Microbial influencers and cotton leaf curl disease (CLCuD) susceptibility: a network perspective.

Biotic stresses, such as plant viruses, e.g., cotton leaf curl virus (CLCuV), can alter root-associated and leaf-associated microbial diversities in plants. There are complex ecological dynamics at play, with each microbe contributing to a multitude of biotic and abiotic interactions, thus deciding the stability of the plant's ecosystem in response to the disease. Deciphering these networks of interactions is a challenging task. The inferential research in microbiome is also at a nascent stage, often constrained by the underlying analytical assumptions and the limitations with respect to the depth of sequencing. There is also no real consensus on network-wide statistics to identify the influential microbial players in a network. Guided by the latest developments in network science, including recently published metrics such as Integrated View of Influence (IVI) and some other centrality measures, this study provides an exposé of the most influential nodes in the rhizospheric and phyllospheric microbial networks of the cotton leaf curl disease (CLCuD) susceptible, partially tolerant, and resistant cotton varieties. It is evident from our results that the CLCuD-resistant Gossypium arboreum possesses an equal share of keystone species, which helps it to withstand ecological pressures. In the resistant variety, the phyllosphere harbors the most influential nodes, whereas in the susceptible variety, they are present in the rhizosphere. Based on hubness score, spreading score, and IVI, the top 10 occurring keystone species in the FDH-228 (resistant) variety include Actinokineospora, Cohnella, Thermobacillus, Clostridium, Desulfofarcimen, and MDD-D21. Elusimicrobia, Clostridium-sensu-stricto_12, Candidatus woesebacteria, and Dyella were identified as the most influential nodes in the PFV-1 (partially tolerant) variety. In the PFV-2 (susceptible) variety, the keystone species were identified as Georginia, Nesterenkonia, Elusimicrobia MVP-88, Acetivibrio, Tepedisphaerales, Chelatococcus, Nitrosospira, and RCP2-54. This concept deciphers the diseased and healthy plant's response to viral disease, which may be microbially mediated.

Efficacy of Zinc Fortified and Fermented Wheat Flour (EZAFFAW): A randomized controlled trial protocol.

BACKGROUND: Zinc deficiency poses significant health risks, particularly in low-income settings. This study aims to evaluate the impact of agronomically zinc biofortified (fermented and non-fermented) and post-harvest wheat flour flatbread on zinc status and metabolic health in adolescents and adult women in rural Pakistan. METHODS: A four-arm triple-blind randomized controlled trial will be conducted in a rural district of Pakistan. Participants (adolescents aged 10-19 and adult women aged 20-40) will be assigned to receive fermented or unfermented high zinc agronomically biofortified wheat flour flatbread, post-harvest zinc-fortified wheat flour flatbread, or low zinc conventional whole wheat flour flatbread. The meal would be served once a day, six days a week for six months. The study aims to enroll 1000 participants and will be analyzed based on the intention-to-treat principle. The trial is registered with number NCT06092515. OUTCOMES: Primary outcomes will include serum zinc concentration and metabolic markers, while secondary outcomes include anthropometric measurements, blood pressure, and dietary intake. CONCLUSION: This trial will provide valuable insights into the efficacy of agronomically zinc biofortified wheat flour in improving zinc status and metabolic health. Findings may inform public health strategies to combat zinc deficiency in resource-limited settings.

Cotton microbiome profiling and Cotton Leaf Curl Disease (CLCuD) suppression through microbial consortia associated with Gossypium arboreum.

The failure of breeding strategies has caused scientists to shift to other means where the new approach involves exploring the microbiome to modulate plant defense mechanisms against Cotton Leaf Curl Disease (CLCuD). The cotton microbiome of CLCuD-resistant varieties may harbor a multitude of bacterial genera that significantly contribute to disease resistance and provide information on metabolic pathways that differ between the susceptible and resistant varieties. The current study explores the microbiome of CLCuD-susceptible Gossypium hirsutum and CLCuD-resistant Gossypium arboreum using 16 S rRNA gene amplification for the leaf endophyte, leaf epiphyte, rhizosphere, and root endophyte of the two cotton species. This revealed that Pseudomonas inhabited the rhizosphere while Bacillus was predominantly found in the phyllosphere of CLCuV-resistant G. arboreum. Using salicylic acid-producing Serratia spp. and Fictibacillus spp. isolated from CLCuD-resistant G. arboreum, and guided by our analyses, we have successfully suppressed CLCuD in the susceptible G. hirsutum through pot assays. The applied strains exhibited less than 10% CLCuD incidence as compared to control group where it was 40% at 40 days post viral inoculation. Through detailed analytics, we have successfully demonstrated that the applied microbes serve as a biocontrol agent to suppress viral disease in Cotton.

