Antifungal Secondary Metabolites Research Articles

Draft genome sequence of Collimonas sp. strain H4R21, an effective mineral-weathering bacterial strain isolated from the beech rhizosphere.

We present the draft genome sequence of Collimonas sp. strain H4R21, isolated from the rhizosphere of Fagus sylvatica in the forest experimental site of Montiers (France). This genome features coding capacity for plant growth promotion, such as the ability to solubilize minerals, to produce siderophores and antifungal secondary metabolites.

A Promising Biocontrol Agent of Bacillus velezensis VC3 against Magnaporthe oryzae and Colletotrichum gloeosporioides in Plants

Fungal diseases of plants are one of the key factors causing global crop losses. In this study, we isolated a Bacillus velezensis strain VC3, which was found to have a broad-spectrum inhibitory effect on a variety of phytopathogenic fungi through in vitro and in planta experiments, especially on Magnaporthe oryzae and Colletotrichum gloeosporioides. Further genomic and transcriptomic analyses revealed that the B. velezensis VC3 has multiple functional gene clusters encoding for the synthesis of a variety of antifungal secondary metabolites, including antimicrobial peptides (AMPs) and lipopeptides (LPs). In addition, AMPs and LPs were isolated and purified from B. velezensis VC3 fermentation broth and their antifungal activities were verified in this study. AMPs and LPs significantly inhibited spore germination, appressorium formation, and disease development, and AMPs have a better potential for controlling M. oryzae and C. gloeosporioides than LPs. These findings open new avenues for utilizing B. velezensis VC3 as biocontrol agents, providing potential sustainable solutions for agricultural production.

Strong antagonism of an endophyte of Fraxinus excelsior towards the ash dieback pathogen, Hymenoscyphus fraxineus, is mediated by the antifungal secondary metabolite PF1140.

Ash dieback, caused by the fungal pathogen Hymenoscyphus fraxineus (Helotiales, Ascomycota), is threatening the existence of the European ash, Fraxineus excelsior. During our search for biological control agents for this devastating disease, endophytic fungi were isolated from healthy plant tissues and co-cultivated with H. fraxineus to assess their antagonistic potential. Among the strains screened, Penicillium cf. manginii DSM 104493 most strongly inhibited the pathogen. Initially, DSM 104493 showed promise in planta as a biocontrol agent. Inoculation of DSM 104493 into axenically cultured ash seedlings greatly decreased the development of disease symptoms in seedlings infected with H. fraxineus. The fungus was thus cultivated on a larger scale in order to obtain sufficient material to identify active metabolites that accounted for the antibiosis observed in dual culture. We isolated PF1140 (1) and identified it as the main active compound in the course of a bioassay-guided isolation strategy. Furthermore, its derivative 2, the mycotoxin citreoviridin (3), three tetramic acids of the vancouverone type (4-6), and penidiamide (7) were isolated by preparative chromatography. The structures were elucidated mainly by NMR spectroscopy and high-resolution mass spectrometry (HRMS), of which compounds 2 and 6 represent novel natural products. Of the compounds tested, not only PF1140 (1) strongly inhibited H. fraxineus in an agar diffusion assay but also showed phytotoxic effects in a leaf puncture assay. Unfortunately, both the latent virulent attributes of DSM 104493 observed subsequent to these experiments in planta and the production of mycotoxins exclude strain Penicillium cf. manginii DSM 104493 from further development as a safe biocontrol agent.IMPORTANCEEnvironmentally friendly measures are urgently needed to control the causative agent of ash dieback, Hymenoscyphus fraxineus. Herein, we show that the endophyte DSM 104493 exhibits protective effects in vitro and in planta. We traced the activity of DSM 104493 to the antifungal natural product PF1140, which unfortunately also showed phytotoxic effects. Our results have important implications for understanding plant-fungal interactions mediated by secondary metabolites, not only in the context of ash dieback but also generally in plant-microbial interactions.

