Identification of sequence mutations in Phytophthora cactorum genome associated with mefenoxam resistance and development of a molecular assay for the mutant detection in strawberry (F. ×ananassa)

Identification of sequence mutations in Phytophthora cactorum genome associated with mefenoxam resistance and development of a molecular assay for the mutant detection in strawberry (F. ×ananassa) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Identification of sequence mutations in Phytophthora cactorum genome associated with mefenoxam resistance and development of a molecular assay for the mutant detection in strawberry (F. ×ananassa) Marcus Vinicius Marin, Juliana S. Baggio, Youngjae Oh, Hyeondae Han, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2097400/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 May, 2023 Read the published version in Scientific Reports → Version 1 posted 9 You are reading this latest preprint version Abstract Phytophthora crown rot (PhCR) caused by P. cactorum is one of the most damaging diseases of strawberry worldwide. Mefenoxam is one of the major fungicides currently applied to manage PhCR. However, the emergence and spread of resistant isolates have made controlling the pathogen in the field problematic. In the present study, using whole genome sequencing analysis, mutations associated with mefenoxam-resistant isolates were identified in six different genomic regions of P. cactorum . The 95.54% reads from a sensitive isolate pool and 95.65% from a resistant isolate pool were mapped to the reference genome of P. cactorum P414. Four point mutations were in coding regions while the other two were in noncoding regions. The genes harboring mutations were functionally unknown. All mutations present in resistant isolates were confirmed by sanger sequencing of PCR products. For the rapid diagnostic assay, SNP-based high-resolution melting (HRM) markers were developed to differentiate mefenoxam-resistant P. cactorum from sensitive isolates. The HRM markers R3-1F/R3-1R and R2-1F/R2-1R were suitable to differentiate both sensitive and resistant profiles using clean and crude DNA extraction. Our findings may contribute to a better understanding of the mechanisms of resistance of mefenoxam in oomycetes as well as contribute to the monitoring of P. cactorum populations for the sustainable use of this product. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Globally, Florida is the largest strawberry winter producer, being responsible for more than 5% of the U.S. production 1 . In 2020, Florida produced approximately 89,000 tons of strawberry over its 4000 harvested hectares raising 240 million dollars 2 . Unfortunately, crop losses caused by plant pathogens represent one of the major threats in strawberry production and agriculture in general and have become even more challenging since organisms are prone to selection for resistance to single-site fungicides commonly used for their control. Phytophthora crown rot (PhCR), caused mainly by Phytophthora cactorum , is an important disease of strawberry worldwide. Symptoms are characterized by wilting and stunting of plants and eventual death due to crown collapse. In Florida, production is based on an annual plasticulture system with mulched, raised beds that are fumigated prior to planting 3 . New transplants acquired every season are the major source of inoculum for this pathogen 4 . After transplanting, daytime overhead irrigation is provided for 10 to 14 days for plant establishment, which creates a conducive environment for disease development immediately at the beginning of the season. Leather rot (LR), also caused by P. cactorum , poses a threat to strawberry production as well. It affects fruit in all development stages and confers discoloration, and unpleasant taste and odor 5 , 6 . Because symptoms on ripped fruit are sometimes subtle, infected fruit could be picked along with healthy fruits and become a post-harvest problem, besides the direct pre-harvest losses 7 . However, epidemics of this disease are sporadic and associated with heavy rainfall events during the fruiting stage 5 . Management of both diseases is based on the combination of resistant cultivars, cultural practices, and chemical applications 7 , 8 . Although resistant cultivars may be available, chemical applications have been widely used since most cultivars are bred and selected based on fruit quality and yield and not for resistance to Phytophthora 9 . Mefenoxam and phosphite products are the most common chemicals used by Florida growers to manage PhCR and LR, but azoxystrobin is also registered to control LR 10 . However, P. cactorum isolates resistant to mefenoxam and azoxystrobin have been found in Florida strawberry fields 11 , 12 . Mefenoxam, the most widely used product, is a site-specific phenylamide fungicide with systemic activity and oomycete specificity, classified as having a high risk for pathogen-resistance selection 13 – 15 . Mefenoxam is the more active enantiomer contained in the racemic fungicide metalaxyl, broadly used in the control of other oomycetes 16 . It is hypothesized that this compound binds to specific sites responsible for ribosomal RNA (rRNA) synthesis, which reduces mycelial growth and zoospore germination 13,17−19 . Mefenoxam efficacy in strawberry production has been threatened by the emergence of fungicide-resistant populations, putting the control of Phytophthora diseases in strawberry fields at risk 11 , 15 . Several mefenoxam-resistant Phytophthora spp. have been reported in multiple hosts, such as P. capsici on cucurbits and peppers, P. infestans on tomato, and P. erythroseptica on potatoes 16,19−23 . Acquired resistance accompanied by fitness penalties on resistant isolates, such as slow growth, have been reported for some species 13 , 14 . However, stable resistance in the subunit of RNA polymerase has been reported providing advantages to P. insfestans in circumventing the fungicide effect without fitness disadvantages 24 . In this scenario, fungicide sensitivity monitoring plays an important role in the implementation of integrated disease management programs. Moreover, comprehending the mechanisms involved with mefenoxam resistance would aid the monitoring process. In P. infestans , for example, eight single nucleotide polymorphisms (SNPs) within the RPA190 gene associated with metalaxyl resistance were identified as causing amino acid changes, such as K267E, R296H, F382Y, T443A, A597T, A871T, P980S, and V1476G 2 5 . More recently, several candidate genes, including a homolog of yeast ribosome synthesis factor Rrp5, which is required for the processing of pre-rRNA transcripts into molecules that form ribosomes 26 , were identified in mefenoxam-resistant isolates of P. capsici 27 . However, because RNA polymerases are multi-subunit complexes and topoisomerases and transcription factors could also influence their activity, the precise mefenoxam target remains unknown 24,28−30 . To our knowledge, studies regarding the mechanisms, genes, and mutations involved with mefenoxam resistance in P. cactorum affecting strawberry have not been published. Species of the genus Phytophthora are known to infect hundreds of different plant species 31 . Genomic studies on Phytophthora spp. have been conducted in several species such as P. sojae , P. megakarya , P. capsica , P. betacei , and P. nicotianae 32 – 37 . A genomic study of P. cactorum was conducted using isolates from European beech ( Fagus sylvatica ), Chinese ginseng ( Panax notoginseng ), and recently from cultivated strawberry ( Fragaria × ananassa ) 38–41 . These data were used in the study of host adaptation within P. cactorum and enabled the study of effector complements within Phytophthora spp. 40 , 41 . Whole genome sequencing of Phytophthora spp. populations could be employed to identify possible SNPs/mutations and candidate genes associated with mefenoxam resistance to compare sensitive and resistant populations. Afterward, PCR and other molecular tools could be utilized to screen these candidate regions to differentiate between sensitive and resistant populations. Currently, mefenoxam resistance monitoring in Phytophthora spp. affecting strawberry has been done exclusively through in vitro screening experiments, in which isolates were grown on fungicide amended and non-amended media 11 , 15 . These methods could be time-consuming and labor-intensive and techniques that provide rapid and accurate information about mefenoxam sensitivity in Phytophthora populations are needed. High-resolution melting (HRM) assay is a suitable and cost-effective method for identifying genetic variation, mutations, and SNP in DNA sequences 42 and could be deployed to distinguish between sensitive and resistant populations of P. cactorum to mefenoxam. In fact, this tool has been frequently used in plant pathology to identify and differentiate pathogens and detect mutations related to fungicide resistance 43 – 47 . Therefore, the main goal of this study was to determine the sequence mutations associated with mefenoxam resistance in P. cactorum of strawberry and differentiate sensitive and resistant populations. In the present study, we searched for mutations/SNPs in the subunits of RNA polymerase I in mefenoxam-resistant isolates and performed whole-genome sequencing of resistant and sensitive isolates to aid the identification of mutations associated with mefenoxam resistance. We further developed the high-throughput HRM diagnostic system to differentiate mefenoxam-sensitive and -resistant isolates for rapid fungicide monitoring and management recommendations. Results Identifying mutations in the RNA polymerase ( RPA190 ), RNA polymerase I subunit I (RPA 1), and RNA polymerase I subunit II (RPA 2) genes. The eight mutations/SNPs responsible for amino acid changes in metalaxyl-resistant isolates of P. infestans were not identified in any of the P. cactorum resistant isolates screened in this study. Sequences of both sensitive and resistant isolates were identical for all the sites where SNPs had been previously reported in P. infestans resistant isolates (Table 1 ). After sequencing the whole RPA 1 (5540 bp) and RPA 2 (3602 bp), mutations/SNPs were not observed in any of the screened P. cactorum isolates resistant to mefenoxam. In fact, sequences of both sensitive and resistant isolates were identical (Fig. 1 ). Table 1 Translation of sequences of Phytophthora cactorum isolates sensitive and resistant to mefenoxam for all the sites where SNPs had been previously reported in P. infestans . [Sequences of P. infestans were extracted from Chen et al. 2018; Sensitivite (S) and resistant (R) profiles were determined by discriminatory doses of 5 and 100 µg/ml as proposed by Marin et al. 2021.] Species Profile Mutation Amino acid sequences P. infestans R K267E DTIRGNVSDN K DENMNGDDSE P. cactorum S DTIRGNVSDK E DENMNGDDSE P. cactorum R DTIRGNVSDK E DENMNGDDSE P. infestans R R296H TYAATEDSSS R SKFLPPLEVQ P. cactorum S TYAATEDSSS R SKFLPPLEVQ P. cactorum R TYAATEDSSS R SKFLPPLEVQ P. infestans R F382Y QNSHLSKIMT Y SESIVQSDYY P. cactorum S QNSHLSKIMT Y SESIVQGDYY P. cactorum R QNSHLSKIMT Y SESIVQGDYY P. infestans R T443A SSKAKPGTDV A QGIKQVIEKK P. cactorum S SSKAKPGTDV A QGIKQVIEKK P. cactorum R SSKAKPGTDV A QGIKQVIEKK P. infestans R A597T LHKPSIMAHT A RVLTNPKMQT P. cactorum S LHKPSIMAHT A RVLTNPKMQT P. cactorum R LHKPSIMAHT A RVLTNPKMQT P. infestans R A871T LLEKKRAGEK A GKKRRMNEEE P. cactorum S LLEKKRAGEK N GKKRRMNEEE P. cactorum R LLEKKRAGEK N GKKRRMNEEE P. infestans R P980S VPILCSGRSL P SFEPFDPAPR P. cactorum S VPILCSGRSL P SFEPFDPAPR P. cactorum R VPILCSGRSL P SFEPFDPAPR P. infestans R V1476G LISREMKKSG V TVSAAAEKNN P. cactorum S LISREMKKSG V TVSAAAEKNN P. cactorum R LISREMKKSG V TVSAAAEKNN Identification of sequence polymorphic variants associated with mefenoxam resistance using whole genome sequencing of P. cactorum isolates. Due to the lack of a high-quality chromosome-scale reference genome of P. cactorum , two approaches were implemented to determine the maximum number of variants related to mefenoxam resistance (Fig. 2 ). The basic whole-genome sequencing statistics are shown in Table 2 . Using the reference genome-guided approach, after trimming and quality filtering of Illumina raw reads, 95.54 and 95.65% reads of sensitive (Spool) and resistant pool (Rpool), respectively, were mapped to the reference genome of P. cactorum P414 41 . A total of 1,009,563 variants were called from Spool Sensitive and Rpool Resistant sequencing libraries. The 669,815 variants were identified in the Spool library and 645,905 variants were present in the Rpool library, respectively. After filtering the variants, 80,104 SNPs and 13,665 InDels were retained. Both, Spool and Rpool genomes are available in the National Center of Biotechnology Information (NCBI), with accession numbers SRR21832435 and SRR21832436, respectively. Table 2 Statistics for whole-genome sequencing of the mefenoxam-resistant strains of Phytophthora cactorum . The resistant strains (Rpool) were compared with the mefenoxam-sensitive strains (Spool). Spool Rpool Total reads 74,540,816 82,532,288 Total mapped reads 71,212,819 78,944,241 Number of variants 131,176 128,796 Number of SNPs 107,883 105,612 Number of insertion 11,344 11,263 Number of deletion 11,949 11,921 Synonymous_variant 17,294 16,842 Non-synonymous_variant 23,077 22,558 Variations in exon 44,566 43,414 Genes with amino acid changes 21,610 21,149 Homozygous genotype 5,597 5,771 It is possible that sequences