草地早熟禾及其矮化突变材料锈病病原菌鉴定及抗病机制初探
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摘要
锈病是草坪草的重要病害,且病原菌种类多,鉴定困难。本研究以草地早熟禾野生型和矮化突变材料中的感病株系为对象,通过观察锈菌的形态特征,并结合ITS和β-tubulin基因序列分析,其中ITS序列的同源比对分析和β-tubulin基因的分子进化分析结果表明,该菌种与小麦禾柄锈菌的同源关系接近,所以初步认定该菌株属于禾柄锈菌,这是国内对禾柄锈菌引起草地早熟禾锈病的首次报道。同时,我们对病原菌诱导后草地早熟禾PR1L、NPR1L基因的表达变化和PRs蛋白表达进行了研究,发现PR1L、NPR1L基因在病菌诱导12 h时的表达量达到了峰值,且在矮化突变植株(A16)中的相对表达量分别达到了8.8-fold、4.5-fold,均大于在对照植株(WT)中的表达量。另外植物的PRs蛋白在禾柄锈菌侵染植物第8天后的表达量明显高于未侵染的植株。初步对草地早熟禾锈病的防御机制进行了探讨,为今后开展草地早熟禾锈病的预防及抗病育种研究奠定基础。
Rust is one of the destructive diseases on turfgrass. The species of rust pathogens are multiple and difficult to identify. In order to determine the rust pathogen species on Kentucky bluegrass wild type and dwarf mutant plant,pathogen morphology,along with ITS and β-tubulin gene sequence analysis were carried out. The analysis of ITS by BLAST and phylogenetic analysis of β-tubulin showed that the strains were related to Puccinia graminis of wheat,so we identified it as Puccinia graminis,which is the first report on Kentucky bluegrass in China. Moreover,transcriptional level of PR1L and NPR1L genes in Kentucky bluegrass inoculated with Puccinia graminis,as well as the expression of PRs protein,were investigated. The results presented that the expression of PR1L and NPR1L genes reached peak at 12 hours induced by Puccinia graminis,and the fold changes in dwarf mutant(A16) were 8. 8 and 4. 5,respectively,greater than in control wild type(WT). Besides,the expression of PRs in P. graminis infected plants 8 days later was higher than in non-infected plants. Defense mechanism of Kentucky bluegrass to rust was preliminary investigated and it presented the related disease-resistant gene and protein induced by rust pathogen. The research provides foundation for rust prevention and disease resistance breeding on Kentucky bluegrass in future.
Rust is one of the destructive diseases on turfgrass. The species of rust pathogens are multiple and difficult to identify. In order to determine the rust pathogen species on Kentucky bluegrass wild type and dwarf mutant plant,pathogen morphology,along with ITS and β-tubulin gene sequence analysis were carried out. The analysis of ITS by BLAST and phylogenetic analysis of β-tubulin showed that the strains were related to Puccinia graminis of wheat,so we identified it as Puccinia graminis,which is the first report on Kentucky bluegrass in China. Moreover,transcriptional level of PR1L and NPR1L genes in Kentucky bluegrass inoculated with Puccinia graminis,as well as the expression of PRs protein,were investigated. The results presented that the expression of PR1L and NPR1L genes reached peak at 12 hours induced by Puccinia graminis,and the fold changes in dwarf mutant(A16) were 8. 8 and 4. 5,respectively,greater than in control wild type(WT). Besides,the expression of PRs in P. graminis infected plants 8 days later was higher than in non-infected plants. Defense mechanism of Kentucky bluegrass to rust was preliminary investigated and it presented the related disease-resistant gene and protein induced by rust pathogen. The research provides foundation for rust prevention and disease resistance breeding on Kentucky bluegrass in future.
引文
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[10]张庆峰,林树燕,张强.南京市观赏型草坪草主要病害发生的特点及防治[J].江苏林业科技,2005,32(3):26--28.ZHANG Q F,LIN S Y,ZHANG Q.Prevention and control measures of main turfgrass diseases in Nanjing City[J].Journal of Jiangsu Forestry Science&Technology,2005,32(3):26-28.
