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

据了解，在不施用化肥和农药的30年以上的自然耕作方式管理的果园中，苹果树表现出明显的防病效果。有人认为，在果园里这么长时间的自然耕作可能引发了一些未知现象，其中一些树木获得了天然免疫力，使它们免受严重疾病的损害，但基于科学的分析是必不可少的。本研究利用下一代DNA测序仪对自然和常规耕作条件下管理的苹果树根际和根际微生物多样性进行了大规模测序分析，并评估了果园微生物多样性在诱导植物天然免疫中的重要作用。从层层真菌的分析来看，4个果园中均以普鲁兰小孢子虫、细枝孢子虫、细枝孢子虫、维多利亚隐球菌和不等温孢子虫为主。苹果赤霉病的致病病菌马利Alternaria mali。斑交蛾和马利双果蛾。在两个天然果园中也检测到马氏斑病。值得注意的是，毛霉属。在自然耕作果园K中，真菌的丰度显著低于其他3个果园，且有一定的病害抑制现象。虽然K果园的优势种是枝孢菌（Cladosporium）或文丘菌（Venturia），而非Aureobasidium，但在K果园以及两个常规耕作果园O和MK中，真菌的优势种相对较少（2-3种），而在其他天然耕作果园MS中，真菌的种类更为多样（4-5种）。与K相似的自然耕作方式在2年前才开始，在抑制病害方面还没有达到K的稳定水平。从层球细菌分析来看，棘皮鞘单胞菌、放射耐受甲基杆菌和丁香假单胞菌占主导地位。8月，天然果园K和MS的细菌种类多样性和丰度均大幅上升；8月份的物种（或序列）数量比6月份增加了20多倍。由于常规栽培果园O和MK的多样性和丰度均未增加，说明化学杀菌剂的施用严重限制了层层非致病性细菌的丰度。细菌多样性对比分析显示，8月份K果园和MS果园两种天然栽培果园的细菌多样性差异无统计学意义，表明层际微生物多样性不是诱导病害抑制的关键因素，仅K果园中观察到。值得注意的是，K果园中假单胞菌属丰富，MS果园中潘氏菌属丰富。接下来，从根际真菌的分析中，在自然耕作果园K和常规耕作果园O分别检测到50和55个属。在K果园中，镰刀菌属、镰孢菌属、摩氏菌属、虫草菌属和莫氏菌属占主导地位；在o果园中，镰刀菌属、隐球菌属、双足菌属、利胡菌属和摩氏菌属占主导地位。在果园K中以黑螺旋体属、慢根瘤菌属、铜菌属、伯克氏菌属、有序假单胞菌属为主，在果园o中以黑螺旋体属、慢根瘤菌属、克特多诺杆菌属、芽孢杆菌属、甲基单胞菌属为主，两果园间优势菌群差异明显。此外，我们收集了天然生长在K和O果园的Nazuna()，提取了poly-A rna，并使用下一代DNA测序仪进行了大规模测序分析。获得的序列数据参照公共DNA数据库中拟南芥cDNA Library中的序列数据，成功地可视化了果园K和果园MS. Less中各基因的差异表达

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