Rational Design of a Potential New Nematicide Targeting Chitin Deacetylase.

In a previously published study, the authors devised a molecular topology QSAR (quantitative structure-activity relationship) approach to detect novel fungicides acting as inhibitors of chitin deacetylase (CDA). Several of the chosen compounds exhibited noteworthy activity. Due to the close relationship between chitin-related proteins present in fungi and other chitin-containing plant-parasitic species, the authors decided to test these molecules against nematodes, based on their negative impact on agriculture. From an overall of 20 fungal CDA inhibitors, six showed to be active against Caenorhabditis elegans. These experimental results made it possible to develop two new molecular topology-based QSAR algorithms for the rational design of potential nematicides with CDA inhibitor activity for crop protection. Linear discriminant analysis was employed to create the two algorithms, one for identifying the chemo-mathematical pattern of commercial nematicides and the other for identifying nematicides with activity on CDA. After creating and validating the QSAR models, the authors screened several natural and synthetic compound databases, searching for alternatives to current nematicides. Finally one compound, the N2-(dimethylsulfamoyl)-N-{2-[(2-methyl-2-propanyl)sulfanyl]ethyl}-N2-phenylglycinamide or nematode chitin deacetylase inhibitor, was selected as the best candidate and was further investigated both in silico, through molecular docking and molecular dynamic simulations, and in vitro, through specific experimental assays. The molecule shows favorable binding behavior on the catalytic pocket of C. elegans CDA and the experimental assays confirm potential nematicide activity.

Suppression of Chitin-Triggered Immunity by Plant Fungal Pathogens: A Case Study of the Cucurbit Powdery Mildew Fungus Podosphaera xanthii.

Fungal pathogens are significant plant-destroying microorganisms that present an increasing threat to the world's crop production. Chitin is a crucial component of fungal cell walls and a conserved MAMP (microbe-associated molecular pattern) that can be recognized by specific plant receptors, activating chitin-triggered immunity. The molecular mechanisms underlying the perception of chitin by specific receptors are well known in plants such as rice and Arabidopsis thaliana and are believed to function similarly in many other plants. To become a plant pathogen, fungi have to suppress the activation of chitin-triggered immunity. Therefore, fungal pathogens have evolved various strategies, such as prevention of chitin digestion or interference with plant chitin receptors or chitin signaling, which involve the secretion of fungal proteins in most cases. Since chitin immunity is a very effective defensive response, these fungal mechanisms are believed to work in close coordination. In this review, we first provide an overview of the current understanding of chitin-triggered immune signaling and the fungal proteins developed for its suppression. Second, as an example, we discuss the mechanisms operating in fungal biotrophs such as powdery mildew fungi, particularly in the model species Podosphaera xanthii, the main causal agent of powdery mildew in cucurbits. The key role of fungal effector proteins involved in the modification, degradation, or sequestration of immunogenic chitin oligomers is discussed in the context of fungal pathogenesis and the promotion of powdery mildew disease. Finally, the use of this fundamental knowledge for the development of intervention strategies against powdery mildew fungi is also discussed.

RNAi Technology: A New Path for the Research and Management of Obligate Biotrophic Phytopathogenic Fungi.

Powdery mildew and rust fungi are major agricultural problems affecting many economically important crops and causing significant yield losses. These fungi are obligate biotrophic parasites that are completely dependent on their hosts for growth and reproduction. Biotrophy in these fungi is determined by the presence of haustoria, specialized fungal cells that are responsible for nutrient uptake and molecular dialogue with the host, a fact that undoubtedly complicates their study under laboratory conditions, especially in terms of genetic manipulation. RNA interference (RNAi) is the biological process of suppressing the expression of a target gene through double-stranded RNA that induces mRNA degradation. RNAi technology has revolutionized the study of these obligate biotrophic fungi by enabling the analysis of gene function in these fungal. More importantly, RNAi technology has opened new perspectives for the management of powdery mildew and rust diseases, first through the stable expression of RNAi constructs in transgenic plants and, more recently, through the non-transgenic approach called spray-induced gene silencing (SIGS). In this review, the impact of RNAi technology on the research and management of powdery mildew and rust fungi will be addressed.

A Loop-Mediated Isothermal Amplification Assay for the Identification of Botrytis fragariae in Strawberry.

