Production Of Antifungal Compounds Research Articles

Selection and application of methanol-utilizing bacteria from tomato leaves for biocontrol of gray mold

Gray mold, caused by Botrytis cinerea, is a significant threat to tomato production. Traditional chemical control methods have become increasingly ineffective because of the development of resistance. This study aimed to isolate methanol-utilizing bacteria from tomato leaves and evaluate their biocontrol potential against gray mold. To obtain bacterial suspensions, tomato leaf samples were collected and washed. We analyzed the microbial communities of these samples using 16S rRNA amplicon sequencing and identified several methylotrophic strains. Among these, 405 isolated strains were cultivated on a solid low-nutrient inorganic salt medium containing methanol, and 7 strains exhibiting considerable antifungal activity against B. cinerea were identified. Greenhouse tests revealed that two strains—SY163 and SY183—significantly reduced the severity of gray mold on tomato leaves. Disease index scores and the area under the disease progress curve values confirmed the efficacy of these strains as biocontrol agents. Statistical analysis indicated the effectiveness of pre- and co-application of these strains with B. cinerea. Phylogenetic analysis identified Serratia rubidaea as the inhibitory strain. The biocontrol activity is likely mediated through the production of antifungal compounds and suppression of B. cinerea sporulation. This study provides the basis for developing a technology of gray mold suppression by controlling the abundance of S. rubidaea in plant microbial communities.

Antarctic Streptomyces: Promising biocontrol agents for combating Fusarium oxysporum f. sp. cubense

Fusarium wilt of Banana (FWB) caused by Fusarium oxysporum f. sp. cubense (Foc) poses a significant threat to the banana industry, with current inadequate control measures. This study evaluated the antifungal potential of nine Streptomyces strains isolated from Antarctic soil samples, using Casein-Starch media to stimulate the production of antifungal compounds. The inhibition spectrum against Foc was assessed under laboratory conditions using the well diffusion on Mueller-Hinton agar, with antifungal activity measured in arbitrary units (AU/mL) and minimum inhibitory concentration (MIC) tested using ethyl acetate extracts. Among the nine isolates, K6 and E7 were closely related to Streptomyces polyrhachis and Streptomyces fildesensis, exhibited significant antifungal activity, with K6 and E7 showing 320 and 80 AU/mL, and MIC values of 250 and >500 ppm, respectively. These findings highlight K6 and E7 as potential biocontrol agents against Foc, offering new avenues for sustainable Fusarium wilt management in banana cultivation.

Antifungal metabolites produced by Pseudomonas hunanensis SPT26 effective in biocontrol of fusarium wilt of Lycopersicum esculentum under saline conditions.

In past few years, salinity has become one of the important abiotic stresses in the agricultural fields due to anthropogenic activities. Salinity is leading towards yield losses due to soil infertility and increasing vulnerability of crops to diseases. Fluorescent pseudomonads are a diverse group of soil microorganisms known for promoting plant growth by involving various traits including protecting crops from infection by the phytopathogens. In this investigation, salt tolerant plant growth promoting bacterium Pseudomonas hunanensis SPT26 was selected as an antagonist against Fusarium oxysporum, causal organism of fusarium wilt in tomato. P. hunanensis SPT26 was found capable to produce various antifungal metabolites. Characterization of purified metabolites using Fourier transform infrared spectroscopy (FT-IR) and liquid chromatography-electron spray ionization-mass spectrometry(LC-ESI/MS) showed the production of various antifungal compounds viz., pyrolnitrin, pyochelin and hyroxyphenazine by P. hunanensis SPT26. In the preliminary examination, biocontrol activity of purified antifungal metabolites was checked by dual culture method and results showed 68%, 52% and 65% growth inhibition by pyrolnitrin, 1- hydroxyphenazine and the bacterium (P. hunanensis SPT26) respectively. Images from scanning electron microscopy (SEM) revealed the damage to the mycelia of fungal phytopathogen due to production of antifungal compounds secreted by P. hunanensis SPT26. Application of bioinoculant of P. hunanensis SPT26 and purified metabolites significantly decreased the disease incidence in tomato and increased the plant growth parameters (root and shoot length, antioxidant activity, number of fruits per plant, etc.) under saline conditions. The study reports a novel bioinoculant formulation with the ability to promote plant growth parameters in tomato in presence of phytopathogens even under saline conditions.

