Exploring the potential of Bacillus for crop productivity and sustainable solution for combating rice false smut disease.

Rice false smut, which is caused by the soil-borne fungal pathogen Ustilaginoidea virens (U. virens), is one of the most threatening diseases in most of the rice-growing countries including India that causes 0.5-75% yield loss, low seed germination, and a reduction in seed quality. The assessment of yield loss helps to understand the relevance of disease severity and facilitates the implementation of appropriate management strategies. This study aimed to mitigate biotic stress in rice by employing a rhizobacterial-based bioformulation, which possesses diverse capabilities as both a plant growth promoter and a biocontrol agent against U. virens. Rhizobacteria were isolated from the soil of the rice rhizospheres from the healthy plant of the false smut affected zone. Furthermore, they were identified as Bacillus strains: B. subtilis (BR_4), B. licheniformis (BU_7), B. licheniformis (BU_8), and B. vallismortis (KU_7) via sequencing. Isolates were screened for their biocontrol potential against U. virens under in vitro conditions. The antagonistic study revealed that B. vallismortis (KU_7) inhibited U. virens the most (44.6%), followed by B. subtilis BR_4 (41.4%), B. licheniformis BU_7 (39.8%), and B. licheniformis BU_8 (43.5%). Various biochemical and plant growth promoting attributes, such as phosphate and Zn solubilization, IAA, ammonium, siderophore, and chitinase production, were also investigated for all the selected isolates. Furthermore, the potential of the isolates was tested in both in vitro and field conditions by employing talc-based bioformulation through bio-priming and root treatment. The application of bioformulation revealed a 20% decrease in disease incidence in plants treated with B. vallismortis (KU_7), a 60.5% increase in the biological yield, and a 45% increase in the grain yield. This eco-friendly approach not only controlled the disease but also improved the grain quality and reduced the chaffiness.

Genetic manipulation of Indian mustard genotypes with WRR-gene(s) confers resistance against Albugo candida.

BACKGROUND: Brassica species is the second most important edible oilseed crop in India. Albugo candida (Pers.) Kuntze, a major oomycete disease of oilseed brassica causing white rust, leads to 60% yield loss globally. The prevalence of A. candida race 2 (Ac2V) that specifically infects B. juncea, coupled with limitations of conventional methods has resulted in a dearth of white rust resistance resources in cultivated varieties. METHODS AND RESULTS: In an effort to develop resistant plants, Agrobacterium mediated genetic transformation of three B. juncea genotypes viz., susceptible host var. Varuna, along with its doubled haploid mutant lines C66 and C69 (showing moderate tolerance to field isolates of A. candida) was initiated to transfer resistance genes (WRR8Sf-2 and WRR9Hi-0) identified in Arabidopsis thaliana against race Ac2V, that encode for Toll-like/interleukin-1 receptor-nucleotide binding-leucine-rich repeat proteins that recognize effectors of the pathogen races. CONCLUSIONS: Our results demonstrate that introduction of resistance genes from a tertiary gene pool by genetic transformation enhances disease resistance in B. juncea genotypes to a highly virulent Ac2V isolate.

Resistance strategies for defense against Albugo candida causing white rust disease.

Albugo candida, the causal organism of white rust, is an oomycete obligate pathogen infecting crops of Brassicaceae family occurred on aerial part, including vegetable and oilseed crops at all growth stages. The disease expression is characterized by local infection appearing on the abaxial region developing white or creamy yellow blister (sori) on leaves and systemic infections cause hypertrophy and hyperplasia leading to stag-head of reproductive organ. To overcome this problem, several disease management strategies like fungicide treatments were used in the field and disease-resistant varieties have also been developed using conventional and molecular breeding. Due to high variability among A. candida isolates, there is no single approach available to understand the diverse spectrum of disease symptoms. In absence of resistance sources against pathogen, repetitive cultivation of genetically-similar varieties locally tends to attract oomycete pathogen causing heavy yield losses. In the present review, a deep insight into the underlying role of the non-host resistance (NHR) defence mechanism available in plants, and the strategies to exploit available gene pools from plant species that are non-host to A. candida could serve as novel sources of resistance. This work summaries the current knowledge pertaining to the resistance sources available in non-host germ plasm, the understanding of defence mechanisms and the advance strategies covers molecular, biochemical and nature-based solutions in protecting Brassica crops from white rust disease.

