Production Of Antimicrobial Metabolites Research Articles

Optimization of Culturing Conditions for the Production of Antimicrobial Metabolite by Bacillus Carotarum

Forest soil antagonistic bacteria Bacillus carotarum was isolated and tested for optimum antimicrobial metabolite production. Maximum antimicrobial metabolite production was found at temperature 30°C and pH 7.0 and on Ist day of incubation at stationary condition. The medium consisting of beef extract - 0.3%, peptone - 0.5% and NaCl - 0.05%, was screened out as a suitable medium for maximum antimicrobial production supplemented individually with four carbon sources of which starch was found as the best carbon source. The active agent was best extracted with petroleum ether. Homogeneity of the crude bioactive metabolites were also tested by paper chromatographic analysis where a single type of bioactive metabolite was found with a Rf value of 0.94 in the solvent system n- butanol: acetic acid : water = 4 : 1 : 5 . The antimicrobial spectrum of the metabolite was wide and showed activity against Shigella dysenteriae (AE14612), Salmonella typhi(AE14296), Vibrio cholerae (A214748), Pseudomonas aeruginosa (CRL, ICDDR’ B), Bacillus cereus (BTCC19), and Bacillus subtilis (BTTC17). The Chittagong Univ. J. B. Sci. 8(1&2): 01-10, 2013

Transcriptome analysis reveals the inhibitory mechanism of 3,4-Dimethoxyphenol from Streptomyces albidoflavus strain ML27 against Xanthomonas oryzae pv. oryzae

Bacterial leaf blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), poses a significant threat to rice cultivation across diverse regions. Growing concerns about pesticide resistance and environmental impact underscore the urgent necessity for eco-friendly biopesticides. Here, the complete genome sequence of Streptomyces albidoflavus strain ML27 revealed substantial antimicrobial activity and secondary metabolite production potential through genome mining. 3,4-dimethoxyphenol (purity 97%) was successfully isolated from the fermentation broth of S. albidoflavus strain ML27, exhibiting broad and pronounced inhibitory effects on the growth of seven different fungi and five tested bacteria. The efficacy of 3,4-dimethoxyphenol in controlling rice bacterial leaf blight was evaluated through pot tests, demonstrating substantial therapeutic (69.39%) and protective (84.53%) effects. Application of 3,4-dimethoxyphenol to Xoo resulted in cells displayed notable surface depressions, wrinkles, distortions, or even ruptures compared to their typical morphology. Transcriptome analysis revealed significant inhibition of membrane structures, protein synthesis and secretion, bacterial secretion system, two-component system, flagellar assembly, as well as various metabolic and biosynthetic pathways by 3,4-dimethoxyphenol. Notably, the down-regulation of the type III secretion system (T3SS) expression was a pivotal finding. Furthermore, validation via quantitative real-time polymerase chain reaction (qRT-PCR) analysis confirmed significant downregulation of 10 genes related to T3SS upon 3,4-dimethoxyphenol treatment. Based on these results, it is promising to develop 3,4-dimethoxyphenol as a novel biopesticide targeting the T3SS of Xoo for controlling bacterial leaf blight in rice.

Screening Plant Growth-Promoting Bacteria with Antimicrobial Properties for Upland Rice.

This study explores beneficial bacteria isolated from the roots and rhizosphere soil of Khao Rai Leum Pua Phetchabun rice plants. A total of 315 bacterial isolates (KK001 to KK315) were obtained. Plant growth-promoting traits (phosphate solubilization and indole-3-acetic acid (IAA) production), and antimicrobial activity against three rice pathogens (Curvularia lunata NUF001, Bipolaris oryzae 2464, and Xanthomonas oryzae pv. oryzae) were assessed. KK074 was the most prolific in IAA production, generating 362.6 ± 28.0 μg/ml, and KK007 excelled in tricalcium phosphate solubilization, achieving 714.2 ± 12.1 μg/ml. In antimicrobial assays using the dual culture method, KK024 and KK281 exhibited strong inhibitory activity against C. lunata, and KK269 was particularly effective against B. oryzae. In the evaluation of antimicrobial metabolite production, KK281 and KK288 exhibited strong antifungal activities in cell-free supernatants. Given the superior performance of KK281, taxonomically identified as Bacillus sp. KK281, it was investigated further. Lipopeptide extracts from KK281 had significant antimicrobial activity against C. lunata and a minimum inhibitory concentration (MIC) of 3.1 mg/ml against X. oryzae pv. oryzae. LC-ESI-MS/MS analysis revealed the presence of surfactin in the lipopeptide extract. The crude extract was non-cytotoxic to the L-929 cell line at tested concentrations. In conclusion, the in vitro plant growth-promoting and disease-controlling attributes of Bacillus sp. KK281 make it a strong candidate for field evaluation to boost plant growth and manage disease in upland rice.

