CbCyp51-mediated DMI resistance is modulated by codon bias.

Cercospora leaf spot (CLS), caused by the fungus Cercospora beticola, is the most destructive foliar disease of sugar beet worldwide. Resistance to the sterol demethylation inhibitor (DMI) fungicide tetraconazole has been previously correlated to synonymous and non-synonymous mutations in CbCyp51. Here, we extend these analyses to the DMI fungicides prothioconazole, difenoconazole, and mefentrifluconazole in addition to tetraconazole to confirm whether the synonymous and nonsynonymous mutations at amino acid positions 144 and 170 are associated with resistance to these fungicides. Nearly half of the 593 isolates of C. beticola collected in the Red River Valley of North Dakota and Minnesota in 2021 were resistant to all four DMIs. Another 20% were resistant to tetraconazole and prothioconazole, but sensitive to difenoconazole and mefentrifluconazole. A total of 13% of isolates were sensitive to all DMIs tested. We found five CbCyp51 haplotypes and associated them with phenotypes to the four DMIs. The most predominant haplotype (E170_A/ L144F_C) correlated to resistance to all four DMIs with up to 97.6% accuracy. The second most common haplotype (E170_A/L144) consisted of isolates associated with resistance phenotypes to tetraconazole and prothioconazole while also exhibiting sensitive phenotypes to difenoconazole and mefentrifluconazole with up to 98.4% accuracy. Quantitative PCR did not identify differences in CbCyp51 expression between haplotypes. This study gives an understanding for the importance of codon usage in fungicide resistance and provides crop management acuity for fungicide application decision-making.

Validation of fungicide spray strategies and selection for fenhexamid resistance in Botrytis cinerea on greenhouse-grown grapevines.

In this study, in planta assays were conducted to assess the effects of fungicide spray tactics, such as the reduction of the labeled fungicide dose and mixture with a multi-site fungicide, on fungicide resistance selection and disease control using Vitis vinifera 'Cabernet Sauvignon' grown in a greenhouse for two years. The entire clusters were inoculated with B. cinerea isolates at varying frequencies of fenhexamid resistance, followed by fungicide sprays, disease and fenhexamid resistance investigations at critical phenological stages. Our findings indicate that the lower dose of the at-risk fungicide, fenhexamid, effectively managed fenhexamid resistance and disease as well as the higher, labeled dose. In addition, mixture with the multi-site fungicide captan generally resulted a net-positive effect on both resistance management and disease control.

Genome-wide association study of fungicide sensitivity in a Fusarium graminearum population collected from North Dakota.

Fusarium head blight (FHB) is a destructive disease of small grains. The disease is predominantly caused by the haploid ascomycete fungus Fusarium graminearum in North America. To understand the genetics of quantitative traits for sensitivity to fungicides in this fungal pathogen, we conducted a genome-wide association study (GWAS) of sensitivity to two demethylation inhibition (DMI) class fungicides, tebuconazole and prothioconazole, using a F. graminearum population of 183 isolates collected between 1981 and 2013 from North Dakota. Baseline sensitivity to tebuconazole and prothioconazole was established using 21 isolates collected between 1981 and 1994. Most fungal isolates were sensitive to both tebuconazole and prothioconazole, however, five isolates showed significantly reduced sensitivity to prothioconazole. GWAS identified one significant marker-trait association (MTA) on chromosome 3 for tebuconazole resistance while six significant MTAs, one on chromosome 1, three on chromosome 2, and two on chromosome 4, were detected for prothioconazole resistance. Functional annotation of the MTA for tebuconazole revealed a candidate gene encoding a basic helix loop helix (bHLH) domain containing protein that reinforces sterol in the fungal membrane. Putative genes for prothioconazole resistance were also identified, which are involved in RNAi, detoxification by ubiquitin-proteasome pathway, and membrane integrity reinforcement. Considering the potential of the pathogen towards overcoming chemical control, continued monitoring of fungal sensitivities to commercially applied fungicides, especially those containing prothioconazole, is warranted to reduce risks of fungicide resistance in the pathogen populations.

