Diversity and Structure of the Endophytic Bacterial Communities Associated With Three Terrestrial Orchid Species as Revealed by 16S rRNA Gene Metabarcoding

none4siThe endophytic microbiota can establish mutualistic or commensalistic interactions within the host plant tissues. We investigated the bacterial endophytic microbiota in three species of Mediterranean orchids (Neottia ovata, Serapias vomeracea, and Spiranthes spiralis) by metabarcoding of the 16S rRNA gene. We examined whether the different orchid species and organs, both underground and aboveground, influenced the endophytic bacterial communities. A total of 1,930 operational taxonomic units (OTUs) were obtained, mainly Proteobacteria and Actinobacteria, whose distribution model indicated that the plant organ was the main determinant of the bacterial community structure. The co-occurrence network was not modular, suggesting a relative homogeneity of the microbiota between both plant species and organs. Moreover, the decrease in species richness and diversity in the aerial vegetative organs may indicate a filtering effect by the host plant. We identified four hub OTUs, three of them already reported as plant-associated taxa (Pseudoxanthomonas, Rhizobium, and Mitsuaria), whereas Thermus was an unusual member of the plant microbiota. Core microbiota analysis revealed a selective and systemic ascent of bacterial communities from the vegetative to the reproductive organs. The core microbiota was also maintained in the S. spiralis seeds, suggesting a potential vertical transfer of the microbiota. Surprisingly, some S. spiralis seed samples displayed a very rich endophytic microbiota, with a large number of OTUs shared with the roots, a situation that may lead to a putative restoring process of the root-associated microbiota in the progeny. Our results indicate that the bacterial community has adapted to colonize the orchid organs selectively and systemically, suggesting an active involvement in the orchid holobiont.openAlibrandi, Pasquale; Schnell, Sylvia; Perotto, Silvia; Cardinale, MassimilianoAlibrandi, Pasquale; Schnell, Sylvia; Perotto, Silvia; Cardinale, Massimilian

Bioactive Metabolite Survey of Actinobacteria Showing Plant Growth Promoting Traits to Develop Novel Biofertilizers

The use of chemical fertilizers and pesticides has caused harmful impacts on the environment with the increase in economic burden. Biofertilizers are biological products containing living microorganisms capable of improving plant growth through eco-friendly mechanisms. In this work, three actinobacterial strains Streptomyces violaceoruber, Streptomyces coelicolor, and Kocuria rhizophila were characterized for multiple plant growth promoting (PGP) traits such as indole acetic acid production, phosphate solubilization, N-2-fixation, and drought and salt tolerance. Then, these strains were investigated for their secreted and cellular metabolome, revealing a rich arsenal of bioactive molecules, including antibiotics and siderophores, with S. violaceoruber being the most prolific strain. Furthermore, the in vivo assays, performed on tomato (Solanum lycopersicum L.), resulted in an improved germination index and the growth of seedlings from seeds treated with PGP actinobacteria, with a particular focus on S. violaceoruber cultures. In particular, this last strain, producing volatile organic compounds having antimicrobial activity, was able to modulate volatilome and exert control on the global DNA methylation of tomato seedlings. Thus, these results, confirming the efficacy of the selected actinobacteria strains in promoting plant growth and development by producing volatile and non-volatile bioactive molecules, can promote eco-friendly alternatives in sustainable agriculture

Integrated omics approach reveals the molecular pathways activated in tomato by Kocuria rhizophila, a soil plant growth-promoting bacterium

Plant microbial biostimulants application has become a promising and eco-friendly agricultural strategy to improve crop yields, reducing chemical inputs for more sustainable cropping systems. The soil dwelling bacterium Kocuria rhizophila was previously characterized as Plant Growth Promoting Bacteria (PGPB) for its multiple PGP traits, such as indole-3-acetic acid production, phosphate solubilization capability and salt and drought stress tolerance. Here, we evaluated by a multi-omics approach, the PGP activity of K. rhizophila on tomato, revealing the molecular pathways by which it promotes plant growth. Transcriptomic analysis showed several up-regulated genes mainly related to amino acid metabolism, cell wall organization, lipid and secondary metabolism, together with a modulation in the DNA methylation profile, after PGPB inoculation. In agreement, proteins involved in photosynthesis, cell division, and plant growth were highly accumulated by K. rhizophila. Furthermore, "amino acid and peptides", "monosaccharides", and "TCA" classes of metabolites resulted the most affected by PGPB treatment, as well as dopamine, a catecholamine neurotransmitter mediating plant growth through S-adenosylmethionine decarboxylase (SAMDC), a gene enhancing the vegetative growth, up-regulated in tomato by K. rhizophila treatment. Interestingly, eight gene modules well correlated with differentially accumulated proteins (DAPs) and metabolites (DAMs), among which two modules showed the highest correlation with nine proteins, including a nucleoside diphosphate kinase, and cytosolic ascorbate peroxidase, as well as with several amino acids and metabolites involved in TCA cycle. Overall, our findings highlighted that sugars and amino acids, energy regulators, involved in tomato plant growth, were strongly modulated by the K. rhizophila-plant interaction