Rumen microbes: Exploring its potential for productivity and commercial use

Ruminant animals are known for their dairy and meat products worldwide. They are the best converters of poor quality fibrous feed ingredients, and presence of rumen, the anaerobic chamber that harbours vast category of microbes, is attributable to this phenomenon. The microbes include bacteria, fungi, protozoa, archaea and bacteriophages that work on synergistically for optimal performance of ruminant animals. These microbes help not only in digestion of fibrous materials, but also involved in various biological functions, such as probiotic activity, antimicrobial metabolite production, synthesis of health promoting bioactive fatty acid molecules, biomass conversion, etc. Earlier, the probiotic organisms used in food animals (calves, sheep, goat, swine and poultry) were mainly originated from dairy products but today organisms of autochthonous origin are being used, as they show better adaptability. Since, rumen do possess organisms with probiotic and fibre utilising activity, these organisms are now explored for their suitability as a probiotic and fibrolytic agent in monogastric food animals. Diversity of rumen microbes was not properly understood through the conventional culture methods, however with advancement in ‘Omic’ technologies, researchers could identify new class of organisms from the rumen and their potential use for the commercial and industrial purposes.

Bioactivity of crude extract produced by endophytic fungi isolated from Ziziphus spina-christi (Nabq) leaves for antimicrobial evaluation as well as optimization of culture medium conditions

Twenty fungal endophytes were isolated from Nabq, and plug diffusion screening revealed four of those isolates to be promising. These isolates were identified molecularly as Aspergillus sp. of Sect. Flavi (ZSEFL2) and Sec. Niger (ZSEFL14), and Penicillium spp. closest related to Penicillum crustosum (ZSEFL15, 19). Investigations were conducted to determine the effects of culture parameters, such as the type of culture medium, incubation temperature, initial pH and agitation, on the production of antimicrobial metabolites in a culture filtrate extract. Using the disk diffusion technique, antimicrobial metabolite synthesis was evaluated. In comparison to the other three distinct culture media (PDB-y, rice, and Cz), PDB demonstrated to be the best culture medium for the four tested fungal endophytes, exhibiting high levels of the Antimicrobial Index (AI). Also, a restricted range of incubation temperatures resulted in the formation of bioactive metabolites, but raising the incubation temperatures from 25 to 30 °C increased the amount of bioactive metabolites produced. Moreover, the best initial hydrogen concentration typically ranged from pH 5 to 6. Finally, when the conditions changed from stationary to shaking, the bioactive secondary metabolite's antimicrobial efficacy reduced. The behavior of the four examined fungal isolates was consistent, and they all required the same conditions to produce the most bioactive metabolites. Moreover, ethyl acetate extract's MIC values against the investigated bacterial and fungal human pathogens ranged from 0.19 to 6.25 mg/mL.

Genomic insights into antimicrobial potential and optimization of fermentation conditions of pig-derived Bacillus subtilis BS21.

Bacillus spp. have been widely used as probiotic supplements in animal feed as alternatives to antibiotics. In the present study, we screened a Bacillus subtilis strain named BS21 from pig feces. Antimicrobial activities, whole genome mining and UHPLC-MS/MS analysis were used to explore its antimicrobial mechanism. Strain BS21 showed Significant growth inhibition against a variety of animal pathogens, including Escherichia coli, Salmonella enterica Pullorum, Salmonella enterica Typhimurium, Citrobacter rodentium, Shigella flexneri and Staphylococcus aureus. Seven gene clusters involved in antimicrobial biosynthesis of secondary metabolites were encoded by strain BS21 genome, including four non-ribosomal peptides (bacillibactin, fengycin, surfactin and zwittermicin A), one ribosomal peptide (subtilosin A), one dipeptide (bacilysin) and one polyketide (bacillaene). Among them, production of surfactin, fengycin, bacillibactin, bacilysin and bacillaene was detected in the supernatant of B. subtilis strain BS21. To develop the potential application of BS21 in animal production, medium components and fermentation parameters optimization was carried out using response surface methodology (RSM). Production of antimicrobial secondary metabolites of strain BS21 was increased by 43.4%, and the best medium formula after optimization was corn flour 2%, soybean meal 1.7% and NaCl 0.5% with optimum culture parameters of initial pH 7.0, temperature 30°C, rotating speed at 220 rpm for 26 h. Our results suggested that strain BS21 has the potential for large-scale production and application as a potential source of probiotics and alternative to antibiotics for animal production.

