The biocontrol effect of the nonpathogenic F. 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.

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.

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.

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.

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.