Graminearum PH-1 Research Articles

Investigating the Structure of the Components of the PolyADP-Ribosylation System in Fusarium Fungi and Evaluating the Expression Dynamics of Its Key Genes

Poly(ADP-ribose) polymerase (PARP) is the key enzyme in polyADP-ribosylation, one of the main post-translational modifications. This enzyme is abundant in eukaryotic organisms. However, information on the PARP structure and its functions in members of the Fungi kingdom is very limited. In this study, we performed a bioinformatic search for homologs of PARP and its antagonist, PARG, in the genomes of four Fusarium strains using their whole-genome sequences annotated and deposited in databases. The F. graminearum PH-1, F. proliferatum ET-1, and F. oxysporum Fo47 strains were shown to possess a single homolog of both PARP and PARG. In addition, the F. oxysporum f. sp. lycopersici strain 4287 contained four additional proteins comprising PARP catalytic domains whose structure was different from that of the remaining identified homologs. Partial nucleotide sequences encoding the catalytic domains of the PARP and PARG homologs were determined in 11 strains of 9 Fusarium species deposited in all-Russian collections, and the phylogenetic properties of the analyzed genes were evaluated. In the toxigenic F. graminearum strain, we demonstrated up-regulation of the gene encoding the PARP homolog upon culturing under conditions stimulating the production of the DON mycotoxin, as well as up-regulation of the gene encoding PARG at later stages of growth. These findings indirectly indicate involvement of the polyADP-ribosylation system in the regulation of the genes responsible for DON biosynthesis.

The biopotential of extremophilic microorganisms isolated from Kuzbass for protection and growth stimulation of oat (Avena sativa L.)

Biological plant protection products are extensively utilized in agriculture owing to their safety, efficacy, and environmental friendliness. The utilization of these funds in locales with challenging weather patterns is constrained. In order to ensure the successful use of biological plant protection products, it is rational to search for microorganisms adapted to the specific soil and climatic conditions of the territory. The objective of the investigation is to evaluate the antagonistic and growth-stimulating properties of the consortium of extremophilic microorganisms of Kuzbass, and to conduct laboratory testing on oats (Avena sativa L.). The work used microorganisms isolated at the initial stages of the research, such as Leclercia sp., Sphingomonas paucimobilis, and Lactobacillus plantarum. The findings revealed that these microorganisms are capable of producing gibberellic acid, dissolving phosphates, and retaining atmospheric nitrogen. Furthermore, the co-cultivation of microorganisms facilitates the enhancement of these indicators. The negative impact of phytopathogens (Alternaria alternata (F-525), Fusarium graminearum (F-877) PH-1, Fusarium graminearum (F-892) and Fusarium sporotrichioides (F-902)) on oats in laboratory conditions was reduced by a consortium based on these microorganisms. Therefore, the consortium has the potential to develop commercial drugs. Field experiments are planned in the future.

Novel Endophytic Pseudescherichia sp. GSE25 Strain Significantly Controls Fusarium graminearum and Reduces Deoxynivalenol in Wheat.

Fusarium heading blight (FHB) is a devastating disease in wheat, primarily caused by field invasion of Fusarium graminearum. Due to the scarcity of resistant wheat varieties, the agricultural sector resorts to chemical fungicides to control FHB incidence. On the other hand, biocontrol represents a promising, eco-friendly approach aligned with sustainable and green agriculture concepts. In the present study, a bacterial endophyte, Pseudescherichia sp. (GSE25), was isolated from wheat seeds and identified through complete genome sequencing and phylogenetic analysis. In vitro testing of this endophytic strain demonstrated strong antifungal activity against F. graminearum PH-1 by inhibiting spore germination, suppressing germ tube growth, and causing cell membrane damage. Under field conditions, the strain GSE25 significantly reduced the FHB incidence and the associated deoxynivalenol mycotoxin accumulation by over 60% and 80%, respectively. These findings highlight the potential of the isolated bacterial endophyte Pseudescherichia sp. GSE25 strain as a biocontrol agent in protecting wheat from FHB-caused F. graminearum. This is the first report showing a biocontrol effect of Pseudescherichia sp. a strain against phytopathogens.

