Pomegranate Rhizosphere Microbial Diversity Revealed by Metagenomics: Toward Organic Farming, Plant Growth Promotion and Biocontrol?

Food production must undergo systems change to meet the sustainable development goals (SDGs). For example, organic farming can be empowered by soil microorganisms with plant growth promotion (PGP) and biocontrol features. In this context, there have been limited studies on pomegranate. We investigated microbial diversity in rhizosphere of the pomegranate "Bhagwa" variety and its potential role in PGP and biocontrol. Both bulk and rhizosphere soil samples were analyzed for their physicochemical properties. Whole metagenome sequencing was conducted using the Illumina NovaSeq6000 platform. Surprisingly, we found that bulk and rhizosphere soil samples had comparable microbial diversity. Metagenome sequencing revealed the abundance of Streptomyces indicus, Bradyrhizobium kalamazoonesis, and Pseudomonas cellulosum in the rhizosphere that are reported here for the first time in agricultural literature. Pathway prediction analysis using KEGG (Kyoto Encyclopedia for Genes and Genomes) and COG (clusters of orthologous genes) databases identified metabolic pathways associated with biocontrol properties against pathogens. We confirmed the metagenome data in vitro, which demonstrated their PGP potential and antimicrobial properties. For instance, S. indicus produced high concentration of indole-3-acetic acid, a PGP phytohormone, that can stimulate plant growth. In addition, an antimicrobial susceptibility assay suggested that bacterial extracts displayed activity against Xanthomonas, a primary pathogen causing the pomegranate wilt disease. In conclusion, this study suggests that S. indicus, B. kalamazoonesis, and P. cellulosum can potentially be PGP and biocontrol agents that may contribute to increased crop productivity in pomegranate cultivation. These agents and their combinations warrant future research with an eye on SDGs and so as to enable and innovate organic farming and pomegranate agricultural practices.

Improved species level bacterial characterization from rhizosphere soil of wilt infected Punica granatum.

Pomegranate crops are prone to wilt complex disease, which is known to severely hamper the crop yield. There have been limited studies that have explored bacteria-plant-host associations in wilt complex disease affecting pomegranate crops. In the present study, wilt infected rhizosphere soil samples (ISI, ASI) in pomegranate were studied in comparison to a healthy control (HSC). The 16S metagenomics sequencing approach using the MinION platform was employed for screening of bacterial communities and predictive functional pathways. Altered physicochemical properties in the soil samples were recorded showing a comparatively acidic pH in the ISI (6.35) and ASI (6.63) soil samples to the HSC soil (7.66), along with higher electrical conductivity in the ISI (139.5 µS/cm), ASI soil (180 µS/cm), HSC soil sample (123.33 µS/cm). While concentration of micronutrients such as Cl and B were significantly higher in the ISI and ASI soil as compared to the HSC, Cu and Zn were significantly higher in the ASI soil. The effectiveness and accuracy of 16S metagenomics studies in identifying beneficial and pathogenic bacterial communities in multi-pathogen-host systems depend on the completeness and consistency of the available 16S rRNA sequence repositories. Enhancing these repositories could significantly improve the exploratory potential of such studies. Thus, multiple 16S rRNA data repositories (RDP, GTDB, EzBioCloud, SILVA, and GreenGenes) were benchmarked, and the findings indicated that SILVA yields the most reliable matches. Consequently, SILVA was chosen for further analysis at the species level. Relative abundance estimates of bacterial species showed variations of growth promoting bacteria, namely, Staphylococcus epidermidis, Bacillus subtilis, Bacillus megatarium, Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas stutzeri and Micrococcus luteus. Functional profiling predictions employing PICRUSt2 revealed a number of enriched pathways such as transporter protein families involved in signalling and cellular processes, iron complex transport system substrate binding protein, peptidoglycan biosynthesis II (staphylococci) and TCA cycle VII (acetate-producers). In line with past reports, results suggest that an acidic pH along with the bioavailability of micronutrients such as Fe and Mn could be facilitating the prevalence and virulence of Fusarium oxysporum, a known causative pathogen, against the host and beneficial bacterial communities. This study identifies bacterial communities taking into account the physicochemical and other abiotic soil parameters in wilt-affected pomegranate crops. The insights obtained could be instrumental in developing effective management strategies to enhance crop yield and mitigate the impact of wilt complex disease on pomegranate crops.

Targeted metagenome sequencing reveals the abundance of Planctomycetes and Bacteroidetes in the rhizosphere of pomegranate.

Agricultural productivity of pomegranate can be enhanced by identifying the crop-associated microbial diversity in the rhizosphere region with respect to plant growth promoters and other beneficial organisms. Traditional culture methods have limitations in microbial screening as only 1-2% of these organisms can be cultured. In the present study, 16S rRNA amplicon-based metagenomics approach using MinION Oxford Nanopore platform was employed to explore the microbial diversity in the rhizosphere of pomegranate Bhagwa variety, across variable soil depths from 0 to 5 cms (R2), 5-10 cms (R4) and 10-15 cms (R6), using bulk soil as the control. Across all the three layers, significant variations in pH, nitrogen content and total fungal count were observed. 16S rRNA analysis showed the abundance of planctomycetes, Pirellula staleyi, followed by bacteroidetes, Flavisolibacter LC59 and Niastella koreensis across the various soil depths in the rhizospheric soil samples. Pathway prediction analysis indicated arginine and proline metabolism (gamma-glutamyl putrescine oxidase) and hydrogen sulfide biosynthesis as the most abundant pathway hits. Comparative abundance analysis across layers showed the R6 layer with the maximum microbial diversity in terms of highest dimension of variation (79.2%) followed by R4 and R2 layers (p < 0.01). Our analysis shows the significant influence of root zone in shaping microbial diversity. This study has reported the presence of Planctomycetes, Pirellula staleyi for the first time in the pomegranate field.

Microbiomics and cloud-based analytics advance sustainable soil management.

Soil constitutes a major component of the agro-ecosystem. Unrestrained uses of chemical pesticides and increased human activities have contributed to unprecedented changes in soil microflora affecting productivity. Modern microbiomics has proven to be an indispensable tool to understand the adaptations underlying complex soil microbial communities and their beneficial applications. In this review, we seek to emphasize the scope of microbiomics in enhancing soil productivity by providing an overview of the various sequencing platforms considering key parameters such as the accuracy, read lengths, reads per run, time involved and weighing out their pros and cons. The advances in modern ultra-high-throughput microbiomics platforms in combination with cloud-based analytics for in-depth exploration of soil-microbe associations can help achieve sustainable soil management contributing to better plant yield and productivity.