四川省山药黑斑病病原菌的鉴定
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摘要
为鉴定引起山药茎蔓黑斑病的病原菌,本研究利用柯赫氏法则验证其致病性,根据菌株的形态特征和基于rpb2基因序列分析进行分类鉴定,并初步测定其寄主范围。结果显示:分离的2株病原菌分生孢子椭圆形,单胞,(3.9～5.1)μm×(1.8～2.7)μm,产孢细胞瓶梗形,分生孢子器近圆形或梨形,无刚毛,具孔口,有时孔口延长形成乳突,NaOH颜色反应呈阳性,参考Phoma鉴定手册,可将其划分到section Peyronellaea。将供试菌的rpb2序列进行BLAST比对分析,其与GenBank中的Epicoccum latusicollum具有最高的同源性,支持率为97%,结合形态学特征可将其鉴定为E.latusicollum,该菌经人工接种后也能侵染番茄、辣椒和花菜幼苗。
To identify the pathogen causing black rot of yam, experiments were performed, including pathogenicity test, host range determination, identification based on morphology and rpb2 sequences. The results showed that conidia of the pathogens were ellipsoidal, aseptate,(3.9-5.1)μm ×(1.8-2.7)μm, with phialidic conidiogenous cells, globose, pyriform and glabrous pycnidia, and sometimes elongated ostioles like a short, slightly papillate neck. The pathogens showed positive reactions in the NaOH test. Based on the morphology, the fungus was classified in section Peyronellaea according to the Phoma identification manual. The analysis of rpb2 sequences showed that the fungus shared the highest homology with Epicoccum latusicollum(up to 97%) in GenBank. Therefore, the fungus was identified as E. latusicollum, which could infect tomato, pepper and cauliflower in the seedling stage after inoculation.
To identify the pathogen causing black rot of yam, experiments were performed, including pathogenicity test, host range determination, identification based on morphology and rpb2 sequences. The results showed that conidia of the pathogens were ellipsoidal, aseptate,(3.9-5.1)μm ×(1.8-2.7)μm, with phialidic conidiogenous cells, globose, pyriform and glabrous pycnidia, and sometimes elongated ostioles like a short, slightly papillate neck. The pathogens showed positive reactions in the NaOH test. Based on the morphology, the fungus was classified in section Peyronellaea according to the Phoma identification manual. The analysis of rpb2 sequences showed that the fungus shared the highest homology with Epicoccum latusicollum(up to 97%) in GenBank. Therefore, the fungus was identified as E. latusicollum, which could infect tomato, pepper and cauliflower in the seedling stage after inoculation.
引文
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[27]LIU Y J,WHELEN S,HALL B D.Phylogenetic relationships among ascomycetes evidence from an RNA polymerase II subunit[J].Molecular Biology and Evolution,1999,16:1799-1808.
[2]王诗军,田福进,田凤环,等.山药褐斑病的发生特点与防治方法[J].中国植保导刊,2004,4(3):42-43.
[3]黄云,王洪波,李庆,等.山药炭疽病病原研究(I):炭疽病的症状及其病原鉴定[J].西北农业大学学报,2004,26(1):44-46.
[4]朱桂宁,蔡健和,胡春锦,等.广西山药炭疽病病原菌的鉴定与ITS序列分析[J].植物病理学报,2007,37(6):572-577.
[5]沈丽淘.山药根腐病的病原学及防治药剂筛选研究[D].成都:四川农业大学,2012.
[6]贾海民,鹿秀云,陈丹,等.麻山药根腐病发生规律及其防治技术[J].北方园艺,2011(1):159-160.
[7]易龙,肖崇刚,杨水英,等.重庆盾叶薯蓣茎腐病发生严重[J].植物保护,2005,31(6):96-97.
[8]陈红岩,刘晓芸.保定市山药病害种类及为害特点调查初报[J].中国植保导刊,2010,30(6):24-26.
