Oligo-DNA Custom Macroarray for Monitoring Major Pathogenic and Non-Pathogenic Fungi and Bacteria in the Phyllosphere of Apple Trees 英文参考文献.docVIP

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Oligo-DNA Custom Macroarray for Monitoring Major Pathogenic and Non-Pathogenic Fungi and Bacteria in the Phyllosphere of Apple Trees 英文参考文献.doc

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Oligo-DNA Custom Macroarray for Monitoring Major Pathogenic and Non-Pathogenic Fungi and Bacteria in the Phyllosphere of Apple Trees 英文参考文献

Oligo-DNACustomMacroarrayforMonitoringMajor PathogenicandNon-PathogenicFungiandBacteriain thePhyllosphereofAppleTrees Ying-HongHe,SayakaIsono,MakotoShibuya,MasaharuTsuji,Charith-RajAdkarPurushothama, KazuakiTanaka,TeruoSano* FacultyofAgricultureandLifeScience,HirosakiUniversity,Hirosaki,Japan Abstract Background:Tomonitortherichnessinmicrobialinhabitantsinthephyllosphereofappletreescultivatedundervarious culturalandenvironmentalconditions,wedevelopedanoligo-DNAmacroarrayformajorpathogenicandnon-pathogenic fungiandbacteriainhabitingthephyllosphereofappletrees. Methods and Findings: First, we isolated culturable fungi and bacteria from apple orchards by an agar-plate culture method, and detected 32 fungal and 34 bacterial species. Alternaria, Aureobasidium, Cladosporium, Rhodotorula, Cystofilobasidium,andEpicoccumgenerawerepredominantamongthefungi,andBacillus,Pseudomonas,Sphingomonas, Methylobacterium,and Pantoeagenerawerepredominantamongthebacteria.Basedonthedata,weselected29major non-pathogenicand12phytopathogenicfungiandbacteriaasthetargetsofmacroarray.Forty-onespecies-specific40-base pairlongoligo-DNAsequenceswereselectedfromthenucleotidesequencesofrDNA-internaltranscribedspacerregionfor fungiand16SrDNAforbacteria.Theoligo-DNAswerefixedonnylonmembraneandhybridizedwithdigoxigenin-labeled cRNA probes prepared for each species. All arrays except those for Alternaria, Bacillus, and their related species, were specificallyhybridized.Thearraywassensitiveenoughtodetect103CFUforAureobasidiumpullulansandBacilluscereus. Nucleotidesequencingof100eachofindependentfungalrDNA-ITSandbacterial16S-rDNAsequencesfromappletreewas inagreementwiththemacroarraydataobtainedusingthesamesample.Finally,weanalyzedtherichnessinthemicrobial inhabitantsinthesamplescollectedfromappletreesinfourorchards.Majorapplepathogensthatcausescab,Alternaria blotch,andMarssoninablotchweredetectedalongwithseveralnon-phytopathogenicfungalandbacterialinhabitants. Conclusions: The macroarray technique presented