Carboxylic Acid Amide but Not Quinone Outside Inhibitor Fungicide Resistance Mutations Show Clade-Specific Occurrence in Pseudoperonospora cubensis Causing Downy Mildew in Commercial and Wild Cucurbits.

Since its reemergence in 2004, Pseudoperonospora cubensis, the causal agent of cucurbit downy mildew (CDM), has experienced significant changes in fungicide sensitivity. Presently, frequent fungicide applications are required to control the disease in cucumber due to the loss of host resistance. Carboxylic acid amides (CAA) and quinone outside inhibitors (QoI) are two fungicide groups used to control foliar diseases in cucurbits, including CDM. Resistance to these fungicides is associated with single nucleotide polymorphism (SNP) mutations. In this study, we used population analyses to determine the occurrence of fungicide resistance mutations to CAA and QoI fungicides in host-adapted clade 1 and clade 2 P. cubensis isolates. Our results revealed that CAA-resistant genotypes occurred more prominently in clade 2 isolates, with more sensitive genotypes observed in clade 1 isolates, while QoI resistance was widespread across isolates from both clades. We also determined that wild cucurbits can serve as reservoirs for P. cubensis isolates containing fungicide resistance alleles. Finally, we report that the G1105W substitution associated with CAA resistance was more prominent within clade 2 P. cubensis isolates while the G1105V resistance substitution and sensitivity genotypes were more prominent in clade 1 isolates. Our findings of clade-specific occurrence of fungicide resistance mutations highlight the importance of understanding the population dynamics of P. cubensis clades by crop and region to design effective fungicide programs and establish accurate baseline sensitivity to active ingredients in P. cubensis populations.

Fifty Years of Fungicide Development, Deployment, and Future Use.

Plant disease management has not significantly changed in the past 50 years, even as great strides have been made in the understanding of fungal biology and the etiology of plant disease. Issues of climate change, supply chain failures, war, political instability, and exotic invasives have created even more serious implications for world food and fiber security, and the stability of managed ecosystems, underscoring the urgency for reducing plant disease-related losses. Fungicides serve as the primary example of successful, widespread technology transfer, playing a central role in crop protection, reducing losses to both yield and postharvest spoilage. The crop protection industry has continued to improve upon previous fungicide chemistries, replacing active ingredients lost to resistance and newly understood environmental and human health risks, under an increasingly stricter regulatory environment. Despite decades of advances, plant disease management continues to be a constant challenge that will require an integrated approach, and fungicides will continue to be an essential part of this effort.

Optimal Resistance Management for Mixtures of High-Risk Fungicides: Robustness to the Initial Frequency of Resistance and Pathogen Sexual Reproduction.

There is a strong consensus that selection for fungicide resistant pathogen strains can be most effectively limited by using applications of mixtures of fungicides designed to balance disease control against selection. However, how to do this in practice is not entirely characterized. Previous work indicates optimal mixtures of pairs of fungicides which are both at a high risk of resistance can be constructed using pairs of doses that select equally for both single resistant strains in the first year of application. What has not been addressed thus far is the important real-world case in which the initial levels of resistance to each fungicide differ, for example because the chemicals have been available for different lengths of time. We show how recommendations based on equal selection in the first year can be suboptimal in this case. We introduce a simple alternative approach, based on equalizing the frequencies of single resistant strains in the year that achieving acceptable levels of control is predicted to become impossible. We show that this strategy is robust to changes in parameters controlling pathogen epidemiology and fungicide efficacy. We develop our recommendation using a preexisting, parameterized model of Zymoseptoria tritici (the pathogen causing Septoria leaf blotch on wheat), which exemplifies the range of plant pathogens that predominantly spread clonally, but for which sexual reproduction forms an important component of the life cycle. We show that pathogen sexual reproduction can influence the rate at which fungicide resistance develops but does not qualitatively affect our optimal resistance management recommendation. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Sensitivity of the U.S. Wheat Powdery Mildew Population to Quinone Outside Inhibitor Fungicides and Determination of the Complete Blumeria graminis f. sp. tritici Cytochrome b Gene.

