Growth enhancement and changes in bacterial microbiome of cucumber plants exhibited by biopriming with some native bacteria.

This study investigated the impact of a mixture of six endophytic bacterial strains isolated from cucumber plants on the growth and microbiome diversity of six cucumber traditional varieties and hybrids. Six bacterial species were isolated and identified by 16 s rRNA sequencing. All the bacteria showed plant growth promoting traits. Bacillus tequilensis showed 80 % inhibition of the mycelia growth of Fusarium oxysporum f.sp. cucumarinum (Foc). Mixed culture of all the bacteria was prepared and applied back to the varieties and hybrids of cucumber plants through seed soaking. Plant growth characteristics indicated that the treated plants showed increased plant growth in terms of plant height, number of leaves, vine length, male:female flower ratio, number of fruits and fruit length. Bacteria treated plants of hybrid HiVeg Chitra recorded 19 cm increase in vine length compared to control plants. The matataxonomic analysis of leaf samples by Illumina sequencing highlighted a diverse bacterial community shift in treated plants, with significant increases in genera like Bacillus and Staphylococcus. The core microbiome analysis identified key genera such as Bacillus, Staphylococcus, Sphingomonas, Methylobacterium, etc that could be pivotal in plant growth promotion. Bacillus and Staphylococcus showed increased abundance in treated varieties, correlating with the observed in plant growth parameters thus indicating their role in growth promotion of cucumber plants. Endophytic bacterial species identified from cucumber plants when re-applied by seed soaking, they promote the plant growth by modulating the microbiome. The bacterial species identified in the study could be potential candidates as microbial bioinputs for cucumber cultivation.

Comparative metataxonamic analyses of seeds and leaves of traditional varieties and hybrids of cucumber (Cucumis sativus L.) reveals distinct and core microbiome.

Profiling the endophytic microbiome of different tissues and varieties of agricultural crops can help to understand i) the tissue specific and varietal specific microbes associated with the plants ii) their potential role in growth, stress tolerance, disease resistance, and yield of the plants. Comparative microbiome profiling across various varieties and hybrids will also be useful to identify the plant's core microbiome. The main objective of the work is to profile and study the microbiome of traditional varieties in comparison with hybrids of cucumber, which would help to understand the microbiome structure in developing consortia to engineer the microbiome of modern hybrids, for useful phenotypes. Metataxonomic sequencing of bacteria and fungi using 16S rRNA gene and ITS regions respectively were carried out in seed and leaf samples of cucumber traditional varieties and modern hybrids. Among bacteria, Prevotella, Bacteroides, Lactobacillus, Dialester, and Fecalibacterium, and among fungal genera, Pichia, Aspergillus, Phaeoisariopsis, Candida, and Malassezia belonged to the core microbiome of cucumber. Modern hybrids were rich in antibiotic producing and toxic pollutant degrading bacteria. Many of the fungi and bacteria observed in the study are well known plant growth promoting microorganisms and play role in offering disease resistance. Some of the bacteria and fungi have beneficial roles in human gut thus revealing the dietary importance of cucumber. The microbes identified in the current study will be useful starting point to develop a consortia to engineer the cucumber microbiome for growth, yield and stress tolerance traits.

Rice WRKY13 TF protein binds to motifs in the promoter region to regulate downstream disease resistance-related genes.