Pilot-scale production of Bacillus subtilis MSCL 897 spore biomass and antifungal secondary metabolites in a low-cost medium.

Bacillus subtilis is a plant growth promoting bacterium (PGPB) that acts as a microbial fertilizer and biocontrol agent, providing benefits such as boosting crop productivity and improving nutrient content. It is able to produce secondary metabolites and endospores simultaneously, enhancing its ability to survive in unfavorable conditions and eliminate competing microorganisms. Optimizing cultivation methods to produce B. subtilis MSCL 897 spores on an industrial scale, requires a suitable medium, typically made from food industry by-products, and optimal temperature and pH levels to achieve high vegetative cell and spore densities with maximum productivity. This research demonstrates successful pilot-scale (100L bioreactor) production of a biocontrol agent B. subtilis with good spore yields (1.5×109 spores mL-1) and a high degree of sporulation (>80%) using a low-cost cultivation medium. Culture samples showed excellent antifungal activity (1.6-2.3cm) against several phytopathogenic fungi. An improved methodology for inoculum preparation was investigated to ensure an optimal seed culture state prior to inoculation, promoting process batch-to-batch repeatability. Increasing the molasses concentration in the medium and operating the process in fed-batch mode with additional molasses feed, did not improve the overall spore yield, hence, process operation in batch mode with 10g molasses L-1 is preferred. Results also showed that the product quality was not significantly impacted for up to 12months of storage at room temperature. An economically-feasible process for B. subtilis-based biocontrol agent production was successfully developed at the pilot scale.

Discovery of antifungal secondary metabolites from an intestinal fungus Fusarium sp.

Intestinal fungi, which are important parts of the gut microbiota, have the ability to produce specialized metabolites that significantly contribute to maintaining the balance of the gut microbiota and promoting the health of the host organism. In the present study, two new glycosides, including fusintespyrone A (1) and cerevisterolside A (4), as well as ten known compounds were isolated from the intestinal fungus Fusarium sp. LE06. The structures of the new compounds were elucidated by a combination of spectroscopic methods, such as mass spectrometry (MS) and nuclear magnetic resonance (NMR), along with chemical reactions and calculations of NMR and ECD spectra. Compounds 1-3 showed significant growth inhibition against Aspergillus fumigatus, Fusarium oxysporum, and Verticillium dahliae with MIC values in the range of 1.56-6.25 μg ml-1.

Whole genome analysis of Bacillus amyloliquefaciens TA-1, a promising biocontrol agent against Cercospora arachidicola pathogen of early leaf spot in Arachis hypogaea L

BackgroundEarly leaf spot disease, caused by Cercospora arachidicola, is a devastating peanut disease that has severely impacted peanut production and quality. Chemical fungicides pollute the environment; however, Bacillus bacteria can be used as an environmentally friendly alternative to chemical fungicides. To understand the novel bacterial strain and unravel its molecular mechanism, De novo whole-genome sequencing emerges as a rapid and efficient omics approach.ResultsIn the current study, we identified an antagonistic strain, Bacillus amyloliquefaciens TA-1. In-vitro assay showed that the TA-1 strain was a strong antagonist against C. arachidicola, with an inhibition zone of 88.9 mm. In a greenhouse assay, results showed that the TA-1 strain had a significant biocontrol effect of 95% on peanut early leaf spot disease. De novo whole-genome sequencing analysis, shows that strain TA-1 has a single circular chromosome with 4172 protein-coding genes and a 45.91% guanine and cytosine (GC) content. Gene function was annotated using non-redundant proteins from the National Center for Biotechnology Information (NCBI), Swiss-Prot, the Kyoto Encyclopedia of Genes and Genomes (KEGG), clusters of orthologous groups of proteins, gene ontology, pathogen-host interactions, and carbohydrate-active enZYmes. antiSMASH analysis predicted that strain TA-1 can produce the secondary metabolites siderophore, tailcyclized peptide, myxochelin, bacillibactin, paenibactin, myxochelin, griseobactin, benarthin, tailcyclized, and samylocyclicin.ConclusionThe strain TA-1 had a significant biological control effect against peanut early leaf spot disease in-vitro and in greenhouse assays. Whole genome analysis revealed that, TA-1 strain belongs to B. amyloliquefaciens and could produce the antifungal secondary metabolites.