Antifungal Potential of Bioactive Metabolites Produced by Pseudomonas aeruginosa against Candida Species

Abstract: Background: The frequency of fungal infections targeting immune-suppressed patients has been prominently rising day by day. Among these infections, Candidiasis is one of the most common life-threatening systemic fungal infections in recent years. These infections are challenging to treat because of the inconvenience of effective antifungal drugs. Therefore, the insufficiency of current drug regimens coupled with constant mutations by the fungi to develop drug resistance can pose a potential problem for future anti-fungal treatments. This frequent increase in drug-resistant fungi has directed attention toward the use of alternative therapy from natural sources. Objective: This study aimed to evaluate Pseudomonas aeruginosa for its antifungal potential against Candida species. Materials and Methods: This study was conducted at the Federal Urdu University of Arts, Science and Technology, Karachi, Pakistan during 2021-2022. For the detection of the antifungal compound, a clinical isolate P. aeruginosa HS 28 (identified conventionally as well as by 16S r RNA analysis) was screened for its bioactivity against Candida species by using agar well diffusion technique. The growth kinetics as well as the effect of different physical and chemical factors were also determined in the study. The compound was also partially purified by ammonium sulfate precipitation. Result: This antifungal compound showed good inhibitory activity against Candida species such as Candida albicans, C. glabrata, C. tropicalis, C. krusei, etc. This compound retained its stability at a high range of temperatures, and varying pH. Moreover, its bioactivity was also conserved when treated with organic solvents, chloroform vapors, metal salts, and different surfactants/detergents. The growth kinetics analysis illustrated that the maximum production of antifungal compounds occurred in the log phase of growth and extended supreme until the late log phase. Furthermore, the highest saturation of this protein was achieved at the concentration of 60% ammonium sulfate. Conclusion: The overall results indicated the promising antifungal potential of the compound produced by Pseudomonas aeruginosa HS 28 against Candida species.

Medium Optimization and Fermentation Kinetics for Antifungal Compounds Production by an Endophytic Paenibacillus polymyxa DS-R5 Isolated from Salvia miltiorrhiza.

An endophytic bacterium Paenibacillus polymyxa DS-R5 which can effectively inhibit the growth of pathogenic fungi was isolated from Salvia miltiorrhiza in our previous study. By using hydrochloric acid precipitation, methanol extraction, silica gel column isolation, dextran gel chromatography column, and HPLC, 3 compounds with antifungal activity were isolated. To further improve the production of antifungal compounds produced by this strain, fermentation medium was optimized using one-factor-at-a-time, Plackett-Burman design, and Box-Behnken design experiments. Through statistical optimization, the optimal medium composition was determined to be as follows: 14.7g/l sucrose, 20.0g/l soluble starch, 7.0g/l corn steep liquor, 10.0g/l(NH4)2SO4, and 0.7g/lKH2PO4. In this optimized medium, the highest titer of antifungal compounds reached 3452 U/ml, which was 123% higher than that in the initial medium. In addition, in order to guide scale-up for production, logistic and Luedeking-Piret equations were proposed to predict the cell growth and antifungal compounds production. The fermentation kinetics and empirical equations of the coefficients (X0, Xm, μm, α, and β) for the two models were reported, which will aid the design and optimization of industrial processes. The degrees of fit between calculated values of the model and the experimental data were 0.989 and 0.973, respectively. The results show that the cell growth and product synthesis models established in this study may better reflect the dynamic process of antifungal compounds production and provide a theoretical basis for further optimization and on-line monitoring of the fermentation process.

Fermentation medium optimization of Streptomyces sp. as an antifungal agent against the Ganoderma boninensis pathogen in oil palm

Ganoderma boninensis is the most common fungus which attacks oil palm trees. However, a significant percentage of inhibition to the problem is found through the use of Streptomyces sp. The optimization of the Streptomyces sp. fermentation medium growth factors affects the secondary metabolites production. This study aimed to identify the best formulation of carbon and nitrogen sources and the optimum concentration of Streptomyces sp. fermentation medium for antifungal compound production. The results showed that the best sources of carbon and nitrogen were liquid glucose and monosodium glutamate in the inhibition zones of 16.7 mm and 6.3 mm, while the best concentration levels were 20 g/L and 14.19 g/L, respectively. The results of the first optimization showed an inhibition zone response and area (%) of the optimum high‐performance liquid chromatography (HPLC) chromatogram of 24.39 mm and 62.68 percent, respectively. Taking the suggestion of the first optimization, the second optimization produced 15.2 g/L and 8.3 g/L. The predicted value of the inhibition zone was 21.47 mm, and the area (%) of the HPLC chromatogram was 53.44 percent. The validation results showed an inhibition zone response of 22.01 mm and an HPLC chromatogram area (%) of 54.86 percent. The difference between the predicted and validation values was less than 5 percent; the validation value was thus in line with the value predicted by Design Expert 10.0.7. The chemical formula of the probable active compound is that of the cyclo(phenylalanyl‐prolyl) compound.

