Recombinant Oxidase from Armillaria tabescens as a Potential Tool for Aflatoxin B1 Degradation in Contaminated Cereal Grain.

Forage grain contamination with aflatoxin B1 (AFB1) is a global problem, so its detoxification with the aim of providing feed safety and cost-efficiency is still a relevant issue. AFB1 degradation by microbial enzymes is considered to be a promising detoxification approach. In this study, we modified an previously developed Pichia pastoris GS115 expression system using a chimeric signal peptide to obtain a new recombinant producer of extracellular AFB1 oxidase (AFO) from Armillaria tabescens (the yield of 0.3 g/L), purified AFO, and selected optimal conditions for AFO-induced AFB1 removal from model solutions. After a 72 h exposure of the AFB1 solution to AFO at pH 6.0 and 30 °C, 80% of the AFB1 was degraded. Treatments with AFO also significantly reduced the AFB1 content in wheat and corn grain inoculated with Aspergillus flavus. In grain samples contaminated with several dozen micrograms of AFB1 per kg, a 48 h exposure to AFO resulted in at least double the reduction in grain contamination compared to the control, while the same treatment of more significantly (~mg/kg) AFB1-polluted samples reduced their contamination by ~40%. These findings prove the potential of the tested AFO for cereal grain decontamination and suggest that additional studies to stabilize AFO and improve its AFB1-degrading efficacy are required.

Some Structural Elements of Bacterial Protein MF3 That Influence Its Ability to Induce Plant Resistance to Fungi, Viruses, and Other Plant Pathogens.

The ability of the MF3 protein from Pseudomonas fluorescens to protect plants by inducing their resistance to pathogenic fungi, bacteria, and viruses is well confirmed both in greenhouses and in the field; however, the molecular basis of this phenomenon remains unexplored. To find a relationship between the primary (and spatial) structure of the protein and its target activity, we analyzed the inducing activity of a set of mutants generated by alanine scanning and an alpha-helix deletion (ahD) in the part of the MF3 molecule previously identified by our group as a 29-amino-acid peptide working as the inducer on its own. Testing the mutants' inducing activity using the "tobacco-tobacco mosaic virus" pathosystem revealed that some of them showed an almost threefold (V60A and V62A) or twofold (G51A, L58A, ahD) reduction in inducing activity compared to the wild-type MF3 type. Interestingly, these mutations demonstrated close proximity in the homology model, probably contributing to MF3 reception in a host plant.

Post-Harvest Prevention of Fusariotoxin Contamination of Agricultural Products by Irreversible Microbial Biotransformation: Current Status and Prospects.

Biological degradation of mycotoxins is a promising environmentally-friendly alternative to chemical and physical detoxification methods. To date, a lot of microorganisms able to degrade them have been described; however, the number of studies determining degradation mechanisms and irreversibility of transformation, identifying resulting metabolites, and evaluating in vivo efficiency and safety of such biodegradation is significantly lower. At the same time, these data are crucial for the evaluation of the potential of the practical application of such microorganisms as mycotoxin-decontaminating agents or sources of mycotoxin-degrading enzymes. To date, there are no published reviews, which would be focused only on mycotoxin-degrading microorganisms with the proved irreversible transformation of these compounds into less toxic compounds. In this review, the existing information about microorganisms able to efficiently transform the three most common fusariotoxins (zearalenone, deoxinyvalenol, and fumonisin B1) is presented with allowance for the data on the corresponding irreversible transformation pathways, produced metabolites, and/or toxicity reduction. The recent data on the enzymes responsible for the irreversible transformation of these fusariotoxins are also presented, and the promising future trends in the studies in this area are discussed.

Risk assessment and molecular mechanism of Fusarium incarnatum resistance to phenamacril.

