Screening and Characterization of Phytases from Bacteria Isolated from Chilean Hydrothermal Environments.

Phytases are enzymes involved in organic phosphorus cycling in nature and widely used as feed additives in animal diets. Thermal tolerance is a desired property of phytases. The objectives of this study were to screen and characterize bacterial phytases from Chilean hydrothermal environments. In this study, 60% (30 of 63) of screened thermophilic (60 °C) isolates showed phytase activity in crude protein extracts. The characterization of phytase from two selected isolates (9B and 15C) revealed that both isolates produce phytases with a pH optimum at 5.0. The temperature optimum for phytate dephosphorylation was determined to be 60 and 50 °C for the phytases from the isolates 9B and 15C, respectively. Interestingly, the phytase from the isolate 15C showed a residual activity of 46% after incubation at 90 °C for 20 min. The stepwise dephosphorylation of phytate by protein extracts of the isolates 9B and 15C was verified by HLPC analysis. Finally, the isolates 9B and 15C were identified by partial sequencing of the 16S rRNA gene as members of the genera Bacillus and Geobacillus, respectively.

Rhizobacterial Community Structures Associated with Native Plants Grown in Chilean Extreme Environments.

Chile is topographically and climatically diverse, with a wide array of diverse undisturbed ecosystems that include native plants that are highly adapted to local conditions. However, our understanding of the diversity, activity, and role of rhizobacteria associated with natural vegetation in undisturbed Chilean extreme ecosystems is very poor. In the present study, the combination of denaturing gradient gel electrophoresis and 454-pyrosequencing approaches was used to describe the rhizobacterial community structures of native plants grown in three representative Chilean extreme environments: Atacama Desert (ATA), Andes Mountains (AND), and Antarctic (ANT). Both molecular approaches revealed the presence of Proteobacteria, Bacteroidetes, and Actinobacteria as the dominant phyla in the rhizospheres of native plants. Lower numbers of operational taxonomic units (OTUs) were observed in rhizosphere soils from ATA compared with AND and ANT. Both approaches also showed differences in rhizobacterial community structures between extreme environments and between plant species. The differences among plant species grown in the same environment were attributed to the higher relative abundance of classes Gammaproteobacteria and Alphaproteobacteria. However, further studies are needed to determine which environmental factors regulate the structures of rhizobacterial communities, and how (or if) specific bacterial groups may contribute to the growth and survival of native plants in each Chilean extreme environments.

Microencapsulation by spray drying of nitrogen-fixing bacteria associated with lupin nodules.

Plant growth promoting bacteria and nitrogen-fixing bacteria (NFB) used for crop inoculation have important biotechnological potential as a sustainable fertilization tool. However, the main limitation of this technology is the low inoculum survival rate under field conditions. Microencapsulation of bacterial cells in polymer matrices provides a controlled release and greater protection against environmental conditions. In this context, the aim of this study was to isolate and characterize putative NFB associated with lupin nodules and to evaluate their microencapsulation by spray drying. For this purpose, 21 putative NFB were isolated from lupin nodules and characterized (16S rRNA genes). Microencapsulation of bacterial cells by spray drying was studied using a mixture of sodium alginate:maltodextrin at different ratios (0:15, 1:14, 2:13) and concentrations (15 and 30% solids) as the wall material. The microcapsules were observed under scanning electron microscopy to verify their suitable morphology. Results showed the association between lupin nodules of diverse known NFB and nodule-forming bacteria belonging to Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria and Bacteroidetes. In microencapsulation assays, the 1:14 ratio of sodium alginate:maltodextrin (15% solids) showed the highest cell survival rate (79%), with a microcapsule yield of 27% and spherical microcapsules of 5-50 µm in diameter. In conclusion, diverse putative NFB genera and nodule-forming bacteria are associated with the nodules of lupine plants grown in soils in southern Chile, and their microencapsulation by spray drying using sodium alginate:maltodextrin represents a scalable process to generate a biofertilizer as an alternative to traditional nitrogen fertilization.

Variovorax sp. strain P1R9 applied individually or as part of bacterial consortia enhances wheat germination under salt stress conditions.

