Activity of OSBPI fungicide fluoxapiprolin against plant-pathogenic oomycetes and its systemic translocation in plants.

Fluoxapiprolin, a novel piperidinyl thiazole isoxazoline fungicide, was developed by Bayer Crop Science in 2012. Despite its well-documented inhibitory activity against plant pathogenic oomycetes such as Phytophthora capsici and Phytophthora infestans, limited information regarding its antifungal spectrum and protective and curative activity is available. Fluoxapiprolin exhibited strong inhibitory activity against Phytophthora spp. and several Pythium spp., with EC50 values ranging from 2.12 × 10-4 to 2.92 µg/mL. It was much more effective against P. capsici in inhibiting mycelial growth, sporangium production, and cystospore germination than at reducing zoospore release. Moreover, fluoxapiprolin displayed both protective and curative activity against P. capsici infection in pepper plants under greenhouse conditions, with systemic translocation capability confirmed by High Performance Liquid Chromatography (HPLC) analysis. The results demonstrated the strong inhibitory activity of fluoxapiprolin against economically important plant oomycete pathogens, including Phytophthora spp. and several Pythium spp., and its certain translocation activity in pepper plants.

Discovery of Novel Oxathiapiprolin Derivatives as Potent Fungicide Candidates.

Oxathiapiprolin (OXA), which targets the oxysterol-binding protein (OSBP), is an outstanding piperidinyl thiazole isoxazoline (PTI) fungicide that can be used to control oomycetes diseases. In this study, starting from the structure of OXA, a series of novel OSBP inhibitors were designed and synthesized by introducing an indole moiety to replace the pyrazole in OXA. Finally, compound b24 was found to exhibit the highest control effect (82%) against cucumber downy mildew (CDM) in the greenhouse at a very low dosage of 0.069 mg/L, which was comparable to that of OXA (88%). Furthermore, it showed better activity against potato late blight (PLB) than other derivatives of indole. The computational results showed that the R-conformation of b24 should be the dominant conformation binding to PcOSBP. The results of the present work indicate that the 3-fluorine-indole ring is a favorable fragment to increasing the electronic energy when binding with PcOSBP. Furthermore, compound b24 could be used as a lead compound for the discovery of new OSBP inhibitors.

Sensitivity of Plasmopara viticola to selected fungicide groups and the occurrence of the G143A mutant in Australian grapevine isolates.

BACKGROUND: Grapevine downy mildew, caused by Plasmopara viticola, is an economically important disease in Australia and worldwide. The application of fungicides is the main tool to control this disease. Frequent fungicide applications can lead to the selection of resistant P. viticola populations, which has negative impacts on the management of the disease. Identification of resistance and its prevalence is necessary to inform resistance management strategies. RESULTS: A total of 86 P. viticola isolates were collected between 2017 and 2022 from vineyards in 15 growing regions across Australia for four fungicide groups; phenylamide (PA, group 4), carboxylic acid amide (CAA, group 40), quinone outside inhibitor (QoI, group 11) and quinone outside inhibitor stigmatellin binding type (QoSI, group 45). Decreased phenotypic sensitivity was detected for all four groups, and resistance to metalaxyl-M (PA) and pyraclostrobin (QoI), was detected. Genetic analysis to detect the G143A (QoI) and G1105S (CAA) mutations using amplicon-based sequencing was performed for 239 and 65 isolates collected in 2014-2017 and 2017-2022, respectively. G143A was detected in 8% and 52% of isolates, respectively, with strong association to phenotypic resistance. However, G1105S was not detected in any isolates. CONCLUSION: Plasmopara viticola isolates in Australia with resistance to at least two fungicide groups have been detected, therefore it is necessary to adopt resistance management strategies where resistance has been detected. Vineyards should continue to be monitored to improve management strategies for downy mildew. © 2024 Society of Chemical Industry.

Picarbutrazox Effectiveness Added to a Seed Treatment Mixture for Management of Oomycetes that Impact Soybean in Ohio.

