Seed inoculation of desert-plant growth-promoting rhizobacteria induce biochemical alterations and develop resistance against water stress in wheat.

Water shortage limits agricultural productivity, so strategies to get higher yields in dry agricultural systems is vital to circumvent the effect of climate change and land-shortage. The plant rhizosphere harbors beneficial bacteria able to confer biotic/abiotic tolerance along with a positive impact on plant growth. Herein, three bacterial strains, Proteus mirabilis R2, Pseudomonas balearica RF-2 and Cronobacter sakazakii RF-4 (accessions: LS975374, LS975373, LS975370, respectively) isolated from native desert-weeds were investigated for their response to improve wheat growth under drought stress. The bacteria showed drought tolerance up to 20% polyethylene glycol (PEG; -0.6 MPa), and salt (65-97 g l-1 ), 1-aminocyclopropane-1-carboxylate (ACC)-deaminase activity, P/Zn/K-solubilization, calcite degradation, IAA, and siderophore production. The plant growth-promoting rhizobacteria (PGPR) were evaluated on wheat under water stress. The P. balearica strain RF-2 primed seeds showed a maximum promptness index and germination index under PEG-stress, that is, 68% and 100%, respectively. Inoculation significantly improved plant growth, leaf area, and biomass under water stress. P. mirabilis R2 inoculated plant leaves showed the highest water contents as compared to the plants inoculated with other strains. C. sakazakii RF-4 inoculated plants showed minimum cell injury, electrolyte leakage, and maximum cell membrane stability at PEG stress. After 13 days exposure to drought, C. sakazakii RF-4 treated plants showed an overall higher expression of cytosolic ascorbate peroxidase (cAPX) and ribulose-bisphosphate carboxylase (rbcL) genes. The activity of stress-induced catalase and polyphenol oxidase was reduced, while that of peroxidase and superoxide dismutase increased after inoculation but the response was temporal. Taken together, this data explains that different PGPR (especially C. sakazakii RF-4) modulate differential responses in wheat that eventually leads towards drought tolerance, hence, it has the potential to enhance crop production in arid regions.

Diversity of Bacillus-like bacterial community in the rhizospheric and non-rhizospheric soil of halophytes (Salsola stocksii and Atriplex amnicola), and characterization of osmoregulatory genes in halophilic Bacilli.

Salinity is one of the major abiotic stresses; a total of 3% of the world's land mass is affected by salinity. Approximately 6.3 million hectares of land in Pakistan is affected by salinity to varying degrees, and most of the areas are arid to semiarid with low annual precipitation. The aim of the present study is to identify and characterize Bacillus and Bacillus-derived bacterial genera from the rhizospheric and non-rhizospheric soil samples from the Khewra Salt Mine, Pakistan, by using culture-independent and -dependent methods. Seven Bacillus-like bacterial genera, Bacillus, Halobacillus, Virgibacillus, Brevibacillus, Paenibacillus, Tumebacillus, and Lysinibacillus, were detected by using pyrosequencing analysis, whereas only four genera, Bacillus, Halobacillus, Oceanobacillus, and Virgibacillus, were identified by culture-dependent methods. Most of the Bacillus-like isolates identified in this study were moderately halophilic, alkaliphilic, and mesophilic bacteria and were considered a good source of hydrolytic enzymes because of their ability to degrade proteins, carbohydrates, and lipids. Eight Bacillus-like strains from the genera Bacillus, Halobacillus, Oceanobacillus, and Virgibacillus showed positive results for the presence of ectABC gene cluster (ectoine), six strains could synthesize betaine from choline, and six strains tested positive for the synthesis of proline from either glutamate or ornithine by using proline dehydrogenase enzyme.

Impact of soil salinity on the microbial structure of halophyte rhizosphere microbiome.

The rhizosphere microbiome plays a significant role in the life of plants in promoting plant survival under adverse conditions. However, limited information is available about microbial diversity in saline environments. In the current study, we compared the composition of the rhizosphere microbiomes of the halophytes Urochloa, Kochia, Salsola, and Atriplex living in moderate and high salinity environments (Khewra salt mines; Pakistan) with that of the non-halophyte Triticum. Soil microbiomes analysis using pyrosequencing of 16S rRNA gene indicated that Actinobacteria were dominant in saline soil samples whereas Proteobacteria predominated in non-saline soil samples. Firmicutes, Acidobacteria, Bacteriodetes and Thaumarchaeota were predominant phyla in saline and non-saline soils, whereas Cyanobacteria, Verrucomicrobia, Gemmatimonadetes and the unclassified WPS-2 were less abundant. Sequences from Euryarchaeota, Ignavibacteriae, and Nanohaloarchaeota were identified only from the rhizosphere of halophytes. Dominant halophilic bacteria and archaea identified in this study included Agrococcus, Armatimonadetes gp4, Halalkalicoccus, Haloferula and Halobacterium. Our analysis showed that increases in soil salinity correlated with significant differences in the alpha and beta diversity of the microbial communities across saline and non-saline soil samples. Having a complete inventory of the soil bacteria from different saline environments in Pakistan will help in the discovery of potential inoculants for crops growing on salt-affected land.

