Fusarium head blight (FHB), a typical climate-dependent disease, not only inflicts substantial yield losses but also the pathogen secretes harmful secondary metabolites, such as deoxynivalenol (DON), posing a serious threat to food and grain safety. Thymol, a representative plant essential oil, is extensively utilized in the biomedical field. Nevertheless, research regarding the application of thymol in the control of plant disease is insufficient. In this study, the biological activity of thymol against Fusarium graminearum and Fusarium asiaticum was evaluated. Additionally, the study further revealed that thymol significantly suppresses DON production by F. asiaticum and alter the morphology of toxisomes and endoplasmic reticulum. Metabolomic analysis of F. asiaticum demonstrated that thymol significantly disrupts its metabolic balance. These results were further verified by measuring intracellular indicators such as ergosterol content and lipid droplet changes. This research can furnish a theoretical basis for the future development and utilization of thymol.

Post-harvest contamination of agroproducts including rice and peanut with aflatoxin B1 (AFB1) produced by Aspergillus flavus is a global problem in conventional and sustainable agriculture. AFB1 degradation by microbial enzymes, which convert this dangerous compound to non-toxic or less toxic derivatives, is considered as a promising detoxification approach that meets the main principles of organic farming. Advanced technologies of protein heterologous expression open wide avenues for large-scale production of such enzymes. In this study, we used a recombinant extracellular AFB1 oxidase (rAFO) from Armillaria tabescens expressed in Pichia pastoris yeast cells to degrade AFB1 in rice grain and peanut seeds artificially inoculated with a toxigenic A. flavus strain. The homogenous enzyme was purified from cell-free culture liquid using immobilized metal affinity chromatography. Treatments of the infected samples with an aqueous solution of freeze-dried rAFO preparation for 72 h at 30 or 20–22 °C resulted in a significant (2.5- and threefold) reduction in the mycotoxin contamination of peanut and rise, respectively. The obtained results showed rAFO may be promising as an environment-friendly tool for providing the safety of organic agricultural production. The possibilities to improve same rAFO characteristics to promote the practical application of the enzyme are discussed.

The ability of a cold-shock protein CspD from Bacillus thuringiensis to protect both dicots and monocots against various pathogens is well confirmed under both greenhouse and field conditions; however, the molecular basis of this phenomenon at the transcriptomic level still remains unexplored. Expression profiles of some marker genes associated with SAR/ISR nonspecific resistance pathways and ROS scavengers were examined in CspD-treated Nicotiana tabacum plants, and the RNA-seq analysis of CspD-treated plants was first carried out. The ISR markers PDF1.2 and PR4 were overexpressed locally in treated tobacco leaves with the maximum 2.4- and 5.7-fold change, respectively, reached 12 h after the leaf treatment with CspD; PDF1.2 was also up-regulated 4.8-fold four days after the inoculation of treated plants with TMV. The ROS scavenger analysis demonstrated overexpression of Cu-Zn superoxide dismutase in both treated (with the maximum 5.4-fold change observed 6 h after the treatment) leaves and leaves from the upper tier (“system” leaves, 6.5-fold change observed 4 days after the treatment). The ROS assay confirmed endogenous accumulation of superoxide in CspD-treated leaves 6 and 24 h after the treatment. An in silico comparative study of Arabidopsis orthologs of highly up-regulated tobacco genes induced by CspD with Arabidopsis genes activated by some other molecular patterns revealed the specific CspD-induced expression of Cu-Zn superoxide dismutase and some other genes associated with light and cold responses. This study may contribute to a better understanding of cross-talking between abiotic stress and nonspecific immunity in plants.

Aflatoxin B1 (AFB1) produced by some Aspergillus species belongs to the most dangerous contaminants of animal feeds. Development of safe and cost efficient decontamination methods saving feed quality and nutritional value are of paramount importance. The use of recombinant AFB1-detoxifying microbial enzymes represents a promising biotechnological approach meeting the aforementioned requirements. In this study, three AFB1-degrading oxidases (AFOs) from edible basidiomycetes Cantharellus cibarius, Lentinula edodes and Pleurotus eryngii as well as AFO from Armillaria tabescens were expressed in E. coli Rosetta (DE3) and purified by immobilized metal-chelate chromatography. The stabilizing effect of the addition of glycerol and β-mercaptoethanol during protein extraction is shown. The catalytic constants of the recombinant AFOs (rAFOs) and other characteristics, which might be important for their practical application (and optimal temperature and pH, thermolability, regulation of the activity by metal ions and chelating agents, storage stability) were investigated. Among the obtained enzymes, rAFO from P. eryngii (Pe-AFO), which was characterized by the highest specific activity, thermostability and pH stability (especially at acidic pH range), the lowest Km, and relative resistance to the inhibition by phytate, showed the best AFB1-degrading efficacy. However, Pe-AFO and all other rAFOs significantly decreased the target activity during heating above 45 °C, storage frozen or lyophilization.