None of the current oomycota fungicides are effective towards all species of Phytophthora, Phytopythium, Globisporangium, and Pythium that affect soybean seed and seedlings in Ohio. Picarbutrazox is a new oomyceticide with a novel mode of action towards oomycete pathogens. Our objectives were to evaluate picarbutrazox to determine (i) baseline sensitivity (EC50) to 189 isolates of 29 species, (ii) the efficacy with a base seed treatment with three cultivars with different levels of resistance in 14 field environments; and (iii) if the rhizosphere microbiome was affected by the addition of the seed treatment on a moderately susceptible cultivar. The mycelial growth of all isolates was inhibited beginning at 0.001 µg, and the EC50 ranged from 0.0013 to 0.0483 µg of active ingredient (a.i.)/ml. The effect of seed treatment was significantly different for plant population and yield in eight of 14 and six of 12 environments, respectively. The addition of picarbutrazox at 1 and 2.5 g of a.i./100 kg seed to the base seed treatment compared to the base alone was associated with higher plant populations and yield in three and one environments, respectively. There was limited impact of the seed treatment mefenoxam 7.5 g of a.i. plus picarbutrazox 1 g of a.i./100 kg seed on the oomycetes detected in the rhizosphere of soybean seedlings collected at the V1 growth stage. Picarbutrazox has efficacy towards a wider range of oomycetes that cause disease on soybean, and this will be another oomyceticide tool to combat early season damping-off in areas where environmental conditions highly favor disease development.

TaqMan-MGB PCR Method for Rapid Detection of QoI Fungicide Resistance in Chinese Populations of Plasmopara viticola.

Grape downy mildew caused by Plasmopara viticola is one of the most devastating diseases of grapevine worldwide. Quinone outside inhibitor (QoI) fungicides are commonly used for the control of the pathogen in grape fields across China. However, their recurrent use could lead to the emergence of resistance against these compounds. Based on the most common mutation in resistant isolates, a glycine to alanine substitution at amino acid position 143 (G143A) in the cytochrome b protein, a TaqMan-MGB PCR was developed for the rapid detection of resistance to the QoI fungicide azoxystrobin in P. viticola. Specificity and sensitivity of this method showed it could specifically detect the point mutations linked with QoI resistance in P. viticola, and the detection limit was 0.2 pg. It could also quantify the resistance allele even in isolate mixtures containing as little as 5% QoI-resistant P. viticola strains. With this method, a large P. viticola population (n = 2,373) was screened, and QoI-resistant isolates were identified for the first time in China. The average frequencies of the resistant genotype from eight major-grapevine regions were up to 66%. Taken together, the results not only provide a novel tool for the rapid distinction and quantification of the QoI-resistant allele in P. viticola but also provide important references for fungicide selection and application, which will facilitate resistance management of grape downy mildew and improve grape production systems in Chinese vineyards.

Synthesis and Anti-Oomycete Activity of 1-Sulfonyloxy/Acyloxydihydroeugenol Derivatives.

Endeavor to discover biorational natural products-based fungicides, two series (26) of novel 1-sulfonyloxy/acyloxydihydroeugenol derivatives (3a-p and 5a-j) were prepared and assessed for their fungicidal activity against P. capsici Leonian, in vitro. Results of fungicidal activity revealed that, among all compounds, especially compounds 3a, 5c, and 5e displayed the most potent anti-oomycete activity against P. capsici with EC50 values of 69.33, 68.81, and 67.77 mg/L, respectively. Overall, the anti-oomycete activities of 1-acyloxydihydroeugenol derivatives (5a-j) were higher than that of 1-sulfonyloxydihydroeugenol derivatives (3a-p). It is proved that the introduction of the acyl group at hydroxy position of dihydroeugenol is more beneficial to improve its anti-oomycete activity than that of the sulfonyl group. These preliminary results will pave the way for further modification of dihydroeugenol in the development of potential new fungicides.

Extracellular metabolites of endophytic fungi from Azadirachta indica inhibit multidrug-resistant bacteria and phytopathogens.

Aim: To evaluate antimicrobial activity of extracellular metabolites (EMs) of endophytic fungal isolates (EFIs) from Azadirachta indica. Materials & methods: EFIs were identified by internal transcribed spacer (ITS) sequencing. Antimicrobial activity, and minimum inhibitor concentration (MIC) and minimum bactericidal concentration (MBC) were determined using agar diffusion and microdilution method, respectively. Results: Seventeen EFIs were isolated from different organs of A. indica. Eight of them were identified based on ITS sequencing. The EMs of EFIs inhibited the growth of six multidrug-resistant (MDR) bacterial superbugs and three phytopathogenic fungi. The MDR bacterial superbugs are resistant to six commercial antibiotics of different generations but susceptible to EMs of EFIs. The MIC (0.125-1.0 µg/µl), MBC (0.5-4.0 µg/µl) and minimum fungicidal concentration (1.0-4.0 µg/µl) of the EMs from EFIs are lower enough. Conclusion: The EMs of the EFIs have promising antimicrobial activity against MDR bacteria and phytopathogenic fungi.

