Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki, Japan.
PLoS One. 2012;7(3):e34249. doi: 10.1371/journal.pone.0034249. Epub 2012 Mar 30.
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.
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 10(3) 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 agreement with the macroarray data obtained using the same sample. Finally, we analyzed the richness in the microbial inhabitants in the samples collected from apple trees in four orchards. Major apple pathogens that cause scab, Alternaria blotch, and Marssonina blotch were detected along with several non-phytopathogenic fungal and bacterial inhabitants.
The macroarray technique presented here is a strong tool to monitor the major microbial species and the community structures in the phyllosphere of apple trees and identify key species antagonistic, supportive or co-operative to specific pathogens in the orchard managed under different environmental conditions.
为了监测在不同文化和环境条件下种植的苹果树叶片微生物群落的丰富度，我们开发了一种用于苹果叶片微生物的主要病原真菌和细菌的寡聚 DNA 微阵列。
首先，我们通过琼脂平板培养法从苹果园中分离可培养的真菌和细菌，并检测到 32 种真菌和 34 种细菌。真菌中优势属为链格孢属、金龟子菌属、枝孢属、红酵母属、Cystofilobasidium 属和层出镰孢属，细菌中优势属为芽孢杆菌属、假单胞菌属、鞘氨醇单胞菌属、甲基杆菌属和泛菌属。基于这些数据，我们选择了 29 种主要的非病原真菌和 12 种植物病原真菌和细菌作为微阵列的目标。从 rDNA 内部转录间隔区的核苷酸序列中选择了 41 种物种特异性的 40 碱基长寡聚 DNA 序列，用于真菌和 16S rDNA 用于细菌。寡聚 DNA 固定在尼龙膜上，并与为每个物种制备的地高辛标记的 cRNA 探针杂交。除了Alternaria、Bacillus 及其相关物种的阵列外，所有阵列都特异性杂交。该阵列足够灵敏，可以检测到 Aureobasidium pullulans 和 Bacillus cereus 的 10（3）CFU。从苹果树中独立提取的 100 个真菌 rDNA-ITS 和细菌 16S-rDNA 序列的核苷酸测序与使用相同样本获得的微阵列数据一致。最后，我们分析了从四个果园的苹果树样本中收集的微生物群落的丰富度。在检测到引起黑星病、褐斑病和灰斑病的主要苹果病原体的同时，还检测到了几种非植物病原真菌和细菌。
本文提出的微阵列技术是一种强大的工具，可以监测苹果叶片微生物的主要物种和群落结构，并识别在不同环境条件下管理的果园中特定病原体的拮抗、支持或合作的关键物种。
