大丽轮枝菌微菌核形成的影响因素研究
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摘要
为了研究大丽轮枝菌微菌核形成的影响因素及其与微菌核形成相关基因之间的关系,本研究通过微菌核形态观察、重量测定及形成相关基因的表达测定,对不同温度、光照、营养及寄主根系诱导条件下,棉花大丽轮枝菌V592菌株微菌核的形成进行了分析。结果表明:以PDA为基质,26℃比22℃更有利于微菌核形成;连续黑暗的条件比自然光及连续光照更有利于微菌核形成;营养丰富的全营养PDA培养基比半营养PDA及水琼脂平板更有利于微菌核形成;棉花根系及其提取物对微菌核的形成具有促进作用。通过q RT-PCR对7个微菌核形成相关基因的表达量进行测定,结果显示,温度、光照及营养条件改变后12 h,7个基因在26℃时的表达量明显高于22℃时的表达量,6个基因在黑暗中的表达量明显高于连续光照条件下的表达量,6个基因在全营养中的表达量明显高于营养缺乏的培养基中的表达量,表明大丽轮枝菌微菌核的产量与温度、光照和营养条件改变后12 h各基因的表达量密切相关。而寄主根系对微菌核形成相关基因的表达在12 h时有短暂的抑制作用,但是48 h后各基因不断被诱导表达。以上结果表明大丽轮枝菌微菌核的产量与微菌核形成相关基因的诱导表达密切相关。
This research aimed to analyze influence factors on microsclerotial development in Verticillium dahliae and the correlation between influence factors and the transcript levels of seven genes involved in microsclerotial formation. Effects of different temperature, illumination and nutrient condition on microsclerotial development in V. dahliae isolate V592 from infected cotton were measured by morphologic observation and weight of microsclerotia. The results showed that V592 produced more microsclerotia under 26 ℃ than under 22 ℃, more microsclerotia under continuous dark than under natural light and continuous illumination, and more microsclerotia on full nutrition PDA medium than on half of nutrition PDA medium and on agar medium. V592 produced more microsclerotia on PDA medium with cotton root extract than on PDA medium, indicating that microsclerotial production by V592 was stimulated by cotton root. The transcript levels of seven genes involved in microsclerotial formation in V592 were determined by q RT-PCR. When the temperature, illumination and nutrition conditions were changed, the transcript levels of seven genes were higher under 26 ℃ than under 22 ℃ during the first 12 hours; the transcript levels of six genes were higher under continuous dark than under continuous illumination, and the transcript levels of six genes were higher on full nutrition PDA medium than on half of nutrition PDA medium and on agar medium, indicating that microsclerotial production by V592 was associated with transcript levels of genes involved in microsclerotial formation within 12 hours. The transcript of six genes within 12 hours were suppressed transiently by cotton root extract, but transcript of genes involved in microsclerotial formation after 48 hours were induced continuously by cotton root extract. In conclusion, microsclerotial production by V. dahliae was associated with inducible expression of genes involved in microsclerotial formation.
This research aimed to analyze influence factors on microsclerotial development in Verticillium dahliae and the correlation between influence factors and the transcript levels of seven genes involved in microsclerotial formation. Effects of different temperature, illumination and nutrient condition on microsclerotial development in V. dahliae isolate V592 from infected cotton were measured by morphologic observation and weight of microsclerotia. The results showed that V592 produced more microsclerotia under 26 ℃ than under 22 ℃, more microsclerotia under continuous dark than under natural light and continuous illumination, and more microsclerotia on full nutrition PDA medium than on half of nutrition PDA medium and on agar medium. V592 produced more microsclerotia on PDA medium with cotton root extract than on PDA medium, indicating that microsclerotial production by V592 was stimulated by cotton root. The transcript levels of seven genes involved in microsclerotial formation in V592 were determined by q RT-PCR. When the temperature, illumination and nutrition conditions were changed, the transcript levels of seven genes were higher under 26 ℃ than under 22 ℃ during the first 12 hours; the transcript levels of six genes were higher under continuous dark than under continuous illumination, and the transcript levels of six genes were higher on full nutrition PDA medium than on half of nutrition PDA medium and on agar medium, indicating that microsclerotial production by V592 was associated with transcript levels of genes involved in microsclerotial formation within 12 hours. The transcript of six genes within 12 hours were suppressed transiently by cotton root extract, but transcript of genes involved in microsclerotial formation after 48 hours were induced continuously by cotton root extract. In conclusion, microsclerotial production by V. dahliae was associated with inducible expression of genes involved in microsclerotial formation.