Wheat powdery mildew, caused by Blumeria graminis f. sp. tritici, is managed primarily with cultivar resistance and foliar fungicides. Quinone outside inhibitors (QoIs), which target the mitochondrial cytochrome b (cytb) gene, are one of the two main fungicide classes used on wheat. While European populations of B. graminis f. sp. tritici are widely insensitive to QoIs, largely because of the cytb mutation G143A, the QoI sensitivity of the U.S. B. graminis f. sp. tritici population had never been evaluated despite years of QoI use on U.S. wheat. A total of 381 B. graminis f. sp. tritici isolates from 15 central and eastern U.S. states were screened for sensitivity to QoI fungicides pyraclostrobin and picoxystrobin. A modest range of sensitivities was observed, with maximum resistance factors of 11.2 for pyraclostrobin and 5.3 for picoxystrobin. The F129L, G137R, and G143A cytb mutations were not detected in the U.S. B. graminis f. sp. tritici population, nor were mutations identified in the PEWY loop, a key part of the Qo site. Thus, no genetic basis for the observed quantitative variation in QoI sensitivity of U.S. B. graminis f. sp. tritici was identified. Isolate sporulation was weakly negatively associated with reduced QoI sensitivity, suggesting a fitness cost. In the course of the study, the complete B. graminis f. sp. tritici cytb gene sequence was determined for the first time in the isolate 96224 v. 3.16 reference genome. Contrary to previous reports, the gene has an intron that appears to belong to intron group II, which is unusual in fungi. The study was the first QoI sensitivity screening of a large, geographically diverse set of U.S. B. graminis f. sp. tritici isolates, and while the population as a whole remains relatively sensitive, some quantitative loss of efficacy was observed.

Occidiofungin Is the Key Metabolite for Antifungal Activity of the Endophytic Bacterium Burkholderia sp. MS455 Against Aspergillus flavus.

Aflatoxin is a secondary metabolite produced by Aspergillus fungi and presents a major food safety concern globally. Among the available methods for prevention and control of aflatoxin, the application of antifungal bacteria has gained favor in recent years. An endophytic bacterium MS455, isolated from soybean, exhibited broad-spectrum antifungal activity against economically important pathogens, including Aspergillus flavus. MS455 was identified as a strain of Burkholderia based on genomic analysis. Random and site-specific mutations were used in discovery of the genes that share high homology to the ocf gene cluster of Burkholderia contaminans strain MS14, which is responsible for production of the antifungal compound occidiofungin. RNA sequencing analysis demonstrated that ORF1, a homolog to the ambR1 LuxR-type regulatory gene, regulates occidiofungin biosynthesis in MS455. Additionally, 284 differentially expressed genes, including 138 upregulated and 146 downregulated genes, suggesting that, in addition to its role in occidiofungin production, ORF1 is involved in expression of multiple genes, especially those involved in ornibactin biosynthesis. Plate bioassays showed the growth of A. flavus was significantly inhibited by the wild-type strain MS455 as compared with the ORF1 mutant. Similarly, corn kernel assays showed that growth of A. flavus and aflatoxin production were reduced significantly by MS455 as compared with buffer control and the ORF1 mutant. Collectively, the results demonstrated that production of occidiofungin is essential for antifungal activity of the endophytic bacterium MS455. This research has provided insights about antifungal mechanisms of MS455 and development of biological approaches to prevent aflatoxin contamination in plant production.

Endophytic Bacillus subtilis TR21 Improves Banana Plant Resistance to Fusarium oxysporum f. sp. cubense and Promotes Root Growth by Upregulating the Jasmonate and Brassinosteroid Biosynthesis Pathways.