specifically present in the genome of strains used in this study could be missed by the P414 reference genome-guided mapping process. Thus, we conducted de novo -based assembly approach to ensure including all possible sequence variants. The high-quality reads from both Rpool Resistant and Spool Sensitive reads were merged to generate a common reference. Further, about 86.05% and 86.86% reads of Spool Sensitive reads and Rpool Resistant reads were mapped separately to the common reference, respectively. A total of 72,748 variants were identified in both Rpool Resistant and Spool sensitive groups. After combining all sequence variants from reference- and de novo -based approaches, we found only six regions that exhibited point mutations in the resistant mutants compared with the mefenoxam-sensitive strains (Fig. 3 ). Four SNPs in different chromosome regions, NHQK01000085.1 (region 1), NHQK01000034.1 (region 3), NHQK01000023.1 (region 4), and NHQK01000001.1 (region 5) were located in genic regions of four functionally unknown genes annotated as Pcac1_g24873, Pcac1_g14675, Pcac1_g11170, and Pcac1_g296. The other two SNPs located in NHQK01000017.1 and NHQK01000087.1 occurred in the non-genic regions (Fig. 3 ). The six regions corresponding to mefenoxam resistance in P. cactorum mutant strains were amplified using the PCR primer sets (fragments of 202, 201, 167, 182, 162, and 192 bp) (Supplementary Table 1) obtained for each of the six regions, respectively. After sequencing the PCR products, we confirmed that the SNPs were present in the six regions identified in the whole genome analysis for all the screened resistant and sensitive isolates (Supplementary Table 3). High-resolution melting (HRM) assay for detection of SNP mutations. HRM markers were designed to amplify the target mutant regions of the genome of P. cactorum for the differentiation of mefenoxam-sensitive and -resistant isolates. Analysis of the HRM curves generated two distinct melting profiles for most of the regions except for region 4, allowing the accurate differentiation of sensitive and resistant isolates (Fig. 4 and Supplementary Fig. 1). Sequences were identical within mefenoxam-profiles, whereas they were different between sensitive and resistant groups. To better differentiate the two populations, the HRM markers R3-1 from region 3 and R2-1 from region 2 were developed by targeting SNP (C/T) in Pcac1_g14675 and SNP (C/A) in the contig NHQK01000017.1 (Fig. 3 ). The HRM results showed that re-designed HRM markers targeting regions 3 and 2 greatly improved the differentiation between mefenoxam-sensitive and -resistant isolates of P. cactorum (Figs. 5 and 6 ). In the primer test using pure DNA, HRM analysis was performed with two HRM markers, R3-1 and R2-1, in a total of 40 isolates consisting of 28 resistance and 12 sensitive isolates, respectively. The 28 resistant and 12 sensitive representative isolates were clearly divided into resistance (blue) and susceptibility (red) curve patterns in the HRM analysis, respectively (Fig. 5 and Supplementary Table 2). For the rapid detection of mefenoxam resistance, two HRM markers, R3-1 and R2-1, were also examined for HRM curve patterns between resistant and sensitive isolates in the crude DNA extracts. In the HRM analysis, out of 16 tested samples, five sensitive-phenotype isolates showed a sensitive curve pattern (red), while the remaining 11 resistant-phenotype isolates had a distinct curve pattern (blue), resembling the separation whenever pure DNA was used (Fig. 6 ). HRM analysis on a mix of resistant and sensitive isolates in the clean DNA condition was also performed. When one resistant isolate, 18–638, and two sensitive isolates, 18–31 and 11 − 03, were mixed in ratios of 1:1, 1:2, and 2:1, respectively, HRM results of R3-1 and R2-1 markers revealed that all mixtures showed a heterozygous HRM pattern (Fig. 6 A and 6 B). However, when one resistant extract, 18–641, and two sensitive mixtures, 18–31 and 11 − 03, were mixed at the same ratios, 1:1, 1:2, and 2:1, a sensitivity HRM pattern with R3-1 and R2-1 markers was observed (Fig. 6 C and 6 D). Discussion Mefenoxam has been considered the gold standard to control strawberry diseases caused by Phytophthora spp. However, the emergence of resistant isolates could threaten the use of this chemical in strawberry commercial fields. In this study, we have identified variations in unknown genes contributing to mefenoxam resistance and developed a high-throughput HRM assay that could rapidly detect mefenoxam-resistant isolates to aid timely management recommendations for strawberry growers and nurseries. Mefenoxam-resistant isolates were first observed in Florida during the 2015–2016 strawberry season, but the sensitive isolates are still predominant within the population. Fortunately, resistance is not widespread throughout strawberry nurseries, but limited to nurseries in North Carolina state 11 . The registration of additional fungicides with different modes of action would allow fruit and nursery growers to alternate products to reduce fungicide resistance risk 48 . However, currently, chemical options to manage PhCR are limited to mefenoxam and phosphite products 10 . Since mefenoxam is considered a premium product, repetitive applications across nursery and fruit production fields may have selected resistant isolates. Although studies aiming to evaluate if there are any fitness penalties on P. cactorum mefenoxam-resistant isolates were not carried out since resistance was found, most of the growers that acquired transplants from the affected North Carolina nurseries experienced failure of mefenoxam control, suggesting that the resistant population had been established in those nurseries. Fitness penalties on resistant isolates, such as slower growth, have been reported on some Phytophthora species 13 , which could be a factor limiting the widespread resistance. However, stable resistance without fitness disadvantages circumventing the fungicide effect of mefenoxam has been reported in P. infestans 24 . Previous studies hypothesized that the efficacy of mefenoxam is related to the binding and inhibition of specific sites responsible for ribosomal RNA (sRNA) synthesis, implying the reduction in mycelial growth and zoospore germination 13,17−19 . However, because RNA polymerases are multi-subunit complexes and topoisomerases and transcription factors could also influence their activity, the precise mefenoxam target remains unknown 24,28−30 . In P. insfestans , resistant isolates were conditioned by variation in the RNA polymerase gene; however, the inheritance profile from crossing a sensitive and resistant isolate suggested the involvement of more than one locus 24 , 25 . Minor effects of several other genes have also been reported to contribute to resistance, such as genes related to efflux pumps and detoxification as ATP binding cassette (ABC) transporters and cytochrome P450 proteins 13 , 49 , 50 . Controversially to the studies carried out with P. infestans , variations within the RPA 1 and RPA 2 genes conferring resistance to mefenoxam were not identified on P. cactorum isolates, but six SNPs associated with resistance were identified after the whole genome sequencing of mutant isolates. Similar findings were reported in P. capsici mefenoxam-resistant isolates, where the major SNPs conferring resistance were not within the RNA polymerase genes, but in a homolog of yeast protein Rrp5 gene required for processing of pre-rRNA transcripts into the cleaved molecule that forms the ribosome 26 , 27 . Unfortunately, due to the lack of a good genome annotation of P. cactorum , the genes harboring these SNPs were not functionally characterized, and therefore their biological function remains unknown. Interestingly, after the discovery of these six SNPs, screening of all resistant isolates revealed that all the SNPs occurred simultaneously. A definitive test to determine whether all alleles are needed to confer resistance to mefenoxam, or which one has a major effect, using genetic transformation to study the interaction and effect of each locus alone would be necessary. Moreover, the detection of all the SNPs in all the resistant isolates could be due to clonal reproduction, since the isolates originated from the same nursery source in North Carolina. Therefore, there is a chance the SNPs found in this study conferring mefenoxam resistance, could vary in P. cactorum isolates selected for resistance in other nurseries, growing systems, or crops. The practical application of determining the SNPs associated with the mefenoxam resistance would be the development of DNA markers for rapid differentiation of sensitive and resistant isolates. Screening isolates for fungicide resistance through in vitro tests is widely used and the use of discriminatory doses for testing mefenoxam was proposed 11 . However, this method is timing-consuming which can delay management recommendations. In this study, an HRM assay is proposed to speed up the process of distinguishing sensitive and resistant populations of P. cactorum to mefenoxam. Primers were designed for all the SNPs identified, and HRM curves generated two distinct melting profiles for most of the regions except for region 4. The issue with region 4 is that the replacement of T (thymine) to A (adenosine), or vice-versa does not change the number of hydrogen bonds (n = 2), which limits the detection of the polymorphism “T/A” in the HRM analysis. The pair of primers R3-1F/R3-1R and R2-1F/R2-1R were suitable to differentiate both sensitive and resistant profiles using clean and crude DNA extraction. However, if there is a mixture of sensitive and resistant populations, shifts in the melting curves could be expected for both primers. The use of crude extraction of symptomatic strawberry crown tissues was proposed by Wang et al. 47 to diagnose Colletotrichum spp., Macrophomina phaseolina , and Phytophthora spp., and the same extracts could be used to detect mefenoxam resistance if samples are positive for P. cactorum , which could faster management recommendations to the strawberry industry and growers. Although strawberry cultivars resistant to PhCR are available, the susceptible ones are widely grown based on their fruit quality and yield. Therefore, the application of chemicals for disease management will continue to provide security for crop production. The determination of genomic regions conditioning fungicide resistance could offer the possibility of accurately monitoring the pathogen population to guide disease control strategies. In this study, we have identified sequence variation in unknown genes that were correlated with the mefenoxam-resistant profile of P. cactorum isolates from strawberry. Furthermore, an HRM assay was designed to be implemented in diagnostic clinics to improve and faster management recommendations. Our findings may contribute to better understanding of the mechanisms of action of mefenoxam in oomycetes as well as contribute to the sustainable use of this product by avoiding its application if resistance is detected. Materials And Methods Collection and storage of P. cactorum isolates. Isolates of P. cactorum were collected between 1997 to 2020 from strawberry samples showing PhCR and LR symptoms received by the Diagnostic Clinic at the University of Florida Gulf Coast Research and Education Center (UF-GCREC). Isolates were purified by hyphal-tipping on cornmeal agar amended with pimaricin, ampicillin, rifampicin, and pentachloronitrobenzene (P5ARP 51 ), and were identified at the species level using the high-resolution melting analysis developed by Wang et al. 47 using genomic DNA and the set of primers Ph29-F and Ph29-R as described by Ratti et al. 46 . Isolates were transferred to 20% V8 media 31 containing 0.03 g/l of β-sitosterol for 7 to 10 days, then stored in deionized water at room temperature (~ 24°C). In total, 54 sensitive and 31 resistant isolates were hyphal-tipped and molecularly screened for mefenoxam sensitivity. Cultures were grown in P 5 ARP and transferred to 20% V8 medium amended with 0.03g/L β-sitosterol for 7–10 days at 25ºC. Mycelial plugs (6-mm-diameter) were used for storage in sterile water at 25ºC and in 30% glycerol at -80ºC in the culture collection of the Strawberry Pathology laboratory at the UF-GCREC. The mefenoxam resistant isolates did not show any growth inhibition on clarified 20% V8 media amended with 5 and 100 mg/µl of mefenoxam, according to Marin et al. 11 (Supplementary Fig. 2). DNA extraction. Isolates were grown on 20% V8 media for 5–7 days and mycelial plugs from actively growing margins of the cultures were collected for DNA extraction using the FastDNA Kit (MP Biomedicals), following the manufacturer’s protocol. The quality and quantity of DNA samples were determined using a NanoDrop 8000 spectrophotometer (ThermoFisher Scientific). DNA samples with a 260/280 ratio between 1.7 and 1.9 and a 260/230 ratio greater than 2.0 were diluted to a final concentration of 10 ng/µL and stored at -20°C. For the P. cactorum whole genome sequencing, a protocol for plant DNA extraction modified from Keb-Llanes et al. 52 by Integrated DNA Technologies (IDT) was used for the pathogen DNA extraction. Colonies were grown on 10% V8 broth for four days, and then mycelia were washed with sterile deionized water, flash frozen in liquid nitrogen, and ground using a mortar and pestle. Subsequent extraction steps followed the protocol above mentioned. DNA concentration was verified using a NanoDrop 8000 spectrophotometer (ThermoFisher Scientific). DNA quality was determined using the Invitrogen Qubit® Fluorometer (Invitrogen Life Technologies, ThermoFisher Scientific), according to the manufacturer’s protocol and concentration was adjusted to 50 ng/µL. Detecting mutations/single nucleotide polymorphisms (SNPs) in the RNA polymerase ( RPA190 ) gene reported in P. infestans . A total of 35 single nucleotide polymorphisms (SNPs) in the RPA190 gene (5433 bp) encoding the large subunit of RNA polymerase I in metalaxyl-resistant isolates of P. infestans were identified by Chen et al. 25 . Eight SNPs caused amino acid mutations associated with metalaxyl resistance in resistant isolates compared with sensitive isolates: K267E, R296H, F382Y, T443A, A597T, A871T, P980S, and V1476G 2 5 . Sequences flanking the mutations from P. infestans were compared with the P. cactorum whole genome sequence published by Armitage et al. 40 . Based on the regions containing the mutations, five sets of primers were developed: RPA190 -F1/R1 to RPA190 -F5/R5 (Supplementary Table 1), using the IDT-PrimerQuest Tool ( https://www.idtdna.com/PrimerQuest/Home/Index ). Ten sensitive and ten resistant P. cactorum isolate were selected. PCR conditions were as follows: 15 µL of 2× AccuStart II PCR ToughMix (Quantabio; Gaithersburg, Maryland - U.S.A.), 1.5 µL of each forward and reverse primers (10 µM), 11 µL of molecular water, and 1 µL of diluted DNA (10 ng/µL), with a total volume of 30 µL. Amplifications were performed according to the following conditions: initial DNA denaturation at 95°C for 4 min; followed by 32 cycles of denaturation at 95°C for 30 s, annealing at 57°C for 30 s, and extension at 72°C for 3.5 min, and a final extension at 72°C for 10 min. PCR products were visualized under UV light in a 1% agarose gel in 1x Tris-acetate-EDTA buffer stained with GelRed™ (Biotium) and sent for purification and sequencing in both directions at Genewiz Inc. (South Plainfield, NJ). Sequences were aligned using Geneious (version 11.1.4) and MEGA (version 7.0.20) software programs and the five amplified regions of sensitive and resistant isolates were compared separately. Identifying mutations/single nucleotide polymorphisms (SNPs) in the RNA polymerase I subunit I (RPA 1) and II (RPA 2). Based on the P. cactorum whole genome sequence published by Armitage et al. (2018), various primers were designed for amplification and sequencing of the whole RNA polymerase I subunit I (RPA 1) and subunit II (RPA 2) genes. In order to locate both genes within the genome, sequences from P. infestans XM_002906849 and XM_002907301 were used for RPA 1 and RPA 2, respectively. Six sets of primers were developed for RPA 1 (RPA1-F1/R1 to RPA1-F6/R6), and four for RPA 2 (RPA2-F1/R1 to RPA2-F4/R4) (Supplementary Table 1). PCR conditions, product visualization, and sequencing were performed for five sensitive and five resistant isolates as described in the previous section. Sequences of RPA 1 and RPA 2 of one sensitive and one resistant isolate were submitted to GenBank (accession numbers: OM273467 – OM273470). Whole genome sequencing and SNP variant calling. One pool containing 18 sensitive isolates and another pool with 18 resistant isolates were submitted for whole genome sequencing by Novogene (Chula Vista, CA) with Illumina Platform PE150, Q30 ≥ 80%. Illumina raw reads adapter trimming and quality filter were performed using CLC genomics workbench 11.0 ( https://www.qiagenbioinformatics.com/ ). Two approaches were implemented to identify sequence variants related to mefenoxam resistance. Variant calling by mapping to reference-based approach. High-quality trimmed reads from mefenoxam-sensitive pooled reads (Spool Sensitive) and mefenoxam-resistant pooled reads (Rpool Resistant) were separately mapped to the two reference genomes of P. cactorum 38 , 53 . Raw reads were trimmed using Trimmomatic v.0.32 54 . Trimmed reads were mapped to the whole genome sequence of ‘P414’ 53 ( https://www.ncbi.nlm.nih.gov/bioproject/PRJNA383548 ) using Minimap2 with default parameters 55 . The file with sequence alignment/map (SAM) format was converted into a binary alignment map (BAM) format with SAMtools v.1.12 56 . DNA variants including SNPs and insertion/deletion (InDel) were called using a genome analysis toolkit (GATK 57 ) according to the manuals with default parameters. Variants were filtered using the following VCF parameters: QD > 2.0, FS > 60, ReadPosRankSum < -8.0, and MQRankSum < -12.5. Alternatively, the low frequency variant caller program of CLC genomics workbench 11.0 was also implemented to call variants from Spool Sensitive and Rpool Resistant reads. Variant filtration was performed by eliminating the common SNPs between Spool Sensitive and Rpool Resistant groups to select only the non-synonymous SNPs. The SNPs with only a high SNP index (> 80%) were considered for further validation. De novo -based approach for calling variants. For this approach, high-quality reads from both “Rpool Resistant” and “Spool Sensitive” were merged for de novo assembly. The software AbySS 2.1.1 58 was chosen for de novo assembly because of its better performance against short reads. Strict parameters were used for de novo assembly and the contigs were made non-redundant using software CD-HIT v4.8.1 59 . The contigs having a length less than 2 kb were discarded and not considered for further analysis. Software Augustus v2.5.5 60 was used for gene prediction. Further, reads from both Rpool Resistant and Spool Sensitive were mapped to the final de novo assembled contigs separately for variant calling. The low frequency variant caller program of CLC genomics workbench 11.0 was used to call variants from Spool Sensitive and Rpool Resistant reads separately. Common variants were removed between Spool Sensitive and Rpool Resistant groups. Only non-synonymous SNPs with a high SNP index were selected for further analysis. Sequencing the genomic regions associated with mefenoxam resistance. Based on the flanking sequence contigs (200 bp) originated from the whole genome sequencing of a pool of sensitive and resistant P. cactorum isolates, seven regions containing the SNPs possibly associated with mefenoxam resistance were amplified through PCR and sequenced. Six sets of primers were developed to amplify the seven regions and designated as 1, 2, 3, 4, 5, and 6: Rdmyl1 F/R to Rdmyl6 F/R (Supplementary Table 1). PCR conditions, product visualization, and sequencing were performed as described in the other PCR sections. High-resolution melting (HRM) assay for detection of SNP mutations. Based on the whole genome sequencing results, markers were designed to amplify the six flanking sequence regions containing the SNPs/mutations that could potentially be associated with mefenoxam resistance in P. cactorum using the IDT-PrimerQuest Tool ( https://www.idtdna.com/PrimerQuest/Home/Index ). Different sets of markers HRM 1F/1R, HRM 3F/3R, HRM 5F/5R, S1F/S1R, R4F/R4R, and R6F/R6R were used to amplify the six respective regions of 54 sensitive and 31 resistant isolates (Supplementary Table 1). PCR and HRM were performed in a Roche LightCycler® 480 Instrument II (Roche Diagnostics, Indianapolis, IN). Reactions had a total volume of 10 µL in a 384-well plate (TempPlate 384-well Full-Skirt PCR plate, White; USA Scientific, Ocala, FL) containing 5 µL of 2× AccuStart II PCR ToughMix (Quantabio; Gaithersburg, Maryland - U.S.A.), 0.5 µL of each forward and reverse primers (10 µM), 0.5 µL of LCGreen Plus dye (BioFire Defense, Salt Lake City, UT), 2.5 µL of molecular water, and 1 µL of diluted DNA (10 ng/µL). PCR amplification began with an initial denaturation at 95°C for 3 min (ramp rate: 4.8°C/s), followed by 35 cycles of 95°C for 20 s (ramp rate: 4.8°C/s), 61°C for 30 s (ramp rate: 2.5°C/s), and 72°C for 30 s (ramp rate: 4.8°C/s). For post-PCR analysis, samples were subjected to the following HRM parameters: 95°C for 1 min (ramp rate: 4.8°C/s), 40°C for 1 min (ramp rate: 2.5°C/s), 65°C for 1 s (ramp rate: 4.8°C/s), and then a continuous fluorescence reading from 65 to 95°C (ramp rate: 0.02°C/s, 25 acquisitions/°C), with a final cooling step at 40°C for 30 s (ramp rate: 2.5°C/s). Melt curve genotyping and gene scanning analysis were performed using the LightCycler® 480 software (version 1.5.1.62). To generate the normalized melting curves, pre-melt and post-melt temperature settings were adjusted according to each region analyzed, and the temperature threshold was set to 0°C. Experiments were conducted in duplicate with three replications per sample. Rapid detection of SNP mutations associated with mefenoxam resistance directly from infected strawberry crowns. To develop HRM markers with more precise melting curve patterns between resistance and sensitive isolates, sequence variations present in the region 3 (NHQK01000034.1) and region 2 (NHQK01000017.1) containing the SNPs/mutations that could potentially be associated with mefenoxam resistance in P. cactorum were used to develop functional markers (Fig. 1 ). Two primer sets, R3-1 and R2-1, were designed from the polymorphic sequences of region 3 and region 2, respectively, using the IDT PrimerQuest tool ( https://www.idtdna.com/PrimerQuest/Home/Index ) (Supplementary Table 1). A total of 40 isolates (28 resistant and 12 sensitive isolates) were used for the primer design test, and a total of 16 crude DNA extracts from symptomatic crowns inoculated with P. cactorum isolates (11 resistant and five sensitive isolates) were used for marker validation (Supplementary Table 2). For the crude DNA extraction of infected strawberry crowns, plants of Sensation® ‘Florida 127’ were inoculated by dipping the roots in a zoospore suspension of each isolate separately (10 4 zoospores/ml) and potted in the greenhouse. Once symptoms of PhCR were observed, the crowns were opened, and symptomatic tissue was used for the crude DNA extraction following the protocol published by Wang et al. 47 . The two primers, R3-1 and R2-1, were also tested in a mixture of resistant and sensitive isolates of the purified DNA. Two resistant and two sensitive isolates diluted at a concentration of 20 ng/µl were used, respectively. The combinations of mixtures were pooled together with resistant and sensitive isolates in ratios of 1:1, 1:2, and 2:1, respectively. PCR reactions were prepared as mentioned above. The PCR and HRM analysis were performed in a LightCycler® 480 system II using a program consisting of an initial denaturation at 95°C for 5 min; 45 cycles of denaturation at 95°C for 10 s, annealing at 62°C for 10 s, and extension at 72°C for 20 s. After PCR amplification, the samples were heated to 95°C for 1 min and cooled to 40°C for 1 min. Melting curves were obtained by melting over the desired range (60–95°C) at a rate of 50 acquisitions per 1°C. Melting data were analyzed using the Melt Curve Genotyping and Gene Scanning Software (Roche Life Science, Germany). Analysis of HRM variants was based on differences in the shape of the melting curves and melting temperature (Tm) values. Declarations COMPLIES WITH INTERNATIONAL, NATIONAL AND/OR INSTITUTIONAL GUIDELINES Experimental research methods used in this study complies with relevant institutional, national, and international guidelines and legislation. ACKNOWLEDGEMENTS This research was supported by grants to SL and NP from the United States Department of Agriculture (http://dx.doi.org/10.13039/100000199) National Institute of Food and Agriculture (NIFA) Specialty Crops Research Initiative (#2017-51181-26833). CONTRIBUTIONS M.M., J.B., N.P. and S.L. conceptualized and designed the experiments. M.M., J.B., Y.O., H.H., S.C., and N-Y.W. conducted the experiments and collected data. M.M., J.B., Y.O., H.H., and S.C., analyzed the data from the experiments and interpreted the findings. M.M., J.B., Y. O., N.P. and S.L. wrote the manuscript and prepared all Tables and Figures, and N.P. and S.L. reviewed and edited the manuscript. AVAILABILITY OF MATERIALS AND DATA The datasets generated and/or analyzed during the current study are available in the GenBank (accession numbers: OM273467 – OM273470), and NCBI (accession numbers: SRR21832435 and SRR21832436). Correspondence and requests for materials should be addressed to N.P. and S.L. Reprints and permissions information is available at www.nature.com/reprints. ETHICS APPROVAL AND CONSENT TO PARTICIPATE We all declare that manuscripts reporting studies do not involve any human participants, human data, or human tissue. So, it is not applicable. ADDITIONAL INFORMATION Competing interests: The authors declare no competing interests. References FAOSTAT 2020. Strawberry production. Food and Agriculture Organization of the United Nations. Statistic division. Accessed: April 7, 2020. Available: http://www.fao.org/faostat/en/#data/QC/visualize USDA. National statistics for strawberries and Florida agricultural overview (2016). United States Department of Agriculture, National Agricultural Statistics Service (USDA, NASS). Data and statistics. (2020). Dittmar, P. J. et al. Vegetable production handbook of Florida . (University of Florida - IFAS Extension, 2017). Baggio, J. S., Marin, M. v & Peres, N. A. 