Efficiency and resistance risk of flusilazole against northern corn leaf blight caused by Setosphaeria turcica.

Northern corn leaf blight (NCLB) infected by Setosphaeria turcica is a devastating disease of corn worldwide. Flusilazole is a broad-spectrum triazole fungicide. However, its resistance risk and field efficiency in controlling NCLB are still unknown. The present research evaluated the antifungal activity of flusilazole against 101 S. turcica isolates, and their EC50 values ranged from 0.0013 to 0.0466 µg/mL, with a mean of 0.0157 µg/mL. Seven S. turcica mutants resistant to flusilazole were obtained from two wild-type isolates by fungicide adaptation. After 10 consecutive transfers on PDA medium without fungicide, their resistance decreased. Cross-resistance was not existed between flusilazole and fluazinam, pyraclostrobin, amobam, epoxiconazole, or fluxapyroxad. Compared to the wild-type isolates, seven flusilazole-resistant mutants showed reduced biological fitness. No point mutation was detected, however, over-expression of StCYP51 and StatrD genes were detected in the resistant mutants. In addition, in the field experiment, flusilazole exhibited over 85 % efficacy against NCLB, significantly higher than amobam. In summary, these results suggested that the resistance risk of S. turcica to flusilazole was low, and the over-expression of StCYP51 and StatrD might be related to the flusilazole resistance against S. turcica. Flusilazole showed great potential as an alternative fungicide for controlling NCLB.

Overexpression of the CcCYP51A and CcCYP51B genes confer Corynespora cassiicola resistance to prochloraz.

Cucumber Corynespora leaf spot caused by Corynespora cassiicola is the primary disease responsible for reducing cucumber yield, and prochloraz is the main fungicide used to control C. cassiicola. This study investigated the sensitivity and resistance mechanism of C. cassiicola isolates to prochloraz, and found that C. cassiicola has developed resistance to prochloraz. The prochloraz EC50 values ranged from 0.02 to 2.33 µg/mL, with a mean of 0.436 ± 0.447 µg/mL. In total, 36 of 146 isolates exhibited prochloraz resistance. The resistant isolates had no fitness cost and could not be completely controlled by 50 µg/mL prochloraz on detached leaves. Prochloraz exhibited positive cross-resistance with propiconazole and tebuconazole but not with difenoconazole, carbendazim, trifloxystrobin and pydiflumetofen. The sensitive isolates had significantly lower ergosterol content than the resistant isolates after prochloraz treatment. Compared to sensitive isolates, prochloraz-resistant isolates had no CcCYP51 gene mutation, but the CcCYP51A and CcCYP51B gene expression levels were significantly higher under the treatment of prochloraz. The overexpression of CcCYP51A and CcCYP51B were associated with prochloraz resistance in C. cassiicola.

Antimalarial mechanism of action of the natural product 9-methoxystrobilurin G.

The natural product 9-methoxystrobilurin G (9MG) from Favolaschia spp basidiomycetes is a potent and selective antimalarial. The mechanism of action of 9MG is unknown. We induced 9MG resistance in Plasmodium falciparum 3D7 and Dd2 strains and identified mutations associated with resistance by genome sequencing. All 9MG-resistant clones possessed missense mutations in the cytochrome b (CYTB) gene, a key component of mitochondrial complex III. The mutations map to the quinol oxidation site of CYTB, which is also the target of antimalarials such as atovaquone. In a complementary approach to identify protein targets of 9MG, a photoactivatable derivative of 9MG was synthesized and applied in chemoproteomic-based target profiling. Three components of mitochondrial complex III (QCR7, QCR9, and COX15) were specifically enriched consistent with 9MG targeting CYTB and complex III function in P. falciparum. Inhibition of complex III activity by 9MG was confirmed by ubiquinone cytochrome c reductase assay using P. falciparum extract. The findings from this study may be useful for developing novel antimalarials targeting CYTB.

