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.

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 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.

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.

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.

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.

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.

Characterization of iprodione resistance in Botrytis cinerea from strawberry and blackberry.

Gray mold, caused by the fungal pathogen Botrytis cinerea, is one of the most destructive diseases of strawberry. Control of the disease in commercial fields is largely dependent on the application of fungicides, including the dicarboximide iprodione. Single-spore isolates were collected from strawberry fields in Florida, North Carolina, and South Carolina and subjected to an assay using conidial germination that distinguished sensitive (S) isolates from isolates with various levels of resistance to iprodione. Of the 245 isolates, 1 was highly resistant (HR), 5 were moderately resistant (MR), and 43 had low resistance (LR) to iprodione. LR and MR strains were found in the Florida population and in 9 of 11 locations from North Carolina and South Carolina, indicating that resistance was widespread but accounted for only a relatively small percentage of the B. cinerea population. Sequence analysis of the target gene bos1, which codes for a class III histidine kinase, revealed that the MR phenotype was associated with Q369P and N373S mutations and that the LR phenotype was associated with either a I365S or a I365N mutation. The I365S and I365N mutations were also present in five additionally included HR isolates from North Carolina and South Carolina blackberry fields and one HR isolate from a Virginia strawberry field but no mutation or mutation combinations in bos1 were uniquely associated with the HR phenotype. Expression analysis of bos1 in S and HR isolates did not reveal convincing evidence of the gene's involvement in HR resistance either. The six HR isolates had three different phenotypes with respect to their sensitivity to fludioxonil; two were S, two were LR, and two were MR. The fludioxonil LR and MR isolates were also resistant to tolnaftate, an indication of multidrug efflux pump activity. These data suggest that, in addition to point mutations in bos1, drug efflux pump activity and potentially a third mechanism of resistance may be contributing to the iprodione HR phenotype. Detached fruit studies showed that field rates of Rovral 4 Flowable (iprodione) did not control iprodione MR and HR isolates.

Fungicide Resistance Profiles in Botrytis cinerea from Strawberry Fields of Seven Southern U.S. States.

The sensitivity to seven chemical classes of fungicides was investigated in 1,810 Botrytis cinerea isolates collected from strawberry blossoms and fruit in 181 strawberry fields from seven southern states in the United States across 2 years. Ten isolates were examined from each field. Fungicide sensitivity assays were carried out based on visual assessment of diametrical mycelial growth after 4 days of incubation on media amended with discriminatory doses of fungicides in microtiter plates. Results of visual assessments were verified with selected isolates using a previously published germination assay and by inoculating representative isolates with resistant phenotypes on fungicide-sprayed fruit. The overall resistance frequencies of 750 isolates collected in 2012 for thiophanate-methyl, pyraclostrobin, boscalid, cyprodinil, fenhexamid, iprodione, and fludioxonil were 76, 42, 29, 27, 25, 3, and 1%, respectively. Frequencies of 1,060 isolates collected in 2013 were 85, 59, 5, 17, 26, 2, and 1%, respectively. Resistance to thiophanate-methyl and pyraclostrobin was found in virtually every location in both years, whereas resistance to iprodione and fludioxonil was rarely found. Resistant isolates were resistant to either one (23%), two (18%), three (19%), four (14%), five (3%), or six (0.1%) chemical classes of fungicides in 2012. In 2013, this distribution was 24, 29, 26, 8, 2, and 0.3%, respectively. Multifungicide-resistant isolates of B. cinerea were widespread in southern states and evidence suggests that the frequency of isolates with multifungicide resistance increased from 2012 to 2013. The data also show that fungicide resistance in B. cinerea was already present in blossoms, indicating that resistance management needs to be implemented early in the season.

Location-Specific Fungicide Resistance Profiles and Evidence for Stepwise Accumulation of Resistance in Botrytis cinerea.