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Supplementary Files SupplementaryMaterial.docx Cite Share Download PDF Status: Published Journal Publication published 06 May, 2023 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Major revision 11 Jan, 2023 Reviews received at journal 29 Dec, 2022 Reviewers agreed at journal 28 Dec, 2022 Reviewers agreed at journal 06 Dec, 2022 Reviewers invited by journal 07 Nov, 2022 Editor assigned by journal 07 Nov, 2022 Editor invited by journal 11 Oct, 2022 Submission checks completed at journal 11 Oct, 2022 First submitted to journal 23 Sep, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2097400","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":143298492,"identity":"5ba008c7-76f4-4929-85b9-6cad22042dc4","order_by":0,"name":"Marcus Vinicius Marin","email":"","orcid":"","institution":"University of Florida, IFAS Gulf Coast Research and Education Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Marcus","middleName":"Vinicius","lastName":"Marin","suffix":""},{"id":143298493,"identity":"be823bae-a7ae-49cc-924b-5450c90c57f1","order_by":1,"name":"Juliana S. Baggio","email":"","orcid":"","institution":"University of Florida, IFAS Gulf Coast Research and Education Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Juliana","middleName":"S.","lastName":"Baggio","suffix":""},{"id":143298494,"identity":"b2b6dc1b-f2ff-40a5-8817-09b3be17df33","order_by":2,"name":"Youngjae Oh","email":"","orcid":"","institution":"Chungbuk National","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Youngjae","middleName":"","lastName":"Oh","suffix":""},{"id":143298495,"identity":"825510f0-7e67-49ab-81d3-6d78898d72dd","order_by":3,"name":"Hyeondae Han","email":"","orcid":"","institution":"University of Florida, IFAS Gulf Coast Research and Education Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hyeondae","middleName":"","lastName":"Han","suffix":""},{"id":143298496,"identity":"0effdd7c-1f80-4cd8-ab6f-7e7122dec4dd","order_by":4,"name":"Saket Chandra","email":"","orcid":"","institution":"North Carolina State University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Saket","middleName":"","lastName":"Chandra","suffix":""},{"id":143298497,"identity":"17e35ccd-0d90-4507-914b-dc7c98e9a6db","order_by":5,"name":"Nan-Yi Wang","email":"","orcid":"","institution":"University of Florida, IFAS Gulf Coast Research and Education Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Nan-Yi","middleName":"","lastName":"Wang","suffix":""},{"id":143298498,"identity":"d143243f-49e0-4020-8dac-0139e6d1a9c9","order_by":6,"name":"Seonghee Lee","email":"","orcid":"","institution":"University of Florida, IFAS Gulf Coast Research and Education Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Seonghee","middleName":"","lastName":"Lee","suffix":""},{"id":143298499,"identity":"733a4fc2-20dc-45a3-aa49-061a67347df9","order_by":7,"name":"Natalia A. Peres","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAqElEQVRIiWNgGAWjYPACGwb2diDFQ4KWNAaewyRqOUyCFnPp5mMfPvw5n9jDzMD44G0bEVos5xxLnjmz7TZIC7PhXGK0GNzIMWbmbbiduJ+ZgU2alzgt+Z+Z//w5B7KF/TeRWnKYgeYfAGlhYyZOy51jxoy9bcnGPcyMzZJzzhGj5XbzY4Yff+xke9ibD354U0aEFgYJOIuxgRj1KFpGwSgYBaNgFOAAAM9oM2SdWHXLAAAAAElFTkSuQmCC","orcid":"","institution":"University of Florida, IFAS Gulf Coast Research and Education Center","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Natalia","middleName":"A.","lastName":"Peres","suffix":""}],"badges":[],"createdAt":"2022-09-23 18:29:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2097400/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2097400/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-023-34271-z","type":"published","date":"2023-05-06T20:43:46+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":27782849,"identity":"9e53ce48-65b2-4342-9985-fb12d3589af7","added_by":"auto","created_at":"2022-10-14 14:46:10","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":402321,"visible":true,"origin":"","legend":"\u003cp\u003eAmino acid sequences of genes RPA 1 and RPA 2, previously found in \u003cem\u003ePhytophthora infestans \u003c/em\u003econferring mefenoxam resistance (Chen et al. 2018), aligned with representative sensitive (09-100) and resistant (16-365) isolates of \u003cem\u003eP. cactorum\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2097400/v1/0ce27ca42e904827cd8c2217.png"},{"id":27782375,"identity":"d03a0580-4a6c-4313-8ee6-3115c4337787","added_by":"auto","created_at":"2022-10-14 14:41:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":86528,"visible":true,"origin":"","legend":"\u003cp\u003eDepiction of two approaches implemented for calling sequence variants linked to the mefenoxam-resistant \u003cem\u003eP. cactorum\u003c/em\u003e isolates.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-2097400/v1/d106d2fece4bbb906955be73.png"},{"id":27782369,"identity":"e79cf680-f393-4525-826d-4799b5d333ef","added_by":"auto","created_at":"2022-10-14 14:41:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":154970,"visible":true,"origin":"","legend":"\u003cp\u003eSelected SNP candidates based on the non-synonymous effect and high SNP index.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-2097400/v1/f9cc4a1281ebe0d7eff27167.png"},{"id":27782374,"identity":"0dc82e96-ccb8-46fc-86ab-cd6849f02296","added_by":"auto","created_at":"2022-10-14 14:41:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1196654,"visible":true,"origin":"","legend":"\u003cp\u003eNormalized high-resolution melting (HRM) curves of regions 1 (A), 2 (B), 3 (C), 4 (D), 5 (E), and 6 (F) for the identification and differentiation of mefenoxam-sensitive (blue) and -resistant (red) isolates of \u003cem\u003eP. cactorum \u003c/em\u003eof strawberry.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-2097400/v1/9c8486c4cfafe231a88c10c1.png"},{"id":27782371,"identity":"61ad4d3d-a7cc-43c9-ab4e-f3aa3e9c9d22","added_by":"auto","created_at":"2022-10-14 14:41:10","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":128140,"visible":true,"origin":"","legend":"\u003cp\u003eHRM analysis of R3-1 and R2-1 HRM markers with clean DNA extracts of 28 resistant and 12 sensitive isolates. (A) and (B), Melt curve genotyping and gene scanning analysis of the R3-1 marker. (C) and (D), Melt curve genotyping and gene scanning analysis of the R2-1 marker.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-2097400/v1/3294ab7ae8cb5cb96868485e.png"},{"id":27782992,"identity":"de5f48cd-ded5-4758-8482-2447969bbcb2","added_by":"auto","created_at":"2022-10-14 14:51:10","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":347048,"visible":true,"origin":"","legend":"\u003cp\u003eHRM analysis on a mix of resistant and sensitive isolates in the clean DNA condition using R3-1 and R2-1 HRM markers. (A) and (B), HRM results of the combinations of resistant isolate, 18-638, and the two sensitive isolates, 11-03 and 18-31, in different ratios with R3-1 and R2-1 markers, respectively. (C) and (D), HRM results of the combinations of resistant isolate, 18-641, and the two sensitive isolates, 18-31 and 11-03, in different ratios with R3-1 and R2-1 markers, respectively.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-2097400/v1/bed3335478204bebea051840.png"},{"id":44728416,"identity":"d0616353-4df7-440b-a60a-9b07c5dc9177","added_by":"auto","created_at":"2023-10-16 21:03:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2475500,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2097400/v1/b8afe643-003a-456a-bdd5-3ea609f5d87a.pdf"},{"id":27782848,"identity":"b83fa40d-20b3-4f39-854c-dd253446b1e5","added_by":"auto","created_at":"2022-10-14 14:46:09","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":624507,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-2097400/v1/cf3100658a88bf1d60522130.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Identification of sequence mutations in Phytophthora cactorum genome associated with mefenoxam resistance and development of a molecular assay for the mutant detection in strawberry (F. ×ananassa)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGlobally, Florida is the largest strawberry winter producer, being responsible for more than 5% of the U.S. production\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. In 2020, Florida produced approximately 89,000 tons of strawberry over its 4000 harvested hectares raising 240\u0026nbsp;million dollars\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Unfortunately, crop losses caused by plant pathogens represent one of the major threats in strawberry production and agriculture in general and have become even more challenging since organisms are prone to selection for resistance to single-site fungicides commonly used for their control.\u003c/p\u003e \u003cp\u003ePhytophthora crown rot (PhCR), caused mainly by \u003cem\u003ePhytophthora cactorum\u003c/em\u003e, is an important disease of strawberry worldwide. Symptoms are characterized by wilting and stunting of plants and eventual death due to crown collapse. In Florida, production is based on an annual plasticulture system with mulched, raised beds that are fumigated prior to planting\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. New transplants acquired every season are the major source of inoculum for this pathogen\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. After transplanting, daytime overhead irrigation is provided for 10 to 14 days for plant establishment, which creates a conducive environment for disease development immediately at the beginning of the season.\u003c/p\u003e \u003cp\u003eLeather rot (LR), also caused by \u003cem\u003eP. cactorum\u003c/em\u003e, poses a threat to strawberry production as well. It affects fruit in all development stages and confers discoloration, and unpleasant taste and odor\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Because symptoms on ripped fruit are sometimes subtle, infected fruit could be picked along with healthy fruits and become a post-harvest problem, besides the direct pre-harvest losses\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. However, epidemics of this disease are sporadic and associated with heavy rainfall events during the fruiting stage\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eManagement of both diseases is based on the combination of resistant cultivars, cultural practices, and chemical applications\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Although resistant cultivars may be available, chemical applications have been widely used since most cultivars are bred and selected based on fruit quality and yield and not for resistance to \u003cem\u003ePhytophthora\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Mefenoxam and phosphite products are the most common chemicals used by Florida growers to manage PhCR and LR, but azoxystrobin is also registered to control LR\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. However, \u003cem\u003eP. cactorum\u003c/em\u003e isolates resistant to mefenoxam and azoxystrobin have been found in Florida strawberry fields\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Mefenoxam, the most widely used product, is a site-specific phenylamide fungicide with systemic activity and oomycete specificity, classified as having a high risk for pathogen-resistance selection\u003csup\u003e\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Mefenoxam is the more active enantiomer contained in the racemic fungicide metalaxyl, broadly used in the control of other oomycetes\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. It is hypothesized that this compound binds to specific sites responsible for ribosomal RNA (rRNA) synthesis, which reduces mycelial growth and zoospore germination\u003csup\u003e13,17\u0026minus;19\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMefenoxam efficacy in strawberry production has been threatened by the emergence of fungicide-resistant populations, putting the control of \u003cem\u003ePhytophthora\u003c/em\u003e diseases in strawberry fields at risk\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Several mefenoxam-resistant \u003cem\u003ePhytophthora\u003c/em\u003e spp. have been reported in multiple hosts, such as \u003cem\u003eP. capsici\u003c/em\u003e on cucurbits and peppers, \u003cem\u003eP. infestans\u003c/em\u003e on tomato, and \u003cem\u003eP. erythroseptica\u003c/em\u003e on potatoes\u003csup\u003e16,19\u0026minus;23\u003c/sup\u003e. Acquired resistance accompanied by fitness penalties on resistant isolates, such as slow growth, have been reported for some species\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. However, stable resistance in the subunit of RNA polymerase has been reported providing advantages to \u003cem\u003eP. insfestans\u003c/em\u003e in circumventing the fungicide effect without fitness disadvantages\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this scenario, fungicide sensitivity monitoring plays an important role in the implementation of integrated disease management programs. Moreover, comprehending the mechanisms involved with mefenoxam resistance would aid the monitoring process. In \u003cem\u003eP. infestans\u003c/em\u003e, for example, eight single nucleotide polymorphisms (SNPs) within the \u003cem\u003eRPA190\u003c/em\u003e gene associated with metalaxyl resistance were identified as causing amino acid changes, such as K267E, R296H, F382Y, T443A, A597T, A871T, P980S, and V1476G\u003csup\u003e2\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. More recently, several candidate genes, including a homolog of yeast ribosome synthesis factor Rrp5, which is required for the processing of pre-rRNA transcripts into molecules that form ribosomes\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, were identified in mefenoxam-resistant isolates of \u003cem\u003eP. capsici\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. However, because RNA polymerases are multi-subunit complexes and topoisomerases and transcription factors could also influence their activity, the precise mefenoxam target remains unknown\u003csup\u003e24,28\u0026minus;30\u003c/sup\u003e. To our knowledge, studies regarding the mechanisms, genes, and mutations involved with mefenoxam resistance in \u003cem\u003eP. cactorum\u003c/em\u003e affecting strawberry have not been published.