The occurrence and mechanism of field resistance to boscalid and pyraclostrobin in Stemphylium solani, the causal agent of tomato gray leaf spot in China.

The destructive disease gray leaf spot, caused by Stemphylium solani, is prevalent in tomato plants in China. A variety of fungicides have been extensively used for controlling the disease, with a particular focus on succinate dehydrogenase inhibitors (SDHIs) and quinone outside inhibitors (QoIs). However, there was a lack of information regarding the resistance of S. solani to boscalid (SDHI) and pyraclostrobin (QoI) in China. In this study, the sensitivity of S. solani to boscalid and pyraclostrobin was monitored. The EC50 values for boscalid ranged from 0.02 to 3.0 µg∙mL-1, with an average value of 0.62 µg∙mL-1, while the EC50 values for pyraclostrobin ranged from 0.21 to 14.71 µg∙mL-1, with an average value of 6.03 µg∙mL-1. Based on these findings, the frequencies of observed resistance were as follows: 36.7% for boscalid and 50% for pyraclostrobin; while the resistance frequency to both boscalid and pyraclostrobin in S. solani was 19.4%. The mutation associated with boscalid resistance in S. solani within tomato fields was identified as SdhB-H277Y, while the mutation related to pyraclostrobin resistance was found in cytochrome b, specifically Cytb-G143A. The resistant mutants displayed diminished fitness in terms of mycelial growth, yet their pathogenicity exhibited no significant disparities. To delay the development of resistance, it is advisable to employ a rotation strategy using alternative fungicides with different modes of action or mix with fungicides with multi-site-contact activity for disease management.

Lignin/Surfactin Coacervate as an Eco-Friendly Pesticide Carrier and Antifungal Agent against Phytopathogen.

Excessive usage of biologically toxic fungicides and their matrix materials poses a serious threat to public health. Leveraging fungicide carriers with inherent pathogen inhibition properties is highly promising for enhancing fungicide efficacy and reducing required dosage. Herein, a series of coacervates have been crafted with lignin and surfactin, both of which are naturally derived and demonstrate substantial antifungal properties. This hierarchically assembled carrier not only effectively loads fungicides with a maximum encapsulation efficiency of 95% but also stably deposits on hydrophobic leaves for high-speed impacting droplets. Intriguingly, these coacervates exhibit broad spectrum fungicidal activity against eight ubiquitous phytopathogens and even act as a standalone biofungicide to replace fungicides. This performance can significantly reduce the fungicide usage and be further strengthened by an encapsulated fungicide. The inhibition rate reaches 87.0% when 0.30 mM pyraclostrobin (Pyr) is encapsulated within this coacervate, comparable to the effectiveness of 0.80 mM Pyr alone. Additionally, the preventive effects against tomato gray mold reached 53%, significantly surpassing those of commercial adjuvants. Thus, it demonstrates that utilizing biosurfactants and biomass with intrinsic antifungal activity to fabricate fully biobased coacervates can synergistically combine the functions of a fungicide carrier and antifungal agent against phytopathogens and guarantee environmental friendliness. This pioneering approach provides deeper insights into synergistically enhancing the effectiveness of agrochemicals from multiple aspects, including fungicide encapsulation, cooperative antifungal action, and droplet deposition.

Cytochrome c Oxidase Influences Pyraclostrobin Sensitivity in Fusarium graminearum by Regulating FgAox Through Transcription Factors FgAod2 and FgAod5.