Investigation of the Antifungal Activity of Bacillus megaterium Against Fusarium Species

Several Fusarium species are emerging as serious pathogens on small grain cereals worldwide. The use of fungicides is a short-term strategy in the fight against Fusarium diseases. The use of biocontrol agents is an attractive alternative strategy by reducing the chemical input to the environment as well as being economical. Bacillus species have received attention as biocontrol agents. In this study, the antagonistic activities of Bacillus megaterium CTBmeg1 and HMA5 strains on Fusarium culmorum UK99 and F. graminearum PH-1 isolates were investigated in vitro and at molecular level. On the 7th day of the dual culture assay, both of B. megaterium strains significantly reduced the mycelial growth of Fusarium isolates, with very high antifungal activity with the inhibition rate between 72.7% and 77.7%, respectively. Similarly, both strains caused high antifungal activity in the volatile organic compound (VOC) analysis between 52.1% and 62.4%, respectively. At the molecular level, in all tested groups, transcript levels of the tri5 gene, which is associated with trichothecene production, decreased, while the transcript levels of cat, an antioxidant gene, and mst20, a gene related to apoptosis, increased. Findings from this study showed that B. megaterium CTBmeg1 and HMA5 strains could be accepted as highly effective biocontrol agents against worldwide phytopathogens F. culmorum and F. graminearum.

Investigation of antifungal activities of myrcene on Fusarium reference strains.

Antifungal effects of myrcene, the plant-based naturel compound, were investigated on Fusarium graminearum PH-1 and Fusarium culmorum FcUK99 references, for the first time. Minimum inhibitory concentration (MIC) and half of MIC (MIC50) of both Fusarium strains against myrcene were found as 25µg/µl and 12.5µg/µl, respectively. MIC50 application decreased the cell viabilities in the ratios of 34.90% and 33.91% in PH-1 and FcUK99, respectively (p < 0.01). The significantly increased catalase (CAT) activity was recorded in MIC50 treated strains (p < 0.01). Apoptosis-like process and cellular oxidative stress were also monitored with acridine orange/ethidium bromide (Ao/Eb) dual staining and 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA) staining. The genomic template stability (GTS) percentages were calculated as 79% for PH-1 and 71% for FcUK99 via random amplified polymorphic DNA (RAPD). Methylation polymorphism values were calculated as 53.8% and 50.6% in PH-1 and 40.4% and 39% in FcUK99 for HapII and MspI, respectively by coupled restriction enzyme digestion-random amplification (CRED-RA). Methylation-sensitive amplified polymorphism (MSAP) revealed that myrcene caused both type I and type III epigenetic modifications in both genomes. MIC50 dose caused up to 13.86 ± 0.42-fold changes in the expressions of cat, mst20, and stuA, whereas downregulation in tri5 was recorded. Myrcene application did not change the retrotransposon movement in both species by the amplifying of idiomorphic retrotransposon patterns through inter-retrotransposon polymorphism-polymerase chain reaction (IRAP-PCR). This study demonstrated that myrcene is an effective compound in the management of phytopathogenic Fusarium species by causing morphological, genetic, epigenetic, and cellular alterations, and has a potential to utilize as an antifungal agent.

Volatile Metabolite Profiling of Wheat Kernels Contaminated by Fusarium graminearum.

Traditional methods used to detect fungi or mycotoxins are time-consuming and prevent real-time monitoring. In this study, solid-phase microextraction combined with full two-dimensional gas chromatography time-of-flight mass spectrometry was utilized to detect volatile organic compounds (VOCs) produced by fungi during grain infestation predictive F. graminearum PH-1 infestation in wheat. The results show that the VOCs emitted by F. graminearum can distinguish strains at different growth stages. The growth matrices (potato dextrose agar medium and wheat kernels) play a large role in VOC production. The infection of wheat sample F. graminearum showed that a specific relationship between VOCs and the composition of fungal flora, for example, 5-pentyl-cyclohexa-1,3-diene, 3-hexanone, and 1,3-octadiene, was positively correlated with the infection rate of PH-1. In the correlation study of fungal mycotoxins and VOCs, zearalenone produced by F. graminearum was predicted based on the VOCs released. Further analysis determined the correlation of three VOCs, 6-butyl-1,4-cycloheptadiene, hexahydro-3-methylenebenzofuran-2(3H)-one, and (E,E)-3,5-octadien-2-one, with zearalenone production, confirming the ability of VOCs as characteristic markers of mycotoxins.