In plants, pathogen resistance is brought about by the binding of certain transcription factor (TF) proteins to the cis-elements of certain target genes. These cis-elements are present upstream in the motif of the promoters of each gene. This ensures the binding of a specific TF to a specific promoter, therefore regulating the expression of that gene. Therefore, the study of each promoter sequence of all the rice genes would help identify the target genes of a specific TF. Rice 1 kb upstream promoter sequences of 55,986 annotated genes were analyzed using the Perl program algorithm to detect WRKY13 binding motifs (bm). The resulting genes were grouped using Gene Ontology and gene set enrichment analysis. A gene with more than 4 TF bm in their promoter was selected. Ten genes reported to have a role in rice disease resistance were selected for further analysis. Cis-acting regulatory element analysis was carried out to find the cis-elements and confirm the presence of the corresponding motifs in the promoter sequences of these genes. The 3D structure of WRKY13 TF and the corresponding ten genes were built, and the interacting residues were determined. The binding capacity of WRKY13 to the promoter of these selected genes was analyzed using docking studies. WRKY13 was considered for docking analysis based on the prior reports of autoregulation. Molecular dynamic simulations provided more details regarding the interactions. Expression data revealed the expression of the genes that helped provide the mechanism of interaction. Further co-expression network helped to characterize the interaction of these selected disease resistance-related genes with the WRKY13 TF protein. This study suggests downstream target genes that are regulated by the WRKY13 TF. The molecular mechanism involving the gene network regulated by WRKY13 TF in disease resistance against rice fungal pathogens is explored.

Proteome Responses to Individual Pathogens and Abiotic Conditions in Rice Seedlings.

Rice plants under field conditions experience various biotic and abiotic stresses and are adapted to survive using a molecular cross-talk of genes and their protein products based on the severity of a given stress. Seedlings of cultivated variety ASD16 (resistant to fungal disease, blast; tolerant to abiotic stress, salinity) were subjected to salt, drought, high temperature and low temperature stress as well as infection by Rhizoctonia solani and Xanthomonas oryzae pv. oryzae (causing reemerging diseases such as sheath blight and leaf blight), respectively, the sheath blight and bacterial leaf blight pathogens. Leaf proteome was analyzed using two-dimensional electrophoresis and differentially expressed proteins were identified using mass spectrometry. In addition to many other differentially expressed proteins, acidic endochitinase was found to be upregulated during fungal infection and drought treatment, and a germin-like protein upregulated during fungal infection and high temperature stress. These two proteins were further validated at the gene expression level using reverse transcription-PCR in dual stress experiments. Pot culture plants were subjected to fungal infection followed by drought and drought followed by fungal infection to validate chitinase gene expression. Similarly, plants subjected to fungal infections followed by high temperature stress and vice versa were used to validate the expression of germin-like protein-coding gene. The results of the present study indicate that chitinase and germin-like protein are potential targets for further exploration to develop rice plants resistant or tolerant to biotic and abiotic stresses.

Sheath blight of rice: a review and identification of priorities for future research.

MAIN CONCLUSION: Rice sheath blight research should prioritise optimising biological control approaches, identification of resistance gene mechanisms and application in genetic improvement and smart farming for early disease detection. Rice sheath blight, caused by Rhizoctonia solani AG1-1A, is one of the most devasting diseases of the crop. To move forward with effective crop protection against sheath blight, it is important to review the published information related to pathogenicity and disease management and to determine areas of research that require deeper study. While progress has been made in the identification of pathogenesis-related genes both in rice and in the pathogen, the mechanisms remain unclear. Research related to disease management practices has addressed the use of agronomic practices, chemical control, biological control and genetic improvement: Optimising nitrogen fertiliser use in conjunction with plant spacing can reduce spread of infection while smart agriculture technologies such as crop monitoring with Unmanned Aerial Systems assist in early detection and management of sheath blight disease. Replacing older fungicides with natural fungicides and use of biological agents can provide effective sheath blight control, also minimising environmental impact. Genetic approaches that show promise for the control of sheath blight include treatment with exogenous dsRNA to silence pathogen gene expression, genome editing to develop rice lines with lower susceptibility to sheath blight and development of transgenic rice lines overexpressing or silencing pathogenesis related genes. The main challenges that were identified for effective crop protection against sheath blight are the adaptive flexibility of the pathogen, lack of resistant rice varieties, abscence of single resistance genes for use in breeding and low access of farmers to awareness programmes for optimal management practices.