Cytochrome c oxidase (Cox) is a crucial terminal oxidase in the electron transport chain. In this study, we generated 14 Cox gene deletion or overexpression mutants in Fusarium graminearum. Fungicide sensitivity tests revealed that 11 Cox gene deletion mutants displayed resistance to pyraclostrobin, while 10 overexpression mutants showed hypersensitivity. RNA-Seq and RT-qPCR analyses demonstrated the upregulation of FgAox (alternative oxidase in F. graminearum), FgAod2, and FgAod5 (alternative oxidase deficiency in F. graminearum) in ΔFgCox4-2 and ΔFgCox17-75 mutants. In 11 Cox gene deletion mutants, FgAox expression was significantly upregulated, whereas in 10 Cox gene overexpression mutants, it was significantly downregulated. FgAox overexpression mutants exhibit resistance to pyraclostrobin, while FgAox deletion mutants show hypersensitivity to pyraclostrobin. FgAod2 and FgAod5 were identified as transcription factors for FgAox. Our findings reveal that FgCox influences pyraclostrobin sensitivity by regulating FgAox through FgAod2 and FgAod5. Understanding pyraclostrobin resistance mechanisms in F. graminearum could help develop better fungicide rotation and application strategies to manage resistance and guide the creation of new fungicides targeting different pathways.

Response of Soil Microbiota, Enzymes, and Plants to the Fungicide Azoxystrobin.

The present study was aimed at assessing the impact of azoxystrobin-a fungicide commonly used in plant protection against pathogens (Amistar 250 SC)-on the soil microbiota and enzymes, as well as plant growth and development. The laboratory experiment was conducted in three analytical terms (30, 60, and 90 days) on sandy clay (pH-7.0). Azoxystrobin was applied to soil in doses of 0.00 (C), 0.110 (F) and 32.92 (P) mg kg-1 d.m. of soil. Its 0.110 mg kg-1 dose stimulated the proliferation of organotrophic bacteria and actinobacteria but inhibited that of fungi. It also contributed to an increase in the colony development index (CD) and a decrease in the ecophysiological diversity index (EP) of all analyzed groups of microorganisms. Azoxystrobin applied at 32.92 mg kg-1 reduced the number and EP of microorganisms and increased their CD. PP952051.1 Bacillus mycoides strain (P), PP952052.1 Prestia megaterium strain (P) bacteria, as well as PP952052.1 Kreatinophyton terreum isolate (P) fungi were identified in the soil contaminated with azoxystrobin, all of which may exhibit resistance to its effects. The azoxystrobin dose of 0.110 mg kg-1 stimulated the activity of all enzymes, whereas its 32.92 mg kg-1 dose inhibited activities of dehydrogenases, alkaline phosphatase, acid phosphatase, and urease and stimulated the activity of catalase. The analyzed fungicide added to the soil at both 0.110 and 32.92 mg kg-1 doses inhibited seed germination and elongation of shoots of Lepidium sativum L., Sinapsis alba L., and Sorgum saccharatum L.

Amino acid mutation of succinate dehydrogenase complex induced resistance to benzovindiflupyr in Magnaporthe oryzae.

Magnaporthe oryzae is a rice blast pathogen that seriously threatens rice yield. Benzovindiflupyr is a succinate dehydrogenase inhibitor (SDHI) fungicide that effectively controls many crop diseases. Benzovindiflupyr has a strong inhibitory effect on M. oryzae; however, control of rice blast by benzovindiflupyr and risk of resistance to benzovindiflupyr are not well studied in this pathogen. In this study, six benzovindiflupyr-resistant strains were obtained by domestication induced in the laboratory. The MoSdhBH245D mutation was the cause of M. oryzae resistance to benzovindiflupyr, which was verified through succinate dehydrogenase (SDH) activity assays, molecular docking, and site-specific mutations. Survival fitness analysis showed no significant difference between the benzovindiflupyr-resistant and parent strains. Positive cross-resistance to benzovindiflupyr and other SDHIs and negative cross-resistance to azoxystrobin were observed. Therefore, the risk of benzovindiflupyr resistance in M. oryzae might be medium to high. It should be combined with other classes of fungicides (tebuconazole and azoxystrobin) to slow the development of resistance.

The G143S mutation in cytochrome b confers high resistance to pyraclostrobin in Fusarium pseudograminearum.