DegQ associated with the degS/degU two-component system regulates biofilm formation, antimicrobial metabolite production, and biocontrol activity in Bacillus velezensis DMW1.

The gram-positive bacterium Bacillus velezensis strain DMW1 produces a high level of antimicrobial metabolites that can suppress the growth of phytopathogens. We investigated the mechanism used by degQ and the degS/degU two-component system to regulate the biocontrol characteristics of DMW1. When degQ and degU were deleted, the biofilm formation, cell motility, colonization activities, and antifungal abilities of ΔdegQ and ΔdegU were significantly reduced compared to wild-type DMW1. The expression levels of biofilm-related genes (epsA, epsB, epsC, and tasA) and swarming-related genes (swrA and swrB) were all down-regulated. We also evaluated the impact on secondary metabolites of these two genes. The degQ and degU genes reduced surfactin and macrolactin production and up-regulated the production of fengycin, iturin, bacillaene, and difficidin metabolites. The reverse transcription-quantitative PCR results were consistent with these observations. Electrophoretic mobility shift assay and microscale thermophoresis revealed that DegU can bind to the promoter regions of these six antimicrobial metabolite genes and regulate their synthesis. In conclusion, we provided systematic evidence to demonstrate that the degQ and degU genes are important regulators of multicellular behaviour and antimicrobial metabolic processes in B. velezensis DMW1 and suggested novel amenable strains to be used for the industrial production of antimicrobial metabolites.

Quantification of Antimicrobial Compounds Produced by Lactic Acid Bacteria Isolated from Pulses

In this study, 11 LAB isolates were isolated from the pulses (green gram and black gram) were tested for biochemical characteristics (catalase, MR-VP, citrate utilization, nitrate reduction) based on these tests four isolates were evaluated for their antimicrobial activity against three prominent pathogens: Escherichia coli, Pseudomonas aeruginosa, and Aspergillus flavus NRRL3357 under different pH 9 (5.5, 6.5, 7.5 and 8.5) and temperature (17ºC, 27ºC, and 40ºC). Among the four isolates, one isolate LB4 exhibited notable antimicrobial efficacy against all three pathogens under pH (6.5) 8.1± 0.6 mm, 8.3 ± 0.5 mm, and 6.6 ± 0.8 mm respectively. Optimum antimicrobial activity showed at temperature 27º against same pathogens the zone of inhibition mean values 10± 0.9 mm, 5.0± 0.6 mm, 8.3± 1.2 mm respectively. LB4 isolate was tested for quantification of key metabolites after three days of incubation, lactic acid, acetic acid, and hydrogen peroxide. Lactic acid (4.6 g/l) and acetic acid (4.8 g/l) were consistently produced in higher amounts, while hydrogen peroxide production remained relatively low (0.58 g/l) by the LB4 isolate. The results indicated the intricate relationship between antimicrobial activity, environmental factors, and metabolite production, emphasizing the promising role of this isolate in combating microbial infections under diverse conditions.

OPTIMIZATION OF PROCESS PARAMETERS FOR IMPROVED PRODUCTION OF ANTIMICROBIAL METABOLITES FROM NOCARDIOPSIS FLAVESCENS VJMS-18 ISOLATED FROM SOUTH-COASTAL REGIONS OF ANDHRA PRADESH, INDIA