Endophytes isolated from extremophile plants are interesting microbes for improving the stress tolerance of agricultural plants. Here, we isolated and characterized endophytic bacteria showing plant growth-promoting (PGP) traits from plants in two extreme Chilean biomes (Atacama Desert and Chilean Patagonia). Forty-two isolates were characterized as both halotolerant auxin producers (2-51 mg L-1) and 1-aminocyclopropane-1-carboxylate (ACC)-degrading bacteria (15-28 µmol αKB mg protein-1 h-1). The most efficient isolates were tested as single strains, in dual and triple consortia, or in combination with previously reported PGP rhizobacteria (Klebsiella sp. 27IJA and 8LJA) for their impact on the germination of salt-exposed (0.15 M and 0.25 M NaCl) wheat seeds. Interestingly, strain P1R9, identified as Variovorax sp., enhanced wheat germination under salt stress conditions when applied individually or as part of bacterial consortia. Under salt stress, plants inoculated with dual consortia containing the strain Variovorax sp. P1R9 showed higher biomass (41%) and reduced lipid peroxidation (33-56%) than uninoculated plants. Although the underlying mechanisms remain elusive, our data suggest that the application of Variovorax sp. P1R9, alone or as a member of PGP consortia, may improve the salt stress tolerance of wheat plants.

Endophytic bacterial communities in ungerminated and germinated seeds of commercial vegetables.

Chile is a prominent seed exporter globally, but the seed microbiome of vegetables (46% of seeds) and its role in the early stages of plant growth have remained largely unexplored. Here, we employed DNA metabarcoding analysis to investigate the composition and putative functions of endophytic bacterial communities in ungerminated and germinated seeds of the commercial vegetables Apiaceae (parsley and carrot), Asteraceae (lettuce), Brassicaceae (cabbage and broccoli), and Solanaceae (tomato). Bacterial quantification showed 104 to 108 copies of the 16S rRNA gene per gram of ungerminated and germinated seeds. Alpha diversity analysis (e.g., Chao1, Shannon, and Simpson indices) did not indicate significant differences (Kruskal-Wallis test) between ungerminated and germinated seeds, except for Solanaceae. However, beta diversity (PCoA) analysis showed distinctions (Adonis test) between ungerminated and germinated seeds, except Apiaceae. Pseudomonadota and Bacillota were identified as the dominant and specialist taxa in both ungerminated and germinated seed samples. Chemoheterotrophy and fermentation were predicted as the main microbial functional groups in the endophytic bacterial community. Notably, a considerable number of the 143 isolated endophytic strains displayed plant growth-promoting traits (10 to 64%) and biocontrol activity (74% to 82%) against plant pathogens (Xanthomonas and Pseudomonas). This study revealed the high variability in the abundance, diversity, composition, and functionality of endophytic bacteria between ungerminated and germinated seeds in globally commercialized vegetables. Furthermore, potential beneficial endophytic bacteria contained in their seed microbiomes that may contribute to the microbiome of the early stages, development, growth and progeny of vegetables were found.

Isolation and Characterization of Cold-Tolerant Hyper-ACC-Degrading Bacteria from the Rhizosphere, Endosphere, and Phyllosphere of Antarctic Vascular Plants.

1-Aminociclopropane-1-carboxylate (ACC)-degrading bacteria having been widely studied for their use in alleviating abiotic stresses in plants. In the present study, we isolated and characterized ACC-degrading bacteria from the rhizosphere, phyllosphere, and endosphere of the Antarctic vascular plants Deschampsia antarctica and Colobanthus quitensis. One hundred and eighty of the 578 isolates (31%) were able to grow on minimal medium containing ACC, with 101 isolates (23, 37, and 41 endosphere-, phyllosphere- and rhizosphere-associated isolates, respectively) identified as being genetically unique by enterobacterial repetitive intergenic consensus (ERIC)-PCR. Subsequently, freeze/thaw treatments and ice-recrystallization-inhibition (IRI) activity assays were performed, the results of which revealed that 77 (13%) of cold-tolerant isolates exhibited putative ACC deaminase activity. Significant (p ≤ 0.05) differences in IRI activity were also observed between the studied plant niches. Surprisingly, all the cold-tolerant isolates showed ACC deaminase activity, independent of the plant niches, with 12 isolates showing the highest ACC deaminase activities of 13.21-39.56 mmol α KB mg protein-1 h-1. These isolates were categorized as 'cold-tolerant hyper-ACC-degrading bacteria', and identified as members of Pseudomonas, Serratia, and Staphylococcus genera. The results revealed the occurrence of cold-tolerant hyper-ACC-degrading bacteria in diverse plant niches of Antarctic vascular plants, that could be investigated as novel microbial inoculants to alleviate abiotic stresses in plants.