BACKGROUND: Cucumber fruit rot (CFR) caused by Fusarium incarnatum is a devastating fungal disease in cucumber. In recent years, CFR has occurred frequently, resulting in serious yield and quality losses in China. Phenamacril exhibits a specific antifungal activity against Fusarium species. However, no data for phenamacril against F. incarnatum is available. RESULTS: The sensitivity of 80 F. incarnatum strains to phenamacril was determined. The half maximal effective concentration (EC50 ) values ranged from 0.1134 to 0.3261 µg mL-1 with a mean EC50 value of 0.2170 ± 0.0496 µg mL-1 . A total of seven resistant mutants were obtained from 450 mycelial plugs by phenamacril-taming on potato dextrose agar (PDA) plates with 10 µg mL-1 of phenamacril, and the resistant frequency was 1.56%. Phenamacril-resistant mutants showed decreased mycelial growth, conidiation and virulence as compared with the corresponding wild-type strains, indicating that phenamacril resistance suffered a fitness penalty in F. incarnatum. In addition, using sequence analysis, the point mutations of S217P or I424S were discovered in Fimyosin-5 (the target of phenamacril). The site-directed mutagenesis of the S217P, P217S, I424S and S424I substitutions were constructed to reveal the relationship between the point mutations and phenamacril resistance. The results strongly demonstrated that the mutations of S217P and I424S in Fimyosin-5 conferred phenamacril-resistance in F. incarnatum. CONCLUSION: Phenamacril-resistant mutants were easily induced and their resistance level was high. The S217P or I424S substitutions in Fimyosin-5 conferring phenamacril resistance were detected and futherly verified by transformation assay with site-directed mutagenesis. Thus, we proposed that the resistance development of F. incarnatum to phenamacril is high risk. © 2022 Society of Chemical Industry.

A Secondary Metabolite Secreted by Penicillium citrinum Is Able to Enhance Parastagonospora nodorum Sensitivity to Tebuconazole and Azoxystrobin.

Parastagonospora nodorum causes glume and leaf blotch of wheat, a harmful disease resulting in serious losses in grain yield. In many countries including Russia, fungicidal formulations based on triazoles and on triazoles combined with strobilurins are used to control this fungus. However, their prolonged application may promote the selection of fungicide-resistant strains of P. nodorum leading to significant attenuation or even loss of fungicidal effect. Chemosensitization of plant pathogenic fungi with natural compounds represents a promising strategy for mitigating fungicide resistance and other negative impacts of fungicides. In this work, we applied a chemosensitization approach towards P. nodorum strains non-resistant or resistant to tebuconazole or azoxystrobin using 6-demethylmevinolin (6-DMM), a metabolite of Penicillium citrinum. The resistant strains were obtained by the mutagenesis and subsequent culturing on agar media incorporated with increasing doses of Folicur® EC 250 (i.e., tebuconazole) or Quadris® SC 250 (i.e., azoxystrobin). Test strains m8-4 and kd-18, most resistant to tebuconazole and azoxystrobin, respectively, were selected for sensitization experiments. These experiments demonstrated that combining 6-DMM with Folicur® enhanced fungicidal effectiveness in vitro and in vivo in addition to attenuating the resistance of P. nodorum to tebuconazole in vitro. 6-DMM was also found to augment Quadris® efficacy towards kd-18 when applied on detached wheat leaves inoculated with this strain. Experiments on P. nodorum sensitization under greenhouse conditions included preventive (applying test compounds simultaneously with inoculation) or post-inoculation spraying of wheat seedlings with 6-DMM together with Folicur® at dose rates (DR) amounting to 10% and 20% of DR recommended for field application (RDR). Combined treatments were run in parallel with using the same DR of the fungicide and sensitizer, alone. A synergistic effect was observed in both preventive and post-inoculation treatments, when the sensitizer was co-applied with the fungicide at 10% of the RDR. In this case, disease reduction significantly exceeded the protective effect of Folicur® at 10% or 20% of the RDR, alone, and also a calculated additive effect. Collectively, our findings suggest that 6-DMM is promising as a putative component for formulations with triazole and strobilurin fungicides. Such new formulations would improve fungicide efficacy and, potentially, lower rates of fungicides needed for plant pathogen control.

Expression and Refolding of the Plant Chitinase From Drosera capensis for Applications as a Sustainable and Integrated Pest Management.