引文
[1]Klosterman S J,Atallah Z K,Vallad G E,et al.Diversity,pathogenicity,and management of verticillium species[J].Annual Review of Phytopathology,2009,47(1):39-62.
[2]Garas N A,Wilhem S,Sagen J E.Relationship to cultivar resistance to distribution of Verticillium dahliae in inoculated cotton plants and to growth to single conidia on excised stem segments[J].Phytopathology,1986,76(10):1005-1010.
[3]Pullman G S,Devay J E.Effect of soil flooding and paddy rice culture on the survival of Verticillium dahliae and incidence of Verticillium wilt in cotton[J].Phytopathology,1982,12(12):1285-1289.
[4]Klimes A,Dobinson K F.A hydrophobin gene,VDH1,is involved in microsclerotial development and spore viability in the plant pathogen Verticillium dahliae[J].Fungal Genetics&Biology,2006,43(4):283-294.
[5]Klimes A,Amyotte S G,Grant S,et al.Microsclerotia development in Verticillium dahliae:Regulation and differential expression of the hydrophobin gene VDH1[J].Fungal Genetics and Biology,2008,45(12):1525-1532.
[6]Zhang T,Jian-Hua,Zhao,et al.Host-induced gene silencing of the target gene in fungal cells confers effective resistaance to the cotton wilt disease pathogen Verticillium dahliae[J].Molecular Plant,2016,9(6):939-942.
[7]Rauyaree P,Ospinagiraldo M D,Kang S,et al.Mutations in VMK1,a mitogen-activated protein kinase gene,affect microsclerotia formation and pathogenicity in Verticillium dahliae[J].Current Genetics,2005,48(2):109-116.
[8]Tzima A K,Paplomatas E J,Rauyaree P,et al.Vd SNF1,the sucrose nonfermenting protein kinase gene of Verticillium dahliae,is required for virulence and expression of genes involved in cell-wall degradation[J].Molecular PlantMicrobe Interactions,2011,24(1),129-142.
[9]Gao F,Zhou B J,Li G Y,et al.A glutamic acidrich protein identified in Verticillium dahliae from an insertional mutagenesis affects microsclerotial formation and pathogenicity [J].PLoS One,2010,5(12):15319.
[10]Tzima A,Paplomatas E J,Rauyaree P,et al.Roles of the catalytic subunit of c AMP-dependent protein kinase A in virulence and development of the soilborne plant pathogen Verticillium dahliae[J].Fungal Genetics&Biology Fg&B,2010,47(5):406-15.
[11]Tzima A K,Paplomatas E J,Tsitsigiannis D I,et al.The G proteinβsubunit controls virulence and multiple growth and development-related traits in Verticillium dahliae[J].Fungal Genetics&Biology,2012,49(4):271-283.
[12]Ben-Yephet Y,Pinkas Y.Germination of individual microsclerotia of Verticillium dahliae[J].Phytoparasitica,1977,5(3):159-166.
[13]Paplomatas E J,Bassett D M,Broome J C,et al.Incidence of Verticillium wilt and yield losses of cotton cultivars based on soil inoculum density of Verticillium dahliae[J].Phytopathology,1993,82(12):1417-1420.
[14]De Vay J E,Pullman G S.Epidemiology and ecology of diseases caused by Verticillium species,with emphosis on Verticillium wilt of cotton[J].Phytopathologia Mediterranea,1984,23(2-3):95-108.
[15]杨家荣,商鸿生,高立强.土壤环境因素对棉花黄萎病菌微菌核存活的影响[J].植物病理学报,2004,34(2):180-183.Yang J R,Shang H S,Gao L Q.The effect of soil habitat factors of microsclerotia of Verticillium dahliae of cotton[J].Acta Phytophathologica Sinica,2004,34(2):180-183.
[16]Tjamos E C,Fravel D R.Detrimental effects of sublethal heating and Talaromyces flavus on microsclerotia of Verticillium dahliae[J].Phytopathology,1995,85(4):388-392.
[17]白应文,胡东芳,胡小平,等.大丽轮枝孢微菌核的形成条件[J].菌物学报,2011,30(5):695-701.Bai Y W,Hu D F,Hu X P,et al.Formation conditions for microsclerotia of Verticillium dahliae[J].Mycosystema,2011,30(5):695-701.
[18]张婷,张博森,刘启,等.不同致病力棉花黄萎病菌乙烯含量及相关基因表达量的测定[J].石河子大学学报(自然科学版),2016,34(5):632-637.Zhang T,Zhang B S,Liu Q,et al.Measurement of ethylene and transcript levels of genes involved in ethylene production by different virulent Verticillim dahliae from cotton[J].Journal of Shihezi University(Natural Science),2016,34(5):632-637.
