1ST-SCRI-SAM Stakeholders Panel Meeting Updates

1st Stakeholder Panel meeting Update- 2024

SCRI-SAM Stakeholder advisory meeting minutes

Objective 1

1. Surveys and single-spore isolations
   1. GA- PD Dutta and Post-doc Kaur (Surveyed 9 fields (n= 230 isolates from watermelon and cucumber in 2024). Sent a subset (n=139 isolates, another set of 38 isolates will be sent soon totaling 177 isolates from GA) to Brewer lab for characterization, population genomics and fungicide resistance studies).
   1. NY- PI Pethybridge/ Grad-student Khmelnitsky (Surveyed 7 fields (n= 17 C. orbiculare isolates from cucumber and 12 were collected from gourds).
   1. VA– Co-PI Higgins/PI Langston (Surveyed 7 fields including a wild watermelon (n =109 Colletotrichum isolates; species identification is underway))
   1. DE- PI Betts (Collected isolates from 12 fields (n=44), multiple Colletotrichum species including C. coccodes, C. chlorophyti, C. orbiculare, and potentially a species from the C. magnum complex were observed; species identification is underway).
   1. NC- PI Quesada [Collected isolates from cucumbers (n=77 from 3 fields and n=17 from 1 watermelon field in 2023) and (n=12 isolates from 2 cucumber fields in 2024; species identification is underway].
   1. SC- PI Keinath/ Grad-student Last (Collected n= 65 (1 field in 2023) and n=122 (2 fields in 2024) from pumpkins, gourd, and squash; species identification is underway).
   1. FL- PI Roberts /Co-PI Vallad (n=42 isolates (8 counties), from watermelon and cucumber in 2024; species identification is underway)

1. C. orbiculare specific primer testing:NY- PI Pethybridge/ Grad-student Khmelnitsky (Completed specificity of C. orbiculare-primers)VA- Co-PI Higgins/PI Langston (screening in process)DE- PI Betts (screening in process)NC- PI Quesada (screening in process)SC- PI Keinath (screening in process)GA- PD Dutta and Post-doc Kaur (Completed screening C. orbiculare isolates along with specificity and sensitivity).  FL- PI Roberts /Co-PI Vallad (screening in process)

• Multilocus sequencing Participants-NY, VA, DE, NC, SC, GA, FL. Currently ITS and GAPDH are used to differentiate C. orbiculare spp. The GS (glutamine synthetase) loci is presently being standardized. Other loci are still being under screening process (CHS-1, HIS3, ACT, TUB2)

• Fungicide sensitivity assays Participants- NY, VA, DE, NC, SC, GA, FL (SC will send isolates to GA)QoI screening (Co-PD Brewer) (QoI (Azoxystrobin), DMI and SDHI) will be screened. PCR primers were developed for cytb for QoI resistance/sensitivity screening. PI Pethybridge and Co-PD Brewer will work together for the standard set of isolates to be used for fungicide resistance/sensitivity screening and protocols. PI Pethybridge has scheduled a meeting on Jan 15 for QoI resistance resistance/sensitivity objective.

• Population genomic analysesParticipants- Co-PD Brewer, PI Pethybridge, PI QuesadaHigh quality DNA extraction and whole genome sequencing for select isolates were conducted. Co-PD Brewer will contact individual state leads for the diverse isolates. Currently sequenced two isolates to construct a high-quality reference genome for population genomic analysis.

• Marker Development- Co-PD Brewer is currently working on developing the allele specific marker for the F129L mutation conferring resistance to the QoIs and will share with the team.

• Host specificity and Race typing (PI Roberts /Co-PI Meru /Co-PI Higgins) Will be conducting this study in VA and FL. FL will receive select isolates from GA, SC and NC. Currently standardization of protocol is in process by PI Roberts and will start accepting and screening isolates once the list of the diverse isolates is finalized. PI Roberts and Co-PI Higgins will coordinate with Co-PD Brewer for the selection of diverse isolates for race typing.PI Roberts and Co-PI Higgins have APHIS permits to receive the isolates from different labs. Seeds for all seven differential cultivars were obtained, and germination studies were conducted to standardize the timeline for the race typing protocol (FL). Co-PI Vallad and Co-PI Kousik volunteered to get in touch with industry personnel to obtain a positive control (Race 2).

Objective 2

1. Crop debris management (Co-PI Sintim/PI Pethybridge). Two locations, (Tifton, GA and Geneva, NY). Both cucumber and watermelon were included in the study in Tifton while only watermelon will be used in NY. In Tifton, three treatments were included, and preliminary observations were made for different soil properties and debris management from 0–6-inch, 6–12-inch soil depth. NY has started the debris management trial with 4 treatments. Soil samples sent to GA and will be planting watermelons in 2025 for the study.

