Production Of Antifungal Metabolites Research Articles

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.

The Biocontrol and Plant Growth-Promoting Properties of Streptomyces alfalfae XN-04 Revealed by Functional and Genomic Analysis.

Fusarium wilt of cotton, caused by the pathogenic fungal Fusarium oxysporum f. sp. vasinfectum (Fov), is a devastating disease of cotton, dramatically affecting cotton production and quality. With the increase of pathogen resistance, controlling Fusarium wilt disease has become a significant challenge. Biocontrol agents (BCAs) can be used as an additional solution to traditional crop breeding and chemical control. In this study, an actinomycete with high inhibitory activity against Fov was isolated from rhizosphere soil and identified as Streptomyces alfalfae based on phylogenetic analyses. Next, an integrative approach combining genome mining and metabolites detection was applied to decipher the significant biocontrol and plant growth-promoting properties of XN-04. Bioinformatic analysis and bioassays revealed that the antagonistic activity of XN-04 against Fov was associated with the production of various extracellular hydrolytic enzymes and diffusible antifungal metabolites. Genome analysis revealed that XN-04 harbors 34 secondary metabolite biosynthesis gene clusters. The ability of XN-04 to promote plant growth was correlated with an extensive set of genes involved in indoleacetic acid biosynthesis, 1-aminocyclopropane-1-carboxylic acid deaminase activity, phosphate solubilization, and iron metabolism. Colonization experiments indicated that EGFP-labeled XN-04 had accumulated on the maturation zones of cotton roots. These results suggest that S. alfalfae XN-04 could be a multifunctional BCA and biofertilizer used in agriculture.

Antifungal metabolite profiling in Chaetomium globosum potential strain Cg2 effective against Bipolaris sorokiniana

The fungus reported to be a potential antagonist of various soil and seed borne plant pathogens. C. globosum mycoparasitizes and produce antifungal metabolites which suppress the growth of the pathogenic fungi. Despite sufficient knowledge available on secondary metabolites of C. globosum and their wide biological activities, limited literatures are available on the polar active constituents and their potential use as antifungal agent. The study was carried out to evaluate chemical composition of secondary metabolites from C. globosum when it interacts with Bipolaris sorokiniana (2017-2020). The volatile compounds identified through GC-MS revealed that, C. globosum strain Cg2 produced a variety of antifungal secondary metabolites, i.e. octadecene trans-limonene oxide, dodecane, tetracosonal, heptacosanol and octadecanoic acid which may be involved in the antagonism. Similarly, UPLC-QToF-ESIMS analysis of chloroform soluble fraction of C. globosum (Cg2), B. sorokiniana (BS112) and their interaction (Cg2-BS112) were undertaken to identify non-volatile metabolites.These metabolites were identified as Chaetomugilin A, D, E, F, Globoxanthone A, Chaetoviridin A, B, E, Chaetoglobin B, Chaetoquadrin A, Chaetocochin B and F, Chaetoglobosin Q and N.The work indicates that the biocontrol agent C. globosum showed high antifungal metabolite production thereby antagonising B. sorokininana pathogen. The obtained data will greatly enrich current C. globosum metabolomic information and provide a good foundation for better understanding of biocontrol mechanism of C. globosum against plant pathogens and facilitating widespread application in the field of bio-control.

Genomic and biochemical characterization of antifungal compounds produced by Bacillus subtilis PMB102 against Alternaria brassicicola

Bacillus subtilis is ubiquitous and capable of producing various metabolites, which make the bacterium a good candidate as a biocontrol agent for managing plant diseases. In this study, a phyllosphere bacterium B. subtilis PMB102 isolated from tomato leaf was found to inhibit the growth of Alternaria brassicicola ABA-31 on PDA and suppress Alternaria leaf spot on Chinese cabbage (Brassica rapa). The genome of PMB102 (Accession no. CP047645) was completely sequenced by Nanopore and Illumina technology to generate a circular chromosome of 4,103,088 bp encoding several gene clusters for synthesizing bioactive compounds. PMB102 and the other B. subtilis strains from different sources were compared in pangenome analysis to identify a suite of conserved genes involved in biocontrol and habitat adaptation. Two predicted gene products, surfactin and fengycin, were extracted from PMB102 culture filtrates and verified by LC–MS/MS. The antifungal activity of fengycin was tested on A. brassicicola ABA-31 in bioautography to inhibit hyphae growth, and in co-culturing assays to elicit the formation of swollen hyphae. Our data revealed that B. subtilis PMB102 suppresses Alternaria leaf spot by the production of antifungal metabolites, and fengycin plays an important role to inhibit the vegetative growth of A. brassicicola ABA-31.