Efficacy of Seed-Biopriming with Trichoderma spp. and Foliar Spraying of ZnO-Nanoparticles Induce Cherry Tomato Growth and Resistance to Fusarium Wilt Disease.

Several microbes that cause plant diseases drastically lower the production of agriculture and jeopardize the safety of the world's food supply. As a result, sustainable agriculture requires disease management tactics based on modern, eco-friendly techniques as alternatives to various agrochemicals. The current study aimed to assess the antifungal activity of ZnO-nanoparticles against Fusarium solani in-vitro, and the ability of two antagonistic Trichoderma isolates, Trichoderma viride and Trichoderma harzianum, to produce antifungal secondary metabolites and identify them using gas chromatography-mass spectrometry, and to evaluate the combined effects of foliar spray of ZnO-nanoparticles and bioprimed seeds of cherry tomato (Solanum lycopersicum L.) with two antagonistic Trichoderma isolates against Fusarium wilt disease caused by Fusarium solani in greenhouse conditions. The results revealed that, in-vitro, the highest concentration of ZnO nanoparticles (3000 ppm) resulted in the greatest decrease in Fusarium solani mycelial growth (90.91% inhibition). The scanning electron microscopy demonstrated the evident distortion in Fusarium solani growing mycelia treated with ZnO-nanoparticles, which might be the source of growth suppression. Additionally, twenty-eight bioactive chemical compounds were isolated and identified from Trichoderma spp. ethyl acetate crude extracts using gas chromatography-mass spectrometry. In a greenhouse experiment, the combination of bioprimed cherry tomato plants with Trichoderma harzianum and foliar spraying of ZnO-nanoparticles at 3000 ppm was the most effective interaction treatment for reducing disease severity index (23.4%) and improving the vegetative growth parameters, micronutrient contents (Mn, Zn, and Fe in leaves), and chlorophyll content (SPAD unit), as well as stimulating phenylalanine ammonia-lyase activity of cherry tomato leaves at 75 days after sowing. In conclusion, the antifungal potential of seed-biopriming with antagonistic Trichoderma isolates and the foliar spraying of ZnO-nanoparticles can boost cherry tomato growth and confer resistance to Fusarium wilt caused by Fusarium solani.

Genome Sequence, assembly and characterization of Bacillus velezensis VR-34 used as biocontrol agent of Peyronellaea arachidicola (peanut web blotch, Pathogen)

Peyronellaea arachidicola, a fungus that specifically targets the foliage of peanut plants, causes web blotch disease, which has an impact on the crop's yield and quality. In the present study, Whole-genome sequencing identified and characterized Bacillus velezensis VR-34, as a biocontrol antagonistic strain. In vitro assay showed that VR-34 had a significant antagonistic effect on P. arachidicola with an inhibition zone of 90.39 mm, which was higher than the inhibition zone of 80.45 mm for Ck. In greenhouse assay, in the pre-inoculated treatment, it was observed that the VR-34 strain exhibited a biocontrol efficacy of 93.52% against peanut web blotch disease, whereas the control group showed a biocontrol efficacy of 95.64%.. However, in the post-inoculated treatment, it was observed that the VR-34 strain exhibited a biocontrol efficacy of 89.89% against peanut web blotch disease, whereas the control group showed a biocontrol efficacy of 88.71%. The SEM analysis revealed that VR-34 caused damaged the hyphal surfaces and broken of spores of P. arachidicola. Whole-genome sequencing revealed twenty functional groups from 2833 annotations in the COG database, most of which were involved in transcription, secondary metabolite biosynthesis, amino acid transport and metabolism, and carbohydrate metabolism. 1694 KEGG annotations link 33 metabolic pathways. Glycoside hydrolases, glycosyl transferases, carbohydrate esterases, auxiliary activities, carbohydrate-binding modules, and polysaccharide lyases were among the 172 annotations in the CAZy database. antiSMASH analysis predicted that strain VR-34 can produce the secondary metabolites difficidin, aurantinin, elansolid, basiliskamide macrobrevin, kalimantacin, sowinholide A, bacillaene, and calyculin. In vitro, and greenhouse studies revealed that VR-34 controlled peanut web blotch disease. The whole genome study showed the VR-34 strain is B. velezensis and could synthesize antifungal secondary metabolites.

