Management of Black Root Disease-Causing Fungus Fusarium solani CRP1 by Endophytic Bacillus siamensis CNE6 through Its Metabolites and Activation of Plant Defense Genes.

Black root rot disease of Cicer arietinum L. is accountable for substantial loss in chickpea production worldwide. Endophytic Bacillus siamensis CNE6 has previously shown multifaceted plant growth-promoting, broad-spectrum antifungal, and chickpea plant-colonizing potential. In the present study, the strain Bacillus siamensis CNE6 was used for controlling black root rot disease caused by Fusarium solani CRP1 in chickpea. CNE6 showed strong antagonistic potential against CRP1 both in vivo and in vitro. Scanning electron microscopic studies indicated cellular deformation of CRP1 due to production of ß-glucanase, protease, and other secondary metabolites. A total of five compounds were detected from the cell-free supernatant (CFS) of the ethyl acetate (EA) fraction of CNE6 through gas chromatography-mass spectrometry analysis. A confocal microscopic study demonstrated strong inhibition of biofilm formation of the pathogen CRP1 by the EA fraction of CFS of CNE6. Molecular docking analysis revealed that one compound, (2E)-6-methoxy-2-[(4-methoxyphenyl)methylidene]-2,3-dihydro-1-benzofuran-3-one, may inhibit the activity of lanosterol 14-alpha demethylase, which is involved in ergosterol biosynthesis and beta-tubulin assembling. In vivo experiments also showed the efficacy of CNE6 for increasing chickpea growth as well as upregulation of four defense genes (CHI1, PAMP, PR2B, and TF1082) upon pathogenic challenge. Thus, our results strongly suggest a positive role for CNE6 as a prospective biocontrol agent for combating Fusarium solani in chickpea. IMPORTANCE The present work was undertaken to explore an effective biocontrol agent against the destructive black root rot disease of chickpea. We have used an efficient bacterial endophyte, CNE6, which can colonize in the chickpea root system, produce secondary metabolites and enzymes to degrade pathogenic cellular integrity, inhibit pathogenic establishment by rupturing biofilm formation, and induce host immunity upon treatment. Interaction of the bacterial metabolite was also observed with lanosterol 14-alpha demethylase, which is an important component in fungal membrane functioning. Being an endophyte, Bacillus siamensis CNE6 fulfills a suitable criterion as a biocontrol agent to control black root rot disease in chickpea and has huge prospects for use commercially.

Pathway to achieve a sustainable food and land-use transition in India.

India has committed to reducing the emissions intensity of GDP by 33-35% from the 2005 level by 2030 in alignment with objectives of the Paris Agreement. This will require a significant reduction in greenhouse gas (GHG) emissions from the food and land-use sector. In this paper, we construct three potential pathways for India to achieve its emissions target by 2050 involving moderate ambitions of mitigation action (BAU), moderate ambitions combined with achieving healthy diets (BAU + NIN), and high levels of mitigation action inclusive of healthy diets (SUSTAINABLE). Using an integrated accounting tool, the FABLE Calculator, that harmonizes various socioeconomic and biophysical data, we project these pathways under the conditions of cross-country balanced trade flows. Results from the projections show that the demand for cereals will increase by 2050, leading to increased GHG emissions under BAU. Under the SUSTAINABLE pathways, GHG emissions will decrease over the same period due to reduced demand for cereals, whereas significant crop productivity and harvest intensity gains would lead to increased crop production. The exercise reveals the indispensability of healthy diets, improved crop, and livestock productivity, and net-zero deforestation in achieving India's mid-century emission targets from the agriculture sector. Supplementary Information: The online version contains supplementary material available at 10.1007/s11625-022-01193-0.

Bacillus siamensis CNE6- a multifaceted plant growth promoting endophyte of Cicer arietinum L. having broad spectrum antifungal activities and host colonizing potential.

