Genomic and metabolic versatility of Pseudomonas aeruginosa contributes to its inter-kingdom transmission and survival.

Pseudomonas aeruginosa is one of the most versatile bacteria with renowned pathogenicity and extensive drug resistance. The diverse habitats of this bacterium include fresh, saline and drainage waters, soil, moist surfaces, taps, showerheads, pipelines, medical implants, nematodes, insects, plants, animals, birds and humans. The arsenal of virulence factors produced by P. aeruginosa includes pyocyanin, rhamnolipids, siderophores, lytic enzymes, toxins and polysaccharides. All these virulent elements coupled with intrinsic, adaptive and acquired antibiotic resistance facilitate persistent colonization and lethal infections in different hosts. To date, treating pulmonary diseases remains complicated due to the chronic secondary infections triggered by hospital-acquired P. aeruginosa. On the contrary, this bacterium can improve plant growth by suppressing phytopathogens and insects. Notably, P. aeruginosa is one of the very few bacteria capable of trans-kingdom transmission and infection. Transfer of P. aeruginosa strains from plant materials to hospital wards, animals to humans, and humans to their pets occurs relatively often. Recently, we have identified that plant-associated P. aeruginosa strains could be pathologically similar to clinical isolates. In this review, we have highlighted the genomic and metabolic factors that facilitate the dominance of P. aeruginosa across different biological kingdoms and the varying roles of this bacterium in plant and human health.

Multifarious characteristics of sulfur-oxidizing bacteria residing in rice rhizosphere.

Sulfur-oxidizing bacteria (SOB) are versatile microorganisms known for their ability to oxidize various reduced sulfur compounds, namely, elemental sulfur (S0), hydrogen sulfide (H2S), tetrathionate (S4O62-), and trithionate (S3O62-) to sulfate (SO42-). In this study, out of twelve SOB isolates from rice rhizosphere, five were screened based on their sulfur oxidation potential, viz., SOB1, SOB2, SOB3, SOB4, and SOB5, and were identified as Ochrobactrum soli SOB1, Achromobacter xylosoxidans SOB2, Stenotrophomonas maltophilia SOB3, Brucella tritici SOB4, and Stenotrophomonas pavanii SOB5, respectively. All the isolates displayed chemolithotrophic nutritional mode by consuming thiosulfate and accumulating trithionate and tetrathionate in the growth medium which is ultimately oxidized to sulfate. The strains were authenticated with the production of thiosulfate oxidizing enzymes such as rhodanese and sulfite oxidase. Despite their tendency to oxidize reduced sulfur compounds, B. tritici SOB4 and S. pavanii SOB5 were also found to possess phosphate and zinc solubilization potential, acetic acid, and indole acetic acid (IAA) production and 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity. The presence of sulfanyl (R-SH) groups was noticed in the A. xylosoxidans SOB2. Elemental sulfur conversion into sulfate was noted in the S. maltophilia SOB3, and hydrogen sulfide conversion into sulfate was observed in the Ochromobacter soli SOB1. Sulfur oxidation potential coupled with beneficial properties of the isolates widen the knowledge on SOB.

Inhibitory effects of epiphytic Kluyveromyces marxianus from Indian senna (Cassia angustifolia Vahl.) on growth and aflatoxin production of Aspergillus flavus.