Whole genome analysis of Gluconacetobacter azotocaptans DS1 and its beneficial effects on plant growth.

Plant-associated bacteria play an important role in the enhancement of plant growth and productivity. Gluconacetobacter azotocaptans is an exceptional bacterium considering that till today it has been isolated and reported only from Mexico and Canada. It is a plant growth-promoting bacterium and can be used as biofertilizer for different crops and vegetables. The objective of the current study was to evaluate the inoculation effect of Gluconacetobacter azotocaptans DS1, Pseudomonas putida CQ179, Azosprillium zeae N7, Azosprillium brasilense N8, and Azosprillium canadense DS2, on the growth of vegetables including cucumber, sweet pepper, radish, and tomato. All strains increased the vegetables' growth; however, G. azotocaptans DS1 showed better results as compared to other inoculated and control plants and significantly increased the plant biomass of all vegetables. Therefore, the whole genome sequence of G. azotocaptans DS1 was analyzed to predict genes involved in plant growth promotion, secondary metabolism, antibiotics resistance, and bioremediation of heavy metals. Results of genome analysis revealed that G. azotocaptans DS1 has a circular chromosome with a size of 4.3 Mbp and total 3898 protein-coding sequences. Based on functional analysis, genes for nitrogen fixation, phosphate solubilization, indole acetic acid, phenazine, siderophore production, antibiotic resistance, and bioremediation of heavy metals including copper, zinc, cobalt, and cadmium were identified. Collectively, our findings indicated that G. azotocaptans DS1 can be used as a biofertilizer and biocontrol agent for growth enhancement of different crops and vegetables. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02996-1.

Comparative Study of the Rhizosphere and Root Endosphere Microbiomes of Cholistan Desert Plants.

Microbial communities associated with the rhizosphere and roots of desert halophytes play an important role in plants' growth and development. Very limited information has been available on the microbial diversity of arid environments of Pakistan. Hence in the current study, the microbial diversity of rhizosphere and root endosphere of desert halophytes, Zygophyllum simplex, Haloxylon salicoricum, Aerva javanica, and Capparis decidua was evaluated. The rhizosphere and root endosphere samples of desert halophytes collected from the three geographic sites of Cholistan desert, Punjab, Pakistan were analyzed by using 16S rRNA based Illumina sequencing. The results showed that Proteobacteria were more abundant in the rhizospheric soils while Actinobacteria were more dominant in the root endosphere of halophytes. Bacteroidetes, Firmicutes, and Deinococcus-Thermus were identified from all rhizospheric soils and roots across the three sites, with variable percentage. Bacillus, Kocuria, Pseudomonas, Halomonas, and Flavobacterium were commonly identified from the rhizosphere and root endosphere of halophytes across all the three sites. At the genus level, microbial diversity from Haloxylon showed the greatest variations between the rhizosphere and root endosphere from the site 2. This study revealed that microbial diversity analysis can be used to study how changes in abiotic factors such as soil moisture content and salinity affect the microbial communities associated with the rhizospheric soils and root endosphere of halophytes across the three sites. This study will also help in the discovery of potential inoculants for crops growing in arid and semi-arid regions of Pakistan.

Gut microbiome dysbiosis and correlation with blood biomarkers in active-tuberculosis in endemic setting.

Tuberculosis (TB) is the largest infectious disease with 10 million new active-TB patients and1.7 million deaths per year. Active-TB is an inflammatory disease and is increasingly viewed as an imbalance of immune responses to M. tb. infection. The mechanisms of a switch from latent infection to active disease is not well worked out but a shift in the immune responses is thought to be responsible. Increasingly, the role of gut microbiota has been described as a major influencer of the immune system. And because the gut is the largest immune organ, we aimed to analyze the gut microbiome in active-TB patients in a TB-endemic country, Pakistan. The study revealed that Ruminococcacea, Enetrobactericeae, Erysipelotrichaceae, Bifidobacterium, etc. were the major genera associated with active-TB, also associated with chronic inflammatory disease. Plasma antibody profiles against several M. tb. antigens, as specific biomarkers for active-TB, correlated closely with the patient gut microbial profiles. Besides, bcoA gene copy number, indicative of the level of butyrate production by the gut microbiome was five-fold lower in TB patients compared to healthy individuals. These findings suggest that gut health in TB patients is compromised, with implications for disease morbidity (e.g., severe weight loss) as well as immune impairment.

