlf F J.Ntsys-pc:Numerical taxonomy and multivariate analysis system version 2.1[Z].Exeter Software,Setauket,New York.2000.
[16]Slippers B,Crous P W,Denman S,et al.Combined multiple gene genealogies and phenotypic characters differentiate several species previously identified as Botryosphaeria dothidea[J].Mycologia,2004,96(1):83-101.
[17]Phillips A,Alves A,Correia A,et al.Two new species of Botryosphaeria with brown,1-septate ascospores and Dothiorella anamorphs[J].Mycologia,2005,97(2):513-529.
[18]Prihastuti H,Cai L,Chen H,et al.Characterization of Colletotrichum species associated with coffee berries in northern Thailand[J].Fungal Diversity,2009,39:89-109.
[19]Motohashi K,Inaba S,Anzai K,et al.Phylogenetic analyses of Japanese species of Phyllosticta sensu stricto[J].Mycoscience,2009,50(4):291-302.