Production Of Antifungal Metabolites Research Articles

Antifungal activity of bio-active cell-free culture extracts and volatile organic compounds (VOCs) synthesised by endophytic fungal isolates of Garden Nasturtium

Antimicrobial resistance in fungal pathogens (both human and plant) is increasing alarmingly, leading to massive economic crises. The existing anti-fungal agents are becoming ineffective, and the situation worsens on a logarithmic scale. Novel antifungals from unique natural sources are highly sought to cope sustainably with the situation. Metabolites from endophytic microbes are the best-fitted alternatives in this case. Endophytes are the untapped sources of ‘plants’ internal microbial population’ and are promising sources of effective bio-therapeutic agents. Fungal endophytes were isolated from Tropaeolum majus and checked for antifungal activity against selected plant and human pathogens. Bioactive metabolites were identified through chromatographic techniques. The mode of action of those metabolites was evaluated through various spectroscopic techniques. The production of antifungal metabolite was optimized also. In particular VOCs (volatile organic compounds) of TML9 were tested in vitro for their anti-phytopathogenic activity. Ethyl acetate (EA) extract of cell-free culture components of Colletotrichum aenigma TML3 exhibited broad-spectrum antifungal activity against four species of Candida and the major constituents reported were 6-pentyl-2H-pyran-2-one, 2-Nonanone, 1 propanol 2-amino. The volatile metabolites, trans-ocimene, geraniol, and 4-terpinyl acetate, produced from Curvularia lunata TML9, inhibited the growth of some selected phyto pathogens. EA extract hampered the biofilm formation, minimised the haemolytic effect, and blocked the transformation of Candida albicans (MTCC 4748) from yeast to hyphal form with a Minimum Fungicidal Concentration (MFC) of 200–600 µg mL−1. Central carbohydrate metabolism, ergosterol synthesis, and membrane permeability were adversely affected and caused the lethal leakage of necessary macromolecules of C. albicans. Volatile metabolites inhibited the growth of phytopathogens i.e., Rhizoctonia solani, Alternaria alternata, Botrytis cinerea, Cercospora beticola, Penicillium digitatum, Aspergillus fumigatus, Ceratocystis ulmi, Pythium ultimum up to 89% with an IC50 value of 21.3–69.6 µL 50 mL−1 and caused leakage of soluble proteins and other intracellular molecules. Citrusy sweet odor volatiles of TML9 cultured in wheat-husk minimised the infections of Penicillium digitatum (green mold), in VOC-exposed sweet oranges (Citrus sinensis). Volatile and non-volatile antifungal metabolites of these two T. majus endophytes hold agricultural and pharmaceutical interests. Metabolites of TML3 have strong anti-Candida activity and require further assessment for therapeutic applications. Also, volatile metabolites of TML9 can be further studied as a source of antifungals. The present investigational outcomes bio-prospects the efficacy of fungal endophytes of Garden Nasturtium.

Type III sourdough: Evaluation of biopreservative potential in bakery products with enhanced antifungal activity

The potential biopreservative role of a Type III sourdough (tIII-SD), produced by starter cultures of Fructilactobacillus sanfranciscensis and Lactiplantibacillus plantarum ATCC 8014, was assessed for its antifungal activity in baking applications. Fermentation was carried out using different substrates to enhance the production of antifungal metabolites for 24 and 48 h. The tIII-SD samples were analyzed in relation to pH, total titratable acidity (TTA) and the production of organic acids. The water/salt-soluble extract of the tIII-SD was evaluated in relation to the inhibition potential against key fungi that contaminate bakery products including Penicillium roqueforti, Penicillium chrysogenum and Aspergillus niger. Finally, breads with 10 % of the tIII-SD were prepared and the fungi contamination was evaluated throughout the shelf life period. The lowest pH value in sourdough was obtained from 48-hour fermentation by L. plantarum. The saline extracts exhibited varying degrees of inhibition in the in vitro test; however, the greatest enhancement of this effect was obtained when whole wheat grain flour was used. The tIII-SD crafted from a blend of wheat and flaxseed flours and fermented with F. sanfranciscensis for 48 h (BSWF48h-FS), demonstrated superior performance compared to other formulations. This variant exhibited a total shelf life of 10 days, suggesting that the utilization of tIII-SD could serve as a viable alternative for natural antifungal agents, proving beneficial for the bakery industry.

