Molecular diagnostics for foodborne pathogen (Salmonella spp.) from poultry

Background: Salmonella species (spp.) are among major food-borne pathogens all over the world. Salmonella typhimurium is the main cause of food poisoning in humans. The fundamental objective of this study is to develop a rapid and reliable method to detect Salmonella (a foodborne pathogen) in raw poultry meat by using molecular approaches. Methods: Total 200 samples of raw poultry meat were collected from different regions of Lahore and analyzed for the presence of Salmonella spp. fimA gene.  Similarly, sent genes were selected for the detection of Salmonella typhimurium and Salmonella enteritidis respectively. PCR technique was optimized for diagnosis of contamination.Results: Out of 200 samples, 2% samples had shown successful amplification of fimA gene representing the presence of serovar Salmonella typhimurium. PCR assay combined with enrichment can enhance the efficiency for detection of Salmonella in poultry. Conclusion: A robust, simple and convenient PCR based method has been developed for the detection of one of the major food-borne pathogen Salmonella typhimurium. 

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

Cotton microbiome profiling and Cotton Leaf Curl Disease (CLCuD) suppression through microbial consortia associated

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

A study of PTF1 interaction with phosphorus stress inducing genes and its influence on root architecture of transgenic Arabidopsis

Background: Phosphorus; an essential macronutrient needed by the plant for its robust growth is inaccessible to the plant as required. Hence, a need arises to develop smart crops capable of utilizing maximum phosphorus from soil. PTF1(Inorganic Phosphorus Starvation Induced Transcription Factor 1) is overexpressed during phosphorus stress and regulates number of genes to combat this abiotic stress.Methods: The current study is the first ever reported case of transforming Arabidopsis thaliana with plant expression binary vector pSB219 harboring PTF1 via floral dip method and analyzing phosphorus stress induced genes interaction, through yeast-one-hybrid. Yeast-one-hybrid analysis was performed on four selected genes namely LPR1 (Low Phosphate Response), PDR2 (Phosphate Deficiency Response 2), PHT1;2 (Phosphate Transporter) and RNS1 (Ribonuclease). The positive transformed lines were expression analyzed for PTF1 by real time PCR and further studied for their root morphology.Results: The results clearly showed direct interaction of LPR1 with PTF1 while other genes, although being overexpressed, were indirectly regulated. Transformation efficiency of 1% was achieved and a maximum 2.5-fold increase in PTF1 expression was observed. Root morphological studies exhibited significantly enhanced root hair and lateral surface area when grown in phosphorus deficient MS medium.Conclusion: The results of the current study may pave path for improved comprehension of gene interactions and root architecture modifications under phosphorus limiting conditions.Keywords: Phosphorus; Arabidopsis; Yeast one hybrid; Roots

Comparison of phytase activity in roots of wheat varieties grown under different phosphorus conditions

Phosphorus (P) is a vital macronutrient for plant growth but it is poorly available to plant roots as a significant amount of soil P is present in the form of phytate. To overcome phosphorus deficiency, wheat is capable of producing phytase. In the present study, six Pakistani wheat varieties (Faisalabad-2008, Sehar-2006, Pak-81, Punjab-11, ASS-11, VII-83) were evaluated for their phytase activity, protein and total phosphorus content of shoots and roots under three phosphorus conditions. All wheat varieties exhibited a differential ability to produce phytase in different P conditions. The highest phytase activity was observed in root extracts (0.781U/g), external root solution (0.0972 U/g) and intact roots (0.0417U/g) of Pak-81 when grown in the presence of phytate. Highest protein content (44 μg), shoot (176.25 μg) and root (111.62 μg) phosphate content was observed in Pb-11 and VIII-83, respectively in the presence of inorganic phosphate. Results indicate that wheat varieties are unable to fulfill their P requirements, thus it is necessary to augment phytase production in wheat via breeding methods or transgenic approach

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, with a total of 3% of the worldâ s land mass being affected by salinity. Approximately 6.3 million hectares of land in Pakistan is affected by salinity to varying degree and most of the areas are arid to semiarid with low annual precipitation. The aim of present study is to identify and characterize Bacillus and Bacillus-derived bacterial genera from the rhizospheric and non-rhizospheric soil samples from Khewra Salt Mine, Pakistan by using culture-independent as well as culture-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 Bacillus-like isolates identified in this study were moderately halophilic, alkaliphilic and mesophilic bacteria and were considered as 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.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Differential response of kabuli and desi chickpea genotypes towards inoculation with PGPR in different soils

Pakistan is among top three chickpea producing countries but the crop is usually grown on marginal lands without irrigation and fertilizer application which significantly hampers its yield. Soil fertility and inoculation with beneficial rhizobacteria play a key role in nodulation and yield of legumes. Four kabuli and six desi chickpea genotypes were, therefore, evaluated for inoculation response with IAA-producing Ochrobactrum ciceri Ca-34T and nitrogen fixing Mesorhizobium ciceri TAL-1148 in single and co-inoculation in two soils. The soil type 1 was previously unplanted marginal soil having low organic matter, P and N contents than soil type 2 which was a fertile routinely legume-cultivated soil. The effect of soil fertility status was pronounced and fertile soil on average, produced 31% more nodules, 62% more biomass and 111 % grain yield than marginal soil. Inoculation either with O. ciceri alone or its co-inoculation with M. ciceri produced on average higher nodules (42%), biomass (31%), grains yield (64%) and harvest index (72%) in both chickpea genotypes over non-inoculated controls in both soils. Soil 1 showed maximum relative effectiveness of Ca-34T inoculation for kabuli genotypes while soil 2 showed for desi genotypes except B8/02. Desi genotype B8/02 in soil type 1 and Pb-2008 in soi

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

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