Exploration of endophytic bacteria with multiple plant growth promoting (PGP) attributes is considered as an eco-friendly and cost-effective alternative to agricultural chemicals for increasing crop productivity. In the present endeavor, healthy chickpea plants (Cicer arietinum L.) collected from district Birbhum, West Bengal, India were subjected for the isolation of endophytic bacteria having multifarious PGP properties. One potent endophytic Gram positive bacterial strain CNE6 was isolated from the nodule of chickpea and was identified as Bacillus siamensis based on 16S rDNA sequence homologies. The isolate showed a number of PGP properties like phosphate solubilization, IAA production, nitrogen fixation, hydroxamate type of siderophore production and ACC deaminase activities. The isolate CNE6 produced 33.27 ± 2.16 µg/mL of IAA in the presence of tryptophan. Production of IAA was also confirmed by HPLC analysis and it was found effective for inducing lateral root branching in chickpea. In addition, the isolate displayed significant antagonistic activity against a number of plant pathogenic fungi when tested by dual culture overlay and agar well diffusion assay. 50 % cell free supernatant of CNE6 was found effective for 60-80 % inhibition of radial growth of pathogenic fungi tested. Scanning electron microscopic observation revealed massive degradation of pathogenic fungal mycelia by the antifungal metabolites of CNE6. LC-MS analysis of bacterial lipopeptides suggested the production of antifungal antibiotics like surfactin, fengycin and iturin by the isolate. The presence of genes encoding antifungal lipopeptides was also confirmed by PCR amplification using specific primers. Green fluorescent protein (GFP) tagging of CNE6 using broad host range plasmid vector (pDSK-GFPuv) followed by colonization study indicated very good host colonization potential of the isolate and its probable movement through xylem vessels. Enhanced shoot and root length and chlorophyll content upon treatment with CNE6 as observed in in vivo pot experiments also supported the positive role of the endophytic isolate on overall development and growth of the chickpea plants. This is the first report of Bacillus siamensis as an endophyte of Cicer arietinum L. which can be successfully applied for improving the productivity of this crop plant.

Demand for Crop Insurance in Developing Countries: New Evidence from India.

Determining farmers' real demand for crop insurance is difficult, especially in developing countries, where there is a lack of formal financial sector integration and a high reliance on informal risk mitigation options. We provide some new estimates of farmers' willingness-to-pay for insurance in the context of a large-scale subsidised programme in India. We conducted a discrete choice experiment with agricultural households across four states in India, enabling us to estimate preferences for specific insurance policy attributes such as coverage period, method of loss assessment, timing of indemnity payments and the cost of insurance. Our results suggest that farmers do value crop insurance under certain conditions and some are willing to pay a premium for such coverage in excess of the subsidised rates they are currently required to pay under this programme. In particular, farmers value the assurances that they will receive timely payouts when they incur losses, and may not have a strong preference for the method with which losses are assessed. On the other hand, farmers are quite sensitive to coverage periods. Our baseline assessment shows that when optimised to farmer requirements, there can be a sizeable demand for crop insurance by developing country farmers.

Inhibition of biofilm- and hyphal- development, two virulent features of Candida albicans by secondary metabolites of an endophytic fungus Alternaria tenuissima having broad spectrum antifungal potential.