Aspergillus flavus infection and subsequent aflatoxin contamination are considered the major constraints in senna (Cassia angustifolia Vahl.) export. Using native epiphytic yeast to control phytopathogens is a successful strategy for managing plant diseases. In the present investigation, we exploited the antagonistic potential of epiphytic yeast isolates obtained from senna against A. flavus growth and aflatoxin B1 (AFB1) production. Four Kluyveromyces marxianus strains (YSL3, YSL16, YSP12, and YSF9) exhibited vigorous antagonistic activity with a maximum inhibition of 64 %. In vivo evaluation of senna pods showed that K. marxianus strains effectively reduced A. flavus colonization with a population range of 5.87 to 7.08 log10 CFU/g. In contrast, the untreated senna pods were found to have severe fungal colonization with a population of 7.84 log10 CFU/g. In addition, HPLC analysis showed that aflatoxin B1 in senna pods was drastically reduced upon yeast treatment up to 14 DAI. Furthermore, we demonstrated the antifungal action mechanisms of K. marxianus, such as surface colonizing ability on pods, production of antifungal volatiles (VOCs), siderophores, extracellular lytic enzymes, and cell wall binding ability to AFB1. All four strains of K. marxianus showed rapid colonization on the senna pod, and YSP12 reached the maximum population of 7.18 log10 CFU/pod at 9 days after inoculation (DAI). The exposure of A. flavus to K. marxianus VOCs significantly reduced the growth by up to 99 and 93.2 % at 7 and 14 DAI, respectively. Scanning electron microscopic images demonstrated severe mycelial damage and hyphal deformities of A. flavus. In addition, yeast VOCs can reduce aflatoxin biosynthesis in A. flavus by up to 99 and 93.2 % at 7 and 14 DAI, respectively. Gas chromatography-mass spectrometry analysis confirmed the presence of antimicrobial compounds such as dimethyl trisulfide, ethyl acetate, ethanol, 3-methyl butanal, 2-methyl-1-butanol, and 3-methyl-1-butanol in the volatiles. K. marxianus strains produced siderophores and hydrolytic enzymes such as chitinase and ß-1,3-glucanase. A higher AFB1 binding ability was observed in the heat-killed cells (47.5 to 70.65 %) than in the viable cells (43.16 to 60.98 %) of K. marxianus. The current study demonstrated that epiphytic K. marxianus isolated from senna could be a successful biocontrol source to reduce aflatoxin contamination in senna pods.

SCAR marker: A potential tool for authentication of agriculturally important microorganisms.

Microbial inoculants are globally recommended for plant growth promotion and control of plant pathogens. These inoculants require stringent quality checks for sustainable field efficacy. Questionable regulatory frameworks constantly deteriorate the reliability of bio-inoculant technology. Existing global regulations do not involve any rapid molecular technique for the routine inspection of microbial preparations. Sequence characterized amplified region (SCAR) marker offers rapid and precise strain-level authentication of target microbes. Such advanced molecular techniques must be exploited to accurately validate the microbial formulations. Besides, the global dissemination of plant pathogenic microbes has always been an alarming threat to food security. SCAR markers could be used at the plant quarantine centers to rapidly detect catastrophic pathogens, thereby circumventing the import and export of contagious plant materials. The current review is focused on promoting the SCAR marker technology to validate commercial bio-inoculants and predict plant pandemics.

Pathogenesis of plant-associated Pseudomonas aeruginosa in Caenorhabditis elegans model.

BACKGROUND: Pseudomonas aeruginosa is a globally dreaded pathogen that triggers fatality in immuno-compromised individuals. The agricultural ecosystem is a massive reservoir of this bacterium, and several studies have recommended P. aeruginosa to promote plant growth. However, there were limited attempts to evaluate the health risks associated with plant-associated P. aeruginosa. The current study hypothesized that agricultural P. aeruginosa strains exhibit eukaryotic pathogenicity despite their plant-beneficial traits. RESULTS: We have demonstrated that feeding with the plant-associated P. aeruginosa strains significantly affects Caenorhabditis elegans health. Out of the 18 P. aeruginosa strain tested, PPA03, PPA08, PPA10, PPA13, PPA14, PPA17, and PPA18 isolated from cucumber, tomato, eggplant, and chili exhibited higher virulence and pathogenicity. Correlation studies indicated that nearly 40% of mortality in C. elegans was triggered by the P. aeruginosa strains with high levels of pyocyanin (> 9 µg/ml) and biofilm to planktonic ratio (> 8). CONCLUSION: This study demonstrated that plant-associated P. aeruginosa could be a potential threat to human health similar to the clinical strains. Pyocyanin could be a potential biomarker to screen the pathogenic P. aeruginosa strains in the agricultural ecosystem.

