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Sustainable Cucurbit Anthracnose Research

Accomplishment Summary -Stakeholders Panel Meeting, 2024

Written by

Pooja Malhotra

Georgia state accomplishment summary (Kaur/Dutta)

Commercial field survey: A total of 177 isolates were collected from 9 commercial fields in 2024. One hundred twenty-four isolates were collected from six cucumber fields and fifty-three isolates were collected from three watermelon fields. Isolates were characterized morphologically and using the PCR based primers.

    Developed Colletotrichum orbiculare specific primers: Conventional PCR primer was developed for detecting C. orbiculare. Primer pair (F5/444R) was tested for the sensitivity and specificity for C. orbiculare. We also shared these primers with co-PIs for screening their isolates.

    Evaluation of conventional and organic fungicides: Four separate field trials were conducted to evaluate the efficacy of conventional and organic products on anthracnose severity in cucumber and watermelon in Georgia. All the trials were inoculated with an aggressive isolate of

    C. orbiculare from GA. In watermelon most of the conventional fungicides (Bravo, Topsin, Cabrio, Tebuconazole, Rhyme, Cevya, Miravis Prime, Switch) were able to reduce foliar anthracnose severity compared to non-treated check except Manzate. Similar observations were also made in cucumber conventional fungicide efficacy trial. All fungicides except Manzate were effective in reducing foliar anthracnose severity to non-treated check. However, pronounced effect of significant reduction in anthracnose severity was observed for Proline, Topsin, Cevya and Rhyme.

    A panel of OMRI-listed fungicides (Guarda, Oxidate, Howler, LifeGuard, Microthio Dispress, OSO, Timorex ACT) were also evaluated in watermelon and cucumber against C. orbiculare foliar severity. Bravo was used as a conventional fungicide standard for both the trials. None of the OMRI-listed fungicides were able to reduce foliar anthracnose severity in watermelon and cucumbers. In both the trials, Bravo performed significantly better than the OMRI-listed products.

    Characterization of C. orbiculare isolates and marker development (Brewer, UGA)

    Multi-locus sequencing: We tested GAPDH, HIS, and GS primers for PCR and sequencing of nine tester strains of C. orbiculare from watermelon, cantaloupe and cucumber. There were no polymorphisms for HIS, but GAPDH and GS showed several SNPs between watermelon and cucumber/cantaloupe isolates. This is further evidence that these are likely distinct populations. We are now sequencing more strains from GA to see if this pattern is consistent. We will be requesting diverse isolates from each state to add to our GA isolates for multi-locus sequencing studies.

    Fungicide target gene sequencing: We developed PCR primers for cytb based on the sequenced

    C. orbiculare 104T genome then amplified and Sanger sequenced cytb for 9 isolates from watermelon, cantaloupe and cucumber with varying EC50 values for sensitivity to the QoI azoxystrobin. We found that all watermelon isolates were wildtype at cytb, but all cucumber and cantaloupe isolates, which were generally less sensitive to azoxystrobin, had the F129L mutation known to provide partial resistance to QoIs.

    Population genomics: We just received the data from PacBio sequencing of a cucumber and watermelon isolate, which will be assembled into reference genomes for use in population genomic analyses. We will be requesting diverse isolates from each state to add to our GA isolates for population genomic studies.

    Marker development: We are developing an allele-specific PCR-based marker for the F129L mutation and plan to test it on a panel of cucumber/cantaloupe and watermelon isolates from multiple states with varying EC50 values.