Fungal resistance against frequently used antifungal medicines used for invasive candidiasis and other fungal infections is directing scientist for searching and developing novel antifungal drugs. An endophytic fungal strain Alternaria tenuissima OE7 has been isolated from leaves of Ocimum tenuiflorum L. which showed antifungal activity against numbers of human pathogenic fungi including Trichophyton rubrum, Microsporum gypseum, Aspergillus parasiticus, A. flavus, A. fumigates, Candida albicans and C. tropicalis. Thermostable, non-proteinacious antifungal metabolites produced zones of inhibition against all pathogenic fungi tested. The ethyl acetate extract of the cell free supernatant was found inhibitory to the radial growth and conidial germination of T. rubrum and M. gypseum. It also showed cidal mode of action against C. albicans at a concentration of 1.0 mg/ml. Most interestingly, inhibition of biofilm formation and hyphal development of C. albicans were observed upon treatment with EA fraction at comparatively lower concentrations (100-500 µg/ml). Release of intracellular contents from treated cells of Candida and scanning electron microscopic observation suggested cellular disruptions by antifungal metabolites. Checkerboard study revealed synergy between EA fraction of OE7 (150 µg/ml) and fluconazole (30 µg/ml) with Æ©FIC of 0.45. Two active fractions viz. band 'C' and band 'G' derived after thin layer chromatographic analysis showed inhibitory activity against C. albicans with MIC values of 80 µg/ml and 130 µg/ml respectively. GCMS analysis suggested presence of numbers of compounds in each active fraction. Overall observations attest the prospective role of the isolate OE7 as a potent candidate for the production of antifungal metabolites against human pathogenic fungi.

Phosphate deficiency induced biofilm formation of Burkholderia on insoluble phosphate granules plays a pivotal role for maximum release of soluble phosphate.

Involvement of biofilm formation process during phosphate (P) solubilization by rhizobacterial strains is not clearly understood. Scanning electron microscopic observations revealed prominent biofilm development on tricalcium phosphate as well as on four different rock phosphate granules by two P solubilizing rhizobacteria viz. Burkholderia tropica P4 and B. unamae P9. Variation in the biofilm developments were also observed depending on the total P content of insoluble P used. Biofilm quantification suggested a strong correlation between the amounts of available P and degrees of biofilm formation. Lower concentrations of soluble P directed both the organisms towards compact biofilm development with maximum substratum coverage. Variation in the production of extracellular polymeric substances (EPS) in the similar pattern also suggested its close relationship with biofilm formation by the isolates. Presence of BraI/R quorum sensing (QS) system in both the organisms were detected by PCR amplification and sequencing of two QS associated genes viz. braR and rsaL, which are probably responsible for biofilm formation during P solubilization process. Overall observations help to hypothesize for the first time that, biofilm on insoluble P granules creates a close environment for better functioning of organic acids secreted by Burkholderia strains for maximum P solubilization during P deficient conditions.

Production of bioactive compounds with bactericidal and antioxidant potential by endophytic fungus Alternaria alternata AE1 isolated from Azadirachta indica A. Juss.

For combating multidrug-resistant microorganisms, exploration of natural compounds from plant endophytes increases the chance of finding novel compounds. An efficient bioactive metabolites producing endophytic fungal strain AE1 was isolated from leaves of Azadirachta indica A. Juss. The metabolites were found to be thermostable, non-proteinacious and produced prominent zones of inhibition against numbers of Gram positive and Gram negative bacteria. Based on 28S rDNA (D1/D2) sequence homology the isolate AE1 was identified as Alternaria alternata. Malt extract broth was found effective for the maximum production of bioactive metabolites by the isolate and was subjected for solvent extraction. The Ethyl acetate (EA) fraction of AE1 showed MIC values of 300-400 µg/ml against Gram positive and Gram negative bacteria tested. The cidal mode of action of EA fraction was detected by treating bacterial cultures at mid log phase. Scanning electron microscopic study supported morphological disintegration of bacterial cells. Release of nucleic acid, protein and potassium ions (K+) also suggested lysis of bacterial cells or leakage of cell membrane upon treatment. In addition, reduction of the activity of EMP pathway, TCA cycle and gluconeogenic enzymes in all bacteria suggested the interference of antibacterial principles with central carbohydrate metabolic pathways. Thin layer chromatographic separation followed by GC-MS analysis of EA fraction suggested numbers of antimicrobial compound production by AE1. In addition, DPPH free radical as well as superoxide radical scavenging assay also suggested strong antioxidant potential of AE1 with an IC50 value of 38.0±1.7 µg/ml and 11.38±1.2 µg/ml respectively. On the basis of above facts it can be concluded that the strain AE1 will be a good source of bioactive compounds having medicinal importance.