Volatiles of antagonistic soil yeasts inhibit growth and aflatoxin production of Aspergillus flavus.

Minimizing Aspergillus flavus growth is an effective strategy to mitigate aflatoxin contamination in food and agricultural products. In the present investigation, we attempted to utilize soil-associated yeasts from the Western and Eastern Ghats of India against A. flavus to reduce aflatoxin contamination. Forty-five yeast isolates were screened against A. flavus using overlay and dual plate assays. Among them, 12 isolates effectively inhibited the growth of A. flavus. The 18S rDNA gene sequence analysis identified the twelve antagonistic isolates as belonging to Saccharomyces cerevisiae, Suhomyces xylopsoci, Pichia kudriavzevii, and Candida tropicalis. From the isolated yeasts, S. cerevisiae strains were selected for further evaluation based on the potential antagonistic activity. Volatiles of S. cerevisiae effectively suppressed the mycelial growth of A. flavus (P < 0.05) up to 92.1 % at 7 DAI. Scanning electron microscopic images of the fungus exposed to volatiles showed hyphal deformity and mycelial damage. Aflatoxin B1 (AFB1) production was drastically reduced up to 99.0 % in the volatile-exposed fungus compared to the control. The yeast strain YKK1 showed consistent Aspergillus flavus growth inhibition (80.7 %) and AFB1 production (98.1 %) for 14 days. Gas chromatography-mass spectrophotometry analysis of the yeast volatiles revealed the presence of antimicrobial compounds, including 1-pentanol, 1-propanol, ethyl hexanol, ethanol, 2-methyl-1-butanol, ethyl acetate, dimethyl trisulfide, p-xylene, styrene, and 1,4-pentadiene. The evaluated compounds of yeast volatiles, including ethyl acetate, hexanal, 1-propanol, 1-heptanol, 1-butanol, and benzothiazole, inhibited the fungal growth and AFB1 production of Aspergillus flavus when applied as pure chemicals. Benzothiazole at 5 mM was responsible for a high level of growth inhibition (23.6 %) and reduction of AFB1 synthesis (93.5 %). Hence, volatile compounds produced by soil yeast strains could be a potential biocontrol mechanism against aflatoxin contamination.

Plant-associated Pseudomonas aeruginosa strains harbour multiple virulence traits critical for human infection.

Introduction. Pseudomonas aeruginosa causes fatal infections in immunocompromised individuals and patients with pulmonary disorders.Gap Statement. Agricultural ecosystems are the vast reservoirs of this dreaded pathogen. However, there are limited attempts to analyse the pathogenicity of P. aeruginosa strains associated with edible plants.Aim. This study aims to (i) elucidate the virulence attributes of P. aeruginosa strains isolated from the rhizosphere and endophytic niches of cucumber, tomato, eggplant and chili;and (ii) compare these phenotypes with that of previously characterized clinical isolates.Methodology. Crystal-violet microtitre assay, swarm plate experiment, gravimetric quantification and sheep blood lysis were performed to estimate the biofilm formation, swarming motility, rhamnolipid production and haemolytic activity, respectively, of P. aeruginosa strains. In addition, their pathogenicity was also assessed based on their ability to antagonize plant pathogens (Xanthomonas oryzae, Pythium aphanidermatum, Rhizoctonia solani and Fusarium oxysporum) and kill a select nematode (Caenorhabditis elegans).Results. Nearly 80 % of the plant-associated strains produced rhamnolipid and exhibited at least one type of lytic activity (haemolysis, proteolysis and lipolysis). Almost 50 % of these strains formed significant levels of biofilm and exhibited swarming motility. The agricultural strains showed significantly higher and lower virulence against the bacterial and fungal pathogens, respectively, compared to the clinical strains. In C. elegans, a maximum of 40 and 100% mortality were induced by the agricultural and clinical strains, respectively.Conclusion. This investigation shows that P. aeruginosa in edible plants isolated directly from the farm express virulence and pathogenicity. Furthermore, clinical and agricultural P. aeruginosa strains antagonized the tested fungal phytopathogens, Pythium aphanidermatum, Rhizoctonia solani and Fusarium oxysporum. Thus, we recommend using these fungi as simple eukaryotic model systems to test P. aeruginosa pathogenicity.

