Revealing the diversity of Jojoba-associated fungi using amplicon metagenome approach and assessing the in vitro biocontrol activity of its cultivable community.

Jojoba shrubs are wild plants cultivated in arid and semiarid lands and characterized by tolerance to drought, salinity, and high temperatures. Fungi associated with such plants may be attributed to the tolerance of host plants against biotic stress in addition to the promotion of plant growth. Previous studies showed the importance of jojoba as jojoba oil in the agricultural field; however, no prior study discussed the role of jojoba-associated fungi (JAF) in reflecting plant health and the possibility of using JAF in biocontrol. Here, the culture-independent and culture-dependent approaches were performed to study the diversity of the jojoba-associated fungi. Then, the cultivable fungi were evaluated for in-vitro antagonistic activity and in vitro plant growth promotion assays. The metagenome analysis revealed the existence of four fungal phyla: Ascomycota, Aphelidiomycota, Basidiomycota, and Mortierellomycota. The phylum Ascomycota was the most common and had the highest relative abundance in soil, root, branch, and fruit samples (59.7%, 50.7%, 49.8%, and 52.4%, respectively). Alternaria was the most abundant genus in aboveground tissues: branch (43.7%) and fruit (32.1%), while the genus Discosia had the highest abundance in the underground samples: soil (24%) and root (30.7%). For the culture-dependent method, a total of 14 fungi were isolated, identified, and screened for their chitinolytic and antagonist activity against three phytopathogenic fungi (Fusarium oxysporum, Alternaria alternata and Rhizoctonia solani) as well as their in vitro plant growth promotion (PGP) activity. Based on ITS sequence analysis, the selected potent isolates were identified as Aspergillus stellatusEJ-JFF3, Aspergillus flavus EJ-JFF4, Stilbocrea sp. EJ-JLF1, Fusarium solani EJ-JRF3, and Amesia atrobrunneaEJ-JSF4. The endophyte strain A. flavus EJ-JFF4 exhibited the highest chitinolytic activity (9 Enzyme Index) and antagonistic potential against Fusarium oxysporum, Alternaria alternata, and Rhizoctonia solani phytopathogens with inhibitory percentages of 72, 70, and 80 respectively. Also, A. flavus EJ-JFF4 had significant multiple PGP properties, including siderophore production (69.3%), phosphate solubilization (95.4 µg ml-1). The greatest production of Indol-3-Acetic Acid was belonged to A. atrobrunnea EJ-JSF4 (114.5 µg ml-1). The analysis of FUNGuild revealed the abundance of symbiotrophs over other trophic modes, and the guild of endophytes was commonly assigned in all samples. For the first time, this study uncovered fungal diversity associated with jojoba plants using a culture-independent approach and in-vitro assessed the roles of cultivable fungal strains in promoting plant growth and biocontrol. The present study indicated the significance of jojoba shrubs as a potential source of diverse fungi with high biocontrol and PGP activities.

Streptomyces biostimulants: an effective sustainable approach to reduce inorganic N input and maintain high yield of wheat crop in different soil types.

AIMS: This study aimed to isolate and characterize endophytic plant growth-promoting (PGP) actinomycetes from the wild medicinal plant Zygophyllum album. METHODS AND RESULTS: Eight actinomycetes were isolated, identified, and screened for their PGP activities to improve the growth and production of wheat plants under low N-inputs. Based on 16S rRNA analysis, the isolated actinobacteria showed high diversity and had multiple in vitro PGP attributes. In pot experiments, Streptomyces sp. NGB-Act4 and NGB-Act6 demonstrated the highest significant PGP activities to enhance the growth of wheat plants under reduced N-inputs. Under various field conditions (high-fertility clay soils and low-fertility sandy soils), in combination with 50% N-dose, the two streptomycetes showed significant increases in grain N% and grain yield of the wheat crop compared with the 50% N-fertilized treatment. Irrespective of soil type, wheat plants inoculated with strain NGB-Act4 produced grain yield and grain N% significantly greater than or comparable to the full N-dose treatment. CONCLUSIONS: This is the first field report on the successful use of endophytic streptomycetes as an effective strategy to improve wheat yield and reduce the use of synthetic N fertilizers.

A new source of bacterial myrosinase isolated from endophytic Bacillus sp. NGB-B10, and its relevance in biological control activity.

Plant metabolism interacts strongly with the plant microbiome. Glucosinolates, secondary metabolites synthesized by Brassica plants, are hydrolyzed by myrosinase into bioactive compounds of great importance in human health and plant protection. Compared with myrosinase from plant sources, myrosinase enzymes of microbial origin have not been extensively investigated. Therefore, seven endophytic strains corresponding to Bacillus sp. were isolated from Eruca vesicaria ssp. sativa plants that could hydrolyse glucosinolates (sinigrin) in the culture medium and showed myrosinase activity (0.08-19.92 U mL-1). The bglA myrosinase-related gene encoding the 6-phospho-ß-glucosidase (GH 1) from Bacillus sp. NGB-B10, the most active myrosinase-producing bacterium, was successfully identified. Response surface methodology (RSM) was applied to statistically optimize culture conditions for myrosinase production from Bacillus sp. strain NGB-B10. The Plackett-Burman design indicated that nitrogen concentration, incubation period, and agitation speed were the significant parameters in myrosinase production. The application of the Box-Behnken design of RSM resulted in a 10.03-fold increase in enzyme activity as compared to the non-optimized culture conditions. The myrosinase was partially purified by 40% fractionation followed by SDS-PAGE analysis which yielded two subunits that had a molecular weight of 38.6 and 35.0 KDa. The purified enzyme was stable under a broad range of pH (5.5-10) and temperatures (10-65 °C). The hydrolysis products released by bacterial myrosinase from some glucosinolate extracts had higher and/or equivalent in vitro antagonistic activity against several phytopathogenic fungi compared to the nystatin (a broad-spectrum antifungal agent). This study provides original information about a new source of bacterial myrosinase and affords an optimized method to enhance myrosinase production.