Longterm storage of post-packaged bread by controlling spoilage pathogens using Lactobacillus fermentum C14 isolated from homemade curd.

One potent lactic acid bacterial strain C14 with strong antifungal activity was isolated from homemade curd. Based on morphological as well as biochemical characters and 16S rDNA sequence homology the strain was identified as Lactobacillus fermentum. It displayed a wide antimicrobial spectrum against both Gram-positive and Gram-negative pathogenic bacteria, and also against number of food spoilage, plant and human pathogenic fungi. The cell free supernatant (CFS) of the strain C14 was also effective against the fungi tested. Inhibition of radial growth of Penicillium digitatum, Trichophyton rubrum and Mucor sp. was noticed in the presence of CFS of C14 even at low concentration (1%). More than 94.3 ± 1.6% and 91.5 ± 2.2% inhibition of conidial germination of P. digitatum and Mucor sp. were noticed in the presence of 10-fold-concentrated CFS of C14. Massive deformation of the fungal mycelia was observed by SEM studies, and losses of cellular proteins and DNA are also evident upon its treatment with C14. HPLC analysis revealed the presence of phenyl lactic acid, lactic acid along with some unidentified compounds in the antifungal extract. Challenge experiment showed immense potential of the strain C14 in preventing the spoilage of bread samples caused by Mucor sp. and Bacillus subtilis. The bread samples remained fresh upto 25 days even after inoculation with Mucor sp. (3.7 × 104 spores /ml) and B. subtilis (4.6 × 104 CFU /ml). Along with the antifungal properties, the isolated lactic acid bacterial strain also showed very good antioxidant activities. Unchanged level of liver enzymes serum glutamic pyruvic transaminase and serum glutamic oxaloacetic transaminase in albino mice upon feeding with C14 also suggested non-toxic nature of the bacterial isolate.

Role of phosphate solubilizing Burkholderia spp. for successful colonization and growth promotion of Lycopodium cernuum L. (Lycopodiaceae) in lateritic belt of Birbhum district of West Bengal, India.

Profuse growth of Lycpodium cernuum L. was found in phosphate deficient red lateritic soil of West Bengal, India. Interaction of vesicular-arbuscular mycorrhiza (VAM) with Lycopodium rhizoids were described earlier but association of PGPR with their rhizoids were not studied. Three potent phosphate solubilizing bacterial strains (P4, P9 and P10) associated with L. cernuum rhizoids were isolated and identified by 16S rDNA homologies on Ez-Taxon database as Burkholderia tropica, Burkholderia unamae and Burkholderia cepacia respectively. Day wise kinetics of phosphate solubilization against Ca3(PO4)2 suggested P4 (580.56±13.38 µg ml(-1)) as maximum mineral phosphate solubilizer followed by P9 (517.12±17.15 µg ml(-1)) and P10 (485.18±14.23 µg ml(-1)) at 28 °C. Release of bound phosphates by isolated strains from ferric phosphate (FePO4), aluminum phosphate (AlPO4) and four different complex rock phosphates indicated their very good phosphate solubilizng efficacy. Nitrogen independent solubilizition also supports their nitrogen fixing capabilities. Inhibition of P solubilization by calcium salts and induction by EDTA suggested pH dependent chelation of metal cations by all of the isolates. Rhizoidal colonization potentials of Burkholderia spp. were confirmed by in planta experiment and also using scanning electron microscope (SEM). Increases of total phosphate content in Lycopodium plants upon soil treatment with these isolates were also recorded. In addition siderophore production on CAS agar medium, tryptophan dependent IAA production and antifungal activities against pathogenic fungi by rhizospheric isolates deep-rooted that they have definite role in nutrient mobilization for successful colonization of L. cernuum in nutrient deficient lateritic soil.

Sunlight-induced rapid and efficient biogenic synthesis of silver nanoparticles using aqueous leaf extract of Ocimum sanctum Linn. with enhanced antibacterial activity.