Variations in the Structure and Evolution of Rice WRKY Genes in Indica and Japonica Genotypes and their Co-expression Network in Mediating Disease Resistance.

WRKY transcription factor (TF) family regulates many functions in plant growth and development and also during biotic and abiotic stress. In this study, 101 WRKY TF gene models in indica and japonica rice were used to conduct evolutionary analysis, gene structure analysis, and motif composition. Co-expression analysis was carried out first by selecting the differentially expressing genes that showed a significant change in response to the pathogens from Rice Oligonucleotide Array Database (ROAD). About 82 genes showed responses to infection by Magnaporthe oryzae or Xanthomonas oryzae pv. oryzae. Co-expression gene network was constructed using direct neighborhood and context associated inbuilt mode in RiceNetv2 tool. Only 41 genes showed interaction with 2299 non-WRKY genes. Variations exist in the structure and evolution of WRKY genes among indica and japonica genotypes which have important implications in their differential roles including disease resistance. WRKY genes mediate a complex networking and co-express along with other WRKY and non-WRKY genes to mediate resistance against fungal and bacterial pathogens in rice.

Insights into the interaction of key biofilm proteins in Pseudomonas aeruginosa PAO1 with TiO2 nanoparticle: An in silico analysis.

Pseudomonas aeruginosa is a pathogenic biofilm forming bacteria which exist in wide range of environments such as water, soil and human body. In an earlier study, we used a system biology approach based analysis of biofilm forming genes of P. aeruginosa and their possible role in TiO2 nanoparticle binding. The major protein of P. aeruginosa targeted by TiO2 was found to be KatA, a major catalase required for H2O2 resistance and acute virulence and the direct interacting protein partners of KatA were found to be DnaK, Hfq, RpoA and RpoS. To understand the protein-protein physical interaction characteristic of these key proteins involved in biofilm related processes, homology modeling, docking and molecular dynamic simulation were performed. For all these proteins, physical and chemical properties, amino acid composition, nest and cleft analysis were performed using online tools. The interactions between TiO2NPs-KatA and four protein-protein complexes such as KatA-DnaK, KatA-Hfq, KatA-RpoA and KatA-RpoS were studied. Our results indicate that all four key proteins and TiO2NPs can have stable complexation with KatA. The study has given enough clues to understand the interaction of TiO2NPs with P. aeruginosa biofilm in natural environment. Further investigations could lead to development of TiO2NPs based therapeutic and sanitary interventions to combat this pathogenic bacterium.

Environmental proteomic studies: closer step to understand bacterial biofilms.

Advancement in proteome analytical techniques and the development of protein databases have been helping to understand the physiology and subtle molecular mechanisms behind biofilm formation in bacteria. This review is to highlight how the evolving proteomic approaches have revealed fundamental molecular processes underlying the formation and regulation of bacterial biofilms. Based on the survey of research reports available on differential expression of proteins in biofilms of bacterial from wide range of environments, four important cellular processes viz. metabolism, motility, transport and stress response that contribute to formation of bacterial biofilms are discussed. This review might answer how proteins related to these cellular processes contribute significantly in stabilizing biofilms of different bacteria in diverse environmental conditions.

Cross-regulatory network in Pseudomonas aeruginosa biofilm genes and TiO2 anatase induced molecular perturbations in key proteins unraveled by a systems biology approach.