Tailored Bioactive Compost from Agri-Waste Improves the Growth and Yield of Chili Pepper and Tomato.

An extensive use of chemical fertilizers has posed a serious impact on food and environmental quality and sustainability. As the organic and biofertilizers can satisfactorily fulfill the crop's nutritional requirement, the plants require less chemical fertilizer application; hence, the food is low in chemical residues and environment is less polluted. The agriculture crop residues, being a rich source of nutrients, can be used to feed the soil and crops after composting and is a practicable approach to sustainable waste management and organic agriculture instead of open-field burning of crop residues. This study demonstrates a feasible strategy to convert the wheat and rice plant residues into composted organic fertilizer and subsequent enrichment with plant-beneficial bacteria. The bioactive compost was then tested in a series of in vitro and in vivo experiments for validating its role in growing organic vegetables. The compost was enriched with a blend of micronutrients, such as zinc, magnesium, and iron, and a multi-trait bacterial consortium AAP (Azospirillum, Arthrobacter, and Pseudomonas spp.). The bacterial consortium AAP showed survival up to 180 days post-inoculation while maintaining their PGP traits. Field emission scanning electron microscopic analysis and fluorescence in situ hybridization (FISH) of bioactive compost further elaborated the morphology and confirmed the PGPR survival and distribution. Plant inoculation of this bioactive compost showed significant improvement in the growth and yield of chilies and tomato without any additional chemical fertilizer yielding a high value to cost ratio. An increase of ≈35% in chlorophyll contents, ≈25% in biomass, and ≈75% in yield was observed in chilies and tomatoes. The increase in N was 18.7 and 25%, while in P contents were 18.5 and 19% in chilies and tomatoes, respectively. The application of bioactive compost significantly stimulated the bacterial population as well as the phosphatase and dehydrogenase activities of soil. These results suggest that bioactive compost can serve as a source of bioorganic fertilizer to get maximum benefits regarding vegetable yield, soil quality, and fertilizer saving with the anticipated application for other food crops. It is a possible win-win situation for environmental sustainability and food security.

Isolation and characterization of bacteria associated with the rhizosphere of halophytes (Salsola stocksii and Atriplex amnicola) for production of hydrolytic enzymes.

Microbes from hypersaline environments are useful in biotechnology as sources of novel enzymes and proteins. The current study aimed to characterize halophilic bacteria from the rhizosphere of halophytes (Salsola stocksii and Atriplex amnicola), non-rhizospheric, and brine lake-bank soils collected from Khewra Salt Mine and screening of these bacterial strains for industrially important enzymes. A total of 45 bacterial isolates from the rhizosphere of Salsola, 38 isolates from Atriplex, 24 isolates from non-rhizospheric, and 25 isolates from lake-bank soils were identified by using 16S rRNA gene analysis. Phylogenetic analysis showed that bacterial strains belonging to Bacillus, Halobacillus, and Kocuria were dominant in the rhizosphere of halophytes (Salsola and Atriplex), and Halobacillus and Halomonas were dominating genera from non-rhizospheric and lake-bank soils. Mostly identified strains were moderately halophilic bacteria with optimum growth at 1.5-3.0 M salt concentrations. Most of the bacterial exhibited lipase, protease, cellulase, amylase, gelatinase, and catalase activities. Halophilic and halotolerant Bacilli (AT2RP4, HL1RS13, NRS4HaP9, and LK3HaP7) identified in this study showed optimum lipase, protease, cellulase, and amylase activities at 1.0-1.5 M NaCl concentration, pH 7-8, and temperature 37 °C. These results indicated that halophilic and halotolerant bacteria can be used for bioconversion of organic compounds to useful products under extreme conditions.

Identification of plasmid encoded osmoregulatory genes from halophilic bacteria isolated from the rhizosphere of halophytes.