[11]侯丽冰,贺伟,刘小勇,等.我国几种松干锈菌亲缘关系的ITS序列分析[J].北京林业大学学报,2002,24(5/6):179-186.HOU L B,HE W,LIU X Y,et al.Sequence analysis of ITS region in relationships of Chinese isolates of Cronartium[J].Journal of Beijing Forestry University,2002,24(5/6):179-186.
[12]郑素娇.红小豆锈病菌鉴定和越冬及侵染过程的研究[D].大庆:黑龙江八一农垦大学,2015.ZHENG S J.Identification,overwintering and infection process of the pathogen causing adzuki bean rust[D].Daqing:Heilongjiang Bayi Agricultural University,2015.
[13]代君丽,于思勤,韩帅,等.一种从感病叶片中高效提取条锈菌基因组DNA的方法[J].西北农业学报,2009,18(2):304--306.DAI J L,YU S Q,HAN S,et al.An efficient protocol for isolatiing genomic DNA of Puccinia striiformis f.sp.tritici from infected leaves[J].Acta Agriculturae Boreali-occidentalis Sinica,2009,18(2):304-306.
[14]SOHPAL V K,DEY A,SINGH A.MEGA biocentric software for sequence and phylogenetic analysis:a review[J].International Journal of Bioinformatics Research and Applications,2010,6(3):230--240.
[15]TIAN B,HARRISON R,MORTON J,et al.Proteomic analysis of sauvignon blanc grape skin,pulp and seed and relative quantification of pathogenesis-related proteins[J/OL].PLo S One,2015,10(6):e130132[2016-05-04].DOI:10.1371/journal.pone.0130132.
[16]CARUSO C,CHILOSI G,CAPORALE C,et al.Induction of pathogenesis-related proteins in germinating wheat seeds infected with Fusarium culmorum[J].Plant Science,1999,140(1):87--97.
[17]ZAMBINO P J,SZABO L J.Phylogenetic relationships of selected cereal and grass rusts based on r DNA sequence analysis[J].Mycologia,1993,85(3):401--414.
[18]MA G B.Functional analysis of plant disease resistance genes and their downstream effectors.[J].Current Opinion in Plant Biology,1999,2(4):273-279.
[19]DURRANT W E,DONG X.Systemic acquired resistance[J].Annual Review of Phytopathology,2004,42(1):185--209.
[20]CAO H,GLAZEBROOK J,CLARKE J D,et al.The arabidopsis NPR1 gene that controls systemic acquired resistance encodes a novel protein containing ankyrin repeats[J].Cell,1997,88(1):57--63.
[21]KINKEMA M,FAN W,DONG X.Nuclear localization of NPR1 is required for activation of PR gene expression[J].The Plant Cell,2000,12(12):2339-2350.
[2]CPUCH H B.Diseases of turfgrasses[M].New York:Reinhold Pub.Corp.,1962.
[3]陈吉虎,赵永军,高旭彪,等.北京地区冷季型草坪草病害调查及防治[J].草业科学,2007,24(12):103--106.CHEN J H,ZHAO Y J,GAO X B,et al.Investigation and control of cold-season turfgrass diseases in Beijing areas[J].Pratacultural Science,2007,24(12):103--106.
[4]李春杰,南志标.甘肃草坪草真菌病害初报[J].草业科学,1998,15(1):49-51.LI C J,NAN Z B.Primary report on fungal diseases of turfgrass in Gansu Province,China[J].Pratacultural Science,1998,15(1):49--51.
[5]薛福祥,姚拓,席琳乔.成都高尔夫球场草坪草主要病害病原鉴定及防治对策初报[J].草原与草坪,2003(2):23-25.XUE F X,YAO T,XI L Q.Preliminary study on identification and control of turfgrass diseases in Chengdu golf course[J].Grassland and Turf,2003(2):23--25.
[6]薛福祥.兰州地区冷季型草坪真菌病害研究[J].草业科学,2003,20(3):66-70.XUE F X.Study on cold-season turfgrass diseases in Lanzhou rejoin[J].Pratacultural Science,2003,20(3):66--70.
