Defining the Rhizobium leguminosarum Species Complex

Bacteria currently included in Rhizobium leguminosarum are too diverse to be considered a single species, so we can refer to this as a species complex (the Rlc). We have found 429 publicly available genome sequences that fall within the Rlc and these show that the Rlc is a distinct entity, well separated from other species in the genus. Its sister taxon is R. anhuiense. We constructed a phylogeny based on concatenated sequences of 120 universal (core) genes, and calculated pairwise average nucleotide identity (ANI) between all genomes. From these analyses, we concluded that the Rlc includes 18 distinct genospecies, plus 7 unique strains that are not placed in these genospecies. Each genospecies is separated by a distinct gap in ANI values, usually at approximately 96% ANI, implying that it is a ‘natural’ unit. Five of the genospecies include the type strains of named species: R. laguerreae, R. sophorae, R. ruizarguesonis, “R. indicum” and R. leguminosarum itself. The 16S ribosomal RNA sequence is remarkably diverse within the Rlc, but does not distinguish the genospecies. Partial sequences of housekeeping genes, which have frequently been used to characterize isolate collections, can mostly be assigned unambiguously to a genospecies, but alleles within a genospecies do not always form a clade, so single genes are not a reliable guide to the true phylogeny of the strains. We conclude that access to a large number of genome sequences is a powerful tool for characterizing the diversity of bacteria, and that taxonomic conclusions should be based on all available genome sequences, not just those of type strains

Defining the Rhizobium leguminosarum Species Complex

Bacteria currently included in Rhizobium leguminosarum are too diverse to be considered a single species, so we can refer to this as a species complex (the Rlc). We have found 429 publicly available genome sequences that fall within the Rlc and these show that the Rlc is a distinct entity, well separated from other species in the genus. Its sister taxon is R. anhuiense. We constructed a phylogeny based on concatenated sequences of 120 universal (core) genes, and calculated pairwise average nucleotide identity (ANI) between all genomes. From these analyses, we concluded that the Rlc includes 18 distinct genospecies, plus 7 unique strains that are not placed in these genospecies. Each genospecies is separated by a distinct gap in ANI values, usually at approximately 96% ANI, implying that it is a ‘natural’ unit. Five of the genospecies include the type strains of named species: R. laguerreae, R. sophorae, R. ruizarguesonis, “R. indicum” and R. leguminosarum itself. The 16S ribosomal RNA sequence is remarkably diverse within the Rlc, but does not distinguish the genospecies. Partial sequences of housekeeping genes, which have frequently been used to characterize isolate collections, can mostly be assigned unambiguously to a genospecies, but alleles within a genospecies do not always form a clade, so single genes are not a reliable guide to the true phylogeny of the strains. We conclude that access to a large number of genome sequences is a powerful tool for characterizing the diversity of bacteria, and that taxonomic conclusions should be based on all available genome sequences, not just those of type strains

Genetic diversity of plant growth promoting rhizobacteria and their effects on the growth of maize plants under greenhouse conditions

Engineering of plant rhizosphere with beneficial plant growth promoting (PGP) bacteria offers a great promise for sustainable crop productivity. In this context, 49 rhizospheric/endophytic bacterial isolates were purified using N-free medium, screened in vitro for PGP characteristics and evaluated for their beneficial effects on the early growth of maize (Zea mays L.). The biodiversity of isolated bacteria was analyzed by amplified ribosomal DNA restriction analysis (ARDRA) using four restriction enzymes. Out of the 49 isolates, 7 produced high levels (32.1–82.8 μg mL−1) of indole-3-acetic acid (IAA); 11 had potential phosphate solubilizing abilities (101–163 μg mL−1), while significant acetylene reduction activities (100–1800 nmole C2H4 mg−1 protein h−1) were observed in 12 isolates. Five ribogroups (A-E) were identified using ARDRA. The 16S rRNA-sequence analysis of bacterial representatives from different ribogroups revealed that, 89% of isolates belonged to phylum Proteobacteria, while 11% of them were assigned into phylum Bacteroidetes. Phylum Proteobacteria included Achromobacter, Agrobacterium, Bordetella, Cupriavidus, Ochrobactrum, Pseudoxanthomonas and Stenotrophomonas genera. While, phylum Bacteroidetes was represented by Chryseobacterium and Flavobacterium genera. Under greenhouse conditions, all the selected isolates significantly increased shoot and root fresh and dry biomass of maize plants, compared to the non-inoculated control. Inoculation with Chryseobacterium sp. NGB-29 and Flavobacterium sp. NGB-31 showed the highest beneficial effects on plant growth parameters that were tentatively associated with the high efficiency of these isolates to fix nitrogen and produce high amounts of IAA. The study indicates the potential use of these isolates for production of maize biofertilizers under field conditions. Keywords: Isolation, PGPR, Zea mays, Diversity, IAA, N2- fixation, P- solubilization, Plant inoculation, Plant biomass productio