A systems biology approach was used to study all the biofilm related genes of P. aeruginosa PAO1, and the interaction of titanium dioxide (TiO2) anatase with biofilm related proteins. Among the 71 genes, the interactions of all the nodal pairs were extracted by STRING 10.5 database. The inter-relationship among these genes was predicted by constructing complete PPI network and visualized in Cytoscape v 3.4.0. Total number of nodes of the network was found to be 335 and edges were 795. The network was further investigated for its clusters and the best cluster was further analyzed for the hub proteins which significantly contribute in cross-regulation of the biofilm related process. The hub proteins were identified based on four topological parameters of degree, closeness, betweeness and radiality. Four major hub proteins of P. aeruginosa PAO1 were identified to be algD, gacS, rpoS and rpoN which were common in all the hubs. Further, we have also elucidated the probable mechanism of TiO2 interaction with P. aeruginosa PAO1 at molecular level. Using STITCH server, the major target gene of TiO2 was identified as katA which also appeared commonly in our main dataset and this gene has been focused for the further study because of its unique common appearance in gene-gene network as well as gene-anatase network. The direct interacting partners of katA were found to be dnaK, hfq, rpoS and rpoA. Based on these findings and available gene regulatory information, probable TiO2 interacting cascade has been represented. This in silico study of P. aeruginosa PAO1 biofilm genes and the interaction of protein products with TiO2 might be significant to understand the perspective pathogenic resistance as well as the toxicity research pertaining to nanoparticles.

Gene-centric metegenome analysis reveals diversity of Pseudomonas aeruginosa biofilm gene orthologs in fresh water ecosystem.

Metagenomic analysis of biofilm forming bacteria in environmental samples remains challenging due to the non-availability of gene sequences of most of the uncultivable bacteria. Sequences of Pseudomonas aeruginosa PAO1-UW genes involved either directly or indirectly in biofilm formation were analyzed using BLASTn to obtain matching sequences from different strain, species and genus. Conserved regions in the functional domain of the amino acid sequences were used to design common primers for direct PCR analysis of freshwater metagenomes. Seven key genes such as aceA, clpP, typA, cbrA, phoR, rpoS and gacA involved in biofilm formation were validated. The ortholog genes belonged to wide range of Pseudomonas sp. indicating the diversity of biofilm genes and the conservation of protein functional domains. The approach would also help in analyzing the expression of biofilm genes in different bacteria of freshwater systems for monitoring toxic contaminations such as organic or inorganic pollutants.

Compatibility of Azospirillum brasilense and Pseudomonas fluorescens in growth promotion of groundnut ( Arachis hypogea L)

ABSTRACT We attempted to study the compatibility among plant beneficial bacteria in the culture level by growing them near in the nutrient agar plates. Among all the bacteria tested, Rhizobium was found to inhibit the growth of other bacteria. From the compatible group of PGPR, we have selected one biofertilizer (Azospirillum brasilense strain TNAU) and one biocontrol agent (Pseudomonas fluorescens strain PF1) for further studies in the pot culture. We have also developed a bioformulation which is talc powder based, for individual bacteria and mixed culture. This formulation was used as seed treatment, soil application, seedling root dip and foliar spray in groundnut crop in vitro germination conditions. A. brasilense was found to enhance the tap root growth and P. fluorescens, the lateral root growth. The other growth parameters like shoot growth, number of leaves were enhanced by the combination of both of the bacteria than their individual formulations. Among the method of application tested in our study, soil application was found to be the best in yielding better results of plant growth promotion.

Compatibility of Azospirillum brasilense and Pseudomonas fluorescens in growth promotion of groundnut ( Arachis hypogea L.).

We attempted to study the compatibility among plant beneficial bacteria in the culture level by growing them near in the nutrient agar plates. Among all the bacteria tested, Rhizobium was found to inhibit the growth of other bacteria. From the compatible group of PGPR, we have selected one biofertilizer (Azospirillum brasilense strain TNAU) and one biocontrol agent (Pseudomonas fluorescens strain PF1) for further studies in the pot culture. We have also developed a bioformulation which is talc powder based, for individual bacteria and mixed culture. This formulation was used as seed treatment, soil application, seedling root dip and foliar spray in groundnut crop in vitro germination conditions. A. brasilense was found to enhance the tap root growth and P. fluorescens, the lateral root growth. The other growth parameters like shoot growth, number of leaves were enhanced by the combination of both of the bacteria than their individual formulations. Among the method of application tested in our study, soil application was found to be the best in yielding better results of plant growth promotion.