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.

Purification and structure elucidation of Penicillium chrysogenum derived antifungal compound with potential anti-Candida property: in silico and in vitro evidence

Following preliminary bioactivity testing, the fungal strain identified as Penicillium crysogenum was cultured in a modified Czapec Yeast Broth medium (CYB) for the production of antifungal compounds. Several chromatographic techniques including HPLC were used to purify the fungal metabolites from the crude extract. The mass determination of the purified compound was performed using Water’s LCMS system while the structure of the compound was elucidated using 400 and 500 Varian NMR machines. The chemical name of the purified compound is (2 R, 4S) −2, 4-dimethyl-4-((E)-2-((3S, 4S)-2, 4, 5-trihydroxy-3-methoxy-4-phenyl–1, 2, 3, 4-tetrahydroquinolin-6-yl) vinyl) cyclohexanone with the chemical formula C26H31NO5 and exact mass of 437.2. Molecular docking predicted compound docking score with dihydrofolate reductase enzyme and lanosterol 14α-demethylase enzyme as −8.1 kcal/mol and −9.8 kcal/mol respectively. Further, the compounds showed stable binding mode with the enzymes and reported robust binding energies. After insilico analysis, the compound with mass 437 was tested for its antifungal potential in vitro against two pathogenic yeast species (i.e. Candida albicans and Candida glaberata) using the agar tube diffusion method. Using sterile di-methyl sulfoxide (DMSO) the compound was prepared in four dose concentrations (100, 250, 500, 1000 µg mL−1) and mixed with autoclaved semisolid Potato Dextrose Agar (PDA) medium in screw-capped test tubes labelled with the corresponding dose concentration. The fungal strains were inoculated on this medium and linear growth inhibition of the fungal strains was calculated using fluconazole as the control drug. The results from in vitro experiments were encouraging as at concentrations of 500 and 1000 μg mL−1 the compound inhibited the growth of C. albicans by 17% and 38% while 19% and 41% inhibition were recorded against C. glaberata. The compound showed antifungal activity in silico and in vitro against both the Candida species and can act as a potent antifungal candidate in the future upon further investigation. Communicated by Ramaswamy H. Sarma

Characterization of a Haloalkaline Nocardiopsis sp. Strain S10 Isolated from Wastewater and Optimization of Culture Medium for Improving Production of Antifungal Compounds

Background: The emerging antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) are a pressing global concern. Furthermore, the limited number of available antifungal agents requires the discovery of new antimicrobials. Introduction: This paper focused on the isolation and characterization of an actinobacterium designated S10, the study of its antimicrobial activity, on the one hand, and the optimization of antifungal production using statistical methods, on the other hand. Methods and Results: A haloalkaliphilic actinobacterium designated S10 was isolated from a wastewater treatment plant in the Souk-El-Tenine region (Bejaia, Algeria). This strain showed a broad spectrum of activity against bacteria (B. subtilis, L. innocua, M. luteus, S. aureus, MRSA, E. coli, P. aeruginosa, S. Typhi, and V. cholerae), molds (A. niger, G. geotricum, and F. oxysporum) and a pathogenic yeast (C. albicans). The extracellular enzymes producer Nocardiopsis sp. S10 was identified based on morphological, chemical, and 16S rRNA gene sequencing analysis. The best antimicrobial production was achieved on Mincer-Sea-Water (MSW) solid medium, and the antibiotics produced were extracted with ethyl acetate. For antifungal production, central composite design (CCD) and response surface methodology (RSM) were used to optimize the culture medium formulation. Conclusion: Optimal antifungal activity was achieved using a mixture of 2 g. L-1 starch, 2 g. L-1 of yeast extract and 2.75 g. L-1 of peptone at pH = 9.2. The application of the statistical approach stimulated a good yield of anti-C. albicans activity, corresponding to an increase of 72.22% compared to the non-optimized conditions.