The banana (Musa spp.) industry experiences dramatic annual losses from Fusarium wilt of banana disease, which is caused by the fungus Fusarium oxysporum f. sp. cubense (FOC). Pisang Awak banana 'Fenza No. 1' (Musa spp. cultivar Fenza No. 1), a major banana cultivar with high resistance to F. oxysporum f. sp. cubense race 4, is considered to be ideal for growth in problematic areas. However, 'Fenza No. 1' is still affected by F. oxysporum f. sp. cubense race 1 in the field. TR21 is an endophytic Bacillus subtilis strain isolated from orchids (Dendrobium sp.). Axillary spraying of banana plants with TR21 controls Fusarium wilt of banana, decreasing the growth period and increasing yields in the field. In this study, we established that TR21 increases root growth in different monocotyledonous plant species. By axillary inoculation, TR21 induced a similar transcriptomic change as that induced by F. oxysporum f. sp. cubense race 1 but also upregulated the biosynthetic pathways for the phytohormones brassinosteroid and jasmonic acid in 'Fenza No. 1' root tissues, indicating that TR21 increases Fusarium wilt of banana resistance, shortens growth period, and increases yield of banana by inducing specific transcriptional reprogramming and modulating phytohormone levels. These findings will contribute to the identification of candidate genes related to plant resistance against fungi in a nonmodel system and facilitate further study and exploitation of endophytic biocontrol agents.

Characterization of High Fludioxonil Resistance in Botrytis cinerea Isolates from Calibrachoa Flowers.

The fungicide fludioxonil is one of the most effective single-site fungicides available for managing flower blight caused by Botrytis cinerea on fruit and ornamental crops. Although low and moderate levels of resistance to fludioxonil have been reported in the pathogen across the United States and Europe, high resistance has been reported only from greenhouses in China. In this study, two B. cinerea isolates with high resistance (half maximal effective concentration >100 µg/ml) to fludioxonil were detected on ornamental calibrachoa flowers grown in a greenhouse. These isolates exhibited stable resistance for >20 generations, produced symptoms on calibrachoa flowers sprayed with label rates of fludioxonil, and displayed in vitro fitness penalties with decreased mycelial growth (P < 0.0001) and sporulation (P < 0.0001) compared with sensitive isolates. Highly resistant isolates were identified as MDR1h, containing the ΔL/V497 deletion in mrr1. However, resistance levels and in vitro fitness parameter characteristics were not consistent with this phenotype. One isolate contained the mutation L267V between HAMP domains 1 and 2 of the Bos-1 gene, and both isolates exhibited high osmotic sensitivity and reduced glycerol accumulation in the presence of fludioxonil, indicating that high resistance of these isolates may be associated with the high-osmolarity glycerol mitogen-activated protein kinase pathway.

ATP-Dependent Protease ClpP and Its Subunits ClpA, ClpB, and ClpX Involved in the Field Bismerthiazol Resistance in Xanthomonas oryzae pv. oryzae.

Resistance of Xanthomonas oryzae pv. oryzae, which causes rice bacterial leaf blight, to bismerthiazol has been detected in China since the 1990s. The strains resistant to bismerthiazol on rice plants were more sensitive to bismerthiazol than wild-type (WT) strains in vitro. Here, quantitative PCR was applied to detect the fold expression of adenosine triphosphate-dependent proteases, ClpP and its subunits, which withstand stresses including bactericides in bismerthiazol-resistant strains and their parental susceptible WT strain (ZJ173). Results showed that the expression of ClpP and its subunits was higher in bismerthiazol-resistant strains than in ZJ173. They were upregulated during the early growth phase and downregulated during the middle growth phase in ZJ173 treated with bismerthiazol but did not change in the resistant strains. ClpP and its subunits were overexpressed in X. oryzae pv. oryzae in this study; the higher expression of these genes increased sensitivity in vitro and increased resistance in vivo to bismerthiazol. Bismerthiazol inhibition of exopolysaccharide (EPS) production, biofilm production, and motility was also lower in ClpP and its subunits' overexpression mutants of X. oryzae pv. oryzae. The deletion mutants of ClpP and its subunits in ZJ173 decreased pathogenicity, biofilm production, swimming ability, EPS production, and growth in low-nutrient environments. Moreover, ClpP and its subunits may act downstream of the histidine utilization pathway, which could be inhibited by bismerthiazol in X. oryzae pv. oryzae. Taken together, our results indicated that ClpP and its subunits of X. oryzae pv. oryzae influenced resistance to bismerthiazol.