The Wheat Head Blight Pathogen Fusarium graminearum Can Recruit Collaborating Bacteria from Soil.

In nature, fungal endophytes often have facultative endohyphal bacteria (FEB). Can a model plant pathogenic fungus have them, and does it affect their phenotype? We constructed a growth system/microcosm to allow an F. graminearum isolate to grow through natural soil and then re-isolated it on a gentamicin-containing medium, allowing endohyphal growth of bacteria while killing other bacteria. F. graminearum PH-1 labelled with a His1mCherry gene staining the fungal nuclei fluorescent red was used to confirm the re-isolation of the fungus. Most new re-isolates contained about 10 16SrRNA genes per fungal mCherry gene determined by qPCR. The F. graminearum + FEB holobiont isolates containing the bacteria were sub-cultured several times, and their bacterial contents were stable. Sequencing the bacterial 16SrRNA gene from several Fg-FEB holobiont isolates revealed endophytic bacteria known to be capable of nitrogen fixation. We tested the pathogenicity of one common Fg-FEB holobiont association, F. graminearum + Stenatrophomonas maltophilia, and found increased pathogenicity. The 16SrRNA gene load per fungal His1mCherry gene inside the wheat stayed the same as previously found in vitro. Finally, strong evidence was found for Fg-S. maltophilia symbiotic nitrogen fixation benefitting the fungus.

Gene Regulatory Network Inference and Gene Module Regulating Virulence in Fusarium oxysporum.

In this work, we inferred the gene regulatory network (GRN) of the fungus Fusarium oxysporum by using the regulatory networks of Aspergillus nidulans FGSC A4, Neurospora crassa OR74A, Saccharomyces cerevisiae S288c, and Fusarium graminearum PH-1 as templates for sequence comparisons. Topological properties to infer the role of transcription factors (TFs) and to identify functional modules were calculated in the GRN. From these analyzes, five TFs were identified as hubs, including FOXG_04688 and FOXG_05432, which regulate 2,404 and 1,864 target genes, respectively. In addition, 16 communities were identified in the GRN, where the largest contains 1,923 genes and the smallest contains 227 genes. Finally, the genes associated with virulence were extracted from the GRN and exhaustively analyzed, and we identified a giant module with ten TFs and 273 target genes, where the most highly connected node corresponds to the transcription factor FOXG_05265, homologous to the putative bZip transcription factor CPTF1 of Claviceps purpurea, which is involved in ergotism disease that affects cereal crops and grasses. The results described in this work can be used for the study of gene regulation in this organism and open the possibility to explore putative genes associated with virulence against their host.

A Novel In Planta Enrichment Method Employing Fusarium graminearum-Infected Wheat Spikes to Select for Competitive Biocontrol Bacteria.

