Dynamics of rice microbiomes reveal core vertically transmitted seed endophytes.

BACKGROUND: Plants and their associated microbiota constitute an assemblage of species known as holobionts. The plant seed microbiome plays an important role in nutrient uptake and stress attenuation. However, the core vertically transmitted endophytes remain largely unexplored. RESULTS: To gain valuable insights into the vertical transmission of rice seed core endophytes, we conducted a large-scale analysis of the microbiomes of two generations of six different rice varieties from five microhabitats (bulk soil, rhizosphere, root, stem, and seed) from four geographic locations. We showed that the microhabitat rather than the geographic location and rice variety was the primary driver of the rice microbiome assemblage. The diversity and network complexity of the rice-associated microbiome decreased steadily from far to near the roots, rice exterior to interior, and from belowground to aboveground niches. Remarkably, the microbiomes of the roots, stems, and seeds of the rice interior compartments were not greatly influenced by the external environment. The core bacterial endophytes of rice were primarily comprised of 14 amplicon sequence variants (ASVs), 10 of which, especially ASV_2 (Pantoea) and ASV_48 (Xanthomonas), were identified as potentially vertically transmitted taxa because they existed across generations, were rarely present in exterior rice microhabitats, and were frequently isolated from rice seeds. The genome sequences of Pantoea and Xanthomonas isolated from the parental and offspring seeds showed a high degree of average nucleotide and core protein identity, indicating vertical transmission of seed endophytes across generations. In silico prediction indicated that the seed endophytes Pantoea and Xanthomonas possessed streamlined genomes with short lengths, low-complexity metabolism, and various plant growth-promoting traits. We also found that all strains of Pantoea and Xanthomonas exhibited cellulase activity and produced indole-3-acetic acid. However, most strains exhibited insignificant antagonism to the major pathogens of rice, such as Magnaporthe oryzae and X. oryzae pv. oryzae. CONCLUSION: Overall, our study revealed that microhabitats, rather than site-specific environmental factors or host varieties, shape the rice microbiome. We discovered the vertically transmitted profiles and keystone taxa of the rice microbiome, which led to the isolation of culturable seed endophytes and investigation of their potential roles in plant-microbiome interactions. Our results provide insights on vertically transmitted microbiota and suggest new avenues for improving plant fitness via the manipulation of seed-associated microbiomes.  Video Abstract.

Gracilibacillus oryzae sp. nov., isolated from rice seeds.

A Gram-stain-positive, facultatively anaerobic, endospore-forming bacterium, designated strain TD8T, was isolated from surface-sterilized rice seeds (Oryza sativa L.). Phylogenetic analysis of the 16S rRNA gene indicated that strain TD8T should be placed within the genus Gracilibacillus (95.2-99.0 % sequence similarity); it exhibited highest similarities to Gracilibacillus ureilyticus CGMCC 1.7727T (99.0 %), 'Gracilibacillus xinjiangensis' CGMCC 1.12449T (98.9 %) and Gracilibacillus dipsosauri CGMCC 1.3642T (97.5 %). Chemotaxonomic analysis showed that menaquinone-7 (MK-7) was the major isoprenoid quinone. Diphosphatidylglycerol, phosphatidylglycerol and one unidentified phospholipid were the major cellular polar lipids, and the major fatty acids were anteiso-C15 : 0, anteiso-C17 : 0, iso-C15 : 0, C16 : 0 and iso-C16 : 0, which supported the allocation of the strain to the genus Gracilibacillus. The digital DNA-DNA hybridization value between strain TD8T and Gracilibacillus ureilyticus CGMCC 1.7727T was lower than 70 % (22.60 %), and the average nucleotide identity score was 79.54±5.09 %, suggesting that strain TD8T represented a novel species in the genus Gracilibacillus. The genomic DNA G+C content was 37.5 %. Based on physiological and biochemical characteristics and genotypic data, strain TD8T represents a novel species of the genus Gracilibacillus, for which the name Gracilibacillus oryzae sp. nov. is proposed. The type strain is TD8T (=ACCC 61556T=CICC 24889T=JCM 33537T).

Dickeya oryzae sp. nov., isolated from the roots of rice.