Evaluation of Bacillus isolates as a biological control agents against soilborne phytopathogenic fungi.

Pesticides, used in agriculture to control plant diseases, pose risks to the environment and human health. To address this, there's a growing focus on biocontrol, using microorganisms instead of chemicals. In this study, we aimed to identify Bacillus isolates as potential biological control agents. We tested 1574 Bacillus isolates for antifungal effects against pathogens like Botrytis cinerea, Fusarium solani, and Rhizoctonia solani. Out of these, 77 isolates formed inhibition zones against all three pathogens. We then investigated their lytic enzyme activities (protease, chitinase, and chitosanase) and the production of antifungal metabolites (siderophore and hydrogen cyanide). Coagulase activity was also examined to estimate potential pathogenicity in humans and animals. After evaluating all mechanisms, 19 non-pathogenic Bacillus isolates with significant antifungal effects were chosen. Molecular identification revealed they belonged to B. subtilis (n = 19) strains. The 19 native Bacillus strains, demonstrating strong antifungal effects in vitro, have the potential to form the basis for biocontrol product development. This could address challenges in agricultural production, marking a crucial stride toward sustainable agriculture.

Identification, Characterization, and Production Optimization of 6-Methoxy-1H-Indole-2-Carboxylic Acid Antifungal Metabolite Produced by Bacillus toyonensis Isolate OQ071612.

Fungal infections currently pose a real threat to human lives. In the current study, soil bacterial isolates were screened for the production of antifungal compounds to combat human fungal pathogens. Notably, the bacterial F1 isolate exhibited antimycotic action towards the Candida albicans ATCC 10231 and Aspergillus niger clinical isolates. By employing phenotypic and molecular techniques, we identified the F1 isolate as the Bacillus toyonensis isolate OQ071612. The purified extract showed stability within a pH range of 6-7 and at temperatures of up to 50 °C. It demonstrated potential antifungal activity in the presence of various surfactants, detergents, and enzymes. The purified extract was identified as 6-methoxy-1H-Indole-2-carboxylic acid using advanced spectroscopic techniques. To optimize the antifungal metabolite production, we utilized response surface methodology (RSM) with a face-centered central composite design, considering nutritional and environmental variables. The optimal conditions were as follows: starch (5 g/L), peptone (5 g/L), agitation rate of 150 rpm, pH 6, and 40 °C temperature. A confirmatory experiment validated the accuracy of the optimization process, resulting in an approximately 3.49-fold increase in production. This is the first documented report on the production and characterization of 6-methoxy-1H-Indole-2-carboxylic acid (MICA) antifungal metabolite from Bacillus toyonensis.

Strain improvement of Streptomyces hydrogenans KMF-A1 for Antifungal Metabolite Production

Mangroves are found to be an excellent source for the isolation of novel bioactive metabolite producing microorganisms. Streptomyces hydrogenans KMFA-1, isolated from mangrove soil sample is exclusively known for its potent activity against various pathogenic fungi. The present study is focused on strain improvement of Streptomyces hydrogenans KMFA-1 by physical and chemical mutagens such as ultraviolet irradiation and nitrous acid (HNO2) respectively, for enhancing the production of antifungal metabolite against C. albicans and Pectinotrichum llanense. The parent isolate KMF-A1 produced 20mm inhibition zone diameter of antifungal activity while, the UV mutant designated as UV7, yielded 28mm and the nitrous acid mutant. UV7N4 (KMF-A1) exhibited 30mm inhibition zone diameter. The mutant strain produced improved antifungal activity by 1.5-fold increase with respect to wild strain.

Genomic, LC-MS, and FTIR Analysis of Plant Probiotic Potential of Bacillus albus for Managing Xanthomonas oryzae via Different Modes of Application in Rice (Oryza sativa L.).