    Effect of irrigation on anthracnose severity (Coolong, UGA)

    In Summer 2024 the irrigation × timing objective was carried out. Treatments included 3 irrigation/misting times (6 AM, 12 PM, 6 PM) and a control where overhead irrigation was not employed. All plots were drip-irrigated with supplemental overhead irrigation that was initiated daily for 10 mins in order to wet the leaves of plants at the desired times. The variety Crimson Sweet, was used. Plots consisted of 10 plants each, with 4 replications per treatment. Empty rows were placed between plots to ensure there was no drift of irrigation water between treatments. Irrigations were controlled by timers in the field. Leaf wetness sensors were deployed in every plot. Plots were all inoculated with a 105 suspension of conidia per ml from a locally obtained isolate of C. orbiculare at approximately 3 weeks after plant emergence (plants were direct seeded through black plastic mulch). Subsequently, disease ratings were conducted approximately every week through the completion of harvest. At maturity, melons were harvested and individually weighed, and any anthracnose lesions were noted. Data were collected for yield, disease incidence and leaf wetness.

    SCRI-SAM Anthracnose Summary, Georgia (McAvoy and Kumari, UGA)

    Objective:

    The primary objective of these experimental trials was to conduct a comprehensive evaluation of watermelon and cucumber varieties to determine their performance with resistance against anthracnose disease, overall yield, and fruit quality parameters.

    Methodology:

    The study was conducted over one year, encompassing the spring season of 2024. The research site was located at Hort Hill, UGA Tifton campus. Eight watermelon (Black Diamond and Charleston Grey checks) and cucumber (Marketer and H-19 checks) varieties were grown in a randomized block design with four replications, respectively. Relevant agronomic practices, such as irrigation, fertilization, and pest management, were implemented uniformly across all plots. Crimson sweet variety was used for the watermelon irrigation trial.

    Results:

    Anthracnose Watermelon Variety Trial: The study exhibited that there were no statistically significant differences in disease severity among watermelon cultivars. However, significant differences were found in fruit size distribution categories. In the 60-count category, ‘Captivation’ (7886.2 lbs/A) and ‘Valor’ (7873.5 lbs/A) had the highest yields, whereas ‘Charleston Grey’ (825.8 lbs/A) had the lowest yield. ‘Captivation’ (13,741.8 lbs/A) also yielded the highest in the 45-count category, while ‘Charleston Grey’ (5036.6 lbs/A) and ‘Troubadour’ (6564.9 lbs/A) performed the worst in this category. In the 36-count category, ‘Excursion’ (13576.2 lbs/A) significantly outperformed ‘Black Diamond’ (4412.7 lbs/A) and ‘Troubadour’ (3920.4 lbs/A). In the 30-count category, ‘Charleston Grey’ (10935.4 lbs/A) had the highest yield, significantly surpassing ‘Captivation’ (1050.4 lbs/A). ‘Black Diamond’ had the highest unmarketable yield at 12589.3 lbs/A, indicating a significant portion of its total yield was not suitable for the market. In contrast, ‘Captivation’ ‘Excursion’, and ‘Charleston Grey’ had much lower unmarketable yields, at 4689.5 lbs/A, 3986.2 lbs/A, and 3605.0 lbs/A respectively, suggesting better overall fruit quality and fewer defects. ‘Black Diamond’ had the highest number of misshapen fruit, with 8673.4 lbs/A, accounting for 24.3% of its total yield.

    Anthracnose Cucumber Variety Trial: This study evaluated eight cucumber cultivars, revealing significant differences in total, marketable, unmarketable, and percent marketable yields. However, there was no difference in anthracnose disease severity. ‘Python’ had the highest total (46,177.2 lbs/A) and marketable yields (37,232.9 lbs/A), while ‘H-19’ achieved the highest percentage of marketable produce (90%). ‘Diamondback’, ‘Brickyard’, ‘Raceway’ and ‘H-19’, had the lowest total yields, with 33053 lbs/A, 25,332 lbs/A, 26,823.9 lbs/A, and 24564.2 lbs/A, respectively. Unmarketable yields were highest in ‘Python’ (8,944.3 lbs/A). The percent marketable fruit ranged from 76.5% (‘Raceway’) to 90% (‘H-19’). In terms of average weight, H- 19 had the lowest at 0.3 lbs., whereas most other varieties, including Bristol, Mamba, Python, and Raceway, had an average weight of 0.6 lbs.