Gray mold in strawberry is caused by multiple species of Botrytis, including Botrytis cinerea, B. pseudocinerea, B. fragariae, and B. mali. The species B. cinerea and B. fragariae are widespread in production regions of the eastern United States and Germany, and their distinction is important for disease management strategies. Currently, the only way to differentiate these species in field samples is by PCR, which is time consuming, labor intensive, and costly. In this study, a loop-mediated isothermal amplification (LAMP) technique was developed based on species-specific NEP2 gene nucleotide sequences. The designed primer set specifically amplified B. fragariae DNA and no other Botrytis spp. (B. cinerea, B. mali, and B. pseudocinerea) or plant pathogens. The LAMP assay was able to amplify fragments from DNA extracted from infected fruit using a rapid DNA extraction protocol, confirming its ability to detect low amounts of B. fragaria DNA from field-infected fruit. In addition, a blind test was performed to identify B. fragariae in 51 samples collected from strawberry fields in the eastern United States using the LAMP technique. The B. fragariae samples were identified with a reliability of 93.5% (29 of 32), and none of the B. cinerea, B. pseudocinerea, or B. mali samples included in the test were amplified in 10 min. Our results show that the LAMP technique is a specific and reliable method for the detection of B. fragariae from infected fruit tissue and can help to control this important disease in the field.

Rational Design of Chitin Deacetylase Inhibitors for Sustainable Agricultural Use Based on Molecular Topology.

Fungicide resistance is a major concern in modern agriculture; therefore, there is a pressing demand to develop new, greener chemicals. Chitin is a major component of the fungal cell wall and a well-known elicitor of plant immunity. To overcome chitin recognition, fungal pathogens developed different strategies, with chitin deacetylase (CDA) activity being the most conserved. This enzyme is responsible for hydrolyzing the N-acetamido group in N-acetylglucosamine units of chitin to convert it to chitosan, a compound that can no longer be recognized by the plant. In previous works, we observed that treatments with CDA inhibitors, such as carboxylic acids, reduced the symptoms of cucurbit powdery mildew and induced rapid activation of chitin-triggered immunity, indicating that CDA could be an interesting target for fungicide development. In this work, we developed an in silico strategy based on QSAR (quantitative structure-activity relationship) and molecular topology (MT) to discover new, specific, and potent CAD inhibitors. Starting with the chemical structures of few carboxylic acids, with and without disease control activity, three predictive equations based on the MT paradigm were developed to identify a group of potential molecules. Their fungicidal activity was experimentally tested, and their specificity as CDA inhibitors was studied for the three best candidates by molecular docking simulations. To our knowledge, this is the first time that MT has been used for the identification of potential CDA inhibitors to be used against resistant powdery mildew strains. In this sense, we consider of special interest the discovery of molecules capable of stimulating the immune system of plants by triggering a defensive response against fungal species that are highly resistant to fungicides such as powdery mildew.

Suppression of Chitin-Triggered Immunity by a New Fungal Chitin-Binding Effector Resulting from Alternative Splicing of a Chitin Deacetylase Gene.

Phytopathogenic fungi have evolved mechanisms to manipulate plant defences, such as chitin-triggered immunity, a plant defensive response based on the recognition of chitin oligomers by plant-specific receptors. To cope with chitin resistance, fungal pathogens have developed different strategies to prevent chitin recognition, such as binding, breaking, or modifying immunogenic oligomers. In powdery mildew fungi, the activity of chitin deacetylase (CDA) is crucial for this purpose, since silencing of the CDA gene leads to a rapid activation of chitin signalling and the subsequent suppression of fungal growth. In this work, we have identified an unusually short CDA transcript in Podosphaera xanthii, the cucurbit powdery mildew pathogen. This transcript, designated PxCDA3, appears to encode a truncated version of CDA resulting from an alternative splicing of the PxCDA gene, which lacked most of the chitin deacetylase activity domain but retained the carbohydrate-binding module. Experiments with the recombinant protein showed its ability to bind to chitin oligomers and prevent the activation of chitin signalling. Furthermore, the use of fluorescent fusion proteins allowed its localization in plant papillae at pathogen penetration sites. Our results suggest the occurrence of a new fungal chitin-binding effector, designated CHBE, involved in the manipulation of chitin-triggered immunity in powdery mildew fungi.

Chitin Deacetylase, a Novel Target for the Design of Agricultural Fungicides.