\u003c/p\u003e \u003cp\u003eSpecies of the genus \u003cem\u003ePhytophthora\u003c/em\u003e are known to infect hundreds of different plant species\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Genomic studies on \u003cem\u003ePhytophthora\u003c/em\u003e spp. have been conducted in several species such as \u003cem\u003eP. sojae\u003c/em\u003e, \u003cem\u003eP. megakarya\u003c/em\u003e, \u003cem\u003eP. capsica\u003c/em\u003e, \u003cem\u003eP. betacei\u003c/em\u003e, and \u003cem\u003eP. nicotianae\u003c/em\u003e\u003csup\u003e\u003cspan additionalcitationids=\"CR33 CR34 CR35 CR36\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. A genomic study of \u003cem\u003eP. cactorum\u003c/em\u003e was conducted using isolates from European beech (\u003cem\u003eFagus sylvatica\u003c/em\u003e), Chinese ginseng (\u003cem\u003ePanax notoginseng\u003c/em\u003e), and recently from cultivated strawberry (\u003cem\u003eFragaria\u003c/em\u003e \u0026times;\u003cem\u003eananassa\u003c/em\u003e)\u003csup\u003e38\u0026ndash;41\u003c/sup\u003e. These data were used in the study of host adaptation within \u003cem\u003eP. cactorum\u003c/em\u003e and enabled the study of effector complements within \u003cem\u003ePhytophthora\u003c/em\u003e spp.\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Whole genome sequencing of \u003cem\u003ePhytophthora\u003c/em\u003e spp. populations could be employed to identify possible SNPs/mutations and candidate genes associated with mefenoxam resistance to compare sensitive and resistant populations. Afterward, PCR and other molecular tools could be utilized to screen these candidate regions to differentiate between sensitive and resistant populations.\u003c/p\u003e \u003cp\u003eCurrently, mefenoxam resistance monitoring in \u003cem\u003ePhytophthora\u003c/em\u003e spp. affecting strawberry has been done exclusively through \u003cem\u003ein vitro\u003c/em\u003e screening experiments, in which isolates were grown on fungicide amended and non-amended media\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. These methods could be time-consuming and labor-intensive and techniques that provide rapid and accurate information about mefenoxam sensitivity in \u003cem\u003ePhytophthora\u003c/em\u003e populations are needed. High-resolution melting (HRM) assay is a suitable and cost-effective method for identifying genetic variation, mutations, and SNP in DNA sequences\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e and could be deployed to distinguish between sensitive and resistant populations of \u003cem\u003eP. cactorum\u003c/em\u003e to mefenoxam. In fact, this tool has been frequently used in plant pathology to identify and differentiate pathogens and detect mutations related to fungicide resistance\u003csup\u003e\u003cspan additionalcitationids=\"CR44 CR45 CR46\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTherefore, the main goal of this study was to determine the sequence mutations associated with mefenoxam resistance in \u003cem\u003eP. cactorum\u003c/em\u003e of strawberry and differentiate sensitive and resistant populations. In the present study, we searched for mutations/SNPs in the subunits of RNA polymerase I in mefenoxam-resistant isolates and performed whole-genome sequencing of resistant and sensitive isolates to aid the identification of mutations associated with mefenoxam resistance. We further developed the high-throughput HRM diagnostic system to differentiate mefenoxam-sensitive and -resistant isolates for rapid fungicide monitoring and management recommendations.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eIdentifying mutations in the RNA polymerase (\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eRPA190\u003c/span\u003e \u003cb\u003e), RNA polymerase I subunit I (RPA 1), and RNA polymerase I subunit II (RPA 2) genes.\u003c/b\u003e The eight mutations/SNPs responsible for amino acid changes in metalaxyl-resistant isolates of \u003cem\u003eP. infestans\u003c/em\u003e were not identified in any of the \u003cem\u003eP. cactorum\u003c/em\u003e resistant isolates screened in this study. Sequences of both sensitive and resistant isolates were identical for all the sites where SNPs had been previously reported in \u003cem\u003eP. infestans\u003c/em\u003e resistant isolates (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). After sequencing the whole RPA 1 (5540 bp) and RPA 2 (3602 bp), mutations/SNPs were not observed in any of the screened \u003cem\u003eP. cactorum\u003c/em\u003e isolates resistant to mefenoxam. In fact, sequences of both sensitive and resistant isolates were identical (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTranslation of sequences of \u003cem\u003ePhytophthora cactorum\u003c/em\u003e isolates sensitive and resistant to mefenoxam for all the sites where SNPs had been previously reported in \u003cem\u003eP. infestans\u003c/em\u003e. [Sequences of \u003cem\u003eP. infestans\u003c/em\u003e were extracted from Chen et al. 2018; Sensitivite (S) and resistant (R) profiles were determined by discriminatory doses of 5 and 100 \u0026micro;g/ml as proposed by Marin et al. 2021.]\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProfile\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMutation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAmino acid sequences\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. infestans\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eK267E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDTIRGNVSDN\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eK\u003c/span\u003eDENMNGDDSE\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. cactorum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDTIRGNVSDK\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eE\u003c/span\u003eDENMNGDDSE\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. cactorum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDTIRGNVSDK\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eE\u003c/span\u003eDENMNGDDSE\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. infestans\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eR296H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTYAATEDSSS\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eR\u003c/span\u003eSKFLPPLEVQ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. cactorum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTYAATEDSSS\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eR\u003c/span\u003eSKFLPPLEVQ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. cactorum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTYAATEDSSS\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eR\u003c/span\u003eSKFLPPLEVQ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. infestans\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eF382Y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eQNSHLSKIMT\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eY\u003c/span\u003eSESIVQSDYY\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. cactorum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eQNSHLSKIMT\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eY\u003c/span\u003eSESIVQGDYY\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. cactorum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eQNSHLSKIMT\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eY\u003c/span\u003eSESIVQGDYY\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. infestans\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eT443A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSSKAKPGTDV\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eA\u003c/span\u003eQGIKQVIEKK\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. cactorum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSSKAKPGTDV\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eA\u003c/span\u003eQGIKQVIEKK\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. cactorum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSSKAKPGTDV\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eA\u003c/span\u003eQGIKQVIEKK\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. infestans\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eA597T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLHKPSIMAHT\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eA\u003c/span\u003eRVLTNPKMQT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. cactorum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLHKPSIMAHT\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eA\u003c/span\u003eRVLTNPKMQT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. cactorum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLHKPSIMAHT\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eA\u003c/span\u003eRVLTNPKMQT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. infestans\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eA871T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLLEKKRAGEK\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eA\u003c/span\u003eGKKRRMNEEE\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. cactorum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLLEKKRAGEK\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eN\u003c/span\u003eGKKRRMNEEE\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. cactorum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLLEKKRAGEK\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eN\u003c/span\u003eGKKRRMNEEE\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. infestans\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eP980S\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eVPILCSGRSL\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eP\u003c/span\u003eSFEPFDPAPR\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. cactorum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eVPILCSGRSL\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eP\u003c/span\u003eSFEPFDPAPR\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. cactorum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eVPILCSGRSL\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eP\u003c/span\u003eSFEPFDPAPR\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. infestans\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eV1476G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLISREMKKSG\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eV\u003c/span\u003eTVSAAAEKNN\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. cactorum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLISREMKKSG\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eV\u003c/span\u003eTVSAAAEKNN\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. cactorum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLISREMKKSG\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eV\u003c/span\u003eTVSAAAEKNN\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIdentification of sequence polymorphic variants associated with mefenoxam resistance using whole genome sequencing of\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eP. cactorum\u003c/span\u003e \u003cb\u003eisolates.\u003c/b\u003e Due to the lack of a high-quality chromosome-scale reference genome of \u003cem\u003eP. cactorum\u003c/em\u003e, two approaches were implemented to determine the maximum number of variants related to mefenoxam resistance (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The basic whole-genome sequencing statistics are shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Using the reference genome-guided approach, after trimming and quality filtering of Illumina raw reads, 95.54 and 95.65% reads of sensitive (Spool) and resistant pool (Rpool), respectively, were mapped to the reference genome of \u003cem\u003eP. cactorum\u003c/em\u003e P414\u003csup\u003e41\u003c/sup\u003e. A total of 1,009,563 variants were called from Spool Sensitive and Rpool Resistant sequencing libraries. The 669,815 variants were identified in the Spool library and 645,905 variants were present in the Rpool library, respectively. After filtering the variants, 80,104 SNPs and 13,665 InDels were retained. Both, Spool and Rpool genomes are available in the National Center of Biotechnology Information (NCBI), with accession numbers SRR21832435 and SRR21832436, respectively.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eStatistics for whole-genome sequencing of the mefenoxam-resistant strains of \u003cem\u003ePhytophthora cactorum\u003c/em\u003e. The resistant strains (Rpool) were compared with the mefenoxam-sensitive strains (Spool).