Functional Genome Analysis of a Conditionally Pathogenic Rhizobacterial Strain, Pseudomonas putida AKMP7.

This manuscript reports the whole genome sequence of a conditionally pathogenic rhizobacterial strain, Pseudomonas putida AKMP7, which has been previously reported by us to be beneficial to Arabidopsis thaliana under well-watered conditions and pathogenic to the plant under water stress. As part of a study to understand this unique behavior, the whole genome sequence of this strain was analyzed. Based on the results, it was identified that the total length of the AKMP7 genome is 5,764,016 base pairs, and the total GC content of the genome is 62.93% (typical of P. putida). Using RAST annotation pipeline, it was identified that the genome has 5605 coding sequences, 80 repeat regions, 71 tRNA genes, and 22 rRNA genes. A total of 4487 functional proteins and 1118 hypothetical proteins were identified. Phylogenetic analysis has classified it as P. putida species, with a P value of 0.03. In order to identify close relatives of this strain, comparative genomics was performed with 30 other P. putida strains, taken from publicly available genome databases, using Average Nucleotide Identity (ANI) analysis. Whole genome comparison with these strains reveals that AKMP7 possesses Type-IV Secretion System (T4SS) with conjugative transfer functionality. Interestingly, the T4SS feature is absent in all the beneficial/harmless strains of P. putida that we analyzed. All the plant pathogenic bacteria that were analyzed had the T4SS feature in their genome, indicating its role in pathogenesis. This study aims to address important gaps in understanding the molecular mechanisms involved in the conditional/opportunistic pathogenesis of plant-associated, beneficial soil bacteria, using genomics approaches.

Azospirillum-Bacillus associations: synergistic effects on in vitro PGP traits and growth of pearl millet at early seedling stage under limited moisture conditions.

The association of plant beneficial Azospirillum and Bacillus spp. strains expressing different sets of PGP traits may have complementary or supplementary effects on host plants. In the present investigation, A. formosense and Bacillus spp. strains showing diverse PGP traits (IAA production, nitrogenase activity, phosphate, zinc and potassium solubilization, siderophores, antagonism against phytopathogens, osmotic stress tolerance, etc.) were assessed for compatibility by cross-streaking and co-culturing. Under co-culture (Azospirillum + Bacillus), a significant increase in the expression of PGP traits, nitrogenase activity (up to 89%), phosphate solubilization (upto 236%), siderophore production (upto 20%) was observed as compared to individual Azospirillum culture, indicating synergistic effect of co-culture. IAA production was higher in Azospirillum sp. strains as compared to Bacillus spp. strains, when cultured individually; however, when co-cultured, the IAA levels were in the mid-range indicating the contributory effects of compatible strains. The effect of individual Azospirillum and Bacillus strains and their co-inoculation was also assessed on the growth of pearl millet at early stages under moisture-deficit stress imposed using PEG6000 (0, 10, and 20%). Co-inoculation enhanced seed germination (up to 10, 3, and 6% increase under 0, 10, and 20% PEG, respectively, over individual Azospirillum treatment), root traits (increased root hair density and lateral branches), and seedling vigor indices (up to 22, 32, 43% increase in seed vigor index I and 8, 14, and 10% increase in seed vigor index II under 0, 10, 20% PEG, respectively, over individual Azospirillum treatment) under normal as well as moisture-deficit conditions suggesting the role of Bacillus spp. strains in better adaptation of the plants to stress and higher yield potential. The synergistic effect of co-cultured Azospirillum and Bacillus strains on PGP traits indicated metabolic interplay between the two strains which needs to be further understood. The positive effect of co-inoculation on plant growth under moisture-deficit stress indicated the promise of Azospirillum and Bacillus as a synergistic bioformulation for combating nutrient and drought stress in pearl millet, particularly in nutrient-poor dryland agricultural systems. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03503-4.

Production of ethanol, lipid and lactic acid from mixed agrowastes hydrolysate.

To combat the shortage of single agro-residue and overcome the problem of seasonal availability, it is beneficial to use mixture of lignocellulosic biomasses. In the present study, efforts were made to use mixed lignocellulosic biomass for production of bioethanol, along with microbial lipids and lactic acid. Upon enzymatic hydrolysis of mixed biomass at varied proportions it was observed that mixture of paddy straw and jute in the ratio 3:1 resulted in best sugar yield (41.50 g/L) at 10% substrate loading. Ethanolic fermentation of mixed substrate hydrolysate by thermotolerant yeast, Saccharomyces cerevisiae JRC6 resulted in 8.39 g/L of ethanol. To maintain sustainability and economic impact, oleaginous yeast (Trichosporon mycotoxinivorans S2) and lactic acid bacteria (Lactobacillus plantarum LP-9) were used for lipid production (14.5 g/L) and lactic acid production (11.08 g/L), respectively. Therefore, this study explored the potential of mixed lignocellulosic biomass to be exploited for production of various value-added products.