Transcription in fungal conidia before dormancy produces phenotypically variable conidia that maximize survival in different environments.

Fungi produce millions of clonal asexual conidia (spores) that remain dormant until favourable conditions occur. Conidia contain abundant stable messenger RNAs but the mechanisms underlying the production of these transcripts and their composition and functions are unknown. Here, we report that the conidia of three filamentous fungal species (Aspergillus nidulans, Aspergillus fumigatus, Talaromyces marneffei) are transcriptionally active and can synthesize mRNAs. We find that transcription in fully developed conidia is modulated in response to changes in the environment until conidia leave the developmental structure. Environment-specific transcriptional responses can alter conidial content (mRNAs, proteins and secondary metabolites) and change gene expression when dormancy is broken. Conidial transcription affects the fitness and capabilities of fungal cells after germination, including stress and antifungal drug (azole) resistance, mycotoxin and secondary metabolite production and virulence. The transcriptional variation that we characterize in fungal conidia explains how genetically identical conidia mature into phenotypically variable conidia. We find that fungal conidia prepare for the future by synthesizing and storing transcripts according to environmental conditions present before dormancy.

English

Macrophomina phaseolina is a highly destructive pathogen of more than 500 plant species. It is difficult to eradicate it through chemical means as no patented fungicide is available against this pathogen. Biological control is the possible alternative method for its suitable management. The present study was carried out to evaluate the biocontrol potential of five Trichoderma spp. against M. phaseolina and the possible mechanisms of action. Identifications of all the Trichoderma spp. viz. T. hamatum, T. harzianum, T. koningii, T. longipile and T. viride were confirmed on molecular basis by using two universal primer pairs namely ITS and EF1. Their biocontrol potential was evaluated in dual culture plate method where T. viride showed the highest inhibitory efficacy (63%) against M. phaseolina. T. koningii, T. hamatum and T. longipile showed akin effects by arresting growth of the pathogen by 46–47% followed by T. harzianum (28%). To find out the mechanisms of action, secondary extrolites of the best biocontrol fungus T. viride were tested against the pathogenic genomic DNA where all the concentrations partially degraded DNA bands after 24 h of incubation and a complete DNA band disappearance was noted after 48 h incubation. In addition, T. viride culture filtrates were partitioned with chloroform and ethyl acetate and subjected to GC-MS analysis for identification of potential antifungal constituents. The most abundant identified volatile compounds in the two organic solvent fractions were 9,12-octadecadienoic acid (Z,Z)- (44.54%), n-hexadecanoic acid (24.02%), hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl) ethyl ester (14.25%), 9-tricosene, (Z)- (10.43%) and [1,1'-bicyclopropyl]-2-octanoic acid, 2'-hexyl-, methyl ester (10.43%). To conclude, T. viride was the best biocontrol agent against M. phaseolina and acts against the pathogen by DNA disintegration and production of antifungal secondary metabolites. © 2021 Friends Science Publishers

Evaluation of the production of antifungal metabolites against Colletotrichum gloeosporioides in Streptomyces 5.1 by random mutagenesis

Streptomyces 5.1 is a bacterium isolated from rice soils in the south of the Tolima department (Colombia). This microorganism is characterized by its antagonistic activity against rubber tree phytopathogens like Colletotrichum gloeosporioides, the causal agent of leaf anthracnose. The antifungal activity of this Streptomyces isolate has been associated with secondary metabolites production. However, the identity of those metabolites is unknown because its purification and identification have not been possible through classic chemical studies. Therefore, aiming to contribute in the study of the secondary metabolites produced by 5.1 from a molecular approach, this research seeks to identify -preliminarily- the genomic fingerprint changes associated with the production of antifungal secondary metabolites produced by Streptomyces 5.1 through the evaluation of a mutant library of 5.1 obtained by random mutagenesis using controlled ultraviolet light exposure. The antifungal activity of obtained mutants was evaluated using Colletotrichum gloeosporioides (C1) fungus as a biosensor, isolated by the Biotechnology Institute of Universidad Nacional de Colombia. In this way, the library of mutants of 5.1, initially formed by 300 isolations, was classified into two phenotypic groups of interest: enhanced mutants (1 isolate) and null mutants (11 isolates) of secondary metabolites. The genomic changes in both groups were analyzed by obtaining the genomic profile of the isolates using Repetitive Extragenic Palindromic (Rep-PCR). The obtained profiles evidenced the presence of one additional band in the enhanced mutant, and the absence of a specific band in the non-producing mutants, both in comparison with the original strain. These bands are proposed for a future sequencing study which will define their role in the production process of metabolites with antifungal activity in Streptomyces 5.1.