Recently, the study of chitinases has become an important target of numerous research projects due to their potential for applications, such as biocontrol pest agents. Plant chitinases from carnivorous plants of the genus Drosera are most aggressive against a wide range of phytopathogens. However, low solubility or insolubility of the target protein hampered application of chitinases as biofungicides. To obtain plant chitinase from carnivorous plants of the genus Drosera in soluble form in E.coli expression strains, three different approaches including dialysis, rapid dilution, and refolding on Ni-NTA agarose to renaturation were tested. The developed « Rapid dilution ¼ protocol with renaturation buffer supplemented by 10% glycerol and 2M arginine in combination with the redox pair of reduced/oxidized glutathione, increased the yield of active soluble protein to 9.5 mg per 1 g of wet biomass. A structure-based removal of free cysteines in the core domain based on homology modeling of the structure was carried out in order to improve the soluble of chitinase. One improved chitinase variant (C191A/C231S/C286T) was identified which shows improved expression and solubility in E. coli expression systems compared to wild type. Computational analyzes of the wild-type and the improved variant revealed overall higher fluctuations of the structure while maintaining a global protein stability. It was shown that free cysteines on the surface of the protein globule which are not involved in the formation of inner disulfide bonds contribute to the insolubility of chitinase from Drosera capensis. The functional characteristics showed that chitinase exhibits high activity against colloidal chitin (360 units/g) and high fungicidal properties of recombinant chitinases against Parastagonospora nodorum. Latter highlights the application of chitinase from D. capensis as a promising enzyme for the control of fungal pathogens in agriculture.

Molecular Insights into the Role of Cysteine-Rich Peptides in Induced Resistance to Fusarium oxysporum Infection in Tomato Based on Transcriptome Profiling.

Cysteine-rich peptides (CRPs) play an important role in plant physiology. However, their role in resistance induced by biogenic elicitors remains poorly understood. Using whole-genome transcriptome sequencing and our CRP search algorithm, we analyzed the repertoire of CRPs in tomato Solanum lycopersicum L. in response to Fusarium oxysporum infection and elicitors from F. sambucinum. We revealed 106 putative CRP transcripts belonging to different families of antimicrobial peptides (AMPs), signaling peptides (RALFs), and peptides with non-defense functions (Major pollen allergen of Olea europaea (Ole e 1 and 6), Maternally Expressed Gene (MEG), Epidermal Patterning Factor (EPF)), as well as pathogenesis-related proteins of families 1 and 4 (PR-1 and 4). We discovered a novel type of 10-Cys-containing hevein-like AMPs named SlHev1, which was up-regulated both by infection and elicitors. Transcript profiling showed that F. oxysporum infection and F. sambucinum elicitors changed the expression levels of different overlapping sets of CRP genes, suggesting the diversification of functions in CRP families. We showed that non-specific lipid transfer proteins (nsLTPs) and snakins mostly contribute to the response of tomato plants to the infection and the elicitors. The involvement of CRPs with non-defense function in stress reactions was also demonstrated. The results obtained shed light on the mode of action of F. sambucinum elicitors and the role of CRP families in the immune response in tomato.

Studying the Ability of Thymol to Improve Fungicidal Effects of Tebuconazole and Difenoconazole Against Some Plant Pathogenic Fungi in Seed or Foliar Treatments.

Thymol, a secondary plant metabolite possessing antifungal and chemosensitizing activities, disrupts cell wall or membrane integrity and interferes with ergosterol biosynthesis. Thymol also functions as a redox-active compound inducing generation of reactive oxygen species and lipid peroxidation in fungal cells. Previously, we showed thymol significantly enhanced the in vitro growth inhibitory effect of difenoconazole against Bipolaris sorokiniana and Parastagonospora nodorum. More recently, we demonstrated a possibility to use thymol to overcome the resistance of a P. nodorum strain able to grow on difenoconazole-containing media. However, potential for thymol to serve as a chemosensitizing agent in seed or plant treatments, to provide an effective suppression of the above-mentioned plant pathogens by triazole fungicides applied in lowered dosages, had yet to be tested. In the work presented here, we showed combined treatments of naturally infected barley seeds with thymol and difenoconazole (Dividend® 030 FS) synergistically exacerbated the protective effect against common root rot agent, B. sorokiniana, and other fungi (Fusarium spp. and Alternaria spp.). Similarly, co-applied treatment of wheat seeds, artificially inoculated with Fusarium culmorum, resulted in equivalent reduction of disease incidence on barley seedlings as application of Dividend®, alone, at a ten-fold higher dosage. In foliar treatments of wheat seedlings, thymol combined with Folicur® 250 EC (a.i. tebuconazole) enhanced sensitivity of P. nodorum, a glume/leaf blotch pathogen, to the fungicide and provided a significant mitigation of disease severity on treated seedlings, compared to controls, without increasing Folicur® dosages. Folicur® co-applied with thymol was also significantly more effective against a strain of P. nodorum tolerant to Folicur® alone. No additional deoxynivalenol or zearalenone production was found when a toxigenic F. culmorum was cultured in a nutrient medium containing thymol at a concentration used for chemosensitization of root rot agents. Accordingly, F. culmorum exposure to thymol at the sensitizing concentration did not up-regulate key genes associated with the biosynthesis of trichothecene or polyketide mycotoxins in this pathogen. Further studies using field trials are necessary to determine if thymol-triazole co-applications result in sensitization of seed- and foliar-associated plant pathogenic fungi, and if thymol affects production of fusarial toxins under field conditions.