No-Tillage with Residue Retention and Foliar Sulphur Nutrition Enhances Productivity, Mineral Biofortification and Crude Protein in Rainfed Pearl Millet under Typic Haplustepts: Elucidating the Responses Imposed on an Eight-Year Long-Term Experiment.

Yield limitation and widespread sulphur (S) deficiency in pearl-millet-nurturing dryland soils has emerged as a serious threat to crop productivity and quality. Among diverse pathways to tackle moisture and nutrient stress in rainfed ecologies, conservation agriculture (CA) and foliar nutrition have the greatest potential due to their economic and environmentally friendly nature. Therefore, to understand ammonium thiosulphate (ATS)-mediated foliar S nutrition effects on yield, protein content, mineral biofortification, and sulphur economy of rainfed pearl millet under diverse crop establishment systems, a field study was undertaken. The results highlighted that pearl millet grain and protein yield was significantly higher under no-tillage +3 t/ha crop residue mulching (NTCRM) as compared to no-tillage without mulch (NoTill) and conventional tillage (ConvTill), whereas the stover yield under NTCRM and ConvTill remained at par. Likewise, grain and stover yield in foliar S application using ATS 10 mL/L_twice was 19.5% and 13.2% greater over no S application. The sulphur management strategy of foliar-applied ATS 10 mL/L_twice resulted in significant improvement in grain protein content, protein yield, micronutrient fortification, and net returns (₹ 54.6 × 1000) over the control. Overall, ATS-mediated foliar S nutrition can be an alternate pathway to S management in pearl millet for yield enhancement, micronutrient biofortification and grain protein content increase under ConvTill, as well as under the new NTCRM systems.

Foliar nutrient supplementation with micronutrient-embedded fertilizer increases biofortification, soil biological activity and productivity of eggplant.

Micronutrient malnutrition or hidden hunger remains a major global challenge for human health and wellness. The problem results from soil micro- and macro-nutrient deficiencies combined with imbalanced fertilizer use. Micronutrient-embedded NPK (MNENPK) complex fertilizers have been developed to overcome the macro- and micro-element deficiencies to enhance the yield and nutritive value of key crop products. We investigated the effect of foliar applications of an MNENPK fertilizer containing N, P, K, Fe, Zn and B in combination with traditional basal NPK fertilizers in terms of eggplant yield, fruit nutritive quality and on soil biological properties. Applying a multi-element foliar fertilizer improved the nutritional quality of eggplant fruit, with a significant increases in the concentration of Fe (+ 26%), Zn (+ 34%), K (+ 6%), Cu (+ 24%), and Mn (+ 27%), all of which are essential for human health. Increasing supply of essential micronutrients during the plant reproductive stages increased fruit yield, as a result of improved yield parameters. The positive effect of foliar fertilizing with MNENPK on soil biological parameters (soil microbial biomass carbon, dehydrogenase, alkaline phosphatase) also demonstrated its capacity to enhance soil fertility. This study suggests that foliar fertilizing with a multi-nutrient product such as MNENPK at eggplant flowering and fruiting stages, combined with the recommended-doses of NPK fertilizers is the optimal strategy to improve the nutritional quality of eggplant fruits and increase crop yields, both of which will contribute to reduce micronutrient malnutrition and hunger globally.

One-pot microbial bioconversion of wheat bran ferulic acid to biovanillin.

Due to growing consumer preference towards natural ingredients in food products, the production of flavors by microbial biotransformation of agrowastes provides an eco-friendly, cost-effective and sustainable pathway for biovanillin production. In the present study, biovanillin was produced by microbial biotransformation of ferulic acid (FA) using Streptomyces sp. ssr-198. The strain was able to grow in glucose medium supplemented with 1 g/L FA and produce 20.91 ± 1.11 mg/L vanillin within 96 h, along with 5.78 ± 0.13 mg/L vanillic acid in 144 h. Estimation of enzymes involved in FA degradation detected maximum feruloyl-CoA synthetase activity (1.21 ± 0.03 U/mg protein) at 96 h and maximum vanillin dehydrogenase activity (0.31 ± 0.008 U/mg protein) at 168 h, with small amounts of ferulic acid esterase activity (0.13 ± 0.002 U/mg protein) in the fermentation medium. Further, the glucose deficient production medium supplemented with 3 g/L of ferulic acid when inoculated with Streptomyces sp. ssr-198 (6% wet weight) produced maximum vanillin (685 ± 20.11 mg/L) within 72 h at 37 °C under agitation (150 rpm) and declined thereafter. Furthermore, in a one-pot experiment, wherein crude ferulic acid esterase (700 IU/g of substrate) from Enterococcus lactis SR1 was added into 10% w/v wheat bran (natural source of ferulic acid) based medium and was inoculated with 1% w/v of Streptomyces sp. ssr-198 resulted in maximum vanillin production (1.02 ± 0.02 mg/g of substrate) within 60 h of incubation. The study provides an insight into synergistic effect of using FAE of E. lactis SR1 and Streptomyces sp. ssr-198 for bioproduction of biovanillin using agro residues. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-03006-0.