Comparative Analysis of the Cultured and Total Bacterial Community in the Wheat Rhizosphere Microbiome Using Culture-Dependent and Culture-Independent Approaches

Rhizosphere and root-associated bacteria are key components of crop production and sustainable agriculture. However, utilization of these beneficial bacteria is often limited by conventional culture techniques because a majority of soil microorganisms cannot be cultured using standard laboratory media. Therefore, the purpose of this study was to improve culturability and investigate the diversity of the bacterial communities from the wheat rhizosphere microbiome collected from three locations in Egypt with contrasting soil characteristics by using metagenomic analysis and improved culture-based methods. The improved strategies of the culture-dependent approach included replacing the agar in the medium with gellan gums and modifying its preparation by autoclaving the phosphate and gelling agents separately. Compared to the total operational taxonomic units (OTUs) observed from the metagenomic data sets derived from the three analyzed soils, 1.86 to 2.52% of the bacteria were recovered using the modified cultivation strategies, whereas less than 1% were obtained employing the standard cultivation protocols. Twenty-one percent of the cultivable isolates exhibited multiple plant growth-promoting (PGP) properties, including P solubilization activity and siderophore production. From the metagenomic analysis, the most abundant phyla were Proteobacteria, Actinobacteria, Chloroflexi, Bacteroidetes, and Firmicutes. Moreover, the relative abundance of the specific bacterial taxa was correlated with the soil characteristics, demonstrating the effect of the soil in modulating the plant rhizosphere microbiome. IMPORTANCE Bacteria colonizing the rhizosphere, a narrow zone of soil surrounding the root system, are known to have beneficial effects in improving the growth and stress tolerance of plants. However, most bacteria in natural environments, especially those in rhizosphere soils, are recalcitrant to cultivation using traditional techniques, and thus their roles in soil health and plant growth remain unexplored. Hence, investigating new culture media and culture conditions to bring "not-yet-cultured" species into cultivation and to identify new functions is still an important task for all microbiologists. To this end, we describe improved cultivation protocols that increase the number and diversity of cultured bacteria from the rhizosphere of wheat plants. Using such approaches will lead to new insights into culturing more beneficial bacteria that live in the plant rhizosphere, in so doing creating greater opportunities not only for field application but also for promoting sustainability

Native Rhizospheric and Endophytic Fungi as Sustainable Sources of Plant Growth Promoting Traits to Improve Wheat Growth under Low Nitrogen Input

Wheat crops require effective nitrogen fertilization to produce high yields. Only half of chemical N2 fertilizers are absorbed into plants while the rest remains in the soil, causing environmental problems. Fungi could maximize nitrogen absorption, and from an environmental and biodiversity point of view, there is an urgent necessity for bioprospecting native fungi associated with wild plants growing in harsh environments, e.g., St. Katherine Protectorate (SKP) in the arid Sinai. Recovered taxa, either endophytic and/or rhizospheric, were screened for their plant growth-promoting (PGP) traits. Eighteen fungal isolates (15 rhizospheric and 3 endophytic) belonging to anamorphic ascomycetes were recovered from 9 different wild plants, and their PGP traits (indole-3-acetic acid [IAA] production, phosphate solubilization, siderophore production, and hydrolytic enzyme production) were measured. Rhizospheric isolate NGB-WS14 (Chaetosphaeronema achilleae) produced high levels of IAA (119.1 μg mL−1) in the presence of tryptophan, while NGB-WS 8 (Acrophialophora levis) produced high IAA levels (42.4 μg mL−1) in the absence of tryptophan. The highest phosphate-solubilizing activity (181.9 μg mL−1) was recorded by NGB-WFS2 (Penicillium chrysogenum). Endophytic isolate NGB-WFE16 (Fusarium petersiae) exhibited a high percentage level of Siderophore Unit (96.5% SU). All isolates showed variability in the secretion of extracellular hydrolytic enzymes. Remarkably, all isolates had antagonistic activity (55.6% to 87.3% suppression of pathogen growth) against the pathogenic taxon Alternaria alternata (SCUF00001378) in the dual-assay results. Out of the 18 isolates, 4 rhizospheric and 1 endophytic isolate showed significant increases in shoot dry weight and shoot nitrogen and chlorophyll content of wheat plants subjected to low inputs of chemical nitrogen (N) fertilizers (50% reduction) compared with the non-inoculated control in a pot experiment. Potent taxa were subjected to sequencing for molecular confirmation of phenotypic identification. The retrieved ITS sequences in this study have been deposited in GenBank under accession numbers from LC642736 to LC642740. This study considered the first report of endophytic fungi of Cheilanthes vellea, a wild plant with PGPF which improves wheat growth. These results recommend using PGPF as inoculants to alleviate low nitrogen fertilization