oligo-dna custom macroarray for monitoring major pathogenic and non-pathogenic fungi and bacteria in the phyllosphere of apple treesoligo-dna定制macroarray监测主要致病性和非致病性真菌和细菌叶围的苹果树.pdfVIP

Oligo-DNA Custom Macroarray for Monitoring Major Pathogenic and Non-Pathogenic Fungi and Bacteria in the Phyllosphere of Apple Trees Ying-Hong He, Sayaka Isono, Makoto Shibuya, Masaharu Tsuji, Charith-Raj Adkar Purushothama, Kazuaki Tanaka, Teruo Sano* Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki, Japan Abstract Background: To monitor the richness in microbial inhabitants in the phyllosphere of apple trees cultivated under various cultural and environmental conditions, we developed an oligo-DNA macroarray for major pathogenic and non-pathogenic fungi and bacteria inhabiting the phyllosphere of apple trees. 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, and Epicoccum genera were predominant among the fungi, and Bacillus, Pseudomonas, Sphingomonas, Methylobacterium, and Pantoea genera were predominant among the bacteria. Based on the data, we selected 29 major non-pathogenic and 12 phytopathogenic fungi and bacteria as the targets of macroarray. Forty-one species-specific 40-base pair long oligo-DNA sequences were selected from the nucleotide sequences of rDNA-internal transcribed spacer region for fungi and 16S rDNA for bacteria. The oligo-DNAs were fixed on nylon membrane and hybridized with digoxigenin-labeled cRNA probes prepared for each species. All arrays except those for Alternaria, Bacillus, and their related species, were specifically hybridized. The array was sensitive enough to detect 103 CFU for Aureobasidium pullulans and Bacillus cereus. Nucleotide sequencing of 100 each of independent fungal rDNA-ITS and bacterial 16S-rDNA sequences from apple tree was in agre