Preparation of Sourdoughs Fermented with Isolated Lactic Acid Bacteria and Characterization of Their Antifungal Properties

Traditional sourdough is obtained using a mixture of flour and water stored at room temperature until acidification. Therefore, adding lactic acid bacteria (LAB) can improve the quality and safety of sourdough bread. Faced with this problem, four drying techniques-freeze-drying, spray-drying, low-temperature drying, and drying at low humidity-have been applied. Our goals were to isolate LAB strains with antifungal potential against Aspergillus and Penicillium fungi. The antifungal capacity was evaluated with agar diffusion, co-culture in overlay agar, and a microdilution susceptibility assay. In addition, the antifungal compounds generated in sourdough were analyzed. As a result, dried sourdoughs were prepared with Lactiplantibacillus plantarum TN10, Lactiplantibacillus plantarum TF2, Pediococcus pentosaceus TF8, Pediococcus acidilactici TE4, and Pediococcus pentosaceus TI6. The minimum fungicidal concentrations ranged from 25 g/L versus P. verrucosum and 100 g/L against A. flavus. A total of 27 volatile organic compounds were produced. Moreover, the lactic acid content reached 26 g/kg of dry product, and the phenyllactic concentration was significantly higher than the control. The P. pentosaceus TI6 exhibited a higher antifungal capacity in vitro and demonstrated a higher production of antifungal compounds compared to the other strains; therefore, further studies will evaluate the impact of this sourdough in bread manufacture.

Amycolatopsis from Desert Specialist Fungus-Growing Ants Suppresses Contaminant Fungi Using the Antibiotic ECO-0501.

Symbiotic Actinobacteria help fungus-growing ants suppress fungal pathogens through the production of antifungal compounds. Trachymyrmex ants of the southwest desert of the United States inhabit a unique niche far from the tropical rainforests in which most fungus-growing ant species are found. These ants may not encounter the specialist fungal pathogen Escovopsis known to threaten colonies of other fungus-growing ants. It is unknown whether Actinobacteria associated with these ants antagonize contaminant fungi and, if so, what the chemical basis of such antagonism is. We find that Pseudonocardia and Amycolatopsis strains isolated from three desert specialist Trachymyrmex species do antagonize diverse contaminant fungi isolated from field-collected ant colonies. We did not isolate the specialist fungal pathogen Escovopsis in our sampling. We trace strong antifungal activity from Amycolatopsis isolates to the molecule ECO-0501, an antibiotic that was previously under preclinical development as an antibacterial agent. In addition to suppression of contaminant fungi, we find that this molecule has strong activity against ant-associated Actinobacteria and may also play a role in bacterial competition in this niche. By studying interspecies interactions in a previously unexplored niche, we have uncovered novel bioactivity for a structurally unique antibiotic. IMPORTANCE Animal hosts often benefit from chemical defenses provided by microbes. These molecular defenses are a potential source of novel antibiotics and offer opportunities for understanding how antibiotics are used in ecological contexts with defined interspecies interactions. Here, we recover contaminant fungi from nests of Trachymyrmex fungus-growing ants of the southwest desert of the United States and find that they are suppressed by Actinobacteria isolated from these ants. The antibiotic ECO-0501 is an antifungal agent used by some of these Amycolatopsis bacterial isolates. This antibiotic was previously investigated in preclinical studies and known only for antibacterial activity.

Production of antifungal compounds from Bacillus paramycoides LBKURCC218 and Aspergillus fumigatus LBKURCC269 co-cultivation

Production of antifungal compounds from Bacillus paramycoides LBKURCC218 and Aspergillus fumigatus LBKURCC269 co-cultivation

Leveduras como agentes de controle biológico de patógenos de pós-colheita em citros

Considering the importance of citriculture for the country, this segment of Brazilian agribusiness faces phytosanitary problems that affect quantity and quality of the fruits, reducing nutritional and market values. Fungi are considered the main cause of fruit losses in the post-harvest, and the main diseases that affect citrus are: blue mold (Penicillium italicum), green mold (P. digitatum) and sour rot (Geotrichum citri-aurantii). Chemical control using Imazalil is the most effective method for controlling molds, however, its excessive and indiscriminate use has led to the proliferation of resistant strains, in addition to the persistence of residues of this fungicide on the fruits. Although the fungicides guazatine and propiconazole control the sour rot, they are only registered in countries in Europe, South Africa and Australia. Our review deals with the use of yeasts as biological control agents as an alternative to chemical control, covering: (i) their main mechanisms of action, such as competition for space and nutrients; (ii) production of siderophores; (iii) biofilm formation; (iv) production of antifungal compounds, hydrolytic enzymes and killer toxin; (v) mycoparasitism; and (vi) resistance induction. In addition, procedures for the development and registration of bioproducts are presented and discussed. KEYWORDS: Geotrichum citri-aurantii, mechanisms of action, Penicillium

The 1-aminocyclopropane-1-carboxylic acid deaminase-producing Streptomyces violaceoruber UAE1 can provide protection from sudden decline syndrome on date palm.