This work introduces an alternative workflow for the discovery of novel bacterial biocontrol agents in wheat against Fusarium head blight (FHB). Unlike the mass testing of isolate collections, we started from a diverse inoculum by extracting microbiomes from ears of field-grown plants at grain filling stage. Four distinct microbial communities were generated which were exposed to 3 14-day culture-independent experimental enrichments on detached wheat spikes infected with F. graminearum PH1. We found that one bacterial community reduced infection symptoms after 3 cycles, which was chosen to subsequently isolate bacteria through limiting dilution. All 94 isolates were tested in an in vitro and in planta assay, and a selection of 14 isolates was further tested on detached ears. The results seem to indicate that our enrichment approach resulted in bacteria with different modes-of-action in regard to FHB control. Erwinia persicina isolate C3 showed a significant reduction in disease severity (Fv/Fm), and Erwinia persicina C3 and Pseudomonas sp. B3 showed a significant reduction in fungal biomass (cGFP). However, the mycotoxin analysis of both these treatments showed no reduction in DON levels. Nevertheless, Pantoea ananatis H3 and H11 and Erwinia persicina H2 were able to reduce DON concentrations by more than 50%, although these effects were not statistically significant. Lastly, Erwinia persicina H2 also showed a significantly greater glucosylation of DON to the less phytotoxic DON-3G. The bacterial genera isolated through the enrichment cycles have been reported to dominate microbial communities that develop in open habitats, showing strong indications that the isolated bacteria can reduce the infection pressure of F. graminearum on the spike phyllosphere.

Rapid Detection of Peach Shoot Blight Caused by Phomopsis amygdali Utilizing a New Target Gene Identified from Genome Sequences Within Loop-Mediated Isothermal Amplification.

Peach shoot blight (PSB), caused by Phomopsis amygdali, is a serious threat to the healthy development of the peach industry and leads to 30 to 50% damage to peach production in southern China. In this study, loop-mediated isothermal amplification (LAMP) technology was used to detect the P. amygdali target of a gene of GME6801 that was unique in the whole genome of the pathogen compared with that of Diaporthe (Phomopsis) longicolla TWH P74, Fusarium graminearum PH-1, Colletotrichum gloeosporioides SMCG1 and Magnaporthe oryzae 70-15. Blast comparison of this gene sequence in NCBI database showed that no homologous sequences were found. Therefore, the gene sequence of GME6801 was used to design two pairs of LAMP primers and one pair of PCR primers. The results showed that both primer sets were specific to the 15 strains of P. amygdali, and the other 15 fungal strains presented negative reactions, similar to the control. In addition, 50 pg of genomic DNA of P. amygdali in a 25-μl reaction system could be detected by LAMP assay, which was 100 times more sensitive than PCR. Furthermore, the GME6801 LAMP assay was used to detect artificially inoculated twigs of the pathogen, disease twigs within significantly symptomatic PSB in the fields, and healthy twigs in the same orchard, with detection rates of 100, 75, and 20.8%, respectively. However, detection rates of conventional PCR were separately 100, 62.5, and 16.7%. The results indicated that GME6801-based LAMP could be used for P. amygdali detection as its specificity, sensitivity, and simplicity. This study provides a rapid experimental basis for the identification and prediction of P. amygdali that causes PSB and is beneficial for precise prevention and control of the disease.

Mycosubtilin Produced by Bacillus subtilis ATCC6633 Inhibits Growth and Mycotoxin Biosynthesis of Fusarium graminearum and Fusarium verticillioides.

Fusarium graminearum and Fusarium verticillioides are fungal pathogens that cause diseases in cereal crops, such as Fusarium head blight (FHB), seedling blight, and stalk rot. They also produce a variety of mycotoxins that reduce crop yields and threaten human and animal health. Several strategies for controlling these diseases have been developed. However, due to a lack of resistant cultivars and the hazards of chemical fungicides, efforts are now focused on the biocontrol of plant diseases, which is a more sustainable and environmentally friendly approach. In the present study, the lipopeptide mycosubtilin purified from Bacillus subtilis ATCC6633 significantly suppressed the growth of F. graminearum PH-1 and F. verticillioides 7600 in vitro. Mycosubtilin caused the destruction and deformation of plasma membranes and cell walls in F. graminearum hyphae. Additionally, mycosubtilin inhibited conidial spore formation and germination of both fungi in a dose-dependent manner. In planta experiments demonstrated the ability of mycosubtilin to control the adverse effects caused by F. graminearum and F. verticillioides on wheat heads and maize kernels, respectively. Mycosubtilin significantly decreased the production of deoxynivalenol (DON) and B-series fumonisins (FB1, FB2 and FB3) in infected grains, with inhibition rates of 48.92, 48.48, 52.42, and 59.44%, respectively. The qRT-PCR analysis showed that mycosubtilin significantly downregulated genes involved in mycotoxin biosynthesis. In conclusion, mycosubtilin produced by B. subtilis ATCC6633 was shown to have potential as a biological agent to control plant diseases and Fusarium toxin contamination caused by F. graminearum and F. verticillioides.