Deciphering the Role of Trehalose in Tripartite Symbiosis Among Rhizobia, Arbuscular Mycorrhizal Fungi, and Legumes for Enhancing Abiotic Stress Tolerance in Crop Plants.

Drought is a critical factor limiting the productivity of legumes worldwide. Legumes can enter into a unique tripartite symbiotic relationship with root-nodulating bacteria of genera Rhizobium, Bradyrhizobium, or Sinorhizobium and colonization by arbuscular mycorrhizal fungi (AMF). Rhizobial symbiosis provides nitrogen necessary for growth. AMF symbiosis enhances uptake of diffusion-limited nutrients such as P, Zn, Cu, etc., and also water from the soil via plant-associated fungal hyphae. Rhizobial and AMF symbioses can act synergistically in promoting plant growth and fitness, resulting in overall yield benefits under drought stress. One of the approaches that rhizobia use to survive under stress is the accumulation of compatible solutes, or osmolytes, such as trehalose. Trehalose is a non-reducing disaccharide and an osmolyte reported to accumulate in a range of organisms. High accumulation of trehalose in bacteroids during nodulation protects cells and proteins from osmotic shock, desiccation, and heat under drought stress. Manipulation of trehalose cell concentrations has been directly correlated with stress response in plants and other organisms, including AMF. However, the role of this compound in the tripartite symbiotic relationship is not fully explored. This review describes the biological importance and the role of trehalose in the tripartite symbiosis between plants, rhizobia, and AMF. In particular, we review the physiological functions and the molecular investigations of trehalose carried out using omics-based approaches. This review will pave the way for future studies investigating possible metabolic engineering of this biomolecule for enhancing abiotic stress tolerance in plants.

Maize seed endophytic bacteria: dominance of antagonistic, lytic enzyme-producing Bacillus spp.

Beneficial aspects of endophytic microorganisms have motivated researchers to explore plant endophytic world. The present study was aimed to isolate and characterize the seed-borne endophytic bacteria from diverse maize genotypes. Eighty maize seed endophytic bacteria (MSEB), isolated from 30 maize genotypes, were characterized using polyphasic approach. The dendrograms and phylogenetic tree generated on the basis of ARDRA analysis and metabolic profiling of endophytic bacteria revealed genotypic and biochemical diversity among MSEB. The 16S rDNA sequence analysis revealed Bacillus as the most dominant encountered genus affiliated with Phylum Firmicutes. Few isolates belonged to genus Staphylococcus, whereas one isolate was identified as Corynebacterium sp. under Phylum Actinobacteria. Majority of the MSEB isolates exhibited antagonism against phytopathogenic fungi, production of ammonia, and secretion of lytic enzymes; some isolates also exhibited indole acetic acid production, the traits of which can be helpful in endophytic establishment and advantageous to the host plant. Besides, many MSEB exhibited tolerance to salinity (10%), osmotic stress (40% PEG6000), and temperature (60 °C), indicating their possible application under stress conditions. Endophytic nature of the selected MSEB isolates was confirmed by tracking their presence in shoots, leaves, and roots of the host seedlings with the help of biochemical marker (rifampicin resistance). Thus, the MSEB identified in the present study can be explored as potential bioinputs for improving plant growth and productivity under stressed conditions, besides helping in understanding the plant-endophyte interactions.

Influence of Bacillus spp. strains on seedling growth and physiological parameters of sorghum under moisture stress conditions.

Microorganisms isolated from stressed ecosystem may prove as ideal candidates for development of bio-inoculants for stressed agricultural production systems. In the present study, moisture stress tolerant rhizobacteria were isolated from the rhizosphere of sorghum, pigeonpea, and cowpea grown under semiarid conditions in India. Four isolates KB122, KB129, KB133, and KB142 from sorghum rhizosphere exhibited plant growth promoting traits and tolerance to salinity, high temperature, and moisture stress. These isolates were identified as Bacillus spp. by 16S rDNA sequence analysis. The strains were evaluated for growth promotion of sorghum seedlings under two different moisture stress conditions (set-I, continuous 50% soil water holding capacity (WHC) throughout the experiment and set-II, 75% soil WHC for 27 days followed by no irrigation for 5 days) under greenhouse conditions. Plate count and scanning electron microscope studies indicated successful root surface colonization by inoculated bacteria. Plants inoculated with Bacillus spp. strains showed better growth in terms of shoot length and root biomass with dark greenish leaves due to high chlorophyll content while un-inoculated plants showed rolling of the leaves, stunted appearance, and wilting under both stress conditions. Inoculation also improved leaf relative water content and soil moisture content. However, variation in proline and sugar content in the different treatments under two stress conditions indicated differential effect of microbial treatments on plant physiological parameters under stress conditions.