Authentication of putative competitive bacterial endophytes of rice by re-isolation and DNA fingerprinting assay.

AIM: The plant-growth-promoting putative competitive endophytes offer significant benefits to sustainable agriculture. The unworthy opportunistic and passenger endophytes are inevitable during the isolation of putative competitive endophytes. This study aimed to discriminate the putative competitive endophytes undoubtedly from the opportunistic and passenger endophytes. METHODS AND RESULTS: The newly isolated endophytes from field-grown rice were inoculated to 5-days old rice seedlings under gnotobiotic conditions. Re-isolation of the inoculated strains from the root surface, inner tissues of the whole plant, root and shoot was performed after 5-days. All the re-isolated colonies were compared with native isolates for homology by BOX-A1R-based repetitive extragenic palindromic-PCR (BOX-PCR) and enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) DNA fingerprints. The results revealed that the putative competitive endophytes (RE25 and RE10) showed positive for re-isolation and BOX and ERIC fingerprints for the whole plant, root and shoot. The opportunistic (RE27 and RE8) and passenger endophytes (RE44 and RE18) failed in re-isolation either from root or shoot. The epiphytes (ZSB15 and Az204) showed negative for endophytic re-isolation and positive for surface colonization. CONCLUSION: This modified procedure can discriminate the putative competitive endophytes from others. SIGNIFICANCE AND IMPACT OF THE STUDY: Eliminating the opportunistic and passenger endophytes and epiphytes early by this method would help develop endophytic inoculants to enhance rice productivity.

Rhizospheric and endophytic Pseudomonas aeruginosa in edible vegetable plants share molecular and metabolic traits with clinical isolates.

AIM: Pseudomonas aeruginosa, a leading opportunistic pathogen causing hospital-acquired infections, is also commonly found in agricultural settings. However, there are minimal attempts to examine the molecular and functional attributes shared by agricultural and clinical strains of P. aeruginosa. This study investigates the presence of P. aeruginosa in edible vegetable plants (including salad vegetables) and analyses the evolutionary and metabolic relatedness of the agricultural and clinical strains. METHODS AND RESULTS: Eighteen rhizospheric and endophytic P. aeruginosa strains were isolated from cucumber, tomato, eggplant, and chili directly from the farms. The identity of these strains was confirmed using biochemical and molecular assays. The genetic and metabolic traits of these plant-associated P. aeruginosa isolates were compared with clinical strains. DNA fingerprinting and 16S rDNA-based phylogenetic analyses revealed that the plant- and human-associated strains are evolutionarily related. Both agricultural and clinical isolates possessed plant-beneficial properties, including mineral solubilization to release essential nutrients (phosphorous, potassium, and zinc), ammonification, and the ability to release extracellular pyocyanin, siderophore, and indole-3 acetic acid. CONCLUSION: These findings suggest that rhizospheric and endophytic P. aeruginosa strains are genetically and functionally analogous to the clinical isolates. In addition, the genotypic and phenotypic traits do not correlate with plant sources or ecosystems. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reconfirms that edible plants are the potential source for human and animal transmission of P. aeruginosa.

Interaction of water activity and temperature on growth, gene expression, and aflatoxin B1 production in Aspergillus flavus on Indian senna (Cassia angustifolia Vahl.).

