甜瓜靶斑病病原菌鉴定
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摘要
甜瓜(Cucumis melo L.)是我国重要的瓜类经济作物。2015年10月在我国广西武鸣甜瓜主要种植区进行病害调查时发现一种新的叶斑病。该病原菌疑似通常危害植物叶片引起叶斑病的多主棒孢菌。采用形态学观察,多基因鉴定和致病性试验的方法对引起该病的病原菌进行了鉴定。病原菌菌落中间墨绿色,边缘浅褐色,有明显的生长圈,气生菌丝茂密。分生孢子呈圆柱形或倒棍棒形,单生或串生,直立或稍微弯曲,具0～13个隔膜,大小为(3.1～6.4)μm×(14.0～138.9)μm。克隆测定该病原菌的4个保守序列ITS1,2、ITS4,5、ACT和TUB2。分离纯化的病原菌回接到甜瓜幼苗,表现出与田间相似的症状,符合柯赫氏法则,鉴定该病原菌为多主棒孢。该病原菌也可侵染西瓜(Citrullus vulgaris Schrad.)和黄瓜(Cucumis sativus L.)幼苗及离体叶片使其发病。
Muskmelon(Cucumis melo L.)is one of the most economically important cucurbit crops grown commercially in China. A new leaf spot disease was observed on muskmelon in October 2015, in Wuming, Guangxi province, China.The disease exhibited symptoms of target spot. Morphological characteristics, multiple-gene sequencing, and pathogenicity tests were used to identify the causal organism. The fungal isolate produced a colony with brownish or greenish concentric growth rings and abundant aerial mycelia. The conidia were solitary, hyaline, cylindrical, oval or obclavate, straight or curve, 0-13-septate and(3.1-6.4)μm ×(14.0-138.9)μm. The isolate was sequenced using four genomic loci, from ribosomal DNA internal transcribed spacer sequences [ITS1, ITS2(ITS1,2), ITS4 and ITS5(ITS4,5)], and the actin(ACT), β-tubulin(TUB2)genes. The purified Guangxi isolate infected all tested genotypes of muskmelon, watermelon(Citrullus vulgaris Schrad.), and cucumber(Cucumis sativus L.), in seedlings and detached leaves, and was consistently reisolated from diseased leaves, satisfying Koch's postulates. Our results identified the pathogen as C. cassiicola.
Muskmelon(Cucumis melo L.)is one of the most economically important cucurbit crops grown commercially in China. A new leaf spot disease was observed on muskmelon in October 2015, in Wuming, Guangxi province, China.The disease exhibited symptoms of target spot. Morphological characteristics, multiple-gene sequencing, and pathogenicity tests were used to identify the causal organism. The fungal isolate produced a colony with brownish or greenish concentric growth rings and abundant aerial mycelia. The conidia were solitary, hyaline, cylindrical, oval or obclavate, straight or curve, 0-13-septate and(3.1-6.4)μm ×(14.0-138.9)μm. The isolate was sequenced using four genomic loci, from ribosomal DNA internal transcribed spacer sequences [ITS1, ITS2(ITS1,2), ITS4 and ITS5(ITS4,5)], and the actin(ACT), β-tubulin(TUB2)genes. The purified Guangxi isolate infected all tested genotypes of muskmelon, watermelon(Citrullus vulgaris Schrad.), and cucumber(Cucumis sativus L.), in seedlings and detached leaves, and was consistently reisolated from diseased leaves, satisfying Koch's postulates. Our results identified the pathogen as C. cassiicola.
引文
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[3]SILVA W P K,MULTANI D S,DEVERALL B J,et al.RFLPand RAPD analyses in the identification and differentiation of isolates of the leaf spot fungus Corynespora cassiicola[J].Australian Journal of Botany,1995,43(6):609-618.
[4]SILVA W P K,WIJESUNDERA R L C,KARUNANAYAKE EH,et al.New hosts of Corynespora cassiicola in Sri Lanka[J].Plant Disease,2000,84(2):202-202.
