March 2021Objective 1: Evaluate the optimal spray timing for Florida and investigate if tree skirting or alternative products improves fungicidal control of citrus black spot.Objective 3: A MAT-1-1 isolate may enter Florida and allow for the production of ascospores. The industry needs to know if this happens, as it will affect management practices. Additionally, the existing asexual population may be more diverse than currently measured. If multiple clonal linages exist, then there may be different sensitivities to fungicides or other phenotypic traits. We also need to determine whether P. paracitricarpa or P. paracapitalensis are present in Florida for regulatory concerns due to misidentification. We plan to survey for the MAT-1-1 mating type, unique clonal lineages, and two closely related Phyllosticta spp. We collected data from the large spray timing and skirting trial in March. We evaluated 50 fruit each for disease severity on approximately 125 trees in 32 rows. We made significant progress on the incidence analysis of the data and are close to finalizing the analysis. We found that fungicide program significantly reduced the black spot incidence compared to the control. The greatest reduction was from the Florida standard timing with applications from May to September. Skirting had no significant effect on the disease incidence but the interaction between fungicide timing and skirting did have a significant effect. The Florida standard timing with skirting was the best performing treatment. For the late fungicide timing (May to October), there was a slight decrease in incidence with the addition of skirting. It should be noted that the 2019-2020 season was light for black spot incidence and severity. From initial scouting, it appears that black spot severity is greater this year and there may be more differences among the treatments that will hopefully support the conclusions from the first year. The minor plots were re-randomized within the main plots and we were able to get the trial re-flagged just in time for the early spray (delayed by 2 weeks but no rain occurred from the first of April until after the early application). All applications were made on time in the spray trial. We will be collecting the data at the end of March, 2021. We were unable to set up the second planned fungicide trial this year because of the COVID-19 shut down. We will be collecting pre-treatment data at the end of March and plan to move forward with the trial. The second season of trials in which different fungicide products are tested for their efficacy to protect citrus fruit from CBS infection, is currently underway. Products being evaluated include Enable (Indar) and Luna experience sprayed on their own as well as Copper hydroxide sprayed in alternation with either Amistar Top or Headline (Cabrio). Fungicides are applied every 4 weeks from October 2020 until March 2021. The fungicides are being tested in a ‘Valencia’ orange orchard with a history of CBS. The trials will be evaluated at the end of August 2021. An additional 8 South African and 8 global (from Argentina and Swaziland) isolates have been sequenced. As the next-generation sequencing data becomes available, the analysis and results are continuously updated to include the new data. DNA from 16 isolates (Argentina, Australia, Brazil and China) passed QC and is in the process of being sequenced. More isolates from Brazil and China are being cultured for DNA extraction and sequencing. We aim to sequence 6 isolates from each of the 5 South African provinces where CBS is found (North West, Limpopo, Mpumalanga, Kwa-Zulu Natal and Eastern Cape) and 6 each from the other countries (Argentina, Australia, Brazil, China, Swaziland and USA) by end of March. Objective 3 (Survey for the MAT-1-1 mating type and two closely related Phyllosticta spp.). AStudies on the diversity of Phyllosticta spp. associated with citrus in Florida has progressed. Our collection of P. citricarpa isolates were obtained from citrus fruit in different areas under quarantine from 2010 to 2020. These isolates were previously screened by morphology to remove nonpathogenic P. capitalensis isolates. We are now screening the remaining 202 isolates using molecular techniques to determine if we have cryptic species that may have been misidentified as P. citricarpa. To date we have screened 125 isolates by amplifying and DNA sequencing the tef-1a (Translation elongation factor-alpha) locus. This screen has revealed that two isolates (Gc-6 and Gc-7) match to a Phyllosticta species not previously reported in association with citrus. The best sequence match based on ITS and tef-1a is to a species reported previously as a pathogen on a member of the Amaryllidaceae family (Hymenocallis littoralis) in Australia. Numerous inoculations of citrus have determined that these isolates do not cause citrus black spot. Multilocus analysis and leaf inoculations on Hymenocallis species are ongoing. These studies will allow us to conclusively determine the identity of these two isolates. Vegetative and sexual compatibility assays (sandwich mating) were performed to determine the mating type of the two isolates. The results showed that Gc6 and Gc-7 isolates are heterothallic and belong to the same mating type, as neither isolate was a capable of producing pseudothecia in solo cultures or in paired cultures with one another. The screening and further characterization of these new species is continuing to obtain robust information on the diversity of Phyllosticta species and determine the presence of cryptic species in Florida.We screened an additional 26 isolates of our P. citricarpa isolates for mating-type (MAT1-1 and MAT1-2). During this period we have screened an additional 26 new isolates and, as shown in Figures 1, only the MAT1-2-1 mating type is detected in new isolates collected in 2020. We conclude that the MAT1-1 mating type is still absent in the Floridian population.To determine the phenology of fruit susceptibility inoculation studies of citrus fruit (Meyer lemon) were performed in a quarantine greenhouse at the Florida Department of Agriculture and Consumer Services (DPI) in Gainesville. A total of 97 fruit were used in this experiment. Of these 97 fruits, 25 served as controls and 72 were inoculated with the Gc-12 isolate of P. citricarpa. Disease assessments were performed weekly for a full calendar year. A total of 50 fruit produced symptoms in this period. All 50 were from fruit inoculated with the Gc-12 isolate, and no symptoms were observed in the control treatment. Therefore, from all inoculated citrus fruit, 69.4% produced symptoms, and 30.5% remained asymptomatic. The indications from this one experiment suggest that citrus fruit are susceptible at all stages of their development regardless of their maturation time. A second trial will be conducted in 2021 to determine the period of fruit susceptibility to P. citricarpa.