Comparative transcriptome analysis reveals the biocontrol mechanism of Bacillus velezensis E68 against Fusarium graminearum DAOMC 180378, the causal agent of Fusarium head blight.

Fusarium graminearum is the causal agent of Fusarium Head Blight, a serious disease affecting grain crops worldwide. Biological control involves the use of microorganisms to combat plant pathogens such as F. graminearum. Strains of Bacillus velezensis are common biological control candidates for use against F. graminearum and other plant pathogens, as they can secrete antifungal secondary metabolites. Here we study the interaction between B. velezensis E68 and F. graminearum DAOMC 180378 by employing a dual RNA-seq approach to assess the transcriptional changes in both organisms. In dual culture, B. velezensis up-regulated genes related to sporulation and phosphate stress and down-regulated genes related to secondary metabolism, biofilm formation and the tricarboxylic acid cycle. F. graminearum up-regulated genes encoding for killer protein 4-like proteins and genes relating to heavy metal tolerance, and down-regulated genes relating to trichothecene biosynthesis and phenol metabolism. This study provides insight into the molecular mechanisms involved in the interaction between a biocontrol bacterium and a phytopathogenic fungus.

Uniting the Role of Endophytic Fungi against Plant Pathogens and Their Interaction.

Endophytic fungi are used as the most common microbial biological control agents (MBCAs) against phytopathogens and are ubiquitous in all plant parts. Most of the fungal species have roles against a variety of plant pathogens. Fungal endophytes provide different services to be used as pathogen control agents, using an important aspect in the form of enhanced plant growth and induced systemic resistance, produce a variety of antifungal secondary metabolites (lipopeptides, antibiotics and enzymes) through colonization, and compete with other pathogenic microorganisms for growth factors (space and nutrients). The purpose of this review is to highlight the biological control potential of fungal species with antifungal properties against different fungal plant pathogens. We focused on the introduction, biology, isolation, identification of endophytic fungi, and their antifungal activity against fungal plant pathogens. The endosymbionts have developed specific genes that exhibited endophytic behavior and demonstrated defensive responses against pathogens such as antibiosis, parasitism, lytic enzyme and competition, siderophore production, and indirect responses by induced systemic resistance (ISR) in the host plant. Finally, different microscopic detection techniques to study microbial interactions (endophytic and pathogenic fungal interactions) in host plants are briefly discussed.

Safety Evaluation of Bacillus subtilis IDCC1101, Newly Isolated from Cheonggukjang, for Industrial Applications.

The present study aimed to evaluate the safety of Bacillus subtilis (BS) IDCC1101, newly isolated from Cheonggukjang in Korea. Genome sequencing of BS IDCC1101 was performed to investigate the presence of secondary metabolites, virulence, antibiotic resistance, and mobile elements. Its phenotypic safety analyses included antibiotic susceptibility, enzyme activity, carbohydrate utilization, production of biogenic amines (BAs) and D-/L-lactate, hemolytic activity, and toxicities in HaCaT cells and rats. The genome of BS IDCC1101 consisted of 4,118,950 bp with 3077 functional genes. Among them, antimicrobial and antifungal secondary metabolites were found, such as fengycin, bacillibactin, and bacilysin. Antibiotic resistance and virulence genes did not exhibit transferability since they did not overlap with mobile elements in the genome. BS IDCC1101 was susceptible to almost all antibiotics suggested for assessment of BS's antibiotic susceptibility by EFSA guidelines, except for streptomycin. BS IDCC1101 showed the utilization of a wide range of 27 carbohydrates, as well as enzyme activities such as alkaline phosphatase, esterase, esterase lipase, naphthol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase, α-glucosidase, and β-glucosidase activities. Additionally, BS IDCC1101 did not exhibit the production of D-/L-lactate and hemolytic activities. Its toxicity in HaCaT cells and rats was also not detected. Thus, these genotypic and phenotypic findings indicate that BS IDCC1101 can be safely used for industrial applications.