Fragments of a Wheat Hevein-Like Antimicrobial Peptide Augment the Inhibitory Effect of a Triazole Fungicide on Spore Germination of Fusarium oxysporum and Alternaria solani.

There are increasing environmental risks associated with extensive use of fungicides for crop protection. Hence, the use of new approaches using natural plant defense mechanisms, including application of plant antimicrobial peptides (AMPs), is of great interest. Recently, we studied the structural-function relationships between antifungal activity and five hevein-like AMPs from the WAMP (wheat AMP) family of Triticum kiharae Dorof. et Migush. We first discovered that short peptides derived from the central, N-, and C-terminal regions of one of the WAMPs (WAMP-2) were able to augment the inhibitory effect of Folicur® EC 250, a triazole fungicide, on spore germination of the wheat pathogenic fungi, including Fusarium spp. and Alternaria alternata. In this research, we explored the ability of chemically synthesized WAMP-2-derived peptides for enhancing the sensitivity of two other Fusarium and Alternaria species, F. oxysporum and A. solani, causing wilt and early blight of tomato, respectively, to Folicur®. The synthesized WAMP-2-derived peptides synergistically interacted with the fungicide and significantly increased its efficacy, inhibiting conidial germination at much lower Folicur® concentrations than required for the same efficiency using the fungicide alone. The experiments on co-applications of some of WAMP-2-fragments and the fungicide on tomato leaves and seedlings, which confirmed the results obtained in vitro, are described.

Assessment of the Sensitivity of Some Plant Pathogenic Fungi to 6-Demethylmevinolin, a Putative Natural Sensitizer Able to Help Overcoming the Fungicide Resistance of Plant Pathogens.

Agricultural fungicides contaminate the environment and promote the spread of fungicide-resistant strains of pathogenic fungi. The enhancement of pathogen sensitivity to these pesticides using chemosensitizers allows the reducing of fungicide dosages without a decrease in their efficiency. Using Petri plate and microplate bioassays, 6-demethylmevinolin (6-DMM), a putative sensitizer of a microbial origin, was shown to affect both colony growth and conidial germination of Alternaria solani, A. alternata, Parastagonospora nodorum, Rhizoctonia solani, and four Fusarium species (F. avenaceum, F. culmorum, F. oxysporum, F. graminearum) forming a wheat root rot complex together with B. sorokiniana. Non- or marginally toxic 6-DMM concentrations suitable for sensitizing effect were determined by the probit analysis. The range of determined concentrations confirmed a possibility of using 6-DMM as a putative sensitizer for the whole complex of root rot agents, other cereal pathogens (A. alternata, P.nodorum), and some potato (R. solani, A. solani) and tomato (A. solani) pathogens. Despite the different sensitivities of the eight tested pathogens, 6-DMM lacked specificity to fungi and possessed a mild antimycotic activity that is typical of other known pathogen-sensitizing agents. The pilot evaluation of the 6-DMM sensitizing first confirmed a principal possibility of using it for the sensitization of B. sorokiniana and R. solani to triazole- and strobilurin-based fungicides, respectively.

Hevein-Like Antimicrobial Peptides Wamps: Structure-Function Relationship in Antifungal Activity and Sensitization of Plant Pathogenic Fungi to Tebuconazole by WAMP-2-Derived Peptides.

Keywords: hevein-like antimicrobial peptides; antifungal activity; antifungal determinants; synergy; chemosensitization; tebuconazole; plant pathogenic fungi.

Effective Zearalenone Degradation in Model Solutions and Infected Wheat Grain Using a Novel Heterologous Lactonohydrolase Secreted by Recombinant Penicillium canescens.

