Genome-Wide Association Mapping and Genomic Prediction of Fusarium Wilt Race 2 Resistance in the USDA Citrullus amarus Collection.

Fusarium wilt caused by Fusarium oxysporum f. sp. niveum (Fon) race 2 is a serious disease in watermelon and can reduce yields by 80%. Genome-wide association studies (GWAS) are a valuable tool in dissecting the genetic basis of traits. Citrullus amarus accessions (n = 120) from the USDA germplasm collection were genotyped with whole-genome resequencing, resulting in 2,126,759 single nucleotide polymorphic (SNP) markers that were utilized for GWAS. Three models were used for GWAS with the R package GAPIT. Mixed linear model (MLM) analysis did not identify any significant marker associations. FarmCPU identified four quantitative trait nucleotides (QTN) on three different chromosomes (i.e., chromosomes 1, 5, and 9), and Bayesian-information and linkage-disequilibrium iteratively nested keyway (BLINK) identified one QTN on chromosome 10 as significantly associated with Fon race 2 resistance. FarmCPU identified four QTN that explained 60% of Fon race 2 resistance, and the single QTN from BLINK explained 27%. Relevant candidate genes were found within the linkage disequilibrium (LD) blocks of these significant SNPs, including genes encoding aquaporins, expansins, 2S albumins, and glutathione S-transferases which have been shown to be involved in imparting resistance to Fusarium spp. Genomic predictions (GP) for Fon race 2 resistance using all 2,126,759 SNPs resulted in a mean prediction accuracy of 0.08 with five-fold cross-validation employing genomic best linear unbiased prediction (gBLUP) or ridge-regression best linear unbiased prediction (rrBLUP). Mean prediction accuracy with gBLUP leave-one-out cross-validation was 0.48. Thus, along with identifying genomic regions associated with Fon race 2 resistance among the accessions, this study observed prediction accuracies that were strongly influenced by population size.

Widespread Resistance to Tebuconazole and Cross-Resistance to Other DMI Fungicides in Stagonosporopsis citrulli Isolated from Watermelon in South Carolina.

Tebuconazole, a demethylation-inhibitor (DMI) fungicide, is widely used on watermelon and muskmelon because it is inexpensive and has been effective against Stagonosporopsis citrulli, the primary causal agent of gummy stem blight in the southeastern United States. Most isolates (94% of 251) collected from watermelon in South Carolina in 2019 and 2021 were moderately resistant to tebuconazole at 3.0 mg/liter in vitro. Ninety isolates were identified as S. citrulli, and no isolates of S. caricae were found in this study. On watermelon and muskmelon seedlings treated with the field rate of tebuconazole, sensitive, moderately resistant, and highly resistant isolates were controlled 99, 74, and 45%, respectively. In vitro, tebuconazole-sensitive isolates were moderately resistant to tetraconazole and flutriafol but sensitive to difenoconazole and prothioconazole, while highly resistant isolates were highly resistant to tetraconazole and flutriafol and moderately resistant to difenoconazole and prothioconazole. On watermelon seedlings treated with field rates of five DMI fungicides in the greenhouse, severity of gummy stem blight did not differ significantly from the nontreated control when seedlings were inoculated with a highly resistant isolate, while severity was lower with all DMIs on seedlings inoculated with a sensitive isolate, although severity was greater with tetraconazole than with the other four DMIs. In the field, tetraconazole rotated with mancozeb did not reduce severity of gummy stem blight caused by a tebuconazole-sensitive isolate when compared to the nontreated control, while the other four DMIs did. With a highly resistant isolate, all DMIs rotated with mancozeb reduced severity of gummy stem blight compared to the nontreated control, but severity with tetraconazole and tebuconazole was greater than with mancozeb alone, and severity with flutriafol, difenoconazole, prothioconazole, and difenoconazole plus cyprodinil did not differ from mancozeb applied alone. Results from in vitro, greenhouse, and field experiments with the five DMI fungicides were highly correlated with each other. Thus, determining relative colony diameters with a discriminatory dose of 3 mg/liter of tebuconazole is an effective way to identify isolates of S. citrulli highly resistant to tebuconazole.

Genome-Wide Association Study of Resistance to Pseudomonas syringae in the USDA Collection of Citrullus amarus.