Objective: The objective of the present study is to optimize the cultural parameters for Nocardiopsis flavescens VJMS-18 strain isolated from marine sediment samples of the south-coast of Andhra Pradesh, India. Methods: The strain N. flavescensVJMS-18 was identified based on morphological, physiological, biochemical and molecular approaches. The effect of environmental parameters such as incubation period, pH, temperature and salt concentration and the effect of various nutrients such as carbon and nitrogen sources and minerals on the bioactive metabolite production by N. flavescens VJMS-18 was evaluated by employing agar well diffusion assay. Results: The nutritional requirements and cultural conditions to enhance the yield of secondary metabolites are optimized under shake flask conditions. ISP-2 medium supplemented with sodium chloride at 3% maintained at pH 7.0 supported the maximum yield of secondary metabolites by the strain when incubated at 35°C for 8 days. The strain exhibited a broad spectrum of antagonistic activity against Gram-positive (Staphylococcus aureus and Bacillus megaterium) and Gram-negative bacteria (Klebsiella pneumoniae, Escherichia coli and Pseudomonas aeruginosa) as well as fungus (Candida albicans). Conclusion: It was found that the antimicrobial metabolite production by the strain was positively influenced by carbohydrates, nitrogen sources and minerals.

Biomass from phytopathogens and culture conditions improve Penicillium chrysogenum antimicrobial activity and antifungal compounds production

AbstractThe present study evaluated the effect of culture conditions and phytopathogenic strain co‐culture on the production of antimicrobial metabolites and antifungal activity of Penicillium chrysogenum R1, which PCR identified. Antimicrobial activity was determined using the Hunter‐Hunter experimental design with three factors (pH, incubation temperature, and inoculum, at two levels each). The antifungal metabolites, β 1‐3 glucanase and chitinase, produced in the presence of live and inactivated Fusarium oxysporum Fsox C11 biomass, were evaluated using HPLC‐MS and GC‐MS. Results showed that P. chrysogenum inhibited the growth of five phytopathogenic fungal strains, and the most significant inhibition was observed for F. oxysporum Fsox C11. The best conditions to achieve the highest antifungal activity of the cell‐free extract were pH 7, 28°C, 1 × 106 spores/mL, and 144 h of fermentation, observing 86% inhibition of F. oxysporum Fsox C11 growth. Production of antifungal metabolites such as 1,4‐benzoquinone imine, viridicatic acid, phenol‐5‐methyl‐2‐(1‐methyl ethyl), and hydrolytic enzymes β 1‐3 glucanase and chitinase was detected. The results define the perspective in designing new processes and products for biocontrol phytopathogens.

Exploring the Inhibitory Activity of Selected Lactic Acid Bacteria against Bread Rope Spoilage Agents

In this study, a wide pool of lactic acid bacteria strains deposited in two recognized culture collections was tested against ropy bread spoilage bacteria, specifically belonging to Bacillus spp., Paenibacillus spp., and Lysinibacillus spp. High-throughput and ex vivo screening assays were performed to select the best candidates. They were further investigated to detect the production of active antimicrobial metabolites and bacteriocins. Moreover, technological and safety features were assessed to value their suitability as biocontrol agents for the production of clean-label bakery products. The most prominent inhibitory activities were shown by four strains of Lactiplantibacillus plantarum (NFICC19, NFICC 72, NFICC163, and NFICC 293), two strains of Pediococcus pentosaceus (NFICC10 and NFICC341), and Leuconostoc citreum NFICC28. Moreover, the whole genome sequencing of the selected LAB strains and the in silico analysis showed that some of the strains contain operons for bacteriocins; however, no significant evidence was observed phenotypically.

Metabolite Signature and Differential Expression of Genes in Washington Navel Oranges (Citrus sinensis) Infected by Spiroplasma citri.

Spiroplasma citri is the pathogen that causes citrus stubborn disease (CSD). Infection of citrus with S. citri has been shown to cause leaf mottling, reduce fruit yield, and stunt tree growth. Fruit from trees exhibiting symptoms of CSD are misshapen and discolored. The symptoms of CSD are easily confused with nutrient deficiencies or symptoms of citrus greening disease. In this study, young Washington navel oranges (Citrus sinensis) were graft-inoculated with budwood originating from trees confirmed to be infected with S. citri. Leaf samples were collected monthly for 10 months for metabolomics and differential gene expression analyses. Significant differences in the concentration of metabolites and expressed genes were observed between control and S. citri-infected trees throughout the experiment. Metabolites and genes associated with important defense and stress pathways, including jasmonic acid signaling, cell wall modification, amino acid biosynthesis, and the production of antioxidant and antimicrobial secondary metabolites, were impacted by S. citri throughout the study, and even prior to symptom development. This work fills a current gap in knowledge surrounding the pathogenicity of S. citri and provides an updated mechanistic explanation for the development of CSD symptoms in S. citri-infected plants.