This paper reports the first results on obtaining an enzyme preparation that might be promising for the simultaneous decontamination of plant feeds contaminated with a polyketide fusariotoxin, zearalenone (ZEN), and enhancing the availability of their nutritional components. A novel ZEN-specific lactonohydrolase (ZHD) was expressed in a Penicillium canescens strain PCA-10 that was developed previously as a producer of different hydrolytic enzymes for feed biorefinery. The recombinant ZHD secreted by transformed fungal clones into culture liquid was shown to remove the toxin from model solutions, and was able to decontaminate wheat grain artificially infected with a zearalenone-producing Fusarium culmorum. The dynamics of ZEN degradation depending on the temperature and pH of the incubation media was investigated, and the optimal values of these parameters (pH 8.5, 30 °C) for the ZHD-containing enzyme preparation (PR-ZHD) were determined. Under these conditions, the 3 h co-incubation of ZEN and PR-ZHD resulted in a complete removal of the toxin from the model solutions, while the PR-ZHD addition (8 mg/g of dried grain) to flour samples prepared from the infected ZEN-polluted grain (about 16 µg/g) completely decontaminated the samples after an overnight exposure.

Non-Specific Lipid Transfer Proteins in Triticum kiharae Dorof. et Migush.: Identification, Characterization and Expression Profiling in Response to Pathogens and Resistance Inducers.

Non-specific lipid-transfer proteins (nsLTPs) represent a family of plant antimicrobial peptides (AMPs) implicated in diverse physiological processes. However, their role in induced resistance (IR) triggered by non-pathogenic fungal strains and their metabolites is poorly understood. In this work, using RNA-seq data and our AMP search pipeline, we analyzed the repertoire of nsLTP genes in the wheat Triticum kiharae and studied their expression in response to Fusarium oxysporum infection and treatment with the intracellular metabolites of Fusarium sambucinum FS-94. A total of 243 putative nsLTPs were identified, which were classified into five structural types and characterized. Expression analysis showed that 121 TkLTPs including sets of paralogs with identical mature peptides displayed specific expression patters in response to different treatments pointing to their diverse roles in resistance development. We speculate that upregulated nsLTP genes are involved in protection due to their antimicrobial activity or signaling functions. Furthermore, we discovered that in IR-displaying plants, a vast majority of nsLTP genes were downregulated, suggesting their role as negative regulators of immune mechanisms activated by the FS-94 elicitors. The results obtained add to our knowledge of the role of nsLTPs in IR and provide candidate molecules for genetic engineering of crops to enhance disease resistance.

Defensin-like peptides in wheat analyzed by whole-transcriptome sequencing: a focus on structural diversity and role in induced resistance.

Antimicrobial peptides (AMPs) are the main components of the plant innate immune system. Defensins represent the most important AMP family involved in defense and non-defense functions. In this work, global RNA sequencing and de novo transcriptome assembly were performed to explore the diversity of defensin-like (DEFL) genes in the wheat Triticum kiharae and to study their role in induced resistance (IR) mediated by the elicitor metabolites of a non-pathogenic strain FS-94 of Fusarium sambucinum. Using a combination of two pipelines for DEFL mining in transcriptome data sets, as many as 143 DEFL genes were identified in T. kiharae, the vast majority of them represent novel genes. According to the number of cysteine residues and the cysteine motif, wheat DEFLs were classified into ten groups. Classical defensins with a characteristic 8-Cys motif assigned to group 1 DEFLs represent the most abundant group comprising 52 family members. DEFLs with a characteristic 4-Cys motif CX{3,5}CX{8,17}CX{4,6}C named group 4 DEFLs previously found only in legumes were discovered in wheat. Within DEFL groups, subgroups of similar sequences originated by duplication events were isolated. Variation among DEFLs within subgroups is due to amino acid substitutions and insertions/deletions of amino acid sequences. To identify IR-related DEFL genes, transcriptional changes in DEFL gene expression during elicitor-mediated IR were monitored. Transcriptional diversity of DEFL genes in wheat seedlings in response to the fungus Fusarium oxysporum, FS-94 elicitors, and the combination of both (elicitors + fungus) was demonstrated, with specific sets of up- and down-regulated DEFL genes. DEFL expression profiling allowed us to gain insight into the mode of action of the elicitors from F. sambucinum. We discovered that the elicitors up-regulated a set of 24 DEFL genes. After challenge inoculation with F. oxysporum, another set of 22 DEFLs showed enhanced expression in IR-displaying seedlings. These DEFLs, in concert with other defense molecules, are suggested to determine enhanced resistance of elicitor-pretreated wheat seedlings. In addition to providing a better understanding of the mode of action of the elicitors from FS-94 in controlling diseases, up-regulated IR-specific DEFL genes represent novel candidates for genetic transformation of plants and development of pathogen-resistant crops.