An acidic shift in the pH profile of Clonostachys rosea zearalenone hydrolase (ZHD), the most effective and well-studied zearalenone-specific lactone hydrolase, is required to extend the range of applications for the enzyme as a decontamination agent in the feed and food production industries. Amino acid substitutions were engineered in the active center of the enzyme to decrease the pKa values of the catalytic residues E126 and H242. The T216K substitution provided a shift in the pH optimum by one unit to the acidic region, accompanied by a notable expansion in the pH profile under acidic conditions. The engineered enzyme demonstrated enhanced activity within the pH range of 3–5 and improved the activity within the pH ranging from 6 to 10. The D31N and D31A substitutions also resulted in a two-unit shift in the pH optimum towards acidic conditions, although this was accompanied by a significant reduction in the enzyme activity. The D31S substitution resulted in a shift in the pH profile towards the alkaline region. The alterations in the enzyme properties observed following the T216K substitution were consistent with the conditions required for the ZHD application as decontamination enzymes at acidic pH values (from 3.0 to 6.0).

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.

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.

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.

The protective effect of the MF3 protein from Pseudomonas fluorescens against Rhizoctonia solani was evaluated on potato plants, as well as its ability to augment fungicidal efficacy of difenoconazole-containing Score® EC 250 and Celest® Top FS 312.5 commercial preparations when applied jointly with these fungicides. It has been first found that the pre-planting treatment of black scurf-infected potato tubers with MF3 significantly reduces the number of affected plants comparing to the control. In parallel, MF3 implementation together with fungicides allows tenfold decreasing the fungicides dosage without reducing their protective effect. The revealed augmentation of the protective effect of aforementioned fungicidal formulations combined with MF3 achieved due to the synergism between this protein and the fungicides. The further study on the MF3 capability for improving the efficacy of triazole-based and other fungicides used against other plant pathogenic fungi is under progress.

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.

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 mu g mL(-1) with a mean EC50 value of 0.2170 +/- 0.0496 mu g 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 mu g 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. (c) 2022 Society of Chemical Industry.

Intensive use of pesticides results in environmental pollution and food contamination. Use of microbial elicitors, enhancing plant immunity, is a promising alternative to fungicidal treatments. MF3, an elicitor protein from Pseudomonas fluorescens, has a strong protective action toward a range of plant pathogens, but can be degraded by UV radiation and plant proteases under field conditions. MF3 encapsulation in biodegradable poly-3-hydroxybutyrate (PHB) protects it against the adverse external factors. Using a model plant-pathogen system, we demonstrated that the treatment of wheat leaf fragments with a MF3-PHB complex prior artificial inoculation with Parastagonospora nodorum, a major wheat pathogen, provides a higher suppression of the infection (41.5%) than the treatment with MF3 alone (28.9%) or PHB (2.5%). Thus, encapsulating this protein elicitor may provide prolonged and more efficient activity under field conditions and is able to promote the development of MF3-based biopesticides as an efficient alternative to commercial fungicides.

Fungicides used in agriculture contaminate the environment and promote the spreading of fungicide-resistant strains. Enhancement of pathogen sensitivity to pesticides using the so-called biosensitizers allows reducing of fungicide dosages with no negative effect on their efficiency. A microbial metabolite 6-demethylmevinolin (6-DMM) already proved its sensitizing activity in Bipolaris sorokiniana towards triazole fungicides. Using Petri dish bioassay and microplate assay combined with the probit analysis, non- or marginally toxic concentrations of this compound, which are used for sensitizing effect, were determined for four Fusarium fungi (F. avenaceum, F. culmorum, F. oxysporum, F. graminearum) forming a wheat root rot complex together with B. sorokiniana. The range of the determined concentrations confirmed a principal possibility to use 6-DMM as a putative sensitizer for the whole of this complex of pathogens.

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.

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 » 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.

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.

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.

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

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.