Niche Differentiation in the Composition, Predicted Function, and Co-occurrence Networks in Bacterial Communities Associated With Antarctic Vascular Plants.

Climate change directly affecting the Antarctic Peninsula has been reported to induce the successful colonization of ice-free lands by two Antarctic vascular plants (Deschampsia antarctica and Colobanthus quitensis). While studies have revealed the importance of microbiota for plant growth and stress tolerance in temperate climates, the role that plant-associated microbes play in the colonization of ice-free lands remains unknown. Consequently, we used high-throughput DNA sequence analyses to explore the composition, predicted functions, and interactive networks of plant-associated microbial communities among the rhizosphere, endosphere, and phyllosphere niches of D. antarctica and C. quitensis. Here we report a greater number of operational taxonomic units (OTUs), diversity, and richness in the microbial communities from the rhizosphere, relative to endosphere and phyllosphere. While taxonomic assignments showed greater relative abundances of Proteobacteria, Bacteroidetes, and Actinobacteria in plant niches, principal coordinate analysis revealed differences among the bacterial communities from the other compartments examined. More importantly, however, our results showed that most of OTUs were exclusively found in each plant niche. Major predicted functional groups of these microbiota were attributed to heterotrophy, aerobic heterotrophy, fermentation, and nitrate reduction, independent of plant niches or plant species. Co-occurrences network analyses identified 5 (e.g., Microbacteriaceae, Pseudomonaceae, Lactobacillaceae, and Corynebacteriaceae), 23 (e.g., Chitinophagaceae and Sphingomonadaceae) and 7 (e.g., Rhodospirillaceae) putative keystone taxa present in endosphere, phyllosphere, and rhizosphere, respectively. Our results revealed niche differentiation in Antarctic vascular plants, highlighting some putative microbial indicators and keystone taxa in each niche. However, more studies are required to determine the pivotal role that these microbes play in the successful colonization of ice-free lands by Antarctic plants.

Endophytic Bacterial Communities Associated with Roots and Leaves of Plants Growing in Chilean Extreme Environments.

Several studies have demonstrated the relevance of endophytic bacteria on the growth and fitness of agriculturally-relevant plants. To our knowledge, however, little information is available on the composition, diversity, and interaction of endophytic bacterial communities in plants struggling for existence in the extreme environments of Chile, such as the Atacama Desert (AD) and Patagonia (PAT). The main objective of the present study was to analyze and compare the composition of endophytic bacterial communities associated with roots and leaves of representative plants growing in Chilean extreme environments. The plants sampled were: Distichlis spicate and Pluchea absinthioides from the AD, and Gaultheria mucronata and Hieracium pilosella from PAT. The abundance and composition of their endophytic bacterial communities was determined by quantitative PCR and high-throughput sequencing of 16S rRNA, respectively. Results indicated that there was a greater abundance of 16S rRNA genes in plants from PAT (1013 to 1014 copies g-1 DNA), compared with those from AD (1010 to 1012 copies g-1 DNA). In the AD, a greater bacterial diversity, as estimated by Shannon index, was found in P. absinthioides, compared with D. spicata. In both ecosystems, the greater relative abundances of endophytes were mainly attributed to members of the phyla Proteobacteria (14% to 68%), Firmicutes (26% to 41%), Actinobacteria (6 to 23%) and Bacteroidetes (1% to 21%). Our observations revealed that most of operational taxonomic units (OTUs) were not shared between tissue samples of different plant species in both locations, suggesting the effect of the plant genotype (species) on the bacterial endophyte communities in Chilean extreme environments, where Bacillaceae and Enterobacteriacea could serve as keystone taxa as revealed our linear discriminant analysis.