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.

Hevein-like antimicrobial peptides (AMPs) comprise a family of plant AMPs with antifungal activity, which harbor a chitin-binding site involved in interactions with chitin of fungal cell walls. However, the mode of action of hevein-like AMPs remains poorly understood. This work reports the structure–function relationship in WAMPs—hevein-like AMPs found in wheat (Triticum kiharae Dorof. et Migush.) and later in other Poaceae species. The effect of WAMP homologues differing at position 34 and the antifungal activity of peptide fragments derived from the central, N- and C-terminal regions of one of the WAMPs, namely WAMP-2, on spore germination of different plant pathogenic fungi were studied. Additionally, the ability of WAMP-2-derived peptides to potentiate the fungicidal effect of tebuconazole, one of the triazole fungicides, towards five cereal-damaging fungi was explored in vitro by co-application of WAMP-2 fragments with Folicur^® EC 250 (25% tebuconazole). The antifungal activity of WAMP homologues and WAMP-2-derived peptides varied depending on the fungus, suggesting multiple modes of action for WAMPs against diverse pathogens. Folicur^® combined with the WAMP-2 fragments inhibited the spore germination at a much greater level than the fungicide alone, and the type of interactions was either synergistic or additive, depending on the target fungus and concentration combinations of the compounds. The combinations, which resulted in synergism and drastically enhanced the sensitivity to tebuconazole, were revealed for all five fungi by a checkerboard assay. The ability to synergistically interact with a fungicide and exacerbate the sensitivity of plant pathogenic fungi to a commercial antifungal agent is a novel and previously uninvestigated property of hevein-like AMPs.

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.

Create a method for highly sensitive, selective, rapid, and easy-to-use detection and identification of economically significant potato pathogens, including viruses, bacteria and oomycetes, be it single pathogen, or a range of various pathogens occurring simultaneously. Test-systems for real-time PCR, operating in the unified amplification regime, have been developed for Phytophthora infestans, Pectobacterium atrosepticum, Dickeya dianthicola, Dickeya solani, Ralstonia solanacearum, Pectobacterium carotovorum, Clavibacter michiganensis subsp. sepedonicus, potato viruses Y (ordinary and necrotic forms as well as indiscriminative test system, detecting all forms), A, X, S, M, potato leaf roll virus, potato mop top virus, and potato spindle tuber viroid. The test-systems (including polymerase and revertase) were immobilized and lyophilized in miniature microreactors (1.2 μl) on silicon DNA/RNA microarrays (micromatrices) to be used with a mobile AriaDNA^® amplifier. Preloaded 30-reaction micromatrices having shelf life of 3 and 6 months (for RNA- and DNA-based pathogens, respectively) at room temperature with no special conditions were successfully tested on both reference and field samples in comparison with traditional ELISA and microbiological methods, showing perfect performance and sensitivity (1 pg). The accurate, rapid and user-friendly diagnostic system in a micromatrix format may significantly contribute to pathogen screening and phytopathological studies.

Viral and bacterial diseases of potato cause significant yield loss worldwide. The current data on the occurrence of these diseases in Russia do not provide comprehensive understanding of the phytosanitary situation. Diagnostic systems based on disposable stationary open qPCR micromatrices intended for the detection of eight viral and seven bacterial/oomycetal potato diseases have been used for wide-scale screening of target pathogens to estimate their occurrence in 11 regions of Russia and to assess suitability of the technology for high-throughput diagnostics under conditions of field laboratories. Analysis of 1025 leaf and 725 tuber samples confirmed the earlier reported data on the dominance of potato viruses Y, S, and M in most regions of European Russia, as well as relatively high incidences of Clavibacter michiganensis subsp. sepedonicus, Pectobacterium atrosepticum, and P. carotovorum subsp. carotovorum, and provided detailed information on the phytosanitary status of selected regions and geographical spread of individual pathogens. Information on the occurrence of mixed infections, including their composition, was the first data set of this kind for Russia. The study is the first large-scale screening of a wide range of potato pathogens conducted in network mode using unified methodology and standardized qPCR micromatrices. The data represent valuable information for plant pathologists and potato producers and indicate the high potential of the combined use of matrix PCR technology and network approaches to data collection and analysis with the view to rapidly and accurately assess the prevalence of certain pathogens, as well as the phytosanitary state of large territories.

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: Phomaglomerata 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. Keywords: Aspergillus flavus; Phoma glomerata; Aflatoxin B1; Decontamination