Isolation, Identification, and Complete Genome Assembly of an Endophytic Bacillus velezensis YB-130, Potential Biocontrol Agent Against Fusarium graminearum.

Wheat scab caused by F. graminearum is a highly destructive disease that leads to yield reduction and mycotoxin contamination of grains. In this study, an endophytic bacterium of strain YB-130 was isolated from surface sterilized wheat spikes with scab symptoms and identified as Bacillus velezensis by whole genome annotation, 16S rRNA gene and average nucleotide identities analysis. The whole-genome sequence of strain YB-130 was obtained by PacBio sequencing. 88 putative Carbohydrate-Active Enzymes and 12 gene clusters encoding for secondary metabolites were identified in the YB-130 genome, including one gene cluster for the synthesis of lanthipeptide only found in strain YB-130 genome. In dual cultures, strain YB-130 significantly inhibited the growth of F. graminearum PH-1 and other eight fungal plant pathogens, indicating a broad antifungal activity. Furthermore, strain YB-130 was able to significantly inhibit spore morphology and hyphal development of F. graminearum PH-1. Strain YB-130 also reduced deoxynivalenol production by F. graminearum PH-1 in dual cultures, possibly due to its ability to suppress the expression of tri5, tri3, and tri8 that are required for deoxynivalenol production in F. graminearum. Overall, B. velezensis YB-130 is a promising biological control agent of both F. graminearum infection and mycotoxin production.

One Small RNA of Fusarium graminearum Targets and Silences CEBiP Gene in Common Wheat

The pathogenic fungus Fusarium graminearum (F. graminearum), causing Fusarium head blight (FHB) or scab, is one of the most important cereal killers worldwide, exerting great economic and agronomic losses on global grain production. To repress pathogen invasion, plants have evolved a sophisticated innate immunity system for pathogen recognition and defense activation. Simultaneously, pathogens continue to evolve more effective means of invasion to conquer plant resistance systems. In the process of co-evolution of plants and pathogens, several small RNAs (sRNAs) have been proved in regulating plant immune response and plant-microbial interaction. In this study, we report that a F. graminearum sRNA (Fg-sRNA1) can suppress wheat defense response by targeting and silencing a resistance-related gene, which codes a Chitin Elicitor Binding Protein (TaCEBiP). Transcriptional level evidence indicates that Fg-sRNA1 can target TaCEBiP mRNA and trigger silencing of TaCEBiP in vivo, and in Nicotiana benthamiana (N. benthamiana) plants, Western blotting experiments and YFP Fluorescence observation proofs show that Fg-sRNA1 can suppress the accumulation of protein coding by TaCEBiP gene in vitro. F. graminearum PH-1 strain displays a weakening ability to invasion when Barley stripe mosaic virus (BSMV) vector induces effective silencing Fg-sRNA1 in PH-1 infected wheat plants. Taken together, our results suggest that a small RNA from F. graminearum can target and silence the wheat TaCEBiP gene to enhance invasion of F. graminearum.

Imaging Gene Expression Dynamics in Pseudomonas fluorescens In5 duringInteractions with the Fungus Fusarium graminearum PH-1.

Genomics, transcriptomics and metabolomics are powerful technologies for studying microbial interactions. The main drawback of these methods is the requirement for destructive sampling. We have established an alternative but complementary technique based on a microplate system combined with promoter fusions for visualizing gene expression in space and time. Here we provide a protocol for measuring spatial and temporal gene expression of a bacterial reporter strain interacting with a fungus on a solid surface.

Genome analysis of the ubiquitous boxwood pathogen Pseudonectria foliicola.