BACKGROUND: Wheat crown rot (WCR), primarily caused by Fusarium pseudograminearum has become more and more prevalent in winter wheat areas in China. However, limited fungicides have been registered for the control of WCR in China so far. Pyraclostrobin is a representative quinone outside inhibitor (QoI) with excellent activity against Fusarium spp. There is currently limited research on the resistance risk and resistance mechanism of F. pseudograminearum to pyraclostrobin. RESULTS: Here, we determined the activity of pyraclostrobin against F. pseudograminearum. The EC50 values ranged from 0.022 to 0.172 µg mL-1 with an average EC50 value of 0.071 ± 0.030 µg mL-1. Four highly pyraclostrobin-resistant mutants were obtained from two sensitive strains by ultraviolet (UV) mutagenesis in the laboratory. The mutants showed decreased mycelial growth rate and virulence as compared with the corresponding wild-type strains, indicating that pyraclostrobin resistance suffered a fitness penalty in F. pseudograminearum. It was found that the high resistance of four mutants was caused by the G143S mutation in Cytb. Molecular docking analysis also further confirms that the G143S mutation in Cytb decreased the binding affinity between pyraclostrobin and Cytb. CONCLUSION: The resistance risk of F. pseudograminearum to pyraclostrobin could be low to medium. Although a mutation at the G143S position of Cytb could potentially occur, this mutation decreases the fitness of the mutant, which may reduce its survival in the environment. Therefore, the negative consequences of a possible mutation are lower. This makes pyraclostrobin a good candidate for controlling crown rot in wheat. © 2024 Society of Chemical Industry.

High resistance risk of Phytophthora colocasiae to azoxystrobin in southeastern of China.

Quinone outside inhibitor (QoI) has been applied to manage taro leaf blight caused by Phytophthora colocasiae in southeastern of China for many years. The risk of P. colocasiae to QoI and the potential resistant mechanism remain unknown. In this study, the 74 P. colocasiae strains were sampled from southeastern of China. Sequence analysis of the QoI target Cytb showed one nucleotide variant in the fragment of this gene in this population, producing two haplotypes. The nucleotide variant leads to codon change at 142 (GGT to GCT) producing A142 (alanine) and G142 (glycine) in Hap_1 and Hap_2 strains, respectively. The sensitivity differentiation to azoxystrobin of two haplotypes were observed in vitro. The Hap_1 and Hap_2 strains were confirmed resistant and sensitive by control efficacy of label rate fungicide application, which was 3.0% and 88.8% treated with 500 µg/mL azoxystrobin, respectively. In addition, 10.0 µg/mL azoxystrobin plus 50 µg/mL salicylhydroxamic acid (SHAM) supplemented in PDA medium was identified as a discriminatory dose for differentiation of these two phenotype strains. The azoxystrobin resistant frequency reached 86.5%, indicating prevalence of QoI resistance in the field. Further fitness related features showed that no significant difference in temperature sensitivity, mycelial growth rate, sporangia production, zoospore release and aggressiveness between azoxystrobin-resistant and sensitive strains indicating no potential fitness cost for azoxystrobin resistance. Taken together, azoxystrobin resistance need to be taken into consideration to manage taro leaf blight in southeastern of China.

Evaluation of Anticholinesterase Activity of the Fungicides Mefentrifluconazole and Pyraclostrobin.