A novel Gram-stain-negative strain, designated ZYY5T, was isolated from rice roots. Results of 16S rRNA gene analysis indicated that strain ZYY5T was a member of the genus Dickeya, with a highest similarity to Dickeya zeae DSM 18068T (98.5%). The major fatty acids were summed feature 3 (C16:1 ω7c and/or C16:1 ω6c), C16:0 and summed feature 8 (C18:1 ω7c and/or C18:1 ω6c). Multi-locus sequence analysis using five concatenated genes (16S rRNA, atpD, infB, recA and gyrB) and phylogenomic analysis based on 2940 core gene sequences showed that strain ZYY5T formed a robust cluster with strains EC1, ZJU1202, DZ2Q, NCPPB 3531 and CSL RW192, while separated from the other strains of D. zeae. The orthologous average nucleotide identity (ANI) and digital DNA-DNAhybridization (dDDH) values among these six strains ranged from 96.8-99.9% and 73.7-99.8%, which supported that they were belonged to the same species. However, strain ZYY5T shared 58.4 of dDDH and 94.5% of ANI values with type strain D. zeae DSM 18068T, which were lower than the proposed species boundary cut-off for dDDH and ANI. The genomic analysis revealed that strain ZYY5T contained virulence-associated genes, which is same as the phylogenetic-related strains of the genus Dickeya. Based on the results of the polyphasic approaches, we propose that strain ZYY5T represents a novel species in the genus Dickeya, for which the name Dickeya oryzae sp. nov. (=JCM 33020 T=ACCC 61554 T) is proposed. Strains EC1, ZJU1202, DZ2Q, NCPPB 3531 and CSL RW192 should also be classified in the same genomospecies of D. oryzae same as ZYY5T.

Bacillus telluris sp. nov. Isolated from Greenhouse Soil in Beijing, China.

A novel Gram-stain-positive, rod-shaped, endospore-forming bacterium, which we designated as strain 03113T, was isolated from greenhouse soil in Beijing, China. Phylogenetic analysis based on 16S rRNA gene sequences showed strain 03113T is in the genus Bacillus and had the highest similarity to Bacillus solani CCTCC AB 2014277T (98.14%). The strain grew at 4 °C-50 °C (optimum 37 °C), with 0-10% (w/v) NaCl (optimum 5%), and in the range of pH 3.0-12.0 (optimum pH 8.0). Menaquinone was identified as MK-7, and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. The main major cellular fatty acids detected were anteiso-C15:0 (51.35%) and iso-C15:0 (11.06%), which are the predominant cellular fatty acids found in all recognized members of the genus Bacillus. The 16S rRNA gene sequence and core-genome analysis, the average nucleotide identity (ANI), and in silico DNA-DNA hybridization (DDH) value between strain 03113T and the most closely related species were 70.5% and 22.6%, respectively, which supported our conclusion that 03113T represented a novel species in the genus Bacillus. We demonstrated that type strain 03113T (=ACCC 03113T=JCM 33017T) was a novel species in the genus Bacillus, and the name Bacillus telluris sp. nov. was proposed. Strain 03113T secreted auxin IAA and carried the nitrogenase iron protein (nifH) gene, which indicated that strain 03113T has the potential to fix nitrogen and promote plant growth. Bacillus telluris sp. nov. 03113T is a potential candidate for the biofertilizers of organic agriculture areas.

Pseudomonas rhizoryzae sp. nov., isolated from rice.

Two yellow-pigmented, Gram-stain-negative and rod-shaped bacterial strains, designated as RY24T and ZYY160, were isolated from rice. Results of phylogenetic analysis based on 16S rRNA gene sequences showed that strains RY24T and ZYY160 belonged to the genus Pseudomonas, and the 16S rRNA gene sequence similarity was 100 % The DNA homology between the two strains was 99.7 %. The 16S rRNA and rpoD gene sequences of the two strains showed highest similarity values to Pseudomonas oryzihabitans CGMCC 1.3392T and Pseudomonas psychrotolerans DSM 15758T (sharing 99.31 and 94.34 %, respectively). The major fatty acids of two strains were identified as summed feature 8 (C18:1ω7c and/or C18:1ω6c), C16;0 and summed feature 3 (C16:1ω7c and/or C16:1ω6c), and the major respiratory quinone was identified as ubiquinone Q-9, which are typical chemotaxonomic features of members of the genus Pseudomonas. The genomic DNA G+C contents of strains RY24T and ZYY160 were determined to be 64.25 and 64.21 mol%, respectively. The DNA-DNA relatedness and average nucleotide identity values between the two strains and their closely related type strains were below 36 and 90 %, which supported that RY24T and ZYY160 represent a novel species in the genus Pseudomonas. Phylogenetic and chemotaxonomic evidence, together with phenotypic characteristics, showed that the two isolates constitute a novel species of the genus Pseudomonas. The type strain is RY24T (JCM 33201T=ACCC 61555T), for which the name Pseudomonas rhizoryzae sp. nov. is proposed.