Fungicide Resistance in Venturia effusa, Cause of Pecan Scab: Current Status and Practical Implications.

Pecan scab, caused by Venturia effusa, is the most economically damaging disease of pecan in the southeastern United States, and annual epidemics are most effectively managed through multiple fungicide applications. The fungicide applications can be the single greatest operating cost for commercial growers and the return on that investment is impacted by fungicide resistance. V. effusa produces multiple generations of conidia per season, exhibits substantial genetic diversity, overwinters as stromata in the tree, and is under immense selection from the applied fungicides, all of which lead to a high risk for developing fungicide resistance. Since the mid-1970s, resistance or reduced sensitivity has been observed in isolates of V. effusa to the methyl benzimidazole carbamates, demethylation inhibitors, quinone outside inhibitors, organotin compounds, and the guanidines. Over the last 10 years, several studies have been conducted that have improved both scab management and fungicide resistance management in V. effusa. The aim of this review is to summarize recent developments in our understanding of fungicide resistance in V. effusa in the context of scab management in southeastern pecan orchards. The history, modes of action, general use of the labeled fungicides, and mechanisms and stability of fungicide resistance in V. effusa are discussed; conclusions and future research priorities are also presented.

A Method for the Examination of SDHI Fungicide Resistance Mechanisms in Phytopathogenic Fungi Using a Heterologous Expression System in Sclerotinia sclerotiorum.

Succinate dehydrogenase inhibitors (SDHIs) are a class of broad-spectrum fungicides used for management of diseases caused by phytopathogenic fungi. In many cases, reduced sensitivity to SDHI fungicides has been correlated with point mutations in the SdhB and SdhC target genes that encode components of the succinate dehydrogenase complex. However, the genetic basis of SDHI fungicide resistance mechanisms has been functionally characterized in very few fungi. Sclerotinia sclerotiorum is a fast-growing and SDHI fungicide-sensitive phytopathogenic fungus that can be conveniently transformed. Given the high amino acid sequence similarity and putative structural similarity of SDHI protein target sites between S. sclerotiorum and other common phytopathogenic ascomycete fungi, we developed an in vitro heterologous expression system that used S. sclerotiorum as a reporter strain. With this system, we were able to demonstrate the function of mutant SdhB or SdhC alleles from several ascomycete fungi in conferring resistance to multiple SDHI fungicides. In total, we successfully validated the function of Sdh alleles that had been previously identified in field isolates of Botrytis cinerea, Blumeriella jaapii, and Clarireedia jacksonii (formerly S. homoeocarpa) in conferring resistance to boscalid, fluopyram, or fluxapyroxad and used site-directed mutagenesis to construct and phenotype a mutant allele that is not yet known to exist in Monilinia fructicola populations. We also examined the functions of these alleles in conferring cross-resistance to more recently introduced SDHIs including inpyrfluxam, pydiflumetofen, and pyraziflumid. The approach developed in this study can be widely applied to interrogate SDHI fungicide resistance mechanisms in other phytopathogenic ascomycetes.

Botrytis spp.: A Contemporary Perspective and Synthesis of Recent Scientific Developments of a Widespread Genus that Threatens Global Food Security.