[5]王爽,黄贵修,李博勋,等.甜瓜棒孢叶斑病病原菌鉴定及其生物学特性研究[J].热带作物学报,2013,34(12):2446-2452.
[6]方中达.植病研究方法[M].北京:中国农业出版社,1988:64-124.
[7]QI Y X,ZHANG X,PU J J,et al.Nested PCR assay for detection of Corynespora leaf fall disease caused by Corynespora cassiicola[J].Australasian Plant Pathology,2009,38(2):141-148.
[8]WHITE T J,BRUNS T,LEE S,et al.Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics[M].London:Academic,1990,18(1):315-322.
[9]陈璐,石延霞,谢学文,等.黄瓜棒孢叶斑病菌PCR检测方法的建立[J].园艺学报,2014,41(3):585-592.
[10]O'DONNELL K,CIGELNIK E.Two divergent intragenomic rDNA ITS2 types within a monophyletic lineage of the fungus Fusarium are nonorthologous[J].Molecular Phylogenetics and Evolution,1997,7(1):103-116.
[11]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.
[12]QI Y X,ZHANG X,PU J J,et al.Morphological and molecular analysis of genetic variability within isolates of Corynespora cassiicola from different hosts[J].European Journal of Plant Pathology,2011,130(1):83-95.
[13]BLAZQUEZ C H.Target spot of tomato[J].Plant Disease Reporter 1972,56(3):243-245.
[14]MOHANTY N N,MOHANTY N W.Target spot of tomatoes[J].Science and Culture,1955,21(6):330-332.
[15]FAJOLA A O,ALASOADURA S O.Corynespora leaf spot,a new disease of tobacco(Nicotiana tabacum)[J].Plant Disease Reporter,1973,57(4):375-378.
[16]JONES J P.A leaf spot of cotton caused by Corynespora cassiicola[J].Phytopathology,1961,51(5):305-308.
[17]SEAMAN W L,SHOEMAKER R A.Corynespora cassiicola on soybean in Ontario[J].Plant Disease Reporter,1964,48(1):69.
[18]OLIVE L S,BAIN D C,LEFEBVRE C L.A leaf spot of cowpea and soybean caused by an undescribed species of Helminthosporium[J].Phytopathology,1945,35(10):822-831.
[19]STONE W J,JONES J P.Corynespora blight of sesame[J].Phytopathology,1960,50(4):263-266.
[20]RAMAKRISHNAN T S,PILLAY P N R.Leaf spot of rubber caused by Corynespora cassiicola(Berk.&Curt.)Wei[J].Rubber Board Bulletin,1961,5(1):32-35.
[21]ELLIS M B,HOLLIDAY P.Corynespora cassiicola[J].C.M.I.Description of Pathogenic Fungi and Bacteria,1971,303:2.
[22]MILLER J W,ALFIERI S A.Leaf spot of Ligustrum sinense caused by Corynespora cassiicola[J].Phytopathology,1973,63:445-446.
[23]ONESIROSAN P T,ARNY D C,DURBIN R D.Host-specificity of Nigerian and North American isolates of Corynespora cassiicola[J].Phytopathology,1974,64(10):1364-1367.
[24]FURUKAWA T,USHIYAMA K,KISHI K.Corynespora leaf spot of scarlet sage caused by corynespora cassiicola[J].Journal of General Plant Pathology,2008,74(2):117-119.
[25]DIXON L J,SCHLUB R L,PERNEZNY K,et al.Host specialization and phylogenetic diversity of Corynespora cassiicola[J].Phytopathology,2009,99(9):1015-1027.
[2]WEI C T.Notes on Corynespora[J].Mycological Papers,1950,34:1-9.
[3]SILVA W P K,MULTANI D S,DEVERALL B J,et al.RFLPand RAPD analyses in the identification and differentiation of isolates of the leaf spot fungus Corynespora cassiicola[J].Australian Journal of Botany,1995,43(6):609-618.