Polymeric micelles and cancer therapy: an ingenious multimodal tumor-targeted drug delivery system.

Since the beginning of pharmaceutical research, drug delivery methods have been an integral part of it. Polymeric micelles (PMs) have emerged as multifunctional nanoparticles in the current technological era of nanocarriers, and they have shown promise in a range of scientific fields. They can alter the release profile of integrated pharmacological substances and concentrate them in the target zone due to their improved permeability and retention, making them more suitable for poorly soluble medicines. With their ability to deliver poorly soluble chemotherapeutic drugs, PMs have garnered considerable interest in cancer. As a result of their remarkable biocompatibility, improved permeability, and minimal toxicity to healthy cells, while also their capacity to solubilize a wide range of drugs in their micellar core, PMs are expected to be a successful treatment option for cancer therapy in the future. Their nano-size enables them to accumulate in the tumor microenvironment (TME) via the enhanced permeability and retention (EPR) effect. In this review, our major aim is to focus primarily on the stellar applications of PMs in the field of cancer therapeutics along with its mechanism of action and its latest advancements in drug and gene delivery (DNA/siRNA) for cancer, using various therapeutic strategies such as crossing blood-brain barrier, gene therapy, photothermal therapy (PTT), and immunotherapy. Furthermore, PMs can be employed as "smart drug carriers," allowing them to target specific cancer sites using a variety of stimuli (endogenous and exogenous), which improve the specificity and efficacy of micelle-based targeted drug delivery. All the many types of stimulants, as well as how the complex of PM and various anticancer drugs react to it, and their pharmacodynamics are also reviewed here. In conclusion, commercializing engineered micelle nanoparticles (MNPs) for application in therapy and imaging can be considered as a potential approach to improve the therapeutic index of anticancer drugs. Furthermore, PM has stimulated intense interest in research and clinical practice, and in light of this, we have also highlighted a few PMs that have previously been approved for therapeutic use, while the majority are still being studied in clinical trials for various cancer therapies.

Biochemical aspects of pathogenic variability in white rust infected Indian mustard.

White rust caused by Albugo candida, an oomycete pathogen, is a devastating disease of Brassica juncea (Indian mustard) worldwide. There is a need to screen virulent white rust isolates to challenge the developed white rust-resistant B. juncea cultivars to screen their resistance potential. The current study explores pathogenic and biochemical response of Indian mustard to white rust isolates collected from three different geographic locations of India. The observations refine our understanding of the disease severity in India. Disease progression and biochemical responses were studied in the cotyledonary as well as true leaf stage of the B. juncea cultivar Varuna at different time points. The biochemical findings highlight the fluctuation of significant biochemical parameters such as total proteins, sugars, and phenols, superoxide dismutase, and hydrogen peroxide during the A. candida infection in B. juncea.

Symbiotic interplay of Piriformospora indica and Azotobacter chroococcum augments crop productivity and biofortification of Zinc and Iron.

In the present study Piriformospora indica (Pi) a phyto-promotional fungus and Azotobacter chroococcumWR5 (AzWR5) a rhizobacterium, were symbiotically evaluated for their role in improving the nutritional quality of wheat (Triticum aestivum L.). Co-inoculation of Pi+AzWR5 modified root system architecture of host and along with increasing the proportion of finer roots by 88% and 92% in C306 and Hd2967 respectively. Furthermore, the synergistic impact of Pi+AzWR5 interplayed for enhanced accumulation of Zn and Fe in different plant parts including grains (3.12 and 1.33 fold respectively). Pi+AzWR5 increased the transfer factor of Zn (62%, 94%, 91% and 213%) and Fe (31%, 54%, 68% and 32%) in root, stem, leaves and grains, respectively, and translocation factor of Zn (20%, 18% and 63%) and Fe (18%, 29% and 29%) for root-stem, root-leaves and root-grains, respectively. In addition to these co-inoculation of endophytes led to several fold increase in expression of four ZIP transporter genes in roots and shoot. In addition to these symbiotic association of endophytes with host led to 3 fold increase in grain yield. We thereby conclude that co-inoculation of Pi+AzWR5 substantially improves mobilization of Zn and Fe from soil and increase its concentration in grains as well as improves crop yield.