Analysis of zntA gene in environmental Escherichia coli and additional implications on its role in zinc translocation.

Escherichia coli strains from sewage sample were screened for the presence and expression of heavy metal-translocating zntA gene by PCR and RT-PCR analysis with type culture of K-12 as standard strain. The strain which showed high level of gene expression (SBVP1) was chosen to further study the growth and heavy metal translocation. This superior strain was grown in the presence of ZnSO4, Pb (CH3COO)2 and mixture of ZnSO4, Pb(CH3COO)2 metal salts and the growth was observed at different time points. The cell pellet fraction was found to have more of zinc than lead as determined by atomic absorption spectroscopy indicating the translocation of these metals from media to the cells. However, the intracellular translocation of zinc is affected by the presence of lead in the media. Expression of the zntA gene in bacteria grown in the presence of ZnSO4 was also studied and the molecular analysis results correlate with spectroscopic observations.

Prevalence of plant beneficial and human pathogenic bacteria isolated from salad vegetables in India.

BACKGROUND: The study aimed at enumerating, identifying and categorizing the endophytic cultivable bacterial community in selected salad vegetables (carrot, cucumber, tomato and onion). Vegetable samples were collected from markets of two vegetable hot spot growing areas, during two different crop harvest seasons. Crude and diluted vegetable extracts were plated and the population of endophytic bacteria was assessed based on morphologically distinguishable colonies. The bacterial isolates were identified by growth in selective media, biochemical tests and 16S rRNA gene sequencing. RESULTS: The endophytic population was found to be comparably higher in cucumber and tomato in both of the sampling locations, whereas lower in carrot and onion. Bacterial isolates belonged to 5 classes covering 46 distinct species belonging to 19 genera. Human opportunistic pathogens were predominant in carrot and onion, whereas plant beneficial bacteria dominated in cucumber and tomato. Out of the 104 isolates, 16.25% are human pathogens and 26.5% are human opportunistic pathogens. CONCLUSIONS: Existence of a high population of plant beneficial bacteria was found to have suppressed the population of plant and human pathogens. There is a greater potential to study the native endophytic plant beneficial bacteria for developing them as biocontrol agents against human pathogens that are harboured by plants.

Identifying a Carotenoid Cleavage Dioxygenase 4a Gene and Its Efficient Agrobacterium-Mediated Genetic Transformation in Bixa orellana L.

Carotenoids are metabolized to apocarotenoids through the pathway catalysed by carotenoid cleavage oxygenases (CCOs). The apocarotenoids are economically important as it is known to have therapeutic as well as industrial applications. For instance, bixin from Bixa orellana and crocin from Crocus sativus are commercially used as a food colourant and cosmetics since prehistoric time. In our present study, CCD4a gene has been identified and isolated from leaves of B. orellana for the first time and named as BoCCD4a; phylogenetic analysis was carried out using CLUSTAL W. From sequence analysis, BoCCD4a contains two exons and one intron, which was compared with the selected AtCCD4, RdCCD4, GmCCD4 and CmCCD4a gene. Further, the BoCCD4a gene was cloned into pCAMBIA 1301, transformed into Agrobacterium tumefaciens EHA105 strain and subsequently transferred into hypocotyledons and callus of B. orellana by agro-infection. Selection of stable transformation was screened on the basis of PCR detection by using GUS and hptII specific primer, which was followed by histochemical characterization. The percent transient GUS expression in hypocotyledons and callus was 84.4 and 80 %, respectively. The expression of BoCCD4a gene in B. orellana was confirmed through RT-PCR analysis. From our results, the sequence analysis of BoCCD4a gene of B. orellana was closely related to the CsCCD4 gene of C. sativus, which suggests this gene may have a role in various processes such as fragrance, insect attractant and pollination.

Studies on interaction of norbixin with DNA: multispectroscopic and in silico analysis.