An Extract Purified from the Mycelium of a Tomato Wilt-Controlling Strain of Fusarium sambucinum Can Protect Wheat against Fusarium and Common Root Rots.

An approach to manage seed-transmitted Fusarium crown-foot-root rot (FCR, Fusarium spp.) and common root rot (CRR, Bipolaris sorokiniana) on wheat, avoiding environmental risks of chemicals, is seed treatments with microbial metabolites. F. sambucinum strain FS-94 that induces resistance to tomato wilt was shown by this study to be a source of non-fungitoxic wheat-protecting metabolites, which were contained in a mycelium extract purified by gel-chromatography and ultrafiltration. Plant-protecting effect of the purified mycelial extract (PME) was demonstrated in vegetation experiments using a rolled-towel assay and by small-plot field trials. To elucidate mechanisms putatively underlying PME protective activity, tests with cultured Triticum aestivum and T. kiharae cells, particularly the extracellular alkalinization assay, as well as gene expression analysis in germinated wheat seeds were used. Pre-inoculation treatments of seeds with PME significantly decreased the incidence (from 30 to 40%) and severity (from 37 to 50%) of root rots on seedlings without any inhibition of the seed germination and potentiation of deoxynivalenol (DON), DON monoacetylated derivatives and zearalenon production in FCR agents. In vegetation experiments, reductions in the DON production were observed with doses of 0.5 and 1 mg/mL of PME. Pre-sowing PME application on seeds of two spring wheat cultivars naturally infected with FCR and CRR provided the mitigation of both diseases under field conditions during four growing seasons (2013⁻2016). PME-induced ion exchange response in cultured wheat cells, their increased survivability, and up-regulated expression of some defensins' genes in PME-exposed seedlings allow the suggestion of the plant-mediated character of disease-controlling effect observed in field.

Effect of Various Compounds Blocking the Colony Pigmentation on the Aflatoxin B1 Production by Aspergillus flavus.

Aflatoxins and melanins are the products of a polyketide biosynthesis. In this study, the search of potential inhibitors of the aflatoxin B1 (AFB1) biosynthesis was performed among compounds blocking the pigmentation in fungi. Four compounds-three natural (thymol, 3-hydroxybenzaldehyde, compactin) and one synthetic (fluconazole)-were examined for their ability to block the pigmentation and AFB1 production in Aspergillus flavus. All compounds inhibited the mycelium pigmentation of a fungus growing on solid medium. At the same time, thymol, fluconazole, and 3-hydroxybenzaldehyde stimulated AFB1 accumulation in culture broth of A. flavus under submerged fermentation, whereas the addition of 2.5 µg/mL of compactin resulted in a 50× reduction in AFB1 production. Moreover, compactin also suppressed the sporulation of A. flavus on solid medium. In vivo treatment of corn and wheat grain with compactin (50 µg/g of grain) reduced the level of AFB1 accumulation 14 and 15 times, respectively. Further prospects of the compactin study as potential AFB1 inhibitor are discussed.

Aflatoxin B1 Degradation by Metabolites of Phoma glomerata PG41 Isolated From Natural Substrate Colonized by Aflatoxigenic Aspergillus flavus.