High level of persister frequency in clinical staphylococcal isolates.

BACKGROUND: Staphylococcus aureus is a notorious human pathogen that causes often lethal systemic conditions that are mostly medical device associated biofilm infections. Similarly, coagulase negative staphylococci are emerging as leading pathogen for nosocomial infections owing to their ability to form biofilm on implanted medical equipment. Chronic in nature, these infections are difficult to treat. Such recalcitrance of these infections is caused mainly due to the presence of persister cells, which exhibit transient yet extreme tolerance to antibiotics. Despite tremendous clinical significance, there is lack of studies on persister cells formation among clinical bacterial isolates. Considering the importance of factors influencing persister formation, in this study, we evaluate the association of antibiotic tolerance with biofilm production, antibiotic stress, growth phase, specimen type, and dependency on staphylococcal species. Biofilm formation was detected among 375 clinical staphylococcal isolates by quantitative tissue culture plate method (TCP) and icaAD genes by genotypic method. The antibiotic susceptibility was determined by Kirby Bauer disc diffusion method while minimum inhibitory concentration values were obtained by agar dilution method. Persister cells were measured in the susceptible staphylococcal isolates in the presence of clinically relevant antibiotics. RESULTS: In the study, 161 (43%) S. aureus and 214 (57%) coagulase negative staphylococci (CNS) were isolated from different clinical samples. TCP method detected biofilm production in 84 (52.2%) S. aureus and 90 (42.1%) CNS isolates. The genotypic method detected icaAD genes in 86 (22.9%) isolates. Majority (> 90%) of both the biofilm producers and non-producers were sensitive to chloramphenicol and tetracycline but were resistant to penicillin. Interestingly, all isolates were sensitive to vancomycin irrespective of biofilm production. While high persister frequency was observed among all staphylococci isolates in the stationary growth phase, the persister frequency in exponential growth phase was statistically high among isolates possessing icaAD genes compared to icaAD negative isolates. CONCLUSION: The research findings provide strong evidence that the clinical staphylococcal isolates exhibit extreme antibiotic tolerance suggesting their causal link with treatment failures. Understanding the factors influencing the formation and maintenance of persister cells are of utmost important aspect to design therapeutics and control recalcitrant bacterial infections.

Role of the blue light receptor gene Icwc-1 in mycelium growth and fruiting body formation of Isaria cicadae.

The Isaria cicadae, is well known highly prized medicinal mushroom with great demand in food and pharmaceutical industry. Due to its economic value and therapeutic uses, natural sources of wild I. cicadae are over-exploited and reducing continuously. Therefore, commercial cultivation in controlled environment is an utmost requirement to fulfill the consumer's demand. Due to the lack of knowledge on fruiting body (synnemata) development and regulation, commercial cultivation is currently in a difficult situation. In the growth cycle of macrofungi, such as mushrooms, light is the main factor affecting growth and development, but so far, specific effects of light on the growth and development of I. cicadae is unknown. In this study, we identified a blue light receptor white-collar-1 (Icwc-1) gene homologue with well-defined functions in morphological development in I. cicadae based on gene knockout technology and transcriptomic analysis. It was found that the Icwc-1 gene significantly affected hyphal growth and fruiting body development. This study confirms that Icwc-1 acts as an upstream regulatory gene that regulates genes associated with fruiting body formation, pigment-forming genes, and related genes for enzyme synthesis. Transcriptome data analysis also found that Icwc-1 affects many important metabolic pathways of I. cicadae, i.e., amino acid metabolism and fatty acid metabolism. The above findings will not only provide a comprehensive understanding about the molecular mechanism of light regulation in I. cicadae, but also provide new insights for future breeding program and improving this functional food production.

Metaproteomics associated with severe early childhood caries highlights the differences in salivary proteins.