BACKGROUND: Nanotechnology is now regarded as a distinct field of research in modern science and technology with multifaceted areas including biomedical applications. Among the various approaches currently available for the generation of metallic nanoparticles, biogenic synthesis is of increasing demand for the purpose of green nanotechnology. Among various natural sources, plant materials are the most readily available template-directing matrix offering cost-effectiveness, eco-friendliness, and easy handling. Moreover, the inherent pharmacological potentials of these medicinal plant extracts offer added biomedical implementations of the synthesized metal nanoparticles. RESULTS: A robust practical method for eco-friendly synthesis of silver nanoparticles using aqueous leaf extract of Ocimum sanctum (Tulsi) as both reducing and capping agent, under the influence of direct sunlight has been developed without applying any other chemical additives. The nanoparticles were characterized with the help of UV-visible spectrophotometer and transmission electron microscopy (TEM). The prepared silver nanoparticles exhibited considerable antibacterial activity. The effects were more pronounced on non-endospore-forming Gram-positive bacteria viz., Staphylococcus aureus, Staphylococcus epidermidis, and Listeria monocytogenes than endospore-forming species Bacillus subtilis. The nanoparticles also showed prominent activity on Gram-negative human pathogenic Salmonella typhimurium, Escherichia coli, Pseudomonas aeruginosa, and plant pathogenic Pantoea ananatis. A bactericidal mode of action was observed for both Gram-positive and Gram-negative bacteria by the nanoparticles. CONCLUSIONS: We have developed a very simple, efficient, and practical method for the synthesis of silver nanoparticles using aqueous leaf extract of O. sanctum under the influence of direct sunlight. The biosynthesis of silver nanoparticles making use of such a traditionally important medicinal plant without applying any other chemical additives, thus offers a cost-effective and environmentally benign route for their large-scale commercial production. The nanoparticles dispersed in the mother solution showed promising antibacterial efficacy. Graphical AbstractSunlight-induced rapid and efficient biogenic synthesis of silver nanoparticles using aqueous leaf extract of Ocimum sanctum Linn. with enhanced antibacterial activity.

Phosphate solubilizing ability of Emericella nidulans strain V1 isolated from vermicompost.

Phosphorus is one of the key factors that regulate soil fertility. Its deficiencies in soil are largely replenished by chemical fertilizers. The present study was aimed to isolate efficient phosphate solubilizing fungal strains from Eisenia fetida vermicompost. Out of total 30 fungal strains the most efficient phosphate solubilizing one was Emericella (Aspergillus) nidulans V1 (MTCC 11044), identified by custom sequencing of beta-tubulin gene and BLAST analysis. This strain solubilized 13 to 36% phosphate from four different rock phosphates. After three days of incubation of isolated culture with black Mussorie phosphate rock, the highest percentage of phosphate solubilization was 35.5 +/- 1.01 with a pH drop of 4.2 +/- 0.09. Kinetics of solubilization and acid production showed a linear relationship until day five of incubation. Interestingly, from zero to tenth day of incubation, solubility of soil phosphate increased gradually from 4.31 +/- 1.57 to 13.65 +/- 1.82 (mg kg(-1)) recording a maximum of 21.23 +/- 0.54 on day 45 in respect of the V1 isolate. Further, enhanced phosphorus uptake by Phaseolus plants with significant pod yield due to soil inoculation of Emericella nidulans V1 (MTCC 11044), demonstrated its prospect as an effective biofertilizer for plant growth.

A new pentacyclic triterpene with potent antibacterial activity from Limnophila indica Linn. (Druce).