Fungicide resistance is a serious problem for agriculture. This is particularly apparent in the case of powdery mildew fungi. Therefore, there is an urgent need to develop new agrochemicals. Chitin is a well-known elicitor of plant immunity, and fungal pathogens have evolved strategies to overcome its detection. Among these strategies, chitin deacetylase (CDA) is responsible for modifying immunogenic chitooligomers and hydrolysing the acetamido group in the N-acetylglucosamine units to avoid recognition. In this work, we tested the hypothesis that CDA can be an appropriate target for antifungals using the cucurbit powdery mildew pathogen Podosphaera xanthii. According to our hypothesis, RNAi silencing of PxCDA resulted in a dramatic reduction in fungal growth that was linked to a rapid elicitation of chitin-triggered immunity. Similar results were obtained with treatments with carboxylic acids such as EDTA, a well-known CDA inhibitor. The disease-suppression activity of EDTA was not associated with its chelating activity since other chelating agents did not suppress disease. The binding of EDTA to CDA was confirmed by molecular docking studies. Furthermore, EDTA also suppressed green and grey mould-causing pathogens applied to oranges and strawberries, respectively. Our results conclusively show that CDA is a promising target for control of phytopathogenic fungi and that EDTA could be a starting point for fungicide design.

Resistance to the SDHI Fungicides Boscalid and Fluopyram in Podosphaera xanthii Populations from Commercial Cucurbit Fields in Spain.

Powdery mildew is caused by Podosphaera xanthii, and is one of the most important diseases that attacks Spanish cucurbit crops. Fungicide application is the primary control tool; however, its effectiveness is hampered by the rapid development of resistance to these compounds. In this study, the EC50 values of 26 isolates were determined in response to the succinate dehydrogenase inhibitor (SDHI) fungicides boscalid and fluopyram. From these data, the discriminatory doses were deduced and used for SDHI resistance monitoring during the 2018 and 2019 growing seasons. Of the 298 isolates analysed, 37.9% showed resistance to boscalid and 44% to fluopyram. Although different phenotypes were observed in leaf disc assays, the resistant isolates showed the same phenotype in plant assays. Compared to sensitive isolates, two amino acid changes were found in the SdhC subunit, A86V and G151R, which are associated mostly with resistance patterns to fluopyram and boscalid, respectively. Furthermore, no significant differences were observed in terms of fitness cost between the selected sensitive and resistant isolates analysed here. Lastly, a loop-mediated isothermal amplification (LAMP) assay was developed to detect A86V and G151R mutations using conidia obtained directly from infected material. Our results show that growers could continue to use boscalid and fluopyram, but resistance management practices must be implemented.

Gene Mining for Conserved, Non-Annotated Proteins of Podosphaera xanthii Identifies Novel Target Candidates for Controlling Powdery Mildews by Spray-Induced Gene Silencing.

The powdery mildew fungus Podosphaera xanthii is one of the most important limiting factors for cucurbit production worldwide. Despite the significant efforts made by breeding and chemical companies, effective control of this pathogen remains elusive to growers. In this work, we examined the suitability of RNAi technology called spray-induced gene silencing (SIGS) for controlling cucurbit powdery mildew. Using leaf disc and cotyledon infiltration assays, we tested the efficacy of dsRNA applications to induce gene silencing in P. xanthii. Furthermore, to identify new target candidate genes, we analyzed sixty conserved and non-annotated proteins (CNAPs) deduced from the P. xanthii transcriptome in silico. Six proteins presumably involved in essential functions, specifically respiration (CNAP8878, CNAP9066, CNAP10905 and CNAP30520), glycosylation (CNAP1048) and efflux transport (CNAP948), were identified. Functional analysis of these CNAP coding genes by dsRNA-induced gene silencing resulted in strong silencing phenotypes with large reductions in fungal growth and disease symptoms. Due to their important contributions to fungal development, the CNAP1048, CNAP10905 and CNAP30520 genes were selected as targets to conduct SIGS assays under plant growth chamber conditions. The spray application of these dsRNAs induced high levels of disease control, supporting that SIGS could be a sustainable approach to combat powdery mildew diseases.

A haustorial-expressed lytic polysaccharide monooxygenase from the cucurbit powdery mildew pathogen Podosphaera xanthii contributes to the suppression of chitin-triggered immunity.

