Large scale sequencing analysis of microbial diversity in phyllo-and rhizo-sphares of apple trees for investigation of the mechanism to acquire natural immunity under natural farming practices

经过30多年的自然栽培，不施化肥、不施农药，果园苹果树的抑病效果显著。有人认为，果园这么长时间的自然耕作可能引发了一些未知的现象，其中一些树已经获得了天然免疫，以防止它们受到严重的病害损害，但科学分析是必要的。本研究利用新一代DNA测序仪对自然和常规栽培条件下苹果树根际和叶圈的微生物多样性进行了大规模测序分析，评价了果园微生物多样性在诱导植物自然免疫中的重要作用。通过对叶圈中真菌的分析，发现细小枝孢子菌(Aureobasidium Pullulans、Cladosporium tenuissimum)、蜡状芽胞杆菌(Cystofobasidium Macerans)、维氏隐球菌(Cryptoins Victoriae)在…中占优势地位更多的是四个果园，还有文丘里。苹果黑星病的病原菌--链格孢菌。黑斑链格孢菌(Alternaria Blotch)和木瓜属(Diplcarpon Mari.)在两个天然果园中也检测到马氏菌斑点病。人们注意到，金黄色葡萄球菌。在其他三个果园中最主要的真菌居住者，在自然耕作果园K中的丰度明显低于其他三个果园，其中以某种病害抑制现象突出。虽然K园的优势种是枝孢属或文氏菌，但不是金黄色葡萄球菌，但在K园以及常规耕作果园O和MK中，真菌数量相对较少(2-3种)。相比之下，另一自然栽培果园MS中的真菌种类较多(4-5种)，与K相似的自然栽培措施两年前才刚刚开始，尚未达到与K一样稳定的抑病水平。从叶圈细菌分析可知，棘球鞘氨醇单胞菌、耐辐射甲基杆菌和丁香假单胞菌为优势种。8月份，K和MS两个自然果园的细菌物种多样性和丰度都有了较大的提高，即8月份的物种(或序列)数量比6月份增加了20多倍。由于两个常规农业果园O和MK的多样性和丰度都没有增加，表明化学杀菌剂的喷洒严重限制了叶圈中非病原菌栖息地的丰度。细菌多样性的比较分析表明，8月份两个自然耕作果园K和MS的细菌多样性没有显著差异，表明叶圈微生物多样性并不是仅在果园K观察到的致病抑制的关键因素。此外，从根际真菌分析来看，自然耕作果园K和常规耕作果园O分别检出50属和55属。从根际细菌分析可知，K园和MS园分别有镰刀菌属、隐球菌属、双歧杆菌属、腐霉属和被孢霉属。在K园和O园分别以Nirospira、慢根瘤菌、铜绿假单胞菌、伯克霍尔德氏菌和假单胞菌为主，O园以Nirospira、慢根瘤菌、克氏杆菌、芽孢杆菌、甲氧菌属为主，两个果园优势菌群明显不同。所获得的序列数据参考公共DNA数据库中的拟南芥基因文库中的数据，并成功地显示了从K园和MS园获得的每个基因的差异表达

It is known that apple trees in an orchard managed under natural farming practices for more than 30 years without applying chemical fertilizer and pesticide exhibit prominent disease suppression effect. It is suggested that such a long years of natural farming practices in the orchard might have incited unknown phenomena in which some of the trees have acquired natural immunity preventing them from severe disease damages, however, science-based analysis is essential. In this research, we have performed large-scale sequencing analysis using next generation DNA sequencer on the microbial diversity of phyllosphere and rhizosphere of apple trees managed under natural and conventional farming conditions, and evaluated how microbial diversity in the orchard plays important roles on the induction of natural immunity in the plants.From analysis of fungi in the phyllosphere, Aureobasidium pullulans、Cladosporium tenuissimum, Cystofilobasidium macerans, Cryptococcus victoriae were predominated in … More all the four orchards, and Venturia inaequalis . the causal pathogen of apple scab, Alternaria mali . alternaria blotch, and Diplocarpon mali . marsonina blotch, were also detected from the two natural farming orchards. It was noted that Aureobasidium . the most predominant fungal inhabitant in the other three orchards, were significantly less abundant in the natural farming orchard K where a certain disease suppression phenomenon was prominent. Although the most predominant species in the orchard K was Cladosporium or Venturia, but not Aureobasidium, relatively small numbers (2-3 species) of fungi was predominated in the orchard K, as well as the two conventional farming orchards O and MK. In contrast, more diverse fungal species (4-5) were inhabiting in the other natural farming orchard MS, where the natural farming practices similar to K has just started two years before and has not yet reached the stable level as K in the disease suppression. From the analysis of bacteria in the phyllosphere, Sphingomonas echinoides, Methylobacterium radiotolerans, and Pseudomonas syringae were predominating. The diversity and abundance of bacterial species were jumped up in August in the two natural farming orchards K and MS ; i.e, the number of species (or sequences) increased more than 20-times in August comparing to June. Since the diversity as well as the abundance was not increased in the two conventional farming orchards O and MK, it was suggested that abundance of non-pathogenic bacterial inhabitants in the phyllosphere was severely restricted by chemical fungicide sprays. Comparative analysis on the bacterial diversity revealed that statistically significant difference was not observed in the two natural farming orchards K and MS in August, suggesting that microbial diversity in the phyllosphere was not the key factor to induce disease suppression observed only in orchard K. It was noted that the genus Pseudomonas and the genus Pantoea was abundant in orchard K and MS, respectively.Next, from the analysis of fungi in the rhizosphere, 50 and 55 genera were detected in the natural farming orchard K and the conventional farming orchard O, respectively. The genera Emericella, Fusarium, Mortierella, Cordyceps, and Leohumicola orderly predominated in the orchard K, and Fusarium, Cryptococcus, Dipodascus, Leohumicola, and Mortierella in the orchard O. From the analysis of bacteria in the rhizosphere, 327 and 356 genera were detected in the orchards K and MS, respectively. The genera Nirospira, Bradyrhizobium, Cupriavidus, Burkholderia, and Pseudomonas orderly predominated in the orchard K, and Nirospira, Bradyrhizobium, Ktedonobacter, Bacillus, Methylosinus in the orchard O. The predominant bacterial inhabitants were apparently different between the two orchards.Furthermore, we have collected Nazuna () naturally growing in the orchards K and O, extracted poly-A RNAs, and used for the large-scale sequencing analysis using next generation DNA sequencer. The sequence data obtained were refer to those in Arabidopsis cDNA Library in the public DNA data bank, and successfully visualized the differential expression of each genes between those obtained from the orchard K and the orchard MS. Less