Folia microbiologica最新文献

The largest obstacle in the promotion of biopesticides is the existence of counterfeit products available in the market. Identification and quantification of antagonistic organisms in biopesticide products are the key to the reduction of spurious microbial pesticides. In this study, we have developed a simple, sensitive, isothermal-based colourimetric assay for specific detection of Bacillus subtilis from the biopesticide formulations and soil samples. A region specific to B. subtilis which codes for shikimate dehydrogenase was identified through in silico analysis. We employed conventional PCR, loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and qPCR for specific detection of B. subtilis in soil samples and biopesticide formulations. Specificity tests showed that the PCR primers amplified an amplicon of 521 bp in four strains of B. subtilis only, and no amplification was found in negative control samples. Similarly, the LAMP assay showed sky blue colour in all four strains of B. subtilis and violet colour in negative control samples. Whereas in the RPA assay, upon the addition of SYBR Green dye, a bright green colour was seen in B. subtilis strains, while a brick-red colour was observed in negative control samples by visualizing under a UV transilluminator. The qPCR assay showed specific amplifications with a Ct value of 12 for B. subtilis strains and no amplification in negative control samples. In the sensitivity test, PCR could amplify DNA of B. subtilis up to 500 pg/µL. DNA concentration as low as 10 pg/µL was enough to show the colour change in the LAMP as well as the RPA assays, whereas the qPCR assay showed sensitivity till 100 pg/µL. All four diagnostic assays developed in the study have been validated in soil samples and B. subtilis-based biopesticides. Compared to conventional PCR, the qPCR assay has the advantage of quantification and visualizing the result in real-time, whereas LAMP and RPA assays have the benefits of being colourimetric and less time-consuming. The other advantages are that the results can be visualized with the naked eye, and these assays do not require a costly thermal cycler and gel documentation system. Hence, LAMP and RPA assays are highly suitable for developing point-of-need diagnostic kits and, in turn, help regulators assess the quality of biopesticides in the market.

Feruloyl esterases (FAEs) are a crucial component of the hemicellulose-degrading enzyme family that facilitates the degradation of lignocellulose while releasing hydroxycinnamic acids such as ferulic acid with high added value. Currently, the low enzyme yield of FAEs is one of the primary factors limiting its application. Therefore, in this paper, we optimized the fermentation conditions for the expression of FAE BpFae^{T132C−D143C} with excellent thermal stability in Escherichia coli by experimental design. Firstly, we explored the effects of 11 factors such as medium type, isopropyl-β-d-thiogalactopyranoside (IPTG) concentration, and inoculum size on BpFae^{T132C−D143C} activity separately by the single factor design. Then, the significance of the effects of seven factors, such as post-induction temperature, shaker rotational speed, and inoculum size on BpFae^{T132C−D143C} activity, was analyzed by Plackett–Burman design. We identified the main factors affecting the fermentation conditions of E. coli expressing BpFae^{T132C−D143C} as post-induction temperature, pre-induction period, and post-induction period. Finally, we used the steepest ascent path design and response surface method to optimize the levels of these three factors further. Under the optimal conditions, the activity of BpFae^{T132C−D143C} was 3.58 U/ml, which was a significant 6.6-fold increase compared to the pre-optimization (0.47 U/ml), demonstrating the effectiveness of this optimization process. Moreover, BpFae^{T132C−D143C} activity was 1.52 U/ml in a 3-l fermenter under the abovementioned optimal conditions. It was determined that the expression of BpFae^{T132C−D143C} in E. coli was predominantly intracellular in the cytoplasm. This study lays the foundation for further research on BpFae^{T132C−D143C} in degrading agricultural waste transformation applications.