    Anthracnose Watermelon Irrigation Trial: The 6 A.M. treatment resulted in the lowest AUDPC value (91), signifying the least disease severity. In contrast, the 12 P.M. treatment exhibited the highest AUDPC value (194), indicating the most severe disease incidence. The 6 P.M. treatment

    had an intermediate AUDPC value of 138. Despite these variations in disease severity, there were no statistically significant differences in marketable yield, unmarketable yield, percent marketable yield, or average fruit weight among treatments. The marketable yield ranged from 14,706.0 lbs/A to 18,188.6 lbs/acre. Unmarketable yield was also similar across treatments, ranging from 7,890.7 lbs/A to 9,363.1 lbs/A. Percent marketable yield was consistent across treatments, ranging from 58.7% to 66.3%. Similarly, the average fruit weight was comparable across treatments, ranging from 17.3 lbs to 18.0 lbs. These findings suggest that while the timing of overhead irrigation significantly influences anthracnose severity, it does not significantly impact marketable yield, unmarketable yield, percent marketable yield, or average fruit weight. The 6 A.M. irrigation schedule appears to be the most effective in reducing disease severity without compromising yield or fruit quality.

    Debris Management Working Group (Sintim, UGA and Pethybridge, Cornell, NY)

    This Working Group assesses debris management for anthracnose control, and the subsequent impact on crop productivity, soil carbon sequestration, and overall soil health. From a regenerative agricultural management perspective, it is recommended to leave crop debris to serve as mulch. The practice protects the soil surface against wind and water erosion and slows evaporation and decomposition of organic matter. However, such management practice may not be a viable option for cucurbit production in the southern United States because of the frequent outbreak of anthracnose disease. Anthracnose is a fungal disease caused by a soilborne pathogen, Colletotrichum spp. The pathogen can overwinter in soil and crop debris, resulting in the outbreak of anthracnose disease in susceptible crops. Anthracnose disease have been found to be less severe in more intensive tillage systems. Nonetheless, intensive tillage systems have a long history of disrupting soil health indicators, prompting the need to consider a sanitation control (crop debris removal), as a compromise between the two extreme systems (retention of crop in the field vs. incorporation of crop debris via tillage).

    To assess the role of crop debris management on anthracnose control, field studies were initiated in Tifton, GA and Geneva, NY. The study entails three crop debris management systems:

    (a) crop debris left on the soil surface; (b) crop debris incorporated via tillage after harvest; and (c) crop debris completely removed after harvest. The treatments were arranged in a randomized complete block design with four replications, and each treatment plot had three beds to ensure adequate buffer. All sampling and data collection were made from the middle bed. The fields were planted to cucumber in Tifton and watermelon in Geneva, and the management systems (treatments) were imposed shortly after harvest. Next year (2025), experimental plots will be planted to cucumber and watermelon, respectively, in Tifton and Geneva, to assess treatment impact on cucurbit anthracnose control. This represents a worst-case scenario for anthracnose outbreak, given that growers would typically plant cucurbit in rotation with other crops. Therefore, a positive treatment response would reflect a very effective management system. We hypothesize that crop debris removal will be effective in minimizing anthracnose outbreak, while having minimal impact on soil carbon and overall soil health, given the low residue biomass and their rapid mineralization. Residues of fresh produce tend to mineralize rapidly because of the high moisture content and low C:N ratio. To test the above hypothesis, soil health assessment and soil carbon measurements were made before treatment imposition. Another assessment will be made in the final year of the study.