[7]文克俭,尚以顺.贵州优良冷季型草坪主要病虫害防治技术[J].贵州畜牧兽医,2003,27(4):34-36.WEN K J,SHAN Y S.Study on pests and disease management control technology of cold-season turf in Guizhou rejoin[J].Guizhou Animal Science and Veterinary Medicine,2003,27(4):34-36.
[8]刘金荣,谢晓蓉.河西走廊草坪草病害调查[J].植物保护,2002,28(5):39-40.LIU J R,XIE X R.Investigation of turfgrass diseases in the Hexi Corridor[J].Plant Protection,2002,28(5):39--41.
[9]侯玉霞,石建宁.宁夏草坪草病害调查及防治[J].植物保护,1999,25(6):28-29.HOU Y X,SHI J N.Identification and control of turfgrass diseases in Ningxia[J].Plant Protection,1999,25(6):28--29.
[10]张庆峰,林树燕,张强.南京市观赏型草坪草主要病害发生的特点及防治[J].江苏林业科技,2005,32(3):26--28.ZHANG Q F,LIN S Y,ZHANG Q.Prevention and control measures of main turfgrass diseases in Nanjing City[J].Journal of Jiangsu Forestry Science&Technology,2005,32(3):26-28.
[11]侯丽冰,贺伟,刘小勇,等.我国几种松干锈菌亲缘关系的ITS序列分析[J].北京林业大学学报,2002,24(5/6):179-186.HOU L B,HE W,LIU X Y,et al.Sequence analysis of ITS region in relationships of Chinese isolates of Cronartium[J].Journal of Beijing Forestry University,2002,24(5/6):179-186.
[12]郑素娇.红小豆锈病菌鉴定和越冬及侵染过程的研究[D].大庆:黑龙江八一农垦大学,2015.ZHENG S J.Identification,overwintering and infection process of the pathogen causing adzuki bean rust[D].Daqing:Heilongjiang Bayi Agricultural University,2015.
[13]代君丽,于思勤,韩帅,等.一种从感病叶片中高效提取条锈菌基因组DNA的方法[J].西北农业学报,2009,18(2):304--306.DAI J L,YU S Q,HAN S,et al.An efficient protocol for isolatiing genomic DNA of Puccinia striiformis f.sp.tritici from infected leaves[J].Acta Agriculturae Boreali-occidentalis Sinica,2009,18(2):304-306.
[14]SOHPAL V K,DEY A,SINGH A.MEGA biocentric software for sequence and phylogenetic analysis:a review[J].International Journal of Bioinformatics Research and Applications,2010,6(3):230--240.
[15]TIAN B,HARRISON R,MORTON J,et al.Proteomic analysis of sauvignon blanc grape skin,pulp and seed and relative quantification of pathogenesis-related proteins[J/OL].PLo S One,2015,10(6):e130132[2016-05-04].DOI:10.1371/journal.pone.0130132.
[16]CARUSO C,CHILOSI G,CAPORALE C,et al.Induction of pathogenesis-related proteins in germinating wheat seeds infected with Fusarium culmorum[J].Plant Science,1999,140(1):87--97.
[17]ZAMBINO P J,SZABO L J.Phylogenetic relationships of selected cereal and grass rusts based on r DNA sequence analysis[J].Mycologia,1993,85(3):401--414.
[18]MA G B.Functional analysis of plant disease resistance genes and their downstream effectors.[J].Current Opinion in Plant Biology,1999,2(4):273-279.
[19]DURRANT W E,DONG X.Systemic acquired resistance[J].Annual Review of Phytopathology,2004,42(1):185--209.
[20]CAO H,GLAZEBROOK J,CLARKE J D,et al.The arabidopsis NPR1 gene that controls systemic acquired resistance encodes a novel protein containing ankyrin repeats[J].Cell,1997,88(1):57--63.
[21]KINKEMA M,FAN W,DONG X.Nuclear localization of NPR1 is required for activation of PR gene expression[J].The Plant Cell,2000,12(12):2339-2350.