Plant growth-promoting bacteria isolated from sugarcane improve the survival of micropropagated plants during acclimatisation

The plant microbiome plays an important role in nutrient acquisition and buffering plant hosts against abiotic and biotic stress. During in vitro propagation of sugarcane, pathogenic microorganisms are eliminated and most of the beneficial endophytic microorganisms. The objective of this study was to isolate and characterise potential plant growth-promoting bacteria (PGPB) from sugarcane and to analyse their ability to improve the survival of micropropagated sugarcane plantlets during the acclimatisation stage. First, bacterial isolates from sugarcane were identified by partial 16S rDNA sequencing and tested for plant growth-promoting (PGP) features, such as inorganic and organic phosphate solubilisation nitrogen fixation, siderophore synthesis, indole-3-acetic acid production, tolerance to abiotic stress and antibiotics production. Then three bacterial strains with multiple PGP traits were independently applied to micropropagated seedlings of the sugarcane variety TUC 03-12 when the plants were transferred to a nursery for ex vitro acclimatisation. The effect of selected PGPB on survival rates of micropropagated plantlets was evaluated in three independent assays, using different batches of seedlings. Thirty days after inoculation, 182-Bacillus and 336-Pseudomonas isolates significantly improved the transferred plants survival rate. High variability in plant survival among independent experiments was observed, but treatments with the 336-Pseudomonas strain showed a low mortality rate (20%) in all assays. This procedure constitutes a biological tool to improve the survival of micropropagated plants during greenhouse acclimatisation. Furthermore, it provides an initial tool for selecting bacteria with possible PGP effects in the field. Highlights - A total of 162 isolates obtained from the rhizosphere, rhizoplane, roots, and stems of sugarcane were characterised for plant growthpromoting features and identified by partial 16S rDNA sequencing. - Two PGPBs strains isolated from sugarcane (182-Bacillus and 336-Pseudomonas) significantly improved survival rates of micropropagated seedlings during the acclimatisation stage. - Under different stress conditions, the 336-Pseudomonas strain improved the survival of micropropagated plants during the acclimatisation stage

Application of PGPB Combined with Variable N Doses Affects Growth, Yield-Related Traits, N-Fertilizer Efficiency and Nutritional Status of Lettuce Grown under Controlled Condition

Nitrogen (N) fertilization is a crucial agricultural practice for boosting production traits in vegetables. However, N synthetic fertilizers—commonly adopted by farmers—have several counterproductive effects on the environment and on humans. The research was performed to assess the combined influence of plant growth promoting bacteria (PGPB) (Azospirillum brasilense DSM 1690, A. brasilense DSM 2298 and Pseudomonas sp. DSM 25356) and various N fertilization doses (0, 30, 60 or 120 kg ha−1) on growth, yield, quality and nitrogen indices of lettuce in protected cultivation. Plant height, root collar diameter, number of leaves and fresh weight were enhanced by A. brasilense DSM 2298 inoculation and N at 30 or 60 kg ha−1. Overall, soluble solids content (SSC), ascorbic acid, total phenolics, carotenoids, total chlorophyll and total sugars were augmented by the combined effect of A. brasilense strains and 30, 60 or 120 kg N ha−1. Furthermore, PGBP inoculation improved potassium (K) and magnesium (Mg) concentrations in leaf tissues. PGPB inoculation increased N leaf concentration; however, it hastened N indices. These results suggest that the PGPB tested can be considered an eco-friendly tool to improve lettuce yield, particularly when combined with N at 30 or 60 kg ha−1