Transcriptional Changes in Potato Sprouts upon Interaction with Rhizoctonia solani Indicate Pathogen-Induced Interference in the Defence Pathways of Potato.

Rhizoctonia solani is the causer of black scurf disease on potatoes and is responsible for high economical losses in global agriculture. In order to increase the limited knowledge of the plants’ molecular response to this pathogen, we inoculated potatoes with R. solani AG3-PT isolate Ben3 and carried out RNA sequencing with total RNA extracted from potato sprouts at three and eight days post inoculation (dpi). In this dual RNA-sequencing experiment, the necrotrophic lifestyle of R. solani AG3-PT during early phases of interaction with its host has already been characterised. Here the potato plants’ comprehensive transcriptional response to inoculation with R. solani AG3 was evaluated for the first time based on significantly different expressed plant genes extracted with DESeq analysis. Overall, 1640 genes were differentially expressed, comparing control (−Rs) and with R. solani AG3-PT isolate Ben3 inoculated plants (+Rs). Genes involved in the production of anti-fungal proteins and secondary metabolites with antifungal properties were significantly up regulated upon inoculation with R. solani. Gene ontology (GO) terms involved in the regulation of hormone levels (i.e., ethylene (ET) and jasmonic acid (JA) at 3 dpi and salicylic acid (SA) and JA response pathways at 8 dpi) were significantly enriched. Contrastingly, the GO term “response to abiotic stimulus” was down regulated at both time points analysed. These results may support future breeding efforts toward the development of cultivars with higher resistance level to black scurf disease or the development of new control strategies.

Profiling of Metabolites of Bacillus spp. and Their Application in Sustainable Plant Growth Promotion and Biocontrol

Bacillus spp. are well-characterized as efficient bioinoculants for sustainable plant growth promotion and biocontrol of phytopathogens. Members of this spp. exhibit the multifaceted beneficial traits that are involved in plant nutrition and antimicrobial activities against phytopathogens. Keeping in view their diverse potential, this study targeted the detailed characterization of three root-colonizing Bacillus strains namely B. amyloliquefaciens, B. subtilis, and B. tequilensis, characterized based on 16S rRNA sequencing homology. The strains exhibited better plant growth promotion and potent broad-spectrum antifungal activities and exerted 43–86% in-vitro inhibition of growth of eight fungal pathogens. All strains produced indole acetic acid (IAA) in the range of 0.067–0.147 μM and were positive for the production of extracellular enzymes such as cellulase, lipase, and protease. Ultra-performance Liquid Chromatography-Electrospray Ionization-Mass Spectrometry (UPLC-ESI-MS/MS) analysis revealed the production of antifungal metabolites (AFMs) such as surfactins, iturins, fengycins, macrolactins, bacillomycin-D, and catechol-based siderophore bacillibactin which were further confirmed by amplifying the genes involved in the biosynthesis of these antimicrobial lipopeptides. When compared for the amounts of different cyclic-peptides produced by three Bacillus strains, B. amyloliquefaciens SB-1 showed the most noticeable amounts of all the antifungal compounds. Plant experiment results revealed that inoculation with phytohormone producing Bacillus spp. strains demonstrated substantial growth improvement of wheat biomass, number of spikes, and dry weight of shoots and roots. Results of this study indicate the biocontrol and biofertilizer potential of Bacillus spp. for sustainable plant nutrient management, growth promotion, and effective biocontrol of crop plants, particularly cultivated in the South Asian region.

Utilization of tofu wastewater and sugar industry by-products as a medium for the production of antifungal metabolites by Paecylomyces Marquand StrainTP4

Paecilomyces Marquand strain TP4 is a saprophytic fungus isolated from soil taken from Tangkuban Perahu Mountain at West Java Province, Indonesia. This research aims to identify the growth profile of P. Marquand strain TP4 and the antifungal activity against Fusarium sp. The antifungal compound is produced by the liquid fermentation method using tofu wastewater and molasses media. The fermentation broth is extracted by liquid-liquid extraction using n-hexane, ethyl acetate and methanol solvents. The result shows that the optimum activity achieved after 120 hours of fermentation. The fermentation broth has antifungal activity against Fusarium sp., where the inhibitory diameter was 20 mm. The total flavonoid content and total phenolic content of freeze-dry fermentation culture were 0.4839 mg/g and 1.171 mg/g, respectively. Ethyl acetate extract shows the highest antifungal activity with MIC 31.25 ppm and MFC 125 ppm. The FTIR spectrum shows the OH, aliphatic CH, CC, aromatic C=C, CH and C-O. Characterization of an antifungal compound based on phytochemical test and FTIR determine to be a phenolic group.