    Seed as a source of inoculum (Walcott and Oakley, UGA)

    The Walcott Lab validated a Colletotrichum orbiculare-specific PCR primer set designed by Dr. Navjot Kaur (Dutta Lab) and observed a limit of detection of 100 picograms. The lab also conducted seedling spray-inoculation and seed vacuum-infiltration assays to preliminarily characterize anthracnose symptoms on cucurbit seedlings and assess the likelihood of seed-to- seedling transmission of C. orbiculare, respectively. In seedling spray inoculation assays using suspensions containing 1.0 × 106 conidia/mL, typical anthracnose symptoms were commonly observed on seedlings. However, anthracnose symptoms were not observed in our seed-to- seedling transmission assay. One explanation for this observation is the conidial suspensions might not be an appropriate source of inoculum for seed transmission assays. Subsequent experiments will evaluate if mycelial fragments are a more effective source of inoculum for seed-to-seedling transmission.

    With the assistance of the Dutta lab, the Walcott lab harvested more than 200 cucumber fruits displaying anthracnose symptoms from a research field plot in Tifton, GA. Fruits were photographed, catalogued, and stored at 4°C until seeds were manually extracted. Seeds from each cucumber fruit were extracted, dried and stored as separate seedlots at 4°C until they are tested for C. orbiculare. A protocol for total DNA extraction from cucurbit seeds is under development and will eventually be used to directly test these seeds for C. orbiculare. Seeds will also be tested by plating assays and correlations will be made between anthracnose fruit symptoms and seedlot infection to infer the mechanisms by which seeds may become infected by the fungus. Infected seedlots will also be used to investigate seed-to-seedling transmission of anthracnose.

    Survey of commercial cucurbits fields and fungicide program on watermelons (Keinath, SC)

    Anthony Keinath, PI; Rob Last, M.S. student; Sierra Zardus, core technician; Anna Mothersbaugh, grant employee

    Clemson University, Coastal Research and Education Center, Charleston, SC

    Objective 1. Survey of commercial watermelon and cucumber crops for Colletotrichum spp.

    In 2024, 5 watermelon fields in Charleston County and 1 community garden in Lexington County were surveyed, but no Colletotrichum isolates were recovered from leaves.

    All together in 2023 and 2024, we surveyed a total of 2 pumpkin fields, 2 bottle gourd fields, and 12 pumpkin patches or retailers. A total of 165 Colletotrichum isolates were collected from pumpkins, winter squash, or gourds. Based on spore shape and size, 53 of the isolates collected in 2023 appear to be C. magna and 2 appear to be C. dematium.

    Objective 2.i. Field trials: Fungicide programs on watermelon

    Not enough anthracnose developed to evaluate fungicides in summer 2024, possibly due to above normal temperatures on 48 of 61 days in May and June. In 2025, experiments will be initiated earlier so anthracnose can develop under more conducive conditions.

    Objective 2.ii. Field trials: Drip vs. over-head irrigation on watermelon cv. Crimson Sweet

    Time Severity % P=0.05 6:00 AM 4.5 a 6:00 PM 3.5 a 12:00 PM 1.9 ab no misting 0.8 b    

    Mini-wobbler sprinkler heads were used to mist 3 of 4 treatments for 15 minutes per day; all treatments received drip irrigation. Severity of foliar anthracnose on July 5, 2024, 73 days after transplanting, was significantly greater on plants misted in the early morning or early evening than on plants misted at noon or not misted.

    Objective 5. Extension activities

    Virginia State Accomplishment Summary (Higgins and Langston, VA)

    Personnel

    Dr. Douglas Higgins, Assistant Professor, Virginia Tech Eastern Shore AREC Dr. David Lanston, Professor, Virginia Tech Tidewater AREC

    Ms. Emma Nieland, M.S. Student, Virginia Tech

    Dr. Emmanual Torres changed positions and left the project.