OBJECTIVE: To investigate the salivary metaproteomic characteristics of the children with and without severe early childhood caries (S-ECC). DESIGN: In this study, we collected unstimulated saliva samples from 34 children (age 3-4 years) with caries free (NC, dmfs (= index of decayed, missing due to caries, or filled tooth surfaces) = 0, n = 23) and with S-ECC (dmfs≥10, n = 11). Salivary proteins were extracted and reduced, and then a Liquid Chromatography/Mass Spectrometry system was used to identify proteins. RESULTS: Nearly 3000 proteins were identified in this study, and about 3.5 % of the proteins originated from human while 86 % were derived from microbes. The salivary protein types in the NC group were statistically greater than those in the S-ECC group (P <0.05). Specifically, the salivary protein types derived from microbes in the NC group were significantly greater than those in the S-ECC group. Three proteins, human lactoferrin, penicillin-binding protein 1C [Burkholderia ubonensis], human alpha-defensin 1 (F28a mutant), were decreased statistically in the NC group compared to the S-ECC group (P < 0.05). Only one protein, 50S ribosomal protein L17 secreted by Haemophilus haemolyticus, was significantly increased in the NC group compared to the S-ECC group. Salivary IgA was the top highest protein in the NC group whereas human lysozyme was the top highest protein in the S-ECC group. CONCLUSIONS: The differential proteins recognized in this study may be conducive for finding a caries biomarker. Understanding the metaproteomic characteristics can help us to control the caries from human origin and microbial origin.

Phenotypic and genotypic characterization of biofilm producing clinical coagulase negative staphylococci from Nepal and their antibiotic susceptibility pattern.

BACKGROUND: Coagulase-negative staphylococci (CNS) survive as commensals of skin, anterior nares and external canals of human and were regarded as non-infectious pathogens. However, they are emerging as a major cause of nosocomial infectious due to their ability to form biofilms and high resistance to several classes of antibiotics. This study examines the biofilm forming abilities of 214 clinical CNS isolates using phenotypic and genotypic methods, and determines their antibiotic susceptibility patterns. METHODS: A total of 214 clinical isolates collected from different clinical samples were identified as CNS and their antibiotic susceptibility determined by CLSI guidelines. The biofilm forming ability of all isolates was determined by three phenotypic methods; Congo red agar (CRA) method, tube adherence method (TM) and tissue culture plate (TCP) method and by genotypic method for the detection of icaAD genes. RESULTS: Among all the isolates, S. epidermidis (57.5%) was found the most frequently, followed by S. saprophyticus (18.7%), S. haemolyticus (11.2%), S. hominis (7%), and S. capitis (5.6%). Antibiotic susceptibility pattern demonstrated 91.6% isolates were resistant to penicillin and 66.8% to cefoxitin while 91.1% isolates were susceptible to chloramphenicol. Constitutive and inducible clindamycin resistant phenotype as measured by D-test was seen among 28% and 14.5% of isolates respectively. Tissue culture plate method detected biofilm production in 42.1% isolate followed by 31.8% through tube method while 20.1% isolates were found to produce slime in Congo red agar method. The genotypic assay revealed presence of icaA and icaD genes in 19.2% isolates. CONCLUSION: The study shows a high prevalence of biofilm formation and inducible clindamycin resistance in CNS isolates, indicating the importance of in-vitro biofilm production test and D-test in routine laboratory diagnostics. Implementation of efficient diagnostic techniques for detection of biofilm production in clinical samples can help manage staphylococcal infections and minimize risks of treatment failures in hospitals.

Study of inhibitory potential and percent inhibition of oil of Syzygium aromaticum and leaves of Ocimum sanctum on ESBL enzyme from Escherichia coli in broilers of Jabalpur.

OBJECTIVE: The inhibitory potential and percent inhibition of Syzygium aromaticum oil and fresh juice of Ocimum sanctum leaves on beta-lactamase enzyme of cecal samples of healthy broilers were studied on samples phenotypically positive for extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli. MATERIALS AND METHODS: Four hundred cecal samples screened for ESBL-producing E. coli were collected from 38 poultry sale outlets located in Jabalpur. The effect of S. aromaticum oil and O. sanctum leaves was seen by colorimetric assay with CENTA and Nitrocefin as chromogenic substrate. RESULTS: Mean absorbance value was inversely propotional to the inhibitory potential. Syzigium aromaticum exhibited 0.4±0.02 and 0.41±0.03 mean absorbance value, 28 per cent and 27 per cent of inhibition with CENTA and Nitrocefin respectively. Ocimum sanctum mean absorbance value and per cent inhibition with CENTA and Nitrocefin was 2.03±0.02 and 10.0 ; 1.97±0.06 and 10.0 respectively (p>0.05) showing non- significant difference in CENTA and Nitrocefin activity. Tazobactum (100 µM) as standard control exhibited a mean absorbance value of 0.12 ± 0.01 and 0.13 ± 0.01 and percent inhibition of 99.88 and 98 against CENTA and Nitrocefin, respectively. Combination of Ocimum sanctum and Syzigium aromaticum showed range of 1.69±0.05 to 1.90±0.08 1.61±0.06 to 1.92±0.08 of absorbance value with per cent inhibition of 14 to 15.9 with CENTA and Nitrocefin respectively. CONCLUSION: The results depicted that the inhibition of beta-lactamase enzyme activity with S. aromaticum oil was higher than that of O. sanctum leaf juice, and combination of both the herbs showed not much difference in activity.