The interaction of food colorant norbixin with calf thymus DNA (CTDNA) was investigated through UV-Visible spectroscopy, Fourier Transform Infrared (FTIR), Circular Dichroism (CD), Nuclear Magnetic Resonance (NMR), DNA melting studies, electrophoretic analysis, histological staining technique and molecular docking studies. The results indicated that norbixin interacted with CTDNA by partial intercalation mode. The binding constant (K) of norbixin with CTDNA was calculated to be 5.08×10(5) Mol(-1) L. FTIR and CD studies were coupled with (1)H NMR spectra revealed that norbixin intercalates partially and binds to the groove's, phosphate group, deoxyribose sugar of DNA and also induces conformational transition of B-form to A-form DNA. Agarose gel electrophoretic and histological staining technique results further prove that, norbixin specifically binds to the DNA in the cell. Moreover, molecular docking studies on the specific binding of norbixin with CTDNA have exhibited lowest conformation energy score of -3.2. Therefore, this food colorant has the ability to interact with DNA and it could emerge as a promising class of natural DNA targeted therapeutic.

Homotypic clustering of OsMYB4 binding site motifs in promoters of the rice genome and cellular-level implications on sheath blight disease resistance.

The promoter regions (1 kb upstream sequences) of 45,836 annotated genes of rice were analyzed for the presence of OsMYB4 binding sites using a Perl program algorithm. Based on the homotypic clustering concept, 113 promoters were found to have more than 4 binding site motifs. Among the downstream genes of these promoters, five genes which are known to have a role in disease resistance were selected and the binding capacity of OsMYB4 protein in the promoter regions was analyzed by docking studies. Expression level of these genes was analyzed by RT-PCR in Rhizoctonia solani infected rice seedlings. Upon pathogen challenge, higher expression of aminotransferase, ankyrin and WRKY 12 genes was observed corresponding to higher expression of Osmyb4. Over-expression of Osmyb4 cDNA in rice leaf tissues by agro-infection failed to result in similar over-expression of aminotransferase, ankyrin and WRKY 12 as expected. Although the role of OsMYB4 in sheath blight resistance was found to be definitive based on our initial results, artificial over-expression of this TF was observed to be insufficient in regulating the disease resistance related genes.

Effect of dissolved inorganic carbon on ß-carotene and fatty acid production in Dunaliella sp.

This study aimed to explore the effect of sodium bicarbonate (0-200 mM) on the production of ß-carotene and lipid content in Dunaliella salina and Dunaliella bardawil. Total carotenoid and chlorophyll content were determined at regular intervals by a UV-VIS spectrophotometer. The ß-carotene and lipid contents were analyzed using high-performance liquid chromatography (HPLC) and gas chromatography coupled with mass spectrometry (GC-MS). The HPLC results revealed a twofold increase of ß-carotene in D. salina and D. bardawil cultures grown with sodium bicarbonate. Moreover, total fatty acid profiles from GC-MS indicated a maximum relative percentage of saturated fatty acids (tetradecanoic acid, 10,13-diethyl, methyl ester and methyl 16-methyl-heptadecanoate) compared to polyunsaturated fatty acids in both algae. Our results indicate that the optimum concentration of bicarbonate (100 to 150 mM) was required to stimulate a positive effect on ß-carotene production as well as the lipid profile in Dunaliella sp.

An in silico model for rapid identification of multiple bacteria in resource limited laboratories.

We have developed an in silico model for simplified detection of five major food borne bacteria using common PCR primer based on conserved region flanking a variable region in the first rRNA operon sequences. The RFLP pattern exhibited on virtual SmaI and MboI digestion were unique to each genus. This design is based on the combination of comparative sequence analysis, conventional PCR and restriction digestion methods. We believe this approach as a better alternative to 16S rRNA sequencing based identification / detection of bacteria and suited for the resource limited academic research laboratories in which variety of bacteria are used in different students' projects.