The fungicide resistance profiles to seven chemical classes of fungicides were investigated in 198 Botrytis cinerea isolates from five blackberry fields and 214 B. cinerea isolates from 10 strawberry fields of North and South Carolina. Populations of B. cinerea tended to have a single dominant, location-specific resistance profile that consisted of resistance to multiple fungicides in fields sprayed weekly with site-specific fungicides. The most prevalent profile in blackberry fields consisted of resistance to thiophanate-methyl, pyraclostrobin, and boscalid. The most prevalent resistance profile found in conventional strawberry fields consisted of resistance to thiophanate-methyl, pyraclostrobin, boscalid, and cyprodinil. A statistical model revealed that multifungicide resistance patterns did not evolve randomly in populations from both crops. Instead, strains resistant to thiophanate-methyl were more likely to acquire resistance to pyraclostrobin, the resulting dual-resistant population was more likely to acquire resistance to boscalid, the resulting triple-resistant population was more likely to acquire resistance to cyprodinil, and the resulting quadruple-resistant population was more likely to acquire resistance to fenhexamid (strawberry population only) compared with random chance. Resistance to iprodione and fludioxonil evolved from a pool of strains with different fungicide resistance profiles. Resistance to thiophanate-methyl, pyraclostrobin, boscalid, and fenhexamid in blackberry isolates was, without exception, based on target gene mutations, including E198A and E198V in ß-tubulin, G143A in cytochrome b, H272Y and H272R in SdhB, and F412I in Erg27, respectively. A new genotype associated with fenhexamid resistance was found in one strain (i.e., Y408H and deletion of P298). Fungicide-resistant strains were present but rare in an unsprayed blackberry field, where some unique phenotypes, including low and medium resistance to fludioxonil, had emerged in the absence of fungicide pressure. The isolates resistant to fludioxonil had effective dose that inhibited 50% of mycelial growth values of 0.16 µg/ml (low resistance) and 0.32 and 0.38 µg/ml (medium resistance) and were also resistant to the anilinopyrimidine fungicide cyprodinil, indicating that this and similar phenotypes will eventually be selected by continued applications of the fludioxonil + cyprodinil premixture Switch. This study shows that multifungicide-resistant phenotypes are common in conventionally maintained strawberry and blackberry fields and that resistance to multiple fungicides evolved from stepwise accumulation of single resistances.

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.

Resistance to Cyprodinil and Lack of Fludioxonil Resistance in Botrytis cinerea Isolates from Strawberry in North and South Carolina.

Chemical control of gray mold of strawberry caused by Botrytis cinerea is essential to prevent pre- and postharvest fruit decay. For more than 10 years, the anilinopyrimidine (AP) cyprodinil and the phenylpyrrole fludioxonil (Switch 62.5WG) have been available to commercial strawberry producers in the United States for gray mold control. Both active ingredients are site-specific inhibitors and, thus, prone to resistance development. In this study, 217 single-spore isolates of B. cinerea from 11 commercial strawberry fields in North and South Carolina were examined for sensitivity to both fungicides. Isolates that were sensitive (53%), moderately resistant (30%), or resistant (17%) to cyprodinil were identified based on germ tube inhibition at discriminatory doses of cyprodinil at 1 and 25 mg/liter at 10 of the 11 locations. None of the isolates was fludioxonil resistant. Phenotypes that were moderately resistant or resistant to cyprodinil were not associated with fitness penalties for mycelial growth rate, spore production, or osmotic sensitivity. Detached fruit assays demonstrated cross resistance between the two AP fungicides cyprodinil and pyrimethanil, and that isolates that were characterized in vitro as moderately resistant or resistant were equivalent in pathogenicity on fruit sprayed with pyrimethanil (currently the only AP registered in strawberry as a solo formulation). This suggests that the in vitro distinction of moderately resistant and resistant isolates is of little if any field relevance. The absence of cross-resistance with fludioxonil, iprodione, cycloheximide, and tolnaftate indicated that multidrug resistance in the form of multidrug resistance phenotypes was unlikely to be involved in conferring resistance to APs in our isolates. Implications for resistance management and disease control are discussed.

Fenhexamid Resistance in Botrytis cinerea from Strawberry Fields in the Carolinas Is Associated with Four Target Gene Mutations.

Botrytis cinerea, the causal agent of gray mold disease, is one of the most important pathogens of strawberry. Its control in commercial strawberry fields is largely dependent on the application of fungicides during bloom and fruit maturation. The hydroxyanilide fenhexamid is one of the most frequently used fungicides in the southeast of the United States for gray mold control. It inhibits the 3-ketoreductase (Erg27) of the ergosterol biosynthesis pathway and, due to this site-specific mode of action, is at risk for resistance development. Single-spore isolates were collected from 11 commercial strawberry fields in North and South Carolina and subjected to a conidial germination assay that distinguished sensitive from resistant phenotypes. Of the 214 isolates collected, 16.8% were resistant to fenhexamid. Resistance was found in three of four locations from North Carolina and in four of seven locations from South Carolina, indicating that resistance was widespread. Mutations in Erg27 (T63I, F412S, F412C, and F412I) were associated with resistance, with F412S the predominant and most widespread mutation. In this study, mutations T63I and F412C in field isolates of B. cinerea are described for the first time. Detached fruit studies showed that field rates of Elevate 50 WDG (fenhexamid) controlled sensitive but not resistant isolates carrying any of the four mutations. Resistant isolates produced the same lesion size and number of sporulating lesions on fruit sprayed with Elevate 50 WDG as on untreated controls, showing the fungicide's loss of efficacy against those isolates. A rapid polymerase chain reaction method was developed to quickly and reliably distinguish isolates sensitive or resistant to fenhexamid in the Carolinas and to determine the mutation associated with resistance. The presence of fenhexamid-resistant strains in B. cinerea from strawberry fields in the Carolinas must be considered in future resistance management practices for sustained gray mold control.