Screening, and Optimization of Fermentation Medium to Produce Secondary Metabolites from Bacillus amyloliquefaciens, for the Biocontrol of Early Leaf Spot Disease, and Growth Promoting Effects on Peanut (Arachis hypogaea L.).

A novel Bacillus amyloliquefaciens BAM strain, with novel fermentation nutrient mediums and compositions, could produce potent antifungal secondary metabolites, as the existing strains face resistance from fungus pathogens. In the current study, we introduced two novel nutrient mediums for the fermentation process, semolina and peanut root extract, as carbon and nitrogen sources in order to maximize the antifungal effects of B. amyloliquefaciens against Cercaspora arachidichola to control early leaf spot disease in peanuts. Based on a single-factor test and the central composite design of response surface methodology, the optimum fermentation medium for Bacillus amyloliquefaciens antagonistic substance was determined, containing 15 gm/L of semolina flour, 12.5 gm/L of beef extract, and 0.5 gm/L of magnesium sulfate, which inhibited the fungal growth by 91%. In vitro, antagonistic activity showed that the fermentation broth of B. amyloliquefaciens BAM with the optimized medium formulation had an inhibition rate of (92.62 ± 2.07)% on the growth of C. arachidichola. Disease control effects in pot experiments show that the pre-infection spray of B. amyloliquefaciens BAM broth had significant efficiency of (92.00 ± 3.79)% in comparison to post-infection spray. B. amyloliquefaciens BAM broth significantly promoted peanut plant growth and physiological parameters and reduced the biotic stress of C. archidechola. Studies revealed that B. amyloliquefaciens BAM with a novel fermentation formulation could be an ideal biocontrol and biofertilizer agent and help in early disease management of early leaf spots in peanuts.

Polarity-guided phytochemical extraction, polyphenolic characterization, and multimode biological evaluation of Seriphidium kurramense (Qazilb.) Y. R. Ling

The undertaken study aims to assess the polyphenolic profile, and antioxidant, antimicrobial, antiviral, antidiabetic, and cytotoxic potential of Seriphidium kurramense (Qazilb.) Y. R. Ling extracts. Extracts of aerial parts were prepared by successive extraction (n-hexane {Sk-nH}, ethyl acetate {Sk-EA}, methanol {Sk-M} and aqueous {Sk-Aq}). Chromogenic assays determined the antioxidant profile while HPLC quantified several polyphenols. Agar well diffusion was employed for antimicrobial potential while brine shrimp and hemolytic assays established the biosafety profile. The results have shown that maximum extract recovery (17.49% w/w), total phenolics content (24.44 ± 0.15 μg GAE/mgE), and total flavonoids content (6.87 ± 0.25 μg QE/mgE) were recorded in Sk-Aq. RP-HPLC quantified a significant amount of syringic acid (1.43 ± 0.05 µg/mgE), caffeic acid (0.48 ± 0.02 µg/mgE), gentisic acid (6.44 ± 0.01 µg/mgE), and quercetin (4.39 ± 0.01 µg/mgE) in Sk-Aq, while maximum amounts of thymoquinone (0.21 ± 0.02 µg/mgE) and luteolin (3.90 ± 0.03 µg/mgE) along with apigenin (3.72 ± 0.03 µg/mgE) existed in Sk-M and highest quantities of ferulic acid (2.98 ± 0.01 µg/mgE), myricetin (1.04 ± 0.02 µg/mgE) and kaempferol (1.23 ± 0.01 µg/mgE) were found to be present in Sk-EA. A substantial free radical scavenging (85.87 ± 1.00%), total reducing power (211.93 ± 0.97 µg AAE/mgE), and urease inhibition activity (87.99 ± 0.19% at 500 µg/ml) were also recorded in the Sk-Aq. The highest antioxidant capacity (243.5 ± 1.12 µg AAE/mgE), antibacterial, antifungal, and antiviral activity (100% reduction in plaque formation at 400 µg/ml) were observed for Sk-EA. Maximum antibacterial and antifungal activities were revealed against Klebsiella pneumoniae (MIC = 25 ± 0.37 µg/ml), and Candida albicans (MIC = 50 ± 0.19 µg/ml) respectively. The prominent antidiabetic potential was displayed by Sk-nH in terms of α-amylase and α-glucosidase inhibition. The results reported, herein suggest that S. kurramense can be a promising candidate for antioxidant, antibacterial, antifungal, antiviral, and antidiabetic secondary metabolites.