In the United Arab Emirates (UAE), sudden decline syndrome (SDS) is one of the major fungal diseases caused by Fusarium solani affecting date palm plantations. To minimize the impact of the causal agent of SDS on date palm, native actinobacterial strains isolated from rhizosphere soils of healthy date palm plants were characterized according to their antifungal activities against F. solani DSM 106836 (Fs). Based on their in vitro abilities, two promising biocontrol agents (BCAs), namely Streptomyces tendae UAE1 (St) andStreptomyces violaceoruber UAE1 (Sv), were selected for the production of antifungal compounds and cell wall degrading enzymes (CWDEs), albeit their variations in synthesizing 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase (ACCD). Although both isolates showed antagonism when applied 7 days before the pathogen in the greenhouse experiments, the ACCD-producing Sv was relatively superior in its efficacy against SDS over the non-ACCD-producing St. This was evident from the symptoms of SDS in diseased date palm seedlings which were greatly reduced by Sv compared to St. On a scale of 5.0, the estimated disease severity indices in Fs-diseased seedlings were significantly (P < 0.05) reduced from 4.8 to 1.5 and 0.5 by St and Sv, respectively. Thus, the number of conidia of Fs recovered from plants pre-treated with both BCAs was comparable, but significantly (P < 0.05) reduced compared to plants without any BCA treatment. In addition, a significant (P < 0.05) decrease in ACC levels of both the root and shoot tissues was detected inSv + Fs seedlings to almost similar levels of healthy seedlings. However, in planta ACC levels highly increased in seedlings grown in soils infested with the pathogen alone or amended with St prior to F. solani infestation (St + Fs). This suggests a major role of ACCD production in relieving the stress of date palm seedlings infected with F. solani, thus supporting the integrated preventive disease management programs against this pathogen. This is the first report of effective rhizosphere actinobacterial BCAs to provide protection against SDS on date palm, and to help increase agricultural productivity in a more sustainable manner in the UAE and the other arid regions.

Lentil-Based Yogurt Alternatives Fermented with Multifunctional Strains of Lactic Acid Bacteria-Techno-Functional, Microbiological, and Sensory Characteristics.

A milk-alternative produced from lentil protein isolate was fermented with three multifunctional strains of lactic acid bacteria, Leuconostoc citreum TR116, Leuconostoc pseudomesenteroides MP070, and Lacticaseibacillus paracasei FST 6.1. As a control, a commercial starter culture containing Streptococcus thermophilus was used. The metabolic performance of these strains and the techno-functional properties of the resulting yogurt alternatives (YA) were studied. Microbial growth was evaluated by cell counts, acidification, and carbohydrate metabolization. The structure of the YA was investigated by textural and rheological analyses and confocal laser scanning microscopy (CLSM). Production of antifungal compounds, the influence of fermentation on the content of FODMAPs, and typical metabolites were analyzed, and a sensory analysis was performed. The results revealed an exponential microbial growth in the lentil base substrate supported by typical acidification, which indicates a suitable environment for the selected strains. The resulting YA showed a gel-like texture typical for non-stirred yogurts, and high water holding capacity. The tested strains produced much higher levels of antifungal phenolic compounds than the commercial control and are therefore promising candidates as adjunct cultures for shelf-life extension. The Leuconostoc strains produced mannitol from fructose and could thus be applied in sugar-reduced YA. Preliminary sensory analysis showed high acceptance for YA produced with Lacticaseibacillus paracasei FST 6.1, and a yogurt-like flavor not statistically different to that produced by the control. Overall, each tested strain possessed promising functionalities with great potential for application in fermented plant-based dairy-alternatives.

Antifungal Mechanisms and Application of Lactic Acid Bacteria in Bakery Products: A Review.

Bakery products are nutritious, but they are susceptible to fungal contamination, which leads to a decline in quality and safety. Chemical preservatives are often used to extend the shelf-life of bakery products, but long-term consumption of these preservatives may increase the risk of chronic diseases. Consumers increasingly demand food with fewer chemical preservatives. The application of lactic acid bacteria (LAB) as a novel biological preservative not only prolongs the shelf-life of bakery products but also improves the baking properties of bakery products. This review summarizes different types and action mechanisms of antifungal compounds produced by LAB, factors affecting the production of antifungal compounds, and the effects of antifungal LAB on bakery products, providing a reference for future applications of antifungal LAB in bakery products.