Application of PGPB Combined with Variable N Doses Affects Growth, Yield-Related Traits, N-Fertilizer Efficiency and Nutritional Status of Lettuce Grown under Controlled Condition

Nitrogen (N) fertilization is a crucial agricultural practice for boosting production traits in vegetables. However, N synthetic fertilizers-commonly adopted by farmers-have several counterproductive effects on the environment and on humans. The research was performed to assess the combined influence of plant growth promoting bacteria (PGPB) (Azospirillum brasilense DSM 1690, A. brasilense DSM 2298 and Pseudomonas sp. DSM 25356) and various N fertilization doses (0, 30, 60 or 120 kg ha(-1)) on growth, yield, quality and nitrogen indices of lettuce in protected cultivation. Plant height, root collar diameter, number of leaves and fresh weight were enhanced by A. brasilense DSM 2298 inoculation and N at 30 or 60 kg ha(-1). Overall, soluble solids content (SSC), ascorbic acid, total phenolics, carotenoids, total chlorophyll and total sugars were augmented by the combined effect of A. brasilense strains and 30, 60 or 120 kg N ha(-1). Furthermore, PGBP inoculation improved potassium (K) and magnesium (Mg) concentrations in leaf tissues. PGPB inoculation increased N leaf concentration; however, it hastened N indices. These results suggest that the PGPB tested can be considered an eco-friendly tool to improve lettuce yield, particularly when combined with N at 30 or 60 kg ha(-1)

Application of PGPB Combined with Variable N Doses Affects Growth, Yield-Related Traits, N-Fertilizer Efficiency and Nutritional Status of Lettuce Grown under Controlled Condition

Nitrogen (N) fertilization is a crucial agricultural practice for boosting production traits in vegetables. However, N synthetic fertilizers—commonly adopted by farmers—have several counterproductive effects on the environment and on humans. The research was performed to assess the combined influence of plant growth promoting bacteria (PGPB) (Azospirillum brasilense DSM 1690, A. brasilense DSM 2298 and Pseudomonas sp. DSM 25356) and various N fertilization doses (0, 30, 60 or 120 kg ha−1) on growth, yield, quality and nitrogen indices of lettuce in protected cultivation. Plant height, root collar diameter, number of leaves and fresh weight were enhanced by A. brasilense DSM 2298 inoculation and N at 30 or 60 kg ha−1. Overall, soluble solids content (SSC), ascorbic acid, total phenolics, carotenoids, total chlorophyll and total sugars were augmented by the combined effect of A. brasilense strains and 30, 60 or 120 kg N ha−1. Furthermore, PGBP inoculation improved potassium (K) and magnesium (Mg) concentrations in leaf tissues. PGPB inoculation increased N leaf concentration; however, it hastened N indices. These results suggest that the PGPB tested can be considered an eco-friendly tool to improve lettuce yield, particularly when combined with N at 30 or 60 kg ha−1

The seed endosphere of Anadenanthera colubrina is inhabited by a complex microbiota, including Methylobacteriumspp. and Staphylococcus spp. with potential plant-growth promoting activities

Background and aims Plant seeds are emerging micro–habitats, whose importance as reservoir and vector of beneficial microbes just begins to be recognized. Here we aimed to characterize the bacterial microbiota of the Anadenanthera colubrina seed endosphere, with special focus to beneficial traits and to the colonization pattern. Methods Cultivation–dependent (isolation from surface–sterilized seeds) and cultivation–independent (pyrosequencing of 16S rRNA gene from metagenomic seed DNA) analyses, functional tests and microscopical investigations (fluorescence in situ hybridization coupled with confocal laser scanning microscopy (FISH-CLSM) were performed. Results We isolated several Methylobacterium and Staphylococcus spp., exhibiting both plant growth promotion and antimicrobial activities. The two taxonomic groups showed complementary traits, which supports a functional selection. Both genera were detected also by pyrosequencing, together with further taxa. The genera Friedmaniella, Bifidobacterium, Delftia, Anaerococcus and Actinomyces appeared here for the first time as seed endophytes. We detected bacterial cells and micro–colonies in seed cryosections by FISHCLSM. Alphaproteobacteria, Firmicutes and other bacteria colonized intercellular spaces of the parenchyma and associated to transport vessels. Conclusions This work sheds light onto the diversity, functions and colonization pattern of the Anadenanthera colubrina seed endophytes, and strongly suggest a role as beneficial partners for seed-associated microbiot