Aspergillus flavus YRB2 from Thymelaea hirsuta (L.) Endl., a non-aflatoxigenic endophyte with ability to overexpress defense-related genes against Fusarium root rot of maize

Fusarium root rot, caused by Fusarium solani (Mart.) Sacc., represents one of the most damaging diseases of maize affecting plant growth and yield. In this study, the antagonistic potential of a non-aflatoxigenic endophytic Aspergillus flavus YRB2, isolated from Thymelaea hirsuta (L.) Endl., was tested against F. solani in vitro. In addition, its biocontrol activity against Fusarium root rot of maize was evaluated under greenhouse conditions. Its impacts on plant molecular, pathological, physiological, and growth levels were also studied. Results obtained revealed a potent antagonistic behavior for A. flavus YRB2 against F. solani in vitro, recording 80% growth inhibition. Seventeen secondary metabolites were detected in the n-hexane extract of A. flavus YRB2 filtered culture broth using GC-MS analysis. Among them, various antifungal secondary metabolites were produced, namely palmitic acid, α-linolenic acid, stearic acid, 2, 4-di-tert-butylphenol, diisobutyl phthalate, and heneicosane. In contrast, HPLC analysis showed that no aflatoxins (B1, B2, G1, and G2) were detected. Under greenhouse conditions, colonization of maize plants with A. flavus YRB2 exhibited a potential biocontrol activity against Fusarium root rot, recording 73.4% reduction in the disease severity. Triggering of transcriptional expression level of the defense-related genes JERF3 (7.2-fold), CHI II (8-fold), and POD (9.1-fold) was reported, indicating the inducing effect on the plant immunity. In addition, an increment in the antioxidant enzymes POD and PPO, and the total phenolic content in maize roots was also observed in response to this treatment. Moreover, a growth-promoting effect was also observed for colonization of maize plants with A. flavus YRB2. Based on the obtained data, we can conclude that A. flavus YRB2 may represent a promising biocontrol and growth-promoting agent for maize plants against Fusarium root rot. Nevertheless, field evaluation is highly requested before the use recommendation.

Identification of polyketide synthase genes required for aspinolide biosynthesis in Trichoderma arundinaceum