Growth Inhibition of Phytopathogenic Fungi and Oomycetes by Basidiomycete Irpex lacteus and Identification of its Antimicrobial Extracellular Metabolites.

In dual culture confrontation assays, basidiomycete Irpex lacteus efficiently antagonized Fusarium spp., Colletotrichum spp., and Phytophthora spp. phytopathogenic strains, with growth inhibition percentages between 16.7-46.3%. Antibiosis assays evaluating the inhibitory effect of soluble extracellular metabolites indicated I. lacteus strain inhibited phytopathogens growth between 32.0-86.7%. Metabolites in the extracellular broth filtrate, identified by UPLC-QTOF mass spectrometer, included nine terpenes, two aldehydes, and derivatives of a polyketide, a quinazoline, and a xanthone, several of which had antifungal activity. I. lacteus strain and its extracellular metabolites might be valuable tools for phytopathogenic fungi and oomycete biocontrol of agricultural relevance.

Quarternary α-cyanobenzylsulfonamides: A new subclass of CAA fungicides with excellent anti-Oomycetes activity.

A bioisosteric carboxamide - sulfonamide replacement explored during the optimization of an insecticide lead compound led to the surprising discovery of a formerly unknown subclass of the Carboxylic Acid Amide (CAA) fungicides, which is the very first CAA fungicide group without a carboxamide function. In this paper we present invention pathway, racemic and stereoselective synthesis routes, structure-activity relationship studies as well as resistance profile of this novel family of fungicides.

Synthesis, fungicidal activity, and 3D-QSAR of tetrazole derivatives containing phenyloxadiazole moieties.

In an effort to discover new agents with good fungicidal activities against CDM (cucumber downy mildew), a series of tetrazole derivatives containing phenyloxadiazole moieties were designed and synthesized. The EC50 values for fungicidal activities against CDM were determined. Bioassay results indicated that most synthesized compounds exhibited potential in vivo fungicidal activity against CDM. A CoMFA (comparative molecular field analysis) model based on the bioactivity was developed to identify some primary structural quality for the efficiency. The values of q2 and r2 for the established model were 0.791 and 0.982 respectively, which reliability and predict abilities were verified. Three analogues (q3, q4, q5) were designed and synthesized based on the model. All these compounds exhibited significant fungicidal activity on CDM with the EC50 of 1.43, 1.52, 1.77 mg·L-1. This work could provide a useful instruction for the further structure optimization.

NoPv1: a synthetic antimicrobial peptide aptamer targeting the causal agents of grapevine downy mildew and potato late blight.

Grapevine (Vitis vinifera L.) is a crop of major economic importance. However, grapevine yield is guaranteed by the massive use of pesticides to counteract pathogen infections. Under temperate-humid climate conditions, downy mildew is a primary threat for viticulture. Downy mildew is caused by the biotrophic oomycete Plasmopara viticola Berl. & de Toni, which can attack grapevine green tissues. In lack of treatments and with favourable weather conditions, downy mildew can devastate up to 75% of grape cultivation in one season and weaken newly born shoots, causing serious economic losses. Nevertheless, the repeated and massive use of some fungicides can lead to environmental pollution, negative impact on non-targeted organisms, development of resistance, residual toxicity and can foster human health concerns. In this manuscript, we provide an innovative approach to obtain specific pathogen protection for plants. By using the yeast two-hybrid approach and the P. viticola cellulose synthase 2 (PvCesA2), as target enzyme, we screened a combinatorial 8 amino acid peptide library with the aim to identify interacting peptides, potentially able to inhibit PvCesa2. Here, we demonstrate that the NoPv1 peptide aptamer prevents P. viticola germ tube formation and grapevine leaf infection without affecting the growth of non-target organisms and without being toxic for human cells. Furthermore, NoPv1 is also able to counteract Phytophthora infestans growth, the causal agent of late blight in potato and tomato, possibly as a consequence of the high amino acid sequence similarity between P. viticola and P. infestans cellulose synthase enzymes.