Biopriming seeds with beneficial bacteria has potential to enhance seed germination. Therefore, in this investigation, five sulphur-oxidizing bacterial cultures, viz., Pantoea dispersa SOB2, Bacillus velezensis SN06, Bacillus cereus SN20, Bacillus tropicus SN16, and Bacillus megaterium SN11, were evaluated for different plant growth-promoting traits and their impact on Vigna radiata L. (mung bean) and Brassica juncea L. (mustard) seed germination. Among these, three bacterial cultures Pantoea dispersa SOB2, Bacillus velezensis SN06, and Bacillus megaterium SN11 evinced potential for mineral solubilization on solid medium where Pantoea dispersa SOB2 had the maximum solubilization indices-3.06, 5.14, and 2.48 for phosphate, zinc, and potassium respectively. The culture also displayed higher indole acetic acid (113.12 µg/mL), gibberellic acid (162.66 µg/mL), ammonia (5.23 µg/mL), and siderophore (69.53%) production than other bacterial cultures whereas Bacillus cereus SN20 showed maximum exopolysaccharide production (9.26 g/L). Bacterial culture Pantoea dispersa SOB2 significantly ameliorated the germination rate (3.73 no./day) and relative seed germination (208%) of Brassica juncea L., while Bacillus velezensis SN06 and Bacillus cereus SN20 followed with germination rate and relative seed germination of 2.86 no./day and 207%, respectively. Pantoea dispersa SOB2 displayed lowest mean germination time 2.91 days followed by Bacillus megaterium SN11 with 3.19 days. Biopriming with sulphur-oxidizing bacterial cultures, germination parameters of Vigna radiata L. were also markedly improved. Pantoea dispersa SOB2 demonstrated the highest germination rate (6.72 no./day), relative seed germination (115.56%), and minimum mean generation time (1.73 days). Bacillus velezensis SN06 inoculation had a beneficial effect on the seedling growth of Vigna radiata L., whereas Pantoea dispersa SOB2 greatly aided the seedling growth of Brassica juncea L. Results corroborated a prominent positive correlation between seed germination and plant growth-promoting traits. This is the first study on Pantoea dispersa as sulphur oxidizer, displaying plant growth promoting traits and seed germination potential. The potent sulphur-oxidizing bacterial cultures possessing plant growth promoting activities enhanced seed germination under in vitro conditions that could be further explored in field as biofertilizers to enhance the growth and yield of Brassica juncea L. and Vigna radiata L. crop.

Present study was aimed to develop an efficient microbial consortium for combating Alternaria blight disease in cumin. The research involved isolating biocontrol agents against Alternaria burnsii, characterizing their biocontrol and growth promotion traits, and assessing compatibility. A pot experiment was conducted during rabi season of 2022-2023 to evaluate the bioefficacy of four biocontrol agents (1F, 16B, 31B, and 223B) individually and in consortium, focusing on disease severity, plant growth promotion, and defense responses in cumin challenged with A. burnsii. Microbial isolates 1F, 16B, 31B, and 223B significantly inhibited A. burnsii growth in dual plate assays (~ 86%), displaying promising biocontrol and plant growth promotion activities. They were identified as Trichoderma afroharzianum 1F, Aneurinibacillus aneurinilyticus 16B, Pseudomonas lalkuanensis 31B, and Bacillus licheniformis 223B, respectively. The excellent compatibility was observed among all selected biocontrol agents. Cumin plants treated with consortia of 1F + 16B + 31B + 223B showed least percent disease index (32.47%) and highest percent disease control (64.87%). Consortia of biocontrol agents significantly enhanced production of secondary metabolites (total phenol, flavonoids, antioxidant, and tannin) and activation of antioxidant-defense enzymes (POX, PPOX, CAT, SOD, PAL, and TAL) compared to individual biocontrol treatment and infected control. Moreover, consortium treatments effectively reduced electrolyte leakage over the individual biocontrol agent and infected control treatment. The four-microbe consortium significantly enhanced chlorophyll (154%), carotenoid content (88%), plant height (78.77%), dry weight (72.81%), and seed yield (104%) compared to infected control. Based on these findings, this environmentally friendly four-microbe consortium may be recommended for managing Alternaria blight in cumin.