    TBD (Advertised Nov 2024), Postdoctoral Researcher, Virginia Tech

    Establishment of Isolate Collection (Objectives 1.i-v)

    For future work on species ID, fungicide sensitivity, genotyping fungicide resistance, and host specificity/race-typing we established an VA isolate collection. Isolates were collected from August to October from seven commercial watermelon fields. We also obtained isolates from a citron melon (Citrullus lanatus var. citroides) and a post-harvest watermelon disease outbreak. In total, 109 unique isolates were collected across all locations. All isolates have been single-spored and are being processed for long-term storage.

    Fungicide Efficacy – Field Trial (Objective 2.iii)

    VA tested 11 products in a conventional fungicide efficacy field trial. Disease pressure was high with nearly 100% disease severity in the untreated plots at the last rating date. Rhyme, Tebuzol and Cevya did not reduce disease compared to the untreated control. Manzate reduced disease severity by 76.2% and was among the products with the lowest disease severity. Manzate, Bravo, Quadris Top, Switch and Proline had lower disease severity than Topsin. Miravis Prime and Carbio had a similar disease severity as Topsin, Bravo, Quadris Top, Switch and Proline. No phytotoxicity was observed.

    Cultivar Evaluation – Field Trial (Objective 2.v.b)

    VA tested 10 cultivars for susceptibility to a local watermelon Colletotrichum isolate in a field trial. All cultivars were highly susceptible, with disease severity reaching 90.5 to 97.5% at the final rating date. Some differences in disease progress were observed among cultivars. AUDPC values for ‘Troubadour’, ‘Talca’, and ‘Embasy’ were lower than ‘Charleston’, ‘Valor’, ‘Cracker Jack’ and ‘El Capitan’. Also, ‘Fasicnation’, ‘Black Diamond’ and ‘Excursion’ had significantly lower AUDPC values than ‘El Capitan’.

    Extension Outreach and Education – Grower Presentations (Objective 5)

    Higgins, D.S. and Langston, D.L. 2024. Downy Mildew, Anthracnose and Gummy Stem Blight Management Update. Watermelon Grower Meeting, Carpon, VA, 17 April.

    Higgins, D.S. 2024. Adaptive Disease Management Strategies for Vegetables. Mid-Atlantic Crop Management School, Princess Royale Oceanfront Resort, Ocean City, MD, 20 Nov.

    New York State Accomplishment Summary (Pethybridge and Khmelnitsky, Cornell)

    • In Year 1 of the SCRI-SAM project, NY completed an OMRI product efficacy trial, a conventional product efficacy trial, a cultivar resistance screening trial and set up a three-year crop residue trial.
    • In Year 1, NY surveyed 7 grower fields and collected isolates from cucurbits with anthracnose. Isolates will undergo species level identification in winter 2024.
    • Both conventional and OMRI-listed fungicide trials were inoculated with three local C. orbiculare isolates at a concentration of 6.3 × 104 conidia/mL and 0.01% Tween-20. Weekly plot- level severity assessments were conducted and canopy health was measured by scanning the entire length of each plot with a hand-held GreenSeeker radiometer. At harvest, marketable and unmarketable fruit were counted and weighed. Marketable fruit were stored at 40 degrees Fahrenheit for 7 days and reassessed afterwards for anthracnose to assess post-harvest losses.
    • Nine conventional fungicides were tested for efficacy for anthracnose control on watermelon (cv. Sugar Baby). Fungicides included Dithane, Topsin, Cabrio, Tebuzol, Rhyme, Proline, Cevya, Miravis Prime and Switch.
      • Dithane, Topsin, Cabrio, Proline and Miravis Prime were effective at controlling anthracnose on watermelon in 2024.
    • Eight OMRI-listed fungicides were tested for efficacy for anthracnose control on watermelon (cv. Sugar Baby). Fungicides included Howler, Lifeguard,Oso, Kocide, Double Nickel, Thymic, Cinaction and Attitude.
      • Oso was the only effective OMRI listed fungicide for controlling anthracnose on watermelon in 2024.
    • Nine cultivars were tested for their susceptibility to anthracnose. Cultivars tested included Troubadour, Valor, Excursion, Fascination, Embasy, Captivation, Charleston Gray, Black Diamond and Sugar Baby. The trial was inoculated with three local C. orbiculare isolates at a concentration of 2.2 × 107 conidia/mL and 0.01% Tween-20. Weekly plot-level severity assessments were conducted, and canopy health was measured by scanning the entire length of each plot with a hand-held GreenSeeker radiometer. At harvest, marketable and unmarketable fruit were counted and weighed. 5 marketable melons from each plot were sliced along the short side and assessed for internal condition. Rating for over ripe, under ripe, bruised and hollow heart.
      • Sugar Baby and Black Diamond cultivars were susceptible to anthracnose, all other cultivars showed significantly reduced susceptability to anthracnose.
    • Seeds were extracted from the most efficacious treatments and the nontreated controls in both OMRI-listed and conventional fungicide trials and sent to Ron Walcott, UGA.
    • Drone flights of all three fields were conducted every 2.5 weeks to capture multiple angles of the fields as well as heat map images.
    • Infested crop residue was moved from the fungicide trials to the crop residue trial plots. 2.2 pounds (~1000 g) of residue was moved to each experimental plot.
    • Soil samples were taken from each plot to calculate the bulk density and porosity and sent to UGA for further biological and chemical analysis. Penetrometer and infiltrometer measurements were taken from each plot as well.