Triazoles are compounds with various biological activities, including fungicidal action. They became popular through cholinesterase studies after the successful synthesis of the dual binding femtomolar triazole inhibitor of acetylcholinesterase (AChE, EC 3.1.1.7) by Sharpless et al. via in situ click chemistry. Here, we evaluate the anticholinesterase effect of the first isopropanol triazole fungicide mefentrifluconazole (Ravystar®), developed to overcome fungus resistance in plant disease management. Mefentrifluconazole is commercially available individually or in a binary fungicidal mixture, i.e., with pyraclostrobin (Ravycare®). Pyraclostrobin is a carbamate that contains a pyrazole ring. Carbamates are known inhibitors of cholinesterases and the carbamate rivastigmine is already in use for the treatment of Alzheimer's disease. We tested the type and potency of anticholinesterase activity of mefentrifluconazole and pyraclostrobin. Mefentrifluconazole reversibly inhibited human AChE and BChE with a seven-fold higher potency toward AChE (Ki = 101 ± 19 µM). Pyraclostrobin (50 µM) inhibited AChE and BChE progressively with rate constants of (t1/2 = 2.1 min; ki = 6.6 × 103 M-1 min-1) and (t1/2 = 1.5 min; ki = 9.2 × 103 M-1 min-1), respectively. A molecular docking study indicated key interactions between the tested fungicides and residues of the lipophilic active site of AChE and BChE. Additionally, the physicochemical properties of the tested fungicides were compared to values for CNS-active drugs to estimate the blood-brain barrier permeability. Our results can be applied in the design of new molecules with a lesser impact on humans and the environment.

Green synthesized lignin nanoparticles for the sustainable delivery of pyraclostrobin to control strawberry diseases caused by Botrytis cinerea.

Lignin, renowned for its renewable, biocompatible, and environmentally benign characteristics, holds immense potential as a sustainable feedstock for agrochemical formulations. In this study, raw dealkaline lignin (DAL) underwent a purification process involving two sequential solvent extractions. Subsequently, an enzyme-responsive nanodelivery system (Pyr@DAL-NPs), was fabricated through the solvent self-assembly method, with pyraclostrobin (Pyr) loaded into lignin nanoparticles. The Pyr@DAL-NPs shown an average particle size of 250.4 nm, demonstrating a remarkable loading capacity of up to 54.70 % and an encapsulation efficiency of 86.15 %. Notably, in the presence of cellulase and pectinase at a concentration of 2 mg/mL, the release of Pyr from the Pyr@DAL-NPs reached 92.66 % within 120 h. Furthermore, the photostability of Pyr@DAL-NPs was significantly improved, revealing a 2.92-fold enhancement compared to the commercially available fungicide suspension (Pyr SC). Bioassay results exhibited that the Pyr@DAL-NPs revealed superior fungicidal activity against Botrytis cinerea over Pyr SC, with an EC50 value of 0.951 mg/L. Additionally, biosafety assessments indicated that the Pyr@DAL-NPs effectively declined the acute toxicity of Pyr towards zebrafish and posed no negative effects on the healthy growth of strawberry plants. In conclusion, this study presents a viable and promising strategy for developing environmentally friendly controlled-release systems for pesticides, offering the unique properties of lignin.

Antimalarial 9-methoxystrobilurin derivatives from cultures of the basidiomycete Favolaschia minutissima.

Investigation of cultures of the basidiomycete Favolaschia minutissima TBRC-BCC 19434 led to the isolation of two undescribed ß-methoxyacrylate metabolites, 9-methoxystrobilurins R (1) and S (2), and a degraded aldehyde derivative, favodehyde E (3). 9-Methoxystrobilurin derivatives 1 and 2 exhibited significant antimalarial activity against Plasmodium falciparum K1 (multidrug-resistant strain) with IC50 values of 0.12 and 0.21 µM, respectively.

Evaluating Treatments for the Protection of Grapevine Pruning Wounds from Natural Infection by Trunk Disease Fungi.