In vitro assessment of the antimicrobial efficacy of chitosan nanoparticles against major fish pathogens and their cytotoxicity to fish cell lines.

Nanotechnology is an emerging avenue employed in disease prevention and treatment. This study evaluated the antimicrobial efficacy of chitosan nanoparticles (CSNPs) against major bacterial and oomycete fish pathogens in comparison with chitosan suspension. Initially, the minimum inhibitory concentrations (MIC, MIC90 ) were determined and the per cent inhibition of bacterial growth was calculated. Subsequently, the minimum bactericidal concentrations (MBCs) were determined. The time-dependent disruptions of CSNP-treated pathogens were observed via transmission electron microscopy (TEM), and the effect of CSNPs on the viability of two fish cell lines was assessed. No antimicrobial effect was observed with chitosan, while CSNPs (105 nm) exhibited a dose-dependent and species-specific antimicrobial properties. They were bactericidal against seven bacterial isolates recording MBC values from 1 to 7 mg/ml, bacteriostatic against four further isolates recording MIC values from 0.125 to 5 mg/ml and fungistatic against oomycetes recording MIC90 values of 3 and 4 mg/ml. TEM micrographs showed the attachment of CSNPs to the pathogenic cell membranes disrupting their integrity. No significant cytotoxicity was observed using 1 mg/ml CSNPs, while low dose-dependent cytotoxicity was elicited by the higher doses. Therefore, it is anticipated that CSNPs are able to compete and reduce using antibiotics in aquaculture.

Lysobacter enzymogenes LE16 autolysates have potential as biocontrol agents-Lysobacter sp. autolysates as biofungicide.

AIMS: Biological techniques can manage plant diseases safely and in environmentally friendly ways, but their efficacy needs improvement. It is of the utmost importance to search for powerful microbes for the effective control of plant diseases. METHODS AND RESULTS: Unheated self-digestive solutions (SDS) that were heated at 100°C for 30 min(H-SDS) or stored for 12 months at room temperature (S-SDS) were prepared from Lysobacter enzymogenes LE16 broth culture to study their potential as biocontrol agents. This bacterium produced protease, phosphatase, lysozyme and siderophores in pure culture as well as 12 secondary metabolites including novel antibiotics lysobactin, WAP-8294A2 and mupirocin determined based on the antiSMASH 5.0.0 blast database. A poison plate assay revealed the antagonistic activities of SDS, H-SDS and S-SDS against an animal pathogenic bacterium Staphylococcus aureus, a phytopathogenic bacterium Pseudomonas syringae pv. tabaci, and numerous plant pathogenic fungi and oomycetes, including Colletotrichum gloeosporioides, Penicillium italicum, Alternaria alternate, Rhizoctonia solani, Didymella bryoniae, Sclerotinia sclerotiorum, Phytophthora nicotianae and Phytophthora capsici. The greenhouse experiment showed that SDS was highly effective in controlling pepper blight disease, which is caused by P. capsici. Compared with only pathogen inoculation, the application of SDS to the soil in preventive or curative treatments significantly reduced the disease incidence and index with relatively high control efficacy of 86·2-93·1%. CONCLUSIONS: SDS enriched lytic enzymes, siderophores and antibiotics, has a wide antimicrobial spectrum, and shows potential as a new, safe and effective biocontrol agent against plant diseases. SIGNIFICANCE AND IMPACT OF THE STUDY: Autolysates of the new biocontrol bacterium L. enzymogenes LE16 demonstrated the potential for industrial production and commercial use as a promising biocontrol agent in agriculture.

Design, Synthesis, and Structure-Activity Relationship of Economical Triazole Sulfonamide Aryl Derivatives with High Fungicidal Activity.