Boxwood (Buxus spp.) are broad-leaved, evergreen landscape plants valued for their longevity and ornamental qualities. Volutella leaf and stem blight, caused by the ascomycete fungi Pseudonectria foliicola and P. buxi, is one of the major diseases affecting the health and ornamental qualities of boxwood. Although this disease is less severe than boxwood blight caused by Calonectria pseudonaviculata and C. henricotiae, its widespread occurrence and disfiguring symptoms have caused substantial economic losses to the ornamental industry. In this study, we sequenced the genome of P. foliicola isolate ATCC13545 using Illumina technology and compared it to other publicly available fungal pathogen genomes to better understand the biology of this organism. A de novo assembly estimated the genome size of P. foliicola at 28.7 Mb (425 contigs; N50 = 184,987 bp; avg. coverage 188×), with just 9,272 protein-coding genes. To our knowledge, P. foliicola has the smallest known genome within the Nectriaceae. Consistent with the small size of the genome, the secretome, CAzyme and secondary metabolite profiles of this fungus are reduced relative to two other surveyed Nectriaceae fungal genomes: Dactylonectria macrodidyma JAC15-245 and Fusarium graminearum Ph-1. Interestingly, a large cohort of genes associated with reduced virulence and loss of pathogenicity was identified from the P. foliicola dataset. These data are consistent with the latest observations by plant pathologists that P. buxi and most likely P. foliicola, are opportunistic, latent pathogens that prey upon weak and stressed boxwood plants.

Wheat microRNA1023 suppresses invasion of Fusarium graminearum via targeting and silencing FGSG_03101

ABSTRACTFusarium graminearum (F. graminearum) is a destructive pathogenic fungus that causes head blight or scab in wheat, barley and other cereals. To repress pathogen invasion, plants have evolved a sophisticated innate immunity system for pathogen recognition and defense activation. In plant immunity signaling pathways, a lot of small RNAs (sRNAs) have been proved in regulating plant immune response and plant–microbial interaction. In this study, we report that a wheat microRNA (miR1023) can suppress the invasion of F. graminearum by targeting and silencing FGSG_03101 which codes an alpha/beta hydrolase gene in F. graminearum. Transcriptional level evidence indicates that Tae-miR1023 can target FGSG_03101 mRNA and trigger silencing of FGSG_03101 in vivo, and translation level proof shows that Tae-miR1023 can suppress the accumulation of alpha/beta Hydrolase coding by FGSG_03101 in vitro. F. graminearum PH-1 FGSG_03101 mutant strain displays a weakening ability to invasion and PH-1 Argonaute like gene mutant strains with transferred artificial Tae-miR1023 show enhancing relative transcript level of FGSG_03101, compared with PH-1 wild-type strain. Taken together, our results suggest that wheat miR1023 can target and silence fungal FGSG_03101 to suppress invasion of F. graminearum.

Evidence of an Unidentified Extracellular Heat-Stable Factor Produced by Lysobacter enzymogenes (OH11) that Degrade Fusarium graminearum PH1 Hyphae.

Lysobacter enzymogenes OH11 produces heat-stable antifungal factor (HSAF) and lytic enzymes possessing antifungal activity. This study bio-prospected for other potential antifungal factors besides those above. The cells and extracellular metabolites of L. enzymogenes OH11 and the mutants ΔchiA, ΔchiB, ΔchiC, Δclp, Δpks, and ΔpilA were examined for antifungal activity against Fusarium graminearum PH1, the causal agent of Fusarium head blight (FHB). Results evidenced that OH11 produces an unidentified extracellular heat-stable degrading metabolite (HSDM) that exhibit degrading activity on F. graminearum PH1 chitinous hyphae. Interestingly, both heat-treated and non-heat-treated extracellular metabolites of OH11 mutants exhibited hyphae-degrading activity against F. graminearum PH1. Enzyme activity detection of heat-treated metabolites ruled out the possibility of enzyme degradation activity. Remarkably, the PKS-NRPS-deficient mutant Δpks cannot produce HSAF or analogues, yet its metabolites exhibited hyphae-degrading activity. HPLC analysis confirmed no HSAF production by Δpks. Δclp lacks hyphae-degrading ability. Therefore, clp regulates HSDM and extracellular lytic enzymes production in L. enzymogenes OH11. ΔpilA had impaired surface cell motility and significantly reduced antagonistic properties. ΔchiA, ΔchiB, and ΔchiC retained hyphae-degrading ability, despite having reduced abilities to produce chitinase enzymes. Ultimately, L. enzymogenes OH11 can produce other unidentified HSDM independent of the PKS-NRPS genes. This suggests HSAF and lytic enzymes production are a fraction of the antifungal mechanisms in OH11. Characterization of HSDM, determination of its biosynthetic gene cluster and understanding its mode of action will provide new leads in the search for effective drugs for FHB management.