Profiling of antimicrobial metabolites of plant growth promoting Pseudomonas spp. isolated from different plant hosts.

In this study, nine strains of Pseudomonas au rantiaca and P. chlororaphis, and two isolates of Pseudomonas sp.: At1RP4 and RS-1, were characterized for the in-vitro production of secondary metabolites in LB, DMB, and King's B media, and of the genes responsible for the production of antagonistic metabolites. Based on 16S rRNA gene sequence, isolates At1RP4 and RS-1 were identified as strains of P. aeruginosa and P. fluorescens. Five phenazine derivatives comprising phenazine, phenazine-1-carboxylic acid (PCA), 2-hydroxyphenazine-1-carboxylic acid (2-OH-Phz-1-COOH), phenazine-1,6-dicarboxylic acid (Phz-1,6-di-COOH), and 2-hydroxyphenazine (2-OH-Phz) were produced by all strains in all three culture media including DMB, King's B and LB. However, 2,8-dihydroxyphenazine, 6-methylphenazine-1-carboxylic acid, pyrrolnitrin, and the ortho-dialkyl-aromatic acids, were produced by the P. aurantiaca and P. chlororaphis strains. In addition, all strains produced 2-acetamidophenol, pyochelin, and diketopiperazine derivatives in variable amounts in all three culture media used. Highest levels of quorum-sensing signal molecules including PQS, 2-Octyl-3-hydroxy-4(1H)-quinolone, and hexahydro-quinoxaline-1,4-dioxide were recorded for P. aeruginosa At1RP4. Moreover, all strains produced volatile hydrogen cyanide (0.95-6.68 µg/L) and the phytohormone indole-3-acetic acid (0.42-13.9 µM). Production of extracellular lipase and protease was recorded in all pseudomonads, whereas, cellulase production and phosphate solubilization were variable. Genes for hydrogen cyanide and phenazine-1-carboxylic acid were detected in all eleven strains while the gene for pyrrolnitrin biosynthesis was amplified in P. aurantiaca and P. chlororaphis strains. Comparative metabolomic analysis provided detailed insights about the strain-specific metabolites in pseudomonads, and their pseudo-relative quantification in different bacterial growth media to be used as single-strain biofertilizer and biocontrol inoculums. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-020-02585-8.

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.

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.

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.

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.

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.

Extracellular Secretion of Phytase from Transgenic Wheat Roots Allows Utilization of Phytate for Enhanced Phosphorus Uptake.

A significant portion of organic phosphorus comprises of phytates which are not available to wheat for uptake. Hence for enabling wheat to utilize organic phosphorus in form of phytate, transgenic wheat expressing phytase from Aspergillus japonicus under barley root-specific promoter was developed. Transgenic events were initially screened via selection media containing BASTA, followed by PCR and BASTA leaf paint assay after hardening. Out of 138 successfully regenerated To events, only 12 had complete constructs and thus further analyzed. Positive T1 transgenic plants, grown in sand, exhibited 0.08-1.77, 0.02-0.67 and 0.44-2.14 fold increase in phytase activity in root extracts, intact roots and external root solution, respectively, after 4 weeks of phosphorus stress. Based on these results, T2 generation of four best transgenic events was further analyzed which showed up to 1.32, 56.89, and 15.40 fold increase in phytase activity in root extracts, intact roots and external root solution, respectively, while in case of real-time PCR, maximum fold increase of 19.8 in gene expression was observed. Transgenic lines showed 0.01-1.18 fold increase in phosphorus efficiency along with higher phosphorus content when supplied phytate or inorganic phosphorus than control plants. Thus, this transgenic wheat may aid in reducing fertilizer utilization and enhancing wheat yield.

Spider venom toxin protects plants from insect attack.

Many of the toxin proteins, that have been heterogeneously expressed in agricultural crops to provide resistance to insect pests, are too specific or are only mildly effective against the major insect pests. Spider venoms are a complex cocktail of toxins that have evolved specifically to kill insects. Here we show that the omega-ACTX-Hv1a toxin (Hvt), a component of the venom of the Australian funnel web spider (Hadronyche versuta) that is a calcium channel antagonist, retains its biological activity when expressed in a heterologous system. Expressed as a fusion protein in E. coli, the purified toxin fusion immobilized and killed Helicoverpa armigera and Spodoptera littoralis caterpillars when applied topically. Transgenic expression of Hvt in tobacco effectively protected the plants from H. armigera and S. littoralis larvae, with 100% mortality within 48 h. We conclude that the Hvt is an attractive and effective molecule for the transgenic protection of plants from herbivorous insects which should be evaluated further for possible application in agriculture.