The fungus Trichoderma arundinaceum exhibits biological control activity against crop diseases caused by other fungi. Two mechanisms that likely contribute to this activity are upregulation of plant defenses and production of two types of antifungal secondary metabolites: the sesquiterpenoid harzianum A (HA) and the polyketide-derived aspinolides. The goal of the current study was to identify aspinolide biosynthetic genes as part of an effort to understand how these metabolites contribute to the biological control activity of T. arundinaceum. Comparative genomics identified two polyketide synthase genes (asp1 and asp2) that occur in T. arundinaceum and Aspergillus ochraceus, which also produces aspinolides. Gene deletion and biochemical analyses in T. arundinaceum indicated that both genes are required for aspinolide production: asp2 for formation of a 10-member lactone ring and asp1 for formation of a butenoyl subsituent at position 8 of the lactone ring. Gene expression and comparative genomics analyses indicated that asp1 and asp2 are located within a gene cluster that occurs in both T. arundinaceum and A. ochraceus. A survey of genome sequences representing 35 phylogenetically diverse Trichoderma species revealed that intact homologs of the cluster occurred in only two other species, which also produced aspinolides. An asp2 mutant inhibited fungal growth more than the wild type, but an asp1 mutant did not, and the greater inhibition by the asp2 mutant coincided with increased HA production. These findings indicate that asp1 and asp2 are aspinolide biosynthetic genes and that loss of either aspinolide or HA production in T. arundinaceum can be accompanied by increased production of the other metabolite(s).Key points• Two polyketide synthase genes are required for aspinolide biosynthesis.• Blocking aspinolide production increases production of the terpenoid harzianum A.• Aspinolides and harzianum A act redundantly in antibiosis of T. arundinaceum.

S3.4c A pipeline toward the identification of novel antifungal compounds derived from the microbial dark matter

S3.4 Free oral paper session, September 21, 2022, 4:45 PM - 6:15 PMBackgroundThe current armamentarium of antifungal drugs and the restricted variety in antifungal drug classes combined with the ever-rising threat of resistant fungal pathogens highlighted the urgent need for novel antifungal compounds. Natural antifungal secondary metabolites have always been the prevalent source for drug development, exemplified by the echinocandin and polyene drug classes. Yet, the golden age discovery platforms were abandoned due to compound rediscovery and its paired economic cost.StudyIn an effort to revive the original success stories, we combined the traditional approach of sampling and screening for active secondary metabolites with present-day advances in sequencing, genome mining, impedance spectroscopy, HPLC, LCMS, and NMR.Soil bacteria and fungi were isolated through in situ cultivation via the iChip method. After application of the OSMAC approach, 389 broths were identified with activity against Candida albicans. To prioritize active extracts, several criteria were set up; low to absent mammalian host cell toxicity, activity against a broad spectrum of fungal pathogens including wild-type reference strains, and established antifungal drug-resistant variants and species identification of the producing strain. Continuing, Lead hits were purified utilizing bioactivity-based semi-preparative HPLC. The resulting pure fractions were analyzed by tandem LCMS-MS, and proposed structures were later confirmed with NMR. In vitro and in vivo validation of the purified compounds will be performed.Additionally, aside from discovering a novel antifungal compound, another project goal is to gauge if impedance spectroscopy can provide an early suggestion regarding the mode of action of the present antifungal agent. For this, a POC study was performed which showed that different antifungal drug classes provide distinct signature response profiles by which they can be classified. As such, when active secretion broths show unique impedance profiles, in comparison with the signature profiles of established antifungal drugs, it suggests that they work through a different mode of action.ResultsSeveral species were identified as producing antifungal secondary metabolites that are currently absent in the literature. Either the compound itself was unknown or literature never described the species as a producer of a known, or variant of a known antimycotic compound. Moreover, several species are novel based on Illumina sequencing. Genera producing our current lead hits include bacteria: Pseudomonas, Tsukamurella, Paraburkholderia, and fungi: Athelia, Penicillium. Within the collection, the Pseudomonas species appear to produce variants of the antimycotic iron-chelating pyoverdine class.

New Approaches to Manage Asian Soybean Rust (Phakopsora pachyrhizi) Using Trichoderma spp. or Their Antifungal Secondary Metabolites.