    North Carolina State Accomplishment Summary (Quesada)

    Posters:

    Alshwaiki, G., Hagerty, A., Cochran S., Quesada-Ocampo L. M. Diversity of Fungal Plant Pathogens on North Carolina Cucumbers. Undergraduate Research and Creativity Symposium. NC State, Raleigh, NC, July 2024.

    Objective 1:

    1. Standardized descriptive survey and isolation protocols:

    Field samples (4 fields total) have been collected based on the classic symptomatology of anthracnose for cucumber and watermelon. A single lesion per leaf was selected and plated onto water agar to isolate Colletotrichum fungi. When mycelium growth was observed, it was then transferred to an antibiotic amended potato dextrose agar plate for morphological identification.

    ii. Species identification:

    For species identification, mycelium was cultured in unclarified V8 broth for several days and then lyophilized for two days. DNA was extracted from the lyophilized samples using Qiagen and Zymo DNA extraction kits. The isolated DNA was subsequently used for ITS and GAPDH PCR amplification and Sanger sequencing. A total of 50 isolates have been successfully recovered so far.

    iii.   Fungicide sensitivity assay:

    We are still collecting isolates to start this objective

    1. Determination of mutations underlying QoI (cytb), MBC (β-tubulin), SDHI (sdhB, sdhC, and sdhD) and DMI (cyp51) resistance:

    We are still collecting isolates to start this objective

    v.  Fungal population genomics:

    We are still collecting isolates to start this objective

    vi.     Development of molecular diagnostic assays for species identification and fungicide resistance:

    We are still collecting isolates to start this objective

    Objective 2:

    1. Fungicide programs.

    The experiment is being conducted at the Cunningham Research Station in Kinston, NC. The plots are single raised beds on 5-ft centers covered with white plastic mulch; each bed is 14 ft long with 5-ft fallow borders at each end and non-treated guard rows on each side. The field was also planted with cucumbers the previous year. Cucumbers were directly seeded and thinned to one plant per hill after emergence (7 plants per plot). Irrigation and fertilization (4-0-8, N-P-K) were applied via drip tape. Treatments were randomized into four complete blocks. Ten fungicide treatments are

    applied using a CO2-pressurized backpack sprayer equipped with a hollow cone nozzle (TXVS- 26) delivering 40 gal/A at 45 psi. Fungicides treatments included Bravo Weather Stik, Dithane, Topsin, Cabrio, Tebuzol, Rhyme, Proline, Cevya, Miravis Prime, and Switch. Disease severity is being assessed weekly as the percentage of leaf area with necrosis per plot. Data will be analyzed using the ARM software (Gylling Data Management, Brookings, SD) with an analysis of variance (AOV) and Fisher’s protected least significant difference (LSD) test to separate the means.

    b.      Commercial variety trials across seven locations.