Pseudomonas leaf spot (PLS), caused by Pseudomonas syringae pv. syringae, is an emerging disease of watermelon in the United States with the potential to severely reduce yield under humid conditions. The genetic basis of resistance to this disease is not known and no resistant germplasm is available. Because Citrullus amarus is an important reservoir of resistance genes for the cultivated watermelon, C. lanatus, we screened the United States Department of Agriculture plant introduction collection of C. amarus for resistance to PLS. Accessions (n = 117) were phenotyped for their level of resistance to PLS in two separate tests. Accession means of percent leaf area affected ranged from 1.5 to 99.4%. The broad-sense heritability for the trait was 0.51. Whole-genome resequencing generated 2,126,759 single-nucleotide polymorphisms (SNPs) which were used to perform a genome-wide association study (GWAS) aimed at discovering molecular markers for resistance. Three different models-BLINK, FarmCPU, and MLM-were included in the GWAS analyses. BLINK and FarmCPU, which are multilocus models, found eight SNPs, located on chromosomes Ca01, Ca05, Ca06, Ca08, and Ca10, that were significantly associated with resistance to PLS. Two of these SNPs were found by both BLINK and FarmCPU. The MLM model did not detect any significant associations. BLINK and FarmCPU estimated an explained phenotypic variance of 43.6 and 28.5%, respectively, for SNP S6_19327000 and 25.0 and 26.0%, respectively, for SNP S1_33362258, the two most significant SNPs found. In total, 43 candidate genes with known involvement in disease resistance were discovered within the genomic intervals of seven of the eight peak SNPs. Eleven of the candidate genes that were found have been reported to be involved in resistance to P. syringae in other plant species. Two significant SNPs were within resistance genes previously documented to play important roles of plant resistance specific to P. syringae in other pathosystems. The SNPs identified in this study will be instrumental in finding causal genes involved in PLS resistance in watermelon and developing resistant germplasm through breeding.

Stachybotriaceae on Cucurbits Demystified: Genetic Diversity and Pathogenicity of Ink Spot Pathogens.

Recently, the incidence of Myrothecium leaf spot, a foliar disease of watermelon, has increased in South Carolina. However, the identity of the fungal species responsible for outbreaks of this disease has not been determined. Sequence data from four partial gene regions were used to conduct Bayesian inference in order to identify 95 isolates of Stachybotriaceae. Isolates were collected in South Carolina between July 2015 and May 2018. In total, six species of Stachybotriaceae were identified on watermelon and two other cucurbits: Albifimbria verrucaria, Gregatothecium humicola, Paramyrothecium foliicola, P. humicola, Xenomyrothecium tongaense, and Xepicula leucotricha. Two species, G. humicola and P. foliicola, were the predominant species found. Within these two species, genetic differences within small spatial scales were detected. Five species (all except Xenomyrothecium tongaense) were tested in experiments to determine their pathogenicity and relative virulence on three hosts grown in rotation in South Carolina. Southern pea plants were less susceptible than watermelon and tomato plants, which were equally susceptible. This constitutes the first reliable report of pathogenicity of any of the five tested species of Stachybotriaceae on these three vegetable crops. Another important finding was that none of the isolates were identified as P. roridum, the species considered to be the only causal agent of Myrothecium leaf spot on cucurbits. We propose the common name "ink spot" for the foliar phase of diseases caused by genera within the family Stachybotriaceae. This name is descriptive and likely to be accepted by growers. To prevent further loss incurred by ink spot, watermelon and tomato crops should be monitored for this disease.

Factors Influencing the Occurrence of Foliar Pathogens in Commercial Watermelon Fields in South Carolina Based on Stratified Cluster Sampling.

The influence of environmental and management factors on the occurrence of foliar pathogens of watermelon was analyzed using survey-sampling data collected from commercial farms in South Carolina in spring 2015 and spring and fall 2016. A stratified two-stage cluster sampling design was used to sample symptomatic watermelon leaves from 56 fields of 27 growers in seven counties representing the main watermelon-producing areas in the state. In the sampling design, counties corresponded to strata, growers to first-stage clusters, and fields to second-stage clusters. Pathogens were identified on 100 leaves collected per field based on reproductive structures that formed on the leaves. Information about previous crops, fruit type, field size, transplanting date, first harvest date, and fungicides applied within 7 days and within 7 to 14 days prior to sampling was obtained from growers. Field alignment was determined with a compass. Survey-specific logistic regression procedures were used to analyze the effect of these factors on the probabilities of pathogen occurrence. Five fungal pathogens, Stagonosporopsis spp., Podosphaera xanthii, Cercospora citrullina, Colletotrichum orbiculare, and Myrothecium sensu lato (s.l.), and the oomycete Pseudoperonospora cubensis were included in the analyses. Among the factors we analyzed, there was a consistent increased probability of occurrence of Stagonosporopsis spp. in fields with a previous cucurbit crop, increasing probabilities of pathogen occurrence with increasing plant age, a lower probability of occurrence of some pathogens on triploid cultivars compared with diploid cultivars, and a decrease in probability of pathogen occurrence in fields aligned toward southwest or west. Application of fungicides significantly reduced the probability of observing C. citrullina, P. cubensis, and Stagonosporopsis spp. in 2015 and P. xanthii in spring 2016. This study emphasizes the importance of crop rotation and fungicide applications to manage foliar diseases of watermelon, particularly gummy stem blight, powdery mildew, and downy mildew. Crop age, cultivar type, and field alignment also were found to significantly influence the probability of pathogen occurrence. To the best of our knowledge, this is the first study examining the influence of various factors on foliar pathogens of watermelon with data collected from commercial fields.