A new pentacyclic triterpenoid constituent, characterized as 3-oxo-olean-12(13),18(19)-dien-29α-carboxylic acid (1) on the basis of detailed spectral studies, was isolated from the aerial parts and roots of Limnophila indica (Scrophulariaceae). Compound 1 exhibited considerable antibacterial activity against three Gram-positive bacteria viz. Bacillus subtilis, Staphylococcus aureus and Listeria monocytogenes (MICs within a range of 25-30 µg/ml) and moderate activity against four Gram-negative bacteria Salmonella typhimurium, Escherichia coli, Pseudomonas aeruginosa, and Pantoea ananatis (MICs within a range of 30-100 µg/ml). The plant pathogenic bacterium P. ananatis and human pathogenic S. typhimurium responded at comparatively higher concentrations of the compound 1, which were 75 and 100 µg/ml respectively. The compound inhibited the growth of Gram-positive B. subtilis and Gram-negative P. aeruginosa completely with a clear bactericidal mode of action at their MIC values. The compound upon treatment on both B. subtilis and P. aeruginosa released substantial amount of nucleic acid in the external medium and also effected the change of morphology towards pleomorphicity, thereby indicating its probable action on cell membrane. Furthermore, the triterpenoid 1 was found not to inhibit a probiotic lactic acid bacterium Lactococcus lactis subsp. lactis LABW4 under in vitro condition and to possess no toxicity in Swiss albino mice.

A comparative antibacterial evaluation of raw and processed Guñja (Abrus precatorius Linn.) seeds.

BACKGROUND: Seed of Guñja (Abrus precatorius Linn.), a known poisonous drug, is used extensively in various ayurvedic formulations with great therapeutic significance. Ayurveda recommends the administration of Guñja in diseases like Indralupta (alopecia), Sotha (edema), Krmi (helminthes), Kustha (skin diseases), Kandu (itching), Prameha (urinary disorders) etc., after being treated with specific Sodhana (purification) procedures. OBJECTIVE: To assess the antimicrobial action of of raw and Shodhita (Processed) Guñja seeds. METHODS: Guñja seeds after being processed with Godugdha (cow's milk), Nimbu swarasa (Lemon juice), Kañji (Sour gruel) and water, as the media, were evaluated for its antibacterial effect against clinically important bacterial strains using agar well diffusion method. RESULTS: Aqueous extracts of raw seeds of Guñja exert its antibacterial effect on both Gram positive, as well as Gram negative bacteria but none of the Sodhita Guñja seeds showed any bactericidal effect on any bacterial strains. Chloroform extracts of all the Sodhita Guñja seed extracts could inhibit bacterial growth but with variations. CONCLUSION: The study displayed that chloroform extracts of raw and sodhita samples for bacterial study were much sensitive than the aqueous extracts.

Correlation of trace elemental profiles in blood samples of Indian patients with leukoplakia and oral submucous fibrosis.

In order to ascertain possible correlation between alterations in trace elemental profile and the progression or regression of two most common potentially malignant disorders affecting oral cavity, namely oral submucous fibrosis and leukoplakia, blood from 60 patients from each group of patients as well from 30 healthy individuals was analyzed for elemental profiling employing EDXRF technique. Out of the 16 detected elements (K, Si, Ca, V, Cr, Ni, Mn, Fe, Cu, Zn, Se, Br, Rb, Sr, Co, and Pb), Mn, Fe, Zn, Br, and Co showed remarkable alteration in their profile in both leukoplakia and oral submucous fibrosis patients with respect to the normal healthy individuals. While Zn, Br, and Fe reflected similar changes--showing gross depletion in both the diseased groups, Mn and Co depicted inverse pattern of alterations in their concentrations in the two types of precancerous disorders when compared to the control subjects.

Dyskeratosis congenita with malignant transformation.

Dyskeratosis congenita (DC) is a rare genodermatosis characterised by a classic triad of dystrophic nails, reticular skin pigmentation and mucous membrane leukoplakic patches, which have a high rate of malignant transformation. The case report presented here deals with a sporadic case of DC without similar clinical presentation in the first-degree and second-degree relatives. Of note in this case, there was rapid malignant transformation in the non-homogeneous nodulo-speckled leukoplakic patch on the dorsum of the tongue.