In this study, lactic acid bacteria (LAB) isolation from fermented foods and molecular identification using magnetic bead technology were performed. And then exopolysaccharide (EPS) production possibility was tested in agar medium, and the positive ones were selected for the next step. The bacteria that could produce higher carbohydrate level were grown in MRS medium fortified with whey and pumpkin waste. In our study, 19 different LAB species were identified from fermented products collected from different places in Hatay (Türkiye) province. In molecular identification, universal primer pairs, p806R/p8FPL, and PEU7/DG74 were used for PCR amplification. After that, PCR products purified using paramagnetic bead technology were sequenced by the Sanger sequencing method. The dominant species, 23.8% of the isolates, were identified as Lactiplantibacillus plantarum. As a technological property of LAB, exopolysaccharide production capability of forty-two LAB isolate was tested in agar medium, and after eleven isolates were selected as positive. Two LAB (Latilactobacillus curvatus SHA2-3B and Loigolactobacillus coryniformis SHA6-3B) had higher EPS production capability when they were grown in MRS broth fortified with pumpkin waste and whey. The highest EPS content (1750 mg/L glucose equivalent) was determined in Loigolactobacillus coryniformis SHA6-3B grown in MRS broth fortified with 10% pumpkin waste. Besides the produced EPS samples were validated with FTIR and SEM methods.

L eishmaniasis is a prevalent disease that impacts 98 countries and territories, mainly in Africa, Asia, and South America. It can cause substantial illness and death, particularly in its visceral manifestation that can be specifically targeted in the development of medications to combat leishmaniasis. This study has found natural compounds with possible inhibitory activity against APX using a reliable and accurate QSAR model. Despite the severe side effects of current treatments and the absence of an effective vaccination, these compounds show promise as a potential treatment for the disease. Nine hit compounds were found, and subsequent molecular docking was performed. Estradiol cypionate showed the lowest binding energy (- 10.5 kcal/mol), thus showing the strongest binding, and also had the strongest binding affinity, with a ΔG_Total of - 26.31 ± 3.01 kcal/mol, second only to the control molecule. Additionally, three hits viz. cloxacillin-sodium (- 16.57 ± 2.89 kcal/mol), cinchonidine (- 16.04 ± 3.27 kcal/mol), and quinine hydrochloride dihydrate (13.38 ± 1.06 kcal/mol) also showed significant binding affinity. Multiple interactions between drugs and active site residues demonstrated a substantial binding affinity with the target protein. The identified compounds exhibited drug-like effects and were orally bioavailable based on their ADME-toxicology features. Overall, estradiol cypionate, cloxacillin sodium, cinchonidine, and quinine hydrochloride dihydrate all exhibited inhibitory effects on the APX enzyme of Leishmania donovani. These results suggest that further investigation is needed to explore the potential of developing novel anti-leishmaniasis drugs using these compounds.

The emergence of biofilm-induced drug tolerance poses a critical challenge to public healthcare management. Pseudomonas aeruginosa, a gram-negative opportunistic bacterium, is involved in various biofilm-associated infections in human hosts. Towards this direction, in the present study, a combinatorial approach has been explored as it is a demonstrably effective strategy for managing microbial infections. Thus, P. aeruginosa has been treated with cuminaldehyde (a naturally occurring phytochemical) and gentamicin (an aminoglycoside antibiotic) in connection to the effective management of the biofilm challenges. It was also observed that the test molecules could show increased antimicrobial activity against P. aeruginosa. A fractional inhibitory concentration index (FICI) of 0.65 suggested an additive interaction between cuminaldehyde and gentamicin. Besides, a series of experiments such as crystal violet assay, estimation of extracellular polymeric substance (EPS), and microscopic images indicated that an enhanced antibiofilm activity was obtained when the selected compounds were applied together on P. aeruginosa. Furthermore, the combination of the selected compounds was found to reduce the secretion of virulence factors from P. aeruginosa. Taken together, this study suggested that the combinatorial application of cuminaldehyde and gentamicin could be considered an effective approach towards the control of biofilm-linked infections caused by P. aeruginosa.