Occurrence of Foliar Pathogens of Watermelon on Commercial Farms in South Carolina Estimated with Stratified Cluster Sampling.

A survey of foliar pathogens of watermelon based on two-stage cluster sampling was conducted on commercial farms in South Carolina in spring 2015, spring and fall 2016, and fall 2017. In total, 60 fields from 27 different growers in seven counties representing the main watermelon-producing areas in the state were sampled, using a stratified two-stage cluster sampling approach. In the sampling design, counties corresponded to strata, growers to first-stage clusters, and fields to second-stage clusters. In each field, 100 symptomatic leaves were collected at five equidistant sampling points along four transects encompassing a square shape of 2,500 m2. After collection, pathogens were identified based on reproductive structures formed on leaves during >12 h incubation. Estimates were obtained for the statewide probability of pathogen occurrence and associations between pathogen pairs. Six fungal pathogens, Stagonosporopsis spp., Podosphaera xanthii, Cercospora citrullina, Colletotrichum orbiculare, Myrothecium sensu lato (s.l.), and Corynespora cassiicola; the oomycete Pseudoperonospora cubensis; and three viral pathogens were identified on the examined leaves. With the exception of fall 2017, Stagonosporopsis spp. was the most prevalent pathogen in every season, followed by P. xanthii. The highest occurrence of P. cubensis was in spring 2015; it did not occur in 2016. The highest occurrence of C. orbiculare was in spring 2016; it did not occur in spring 2015. Myrothecium s.l. was the most common pathogen in fall 2017 and the second most common pathogen occurring by itself in fall 2016. The third most common pathogen in fall 2017, Corynespora cassiicola, was not observed in any other season. Eight of the 80 isolates of Stagonosporopsis spp. collected were identified as S. caricae, the rest as S. citrulli. All isolates of S. caricae were found in spring 2015 and originated from two fields in different counties. A total of three positive and five negative associations were found between pathogen pairs co-occurring on the same leaf. A positive association between Stagonosporopsis spp. and C. citrullina was the only significant association between pathogens found in two seasons, spring 2015 and spring 2016. Based on estimates of probability of pathogen occurrence across seasons, Stagonosporopsis spp. and P. xanthii are the most common pathogens on watermelons in South Carolina. This is the first report of C. cassiicola, S. caricae, and Myrothecium s.l. on watermelon in South Carolina.

Susceptibility of Fourteen New Cucurbit Species to Gummy Stem Blight Caused by Stagonosporopsis citrulli Under Field Conditions.

At least 24 species of cucurbits from 13 genera are known to be susceptible to gummy stem blight, caused by three species of Stagonosporopsis. Cankers that are formed on crowns and stems play an important role in the disease cycle and the survival of the pathogen. Fourteen cucurbit species of unknown susceptibility representing 12 genera, four taxonomic tribes, and four geographic origins were inoculated with Stagonosporopsis citrulli in Charleston, SC, in spring 2015, spring 2016, and fall 2016 to evaluate their level of susceptibility to gummy stem blight and the ability of the pathogen to reproduce on crown cankers. An additional species, Cucumis melo, was included as a reference due to its known high susceptibility. Data sets of area under the disease progress curve (AUDPC) for foliar severity and crown cankers, final percentage of diseased leaf area, final percentage of plants with cankers, final percentage of plants with fruiting bodies, and rates of increase in canker incidence were analyzed to evaluate susceptibility. Results were similar for datasets of AUDPC and final ratings but there were more differences for AUDPC. In all experiments, Apodanthera sagittifolia, Ecballium elaterium, and Kedrostis leloja were at least as susceptible to foliar blight as the reference C. melo. K. leloja was as susceptible to crown cankers as C. melo in all experiments and A. sagittifolia and E. elaterium were among the species most susceptible to crown cankers in two experiments. Coccinia grandis was highly resistant to gummy stem blight and had a few cankers only in fall 2016. Sicana odorifera and Zehneria pallidinervia also consistently grouped with the most resistant species. Incidence of crown cankers on Cucumis melo and K. leloja increased at the fastest rate of all species in all experiments and had, along with E. elaterium, the highest incidence of crowns with fruiting bodies. In general, the most susceptible species also were most suitable for reproduction of the pathogen and had the fastest disease progression. The tribes Benincaseae and Cucurbiteae had consistently lower levels of foliar blight than Bryonieae and Coniandreae. The tribe Benincaseae had a consistently lower AUDPC for canker incidence than Bryonieae and Coniandreae. The species originating from Europe (E. elaterium) was consistently most susceptible to both symptoms, while African species grouped with the least susceptible species in all experiments. To the best of our knowledge, this is the first report of susceptibility to gummy stem blight of 14 species and the first report of susceptibility of the cucurbit tribes Coniandreae and Gomphogyneae. This expands the host range of Stagonosporopsis citrulli to 37 species representing 21 genera and seven tribes in the family Cucurbitaceae. This study demonstrates the importance of crown cankers as reproductive sites for S. citrulli.