    The experiment is being conducted at the Cunningham Research Station in Kinston, NC. The plots are single raised beds on 5-ft centers covered with white plastic mulch; each bed is 14 ft long with 5-ft fallow borders at each end and non-treated guard rows on each side. The field was also planted with cucumbers the previous year. Cucumbers were directly seeded and thinned to one plant per hill after emergence (7 plants per plot). Irrigation and fertilization (4-0-8, N-P-K) were applied via drip tape. Cucumber varieties were randomized into four complete blocks. The varieties included were Bristol, DiamondBack, Mamba, Raceway, Python, Brickyard, Marketer and H-19. Disease severity is being assessed weekly as the percentage of leaf area with necrosis per plot. Data will be analyzed using the ARM software (Gylling Data Management, Brookings, SD) with an analysis of variance (AOV) and Fisher’s protected least significant difference (LSD) test to separate the means.

    Delaware State Accomplishment Summary 2024 (Betts, DE)

    2024 was a very dry season in DE with very limited disease pressure for watermelon across the region.

    Objective 1:

    We were in communication with growers and scouts throughout the season, but due to the dry conditions, we did not begin to obtain samples until September/October. Tissue samples were collected from a total of 12 sampling sites, these included watermelon and pumpkin. 11 of these grower fields were in Delaware and one was in Maryland. These sampling efforts yielded 91 cucurbit isolates, 44 of which were confirmed to be Colletotrichum through sequencing. We are in the process of finalizing all long-term storage.

    For preliminary sequencing ITS4 and ITS5 were utilized. Further sequencing is being done with ACT, CHS-1, TUB2, GS, GAPDH, and HIS3 primers. So far all sequencing has been done using the Phire Tissue Direct PCR Master Mix. Our initial sequencing results indicate the presence of multiple species of Colletotrichum, including C. coccodes, C. chlorophyti, C. orbiculare, and potentially a species from the C. magnum complex. Initial sequencing results have yielded few Colletotrichum orbiculare hits, though additional sequencing is needed to obtain better species resolution. We have begun working with the C. orbiculare specific primers. In general, the number of C. orbiculare isolates was very limited, which will be interesting to observe as we continue with isolate collection in 2025. As we finalize species identification, we will begin fungicide sensitivity assays in spring 2025.

    Objective 2:

    This summer we conducted a fungicide efficacy trial. For the trial, a complete randomized block design with four replicates was utilized. This trial included fourteen treatments, 10 traditional fungicides, 3 nontraditional fungicides, and one control. Fungicides included: Bravo weatherstik, Dithane F-45, Topsin, Cabrio, Tebuzol, Rhyme, Proline, Cevya, Miravis Prime, Switch, Howler EVO, OSO, and Guarda. Spray rates used were based on manufacturer recommendations found on product labels. There were a total of six weekly spray applications that began in mid-July and ended in late August. Harvest data was collected twice on August 13 and August 28. At harvest data was collected on the number and weight of healthy and unhealthy melons. On harvest days plant vigor ratings were also conducted. For these ratings, plots were rated on a 1-9 scale based on the general health of the plants in the plot. Unfortunately, disease pressure was absent in this trial. We did not yet have a local isolate, so we relied upon natural disease pressure for 2024, which was very low due to environmental conditions. Now that we have local isolates, we will be inoculating all future trials associated with this project.

    Host-resistance and breeding (Kousik, USDA)

    Identify sources of resistance to anthracnose in watermelon and cucumber plant introductions belonging to the core collections.