Podosphaera xanthii is the main causal agent of cucurbit powdery mildew and a limiting factor of crop productivity. The lifestyle of this fungus is determined by the development of specialized parasitic structures inside epidermal cells, termed haustoria, that are responsible for the acquisition of nutrients and the release of effectors. A typical function of fungal effectors is the manipulation of host immunity, for example the suppression of pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). Chitin is a major component of fungal cell walls, and chitin oligosaccharides are well-known PAMP elicitors. In this work, we examined the role of PHEC27213, the most highly expressed, haustorium-specific effector candidate of P. xanthii. According to different computational predictions, the protein folding of PHEC27213 was similar to that of lytic polysaccharide monooxygenases (LPMOs) and included a conserved histidine brace; however, PHEC27213 had low sequence similarity with LPMO proteins and displayed a putative chitin-binding domain that was different from the canonical carbohydrate-binding module. Binding and enzymatic assays demonstrated that PHEC27213 was able to bind and catalyse colloidal chitin, as well as chitooligosaccharides, acting as an LPMO. Furthermore, RNAi silencing experiments showed the potential of this protein to prevent the activation of chitin-triggered immunity. Moreover, proteins with similar features were found in other haustorium-forming fungal pathogens. Our results suggest that this protein is a new fungal LPMO that catalyses chitooligosaccharides, thus contributing to the suppression of plant immunity during haustorium development. To our knowledge, this is the first mechanism identified in the haustorium to suppress chitin signalling.

A Hybrid Genome Assembly Resource for Podosphaera xanthii, the Main Causal Agent of Powdery Mildew Disease in Cucurbits.

Podosphaera xanthii is the main causal agent of powdery mildew in cucurbits and, arguably, the most important fungal pathogen of cucurbit crops. Here, we present the first reference genome assembly for P. xanthii. We performed a hybrid genome assembly, using reads from Illumina NextSeq550 and PacBio Sequel S3. The short and long reads were assembled into 1,727 scaffolds with an N50 size of 163,173 bp, resulting in a 142-Mb genome size. The combination of homology-based and ab initio predictions allowed the prediction of 14,911 complete genes. Repetitive sequences comprised 76.2% of the genome. Our P. xanthii genome assembly improves considerably the molecular resources for research on P. xanthii-cucurbit interactions and provides new opportunities for further genomics, transcriptomics, and evolutionary studies in powdery mildew fungi.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Fungicide Resistance in Powdery Mildew Fungi.

Powdery mildew fungi (Erysiphales) are among the most common and important plant fungal pathogens. These fungi are obligate biotrophic parasites that attack nearly 10,000 species of angiosperms, including major crops, such as cereals and grapes. Although cultural and biological practices may reduce the risk of infection by powdery mildew, they do not provide sufficient protection. Therefore, in practice, chemical control, including the use of fungicides from multiple chemical groups, is the most effective tool for managing powdery mildew. Unfortunately, the risk of resistance development is high because typical spray programs include multiple applications per season. In addition, some of the most economically destructive species of powdery mildew fungi are considered to be high-risk pathogens and are able to develop resistance to several chemical classes within a few years. This situation has decreased the efficacy of the major fungicide classes, such as sterol demethylation inhibitors, quinone outside inhibitors and succinate dehydrogenase inhibitors, that are employed against powdery mildews. In this review, we present cases of reduction in sensitivity, development of resistance and failure of control by fungicides that have been or are being used to manage powdery mildew. In addition, the molecular mechanisms underlying resistance to fungicides are also outlined. Finally, a number of recommendations are provided to decrease the probability of resistance development when fungicides are employed.

Monitoring Methyl Benzimidazole Carbamate-Resistant Isolates of the Cucurbit Powdery Mildew Pathogen, Podosphaera xanthii, Using Loop-Mediated Isothermal Amplification.