Attempts have been made to determine the in vitro and in planta suppressive potential of particular Trichoderma strains (T16 and T23) and their secondary metabolites (SMs) against Asian soybean rust (ASR) incited by Phakopsora pachyrhizi. Aside from the previously identified SMs 6-pentyl-α-pyrone (6PAP) and viridiofungin A (VFA), the chemical structures of harzianic acid (HA), iso-harzianic acid (iso-HA), and harzianolide (HZL) were characterized in this study. Our results indicate that exposure of urediospores to 200 ppm 6PAP completely inhibits germination. A slightly higher dosage (250 ppm) of HZL and VFA reduces germination by 53.7% and 44%, respectively. Germ tube elongation seems more sensitive to 6PAP than urediospore germination. On detached leaves, application of conidia of T16 and T23 results in 81.4% and 74.3% protection, respectively. Likewise, 200 ppm 6PAP recorded the highest ASR suppression (98%), followed by HZL (78%) and HA (69%). Treatment of undetached leaves with 6PAP, HA, or HZL reduces ASR severity by 84.2%, 65.8%, and 50.4%, respectively. Disease reduction on the next, untreated trifoliate by T23 (53%), T16 (41%), HZL (42%), and 6PAP (32%) suggests a translocation or systemic activity of the SMs and their producers. To our knowledge, this study provides the first proof for controlling ASR using antifungal SMs of Trichoderma. Our findings strongly recommend the integration of these innovative metabolites, particularly 6PAP and/or their producers in ASR management strategies.

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.

Bacillus spp. as Bio-factories for Antifungal Secondary Metabolites: Innovation Beyond Whole Organism Formulations.

Several fungi act as parasites for crops causing huge annual crop losses at both pre- and post-harvest stages. For years, chemical fungicides were the solution; however, their wide use has caused environmental contamination and human health problems. For this reason, the use of biofungicides has been in practice as a green solution against fungal phytopathogens. In the context of a more sustainable agriculture, microbial biofungicides have the largest share among the commercial biocontrol products that are available in the market. Precisely, the genus Bacillus has been largely studied for the management of plant pathogenic fungi because they offer a chemically diverse arsenal of antifungal secondary metabolites, which have spawned a heightened industrial engrossment of it as a biopesticide. In this sense, it is indispensable to know the wide arsenal that Bacillus genus has to apply these products for sustainable agriculture. Having this idea in our minds, in this review, secondary metabolites from Bacillus having antifungal activity are chemically and structurally described giving details of their action against several phytopathogens. Knowing the current status of Bacillus secreted antifungals is the base for the goal to apply these in agriculture and it is addressed in depth in the second part of this review.

Identification of Putative Elicitors From Plant Root Exudates Responsible for PsoR Activation in Plant-Beneficial Pseudomonas spp. by Docking and Molecular Dynamics Simulation Approaches to Decipher Plant-Microbe Interaction.

Plants and rhizobacteria are coexisting since the beginning, but the exact mechanism of communication between them remains enigmatic. The PsoR protein of plant-beneficial Pseudomonas spp., a group of root-associated bacteria, is known to produce a range of antifungal and insecticidal secondary metabolites like 2,4-diacetyl phloroglucinol (DAPG), pyrrolnitrin, and chitinase making them great biocontrol agents and thus helping in plant growth promotion. To better understand the inter-kingdom signaling between plants and plant growth-promoting rhizobacteria (PGPR), the interaction of PsoR with various root exudates was investigated computationally. For this, we first modeled the PsoR protein and confirmed it using the Ramachandran plot. A total of 59 different low molecular weight phytochemicals, secreted as root exudates by plants, were identified by extensive text mining. They were virtually screened with the PsoR protein by molecular docking. Based on the lowest binding energy, ranging from −7.1 to −6.3 kcal mol−1, the top five exudates were chosen. To analyze the stability of the docked protein–ligand complex, a molecular dynamics (MD) simulation of 100 nanoseconds was done. Two root exudates, saponarin and 2-benzoxazolinone (BOA), showed suitable binding with PsoR by forming hydrogen, hydrophobic, and Van der Waals interactions. To confirm the MD simulation results, RMSF, RG, SASA, and interaction energy were calculated. This computational study first time reports that saponarin and 2-BOA, predominantly present in the root exudates of barley and wheat, respectively, demonstrate effective binding with the modeled PsoR protein and are likely of showing cross-kingdom interactions.