Deciphering the network of interconnected pathways of Chaetomium globosum antagonistic related genes against Bipolaris sorokiniana using RNA seq approach

Chaetomium species are known as potential biocontrol agents against phytopathogens due to their multiple antagonistic mechanisms. Plant disease is controlled by Chaetomium exhibit complex interactions against plant pathogen under varied conditions. Previously, mycoparasitism and antibiosis have been reported as most effective mechanism exhibited by the C. globosum against Bipolaris sorokiniana . In the present study, the examination of major biosynthetic pathways underlying Chaetomium globosum biocontrol activity was elucidated. It was shown that the pathways related to biosynthesis of secondary metabolites, hydrolytic enzymes and other key regulator genes were involved in production of hydrolytic enzymes and antifungal metabolites. We identified various genes of biological function with significant log2 fold change such as phosphoribosyl aminoimidazole carboxylase (9.693), protease (8.18), cyanate hydratase (Cyanase) (6.7), Fe2OG dioxygenase domain-containing protein (5.9), superoxide dismutase (5.55), glycosidase (5.34), carboxylic ester hydrolase (5.27), alpha-1,2-Mannosidase (4.44), alpha-1,4 glucan phosphorylase (3.99), endochitinase (3.87), P53-like transcription factor (Fragment) (3.55), metalloprotease (3.4), polyketide synthase (3.35), Catalase-peroxidase (CP) (3.14), protein kinase domain-containing protein (3.18) and glutamate decarboxylase (2.1) which are involved in biosynthesis of secondary metabolites, polyketide synthase, antibiotic, hydrolytic enzymes and putative fungistatic metabolites. This data provides a good foundation for continued researches into C. globosum Cg2 biocontrol activity for facilitating widespread application under the field conditions.

Optimization of the antifungal metabolite production in Streptomyces libani isolated from northern forests soils in Iran.

Background and Purpose: Soil bacteria have extreme population diversity among natural sources and are able to produce a wide array of antifungal metabolites. This study aimed to isolate and identify the bioactive metabolite-producing bacteria from forest soils and evaluate their antimicrobial potent against some pathogenic organisms.Materials and Methods: In this study, soil samples were screened for antifungal activity against Aspergillus fumigatus on glucose-yeast extract (GY) agar using a visual agar plate assay method. All growing bacteria were examined for antifungal activity, and antagonistic bacteria were identified based on 16S ribosomal RNA sequence analysis. For optimization of the production of antifungal bioactive metabolites, inhibitory bacteria were cultured on different culture conditions, including media, pH, temperature, and incubation time. Results: In total, 110 bacterial strains were isolated from the forest soils and four species with high antifungal activity were identified as Streptomyces libani, Streptomyces angustmyceticus, Bacillus subtilis, and Sphingopyxis spp. on the basis of 16s ribosomal RNA sequencing. Dichloromethane extract of the starch casein broth culture filtrate of the S. libani (incubated at 30° C for five days) showed strong antifungal activity against A. fumigatus, Aspergillus niger, and Aspergillus flavus. Conclusion: Based on the results, forest soils contain organisms with antifungal activity and could be considered as a good source for novel antifungal metabolites as effective and safe therapeutics.

Optimization of cultural conditions for production of antifungal metabolites by Streptomyces gougeroti

Use of antagonist microorganisms against fungal plant pathogens is an attractive and ecologically alternative to the use of chemical pesticides. Streptomyces spp. are beneficial soil bacteria and potential candidates for biocontrol agents. Different Streptomyces species were isolated from different sources sites from soil in Damietta. During primary and secondary screening, some actinomyctes isolates showed antifungal activity against tested phyto pathogenic fungi (Penicillium italicum, Macrophomina phaseolina, Fusarium oxysporium, F. solani, Alternaria alternata, Sclerotina sclerotiorum and Rhizoctonia solani). The results showed that the most active isolate against tested phyto pathogenic fungi Macrophomina phaseolina and Alternaria alternata was identified as Streptomyces gougeroti. Streptomyces gougeroti showed maximum antifungal activity against Alternaria alternata and Macrophomina phaseolina at 35°C ± 2, pH 6.5. Glycerol and beef extract were the best carbon and nitrogen sources used respectively.