Powdery mildew, caused by the fungus Podosphaera xanthii, is one of the most economically important diseases affecting cucurbit crops in Spain. Currently, chemical control offers the most efficient management of the disease; however, P. xanthii isolates resistant to multiple classes of site-specific fungicides have been reported in the Spanish cucurbit powdery mildew population. In previous studies, resistance to the fungicides known as methyl benzimidazole carbamates (MBCs) was found to be caused by the amino acid substitution E198A on ß-tubulin. To detect MBC-resistant isolates in a faster, more efficient, and more specific way than the traditional methods used to date, a loop-mediated isothermal amplification (LAMP) system was developed. In this study, three sets of LAMP primers were designed. One set was designed for the detection of the wild-type allele and two sets were designed for the E198A amino acid change. Positive results were only obtained with both mutant sets; however, LAMP reaction conditions were only optimized with primer set 2, which was selected for optimal detection of the E198A amino acid change in P. xanthii-resistant isolates, along with the optimal temperature and duration parameters of 65°C for 75 min, respectively. The hydroxynaphthol blue (HNB) metal indicator was used for quick visualization of results through the color change from violet to sky blue when the amplification was positive. HNB was added before the amplification to avoid opening the lids, thus decreasing the probability of contamination. To confirm that the amplified product corresponded to the ß-tubulin gene, the LAMP product was digested with the enzyme LweI and sequenced. Our results show that the LAMP technique is a specific and reproducible method that could be used for monitoring MBC resistance of P. xanthii directly in the field.

The Functional Characterization of Podosphaera xanthii Candidate Effector Genes Reveals Novel Target Functions for Fungal Pathogenicity.

Podosphaera xanthii is the main causal agent of powdery mildew disease in cucurbits. In a previous study, we determined that P. xanthii expresses approximately 50 Podosphaera effector candidates (PECs), identified based on the presence of a predicted signal peptide and the absence of functional annotation. In this work, we used host-induced gene silencing (HIGS), employing Agrobacterium tumefaciens as a vector for the delivery of the silencing constructs (ATM-HIGS), to identify genes involved in early plant-pathogen interaction. The analysis of seven selected PEC-encoding genes showed that six of them, PEC007, PEC009, PEC019, PEC032, PEC034, and PEC054, are required for P. xanthii pathogenesis, as revealed by reduced fungal growth and increased production of hydrogen peroxide by host cells. In addition, protein models and protein-ligand predictions allowed us to identify putative functions for these candidates. The biochemical activities of PEC019, PEC032, and PEC054 were elucidated using their corresponding proteins expressed in Escherichia coli. These proteins were confirmed as phospholipid-binding protein, α-mannosidase, and cellulose-binding protein. Further, BLAST searches showed that these three effectors are widely distributed in phytopathogenic fungi. These results suggest novel targets for fungal effectors, such as host-cell plasma membrane, host-cell glycosylation, and damage-associated molecular pattern-triggered immunity.

Heteroplasmy for the Cytochrome b Gene in Podosphaera xanthii and its Role in Resistance to QoI Fungicides in Spain.

In Spain, management of the cucurbit powdery mildew pathogen Podosphaera xanthii is strongly dependent on chemicals such as quinone outside inhibitor (QoI) fungicides. In a previous report, widespread resistance to QoI fungicides in populations of P. xanthii in south-central Spain was documented, but the molecular mechanisms of resistance remained unclear. In this work, the role of the Rieske-FeS (risp) and the cytochrome b (cytb) gene mutations in QoI resistance of P. xanthii were examined. No point mutations in the risp gene were found in the three QoI-resistant isolates analyzed. For cytb, sequence analysis revealed the presence of a G143A substitution that occurs in many QoI-resistant fungi. This mutation was always detected in QoI-resistant isolates of P. xanthii; however, it was also detected in sensitive isolates. To better understand the role of heteroplasmy for cytb in QoI resistance of P. xanthii, an allele-specific quantitative PCR was developed to quantify the relative abundance of the G143 (sensitive) and A143 (resistant) alleles. High relative abundance of A143 allele (70%) was associated with isolates resistant to QoI fungicides; however, QoI-sensitive isolates also carried the mutated allele in frequencies ranged from 10 to 60%. Our data suggest that G143A mutation in cytb is the primary factor involved in QoI resistance of P. xanthii but the proportion of G143 and A143 alleles in an isolate may determine its QoI resistance level.

Resistance to the SDHI Fungicides Boscalid, Fluopyram, Fluxapyroxad, and Penthiopyrad in Botrytis cinerea from Commercial Strawberry Fields in Spain.