Xanthomonas oryzae causes tremendous damage in rice plants (Oryza sativa L). Therefore, this study is focused on siderophore-producing Bacillus albus (CWTS 10) for managing BLB disease caused by X. oryzae. Both B. albus and its crude siderophore (methanolic and diethyl ether) extracts inhibited X. oryzae (10-12mm). Fourier transform infrared spectroscopy (FTIR) analysis of the extracts indicated the presence of catecholate siderophore functional groups. Liquid chromatography-mass spectrometry (LC-MS) analysis revealed the presence of antimicrobial compounds such as 2-deoxystreptamine, miserotoxin, fumitremorgin C, pipercide, pipernonaline, gingerone A, and deoxyvasicinone. Complete genome sequencing revealed the gene clusters for antibiotic, siderophore, antibacterial, antifungal, and secondary metabolite production. An in vivo study revealed that bacteria (CWTS 10) and their siderophore extracts effectively inhibited X. oryzae. The mode of application of bacterial or siderophore extracts in terms of DI and DSI percentage was as follows: soak method > inoculation method > spray method. In addition to providing enhanced antagonistic activity, there was a significant increase in root and shoot length and weight (wet and dry) of treated plants compared to control plants challenged with X. oryzae. Thus, the results clearly indicate that siderophore-producing B. albus and its siderophore extracts strongly inhibited X. oryzae. However, further field experiments are required before being formulated to protect rice crops from X. oryzae.

Bacillus amyloliquefaciens FG14 as a potential biocontrol strain against rusty root rot of Panax ginseng, and its impact on the rhizosphere microbial community

Rusty root rot is the most destructive disease in ginseng production. The bacterial strain FG14 isolated from the rhizosphere of Panax ginseng was characterised as Bacillus amyloliquefaciens. The inhibition rates of the FG14 bacterial suspension against the mycelial growth, spore production and spore germination of Ilyonectria robusta which causes ginseng rusty root rot were all more than 90% at a concentration of 108 cfu/mL. The sterile fermentation broth of strain FG14 also had significant inhibitory ability. The results of plantgrowth promotion showed that the fresh weight and root length of ginseng seedlings treated with 3 × 108 cfu/mL FG14 bacterial suspension were 4.49 times and 2.12 times greater than the seedlings treated with I. robusta. The soil enzyme activities of urease, phosphatase, invertase, and catalase of strain FG14 and the expression of seven lipopeptide biosynthesis genes were up-regulated, and antifungal metabolite production was stable. The diversity and abundance of bacterial and fungal communities in rhizosphere soil changed during different growth stages of ginseng after inoculation with strain FG14. The relative abundance of the Ilyonectria genus was significantly lower than the water control at the leaf spreading stage of P. ginseng. These results indicate the great potential of B. amyloliquefaciens FG14 for use as a biological agent for the green control of ginseng rusty root rot.

Bioprospecting of desert actinobacteria with special emphases on griseoviridin, mitomycin C and a new bacterial metabolite producing Streptomyces sp. PU-KB10–4

BackgroundBioprospecting of actinobacteria isolated from Kubuqi desert, China for antibacterial, antifungal and cytotoxic metabolites production and their structure elucidation.ResultsA total of 100 actinobacteria strains were selectively isolated from Kubuqi desert, Inner Mongolia, China. The taxonomic characterization revealed Streptomyces as the predominant genus comprising 37 different species, along with the rare actinobacterial genus Lentzea. The methanolic extracts of 60.8% of strains exhibited potent antimicrobial activities against Staphylococcus aureus, Micrococcus luteus, Bacillus subtilis, Escherichia coli, Salmonella enterica, Saccharomyces cerevisiae and high to mild in vitro cytotoxicity against PC3 (prostate cancer) and A549 (lung carcinoma) cell lines. The metabolomics analysis by TLC, HPLC-UV/vis, HPLC-MS and NMR showed the presence of compounds with molecular weights ranging from 100 to 1000 Da. The scale-up fermentation of the prioritized anti-Gram-negative strain PU-KB10–4 (Streptomyces griseoviridis), yielded three pure compounds including; griseoviridin (1; 42.0 mgL− 1) with 20 fold increased production as compared to previous reports and its crystal structure as monohydrate form is herein reported for the first time, mitomycin C (2; 0.3 mgL− 1) and a new bacterial metabolite 4-hydroxycinnamide (3; 0.59 mgL− 1).ConclusionsThis is the first report of the bioprospecting and exploration of actinobacteria from Kubuqi desert and the metabolite 4-hydroxycinnamide (3) is first time isolated from a bacterial source. This study demonstrated that actinobacteria from Kubuqi desert are a potential source of novel bioactive natural products. Underexplored harsh environments like the Kubuqi desert may harbor a wider diversity of actinobacteria, particularly Streptomyces, which produce unique metabolites and are an intriguing source to develop medicinally valuable natural products.