Octadecyl 3-(3, 5-di-tert-butyl-4-hydroxyphenyl) propanoate, an antifungal metabolite of Alcaligenes faecalis strain MT332429 optimized through response surface methodology

In the current study, a soil bacterial isolate F2 expressed a significant antagonistic activity against Candida albicans ATCC 10231 and Aspergillus niger clinical isolate confirmed through cross streak, dual culture, and agar well diffusion methods. The isolate F2 was identified using phenotypic and molecular approaches as Alcaligenes (A.) faecalis MT332429. The identification and structural characterization of the antifungal compound was performed using advanced spectroscopic techniques including UV absorbance, 1H and 13C NMR and 2D NMR (COSY, HSQC, and HMBC) and was identified as octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl) propanoate. Response surface methodology (RSM) using a central composite design was employed to optimize the nutritional and cultural variables affecting the antifungal metabolite yield. The optimum conditions were found to be temperature 30 °C, agitation 150 rpm, glucose 1 g/l, peptone 2 g/l, and pH 8. A confirmatory experiment was performed to assess the accuracy of the optimization procedure, where an increase in the antifungal metabolite production by about 2.48-fold was obtained. To the best of our knowledge, this is the first report of octadecyl 3-(3, 5-di-tert-butyl-4-hydroxyphenyl) propanoate recovered from the culture broth of A. faecalis MT332429 with a promising antifungal activity along with its optimized production through RSM. KEY POINTS: • A novel soil bacterial isolate, F2, identified as Alcaligenes faecalis MT332429, showed significant antagonistic activity against Candida albicans ATCC 10231and Aspergillus niger clinical isolate. • This stable fungicidal extracellular metabolite was identified as octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl) propanoate. • Optimization using central composite design resulted in 2.48-fold increase in production reaching 213.82 μg/ml.

Optimized Production of the Allylamine Antifungal "Terbinafine" by Lysinibacillus Isolate MK212927 Using Response Surface Methodology.

PurposeWe aimed to optimize the factors affecting the production of the allylamine antifungal, terbinafine, by Lysinibacillus isolate MK212927, a natural producer of this broad-spectrum fungicidal compound.MethodsWe employed a central composite design to optimize the five most important variables influencing the production of terbinafine which were carbon source, nitrogen source, temperature, pH and agitation.ResultsThe optimum conditions were found to be starch 5 g/L, ammonium chloride 5 g/L, temperature 32°C, agitation 150 rpm and pH 7. The actual response (inhibition zone diameter) was highly comparable to the value predicted by the model, indicating a valid model. Using the standard calibration curve of terbinafine, the optimized conditions resulted in an increase in the antifungal metabolite production (terbinafine) by about 1.6-fold (1814.662 µg/mL compared to 1165.550 µg/mL under standardized conditions).ConclusionThis is the first report, to the best of our knowledge, on optimized production of terbinafine by Lysinibacillus species. Hence, these findings may be useful as baseline data for scaling up the production of terbinafine from a natural microbial source.

Production of plant protection agents in medium containing waste glycerol by Streptomyces hygroscopicus: Bioprocess analysis

The surplus of waste glycerol, by-product of the biodiesel production process, is available at the global market. Some species of the genera Streptomyces have the ability to assimilate glycerol and convert it into valuable metabolic products. In the present study, the ability of Streptomyces hygroscopicus to assimilate waste glycerol and convert it into metabolic compounds with antifungal activity against four phytopathogenic fungi obtained from apple fruit samples expressing rot symptoms, was investigated. Production of antifungal metabolites by S. hygroscopicus was carried out in 3 l stirred tank bioreactor through 7 days. Fermentation was carried out at 27 °C with aeration rate of 1.5 vvm and agitation rate of 100 r.p.m. The aim of this work was to analyse bioprocess parameters and to determine at which stage of bioprocess the production of antifungal metabolites occurs. Activity of the cultivation liquid on two isolates of Alternaria alternata and two isolates of Fusarium avenaceum were determined every 12 h using in vitro well diffusion method. It was found that the maximum production of antifungal metabolites occurred at 108 hour of cultivation. Formed inhibition zones have shown that the produced antifungal metabolites have high efficacy on tested phytopathogenic fungi (inhibition zone diameter higher than 35 mm for all test organisms).