The intensity of biofilm formation by heterotrophic bacteria isolated from soil ferrosphere

The intensity of biofilm formation by heterotrophic bacteria possessed ammonifying ability (Bacillus simplex ChNPU F1, Streptomyces canus NUChC F2, Streptomyces gardneri ChNPU F3), ammonifying and iron-reducing ability (Fictibacillus sp. ChNPU ZVB1) previously isolated from soil ferrosphere was studied. Methods used: indirect measurement of the biomass of the bacterial biofilm using the adsorption/desorption of crystal violet, the aggregation test (to determine aggregation properties), the salt aggregation test (to determine hydrophobicity). The correlation analysis between the intensity of biofilm formation and aggregation of strains showed a significant positive correlation. The studied strains of microorganisms did not show high adhesive properties, they were moderately-adhesive (B. simplex, S. canus and S. gardneri) and weakly-adhesive (Fictibacillus sp.). It is supposed that the role these bacteria in the microbial damage of materials is determined preferably by bioelectrochemical reactions (iron-reducing bacteria) and the production of corrosive and/or antimicrobial metabolites (ammonifying and iron-reducing bacteria), but not by the biofilms formation. The prospect of further research is to analyze the antagonistic properties and biofilm formation of heterotrophic bacteria under co-cultivation conditions, in particular, with sulfate-reducing bacteria.

The Effect of Varied Culture Conditions and Nutritional Requirements in the Production of Antimicrobial Metabolite by Streptomyces

the Effect of Varied Culture Conditions and Nutritional Requirements in the Production of Antimicrobial Metabolite by Streptomyces

An endophytic Streptomyces sp. DARP-7 isolated from coastal lichen and enhancement of its antibacterial metabolite production using response surface methodology

Actinomycete DARP-7 was isolated from the coastal foliose lichen Dirinaria aegilita (Afzel.ex Ach.) B.J.Moore and identified as Streptomyces sp. using 16S rRNA sequencing. Among the eight culture media used for antimicrobial metabolite production, the peptone yeast extract iron medium (ISP-6) showed higher production with the maximum zone of inhibition (12 mm) against Staphylococcus aureus . Further, the ISP-6 medium components were optimized through the Plackett-Burman design (PBD) followed by Response Surface Methodology (RSM). Yeast extract, sodium thiosulphate, and ferric ammonium citrate would play a significant role in antibiotic production. Further, the significant variables from PBD were optimized through Central Composite Design (CCD) of RSM for enhanced antibiotic production. The optimal medium components were found to be peptone - 15 (g/L), protease peptone - 5 (g/L), yeast extract - 2.915 (g/L), ferric ammonium citrate - 0.651 (g/L), K 2 HPO 4 – 1 (g/L) and Na 2 S 2 O 3 – 0.155 (g/L), respectively. The optimized result was validated and the antibacterial activity against S. aureus increased upto 19.9 mm (66.7%). The resazurin dye based quantification of minimum inhibitory concentration of the DARP-7 crude extract was found to be 41.34 µg and 46.44 µg against S. aureus and E.coli , respectively. Further, the compound profiling was done using UV-VIS, FTIR, EEM and GC-MS confirming the presence of polyene conjugates. Thus, the study delivers coastal lichen could be promising source for isolating bioactive metabolites producing rare actinobacteria. Furthermore, the use of a statistical optimization tool to improve bioactive metabolite yield is discussed.

Isolation and bioactive potentials of Streptomyces from Tripura forest soil, North-east India