Infection of grapevines by fungal pathogens causing grapevine trunk diseases (GTDs) primarily arises from annual pruning wounds made during the dormant season. While various studies have showcased the efficacy of products in shielding pruning wounds against GTD infections, most of these investigations hinge on artificial pathogen inoculations, which may not faithfully mirror real field conditions. This study aimed to evaluate and compare the efficacy of various liquid formulation fungicides (pyraclostrobin + boscalid) and paste treatments, as well as biological control agents (BCA: Trichoderma atroviride SC1, T. atroviride I-1237, and T. asperellum ICC012 + T. gamsii ICC080), for their potential to prevent natural infection of grapevine pruning wounds by trunk disease fungi in two field trials located in Samaniego (Northern Spain) and Madiran (Southern France) over three growing seasons. Wound treatments were applied immediately after pruning in February. One year after pruning, canes were harvested from vines and brought to the laboratory for assessment of Trichoderma spp. and fungal trunk pathogens. More than 1,200 fungal isolates associated with five GTDs (esca, Botryosphaeria, Diaporthe and Eutypa diebacks, and Cytospora canker) were collected from the two vineyards each growing season. Our findings reveal that none of the products under investigation exhibited complete effectiveness against all the GTDs. The efficacy of these products was particularly influenced by the specific year of study. A notable exception was observed with the biocontrol agent T. atroviride I-1237, which consistently demonstrated effectiveness against Botryosphaeria dieback infections throughout each year of the study, irrespective of the location. The remaining products exhibited efficacy in specific years or locations against particular diseases, with the physical barrier (paste) showing the least overall effectiveness. The recovery rates of Trichoderma spp. in treated plants were highly variable, ranging from 17 to 100%, with both strains of T. atroviride yielding the highest isolation rates. This study underscores the importance of customizing treatments for specific diseases, taking into account the influence of environmental factors for BCA applications.

Inhibitory Activities of Five Fungicides on Alternaria suffruticosae and Their Field Control Efficacy Against Tree Peony Black Spot.

Tree peony black spot (TPBS), mainly caused by Alternaria suffruticosae, is a common leaf disease on the ornamental peony, which poses a great threat to the flower buds in the current year and the flowering quality in the next year. However, there is only one fungicide registered for the control of this disease, difenoconazole. In order to avoid the severe problem of pathogen resistance caused by long-term use of difenoconazole, it is necessary to screen more chemical fungicides for the prevention and control of TPBS. In this study, the biological activities of flutolanil, phenamacril, pyraclostrobin, and boscalid on mycelial growth, conidial germination, germ tube elongation, and sporulation quantity of A. suffruticosae were determined, and the field control efficacy was tested to evaluate the preventive and therapeutic activities. Difenoconazole was used as a control simultaneously. The results showed that pyraclostrobin had the strongest inhibitory effects on the conidial germination, mycelium growth, germ tube elongation, and sporulation quantity, with the average EC50 values of 0.0517, 0.5343, 0.0008, and 0.8068 µg/ml, respectively. The inhibitory activity of flutolanil on the four developmental stages of A. suffruticosae was weaker than that of the other three fungicides. Compared with flutolanil, boscalid, the other succinate dehydrogenase inhibitor, had more strong inhibitory effects on the mycelial growth and sporulation quantity, with the average EC50 values of 3.8603 and 1.4760 µg/ml, respectively. Phenamacril had a moderate inhibitory level and had more inhibitory activity on conidial germination and germ tube elongation, with the average EC50 values of 31.5349 and 5.2597 µg/ml, respectively. All of the four fungicides had no significant effects on the shape of spores and germ tubes. The control fungicide difenoconazole had the strongest inhibitory activity on mycelial growth, and the average EC50 value was only 0.3297 µg/ml. However, its inhibitory activity on the other three growth stages was not high. In the field trials, pyraclostrobin had high control efficacy on TPBS even at low concentrations, reaching a minimum of 62.6293%, which was higher than that of difenoconazole. The other three fungicides had higher control efficacy at high concentrations but decreased significantly at low concentrations. Considering the dosage and control efficacy, pyraclostrobin was the first choice for the control of TPBS. Pyraclostrobin is the preferred alternative fungicide to difenoconazole for the prevention and control of TPBS in production.

Carboxylesterase and Cytochrome P450 Confer Metabolic Resistance Simultaneously to Azoxystrobin and Some Other Fungicides in Botrytis cinerea.