[4]SILVA W P K,WIJESUNDERA R L C,KARUNANAYAKE EH,et al.New hosts of Corynespora cassiicola in Sri Lanka[J].Plant Disease,2000,84(2):202-202.
[5]王爽,黄贵修,李博勋,等.甜瓜棒孢叶斑病病原菌鉴定及其生物学特性研究[J].热带作物学报,2013,34(12):2446-2452.
[6]方中达.植病研究方法[M].北京:中国农业出版社,1988:64-124.
[7]QI Y X,ZHANG X,PU J J,et al.Nested PCR assay for detection of Corynespora leaf fall disease caused by Corynespora cassiicola[J].Australasian Plant Pathology,2009,38(2):141-148.
[8]WHITE T J,BRUNS T,LEE S,et al.Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics[M].London:Academic,1990,18(1):315-322.
[9]陈璐,石延霞,谢学文,等.黄瓜棒孢叶斑病菌PCR检测方法的建立[J].园艺学报,2014,41(3):585-592.
[10]O'DONNELL K,CIGELNIK E.Two divergent intragenomic rDNA ITS2 types within a monophyletic lineage of the fungus Fusarium are nonorthologous[J].Molecular Phylogenetics and Evolution,1997,7(1):103-116.
[11]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.
[12]QI Y X,ZHANG X,PU J J,et al.Morphological and molecular analysis of genetic variability within isolates of Corynespora cassiicola from different hosts[J].European Journal of Plant Pathology,2011,130(1):83-95.
[13]BLAZQUEZ C H.Target spot of tomato[J].Plant Disease Reporter 1972,56(3):243-245.
[14]MOHANTY N N,MOHANTY N W.Target spot of tomatoes[J].Science and Culture,1955,21(6):330-332.
[15]FAJOLA A O,ALASOADURA S O.Corynespora leaf spot,a new disease of tobacco(Nicotiana tabacum)[J].Plant Disease Reporter,1973,57(4):375-378.
[16]JONES J P.A leaf spot of cotton caused by Corynespora cassiicola[J].Phytopathology,1961,51(5):305-308.
[17]SEAMAN W L,SHOEMAKER R A.Corynespora cassiicola on soybean in Ontario[J].Plant Disease Reporter,1964,48(1):69.
[18]OLIVE L S,BAIN D C,LEFEBVRE C L.A leaf spot of cowpea and soybean caused by an undescribed species of Helminthosporium[J].Phytopathology,1945,35(10):822-831.
[19]STONE W J,JONES J P.Corynespora blight of sesame[J].Phytopathology,1960,50(4):263-266.
[20]RAMAKRISHNAN T S,PILLAY P N R.Leaf spot of rubber caused by Corynespora cassiicola(Berk.&Curt.)Wei[J].Rubber Board Bulletin,1961,5(1):32-35.
[21]ELLIS M B,HOLLIDAY P.Corynespora cassiicola[J].C.M.I.Description of Pathogenic Fungi and Bacteria,1971,303:2.
[22]MILLER J W,ALFIERI S A.Leaf spot of Ligustrum sinense caused by Corynespora cassiicola[J].Phytopathology,1973,63:445-446.
[23]ONESIROSAN P T,ARNY D C,DURBIN R D.Host-specificity of Nigerian and North American isolates of Corynespora cassiicola[J].Phytopathology,1974,64(10):1364-1367.
[24]FURUKAWA T,USHIYAMA K,KISHI K.Corynespora leaf spot of scarlet sage caused by corynespora cassiicola[J].Journal of General Plant Pathology,2008,74(2):117-119.
[25]DIXON L J,SCHLUB R L,PERNEZNY K,et al.Host specialization and phylogenetic diversity of Corynespora cassiicola[J].Phytopathology,2009,99(9):1015-1027.