Senna (Cassia angustifolia Vahl.) is a medicinal crop with laxative properties, and it has significant demand in the global pharmaceutical market. Senna pods are highly susceptible to aflatoxin contamination, and the successful export of pods is hindered due to the regulatory limits of importing countries. The senna pod water activity (aw) from harvest to storage is the key factor determining AFB1 accumulation. The temperature conditions from field to warehouse also interact with pod aw, which influences fungal growth and AFB1 production. The determination of an ideal combination of aw and temperature led to the assessment of the critical control point for AFB1 synthesis in senna. Hence, this study aimed to evaluate the influence of aw (0.99, 0.96, 0.93, 0.90, and 0.87 aw) and temperature (20, 28, and 37 °C) on fungal growth, gene expression (aflR and aflS), and AFB1 production by A. flavus in senna agar medium. The fungus showed the longest lag time (7.7 days) at 20 °C with 0.87 aw. We observed that 0.96 aw (P < 0.01) was optimum for the diametric growth rate at 28 and 37 °C. However, the peak expression of regulatory genes (aflR and aflS) and the maximum AFB1 production were observed only at 28 °C (0.96 aw). The highest growth rate occurred at 37 °C, which did not favor the expression of genes and AFB1 production. However, at 28 °C, it positively correlated with gene expression and AFB1 production. The suppressed expression of regulatory genes and a trace amount of aflatoxin B1 were found at 20 °C with all the tested aw. In our experiments, the low aw (0.87 and 0.90 aw) suppressed the fungal growth, gene expression, and AFB1 production of A. flavus at all of the tested temperatures (20, 28, and 37 °C). The rapid drying of senna pods with a low water activity (≤0.87 aw) and storage at low temperature (20 °C) are ideal conditions to avoid AFB1 and ensure the quality of produce for export.

Harnessing PGPR inoculation through exogenous foliar application of salicylic acid and microbial extracts for improving rice growth.

Plant-growth-promoting rhizobacteria (PGPR) should effectively colonize along the plant root to enhance the plant and soil health. The present investigation aims to improve the PGPR-mediated plant health benefits through above-ground foliar management. A green fluorescent protein-tagged PGPR strain, Pseudomonas chlororaphis (ZSB15-M2) was inoculated in a nonautoclaved agricultural soil before rice culturing. Salicylic acid and cell extracts of Corynebacterium glutamicum and Saccharomyces cerevisiae as a supply of hormonal and inducer compounds were applied on the foliage of the 10-days-old rice plants and subsequently observed the colonizing ability of ZSB15-M2. The cell extracts of Corynebacteria and yeast showed a 100-fold increase in the ZSB15-M2 population in the rhizosphere of rice, whereas salicylic acid had a 10-fold increase in relation to mock control. The rice root exudates collected after the spraying of salicylic acid and microbial extracts showed significantly enhanced release of total carbon, total protein, total sugar, total amino nitrogen, total nitrogen, and phenol content. In vitro assays revealed that these root exudates collected after exogenous spray of these chemicals enhanced the chemotactic motility and biofilm formation of ZSB15-M2 compared to the control plant's root exudate. Metabolomic analysis of root exudates collected from these rice plants by gas chromatography-mass spectrometry revealed that the Corynebacteria and yeast cell extracts enhanced the divergence of metabolites of rice root exudate. Further, due to these cumulative effects in the rice rhizosphere, the total chlorophyll, total protein, total nitrogen, and total phosphorus of rice were significantly improved. These observations provide insights into the rhizosphere functioning of rice plants as modulated by above-ground treatments with improved colonization of inoculant strains as well as the plant growth.

Mitigation of drought stress in rice crop with plant growth-promoting abiotic stress-tolerant rice phyllosphere bacteria.