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.

Resistance to Pyraclostrobin and Boscalid in Botrytis cinerea Isolates from Strawberry Fields in the Carolinas.

Botrytis cinerea, the causal agent of gray mold disease, is one of the most important plant-pathogenic fungi affecting strawberry. During the last decade, control of gray mold disease in the southeastern United States has largely been dependent on captan and the use of at-risk fungicides with single-site modes of action, including a combination of the quinone outside inhibitor (QoI) fungicide pyraclostrobin and succinate dehydrogenase inhibitor (SDHI) fungicide boscalid formulated as Pristine 38WG. Reports about loss of efficacy of Pristine in experimental fields in North Carolina prompted us to collect and examine 216 single-spore isolates from 10 conventional fields and 1 organic field in North Carolina and South Carolina in early summer 2011. Sensitivity to pyraclostrobin or boscalid was determined using a conidial germination assay with previously published discriminatory doses. Pyraclostrobin- and pyraclostrobin+boscalid-resistant isolates were found in all conventional fields (with some populations revealing no sensitive isolates) and in the organic field. Among the isolates collected, 66.7% were resistant to pyraclostrobin and 61.5% were resistant to both pyraclostrobin and boscalid. No isolates were identified that were resistant to boscalid but sensitive to pyraclostrobin, indicating that dual resistance may have derived from a QoI-resistant population. The molecular basis of QoI and SDHI fungicide resistance was determined in a subset of isolates. Polymerase chain reaction-restriction fragment length polymorphism analysis of the partial cytochrome b (CYTB) gene showed that pyraclostrobin-resistant isolates possessed the G143A mutation known to confer high levels of QoI fungicide resistance in fungi. Boscalid-resistant isolates revealed point mutations at codon 272 leading to the substitution of histidine to arginine (H272R) or tyrosine (H272Y), affecting the third Fe-S cluster region of the iron-sulfur protein (SdhB) target of SDHIs. The results of the study show that resistance to QoI fungicides and dual resistance to QoI and SDHI fungicides is common in B. cinerea from strawberry fields in the Carolinas. Resistant strains were more frequent in locations heavily sprayed with QoI and SDHI fungicides. However, resistance to both fungicides was also found in the unsprayed, organic field, indicating that some resistant strains may have been introduced from the nursery.

Identification and Prevalence of Botrytis spp. from Blackberry and Strawberry Fields of the Carolinas.

Gray mold disease of blackberry and strawberry is caused by Botrytis cinerea and B. caroliniana in the southeastern United States. In this study, methods to distinguish both species were established and their prevalence was determined in commercial blackberry and strawberry fields. Using DNA from B. cinerea and B. caroliniana reference strains, a species-differentiating polymerase chain reaction (PCR) amplification was developed that amplified G3PDH gene fragments of two different sizes depending on the species. The PCR is performed with three primers (two species-differentiating forward primers and one universal reverse primer) and amplified a 238-bp product from B. cinerea and a 536-bp fragment from B. caroliniana reference isolates. A total of 400 Botrytis isolates were collected from 6 commercial blackberry and 11 strawberry fields of the Carolinas and identified to the species level by the new PCR method. Both Botrytis spp. were identified in blackberry and strawberry fields, but B. caroliniana was less common than B. cinerea. Only 33 of 202 isolates from blackberry fields were identified as B. caroliniana, and the majority of these isolates came from two fields in South Carolina. Only 1 of 198 isolates from strawberries was identified as B. caroliniana, and this isolate was found in central North Carolina. B. cinerea but not B. caroliniana isolates sporulated on potato dextrose agar and Kings medium B. Our results show that B. cinerea and B. caroliniana coexist in at least some commercial blackberry and strawberry fields of the Carolinas, with B. cinerea being the more prevalent species.