BACKGROUND: Aflatoxin B1 (AFB1), produced by Aspergillus flavus, is one of the most life threatening food contaminants causing significant economic losses worldwide. Biological AFB1 degradation by microorganisms, or preferably microbial enzymes, is considered as one of the most promising approaches. OBJECTIVES: The current work aimed to study the AFB1-degrading metabolites, produced by Phoma glomerata PG41, sharing a natural substrate with aflatoxigenic A. flavus, and the preliminary determination of the nature of these metabolites. MATERIALS AND METHODS: The AFB1-degrading potential of PG41 metabolites was determined by a quantitative high performance liquid chromatography (HPLC) of residual AFB1 after 72 hours incubation at 27ºC. The effects of pH, heat, and protease treatment on the AFB1-destroying activity of extracellular metabolites were examined. RESULTS: The AFB1-degrading activity of protein-enriched fractions, isolated from culture liquid filtrate and cell-free extract, is associated with high-molecular-weight components, is time- and pH-dependent, thermolabile, and is significantly reduced by proteinase K treatment. The AFB1 degradation efficiency of these fractions reaches 78% and 66%, respectively. CONCLUSIONS: Phoma glomerata PG41 strain sharing natural substrate with toxigenic A. flavus secretes metabolites possessing a significant aflatoxin-degrading activity. The activity is associated mainly with a protein-enriched high-molecular-weight fraction of extracellular metabolites and appears to be of enzymatic origin.

Identification of a Novel Small Cysteine-Rich Protein in the Fraction from the Biocontrol Fusarium oxysporum Strain CS-20 that Mitigates Fusarium Wilt Symptoms and Triggers Defense Responses in Tomato.

The biocontrol effect of the non-pathogenic Fusarium oxysporum strain CS-20 against the tomato wilt pathogen F. oxysporum f. sp. lycopersici (FOL) has been previously reported to be primarily plant-mediated. This study shows that CS-20 produces proteins, which elicit defense responses in tomato plants. Three protein-containing fractions were isolated from CS-20 biomass using size exclusion chromatography. Exposure of seedling roots to one of these fractions prior to inoculation with pathogenic FOL strains significantly reduced wilt severity. This fraction initiated an ion exchange response in cultured tomato cells resulting in a reversible alteration of extracellular pH; increased tomato chitinase activity, and induced systemic resistance by enhancing PR-1 expression in tomato leaves. Two other protein fractions were inactive in seedling protection. The main polypeptide (designated CS20EP), which was specifically present in the defense-inducing fraction and was not detected in inactive protein fractions, was identified. The nucleotide sequence encoding this protein was determined, and its complete amino acid sequence was deduced from direct Edman degradation (25 N-terminal amino acid residues) and DNA sequencing. The CS20EP was found to be a small basic cysteine-rich protein with a pI of 9.87 and 23.43% of hydrophobic amino acid residues. BLAST search in the NCBI database showed that the protein is new; however, it displays 48% sequence similarity with a hypothetical protein FGSG_10784 from F. graminearum strain PH-1. The contribution of CS20EP to elicitation of tomato defense responses resulting in wilt mitigating is discussed.

Chemosensitization of plant pathogenic fungi to agricultural fungicides.

A common consequence of using agricultural fungicides is the development of resistance by fungal pathogens, which undermines reliability of fungicidal effectiveness. A potentially new strategy to aid in overcoming or minimizing this problem is enhancement of pathogen sensitivity to fungicides, or "chemosensitization." Chemosensitization can be accomplished by combining a commercial fungicide with a certain non- or marginally fungicidal substance at levels where, alone, neither compound would be effective. Chemosensitization decreases the probability of the pathogen developing resistance, reduces the toxic impact on the environment by lowering effective dosage levels of toxic fungicides, and improves efficacy of antifungal agents. The present study shows that the antifungal activity of azole and strobilurin fungicides can be significantly enhanced through their co-application with certain natural or synthetic products against several economically important plant pathogenic fungi. Quadris (azoxystrobin) combined with thymol at a non-fungitoxic concentration produced much higher growth inhibition of Bipolaris sorokiniana, Phoma glomerata, Alternaria sp. and Stagonospora nodorum than the fungicide alone. The effect of Dividend (difenoconazole) applied with thymol significantly enhanced antifungal activity against B. sorokiniana and S. nodorum. Folicur (tebuconazole) combined with 4-hydroxybenzaldehyde (4-HBA), 2,3-dihydroxybenzaldehyde or thymol significantly inhibited growth of Alternaria alternata, at a much greater level than the fungicide alone. In addition, co-application of Folicur and 4-HBA resulted in a similar enhancement of antifungal activity against Fusarium culmorum. Lastly, we discovered that metabolites in the culture liquid of Fusarium sambucinum biocontrol isolate FS-94 also had chemosensitizing activity, increasing S. nodorum sensitivity to Folicur and Dividend.