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.

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.

Proteomics Analysis Reveals That Caspase-Like and Metacaspase-Like Activities Are Dispensable for Activation of Proteases Involved in Early Response to Biotic Stress in Triticum aestivum L.

Plants, including Triticum aestivum L., are constantly attacked by various pathogens which induce immune responses. Immune processes in plants are tightly regulated by proteases from different families within their degradome. In this study, a wheat degradome was characterized. Using profile hidden Markov model (HMMer) algorithm and Pfam database, comprehensive analysis of the T. aestivum genome revealed a large number of proteases (1544 in total) belonging to the five major protease families: serine, cysteine, threonine, aspartic, and metallo-proteases. Mass-spectrometry analysis revealed a 30% difference between degradomes of distinct wheat cultivars (Khakasskaya and Darya), and infection by biotrophic (Puccinia recondita Rob. ex Desm f. sp. tritici) or necrotrophic (Stagonospora nodorum) pathogens induced drastic changes in the presence of proteolytic enzymes. This study shows that an early immune response to biotic stress is associated with the same core of proteases from the C1, C48, C65, M24, M41, S10, S9, S8, and A1 families. Further liquid chromatography-mass spectrometry (LC-MS) analysis of the detected protease-derived peptides revealed that infection by both pathogens enhances overall proteolytic activity in wheat cells and leads to activation of proteolytic cascades. Moreover, sites of proteolysis were identified within the proteases, which probably represent targets of autocatalytic activation, or hydrolysis by another protease within the proteolytic cascades. Although predicted substrates of metacaspase-like and caspase-like proteases were similar in biotrophic and necrotrophic infections, proteolytic activation of proteases was not found to be associated with metacaspase-like and caspase-like activities. These findings indicate that the response of T. aestivum to biotic stress is regulated by unique mechanisms.

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.

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.

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.

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.

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.

Potato Pathogens in Russia's Regions: An Instrumental Survey with the Use of Real-Time PCR/RT-PCR in Matrix Format.

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.

Preserved Microarrays for Simultaneous Detection and Identification of Six Fungal Potato Pathogens with the Use of Real-Time PCR in Matrix Format.

Fungal diseases of plants are of great economic importance causing 70⁻80% of crop losses associated with microbial plant pathogens. Advanced on-site disease diagnostics is very important to maximize crop productivity. In this study, diagnostic systems have been developed for simultaneous detection and identification of six fungal pathogens using 48-well microarrays (micromatrices) for qPCR. All oligonucleotide sets were tested for their specificity using 59 strains of target and non-target species. Detection limit of the developed test systems varied from 0.6 to 43.5 pg of DNA depending on target species with reproducibility within 0.3-0.7% (standard deviation). Diagnostic efficiency of test systems with stabilized and freeze-dried PCR master-mixes did not significantly differ from that of freshly prepared microarrays, though detection limit increased. Validation of test systems on 30 field samples of potato plants showed perfect correspondence with the results of morphological identification of pathogens. Due to the simplicity of the analysis and the automated data interpretation, the developed microarrays have good potential for on-site use by technician-level personnel, as well as for high-throughput monitoring of fungal potato pathogens.

Evaluation of eliciting activity of peptidil prolyl cys/trans isomerase from Pseudonomas fluorescens encapsulated in sodium alginate regarding plant resistance to viral and fungal pahogens.

Use of chemical pesticides poses a threat for environment and human health, so green technologies of crop protection are of high demand. Some microbial proteins able to activate plant defense mechanisms and prevent the development of resistance in plant pathogens, may be good alternative to chemicals, but practical use of such elicitors is limited due to need to protect them against adverse environment prior their delivery to target receptors of plant cells. In this study we examined a possibility to encapsulate heat resistant FKBP-type peptidyl prolyl cis-trans isomerase (PPIase) from Pseudomonas fluorescens, which possesses a significant eliciting activity in relation to a range of plant pathogens, in sodium alginate microparticles and evaluated the stability of resulted complex under long-term UV irradiation and in the presence of proteinase K, as well as its eliciting activity in three different "plant-pathogen" models comparing to that of free PPIase. The obtained PPIase-containing microparticles consisted of 70% of sodium alginate, 20% of bovine serum albumin, and 10% of PPIase. In contrast to free PPIase, which lost its eliciting properties after 8-h UV treatment, encapsulated PPIase kept its eliciting ability unchanged; after being exposed to proteinase K, its eliciting ability twice exceeded that of free PPIase. Using "tobacco-TMV", "tobacco-Alternaria longipes", and "wheat-Stagonospora nodorum" model systems, we showed that encapsulation process did not influence on the eliciting activity of PPIase. In the case of the "wheat-S. nodorum" model system, we also observed a significant eliciting activity of alginate-albumin complex and almost doubled activity of encapsulated PPIase as compared to the free PPIase. As far as we know, this is the first observation of a synergistic interaction between alginate and other compound possessing any bioactive properties. The results of the study show some prospects for a PPIase use in agriculture.

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.

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.