In the search of effective drought-alleviating and growth-promoting phyllosphere bacteria, a total of 44 bacterial isolates were isolated from the leaf surface of drought-tolerant rice varieties, Mattaikar, Nootripattu, Anna R(4), and PMK3, and screened for their abiotic stress tolerance by subjecting their growth medium to temperature, salinity, and osmotic stress. Only eight isolates were found to grow and proliferate under different abiotic stress conditions. These isolates were identified using 16S ribosomal DNA gene sequence and submitted to the NCBI database. All the bacterial isolates were identified as Bacillus sp., except PB24, which was identified as Staphylococcus sp., and these isolates were further screened for plant growth-promoting (PGP) traits such as IAA production, GA production, ACC deaminase activity, and exopolysaccharide production under three different osmotic stress conditions adjusted using polyethylene glycol (PEG 6000). Additionally, mineral solubilization was measured under the normal condition. Bacillus endophyticus PB3, Bacillus altitudinis PB46, and Bacillus megaterium PB50 were found to have multifarious PGP traits. Consecutively, the performance of an individual strain to improve the plant growth was investigated under the osmotic stress (25% PEG 6000) and nonstress condition by inoculating them into rice seeds using hydroponics culture. Furthermore, the drought-alleviating potency of bacterial strains was assessed in the rice plants using pot experiment (-1.2 MPa) through bacterial foliar application during the reproductive stage. Finally, as a result of seed inoculation and foliar spray, the application of B. megaterium PB50 significantly improved the plant growth under osmotic stress, protected plants from physical drought through stomatal closure, and improved carotenoid, total soluble sugars, and total protein content. Metabolites of PB50 were profiled under both stress and nonstress conditions using gas chromatography-mass spectroscopy.

Rhizosphere engineering through exogenous growth-regulating small molecules improves the colonizing efficiency of a plant growth-promoting rhizobacterium in rice.

Enhancing the rhizosphere colonization and persistence of plant growth-promoting rhizobacteria (PGPR) is necessary for maximizing PGPR-mediated benefits for crop growth and fitness in environmentally friendly agriculture. In the present investigation, we attempted manipulation of the rice rhizosphere by spraying of low molecular weight plant-regulating metabolites on the foliage of rice plants to in turn enhance the colonizing efficiency of soil-inoculated PGPR strain. The green fluorescent protein gene-tagged rhizobacterial strain, Pseudomonas chlororaphis ZSB15-M2, was inoculated in sterile plant growth medium (vermiculite coco peat mixture) and non-autoclaved agricultural soil. We sprayed different plant growth-regulating small molecules on the foliage of rice seedlings and monitored the colonizing efficiency of ZSB15-M2 in the rice rhizosphere. Among the chemicals assessed, salicylic acid (SA) at 1 mM or Corynebacterium glutamicum cell extract (CGCE, 0.2% w/v) or Saccharomyces cerevisiae cell extract (SCCE, 0.2% w/v) showed a tenfold increase in rhizosphere colony-forming units of ZSB15-M2 compared to control with a significant decline in non-rhizosphere bulk soil population. Foliar spray of CGCE enhanced soil organic carbon, microbial biomass carbon and soil protein by 21.86%, 9.68% and 11.57% respectively in the rice rhizosphere as compared to mock control. Additionally, CGCE spray enhanced the key soil enzymes, viz., dehydrogenase and acid- and alkaline phosphatase in the rhizosphere ranging 15-36%. The cumulative effect of this engineered rhizosphere resulted in the elevation of nitrogen, phosphorus, potassium and zinc availability by 21.83%, 28.83%, 23.95% and 61.94%, respectively, in rice rhizosphere as compared to control. On the other hand, SCCE and SA spray had an equal influence on the rhizosphere's biological attributes, which is lower than that of GCGE and higher than that of mock control. From the study, we propose that the aboveground management of rice with microbial-based small molecules will modulate the rice rhizosphere to attract more beneficial PGPR-based inoculants, thus improving the crop and soil health.

Multiplex and quantitative PCR targeting SCAR markers for strain-level detection and quantification of biofertilizers.

Biofertilizers are the eco-friendly bio-input being used to sustain the agriculture by reducing the chemical inputs and improving the soil health. Quality is the major concern of biofertilizer technology which often leads to poor performance in the field and thereby loses the farmers' faith. To authenticate the strain as well as its presumed cell load of a commercial product, sequence characterized amplified region (SCAR) markers were developed for three biofertilizer strains viz., Azospirillum brasilense (Sp7), Bacillus megaterium (Pb1) and Azotobacter chroococcum (Ac1). We evaluated the feasibility of multiplex-PCR and quantitative real-time PCR for SCAR marker-based quality assessment of the product as well as the persistence of the strains during crop growth. We showed that multiplex PCR can concurrently discriminate the strains based on the amplicons' size and detects up to 104 cells per g or per ml of carrier-based or liquid formulation of biofertilizer, respectively. The detection limit of quantitative PCR targeting SCAR markers is 103 cells per g or ml of biofertilizer. Both the PCR methods detected and quantified them in the maize rhizosphere. Hence SCAR marker-based quality assessment would be a sensitive tool to monitor the biofertilizer production as well as its persistence in the inoculated crop rhizosphere.