Plant fungal diseases have caused great decreases in crop quality and yield. As one of the considerable agricultural diseases, cucumber downy mildew (CDM) caused by pseudoperonospora cubensis seriously influences the production of cucumber. Amisulbrom is a commercial agricultural fungicide developed by Nissan Chemical, Ltd., for the control of oomycetes diseases that is highly effective against CDM. However, the synthesis of amisulbrom has a high cost because of the introduction of the bromoindole ring. In addition, the continuous use of amisulbrom might increase the risk of resistance development. Hence, there is an imperative to develop active fungicides with new scaffolds but low cost against CDM. In this study, a series of 1,2,4-triazole-1,3-disulfonamide derivatives were designed, synthesized, and screened. Compound 1j showed a comparable fungicidal activity with amisulbrom, but it was low cost and ecofriendly. It has the potential to be developed as a new fungicide candidate against CDM. Further investigations of structure-activity relationship exhibited the structural requirements of 1,2,4-triazole-1,3-disulfonamide and appropriate modification in N-alkyl benzylamine groups with high fungicidal activity. This research will provide powerful guidance for the design of highly active lead compounds with a novel skeleton and low cost.

Design, Synthesis, and Structure-Activity Relationship of Novel Spiropyrimidinamines as Fungicides against Pseudoperonospora cubensis.

Harmful fungus and the developed resistance to available fungicides seriously threaten the yield and quality of crops; thus, the search for new, highly efficient, and resistance-overcoming fungicides remains a quite urgent goal of agricultural scientists. In this study, a series of novel spiropyrimidinamine derivatives were designed and synthesized by employing the intermediate derivatization method (IDM). Their structures were identified by 1H NMR, elemental analyses, and MS spectra. The structure of compound 5 was further confirmed by X-ray diffraction. Bioassays indicated that a number of the title compounds exhibited some fungicidal activities against Pseudoperonospora cubensis. Especially, compound 5 displayed excellent activity (EC50 = 0.422 mg/L), significantly higher than those of the commercialized fungicides cyazofamid, flumorph, and diflumetorim. The structure-activity relationship was also discussed. It was concluded that compound 5 with super fungicidal potency and a novel structure is a promising agrochemical fungicide candidate for further development.

Forecasting severe grape downy mildew attacks using machine learning.

Grape downy mildew (GDM) is a major disease of grapevine that has an impact on both the yields of the vines and the quality of the harvested fruits. The disease is currently controlled by repetitive fungicide treatments throughout the season, especially in the Bordeaux vineyards where the average number of fungicide treatments against GDM was equal to 10.1 in 2013. Reducing the number of treatments is a major issue from both an environmental and a public health point of view. One solution would be to identify vineyards that are likely to be heavily attacked in spring and then apply fungicidal treatments only to these situations. In this perspective, we use here a dataset including 9 years of GDM observations to develop and compare several generalized linear models and machine learning algorithms predicting the probability of high incidence and severity in the Bordeaux region. The algorithms tested use the date of disease onset and/or average monthly temperatures and precipitation as input variables. The accuracy of the tested models and algorithms is assessed by year-by-year cross validation. LASSO, random forest and gradient boosting algorithms show better performance than generalized linear models. The date of onset of the disease has a greater influence on the accuracy of forecasts than weather inputs and, among weather inputs, precipitation has a greater influence than temperature. The best performing algorithm was selected to evaluate the impact of contrasted climate scenarios on GDM risk levels. Results show that risk of GDM at bunch closure decreases with reduced rainfall and increased temperatures in April-May. Our results also show that the use of fungicide treatment decision rules that take into account local characteristics would reduce the number of treatments against GDM in the Bordeaux vineyards compared to current practices by at least 50%.

Effects of sophorolipids on fungal and oomycete pathogens in relation to pH solubility.

AIMS: The objective of this study was to determine the effects of sophorolipids on several fungal and oomycete plant pathogens and the relationship between sophorolipids at different pH and antimicrobial activities. METHODS AND RESULTS: Sophorolipids had different solubility at different pH with a dramatic increase in solubility when pH was 6 or higher. Inhibition of mycelial growth of Phytophthora infestans by sophorolipids was affected by pH values, showing that when the pH value was higher, the inhibition rate was lower. Sophorolipids inhibited spore germination and mycelial growth of several fungal and oomycete pathogens in vitro including Fusarium sp., F. oxysporum, F. concentricum, Pythium ultimum, Pyricularia oryzae, Rhizoctorzia solani, Alternaria kikuchiana, Gaeumannomyces graminis var. tritici and P. infestans and caused morphological changes in hyphae by microscope observation. Sophorolipids reduced ß-1,3-glucanase activity in mycelia of P. infestans. In greenhouse studies, foliar application of sophorolipids at 3 mg ml-1 reduced severity of late blight of potato caused by P. infestans significantly. CONCLUSION: Sophorolipids influenced spore germination and hyphal tip growth of several plant pathogens and pH solubility of sophorolipids had an effect on their efficacy. Application of sophorolipids reduced late blight disease on potato under greenhouse conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings indicated that sophorolipids have the potential to be developed as a convenient and easy-to-use formulation for managing plant diseases.