Bacterial plasmids carry genes that code for additional traits such as osmoregulation, CO2 fixation, antibiotic and heavy metal resistance, root nodulation and nitrogen fixation. The main objective of the current study was to identify plasmid-conferring osmoregulatory genes in bacteria isolated from rhizospheric and non-rhizospheric soils of halophytes (Salsola stocksii and Atriplex amnicola). More than 55% of halophilic bacteria from the rhizosphere and 70% from non-rhizospheric soils were able to grow at 3 M salt concentrations. All the strains showed optimum growth at 1.5-3.0 M NaCl. Bacterial strains from the Salsola rhizosphere showed maximum (31%) plasmid elimination during curing experiments as compared to bacterial strains from the Atriplex rhizosphere and non-rhizospheric soils. Two plasmid cured strains Bacillus HL2HP6 and Oceanobacillus HL2RP7 lost their ability to grow in halophilic medium, but they grew well on LB medium. The plasmid cured strains also showed a change in sensitivity to specific antibiotics. These plasmids were isolated and transformed into E. coli strains and growth response of wild-type and transformed E. coli strains was compared at 1.5-4 M NaCl concentrations. Chromosomal DNA and plasmids from Bacillus filamentosus HL2HP6 were sequenced by using high throughput sequencing approach. Results of functional analysis of plasmid sequences showed different proteins and enzymes involved in osmoregulation of bacteria, such as trehalose, ectoine synthetase, porins, proline, alanine, inorganic ion transporters, dehydrogenases and peptidases. Our results suggested that plasmid conferring osmoregulatory genes play a vital role to maintain internal osmotic balance of bacterial cells and these genes can be used to develop salt tolerant transgenic crops.

A comparative study of bacterial diversity based on culturable and culture-independent techniques in the rhizosphere of maize (Zea mays L.).

OBJECTIVE: Maize is an important crop for fodder, food and feed industry. The present study explores the plant-microbe interactions as alternative eco-friendly sustainable strategies to enhance the crop yield. METHODOLOGY: Bacterial diversity was studied in the rhizosphere of maize by culture-dependent and culture-independent techniques by soil sampling, extraction of DNA, amplification of gene of interest, cloning of desired fragment and library construction. RESULTS: Culturable bacteria were identified as Achromobacter, Agrobacterium, Azospirillum, Bacillus, Brevibacillus, Bosea, Enterobacter, Microbacterium, Pseudomonas, Rhodococcus, Stenotrophomonas and Xanthomonas genera. For culture-independent approach, clone library of 16S ribosomal RNA gene was assembled and 100 randomly selected clones were sequenced. Majority of the sequences were related to Firmicutes (17%), Acidobacteria (16%), Actinobacteria (17%), Alpha-Proteobacteria (7%), Delta-proteobacteria (4.2%) and Gemmatimonadetes (4.2%) However, some of the sequences (30%) were novel that showed no homologies to phyla of cultured bacteria in the database. Diversity of diazotrophic bacteria in the rhizosphere investigated by analysis of PCR-amplified nifH gene sequence that revealed abundance of sequences belonging to genera Azoarcus (25%), Aeromonas (10%), Pseudomonas (10%). The diazotrophic genera Azotobacter, Agrobacterium and Zoogloea related nifH sequences were also detected but no sequence related to Azospirillum was found showing biasness of the growth medium rather than relative abundance of diazotrophs in the rhizosphere. CONCLUSION: The study provides a foundation for future research on focussed isolation of the Azoarcus and other diazotrophs found in higher abundance in the rhizosphere.

Phosphate solubilizing bacteria with glucose dehydrogenase gene for phosphorus uptake and beneficial effects on wheat.