Biofilm Producing Clinical Staphylococcus aureus Isolates Augmented Prevalence of Antibiotic Resistant Cases in Tertiary Care Hospitals of Nepal.

Staphylococcus aureus, a notorious human pathogen, is a major cause of the community as well as healthcare associated infections. It can cause a diversity of recalcitrant infections mainly due to the acquisition of resistance to multiple drugs, its diverse range of virulence factors, and the ability to produce biofilm in indwelling medical devices. Such biofilm associated chronic infections often lead to increase in morbidity and mortality posing a high socio-economic burden, especially in developing countries. Since biofilm formation and antibiotic resistance function dependent on each other, detection of biofilm expression in clinical isolates would be advantageous in treatment decision. In this premise, we attempt to investigate the biofilm formation and its association with antibiotic resistance in clinical isolates from the patients visiting tertiary health care hospitals in Nepal. Bacterial cells isolated from clinical samples identified as S. aureus were examined for in-vitro biofilm production using both phenotypic and genotypic assays. The S. aureus isolates were also examined for susceptibility patterns of clinically relevant antibiotics as well as inducible clindamycin resistance using standard microbiological techniques and D-test, respectively. Among 161 S. aureus isolates, 131 (81.4%) were methicillin resistant S. aureus (MRSA) and 30 (18.6%) were methicillin sensitive S. aureus (MSSA) strains. Although a majority of MRSA strains (69.6%) showed inducible clindamycin resistance, almost all isolates (97% and 94%) were sensitive toward chloramphenicol and tetracycline, respectively. Detection of in vitro production of biofilm revealed the association of biofilm with methicillin as well as inducible clindamycin resistance among the clinical S. aureus isolates.

ATMT transformation efficiencies with native promoters in Botryosphaeria kuwatsukai causing ring rot disease in pear.

Botryosphaeria kuwatsukai is an important fungal pathogen affecting pear fruits. However, infection processes of this fungus are still unclear. This study seeks to develop the fungal transformation of B. kuwatsukai by Agrobacterium tumefaciens-mediated transformation (ATMT), assess the reliability of appropriate vectors and examine the infection processes in vitro using a GFP labeled strain of B. kuwatsukai. To establish a highly effective transformation system in B. kuwatsukai, binary vectors containing various lengths of H3 promoters and TEF promoters fused with GFP and hygromycin B resistance gene cassettes were constructed. These cassettes were integrated into the genomic DNA of B. kuwatsukai with high transformation frequency by the ATMT method. Transformants showed strong expression of GFP and hygromycin B resistance genes in cells. Furthermore, we investigated if native promoters are more suitable to govern marker genes than other general promoters used in other filamentous fungi. The results obtained herein demonstrate that the vectors constructed in this study can be utilized with high transformation rate. Microscopic examinations also reveal that fungal hyphae undergo morphological changes during the infection process resulting in biotrophic stage of infected host cells. Our results provide genetic insights to further explore the infection processes of B. kuwatsukai.

Evaluation of methods to detect in vitro biofilm formation by staphylococcal clinical isolates.