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eSpool\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eRpool\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTotal reads\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e74,540,816\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e82,532,288\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTotal mapped reads\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e71,212,819\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e78,944,241\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNumber of variants\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e131,176\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e128,796\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNumber of SNPs\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e107,883\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e105,612\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNumber of insertion\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e11,344\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11,263\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNumber of deletion\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e11,949\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11,921\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSynonymous_variant\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e17,294\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16,842\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNon-synonymous_variant\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e23,077\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e22,558\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eVariations in exon\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e44,566\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e43,414\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGenes with amino acid changes\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e21,610\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e21,149\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHomozygous genotype\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e5,597\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5,771\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eIt is possible that sequences specifically present in the genome of strains used in this study could be missed by the P414 reference genome-guided mapping process. Thus, we conducted \u003cem\u003ede novo\u003c/em\u003e-based assembly approach to ensure including all possible sequence variants. The high-quality reads from both Rpool Resistant and Spool Sensitive reads were merged to generate a common reference. Further, about 86.05% and 86.86% reads of Spool Sensitive reads and Rpool Resistant reads were mapped separately to the common reference, respectively. A total of 72,748 variants were identified in both Rpool Resistant and Spool sensitive groups.\u003c/p\u003e \u003cp\u003eAfter combining all sequence variants from reference- and \u003cem\u003ede novo\u003c/em\u003e-based approaches, we found only six regions that exhibited point mutations in the resistant mutants compared with the mefenoxam-sensitive strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Four SNPs in different chromosome regions, NHQK01000085.1 (region 1), NHQK01000034.1 (region 3), NHQK01000023.1 (region 4), and NHQK01000001.1 (region 5) were located in genic regions of four functionally unknown genes annotated as Pcac1_g24873, Pcac1_g14675, Pcac1_g11170, and Pcac1_g296. The other two SNPs located in NHQK01000017.1 and NHQK01000087.1 occurred in the non-genic regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The six regions corresponding to mefenoxam resistance in \u003cem\u003eP. cactorum\u003c/em\u003e mutant strains were amplified using the PCR primer sets (fragments of 202, 201, 167, 182, 162, and 192 bp) (Supplementary Table\u0026nbsp;1) obtained for each of the six regions, respectively. After sequencing the PCR products, we confirmed that the SNPs were present in the six regions identified in the whole genome analysis for all the screened resistant and sensitive isolates (Supplementary Table\u0026nbsp;3).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eHigh-resolution melting (HRM) assay for detection of SNP mutations.\u003c/b\u003e HRM markers were designed to amplify the target mutant regions of the genome of \u003cem\u003eP. cactorum\u003c/em\u003e for the differentiation of mefenoxam-sensitive and -resistant isolates. Analysis of the HRM curves generated two distinct melting profiles for most of the regions except for region 4, allowing the accurate differentiation of sensitive and resistant isolates (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Supplementary Fig.\u0026nbsp;1). Sequences were identical within mefenoxam-profiles, whereas they were different between sensitive and resistant groups. To better differentiate the two populations, the HRM markers R3-1 from region 3 and R2-1 from region 2 were developed by targeting SNP (C/T) in Pcac1_g14675 and SNP (C/A) in the contig NHQK01000017.1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The HRM results showed that re-designed HRM markers targeting regions 3 and 2 greatly improved the differentiation between mefenoxam-sensitive and -resistant isolates of \u003cem\u003eP. cactorum\u003c/em\u003e (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the primer test using pure DNA, HRM analysis was performed with two HRM markers, R3-1 and R2-1, in a total of 40 isolates consisting of 28 resistance and 12 sensitive isolates, respectively. The 28 resistant and 12 sensitive representative isolates were clearly divided into resistance (blue) and susceptibility (red) curve patterns in the HRM analysis, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and Supplementary Table\u0026nbsp;2). For the rapid detection of mefenoxam resistance, two HRM markers, R3-1 and R2-1, were also examined for HRM curve patterns between resistant and sensitive isolates in the crude DNA extracts. In the HRM analysis, out of 16 tested samples, five sensitive-phenotype isolates showed a sensitive curve pattern (red), while the remaining 11 resistant-phenotype isolates had a distinct curve pattern (blue), resembling the separation whenever pure DNA was used (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHRM analysis on a mix of resistant and sensitive isolates in the clean DNA condition was also performed. When one resistant isolate, 18\u0026ndash;638, and two sensitive isolates, 18\u0026ndash;31 and 11\u0026thinsp;\u0026minus;\u0026thinsp;03, were mixed in ratios of 1:1, 1:2, and 2:1, respectively, HRM results of R3-1 and R2-1 markers revealed that all mixtures showed a heterozygous HRM pattern (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). However, when one resistant extract, 18\u0026ndash;641, and two sensitive mixtures, 18\u0026ndash;31 and 11\u0026thinsp;\u0026minus;\u0026thinsp;03, were mixed at the same ratios, 1:1, 1:2, and 2:1, a sensitivity HRM pattern with R3-1 and R2-1 markers was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMefenoxam has been considered the gold standard to control strawberry diseases caused by \u003cem\u003ePhytophthora\u003c/em\u003e spp. However, the emergence of resistant isolates could threaten the use of this chemical in strawberry commercial fields. In this study, we have identified variations in unknown genes contributing to mefenoxam resistance and developed a high-throughput HRM assay that could rapidly detect mefenoxam-resistant isolates to aid timely management recommendations for strawberry growers and nurseries.\u003c/p\u003e \u003cp\u003eMefenoxam-resistant isolates were first observed in Florida during the 2015\u0026ndash;2016 strawberry season, but the sensitive isolates are still predominant within the population. Fortunately, resistance is not widespread throughout strawberry nurseries, but limited to nurseries in North Carolina state\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. The registration of additional fungicides with different modes of action would allow fruit and nursery growers to alternate products to reduce fungicide resistance risk\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. However, currently, chemical options to manage PhCR are limited to mefenoxam and phosphite products\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Since mefenoxam is considered a premium product, repetitive applications across nursery and fruit production fields may have selected resistant isolates. Although studies aiming to evaluate if there are any fitness penalties on \u003cem\u003eP. cactorum\u003c/em\u003e mefenoxam-resistant isolates were not carried out since resistance was found, most of the growers that acquired transplants from the affected North Carolina nurseries experienced failure of mefenoxam control, suggesting that the resistant population had been established in those nurseries. Fitness penalties on resistant isolates, such as slower growth, have been reported on some \u003cem\u003ePhytophthora\u003c/em\u003e species\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, which could be a factor limiting the widespread resistance. However, stable resistance without fitness disadvantages circumventing the fungicide effect of mefenoxam has been reported in \u003cem\u003eP. infestans\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePrevious studies hypothesized that the efficacy of mefenoxam is related to the binding and inhibition of specific sites responsible for ribosomal RNA (sRNA) synthesis, implying the reduction in mycelial growth and zoospore germination\u003csup\u003e13,17\u0026minus;19\u003c/sup\u003e. However, because RNA polymerases are multi-subunit complexes and topoisomerases and transcription factors could also influence their activity, the precise mefenoxam target remains unknown\u003csup\u003e24,28\u0026minus;30\u003c/sup\u003e. In \u003cem\u003eP. insfestans\u003c/em\u003e, resistant isolates were conditioned by variation in the RNA polymerase gene; however, the inheritance profile from crossing a sensitive and resistant isolate suggested the involvement of more than one locus\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Minor effects of several other genes have also been reported to contribute to resistance, such as genes related to efflux pumps and detoxification as ATP binding cassette (ABC) transporters and cytochrome P450 proteins\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eControversially to the studies carried out with \u003cem\u003eP. infestans\u003c/em\u003e, variations within the RPA 1 and RPA 2 genes conferring resistance to mefenoxam were not identified on \u003cem\u003eP. cactorum\u003c/em\u003e isolates, but six SNPs associated with resistance were identified after the whole genome sequencing of mutant isolates. Similar findings were reported in \u003cem\u003eP. capsici\u003c/em\u003e mefenoxam-resistant isolates, where the major SNPs conferring resistance were not within the RNA polymerase genes, but in a homolog of yeast protein Rrp5 gene required for processing of pre-rRNA transcripts into the cleaved molecule that forms the ribosome\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Unfortunately, due to the lack of a good genome annotation of \u003cem\u003eP. cactorum\u003c/em\u003e, the genes harboring these SNPs were not functionally characterized, and therefore their biological function remains unknown. Interestingly, after the discovery of these six SNPs, screening of all resistant isolates revealed that all the SNPs occurred simultaneously. A definitive test to determine whether all alleles are needed to confer resistance to mefenoxam, or which one has a major effect, using genetic transformation to study the interaction and effect of each locus alone would be necessary. Moreover, the detection of all the SNPs in all the resistant isolates could be due to clonal reproduction, since the isolates originated from the same nursery source in North Carolina. Therefore, there is a chance the SNPs found in this study conferring mefenoxam resistance, could vary in \u003cem\u003eP. cactorum\u003c/em\u003e isolates selected for resistance in other nurseries, growing systems, or crops.\u003c/p\u003e \u003cp\u003eThe practical application of determining the SNPs associated with the mefenoxam resistance would be the development of DNA markers for rapid differentiation of sensitive and resistant isolates. Screening isolates for fungicide resistance through \u003cem\u003ein vitro\u003c/em\u003e tests is widely used and the use of discriminatory doses for testing mefenoxam was proposed\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. However, this method is timing-consuming which can delay management recommendations. In this study, an HRM assay is proposed to speed up the process of distinguishing sensitive and resistant populations of \u003cem\u003eP. cactorum\u003c/em\u003e to mefenoxam. Primers were designed for all the SNPs identified, and HRM curves generated two distinct melting profiles for most of the regions except for region 4. The issue with region 4 is that the replacement of T (thymine) to A (adenosine), or vice-versa does not change the number of hydrogen bonds (n\u0026thinsp;=\u0026thinsp;2), which limits the detection of the polymorphism \u0026ldquo;T/A\u0026rdquo; in the HRM analysis. The pair of primers R3-1F/R3-1R and R2-1F/R2-1R were suitable to differentiate both sensitive and resistant profiles using clean and crude DNA extraction. However, if there is a mixture of sensitive and resistant populations, shifts in the melting curves could be expected for both primers. The use of crude extraction of symptomatic strawberry crown tissues was proposed by Wang et al.\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e to diagnose \u003cem\u003eColletotrichum\u003c/em\u003e spp., \u003cem\u003eMacrophomina phaseolina\u003c/em\u003e, and \u003cem\u003ePhytophthora\u003c/em\u003e spp., and the same extracts could be used to detect mefenoxam resistance if samples are positive for \u003cem\u003eP. cactorum\u003c/em\u003e, which could faster management recommendations to the strawberry industry and growers.