Plant pathogens have frequently shown multidrug resistance (MDR) in the field, often linked to efflux and sometimes metabolism of fungicides. To investigate the potential role of metabolic resistance in B. cinerea strains showing MDR, the azoxystrobin-sensitive strain B05.10 and -resistant strain Bc242 were treated with azoxystrobin. The degradation half-life of azoxystrobin in Bc242 (9.63 days) was shorter than that in B05.10 (28.88 days). Azoxystrobin acid, identified as a metabolite, exhibited significantly lower inhibition rates on colony and conidia (9.34 and 11.98%, respectively) than azoxystrobin. Bc242 exhibited higher expression levels of 34 cytochrome P450s (P450s) and 11 carboxylesterase genes (CarEs) compared to B05.10 according to RNA-seq analysis. The expression of P450 genes Bcin_02g01260 and Bcin_12g06380, along with the CarEs Bcin_12g06360 in Saccharomyces cerevisiae, resulted in reduced sensitivity to various fungicides, including azoxystrobin, kresoxim-methyl, pyraclostrobin, trifloxystrobin, iprodione, and carbendazim. Thus, the mechanism of B. cinerea MDR is linked to metabolism mediated by the CarE and P450 genes.

Host Range Characterization of Phytophthora sansomeana Across Corn, Soybean, Wheat, Winter Cereal Rye, Dry Bean, and Oats and an In Vitro Assessment of Seed Treatment Sensitivity.

Formally described in 2009, Phytophthora sansomeana is a pathogen of increasing interest in native, agricultural, and horticulturally important plant species. The objective of this study was to elucidate the symptomatic and asymptomatic host range of P. sansomeana on six agricultural crop species commonly used in field crop rotations in Michigan. In addition, sensitivity to oomicides commonly used in seed treatments, including oxathiapiprolin, mefenoxam, ethaboxam, and pyraclostrobin, was performed to aid in disease management recommendations. Plant biomass, quantity of P. sansomeana DNA in roots, and reisolations were used to assess pathogenicity and virulence of 18 isolates of P. sansomeana on each plant species using an inoculated seedling growth chamber assay. Isolates displayed varying levels of virulence to the hosts tested. Reisolations were completed for each plant species tested, and varying quantities of P. sansomeana DNA were found within all plant species root samples. Corn, wheat, soybean, dry bean, and winter cereal rye plants were symptomatic hosts with significant reduction observed in the total plant biomass. No significant reduction in total plant biomass was observed in oats, and oat roots harbored the least amount of P. sansomeana DNA. No P. sansomeana isolates were insensitive to the oomicide compounds tested with mean absolute inhibition (EC50) values of fungicide required for 50% growth inhibition values of 7.8 × 10-2 µg/ml for mefenoxam, 1.13 × 10-1 µg/ml for ethaboxam, 2.6 × 10-2 µg/ml for oxathiapiprolin, and 3.04 × 10-1 µg/ml for pyraclostrobin. These results suggest that common crop rotations in Michigan may not be a viable option to reduce soilborne inoculum accumulation and oomicide seed treatments could be considered for early-season management of P. sansomeana.

The Combined Effects of Azoxystrobin and the Biosurfactant-Producing Bacillus sp. Kol B3 against the Phytopathogenic Fungus Fusarium sambucinum IM 6525.