    Approach: In this proposed project we will combine research efforts with ARS and University Scientists at University of Georgia and University of Florida to phenotype the core collections of watermelon and cucumber plant introductions (PI) for resistance to anthracnose tat three locations against the prevailing strains o identify potential differential PI. Genome wide analysis will be conducted to identify potential markers conditioning resistance to anthracnose. The differential PI will then be evaluated for resistance in multiple locations (New York, Delaware, Virginia, South Carolina, Georgia, Florida) to determine variants in the pathogen population with respect to resistance in the host (watermelon or cucumber).

    Work Completed: The bulk of the initial work for this part of the project during year 1 entailed assembling all the Plant Introduction in the cucumber and watermelon core collections developed by the USDA NIFA, SCRI CucCAP2 project.

    Watermelon Core Collection: Seeds for majority of the 34 Plant Introductions (PO) in the watermelon core collection have been increased at the USDA ARS, U.S. Vegetable Laboratory facility in Charleston, SC. Seeds for the remaining few will be increased by March 2025. Thirty seeds of each PI in the watermelon core collection will be sent to the cooperators in GA and FL in April 2025 for phenotyping for resistance to Anthracnose. The whole genome sequences for all these 334 PI has been published and is freely available (CuGenDBv2).

    Cucumber core collection: Seeds for a significant number of PI in the cucumber core collection of 384 PI that were sent to us by the CucCAP2 team have been increased. We are continuing to working to increase the seeds of over 100 PI as we did not receive the seeds of these PI. Plus it has been challenging to get some of the PI to fruit in our conditions. The seeds of cucumber PI that have been increased (around 200 PI), will be sent to the cooperators early in 2025.

    Florida State Accomplishment Summary (Meru, Roberts, Vallad)

    Objective 1

    1. Survey & isolate collection

    Four fields in Florida—three watermelon fields in Levy, Gilchrist, and Collier counties and one cucumber field in Collier County—were surveyed for anthracnose. The Florida isolate collection currently comprises 42 isolates representing eight counties (Charlotte, Collier, Desoto, Gilchrist, Hendry, Hillsborough, Levy, and Osceola). Of these, 95% were isolated from watermelon, with the remaining 5% from cucumber. Among the watermelon isolates, 31% originated from fruit tissue, while 69% were from leaf tissue. Efforts to survey additional fields are ongoing, with outreach to county extension agents to expand collections as watermelon production in south Florida peaks and moves into the northern parts of the state. Over 50% of the isolates have been single-spored and stored using several techniques, including filter paper, glycerol, and Microbank tubes.

    1. Species identification

    The PCR procedures were optimized and a universal Colletotrichum primer and a Colletotrichum lagenarium-specific primers were tested. We are testing C. orbiculare primers developed by Dr. Dutta’s lab for species identification

    1. Race Typing

    Seeds for all seven differential cultivars were obtained, and germination studies were conducted to standardize the timeline for the race typing protocol. Preliminary results from the first isolate suggest it belongs to race 2. Additional race typing assays are in progress and will continue until all isolates are analyzed. Isolates are currently being single spored and stored using the protocol provided by the grant.

    v. Commercial Varieties Across Seven Locations

    Seeds for eight commercial varieties each of watermelon and cucurbit were planted at UF-TREC in Fall 2024 and the trial for disease severity and yield are in progress to determine genotypes that perform well under anthracnose disease pressure. A graduate student was recruited for this project.

    Objective 2

    i. Fungicide Field Trials In Spring 2024, two cucumber field trials were conducted to evaluate the efficacy of conventional and biological fungicides. Preliminary results from the conventional trial showed that Cabrio, containing pyraclostrobin (FRAC Group 11), provided the highest level of disease control. In the second trial, which contained several biological products, chlorothalonil was the only treatment that demonstrated a significant impact on disease suppression. Unfortunately, Hurricane Milton destroyed the fall 2024 watermelon field trial. Spring 2025 trials are planned

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    Pooja Malhotra

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