Effects of Bacillus amyloliquefaciens XJ-BV2007 on Growth of Alternaria alternata and Production of Tenuazonic Acid.

Large amounts of processing tomato are grown in Xinjiang, China. Tomato black spot disease, caused by Alternaria spp., and the produced alternaria toxins in tomato products are posing risks to human health. In this study, we isolated a rhizospheric bacterium, XJ-BV2007, from tomato (Solanum lycopersicum) fields, which we identified as Bacillus amyloliquefaciens. We found that this bacterium has a strong antagonistic effect against Alternaria alternata and reduces the accumulation of alternaria toxins in tomatoes. According to the antifungal activity of the bacteria-free filtrate, we revealed that B. amyloliquefaciens XJ-BV2007 suppresses A. alternata by the production of antifungal metabolites. Combining semi-preparative high-performance liquid chromatography, we employed UPLC-QTOF-MS analysis and the Oxford cup experiment to find that fengycin plays an important role in inhibiting A. alternata. This paper firstly reported that B. amyloliquefaciens efficiently controls tomato black spot disease and mycotoxins caused by A. alternata. B. amyloliquefaciens XJ-BV2007 may provide an alternative biocontrol strain for the prevention of tomato black spot disease.

Limiting the production of virulence factors as a mechanism of action for the control of Penicillium expansum by the Antarctic antagonistic yeast Debaryomyces hansenii F9D

Fungal decay, predominantly caused by Penicillium expansum, is the main cause of apple postharvest losses. To overcome this problem, synthetic fungicides are applied to fruit before storage. Public concern about this practice has raised interest in developing alternative biocontrol strategies for postharvest diseases. Debaryomyces hansenii F9D, a psychrotrophic Antarctic yeast, has been previously selected as a promising biocontrol agent due to its ability to reduce blue mold incidence in apples and pears. Previous in vitro studies indicated that the biocontrol mechanism of this yeast does not involve the production of antifungal metabolites or other inhibitory or competitive interactions with the pathogen. In this work, we conducted further studies in order to understand the interaction between Penicillium expansum and D. hansenii F9D within apple wounds. The growth of both microorganisms in apple wounds during cold storage was followed over time by q-PCR and viable cell counts. Scanning electron microscopy observations of co-inoculated and control wounds (inoculated only with the yeast or with the pathogen) were conducted to evaluate if the pathogen conidia could germinate in presence of the yeast and to determine the colonization patterns of P. expansum and the biocontrol yeast. Our results indicate that Debaryomyces hansenii F9D is able to prevent cold stored apples' wounds rot by inhibiting the virulence factors produced by the pathogen without impeding its growth. These findings provide circumstantial evidence for a new biocontrol mechanism associated with the prevention of fungal decay in apples by biocontrol yeasts during cold storage of fruit.