This study aimed to evaluate how the combined presence of the synthetic fungicide azoxystrobin (AZ) and the biosurfactant-producing Bacillus sp. Kol B3 influences the growth of the phytopathogenic fungus Fusarium sambucinum IM 6525. The results showed a noticeable increase in antifungal effectiveness when biotic and abiotic agents were combined. This effect manifested across diverse parameters, including fungal growth inhibition, changes in hyphae morphology, fungal membrane permeability and levels of intracellular reactive oxygen species (ROS). In response to the presence of Fusarium and AZ in the culture, the bacteria changed the proportions of biosurfactants (surfactin and iturin) produced. The presence of both AZ and/or Fusarium resulted in an increase in iturin biosynthesis. Only in 72 h old bacterial-fungal co-culture a 20% removal of AZ was noted. In the fungal cultures (with and without the addition of the bacteria), the presence of an AZ metabolite named azoxystrobin free acid was detected in the 48th and 72nd hours of the process. The possible involvement of increased iturin and ROS content in antifungal activity of Bacillus sp. and AZ when used together are also discussed. Biosurfactants were analyzed by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Microscopy techniques and biochemical assays were also used.

Development of a Greenhouse Assay to Screen Soybean Varieties for Resistance to Aerial Blight Caused by Rhizoctonia solani Anastomosis Group 1-IA.

Aerial blight, caused by the fungus Rhizoctonia solani anastomosis group (AG) 1-IA, is an economically important soybean disease in the mid-Southern United States. Management has relied on fungicide applications during the season, but there is an increasing prevalence of resistance to commonly used strobilurin fungicides and an urgent need to identify soybean varieties resistant to aerial blight. Because the patchy distribution of the pathogen complicates field variety screening, the present study aimed to develop a greenhouse screening protocol to identify soybean varieties resistant to aerial blight. For this, 88 pathogen isolates were collected from commercial fields and research farms across five Louisiana parishes, and 77% were confirmed to be R. solani AG1-IA. Three polymorphic codominant microsatellite markers were used to explore the genetic diversity of 43 R. solani AG1-IA isolates, which showed high genetic diversity, with 35 haplotypes in total and only two haplotypes common to two other locations. Six genetically diverse isolates were chosen and characterized for their virulence and fungicide sensitivity. The isolate AC2 was identified as the most virulent and was resistant to both active ingredients, azoxystrobin and pyraclostrobin, tested. The six isolates were used in greenhouse variety screening trials using a millet inoculation protocol. Of the 31 varieties screened, only Armor 48-D25 was classified as moderately resistant, and plant height to the first node influenced final disease severity. The study provides short-term solutions for growers to choose less susceptible varieties for planting and lays the foundation to characterize host resistance against this important soybean pathogen.

Prevalence of FRAC Group 11 Fungicide Resistance in Stemphylium vesicarium Isolates, but Not S. beticola Isolates, Causing Stemphylium Leaf Spot of Spinach (Spinacia oleracea).

Stemphylium leaf spot of spinach, caused by Stemphylium beticola and S. vesicarium, is a disease of economic importance in fresh market, processing, and seed production. There have been increasing reports of difficulty managing the disease in the southern United States using fungicides in Fungicide Resistance Action Committee (FRAC) group 11. Isolates of S. beticola and S. vesicarium obtained from spinach leaves and seed from 2001 to 2020 were screened for resistance to azoxystrobin and pyraclostrobin in vitro, in vivo, and using PCR assays to detect mutations in cytochrome b associated with resistance in other fungi (F129L, G137R, and G143A). EC50 values for mycelial growth and conidial germination of S. vesicarium isolates in vitro were significantly less (mean of 0.35 µg/ml) than that of S. vesicarium (mean of 14.17 µg/ml) with both fungicides. All isolates were slightly more sensitive to pyraclostrobin than azoxystrobin in both assays. In vivo assays of plants inoculated with the isolates of S. vesicarium demonstrated poor efficacy of fungicides with each of the two active ingredients. Only the G143A mutation was detected in all spinach isolates of S. vesicarium, including an isolate of S. vesicarium collected in 2003 and 82.9% of isolates from spinach seed lots harvested from crops grown in or after 2017 in Europe, New Zealand, and the United States. The FRAC 11 mutations were not detected in any isolates of S. beticola. The in vitro, in vivo, and DNA mutation assays suggest FRAC group 11 fungicide resistance is widespread in spinach isolates of S. vesicarium but not S. beticola.