Gray mold, caused by the necrotrophic fungus Botrytis cinerea., is one of the most economically important diseases of strawberry. Gray mold control involves the application of fungicides throughout the strawberry growing season; however, B. cinerea isolates resistant to multiple classes of site-specific fungicides have been recently reported in the Spanish gray mold population. Succinate dehydrogenase inhibitors (SDHI) constitute a relatively novel class of fungicides registered for gray mold control representing new alternatives for strawberry growers. In the present study, 37 B. cinerea isolates previously characterized for their sensitivity to boscalid and amino acid changes in the SdhB protein were used to determine the effective concentration that reduces mycelial growth by 50% (EC50) to fluopyram, fluxapyroxad, and penthiopyrad. The present study was also conducted to obtain discriminatory doses to monitor SDHI fungicide resistance in 580 B. cinerea isolates collected from 27 commercial fields in Spain during 2014, 2015, and 2016. The EC50 values ranged from 0.01 to >100 µg/ml for fluopyram, <0.01 to 4.19 µg/ml for fluxapyroxad, and, finally, <0.01 to 59.65 µg/ml for penthiopyrad. Based on these results, as well as findings from a previous publication, the discriminatory doses chosen to examine sensitivities to boscalid, fluopyram, fluxapyroxad, and penthiopyrad were 100, 15, 1, and 6 µg/ml, respectively. Over the course of the 3-year monitoring period, the overall frequencies of resistance to the four SDHI were 56.9, 6.9, 12.9, and 24.6%, respectively. The frequency of boscalid-resistant isolates decreased from 73 to 41% over the years; however, the fluopyram-resistant isolates increased from 5 to 10% after 1 year of registration. Four SDHI resistance patterns were observed in our population, which included patterns I (30%; resistance to boscalid), II (13.8%; resistance to boscalid and penthiopyrad), III (5.7%; boscalid, fluxapyroxad, and penthiopyrad), and IV (7.9%; resistance to boscalid, fluopyram, fluxapyroxad, and penthiopyrad). Patterns I and II were associated with the amino acid substitutions H272R and H272Y; pattern III was associated only with the H272Y mutation; and, finally, pattern IV was associated with the N230I allele in the SdhB subunit. For gray mold management, it is suggested that the simultaneous use of boscalid and penthiopyrad should be limited to one application per season; however, fluxapyroxad and, especially, fluopyram could be used as valid SDHI alternatives for gray mold control, although they should be applied with caution.

Characterization of Resistance to Six Chemical Classes of Site-Specific Fungicides Registered for Gray Mold Control on Strawberry in Spain.

Botrytis cinerea, causal agent of the gray mold disease, is one of the most economically important fungal pathogens of strawberry worldwide. In Spain, as in other parts of the world, management of gray mold control primarily involves the application of fungicides. To determine the fungicide resistance of the Spanish strawberry field population, 367 B. cinerea isolates were examined from one organic and 13 conventional strawberry fields in Huelva (Spain) in 2014 and 2015. The sensitivities of these isolates to six fungicides used for gray mold management in Spain were examined using a spore germination assay based on previously published discriminatory doses. The frequency of resistance to pyraclostrobin, boscalid, cyprodinil, fenhexamid, iprodione, and fludioxonil was 74.6, 64.8, 37.0, 23.7, 14.7, and 0.8%, respectively. The majority of isolates (35.1%) were resistant to three different fungicides classes. Within these isolates, the most prevalent resistance profile (55.8%) was resistance to pyraclostrobin, boscalid, and cyprodinil, followed by the resistance profile (30.2%) of resistance to pyraclostrobin, boscalid, and fenhexamid. One isolate collected in 2015 was resistant to all six fungicide classes. Resistance to boscalid, fenhexamid, iprodione, and pyraclostrobin was found to be caused by amino acid substitutions on target proteins, including H272R/Y in SdhB, F412I/S/V in Erg27, I365 N/S in Bos1, and G143A in Cytb, respectively. The presence of multifungicide resistance phenotypes in B. cinerea isolates from strawberry fields in Spain must be considered in the development of future resistance management practices.

Monitoring Resistance to SDHI Fungicides in Botrytis cinerea From Strawberry Fields.