Antifungal cultures and metabolites of lactic acid bacteria for use in dairy fermentations

Fungal spoilage limits the shelf life of fermented dairy products. To address the problem, this study explores the potential of lactic acid bacteria as antifungal adjunct cultures in dairy matrices. Strains of lactic acid bacteria (113) representing 19 species were screened for their activity against Penicillium caseifulvum, Aspergillus clavatus and Mucor racemosus in modified MRS medium, milk, and yogurt. Strains of Lactiplantibacillus plantarum, Furfurilactobacillus milii, and Lentilactobacillus parabuchneri inhibited the growth of mycelial fungi. The inhibitory effects of lactic acid bacteria against yeasts were also determined in yogurt with Candida sake, Saccharomyces bayanus, and Torulaspora delbrueckii as challenge strains. The inhibition of yeasts by lactic acid bacteria was strain-specific and unrelated to the activity towards mycelial fungi. Organic acids and hydroxy fatty acids were quantified by liquid chromatograph coupled with refractive index detector and tandem mass spectrometry, respectively. Principal component analysis indicated 10-OH 18: 1 fatty acids and acetate are the main antifungal metabolites and explained over 50 % of the antifungal activity. The correlation analysis of metabolites and mold-free shelf life of milk and yogurt confirmed the role of these compounds. The genomic study analysed genes related to the production of major antifungal metabolites and predicted the formation of 1,2-propanediol and acetate but not of hydroxy unsaturated fatty acids. The findings provide new perspectives on the selection of antifungal strains, the characterization of antifungal metabolites and the exploration of antifungal mechanisms among different species.

Response Surface Methodology (RSM) Mediated Optimization of Medium Components for Mycelial Growth and Metabolites Production of Streptomyces alfalfae XN-04.

Streptomyces alfalfae XN-04 has been reported for the production of antifungal metabolites effectively to control Fusarium wilt of cotton, caused by Fusarium oxysporum f. sp. vasinfectum (Fov). In this study, we used integrated statistical experimental design methods to investigate the optimized liquid fermentation medium components of XN-04, which can significantly increase the antifungal activity and biomass of XN-04. Seven variables, including soluble starch, KNO3, soybean cake powder, K2HPO4, MgSO4·7H2O, CaCO3 and FeSO4·7H2O, were identified as the best ingredients based on one-factor-at-a-time (OFAT) method. The results of Plackett–Burman Design (PBD) showed that soluble starch, soybean cake powder and K2HPO4 were the most significant variables among the seven variables. The steepest climbing experiment and response surface methodology (RSM) were performed to determine the interactions among these three variables and fine-tune the concentrations. The optimal compositions of medium were as follows: soluble starch (26.26 g/L), KNO3 (1.00 g/L), soybean cake powder (23.54 g/L), K2HPO4 (0.27 g/L), MgSO4·7H2O (0.50 g/L), CaCO3 (1.00 g/L) and FeSO4·7H2O (0.10 g/L). A verification experiment was then carried out under the optimized conditions, and the results revealed the mycelial dry weight of S. alfalfae XN-04 reaching 6.61 g/L. Compared with the initial medium, a 7.47-fold increase in the biomass was achieved using the optimized medium. Moreover, the active ingredient was purified from the methanol extract of S. alfalfae XN-04 mycelium and then identified as roflamycoin (a polyene macrolide antibiotic). The results may provide new insights into the development of S. alfalfae XN-04 fermentation process and the control of the Fusarium wilt of cotton and other plant diseases.

Functional Redundancy in Bat Microbial Assemblage in the Presence of the White Nose Pathogen.

Understanding how host-associated microbial assemblages respond to pathogen invasion has implications for host health. Until recently, most investigations have focused on understanding the taxonomic composition of these assemblages. However, recent studies have suggested that microbial assemblage taxonomic composition is decoupled from its function, with assemblages being taxonomically varied but functionally constrained. The objective of this investigation was to understand how the Tri-colored bat, Perimyotis subflavus cutaneous microbial assemblage responds to fungal pathogen invasion within a functional context. We hypothesized that at a broad scale (e.g., KEGG pathways), there will be no difference in the functional assemblages between the white nose pathogen, Pseudogymnoascus destructans, positive and negative bats; and this pattern will be driven by the functional redundancy of bacterial taxa. At finer scales (e.g., gene models), we postulate differences in function attributed to interactions between bacteria and P. destructans, resulting in the production of antifungal metabolites. To test this, we used a combination of shotgun metagenomic and amplicon sequencing to characterize the bat cutaneous microbial assemblage in the presence/absence of P. destructans. Results showed that while there was a shift in taxonomic assemblage composition between P. destructans positive and negative bats, there was little overall difference in microbial function. Functional redundancy across bacterial taxa was clear at a broad-scale; however, both redundancy and variation in bacterial capability related to defense against pathogens was evident at finer scales. While functionality of the microbial assemblage was largely conserved in relation to P. destructans, the roles of particular functional pathways in resistance to fungal pathogens require further attention.