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.

Purification and characterization of antifungal lipopeptide produced by Bacillus velezensis isolated from raw honey.

Raw honey contains a diverse microbiota originating from honeybees, plants, and soil. Some gram-positive bacteria isolated from raw honey are known for their ability to produce secondary metabolites that have the potential to be exploited as antimicrobial agents. Currently, there is a high demand for natural, broad-spectrum, and eco-friendly bio-fungicides in the food industry. Naturally occurring antifungal products from food-isolated bacteria are ideal candidates for agricultural applications. To obtain novel antifungals from natural sources, we isolated bacteria from raw clover and orange blossom honey to evaluate their antifungal-producing potential. Two Bacillus velezensis isolates showed strong antifungal activity against food-isolated fungal strains. Antifungal compound production was optimized by adjusting the growth conditions of these bacterial isolates. Extracellular proteinaceous compounds were purified via ammonium sulfate precipitation, solid phase extraction, and RP-HPLC. Antifungal activity of purified products was confirmed by deferred overlay inhibition assay. Mass spectrometry (MS) was performed to determine the molecular weight of the isolated compounds. Whole genome sequencing (WGS) was conducted to predict secondary metabolite gene clusters encoded by the two antifungal-producing strains. Using MS and WGS data, we determined that the main antifungal compound produced by these two Bacillus velezensis isolates was iturin A, a lipopeptide exhibiting broad spectrum antifungal activity.

Evaluation of the Antagonistic Effect of Pseudomonas Rhizobacteria on Fusarium Wilt of Chickpea

This study aimed to isolate rhizobacteria belonging to the genus Pseudomonas with plant-growth-promoting properties that can be used in the control of chickpea wilt disease caused by Fusarium oxysporum f. sp. ciceris (Foc). The production of antifungal compounds by the isolated rhizobacteria was assessed against two Foc isolates, coded Foc-S1 and Foc-S2. Strains E1FP13, E1FP4, and E1PP7 were the most effective against Foc-S1, with percentages of 52.77%, 48.37%, and 47.97%, respectively, while E1PP6, E1FP13, and E1PP15 were the most effective against Foc-S2 with percentages of 52.20%, 52.09%, and 45.38%, respectively. All five isolates were identified as Pseudomonas species using 16S rRNA sequencing. The microscopic examination of the impact of the Pseudomonas strains on Foc revealed that all five strains caused morphological changes in Foc, such as granulation and condensation of the cytoplasm, fragmentation, and deformation of the hyphae. The strains produced several plant-growth-promoting compounds, such as cellulase, hydrogen cyanide, indole acetic acid, ammonia, siderophores, lipase, protease, and solubilized phosphate. They were also able to significantly increase chickpea growth and reduce wilt disease, with E1FP13 resulting in the highest disease reductions of 55.77% (Foc-S1) and 53.33% (Foc-S2). The results revealed that our isolates can make promising biocontrol agents for controlling chickpea wilt disease.

Inhibition of the phytopathogenic fungi Curvularia lunata BM and Ganoderma sp. TB4 by antifungal compounds produced by Bacillus siamensis LDR grown on hanjeli (Coix lacryma-jobi L.) starch

Bacillus siamensis LDR was tested for its potential as a biocontrol agent against the phytopathogenic fungi Curvularia lunata BM and Ganoderma sp. TB4. Fermentation of B. siamensis LDR for the production of antifungal compound was performed in modified Czapex-Dox broth using hanjeli (Coix lacryma-jobi L.) starch as carbon source. The Bacillus siamensis LDR inoculum was 105 CFU/ mL, and fermentation was conducted for up to 16 days. Antibiosis assay conducted to test the antifungal activity of filtrate medium. The results showed inhibition of C. lunata BM and Ganoderma sp. TB4 were 47.08% and 85.99%, respectively on 14th day of fermentation. Antifungal assay of the crude extract from filtrate medium revealed growth inhibition of C. lunata BM (60.70%) and Ganoderma sp. TB4 (65.25%). Thin layer chromatography of the crude extract revealed pink-colored spots indicative of lipopeptide compounds. Analysis of the crude extract by ultraperformance liquid chromatography-mass spectrometry was tentatively identified as iturin A, bacillomycin F, and surfactin.