Succinate dehydrogenase inhibitor (SDHI) fungicides have been used to control gray mold of strawberry for more than a decade, and selection for resistance in the causal agent Botrytis cinerea has become a threat to producers. In total, 2,570 B. cinerea isolates were collected from strawberry fields in the eastern United States across three seasons and their sensitivity to the SDHI materials boscalid, fluopyram, fluxapyroxad, and penthiopyrad was assessed. Assays were based on visual assessment of presence or absence of mycelial growth on media amended with discriminatory fungicide doses to distinguish sensitive from resistant isolates, respectively. Overall frequencies of isolates resistant to boscalid, fluopyram, fluxapyroxad, and penthiopyrad increased over the 3 years to 30.0, 1.0, 5.5, and 7.4%, respectively. Four resistance patterns, designated A, B, C, or D, were found. Pattern A isolates were resistant to boscalid with the allele H272R at locus sdhB; pattern B isolates were resistant to boscalid and penthiopyrad with the allele H272R or H272Y at locus sdhB; pattern C isolates were resistant to boscalid, fluxapyroxad, and penthiopyrad with the allele H272Y at locus sdhB; and pattern D isolates were resistant to boscalid, fluopyram, fluxapyroxad, and penthiopyrad with alleles P225F or N230I at locus sdhB. Isolates with alleles H272Y, N230I, or P225F were sensitive to a new SDHI, benzovindiflupyr, with mean effective dose that inhibits 50% of mycelial growth values of less than 0.5 µg/ml for each genotype, suggesting that this fungicide may be useful for resistance management. Our data show an increase of B. cinerea isolates resistant to SDHI fungicides over three consecutive production seasons. Resistance management practices must be implemented for the sustained efficacy of SDHI fungicides against gray mold of strawberry.

Independent Emergence of Resistance to Seven Chemical Classes of Fungicides in Botrytis cinerea.

Gray mold, caused by the fungal pathogen Botrytis cinerea, is one of the most destructive diseases of small fruit crops and control is largely dependent on the application of fungicides. As part of a region-wide resistance-monitoring program that investigated 1,890 B. cinerea isolates from 189 fields in 10 states of the United States, we identified seven isolates (0.4%) from five locations in four different states with unprecedented resistance to all seven Fungicide Resistance Action Committee (FRAC) codes with single-site modes of action including FRAC 1, 2, 7, 9, 11, 12, and 17 registered in the United States for gray mold control. Resistance to thiophanate-methyl, iprodione, boscalid, pyraclostrobin, and fenhexamid was based on target gene mutations that conferred E198A and F200Y in ß-tubulin, I365N/S in Bos1, H272R/Y in SdhB, G143A in Cytb, and T63I and F412S in Erg27. Isolates were grouped into MDR1 and MDR1h phenotypes based on sensitivity to fludioxonil and variations in transcription factor mrr1. MDR1h isolates had a previously described 3-bp deletion at position 497 in mrr1. Expression of ABC transporter atrB was increased in MDR1 isolates but highest in MDR1h isolates. None of the isolates with seven single resistances (SR) had identical nucleotide variations in target genes, indicating that they emerged independently. Multifungicide resistance phenotypes did not exhibit significant fitness penalties for the parameters used in this study, but MDR1h isolates produced more sclerotia at low temperatures and exhibited increased sensitivity to salt stress. In this study we show that current resistance management strategies have not been able to prevent the geographically independent development of resistance to all seven site-specific fungicides currently registered for gray mold control in the United States and document the presence of MDR1h in North America.

Resistance to fludioxonil in Botrytis cinerea isolates from blackberry and strawberry.

Site-specific fungicides, including the phenylpyrrole fludioxonil, are frequently used for gray mold control but are at risk for the development of resistance. In this study, field isolates that were low-resistant (LR) and moderately resistant (MR) to fludioxonil from blackberry and strawberry fields of North Carolina, South Carolina, and Virginia were characterized. Genes involved in osmoregulation, including bcsak1, BcOS4, bos5, and BRRG-1, were cloned and sequenced to detect potential target gene alterations; however, none were found. A previously described mutation (R632I) in transcription factor Mrr1, which is known to increase the expression of ATP-binding cassette transporter AtrB, was found in MR but not in sensitive (S) or LR isolates. Expression of atrB in MR isolates was ≈200-fold increased compared with an S isolate; however, 30- to 100-fold overexpression was also detected in LR isolates. Both MR isolates exhibited increased sensitivity to salt stress in the form of mycelial growth inhibition at 4% NaCl, indicating a disruption of osmoregulatory processes in those strains. However, the glycerol content was indistinguishable between S, LR, and MR isolates with and without exposure to fludioxonil, suggesting that the glycerol synthesis pathway may not be a part of the resistance mechanism in LR or MR strains. An investigation into the origin of LR and MR isolates from blackberry revealed two insertions in the mrr1 gene consistent with those found in the Botrytis clade group S. The emergence of strains overexpressing atrB in European and now in North American strawberry fields underscores the importance of this resistance mechanism for development of resistance to fludioxonil in Botrytis cinerea.