Complete Genome Sequences of Five Gram-Negative Bacterial Strains Comprising Synthetic Bacterial Consortium "The Great Five" with Antagonistic Activity Against Plant-Pathogenic Pectobacterium spp. and Dickeya spp.

Complete Genome Sequences of Five Gram-Negative Bacterial Strains Comprising Synthetic Bacterial Consortium "The Great Five" with Antagonistic Activity Against Plant-Pathogenic Pectobacterium spp. and Dickeya spp.

EppR, a new LysR-family transcription regulator, positively influences phenazine biosynthesis in the plant growth-promoting rhizobacterium Pseudomonas chlororaphis G05

Pseudomonas chlororaphis G05 has the capability to repress the mycelial growth of many phytopathogenic fungi by producing and secreting certain antifungal compounds, including phenazines and pyrrolnitrin. Although some regulatory genes have been identified to be involved in antifungal metabolite production, the regulatory mechanism and pathway of phenazine-1-carboxylic acid biosynthesis remain poorly defined. To identify more new regulatory genes, we applied transposon mutagenesis with the chromosomal lacZ fusion strain G05Δphz::lacZ as an acceptor. In the white conjugant colony G05W05, a novel transcriptional regulator gene, eppR, was verified to be interrupted by the transposon mini-Tn5Kan. To evaluate the specific function of eppR, we created a set of eppR-deletion mutants, including G05ΔeppR, G05Δphz::lacZΔeppR and G05Δprn::lacZΔeppR. By quantifying the production of antifungal compounds and β-galactosidase expression, we found that the expression of the phenazine biosynthetic gene cluster (phz) and the production of phenazine-1-carboxylic acid were markedly reduced in the absence of EppR. Moreover, the pathogen suppression test verified that the yield of phenazine-1-carboxylic acid was significantly decreased when eppR was deleted in frame. At the same time, no changes in the expression of the phzI/phzR quorum-sensing (QS) system and the production of N-acyl homoserine lactones (AHLs) and pyrrolnitrin were found in the EppR-deficient mutant. In addition, chromosomal fusion analyses and quantitative real-time polymerase chain reaction (qRT–PCR) results also showed that EppR could positively mediate the expression of the phz cluster at the posttranscriptional level. In summary, EppR is specifically essential for phenazine biosynthesis but not for pyrrolnitrin biosynthesis in P. chlororaphis.

Antifungal activity of endophytic Fusarium oxysporum from Cola acuminata against Candida albicans isolates from HIV-infected patients

Abstract Background: The Human Immunodeficiency Virus (HIV) infection has exacerbated the occurrence of opportunistic diseases with Candida infection being the most prominent. Unfortunately, the growing ineffectiveness of the available antifungal drugs along with the emergence of new multi-resistant strains further worsen the matter. In the search for new active molecules, the present study aims at investigating the optimal growth conditions for the production of antifungal metabolites from the endophytic fungus Fusarium oxysporum. Methods: Fusarium sp. isolated from Cola acuminata was identified using the ITS1-5.8S rRNA-ITS2 nucleotide sequence. For the production of various metabolites, the fungus was cultivated for ten days in various media supplemented with different sources of carbon, amino acids, nitrogen, ion and organic acids. All extracts were submitted to antifungal activity test against seven Candida albicans isolates using the broth microdilution method. The most active extract was evaluated for cytotoxic activity against the Vero cell line using the MTT assay. Results: The endophytic fungal isolate Casb122 was identified as Fusarium oxysporum based on the ITS sequence. Extracellular extracts showed better activity against Candida albicans (MICs ranges 500-1000 µg/mL) compared to intracellular extracts (2000 µg/mL). The growth media supplemented with carbon, amino acids, nitrogen, ion and organic acids promoted mycelium growth and production of secondary metabolites. The MIC of extracts obtained from certain media supplemented with carbon, amino acids, nitrogen, ion and organic acids were 2 (500 µg/mL), 4 (250 µg/mL), 8 (125µg/mL) and 16 (62.5 µg/mL) times lower than that of the control (extract obtained from unsupplemented media). Active extracts with a Cytotoxic concentration 50 (CC50) >200 µg/mL were not cytotoxic to Vero cell line. Conclusion: The results from this study demonstrate that the culture of endophytic Fusarium oxysporum in supplemented media improves biomass production and antifungal activity against Candida albicans. This isolate could be an excellent source for the discovery of new antifungal compounds. Current investigations are ongoing to isolate and characterize potential antifungal metabolites.