A simple method for direct isolation of N-acyl-L-homoserine lactone mediated biofilm-forming rhizobacteria from roots.

Plant-associated bacteria produce quorum sensing (QS) signals for community (biofilm) formation and functioning in the rhizosphere. The QS-positive biofilm-forming rhizobacteria that excel benefits to the plants are now gaining increased importance for agricultural use due to their high competitiveness. However, there is no method available to distinguish these bacteria from the roots of a plant to ease the isolation. Currently, all the plant-associated bacteria have to be isolated, purified and subsequently screened for the QS activity using biosensor strains. This study describes a direct isolation method for N-acyl-homoserine lactone (AHL) type quorum sensing signal producing bacteria from the plant root. In this method, the root sample collected from the field was overlaid directly with the bacterial growth medium seeded with the biosensor reporter, Chromobacterium violaceum (CV026). The AHL produced by QS positive rhizobacteria residing on the surface of the root will be recognized by violacein production of CV026. The bacterial isolates recovered from rice root using this method were further confirmed for the QS activity and biofilm formation. All the QS-positive strains produced N-butyryl DL-homoserine lactone (a C4-AHL type) signal in the culture medium and had biofilm formation during in vitro culturing. The 16S rRNA gene sequences of these QS-positive biofilm-forming rhizobacteria revealed that these strains are phylogenetically close to Pseudomonas siluiensis, Aeromonas hydrophila and A. caviae. Therefore, this could be a simple, rapid and straightforward procedure for isolation and characterization of quorum-sensing rhizobacteria from plant roots.

Expression of Zinc Transporter Genes in Rice as Influenced by Zinc-Solubilizing Enterobacter cloacae Strain ZSB14.

Zinc (Zn) deficiency in major food crops has been considered as an important factor affecting the crop production and subsequently the human health. Rice (Oryza sativa) is sensitive to Zn deficiency and thereby causes malnutrition to most of the rice-eating Asian populations. Application of zinc solubilizing bacteria (ZSB) could be a sustainable agronomic approach to increase the soil available Zn which can mitigate the yield loss and consequently the nutritional quality of rice. Understanding the molecular interactions between rice and unexplored ZSB is useful for overcoming Zn deficiency problems. In the present study, the role of zinc solubilizing bacterial strain Enterobacter cloacae strain ZSB14 on regulation of Zn-regulated transporters and iron (Fe)-regulated transporter-like protein (ZIP) genes in rice under iron sufficient and deficient conditions was assessed by quantitative real-time reverse transcription PCR. The expression patterns of OsZIP1, OsZIP4, and OsZIP5 in root and shoot of rice were altered due to the Zn availability as dictated by Zn sources and ZSB inoculation. Fe sufficiency significantly reduced the root and shoot OsZIP1 expression, but not the OsZIP4 and OsZIP5 levels. Zinc oxide in the growth medium up-regulated all the assessed ZIP genes in root and shoot of rice seedlings. When ZSB was inoculated to rice seedlings grown with insoluble zinc oxide in the growth medium, the expression of root and shoot OsZIP1, OsZIP4, and OsZIP5 was reduced. In the absence of zinc oxide, ZSB inoculation up-regulated OsZIP1 and OsZIP5 expressions. Zinc nutrition provided to the rice seedling through ZSB-bound zinc oxide solubilization was comparable to the soluble zinc sulfate application which was evident through the ZIP genes' expression and the Zn accumulation in root and shoot of rice seedlings. These results demonstrate that ZSB could play a crucial role in zinc fertilization and fortification of rice.