Characterization of rhizobial isolates nodulating Millettia pinnata in India.

Millettia pinnata (Synonym Pongamia pinnata) is a viable source of oil for the mushrooming biofuel industry, source for agroforestry, urban landscaping, and the bio-amelioration of degraded lands. It also helps in maintaining soil fertility through symbiotic nitrogen fixation. However, not much work is reported on classification and characterization of the rhizobia associated with this plant. In the present study, an attempt was made to isolate rhizobial strains nodulating Millettia from soils collected from southern regions of India. The isolates were characterized using numerical taxonomy, 16S rRNA gene sequencing, and cross nodulation ability. The results showed high phenotypic and genetic diversity among the rhizobia symbiotic with Millattia pinnata. The isolates formed five clusters at similarity level of 0.82 based on the results of numerical taxonomy. Results on 16S rRNA gene sequence analysis revealed that most microsymbionts of M. pinnata belonged to Rhizobium and Bradyrhizobium, which are closely related to Rhizobium sp., B. elkanii and B. yuanmingense. Among these isolates, some isolates could grow in a pH range of 4.0-10.0, some could tolerate a high salt concentration (3% NaCl) and could grow at a maximum temperature between 35 and 45 °C. M. pinnata formed nodules with diverse rhizobia in Indian soils. These results offered the first systematic information about the microsymbionts of M. pinnata grown in the soils from southern part of India.

Effect of osmotic stress on plant growth promoting Pseudomonas spp.

In this study we isolated and screened drought tolerant Pseudomonas isolates from arid and semi arid crop production systems of India. Five isolates could tolerate osmotic stress up to -0.73 MPa and possessed multiple PGP properties such as P-solubilization, production of phytohormones (IAA, GA and cytokinin), siderophores, ammonia and HCN however under osmotic stress expression of PGP traits was low compared to non-stressed conditions. The strains were identified as Pseudomonas entomophila, Pseudomonas stutzeri, Pseudomonas putida, Pseudomonas syringae and Pseudomonas monteilli respectively on the basis of 16S rRNA gene sequence analysis. Osmotic stress affected growth pattern of all the isolates as indicated by increased mean generation time. An increase level of intracellular free amino acids, proline, total soluble sugars and exopolysaccharides was observed under osmotic stress suggesting bacterial response to applied stress. Further, strains GAP-P45 and GRFHYTP52 showing higher levels of EPS and osmolytes (amino acids and proline) accumulation under stress as compared to non-stress conditions, also exhibited higher expression of PGP traits under stress indicating a relationship between stress response and expression of PGP traits. We conclude that isolation and screening of indigenous, stress adaptable strains possessing PGP traits can be a method for selection of efficient stress tolerant PGPR strains.

Molecular and biochemical approaches for characterization of antifungal trait of a potent biocontrol agent Bacillus subtilis RP24.

Bacillus subtilis strain RP24, isolated from rhizoplane of field grown pigeon pea, exhibited in vitro antagonism against a wide range of phytopathogenic fungi. An attempt was made to partially purify and characterize the diffusible antifungal metabolite/s produced by the strain RP24 and its negative mutant (NM) in potato dextrose medium. High performance liquid chromatography (HPLC) of partially purified extract of RP24 showed the presence of lipopeptide antibiotic iturin as a major peak that was comparable to that of standard iturin A (5.230 min) from Sigma-Aldrich whereas the corresponding peak was absent in extract of NM. The structure was further confirmed by liquid chromatographic mass spectrometric (LCMS) analysis as iturin A. LCMS analysis also showed the presence of surfactin and fengycin besides iturin A. Amplification of the lpa-14 (encodes the 4'-phosphopantetheinyl transferase required for the maturation of template enzyme of iturin A) and ituD (encodes a putative malonyl coenzyme A transacylase, whose disruption results in a specific deficiency in iturin A production) genes of iturin operon of strain RP24 was carried out and the sequences obtained were compared with the existing database of NCBI. The sequences of lpa-14 and ituD gene of RP24 showed 98% and 97% homology with lpa-14 and ituD genes of B. subtilis in the existing database. The results indicated that strain RP24 harbors iturin operon in its genome and a chemical mutation in this operon might have resulted in loss of antifungal activity in the negative mutant.