Three endophytic bacteria, namely BvV, BvP and BvL, were newly isolated from the root nodules of bean, pea and lentil plants respectively cultivated in Mascara the northwest of Algeria, and identified by 16S ribosomal RNA gene sequencing as Brevundimonas naejangsanensis. These strains were able to produce hydrolytic enzymes and hydrogen cyanide. All strains produced a growth-promoting hormone, indole acetic acid, varying in concentration from 83.2 to 171.7 µg/mL. The phosphate solubilizing activity of BvV, BvP and BvL varied from 25.5 to 42.02 µg/mL for tricalcium phosphate. The three antagonistic Brevundimonas spp. showed in vitro the most inhibitory effect on mycelial growth of Fusarium redolens FRC (from 78.33 to 85.55%). Strain BvV, BvP and BvL produced also volatile metabolites which inhibited mycelial FRC growth up to 39.2%. All strains showed significant disease reduction in pot experiments. Chickpea Fusarium yellows severity caused by FRC was reduced significantly from 89.3 to 96.6% in the susceptible cultivar ILC 482 treated with antagonistic B. naejangsanensis. The maximum stimulatory effect on chickpea plants growth was observed by inoculation of strain BvV. This treatment resulted in a 7.40-26.21% increase in shoot height as compared to the control plants. It is concluded that the endophytic bacterial strains of B. naejangsanensis having different plant growth promoting (PGP) activities can be considered as beneficial microbes for sustainable agriculture. To our knowledge, this is the first report to use B. naejangsanensis strains as a new biocontrol agent against F. redolens, a new pathogen of chickpea plants causing Fusarium yellows disease in Algeria.

This study profiled the various endophytic fungi isolated from the orchid Cymbidium sp. and their L-asparaginase production and antioxidant potential. The L-asparaginase production was first screened through qualitative plate screening then quantified by the Nesslerization method. The antioxidant potential was quantified via the 2,2-diphenyl-1-picrylhydrazyl assay. A total of 30 endophytic fungi were isolated and all fungal isolates exhibited various degrees of radical scavenging activities (45.28% to 76.4%). Isolate Lasiodiplodia theobromae (C11) had the highest antioxidant capacity, represented by the lowest IC₅₀ value (5.75 mg/mL) and highest ascorbic acid equivalent antioxidant capacity value (12.17 mg/g). Additionally, 16 isolates produced L-asparaginase (53.33%), which includes primarily species of Fusarium proliferatum, Fusarium fujikuroi, Fusarium incarnatum, and Fusarium oxysporum. A new isolate has also been discovered from Cymbidium orchid, Buergenerula spartinae (C28), which showed the highest L-asparaginase activity (1.736 unit/mL). These findings supported the postulation that medicinal species of Orchidaceae such as Cymbidium sp. harbor endophytes that are producers of L-asparaginase and antioxidants with various potential applications.

Brucellosis is a zoonosis caused by Brucella, which poses a great threat to human health and animal husbandry. Pathogen surveillance is an important measure to prevent brucellosis, but the traditional method is time-consuming and not suitable for field applications. In this study, a recombinase polymerase amplification-SYBR Green I (RPAS) assay was developed for the rapid and visualized detection of Brucella in the field by targeting BCSP31 gene, a conserved marker. The method was highly specific without any cross-reactivity with other common bacteria and its detection limit was 2.14 × 10⁴ CFU/mL or g of Brucella at 40 °C for 20 min. It obviates the need for costly instrumentation and exhibits robustness towards background interference in serum, meat, and milk samples. In summary, the RPAS assay is a rapid, visually intuitive, and user-friendly detection that is highly suitable for use in resource-limited settings. Its simplicity and ease of use enable swift on-site detection of Brucella, thereby facilitating timely implementation of preventive measures.