Aim: The bioactive potential of Actinobacteria from Tripura forest soil, Northeast India was investigated in this study. Methodology: Sixty seven actinobacterial isolates recovered from six soil samples were screened for antimicrobial activity against a panel of pathogenic bacteria and fungi. The production of antimicrobial metabolites from the potential strain TFA59 was done by both solid state and submerged fermentation. The antimicrobial compounds were further extracted using ethyl acetate as solvent and the activity was tested against Staphylococcus aureus and Candida albicans. The actinobacterial strain TFA59 was characterized based on its phenotypic and molecular characteristics. Results: Antimicrobial activity was exhibited by 28 actinobacterial cultures against atleast one of the test pathogens. Among the solvents tested for extraction, only ethyl acetate extract exhibited activity against S. aureus (21 mm) and C. albicans (23 mm). The production of antimicrobial compounds by TFA59 was influenced by raffinose, starch, yeast extract and pH 7 when compared with other variables. Based on the phenotypic and molecular characterization, the strain TFA59 was identified as a species of the genus Streptomyces. Interpretation: This study concluded that the Streptomyces strains isolated from Tripura forest soil show potential in producing bioactive metabolites that are effective against wide range of pathogens. Key words: Actinobacteria, Antimicrobial, Bioprospecting, North-east India, Streptomyces

Draft genome sequences of Bacillus pumilus 36R ATNSAL and B. safensis 13L LOBSAL, two potential candidate probiotic strains for shrimp aquaculture

This work aimed to isolate bacterial strains with antagonist activity against Vibrio parahaemolyticus, the causative agent of acute hepatopancreatic necrosis disease (VPAHPND) that was isolated from outbreaks in Mexico. Here, we report the draft genome sequences of two antagonistic strains, isolated from saline sediment in Sonora, Mexico. Cross-streak and well diffusion tests were employed to find the bacterial strains with higher inhibitory activity against VPAHPND. The whole genomes of B. pumilus 36R ATNSAL and B. safensis 13L LOBSAL were sequenced using Ion TorrentTM (PGM) and Illumina MiseqTM platforms, respectively. Annotation was performed using the RAST server, and the genes involved in the biosynthesis of bacterial secondary metabolites were predicted using antiSMASH. Two bacterial isolates, B. safensis 13L LOBSAL and B. pumilus 36R ATNSAL, were chosen based on their strong antagonistic profiles. The genome of 36R ATNSAL was 3.94 Mbp in length and contained 3824 genes and a total of 4116 coding sequences (CDSs); the genome of 13L LOBSAL was 3.68 Mbp and contained 3619 genes and 3688 CDSs. Twenty-eight and 32 biosynthetic gene clusters responsible for putative antimicrobial metabolite production were identified in 36R ATNSAL and 13L LOBSAL, respectively. The two strains 13L LOBSAL and 36R ATNSAL showed excellent probiotic profiles in vitro. The genome sequences will help with the mining and reconstruction of metabolic pathways in Bacillus strains. Genome sequence-guided strain improvement could augment the probiotic potential of Bacillus strains for applications in shrimp aquaculture.

Evaluation of the Antifungal Activity of Bacillusamyloliquefaciens and B. velezensis and Characterization of the Bioactive Secondary Metabolites Produced against Plant Pathogenic Fungi.

Simple SummaryPlant pathogenic fungi cause serious damage in agriculture, resulting in major losses in the yield and the quality of different economic crops. Chemical fungicides are dangerous to human health and the environment. They have many harmful side effects on non-target organisms. Moreover, their residues have been found in human food. Endophytic bacteria could be a valuable and safe alternative method for the biological control of phytopathogenic fungus. The endophyte Bacillus amyloliquefaciens provides a strong prospect as a biocontrol agent against Alternaria sp. on pepper plants.Endophytic bacteria are plant-beneficial bacteria with a broad host range. They provide numerous benefits to their hosts, helping them tolerate several biotic and abiotic stresses. An interest has recently been developed in endophytic bacteria which are producing bioactive compounds that contribute to the biological control of various phytopathogens. This research paper aimed to investigate the potentiality of new local strains of endophytic bacteria such as Bacillus amyloliquefaciens and B. velezensis and the production of several antimicrobial metabolites associated with the biocontrol of Alternaria sp., which cause serious diseases and affect important vegetable crops in Egypt. Twenty-five endophytic bacteria isolates were obtained from different plants cultivated in El-Sharkia Governorate, Egypt. Dual culture technique was used to evaluate the bacterial isolates’ antagonistic potentiality against Alternaria sp. and Helminthosporium sp. The most active bacterial isolates obtained were selected for further screening. The antifungal activity of the most active endophytic bacterial isolate was assessed in vivo on pepper seedlings as a biocontrol agent against Alternaria sp. A significant antifungal activity was recorded with isolates C1 and T5 against Alternaria sp. and Helminthosporium sp. The bacterial endophyte discs of C1 and T5 showed the highest inhibitory effect against Alternaria sp. at 4.7 and 3.1 cm, respectively, and Helminthosporium sp. at 3.9 and 4.0 cm, respectively. The most active endophytic isolates C1 and T5 were identified and the 16S rRNA sequence was submitted to the NCBI GenBank database with accession numbers: MZ945930 and MZ945929 for Bacillus amyloliquefaciens and Bacillus velezensis, respectively. The deformity of pathogenic fungal mycelia of Alternaria sp. and Helminthosporium sp. was studied under the biotic stress of bacteria. The culture filtrates of B. amyloliquefaciens and B. velezensis were extracted with different solvents, and the results indicated that hexane was the most efficient. Gas Chromatography-Mass Spectrometry revealed that Bis (2-ethylhexyl) phthalate, Bis (2-ethylhexyl) ester, and N,N-Dimethyldodecylamine were major constituents of the endophytic crude extracts obtained from B. amyloliquefaciens and B. velezensis. The in vivo results showed that Alternaria sp. infection caused the highest disease incidence, leading to a high reduction in plant height and in the fresh and dry weights of pepper plants. With B. amyloliquefaciens application, DI significantly diminished compared to Alternaria sp. infected pepper plants, resulting in an increase in their morphological parameters. Our findings allow for a reduction of chemical pesticide use and the control of some important plant diseases.