\u003c/p\u003e \u003cp\u003eAlthough strawberry cultivars resistant to PhCR are available, the susceptible ones are widely grown based on their fruit quality and yield. Therefore, the application of chemicals for disease management will continue to provide security for crop production. The determination of genomic regions conditioning fungicide resistance could offer the possibility of accurately monitoring the pathogen population to guide disease control strategies. In this study, we have identified sequence variation in unknown genes that were correlated with the mefenoxam-resistant profile of \u003cem\u003eP. cactorum\u003c/em\u003e isolates from strawberry. Furthermore, an HRM assay was designed to be implemented in diagnostic clinics to improve and faster management recommendations. Our findings may contribute to better understanding of the mechanisms of action of mefenoxam in oomycetes as well as contribute to the sustainable use of this product by avoiding its application if resistance is detected.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e \u003cb\u003eCollection and storage of\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eP. cactorum\u003c/span\u003e \u003cb\u003eisolates.\u003c/b\u003e Isolates of \u003cem\u003eP. cactorum\u003c/em\u003e were collected between 1997 to 2020 from strawberry samples showing PhCR and LR symptoms received by the Diagnostic Clinic at the University of Florida Gulf Coast Research and Education Center (UF-GCREC). Isolates were purified by hyphal-tipping on cornmeal agar amended with pimaricin, ampicillin, rifampicin, and pentachloronitrobenzene (P5ARP\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e), and were identified at the species level using the high-resolution melting analysis developed by Wang et al.\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e using genomic DNA and the set of primers Ph29-F and Ph29-R as described by Ratti et al.\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Isolates were transferred to 20% V8 media\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e containing 0.03 g/l of β-sitosterol for 7 to 10 days, then stored in deionized water at room temperature (~\u0026thinsp;24\u0026deg;C). In total, 54 sensitive and 31 resistant isolates were hyphal-tipped and molecularly screened for mefenoxam sensitivity. Cultures were grown in P\u003csub\u003e5\u003c/sub\u003eARP and transferred to 20% V8 medium amended with 0.03g/L β-sitosterol for 7\u0026ndash;10 days at 25\u0026ordm;C. Mycelial plugs (6-mm-diameter) were used for storage in sterile water at 25\u0026ordm;C and in 30% glycerol at -80\u0026ordm;C in the culture collection of the Strawberry Pathology laboratory at the UF-GCREC. The mefenoxam resistant isolates did not show any growth inhibition on clarified 20% V8 media amended with 5 and 100 mg/\u0026micro;l of mefenoxam, according to Marin et al.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e (Supplementary Fig.\u0026nbsp;2).\u003c/p\u003e \u003cp\u003e \u003cb\u003eDNA extraction.\u003c/b\u003e Isolates were grown on 20% V8 media for 5\u0026ndash;7 days and mycelial plugs from actively growing margins of the cultures were collected for DNA extraction using the FastDNA Kit (MP Biomedicals), following the manufacturer\u0026rsquo;s protocol. The quality and quantity of DNA samples were determined using a NanoDrop 8000 spectrophotometer (ThermoFisher Scientific). DNA samples with a 260/280 ratio between 1.7 and 1.9 and a 260/230 ratio greater than 2.0 were diluted to a final concentration of 10 ng/\u0026micro;L and stored at -20\u0026deg;C.\u003c/p\u003e \u003cp\u003eFor the \u003cem\u003eP. cactorum\u003c/em\u003e whole genome sequencing, a protocol for plant DNA extraction modified from Keb-Llanes et al.\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e by Integrated DNA Technologies (IDT) was used for the pathogen DNA extraction. Colonies were grown on 10% V8 broth for four days, and then mycelia were washed with sterile deionized water, flash frozen in liquid nitrogen, and ground using a mortar and pestle. Subsequent extraction steps followed the protocol above mentioned. DNA concentration was verified using a NanoDrop 8000 spectrophotometer (ThermoFisher Scientific). DNA quality was determined using the Invitrogen Qubit\u0026reg; Fluorometer (Invitrogen Life Technologies, ThermoFisher Scientific), according to the manufacturer\u0026rsquo;s protocol and concentration was adjusted to 50 ng/\u0026micro;L.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDetecting mutations/single nucleotide polymorphisms (SNPs) in the RNA polymerase (\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eRPA190\u003c/span\u003e \u003cb\u003e) gene reported in\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eP. infestans\u003c/span\u003e. A total of 35 single nucleotide polymorphisms (SNPs) in the \u003cem\u003eRPA190\u003c/em\u003e gene (5433 bp) encoding the large subunit of RNA polymerase I in metalaxyl-resistant isolates of \u003cem\u003eP. infestans\u003c/em\u003e were identified by Chen et al.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Eight SNPs caused amino acid mutations associated with metalaxyl resistance in resistant isolates compared with sensitive isolates: K267E, R296H, F382Y, T443A, A597T, A871T, P980S, and V1476G\u003csup\u003e2\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Sequences flanking the mutations from \u003cem\u003eP. infestans\u003c/em\u003e were compared with the \u003cem\u003eP. cactorum\u003c/em\u003e whole genome sequence published by Armitage et al.\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Based on the regions containing the mutations, five sets of primers were developed: \u003cem\u003eRPA190\u003c/em\u003e-F1/R1 to \u003cem\u003eRPA190\u003c/em\u003e-F5/R5 (Supplementary Table\u0026nbsp;1), using the IDT-PrimerQuest Tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.idtdna.com/PrimerQuest/Home/Index\u003c/span\u003e\u003cspan address=\"https://www.idtdna.com/PrimerQuest/Home/Index\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Ten sensitive and ten resistant \u003cem\u003eP. cactorum\u003c/em\u003e isolate were selected. PCR conditions were as follows: 15 \u0026micro;L of 2\u0026times; AccuStart II PCR ToughMix (Quantabio; Gaithersburg, Maryland - U.S.A.), 1.5 \u0026micro;L of each forward and reverse primers (10 \u0026micro;M), 11 \u0026micro;L of molecular water, and 1 \u0026micro;L of diluted DNA (10 ng/\u0026micro;L), with a total volume of 30 \u0026micro;L. Amplifications were performed according to the following conditions: initial DNA denaturation at 95\u0026deg;C for 4 min; followed by 32 cycles of denaturation at 95\u0026deg;C for 30 s, annealing at 57\u0026deg;C for 30 s, and extension at 72\u0026deg;C for 3.5 min, and a final extension at 72\u0026deg;C for 10 min. PCR products were visualized under UV light in a 1% agarose gel in 1x Tris-acetate-EDTA buffer stained with GelRed\u0026trade; (Biotium) and sent for purification and sequencing in both directions at Genewiz Inc. (South Plainfield, NJ). Sequences were aligned using Geneious (version 11.1.4) and MEGA (version 7.0.20) software programs and the five amplified regions of sensitive and resistant isolates were compared separately.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIdentifying mutations/single nucleotide polymorphisms (SNPs) in the RNA polymerase I subunit I (RPA 1) and II (RPA 2).\u003c/b\u003e Based on the \u003cem\u003eP. cactorum\u003c/em\u003e whole genome sequence published by Armitage et al. (2018), various primers were designed for amplification and sequencing of the whole RNA polymerase I subunit I (RPA 1) and subunit II (RPA 2) genes. In order to locate both genes within the genome, sequences from \u003cem\u003eP. infestans\u003c/em\u003e XM_002906849 and XM_002907301 were used for RPA 1 and RPA 2, respectively. Six sets of primers were developed for RPA 1 (RPA1-F1/R1 to RPA1-F6/R6), and four for RPA 2 (RPA2-F1/R1 to RPA2-F4/R4) (Supplementary Table\u0026nbsp;1). PCR conditions, product visualization, and sequencing were performed for five sensitive and five resistant isolates as described in the previous section. Sequences of RPA 1 and RPA 2 of one sensitive and one resistant isolate were submitted to GenBank (accession numbers: OM273467 \u0026ndash; OM273470).\u003c/p\u003e \u003cp\u003e \u003cb\u003eWhole genome sequencing and SNP variant calling.\u003c/b\u003e One pool containing 18 sensitive isolates and another pool with 18 resistant isolates were submitted for whole genome sequencing by Novogene (Chula Vista, CA) with Illumina Platform PE150, Q30\u0026thinsp;\u0026ge;\u0026thinsp;80%. Illumina raw reads adapter trimming and quality filter were performed using CLC genomics workbench 11.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.qiagenbioinformatics.com/\u003c/span\u003e\u003cspan address=\"https://www.qiagenbioinformatics.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Two approaches were implemented to identify sequence variants related to mefenoxam resistance.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eVariant calling by mapping to reference-based approach.\u003c/span\u003e High-quality trimmed reads from mefenoxam-sensitive pooled reads (Spool Sensitive) and mefenoxam-resistant pooled reads (Rpool Resistant) were separately mapped to the two reference genomes of \u003cem\u003eP. cactorum\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Raw reads were trimmed using Trimmomatic v.0.32\u003csup\u003e54\u003c/sup\u003e. Trimmed reads were mapped to the whole genome sequence of \u0026lsquo;P414\u0026rsquo;\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/bioproject/PRJNA383548\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/bioproject/PRJNA383548\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) using Minimap2 with default parameters\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. The file with sequence alignment/map (SAM) format was converted into a binary alignment map (BAM) format with SAMtools v.1.12\u003csup\u003e56\u003c/sup\u003e. DNA variants including SNPs and insertion/deletion (InDel) were called using a genome analysis toolkit (GATK\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e) according to the manuals with default parameters. Variants were filtered using the following VCF parameters: QD\u0026thinsp;\u0026gt;\u0026thinsp;2.0, FS\u0026thinsp;\u0026gt;\u0026thinsp;60, ReadPosRankSum \u0026lt; -8.0, and MQRankSum \u0026lt; -12.5. Alternatively, the low frequency variant caller program of CLC genomics workbench 11.0 was also implemented to call variants from Spool Sensitive and Rpool Resistant reads. Variant filtration was performed by eliminating the common SNPs between Spool Sensitive and Rpool Resistant groups to select only the non-synonymous SNPs. The SNPs with only a high SNP index (\u0026gt;\u0026thinsp;80%) were considered for further validation.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eDe novo\u003c/span\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e-based approach for calling variants.\u003c/span\u003e For this approach, high-quality reads from both \u0026ldquo;Rpool Resistant\u0026rdquo; and \u0026ldquo;Spool Sensitive\u0026rdquo; were merged for \u003cem\u003ede novo\u003c/em\u003e assembly. The software AbySS 2.1.1\u003csup\u003e58\u003c/sup\u003e was chosen for \u003cem\u003ede novo\u003c/em\u003e assembly because of its better performance against short reads. Strict parameters were used for \u003cem\u003ede novo\u003c/em\u003e assembly and the contigs were made non-redundant using software CD-HIT v4.8.1\u003csup\u003e59\u003c/sup\u003e. The contigs having a length less than 2 kb were discarded and not considered for further analysis. Software Augustus v2.5.5\u003csup\u003e60\u003c/sup\u003e was used for gene prediction. Further, reads from both Rpool Resistant and Spool Sensitive were mapped to the final \u003cem\u003ede novo\u003c/em\u003e assembled contigs separately for variant calling. The low frequency variant caller program of CLC genomics workbench 11.0 was used to call variants from Spool Sensitive and Rpool Resistant reads separately. Common variants were removed between Spool Sensitive and Rpool Resistant groups. Only non-synonymous SNPs with a high SNP index were selected for further analysis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSequencing the genomic regions associated with mefenoxam resistance.\u003c/b\u003e Based on the flanking sequence contigs (200 bp) originated from the whole genome sequencing of a pool of sensitive and resistant \u003cem\u003eP. cactorum\u003c/em\u003e isolates, seven regions containing the SNPs possibly associated with mefenoxam resistance were amplified through PCR and sequenced. Six sets of primers were developed to amplify the seven regions and designated as 1, 2, 3, 4, 5, and 6: Rdmyl1 F/R to Rdmyl6 F/R (Supplementary Table\u0026nbsp;1). PCR conditions, product visualization, and sequencing were performed as described in the other PCR sections.\u003c/p\u003e \u003cp\u003e \u003cb\u003eHigh-resolution melting (HRM) assay for detection of SNP mutations.