Impact of environmental factors on Streptomyces spp. metabolites against Botrytis cinerea.

Botrytis cinerea is an economically important disease on numerous vegetables including tomato. From our previous studies, a spore suspension of Streptomyces philanthi RL-1-178 and RM-1-138 and Streptomyces mycarofaciens SS-2-243 showed strong inhibition against B. cinerea. In this study, the efficacy of their antifungal metabolites against B. cinerea was investigated after enhancing the production through the optimum culture medium and environmental conditions (temperature, light/dark cycle). In vitro studies indicated that glucose yeast-malt (GYM) agar and incubation at 28°C were optimal for growth and mass spore production of all three Streptomyces strains. Moreover, light/dark conditions had a positive effect on the growth and spore production of S. philanthi RM-1-138 and RL-1-178 but not on S. mycarofaciens SS-2-243. Both strains of S. philanthi possessed an antifungal activity against B. cinerea (100% inhibition) while S. mycarofaciens showed different results on PDA (83% inhibition) and GYM (88% inhibition) at the optimum incubation temperature at 21°C. The antifungal compounds from S. philanthi RM-1-138 exhibited the highest protection efficacy against B. cinerea on tomato leaves (82.89% and 0.33 cm2 lesion areas symptoms). The antifungal compounds RM-1-138, identified by GC-MS, were greatly altered based on components concentration under various temperatures and light/dark conditions. The anti-B. cinerea of S. philanthi RM-1-138 was established at a higher level in several metabolic compounds in the dark condition (11 and 32 antifungal compounds after incubation at 21°C and 28°C, respectively) than in the light condition (11 and 19 antifungal compounds after incubation at 21°C and 28°C, respectively). At 21°C, the dominant component was acetic acid (67.41% and 68.77% in light and dark conditions, respectively) while at 28°C, benzeneacetamide (43.58% in light) and propanamide (20.68% in the dark) were dominant. The results clearly demonstrated the significant influence of environmental factors on the production of antifungal metabolites of Streptomyces spp.

Optimization of glycerol-based medium composition for antifungal metabolites production by Bacillus subtilis

Bacillus species are promising producers of various compounds that have pronounced antimicrobial, antiviral and antitumor activities. Due to its GRAS status, Bacillus subtilis represents an excellent candidate for the usage in plant pathogens biocontrol. In this research, evaluation of antifungal metabolites biosynthesis by Bacillus subtilis ATCC 6633 and optimization of glycerol-based medium composition, using response surface methodology, for the production of compounds effective against Neurospora crassa were investigated. The results of disc-diffusion method indicate that applied Bacillus strain produces compounds with antifungal activity against tested fungus. In order to find optimal cultivation medium composition, the experiments were carried out in accordance with Box-Behnken design, and optimization was performed using the concept of desirability function combined with previously defined mathematical equation, which describes examined bioprocess. The optimization model predicts that maximum inhibition zone diameter against Neurospora crassa of 32.24 mm is achieved when initial content of glycerol, NaNO2 and K2HPO4 were 49.68 g/L, 2.90 g/L and 6.49 g/L, respectively. Additionally, the second optimization set was made to minimize the consumption of medium components and costs of medium preparation. The obtained results are the basis for further research aimed to develop medium appropriate for economically justified production of bioactive compounds at industrial scale.