Sequential interspecies interactions affect production of antimicrobial secondary metabolites in Pseudomonas protegens DTU9.1.

Soil and rhizosphere microbiomes play important roles in suppression of plant pathogens through production of antagonistic secondary metabolites, yet mechanisms that determine the strength of pathogen control are not well understood. Many Pseudomonas species are associated with soil and rhizosphere microbiomes, and their ability to suppress pathogens is well documented. Here, we investigate how interactions within the Pseudomonas genus affect their production of antimicrobial metabolites. From a biosensor-based screen, we identify P. capeferrum species as capable of modulating secondary metabolite production in P. protegens. We show that P. capeferrum alters production of pyoluteorin and 2,4-diacetylphloroglucinol (DAPG) in P. protegens via two distinct and sequential mechanisms that depends on spatial proximity of the two species. Specifically, P. capeferrum secretes a diffusible signal that induce pyoluteorin production up to 100-fold in neighboring P. protegens colonies. In contrast, the interaction results in reduced DAPG production, but only within mixed-species colonies. Additionally, we found that increased pyoluteorin production and cell lysis of P. capeferrum is required for inhibition of DAPG production, suggesting that pyoluteorin-facilitated antibiosis of P. protegens on P. capeferrum leads to release of cell-associated metabolites and subsequent inhibition of DAPG production in P. protegens. As the interaction modulates in vitro bioactivity of the species, genus-specific interactions may assist in improving efficacy of biocontrol strains and consortia.

Antimicrobial potential of filamentous fungi growing on renewable substrates

The bioprospecting of secondary metabolites with antimicrobial action produced by filamentous fungi isolated from various ecosystems using agro-industrial residues is of considerable importance, since it reconciles the discovery of new antimicrobial agents with the reduction of environmental impacts adding economic value to wastes. The present paper investigated the potential of filamentous fungi for antimicrobial metabolites production using alternative media based on corn steep liquor, cassava wastewater and glycerol as substrates in different combinations. From 15 filamentous fungi assayed in preliminary screening, Paecilomyces variotii UCP 0334, Aspergillus flavus UCP 0316, Aspergillus foetidus UCP 0360, Aspergillus niger UCP 1064 e Aspergillus sp. 74M4 showed higher activity against all tested bacteria. After submerged fermentation in media containing corn steep liquor, cassava wastewater and glycerol, P. variotii UCP 0334 exhibitted greater activity and spectrum of action with inhibition halos ranging from 7 to 28 mm in diameter against all Gram negative and positive bacteria tested regardless of the culture medium type. It is worth mentioning that all fungi in alternative medium containing cassava wastewater and glycerol (CWWG) showed inhibitory activity against 3 bacteria with inhibition halos ranging from 9 to 15 mm. On the other hand, the medium containing corn steep liquor and glycerol (CSLG) favored higher biomass yield for all fungi. These results stimulate further studies of identification and characterization of the secondary metabolites produced by the selected fungi with a view to discovering new antimicrobial drugs.