\u003c/b\u003e Based on the whole genome sequencing results, markers were designed to amplify the six flanking sequence regions containing the SNPs/mutations that could potentially be associated with mefenoxam resistance in \u003cem\u003eP. cactorum\u003c/em\u003e using the IDT-PrimerQuest Tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.idtdna.com/PrimerQuest/Home/Index\u003c/span\u003e\u003cspan address=\"https://www.idtdna.com/PrimerQuest/Home/Index\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Different sets of markers HRM 1F/1R, HRM 3F/3R, HRM 5F/5R, S1F/S1R, R4F/R4R, and R6F/R6R were used to amplify the six respective regions of 54 sensitive and 31 resistant isolates (Supplementary Table\u0026nbsp;1). PCR and HRM were performed in a Roche LightCycler\u0026reg; 480 Instrument II (Roche Diagnostics, Indianapolis, IN). Reactions had a total volume of 10 \u0026micro;L in a 384-well plate (TempPlate 384-well Full-Skirt PCR plate, White; USA Scientific, Ocala, FL) containing 5 \u0026micro;L of 2\u0026times; AccuStart II PCR ToughMix (Quantabio; Gaithersburg, Maryland - U.S.A.), 0.5 \u0026micro;L of each forward and reverse primers (10 \u0026micro;M), 0.5 \u0026micro;L of LCGreen Plus dye (BioFire Defense, Salt Lake City, UT), 2.5 \u0026micro;L of molecular water, and 1 \u0026micro;L of diluted DNA (10 ng/\u0026micro;L). PCR amplification began with an initial denaturation at 95\u0026deg;C for 3 min (ramp rate: 4.8\u0026deg;C/s), followed by 35 cycles of 95\u0026deg;C for 20 s (ramp rate: 4.8\u0026deg;C/s), 61\u0026deg;C for 30 s (ramp rate: 2.5\u0026deg;C/s), and 72\u0026deg;C for 30 s (ramp rate: 4.8\u0026deg;C/s). For post-PCR analysis, samples were subjected to the following HRM parameters: 95\u0026deg;C for 1 min (ramp rate: 4.8\u0026deg;C/s), 40\u0026deg;C for 1 min (ramp rate: 2.5\u0026deg;C/s), 65\u0026deg;C for 1 s (ramp rate: 4.8\u0026deg;C/s), and then a continuous fluorescence reading from 65 to 95\u0026deg;C (ramp rate: 0.02\u0026deg;C/s, 25 acquisitions/\u0026deg;C), with a final cooling step at 40\u0026deg;C for 30 s (ramp rate: 2.5\u0026deg;C/s). Melt curve genotyping and gene scanning analysis were performed using the LightCycler\u0026reg; 480 software (version 1.5.1.62). To generate the normalized melting curves, pre-melt and post-melt temperature settings were adjusted according to each region analyzed, and the temperature threshold was set to 0\u0026deg;C. Experiments were conducted in duplicate with three replications per sample.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRapid detection of SNP mutations associated with mefenoxam resistance directly from infected strawberry crowns.\u003c/b\u003e To develop HRM markers with more precise melting curve patterns between resistance and sensitive isolates, sequence variations present in the region 3 (NHQK01000034.1) and region 2 (NHQK01000017.1) containing the SNPs/mutations that could potentially be associated with mefenoxam resistance in \u003cem\u003eP. cactorum\u003c/em\u003e were used to develop functional markers (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Two primer sets, R3-1 and R2-1, were designed from the polymorphic sequences of region 3 and region 2, respectively, using the IDT PrimerQuest tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.idtdna.com/PrimerQuest/Home/Index\u003c/span\u003e\u003cspan address=\"https://www.idtdna.com/PrimerQuest/Home/Index\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Supplementary Table\u0026nbsp;1). A total of 40 isolates (28 resistant and 12 sensitive isolates) were used for the primer design test, and a total of 16 crude DNA extracts from symptomatic crowns inoculated with \u003cem\u003eP. cactorum\u003c/em\u003e isolates (11 resistant and five sensitive isolates) were used for marker validation (Supplementary Table\u0026nbsp;2). For the crude DNA extraction of infected strawberry crowns, plants of Sensation\u0026reg; \u0026lsquo;Florida 127\u0026rsquo; were inoculated by dipping the roots in a zoospore suspension of each isolate separately (10\u003csup\u003e4\u003c/sup\u003e zoospores/ml) and potted in the greenhouse. Once symptoms of PhCR were observed, the crowns were opened, and symptomatic tissue was used for the crude DNA extraction following the protocol published by Wang et al.\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. The two primers, R3-1 and R2-1, were also tested in a mixture of resistant and sensitive isolates of the purified DNA. Two resistant and two sensitive isolates diluted at a concentration of 20 ng/\u0026micro;l were used, respectively. The combinations of mixtures were pooled together with resistant and sensitive isolates in ratios of 1:1, 1:2, and 2:1, respectively. PCR reactions were prepared as mentioned above. The PCR and HRM analysis were performed in a LightCycler\u0026reg; 480 system II using a program consisting of an initial denaturation at 95\u0026deg;C for 5 min; 45 cycles of denaturation at 95\u0026deg;C for 10 s, annealing at 62\u0026deg;C for 10 s, and extension at 72\u0026deg;C for 20 s. After PCR amplification, the samples were heated to 95\u0026deg;C for 1 min and cooled to 40\u0026deg;C for 1 min. Melting curves were obtained by melting over the desired range (60\u0026ndash;95\u0026deg;C) at a rate of 50 acquisitions per 1\u0026deg;C. Melting data were analyzed using the Melt Curve Genotyping and Gene Scanning Software (Roche Life Science, Germany). Analysis of HRM variants was based on differences in the shape of the melting curves and melting temperature (Tm) values.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCOMPLIES WITH INTERNATIONAL, NATIONAL AND/OR INSTITUTIONAL GUIDELINES\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExperimental research methods used in this study complies with relevant institutional, national, and international guidelines and legislation.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by grants to SL and NP from the United States Department of Agriculture (http://dx.doi.org/10.13039/100000199) National Institute of Food and Agriculture (NIFA) Specialty Crops Research Initiative (#2017-51181-26833).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.M., J.B., N.P. and S.L. conceptualized and designed the experiments. M.M., J.B., Y.O., H.H., S.C., and N-Y.W. conducted the experiments and collected data. M.M., J.B., Y.O., H.H., and S.C., analyzed the data from the experiments and interpreted the findings. M.M., J.B., Y. O., N.P. and S.L. wrote the manuscript and prepared all Tables and Figures, and N.P. and S.L. reviewed and edited the manuscript.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAVAILABILITY OF MATERIALS AND DATA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analyzed during the current study are available in the GenBank (accession numbers: OM273467 \u0026ndash; OM273470), and NCBI (accession numbers: SRR21832435 and SRR21832436). Correspondence and requests for materials should be addressed to N.P. and S.L. Reprints and permissions information is available at www.nature.com/reprints.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eETHICS APPROVAL AND CONSENT TO PARTICIPATE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe all declare that manuscripts reporting studies do not involve any human participants, human data, or human tissue. So, it is not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eADDITIONAL INFORMATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCompeting interests: The authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFAOSTAT 2020. Strawberry production. Food and Agriculture Organization of the United Nations. Statistic division. Accessed: April 7, 2020. Available: http://www.fao.org/faostat/en/#data/QC/visualize\u003c/li\u003e\n\u003cli\u003eUSDA. National statistics for strawberries and Florida agricultural overview (2016). United States Department of Agriculture, National Agricultural Statistics Service (USDA, NASS). Data and statistics. (2020).\u003c/li\u003e\n\u003cli\u003eDittmar, P. J. \u003cem\u003eet al.\u003c/em\u003e \u003cem\u003eVegetable production handbook of Florida\u003c/em\u003e. (University of Florida - IFAS Extension, 2017).\u003c/li\u003e\n\u003cli\u003eBaggio, J. S., Marin, M. v \u0026amp; Peres, N. A. Phytophthora crown rot of Florida strawberry: inoculum sources and thermotherapy of transplants for disease management. \u003cem\u003ePlant Disease\u003c/em\u003e \u003cstrong\u003e105\u003c/strong\u003e, 3496\u0026ndash;3502 (2021).\u003c/li\u003e\n\u003cli\u003eMadden, L. V. Epidemiology and control of leather rot of strawberries. \u003cem\u003ePlant Disease\u003c/em\u003e \u003cstrong\u003e75\u003c/strong\u003e, 439-446 (1991).\u003c/li\u003e\n\u003cli\u003eEllis, M. A. \u0026amp; Grove, G. G. Leather rot in Ohio. \u003cem\u003ePlant Disease\u003c/em\u003e \u003cstrong\u003e67\u003c/strong\u003e, 549\u0026ndash;549 (1983).\u003c/li\u003e\n\u003cli\u003eRebollar-Alviter, A. \u0026amp; Ellis, M. A. Efficacy of azoxystrobin, pyraclostrobin, potassium phosphite, and mefenoxam for control of strawberry leather rot caused by \u003cem\u003ePhytophthora cactorum\u003c/em\u003e. \u003cem\u003ePlant Health Progress\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 17 (2005).\u003c/li\u003e\n\u003cli\u003eRebollar-Alviter, A., Madden, L. v, Jeffers, S. N. \u0026amp; Ellis, M. A. Baseline and differential sensitivity to two QoI fungicides among isolates of \u003cem\u003ePhytophthora cactorum\u003c/em\u003e that cause leather rot and crown rot on strawberry. \u003cem\u003ePlant Disease\u003c/em\u003e \u003cstrong\u003e91\u003c/strong\u003e, 1625\u0026ndash;1637 (2007).\u003c/li\u003e\n\u003cli\u003eMarin, M. V., Seijo, T. E., Baggio, J. S., Whitaker, V. M. \u0026amp; Peres, N. A. 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Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. \u003cem\u003eBioinformatics\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 1658\u0026ndash;1659 (2006).\u003c/li\u003e\n\u003cli\u003eStanke, M. \u003cem\u003eet al.\u003c/em\u003e AUGUSTUS: ab initio prediction of alternative transcripts. \u003cem\u003eNucleic Acids Research\u003c/em\u003e \u003cstrong\u003e34\u003c/strong\u003e, W435\u0026ndash;W439 (2006).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-2097400/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2097400/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePhytophthora crown rot (PhCR) caused by \u003cem\u003eP. cactorum\u003c/em\u003e is one of the most damaging diseases of strawberry worldwide. Mefenoxam is one of the major fungicides currently applied to manage PhCR. However, the emergence and spread of resistant isolates have made controlling the pathogen in the field problematic. In the present study, using whole genome sequencing analysis, mutations associated with mefenoxam-resistant isolates were identified in six different genomic regions of \u003cem\u003eP. cactorum\u003c/em\u003e. The 95.54% reads from a sensitive isolate pool and 95.65% from a resistant isolate pool were mapped to the reference genome of \u003cem\u003eP. cactorum\u003c/em\u003e P414. Four point mutations were in coding regions while the other two were in noncoding regions. The genes harboring mutations were functionally unknown. All mutations present in resistant isolates were confirmed by sanger sequencing of PCR products. For the rapid diagnostic assay, SNP-based high-resolution melting (HRM) markers were developed to differentiate mefenoxam-resistant \u003cem\u003eP. cactorum\u003c/em\u003e from sensitive isolates. The HRM markers R3-1F/R3-1R and R2-1F/R2-1R were suitable to differentiate both sensitive and resistant profiles using clean and crude DNA extraction. Our findings may contribute to a better understanding of the mechanisms of resistance of mefenoxam in oomycetes as well as contribute to the monitoring of \u003cem\u003eP. cactorum\u003c/em\u003e populations for the sustainable use of this product.\u003c/p\u003e","manuscriptTitle":"Identification of sequence mutations in Phytophthora cactorum genome associated with mefenoxam resistance and development of a molecular assay for the mutant detection in strawberry (F. ×ananassa)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-10-14 14:41:07","doi":"10.21203/rs.3.rs-2097400/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2023-01-11T09:39:58+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-12-30T03:42:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"5e332fd5-e10c-4940-b8a1-a0022d31b6ec","date":"2022-12-29T02:47:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"b6df5f88-23e4-4dfa-81c2-69a3c04247f5","date":"2022-12-06T05:30:30+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-11-07T07:33:52+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-11-07T07:10:10+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2022-10-11T05:54:31+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-10-11T05:51:24+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2022-09-23T18:22:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8c6b5028-f920-440c-88a7-196d07aeb091","owner":[],"postedDate":"October 14th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-10-16T20:53:44+00:00","versionOfRecord":{"articleIdentity":"rs-2097400","link":"https://doi.org/10.1038/s41598-023-34271-z","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2023-05-06 20:43:46","publishedOnDateReadable":"May 6th, 2023"},"versionCreatedAt":"2022-10-14 14:41:07","video":"","vorDoi":"10.1038/s41598-023-34271-z","vorDoiUrl":"https://doi.org/10.1038/s41598-023-34271-z","workflowStages":[]},"version":"v1","identity":"rs-2097400","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2097400","identity":"rs-2097400","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}