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

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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