This perspective presents a synopsis of the topics contained in the Phytopathology Pathogen Spotlight on Botrytis spp. causing gray mold, including pathogen biology and systematics, genomic characterization of new species, perspectives on genome editing, and fungicide resistance. A timely breakthrough to engineer host plant resistance against the gray mold fungus has been demonstrated in planta and may augment chemical controls in the near future. While B. cinerea has garnered much of the research attention, other economically important Botrytis spp. have been identified and characterized via morphological and genome-based approaches. Gray mold control is achieved primarily through fungicide applications but resistance to various chemical classes is a major concern that threatens global plant health and food security. In this issue, new information on molecular mechanism(s) of fungicide resistance and ways to manage control failures are presented. Finally, a significant leap in fundamental pathogen biology has been achieved via development of CRISPR/Cas9 to assess gene function in the fungus which likely will spawn new control mechanisms and facilitate gene discovery studies.

Lack of an Intron in Cytochrome b and Overexpression of Sterol 14α-Demethylase Indicate a Potential Risk for QoI and DMI Resistance Development in Neophysopella spp. on Grapes.

Asian grapevine leaf rust, caused by Neophysopella meliosmae-myrianthae and N. tropicalis, is often controlled by quinone outside inhibitor (QoI) and demethylation inhibitor (DMI) fungicides in Brazil. Here, we evaluated the sensitivity of 55 Neophysopella spp. isolates to pyraclostrobin (QoI) and tebuconazole (DMI). To elucidate the resistance mechanisms, we analyzed the sequences of the cytochrome b (CYTB) and cytochrome P450 sterol 14α-demethylase (CYP51) target proteins of QoI and DMI fungicides, respectively. The CYP51 expression levels were also determined in a selection of isolates. In leaf disc assays, the mean 50% effective concentration (EC50) value for pyraclostrobin was about 0.040 µg/ml for both species. CYTB sequences were identical among all 55 isolates, which did not contain an intron immediately after codon 143. No amino acid substitution was identified at codons 129, 137, and 143. The mean EC50 value for tebuconazole was 0.62 µg/ml for N. tropicalis and 0.46 µg/ml for N. meliosmae-myrianthae, and no CYP51 sequence variation was identified among isolates of the same species. However, five N. meliosmae-myrianthae isolates grew on leaf discs treated at 10 µg/ml tebuconazole, and these were further exposed to tebuconazole selection pressure. Tebuconazole-adapted laboratory isolates of N. meliosmae-myrianthae showed an eight- to 25-fold increase in resistance after four rounds of selection that was not associated with CYP51 target alterations. In comparison with sensitive isolates, CYP51 expression was induced in the presence of tebuconazole in three out of four tebuconazole-adapted isolates tested. These results suggest a potential risk for QoI and DMI resistance development in Neophysopella spp.

The FgCYP51B Y123H Mutation Confers Reduced Sensitivity to Prochloraz and Is Important for Conidiation and Ascospore Development in Fusarium graminearum.

Fusarium graminearum is one of the most important causal agents of Fusarium head blight disease and is controlled mainly by chemicals such as demethylation inhibitor (DMI) fungicides. FgCYP51B is one of the DMI targets in F. graminearum, and Tyrosine123 (Y123) is an important amino acid in F. graminearum CYP51B, located in one of predicted substrate binding pockets based on the binding mode between DMIs and CYP51B. Previous studies suggest that resistance to DMI fungicides is attributed primarily to point mutations in the CYP51 gene and that the Y123H mutation in F. verticillioides CYP51 confers prochloraz resistance in the laboratory. To investigate the function of FgCYP51B Y123 residue in the growth and development, pathogenicity, and DMI resistance, we generated and analyzed the FgCYP51B Y123H mutant. Results revealed that the Y123H mutation led to reduced conidial sporulation and affected ascospore development; moreover, the mutation conferred reduced sensitivity to prochloraz. Quantitative PCR and molecular docking were performed to investigate the resistance mechanism. Results indicated that Y123H mutation changed the target gene expression and decreased the binding affinity of FgCYP51 to prochloraz. These results will attract more attention to the potential DMI-resistant mutation of F. graminearum and increase our understanding of the DMI resistance mechanism.