Design, Synthesis, and Structure-Activity Relationship of New Arylpyrazole Pyrimidine Ether Derivatives as Fungicides.

To explore a novel fungicide effectively against cucumber downy mildew (CDM), a series of new arylpyrazole containing pyrimidine ether derivatives were designed and synthesized by employing the intermediate derivatization method (IDM). The structures of synthesized compounds were identified by 1H NMR, 13C NMR, elemental analyses, MS, and X-ray diffraction. Bioassays demonstrated that some of the title compounds exhibited excellent fungicidal activities against CDM. Especially, compound 7 (EC50 = 1.22 mg/L) displayed significantly higher bioactivity than that of commercial fungicides diflumetorim and flumorph and nearly equal effect to that of cyazofamid. The relationship between the structure and fungicidal activity of the synthesized compounds was discussed as well. The study showed that compound 7 was a promising fungicide candidate for further development.

Disease Control Attributes of Oxathiapiprolin Fungicides for Management of Cucurbit Downy Mildew.

Oxathiapiprolin, a novel oomycete fungicide recently registered by DuPont, was reported to have high intrinsic activity against cucurbit downy mildew (Pseudoperonospora cubensis). The goal of this study was to characterize disease control attributes of oxathiapiprolin-based fungicides critical to effective management of cucurbit downy mildew. In growth chamber and greenhouse studies, oxathiapiprolin-based fungicides were compared with mandipropamid, mefenoxam + mancozeb, fluopicolide + propamocarb, cymoxanil + mancozeb, and ametoctradin + dimethomorph products for pre- and postinfection activity, local systemic movement, and protection of new growth produced after fungicide application. In preventive application, oxathiapiprolin-based fungicides significantly (P < 0.0001) inhibited downy mildew development, with the highest level of disease observed being 0.4% compared with 86.7% observed for mandipropamid. When applied postinfection, oxathiapiprolin-based fungicides significantly (P < 0.0001) suppressed disease development, but disease control was reduced relative to that observed for preventive application. There was a significant effect of formulation on the postinfection activity of oxathiapiprolin, whereby the oil dispersion (OD) formulation was more inhibitory than the water-dispersible granule formulation (0.001 ≤ P ≤ 0.049). Disease severity on the outer half leaf portion protected from spray deposition during fungicide application was lower for oxathiapiprolin-based fungicides (1.6 to 6.6%) than observed for fluopicolide + propamocarb (10.9 to 23.7%), mefenoxam + mancozeb (40.3 to 51.4%), and the nontreated controls (83.3 to 84.9%), which indicates significant acropetal movement within the leaf. Postinfection applications of oxathiapiprolin-based formulations had the greatest effect on lesion growth and sporangia production compared with the other fungicides in the experiment. When applied preventively to rapidly growing plants in a greenhouse, oxathiapiprolin-based fungicides consistently protected new growth that was not present at the time of application, with the OD formulation reducing disease severity by >75% relative to nontreated plants. The practical implications of these observations are discussed.

Novel synergistic fungicidal mixtures of oxathiapiprolin protect sunflower seeds from downy mildew caused by Plasmopara halstedii.

Plenaris (oxathiapiprolin) applied to sunflower seedlings was highly effective in controlling downy mildew incited by the oomycete Plasmopara halstedii. In vitro assays revealed strong suppression of zoospore release and cystospore germination of P.halstedii by Plenaris. Bion (acibenzolar-S-methyl) and Apron (mefenoxam) were partially effective when used singly, but performed synergistically when mixed with Plenaris. Seed treatment (coating) with Plenaris provided dose-dependent control of the disease whereas Bion and Apron provided partial or poor control. However, seeds treated with mixtures containing reduced rates of Plenaris and full rates of Bion and/or Apron provided complete control of the disease due to the synergistic interaction between these components. Such mixtures should be used for seed treatment in the field to minimize selection pressure imposed on the pathogen.