The aim of this study was to isolate, characterize and use phosphate solubilizing bacteria to enhance the bioavailability of insoluble Ca-phosphate for wheat plants. For this purpose, 15 phosphorus solubilizing bacteria (PSB) were isolated from wheat rhizospheric soils of Peshawar and southern Punjab region, Pakistan. These isolates were identified using light microscopy and 16S rRNA gene. Among the isolated bacteria, two strains (Pseudomonas sp. MS16 and Enterobacter sp. MS32) were the efficient P solubilizers based on their P solubilization activity determined qualitatively (solubilization index 3.2-5.8) as well as quantitatively (136-280 µg mL-1). These two strains produced indole-3-acetic acid (25.6-28.1 µg mL-1), gibberellic acid (2.5-11.8), solubilized zinc compounds (SI 2.8-3.3) and showed nitrogenase and 1-Aminocyclopropane-1-carboxylic acid deaminase activity in vitro. Phosphate solubilization activity of Pseudomonas sp. MS16 was further validated by amplification, sequencing and phylogenetic analysis of glucose dehydrogenase (gcd) gene (LT908484) responsible for P solubilization. Response Surface Methodology for large-scale production was used to find optimal conditions (Temperature 22.5°C, pH 7) for P solubilization. Glucose was found to support higher P solubilization in vitro. In an in vitro experiment, PSB treated wheat seedlings improved germination and seedling vigor (11% increases) as compared to un-inoculated control. Rhizoscanning of these seedlings showed an increase in various root growth parameters. Wheat inoculation with selected strain MS16 showed pronounced effect on grain yield in pot (38.5% increase) and field (17-18% increase) experiments compared to non-inoculated control. Root colonization by PSB through Florescent in situ Hybridization and Confocal Laser Scanning Microscopy confirmed their rhizosphere competence in soil. BOX-PCR confirmed the re-isolated colonies of Pseudomonas sp. MS16. The results indicated that gluconic acid producing Pseudomonas sp. MS16 from un-explored soils may be cost effective and environment friendly candidate to improve plant growth and phosphorous uptake by wheat plants.

Phylogeny of the 1-aminocyclopropane-1-carboxylic acid deaminase-encoding gene acdS in phytobeneficial and pathogenic Proteobacteria and relation with strain biogeography.

Deamination of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) is a key plant-beneficial trait found in plant growth-promoting rhizobacteria (PGPR) and phytosymbiotic bacteria, but the diversity of the corresponding gene (acdS) is poorly documented. Here, acdS sequences were obtained by screening putative ACC deaminase sequences listed in databases, based on phylogenetic properties and key residues. In addition, acdS was sought in 71 proteobacterial strains by PCR amplification and/or hybridization using colony dot blots. The presence of acdS was confirmed in established AcdS+ bacteria and evidenced noticeably in Azospirillum (previously reported as AcdS-), in 10 species of Burkholderia and six Burkholderia cepacia genomovars (which included PGPR, phytopathogens and opportunistic human pathogens), and in five Agrobacterium genomovars. The occurrence of acdS in true and opportunistic pathogens raises new questions concerning their ecology in plant-associated habitats. Many (but not all) acdS+ bacteria displayed ACC deaminase activity in vitro, including two Burkholderia clinical isolates. Phylogenetic analysis of partial acdS and deduced AcdS sequences evidenced three main phylogenetic clusters, each gathering pathogens and plant-beneficial strains of contrasting geographic and habitat origins. The acdS phylogenetic tree was only partly congruent with the rrs tree. Two clusters gathered both Betaprotobacteria and Gammaproteobacteria, suggesting extensive horizontal transfers of acdS, noticeably between plant-associated Proteobacteria.

Isolation and identification by 16S rRNA sequence analysis of plant growth-promoting azospirilla from the rhizosphere of wheat.

The main objective of the present study was to isolate phytohormone-producing, phosphate-solubilizing strains of Azospirillum from wheat to be used as inoculants for plant growth promotion. Five Azospirillum strains were isolated from the rhizosphere of field-grown wheat (Triticum aestivum L.), and it was confirmed by BOX-polymerase chain reaction (PCR) that the isolates were different and not re-isolates of the same strain. Sequence analysis of the PCR-amplified 16S rRNA gene indicated that four isolates showed maximum similarity to Azospirillum brasilense and one isolate showed maximum similarity to Azospirillum zeae. This is the first report indicating the presence of an A. zeae like isolate in the wheat rhizosphere in Pakistan. The bacterial isolates were characterized for their plant growth-promoting traits, phosphate solubilization, and indole-3-acetic acid (IAA) production. None of the isolates showed phosphate solubilization activity in the commonly used Pikovskaya medium. However, all strains (except AzoK4) exhibited ability to solubilize tricalcium phosphate (TCP) in modified Pikovskaya medium in which sucrose was replaced by Na-malate, as well as in TCP-supplemented Luria-Bertani (LB) medium. Organic acids, such as acetic, citric, lactic, malic, and succinic acids, were detected in culture supernatants of the tested Azospirillum strains. All strains exhibited ability to produce IAA in the growth medium, except Azospirillum sp. AzoK1. Among the strains tested, the maximum IAA production (30.49±1.04mgL(-1)) and phosphate solubilization (105.50±4.93mgL(-1)) were shown by a pure culture of Azospirillum sp. AzoK2. In pot experiments, single-strain inocula of Azospirillum sp. AzoK1 and AzoK2 improved wheat plant growth.

Plant Growth Promotion and Suppression of Bacterial Leaf Blight in Rice by Inoculated Bacteria.