Development of sequence-characterized amplified region (SCAR) markers as a quality standard of inoculants based on Azospirillum.

An attempt was made in this work to develop a strain-level molecular marker for unambiguous authentication of two Azospirillum inoculants, viz. A. lipoferum (strain Az204) and A. brasilense (strain Sp7). The sequence-characterized amplified region (SCAR) markers obtained from DNA fingerprints were designed for discrete detection of the strains. The SCAR primers could successfully amplify the target strain without cross-reaction with other Azospirillum strains, native isolates and other inoculants. The detection limit of SCAR primer for Az204 was 8.00 pg of DNA (approximately 10(5) cells per mL), and for Sp7, it was 0.49 pg of DNA (equal to 10(4) cells per mL). A simplified Sephadex G100-based crude DNA extraction protocol developed in this study was found suitable for SCAR marker-based strain authentication. Further, SCAR primers were assessed for simultaneous authentication as well as quantification of commercially prepared Azospirillum inoculants by quantitative real-time PCR (RT-PCR) and most-probable-number PCR (MPN-PCR). The RT-PCR assay can be able to quantify the commercial formulations as equal to culturable MPN method, while MPN-PCR failed for Az204. The SCAR marker-based strain authentication and presumptive quantification developed in the present work can contribute to improving the quality standard of commercial inoculants.

Molecular profiling of rhizosphere bacterial communities associated with Prosopis juliflora and Parthenium hysterophorus.

Prosopis juliflora and Parthenium hysterophorus are the two arid, exotic weeds of India that are characterized by distinct, profuse growth even in nutritionally poor soils and environmentally stressed conditions. Owing to the exceptional growth nature of these two plants, they are believed to harbor some novel bacterial communities with wide adaptability in their rhizosphere. Hence, in the present study, the bacterial communities associated with the rhizosphere of Prosopis and Parthenium were characterized by clonal 16S rRNA gene sequence analysis. The culturable microbial counts in the rhizosphere of these two plants were higher than bulk soils, possibly influenced by the root exudates of these two plants. The phylogenetic analysis of V1_V2 domains of the 16S rRNA gene indicated a wider range of bacterial communities present in the rhizosphere of these two plants than in bulk soils and the predominant genera included Acidobacteria, Gammaproteobacteria, and Bacteriodetes in the rhizosphere of Prosopis, and Acidobacteria, Betaproteobacteria, and Nitrospirae in the Parthenium rhizosphere. The diversity of bacterial communities was more pronounced in the Parthenium rhizosphere than in the Prosopis rhizosphere. This culture-independent bacterial analysis offered extensive possibilities of unraveling novel microbes in the rhizospheres of Prosopis and Parthenium with genes for diverse functions, which could be exploited for nutrient transformation and stress tolerance in cultivated crops.

Diversity analysis of pseudomonas in rice rhizosphere for multifaceted plant growth promotion.

This investigation was carried out based on the hypothesis that there may be some pseudomonad strains, which could exist in rhizosphere of plant species contributing multifaceted beneficial activities. For this purpose, 21 pseudomonad isolates from the rhizosphere of rice, cultivated in western parts of Tamil Nadu were screened. All the 21 isolates were authenticated as pseudomonads by a genus-specific PCR screening. The molecular diversity of these isolates was investigated by Amplified Ribosomal DNA Restriction Analysis (ARDRA) and the dendrogram obtained from the analysis revealed that all the 21 isolates clustered into seven groups. Further, these isolates were screened for plant growth promoting activities such as diazotrophy (PCR amplification of nifH gene and acetylene reduction assay), Indole acetic acid (IAA) and siderophore production (spectrometrically), 1-Aminocyclopropane-1-carboxylic acid (ACC) deaminase for ethylene regulation (PCR screening), mineral solubilization (biochemically) and antagonistic potential against soil pathogenic fungi (dual culture assay). Based on the results, two elite Pseudomonas isolates (S9 and O3) were chosen as multi-functional plant growth-promoting rhizobacteria, paving way for potential use as bioinoculants in rice.