OBJECTIVE: Staphylococcus genus comprising both Staphylococcus aureus and coagulase negative staphylococci (CoNS) are widely distributed in nature and can infect diversity of hosts. Indeed, staphylococci are the major pathogens causing biofilm associated infections caused by contaminated hospital indwelling devices. These infections are persistent in nature being highly refractory to various stresses including antibiotics. Implementation of efficient diagnostic techniques for the biofilm production would help minimize the disease burden. Thus, early detection of pathogenic strains producing biofilms warrant the utmost importance in diagnostic laboratories especially in resource limited settings. RESULT: Among 375 isolates collected from different clinical specimens, 214 (57%) were identified as coagulase negative staphylococci and 161 (43%) S. aureus. Detection of In-vitro biofilm formation in these isolates were carried out by three commonly used phenotypic assays and a genotypic assay. While evaluating the results, tissue-culture method with supplemented glucose and sucrose showed the best correlation with the results of genotypic assay.

Proteomic approach to understand the molecular physiology of symbiotic interaction between Piriformospora indica and Brassica napus.

Many studies have been now focused on the promising approach of fungal endophytes to protect the plant from nutrient deficiency and environmental stresses along with better development and productivity. Quantitative and qualitative protein characteristics are regulated at genomic, transcriptomic, and posttranscriptional levels. Here, we used integrated in-depth proteome analyses to characterize the relationship between endophyte Piriformospora indica and Brassica napus plant highlighting its potential involvement in symbiosis and overall growth and development of the plant. An LC-MS/MS based label-free quantitative technique was used to evaluate the differential proteomics under P. indica treatment vs. control plants. In this study, 8,123 proteins were assessed, of which 46 showed significant abundance (34 downregulated and 12 upregulated) under high confidence conditions (p-value ≤ 0.05, fold change ≥2, confidence level 95%). Mapping of identified differentially expressed proteins with bioinformatics tools such as GO and KEGG pathway analysis showed significant enrichment of gene sets involves in metabolic processes, symbiotic signaling, stress/defense responses, energy production, nutrient acquisition, biosynthesis of essential metabolites. These proteins are responsible for root's architectural modification, cell remodeling, and cellular homeostasis during the symbiotic growth phase of plant's life. We tried to enhance our knowledge that how the biological pathways modulate during symbiosis?

Prevalence and characterization of Panton-Valentine leukocidin-positive Staphylococcus aureus in bovine milk in Jabalpur district of Madhya Pradesh, India.

AIM: The study aimed to investigate the Panton-Valentine leukocidin (PVL)-positive Staphylococcus aureus in bovine milk due to its public health significance. MATERIALS AND METHODS: A total of 400 milk samples of bovines taken from different dairy farms and outlets of Jabalpur were screened for the S. aureus and methicillin-resistant S. aureus (MRSA). The strains were tested for the PVL gene and antimicrobial sensitivity toward 10 different classes of antimicrobial agents. The PVL-positive S. aureus strains were further characterized by staphylococcal protein A or spa typing. RESULT: The prevalence of PVL-positive S. aureus was 10.53%. All the isolates positive for the PVL were resistant to methicillin, while the methicillin-sensitive S. aureus isolates were negative for the PVL. Five different spa types were found. CONCLUSION: The presence of PVL-positive MRSA in bovine milk close to consumer poses a potential public health risk to the community.

Piriformospora indica promotes growth, seed yield and quality of Brassica napus L.

In current scenario, crop productivity is being challenged by decreasing soil fertility. To cope up with this problem, different beneficial microbes are explored to increase the crop productivity with value additions. In this study, Brassica napus L., an important agricultural economic oilseed crop with rich source of nutritive qualities, was interacted with Piriformospora indica, a unique root colonizing fungus with wide host range and multifunctional aspects. The fungus-treated plants showed a significant increase in agronomic parameters with plant biomass, lodging-resistance, early bolting and flowering, oil yield and quality. Nutritional analysis revealed that plants treated by P. indica had reduced erucic acid and glucosinolates contents, and increased the accumulation of N, Ca, Mg, P, K, S, B, Fe and Zn elements. Low erucic acid and glucosinolates contents are important parameters for high quality oil, because oils high in erucic acid and glucosinolates are considered undesirable for human nutrition. Furthermore, the expression profiles of two encoding enzyme genes, Bn-FAE1 and BnECR, which are responsible for regulating erucic acid biosynthesis, were down-regulated at mid- and late- life stages during seeds development in colonized plants. These results demonstrated that P. indica played an important role in enhancing plant growth, rapeseed yield and quality improvement of B. napus.