The Fungus Trichoderma Regulates Submerged Conidiation Using the Steroid Pregnenolone.

In previous work, we evolved a population of Trichoderma citrinoviride in liquid cultures to speed up its asexual development cycle. The evolved population, called T-6, formed conidia 3 times sooner and in >1000-fold greater numbers. Here, we identify the steroid pregnenolone as a molecular signal for this different behavior. Media in which the ancestral T. citrinoviride population was grown (called ancestral spent media) contained a submerged conidiation inhibitor. Growing the evolved population T-6 in ancestral spent media eliminated the abundant formation of conidia. This inhibition depended on the amount and age of the ancestral spent medium and the time that the ancestral spent medium was added to the T-6 culture. Fractionation of the ancestral spent medium identified a hydrophobic inhibiting compound with a molecular weight less than 2000 g/mol. A combination of GC-MS, ELISA, and reaction with cholesterol oxidase identified it as pregnenolone. The addition of pregnenolone to cultures of T-6 inhibited submerged conidiation by inhibiting formation of conidiophores, while 10 other analogous steroids did not. Pregnenolone also inhibited submerged conidiation of Fusarium graminearum PH-1, a plant pathogen that causes head blight in wheat and barley. This identification of steroids as signal molecules in fungi creates opportunities to disrupt this signaling to control fungal behavior.

Real-time imaging of the growth-inhibitory effect of JS399-19 on Fusarium

Real-time imaging was used to study the effects of a novel Fusarium-specific cyanoacrylate fungicide (JS399-19) on growth and morphology of four Fusarium sp. This fungicide targets the motor domain of type I myosin. Fusarium graminearum PH-1, Fusarium solani f. sp. pisi 77-13-4, Fusarium avenaceum IBT8464, and Fusarium avenaceum 05001, which has a K216Q amino-acid substitution at the resistance-implicated site in its myosin type I motor domain, were analyzed. Real-time imaging shows that JS399-19 inhibits fungal growth but not to the extent previously reported. The fungicide causes the hypha to become entangled and unable to extend vertically. This implies that type I myosin in Fusarium is essential for hyphal and mycelia propagation. The K216Q substitution correlates with reduced susceptibility in F. avenaceum.

Separation and purification of deoxynivalenol (DON) mycotoxin from wheat culture using a simple two-step silica gel column chromatography

Deoxynivalenol (DON) is a type B trichothecenes mycotoxin produced by several Fusarium species, often found in foodstuffs for humans and animals. DON is in great demand for the toxicological researches both in vivo and in vitro. In this work, wheat culture was inoculated with a Fusarium graminearum PH-1 strain for DON production. The solvent system for crude extraction was acetonitrile-water (84:16, v/v). A simple two-step silica gel column chromatography was employed to separate the DON mycotoxin from wheat culture, combined with preparative high performance liquid chromatography (preparative HPLC) to purify the compound. The solvent system for the second silica gel column chromatography was methylene chloride-methanol (17:1, v/v), which provided a good elution effect selected on thin layer chromatography (TLC). The target compound was identified by HPLC, and the chemical structure was confirmed by mass spectrometry (MS) and 1H and 13C nuclear magnetic resonance (NMR) spectroscopy. A total of 433 mg of purified DON was obtained from 1 kg of wheat culture, with a purity of 99.01%. The study had provided an easy-operating and cost-effective method to isolate an expensive compound in a simple way.