• Fungicide trials updateGA- PD Dutta (Conducted both conventional and organic fungicide trial in 2024). Four separate trials were conducted in cucumber and watermelon. All fungicides except Manzate were effective in reducing foliar anthracnose severity. However, none of the OMRI-listed fungicides had efficacy in reducing foliar disease severity and fruit rot. FL- Co-PI Vallad / PI Roberts (Conducted both conventional and organic fungicide trials in 2024, Hurricane Milton unfortunately destroyed the watermelon trials). Two trials were conducted in cucumber. Preliminary results from the conventional fungicide trial showed that Cabrio, provided the highest level of disease control. Among OMRI-listed products, chlorothalonil was the only treatment that demonstrated a significant impact on disease suppression.VA- Co-PI Higgins/PI Langston (Conducted one conventional fungicide trial each in watermelon and cucumber). Manzate, Bravo, Quadris Top, Switch and Proline had lower disease severity than Topsin.NY- PI Pethybridge/ Grad-student Khmelnitsky (Conducted both conventional and organic fungicide trials in watermelon in 2024). Among conventional fungicides, Dithane, Topsin, Cabrio, Proline and Miravis Prime were effective. While Oso was the only effective OMRI-listed fungicide that provided significantly better efficacy in reducing foliar disease severity. NC- PI Quesada (Conducted one conventional fungicide trial in cucumber). Analysis is ongoing; however, no disease pressure was observed. SC- PI Keinath/ Grad-student Last (Not enough anthracnose pressure developed to evaluate fungicides in Summer 2024, will conduct fungicide trials in 2025).DE- PI Betts (Conducted one conventional fungicide field trial in watermelon but no disease pressure was observed, relied on natural inoculum. Will inoculate trials in 2025).
• Irrigation Trials update
• GA- Co-PI Coolong/ Grad-student Burnett (Irrigation trial including three 3 irrigation/misting times (6 AM, 12 PM, 6 PM) and a control was conducted in Athens, GA). No significant difference for final disease severity, yield was observed. However, there was significantly high disease in the 6 PM treatment. Leaf wetness readings were taken and will be compiled.
• GA- Co-PI McaVoy / Post-doc Kumari (Irrigation trial including three 3 irrigation/misting times (6 AM, 12 PM, 6 PM) and a control was conducted in Tifton, GA). No significant difference was observed for fruit size distribution in different categories (60-count, 45-count, 36-count and 30 count). No significant differences for yield components (marketable yield, unmarketable yield, average weight). The 6 A.M. treatment had significantly lower disease severity compared to other treatments.
• SC- PI Keinath (Irrigation trial including three 3 irrigation/misting times (6 AM, 12 PM, 6 PM) and a control was conducted). No significant differences were observed for the final disease severity, AUDPC and percent symptomatic fruits. However, more disease was observed in the early morning or early evening treatments.

• Varietal trials update-
  • GA- Co-PI McaVoy / Post-doc Kumari (Conducted watermelon (n=8 commercial lines) and cucumber (n=8 commercial lines) varietal trials in 2024). No statistically significant differences in disease severity and yield among watermelon cultivars were observed. However, significant differences were found in fruit size distribution categories. No difference in anthracnose disease severity in cucumber varietal trials but yield components including yield, marketable and unmarketable yield and average weight varied significantly among cucumber cultivars.
  • NY- PI Pethybridge/ Grad-student Khmelnitsky (Conducted a watermelon (n=9 commercial lines) varietal trial in 2024). Sugar Baby and Black Diamond cultivars were susceptible to anthracnose, all other cultivars had significantly reduced susceptibility to anthracnose.
  • VA- Co-PI Higgins/PI Langston (Conducted a watermelon (n=10 commercial lines) varietal trial in 2024). All cultivars were highly susceptible but Fascination’, ‘Black Diamond’ and ‘Excursion’ had significantly lower AUDPC values than ‘El Capitan’.
  • DE- PI Betts will evaluate few commercial watermelon lines in 2025.
  • NC- PI Quesada (Conducted a cucumber (n=10 commercial lines) varietal trial in 2024). No disease was observed.
  • FL- Co-PI Meru (Ongoing watermelon and cucumber varietal trials by Co-PI Meru).

• Seed pathology- Co-PI Walcott/ Post-doc Oakley. Confirmed the sensitivity of the C. orbiculare specific primers (100 pg). Harvested 200 cucumber seedlots (infected) and stored for detection of C. orbiculare in seeds. Seedling spray inoculation was tested in watermelon and rapid symptom progression was observed in 24-48 h and severe disease severity was observed at 7 dpi. Seed-to-seedling transmission was tested using vacuum infiltration; however, symptom development did not occur until 28 dpi. Further mycelial fragments will be used to inoculate seeds and will be evaluated for seed-to-seedling transmission. Seed treatments strategies such as cold plasma and a NO donor molecule will be investigated. Lab is ready to receive the infested seeds from different collaborators.
• Breeding aspects– Co-PI Kousik /Co-PI Meru/ Co-PI Mcgregor. Seeds of 287/334 watermelon PI lines and 214/265 Cucumber PI lines have been increased and are ready to be shipped to collaborators (GA, FL). Evaluate local (FL, GA, SC) isolates using the standard differential cultivars (Marketer, H-19, Charleston Grey and Black Diamond). Phenotyping of Watermelon Core collection at three locations using local (FL, GA, SC) C. orbiculare isolates under controlled conditions will be conducted. SK also evaluated a RIL (183) population in the field and conducted QTL mapping. They identified potential QTL associated with the anthracnose resistance at chromosome 6 and currently evaluating KASP markers. Further field screening of the RILs will be conducted in 2025 to confirm the field results.
• Economic analysis- Co-PI Colson requested the team to share the data in any format including presentations, PDMR, reports, excel files when the data is generated. The components including yield, labor usage etc.
• Coordinate isolate sharing- Co-PD Brewer lab has APHIS permit and will be contacting groups for diverse isolates for population genomic analysis.
• Isolation protocol – Post-doc Kaur will put together a Colletotrichum isolation protocol with more details for the groups with difficulty in isolating the pathogen from both leaf and fruit tissues (in shared one drive folder).