The present study was conducted to evaluate the potential of rice rhizosphere associated antagonistic bacteria for growth promotion and disease suppression of bacterial leaf blight (BLB). A total of 811 rhizospheric bacteria were isolated and screened against 3 prevalent strains of BLB pathogen Xanthomonas oryzae pv. oryzae (Xoo) of which five antagonistic bacteria, i.e., Pseudomonas spp. E227, E233, Rh323, Serratia sp. Rh269 and Bacillus sp. Rh219 showed antagonistic potential (zone of inhibition 1-19 mm). Production of siderophores was found to be the common biocontrol determinant and all the strains solubilized inorganic phosphate (82-116 µg mL-1) and produced indole acetic acid (0.48-1.85 mg L-1) in vitro. All antagonistic bacteria were non-pathogenic to rice, and their co-inoculation significantly improved plant health in terms of reduced diseased leaf area (80%), improved shoot length (31%), root length (41%) and plant dry weight (60%) as compared to infected control plants. Furthermore, under pathogen pressure, bacterial inoculation resulted in increased activity of defense related enzymes including phenylalanine ammonia-lyase and polyphenol oxidase, along with 86% increase in peroxidase and 53% increase in catalase enzyme activities in plants inoculated with Pseudomonas sp. Rh323 as well as co-inoculated plants. Bacterial strains showed good colonization potential in the rice rhizosphere up to 21 days after seed inoculation. Application of bacterial consortia in the field resulted in an increase of 31% in grain yield and 10% in straw yield over non-inoculated plots. Although, yield increase was statistically non-significant but was accomplished with overall saving of 20% chemical fertilizers. The study showed that Pseudomonas sp. Rh323 can be used to develop dual-purpose inoculum which can serve not only to suppress BLB but also to promote plant growth in rice.

Inoculum pretreatment affects bacterial survival, activity and catabolic gene expression during phytoremediation of diesel contaminated soil.

Plant-bacteria partnership is a promising approach for remediating soil contaminated with organic pollutants. The colonization and metabolic activity of an inoculated microorganism depend not only on environmental conditions but also on the physiological condition of the applied microorganisms. This study assessed the influence of different inoculum pretreatments on survival, gene abundance and catabolic gene expression of an applied strain (Pantoea sp. strain BTRH79) in the rhizosphere of ryegrass vegetated in diesel contaminated soil. Maximum bacterium survival, gene abundance and expression were observed in the soil inoculated with bacterial cells that had been pregrown on complex medium, and hydrocarbon degradation and genotoxicity reduction were also high in this soil. These findings propose that use of complex media for growing plant inocula may enhance bacterial survival and colonization and subsequently the efficiency of pollutant degradation.

Isolation and identification by 16S rRNA sequence analysis of plant growth-promoting azospirilla from the rhizosphere of wheat

ABSTRACT The main objective of the present study was to isolate phytohormone-producing, phosphate-solubilizing strains of Azospirillum from wheat to be used as inoculants for plant growth promotion. Five Azospirillum strains were isolated from the rhizosphere of field-grown wheat (Triticum aestivum L.), and it was confirmed by BOX-polymerase chain reaction (PCR) that the isolates were different and not re-isolates of the same strain. Sequence analysis of the PCR-amplified 16S rRNA gene indicated that four isolates showed maximum similarity to Azospirillum brasilense and one isolate showed maximum similarity to Azospirillum zeae. This is the first report indicating the presence of an A. zeae like isolate in the wheat rhizosphere in Pakistan. The bacterial isolates were characterized for their plant growth-promoting traits, phosphate solubilization, and indole-3-acetic acid (IAA) production. None of the isolates showed phosphate solubilization activity in the commonly used Pikovskaya medium. However, all strains (except AzoK4) exhibited ability to solubilize tricalcium phosphate (TCP) in modified Pikovskaya medium in which sucrose was replaced by Na-malate, as well as in TCP-supplemented Luria-Bertani (LB) medium. Organic acids, such as acetic, citric, lactic, malic, and succinic acids, were detected in culture supernatants of the tested Azospirillum strains. All strains exhibited ability to produce IAA in the growth medium, except Azospirillum sp. AzoK1. Among the strains tested, the maximum IAA production (30.49 ± 1.04 mg L-1) and phosphate solubilization (105.50 ± 4.93 mg L-1) were shown by a pure culture of Azospirillum sp. AzoK2. In pot experiments, single-strain inocula of Azospirillum sp. AzoK1 and AzoK2 improved wheat plant growth.