[2]Garas N A,Wilhem S,Sagen J E.Relationship to cultivar resistance to distribution of Verticillium dahliae in inoculated cotton plants and to growth to single conidia on excised stem segments[J].Phytopathology,1986,76(10):1005-1010.
[3]Pullman G S,Devay J E.Effect of soil flooding and paddy rice culture on the survival of Verticillium dahliae and incidence of Verticillium wilt in cotton[J].Phytopathology,1982,12(12):1285-1289.
[4]Klimes A,Dobinson K F.A hydrophobin gene,VDH1,is involved in microsclerotial development and spore viability in the plant pathogen Verticillium dahliae[J].Fungal Genetics&Biology,2006,43(4):283-294.
[5]Klimes A,Amyotte S G,Grant S,et al.Microsclerotia development in Verticillium dahliae:Regulation and differential expression of the hydrophobin gene VDH1[J].Fungal Genetics and Biology,2008,45(12):1525-1532.
[6]Zhang T,Jian-Hua,Zhao,et al.Host-induced gene silencing of the target gene in fungal cells confers effective resistaance to the cotton wilt disease pathogen Verticillium dahliae[J].Molecular Plant,2016,9(6):939-942.
[7]Rauyaree P,Ospinagiraldo M D,Kang S,et al.Mutations in VMK1,a mitogen-activated protein kinase gene,affect microsclerotia formation and pathogenicity in Verticillium dahliae[J].Current Genetics,2005,48(2):109-116.
[8]Tzima A K,Paplomatas E J,Rauyaree P,et al.Vd SNF1,the sucrose nonfermenting protein kinase gene of Verticillium dahliae,is required for virulence and expression of genes involved in cell-wall degradation[J].Molecular PlantMicrobe Interactions,2011,24(1),129-142.
[9]Gao F,Zhou B J,Li G Y,et al.A glutamic acidrich protein identified in Verticillium dahliae from an insertional mutagenesis affects microsclerotial formation and pathogenicity [J].PLoS One,2010,5(12):15319.
[10]Tzima A,Paplomatas E J,Rauyaree P,et al.Roles of the catalytic subunit of c AMP-dependent protein kinase A in virulence and development of the soilborne plant pathogen Verticillium dahliae[J].Fungal Genetics&Biology Fg&B,2010,47(5):406-15.
[11]Tzima A K,Paplomatas E J,Tsitsigiannis D I,et al.The G proteinβsubunit controls virulence and multiple growth and development-related traits in Verticillium dahliae[J].Fungal Genetics&Biology,2012,49(4):271-283.
[12]Ben-Yephet Y,Pinkas Y.Germination of individual microsclerotia of Verticillium dahliae[J].Phytoparasitica,1977,5(3):159-166.
[13]Paplomatas E J,Bassett D M,Broome J C,et al.Incidence of Verticillium wilt and yield losses of cotton cultivars based on soil inoculum density of Verticillium dahliae[J].Phytopathology,1993,82(12):1417-1420.
[14]De Vay J E,Pullman G S.Epidemiology and ecology of diseases caused by Verticillium species,with emphosis on Verticillium wilt of cotton[J].Phytopathologia Mediterranea,1984,23(2-3):95-108.
[15]杨家荣,商鸿生,高立强.土壤环境因素对棉花黄萎病菌微菌核存活的影响[J].植物病理学报,2004,34(2):180-183.Yang J R,Shang H S,Gao L Q.The effect of soil habitat factors of microsclerotia of Verticillium dahliae of cotton[J].Acta Phytophathologica Sinica,2004,34(2):180-183.
[16]Tjamos E C,Fravel D R.Detrimental effects of sublethal heating and Talaromyces flavus on microsclerotia of Verticillium dahliae[J].Phytopathology,1995,85(4):388-392.
[17]白应文,胡东芳,胡小平,等.大丽轮枝孢微菌核的形成条件[J].菌物学报,2011,30(5):695-701.Bai Y W,Hu D F,Hu X P,et al.Formation conditions for microsclerotia of Verticillium dahliae[J].Mycosystema,2011,30(5):695-701.
[18]张婷,张博森,刘启,等.不同致病力棉花黄萎病菌乙烯含量及相关基因表达量的测定[J].石河子大学学报(自然科学版),2016,34(5):632-637.Zhang T,Zhang B S,Liu Q,et al.Measurement of ethylene and transcript levels of genes involved in ethylene production by different virulent Verticillim dahliae from cotton[J].Journal of Shihezi University(Natural Science),2016,34(5):632-637.