[9]王飞,刘红彦,文艺,等.山药漆腐叶斑病病原菌的鉴定及其生物学特性研究[J].园艺学报,2017,44(5):972-978.
[10]CROUS P W,GAMS W,STAPLERS J A,et al.MycoBank:an online initiative to launch mycology into the 21st century[J].Studies in Mycology,2004,50:19-22.
[11]BOEREMA G H,DE GRUYER J,NOORDELOOS M E,et al.Phomaidentification manual[M].CABI publishing,2004.
[12]AVESKAMP M M,VERKLEY G J M,DE GRUYTER J,et al.DNA phylogeny reveals polyphyly of Phoma section Peyronellaea and multiple taxonomic novelties[J].Mycologia,2009,101:363-382.
[13]AVESKAMP M M,DE GRUYTER J,WOUDENBERG J HC,et al.Highlights of the Didymellaceae:A polyphasic approach to characterise Phomaand related pleosporalean genera[J].Studies in Mycology,2010,65:1-60.
[14]CHEN Qian,JIANG J R,ZHANG G Z,et al.Resolving the Phoma enigma[J].Studies in Mycology,2015,82:137-217.
[15]CHEN Q,HOU L W,HOU W J,et al.Didymellaceae revisited[J].Studies in Mycology,2017,87:105-159.
[16]CAMPBELL W P.The influence of associated microorganisms on the pathogenicity of Helminthosporium sativum[J].Canadian Journal of Botany,1956,34:865-874.
[17]MERCIER J,REELEDER R D.Interactions between Sclerotinia sclerotiorumand other fungi on the phylloplane of lettuce[J].Canadian Journal of Botany,1987,65(8):1633-1637.
[18]MELGAREJO P,CARRILLO R,SAGASTA E.Potential for biological control of Monilinia laxa in peach twigs[J].Crop Protection,1986,5(6):422-426.
[19]李宝年,徐树辉,杨传波,等.樟子松枯梢病拮抗真菌的筛选[J].东北林业大学学报,2004,32(5):97-99.
[20]STOKHOLMA M S,WULFFB E G,ZIDAC E P,et al.DNAbarcoding and isolation of vertically transmitted ascomycetes in sorghum from Burkina Faso:Epicoccum sorghinum is dominant in seedlings and appears as a common root pathogen[J].Microbiological Research,2016,191:38-50.
[21]RISTIC′D,STANKOVIC′I,VUCˇUROVIC′A,et al.Epicoccum nigrumthe new pathogen of sorghum seed in Serbia[J].Ratarstvo I Povrtarstvo,2012,49(2):160-166.
[22]WU Di,ZHANG Danhua,TIMKO M P,et al.First report of Epicoccum nigrumcausing brown leaf spot of loquat in Southwestern China[J].Plant Disease,2017,101(8):1553.
[23]方中达.植病研究方法[M].第3版.北京:中国农业出版社,1998.
[24]WHITE T J,BRUNS T,LEE S,et al.Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics[M]∥INNIS M A,GELFAND D,SNINSKY J J,et al.PCR Protocols:aguide to methods and applications.San Diego,California,USA:Academic Press,1990:315-322.
[25]SUNG G H,SUNG J M,HYWEL-JONES N L,et al.Amulti-gene phylogeny of Clavicipitaceae(Ascomycota,Fungi):identification of localized incongruence using a combinational bootstrap approach[J].Molecular Phylogenetics and E-volution,2007,44:1204-1223.
[26]DE GRUYTER J,NOORDELOOS M E.Contributions towards a monograph of Phoma(Coelomycetes)-I.1.Section Phoma:Taxa with conidia very small conidia in vitro[M].Rijksherbarium/Hortus Botanicus,Leiden,Volume 15,Part 1,1992:71-92.
[27]LIU Y J,WHELEN S,HALL B D.Phylogenetic relationships among ascomycetes evidence from an RNA polymerase II subunit[J].Molecular Biology and Evolution,1999,16:1799-1808.