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.

Plant-microbe-microbe interactions influence the faba bean nodule colonization by diverse endophytic bacteria.

Legume root nodules harbor rhizobia and other non-nodulating endophytes known as nodule-associated bacteria (NAB) whose role in the legume symbiosis is still unknown. We analysed the genetic diversity of 34 NAB isolates obtained from the root nodules of faba bean grown under various soil conditions in Egypt using 16S rRNA and concatenated sequences of three housekeeping genes. All isolates were identified as members of the family Enterobacteriaceae belonging to the genera Klebsiella, Enterobacter and Raoultella. We identified nine enterobacterial genospecies, most of which have not been previously reported as NAB. All isolated strains harbored nifH gene sequences and most of them possessed plant growth-promoting (PGP) traits. Upon co-inoculation with an N2 fixing rhizobium (Rlv NGB-FR128), two strains (Enterobacter sichanensis NGB-FR97 and Klebsiella variicola NGB-FR116) significantly increased nodulation, growth and N-uptake of faba bean plants over the single treatments or the uninoculated control. The presence of these enterobacteria in nodules was significantly affected by the host plant genotype, symbiotic rhizobium genotype and endophyte genotype, indicating that the nodule colonization process is regulated by plant-microbe-microbe interactions. This study emphasizes the importance of nodule-associated enterobacteria and suggests their potential role in improving the effectiveness of rhizobial inoculants.

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.

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.

Diverse Rhizobium strains isolated from root nodules of Trifolium alexandrinum in Egypt and symbiovars.

Berseem clover (T. alexandrinum) is the main forage legume crop used as animal feed in Egypt. Here, eighty rhizobial isolates were isolated from root nodules of berseem clover grown in different regions in Egypt and were grouped by RFLP-16S rRNA ribotyping. Representative isolates were characterized using phylogenetic analyses of the 16S rRNA, rpoB, glnA, pgi, and nodC genes. We also investigated the performance of these isolates using phenotypic tests and nitrogen fixation efficiency assays. The majority of strains (<90%) were closely related to Rhizobium aegyptiacum and Rhizobium aethiopicum and of the remaining strains, six belonged to the Rhizobium leguminosarum genospecies complex and only one strain was assigned to Agrobacterium fabacearum. Despite their heterogeneous chromosomal background, most of the strains shared nodC gene alleles corresponding to symbiovar trifolii. Some of the strains closely affiliated to R. aegyptiacum and R. aethiopicum had superior nodulation and nitrogen fixation capabilities in berseem clover, compared to the commercial inoculant (Okadein®) and N-added treatments. R. leguminosarum strain NGB-CR 17 that harbored a nodC allele typical of symbiovar viciae, was also able to form an effective symbiosis with clover. Two strains with nodC alleles of symbiovar trifolii, R. aegyptiacum strains NGB-CR 129 and 136, were capable of forming effective nodules in Phaseolus vulgaris in axenic greenhouse conditions. This adds the symbiovar trifolii which is well-established in the Egyptian soils to the list of symbiovars that form nodules in P. vulgaris.

Phylogenetic multilocus sequence analysis of native rhizobia nodulating faba bean (Vicia faba L.) in Egypt.

The taxonomic diversity of forty-two Rhizobium strains, isolated from nodules of faba bean grown in Egypt, was studied using 16S rRNA sequencing, multilocus sequence analyses (MLSA) of three chromosomal housekeeping loci and one nodulation gene (nodA). Based on the 16S rRNA gene sequences, most of the strains were related to Rhizobium leguminosarum, Rhizobium etli, and Rhizobium radiobacter (syn. Agrobacterium tumefaciens). A maximum likelihood (ML) tree built from the concatenated sequences of housekeeping proteins encoded by glnA, gyrB and recA, revealed the existence of three distinct genospecies (I, II and III) affiliated to the defined species within the genus Rhizobium/Agrobacterium. Seventeen strains in genospecies I could be classified as R. leguminosarum sv. viciae. Whereas, a single strain of genospecies II was linked to R. etli. Interestingly, twenty-four strains of genospecies III were identified as A. tumefaciens. Strains of R. etli and A. tumefaciens have been shown to harbor the nodA gene and formed effective symbioses with faba bean plants in Leonard jar assemblies. In the nodA tree, strains belonging to the putative genospecies were closely related to each other and were clustered tightly to R. leguminosarum sv. viciae, supporting the hypothesis that symbiotic and core genome of the species have different evolutionary histories and indicative of horizontal gene transfer among these rhizobia.