Comparative Genome Analysis of Bacillus amyloliquefaciens Focusing on Phylogenomics, Functional Traits, and Prevalence of Antimicrobial and Virulence Genes.

Bacillus amyloliquefaciens is a gram-positive, nonpathogenic, endospore-forming, member of a group of free-living soil bacteria with a variety of traits including plant growth promotion, production of antifungal and antibacterial metabolites, and production of industrially important enzymes. We have attempted to reconstruct the biogeographical structure according to functional traits and the evolutionary lineage of B. amyloliquefaciens using comparative genomics analysis. All the available 96 genomes of B. amyloliquefaciens strains were curated from the NCBI genome database, having a variety of important functionalities in all sectors keeping a high focus on agricultural aspects. In-depth analysis was carried out to deduce the orthologous gene groups and whole-genome similarity. Pan genome analysis revealed that shell genes, soft core genes, core genes, and cloud genes comprise 17.09, 5.48, 8.96, and 68.47%, respectively, which demonstrates that genomes are very different in the gene content. It also indicates that the strains may have flexible environmental adaptability or versatile functions. Phylogenetic analysis showed that B. amyloliquefaciens is divided into two clades, and clade 2 is further dived into two different clusters. This reflects the difference in the sequence similarity and diversification that happened in the B. amyloliquefaciens genome. The majority of plant-associated strains of B. amyloliquefaciens were grouped in clade 2 (73 strains), while food-associated strains were in clade 1 (23 strains). Genome mining has been adopted to deduce antimicrobial resistance and virulence genes and their prevalence among all strains. The genes tmrB and yuaB codes for tunicamycin resistance protein and hydrophobic coat forming protein only exist in clade 2, while clpP, which codes for serine proteases, is only in clade 1. Genome plasticity of all strains of B. amyloliquefaciens reflects their adaption to different niches.

LysR-type transcriptional regulator FinR is required for phenazine and pyrrolnitrin biosynthesis in biocontrol Pseudomonas chlororaphis strain G05.

Phenazine-1-carboxylic acid and pyrrolnitrin, the two secondary metabolites produced by Pseudomonas chlororaphis G05, serve as biocontrol agents that mainly contribute to the growth repression of several fungal phytopathogens. Although some regulators of phenazine-1-carboxylic acid biosynthesis have been identified, the regulatory pathway involving phenazine-1-carboxylic acid synthesis is not fully understood. We isolated a white conjugant G05W03 on X-Gal-containing LB agar during our screening of novel regulator candidates using transposon mutagenesis with a fusion mutant G05Δphz::lacZ as a recipient. By cloning of DNA adjacent to the site of the transposon insertion, we revealed that a LysR-type transcriptional regulator (LTTR) gene, finR, was disrupted in the conjugant G05W03. To confirm the regulatory function of FinR, we constructed the finR-knockout mutant G05ΔfinR, G05Δphz::lacZΔfinR, and G05Δprn::lacZΔfinR, using the wild-type strain G05 and its fusion mutant derivatives as recipient strains, respectively. We found that the expressions of phz and prn operons were dramatically reduced in the finR-deleted mutant. With quantification of the production of antifungal metabolites biosynthesized by the finR-negative strain G05ΔfinR, it was shown that FinR deficiency also led to decreased yield of phenazine-1-carboxylic acid and pyrrolnitrin. In addition, the pathogen inhibition assay confirmed that the production of phenazine-1-carboxylic acid was severely reduced in the absence of FinR. Transcriptional fusions and qRT-PCR verified that FinR could positively govern the transcription of the phz and prn operons. Taken together, FinR is required for antifungal metabolite biosynthesis and crop protection against some fungal pathogens.Key points• A novel regulator FinR was identified by transposon mutagenesis.• FinR regulates antifungal metabolite production.• FinR regulates the phz and prn expression by binding to their promoter regions.