June 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. 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. Data was collected from the trial and has been entered into the computer (~188,000 entries) and is being verified for accuracy. As noted previously, there was far more disease this year than 2020. We are examining the weather data from both seasons to see what factors rainfall and temperature may have played. Pre-treatment data were collected at the end of March for the CBS spray trial. We have 15 treatments included in the trial. One application has been completed of a planned 6 applications with the 2nd scheduled for this week. 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 in South Africa. 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. Sixty-five P. citricarpa isolates (36 from South Africa, representing the five provinces where CBS is found, and 29 from other countries, Argentina, Australia, Brazil, China, Eswatini and USA) have been sequenced. Sequence data for 6 isolates, 1 from China, 1 from Brazil and 4 from USA are in the process of being generated. The sequencing data for the 65 isolates were analysed in the same manner as previously described, namely read mapping and variant calling, and in silico genotyping. Both analyses approaches showed the same patterns in terms of genetic distribution of isolates. Isolates from China are the most genetically distinct, while there is different degrees of genetic connectivity between isolates from Argentina, Australia, Brazil, Eswatini, USA and those form South Africa, and corresponds with previously published results (Carstens et al., 2017). Objective 3 (Survey for the MAT-1-1 mating type and two closely related Phyllosticta spp.). Studies to determine what Phyllosticta spp. associated with citrus are present in Florida are on going. From 125 screened isolates using DNA sequencing of the translation elongation factor-alpha (tef-1a) locus, three isolates grew distinctly from species previously reported in Florida. Within the three new isolates identified, only two were screened using the internal transcribed spacer (ITS) and the actin (ACT). The result demonstrated that both isolates (Gc-6 and Gc-7) are from distant lineage from. P. citricarpa. and. P. capitalensis.a nd grouped with. P. hymenocallidicola, a. Phyllosticta. species not previously associated with citrus. An assay was conducted on citrus fruit (Valencia and Meyer lemon) using isolates Gc-6, Gc-7, Gm33 (P. capitalensis), Gc-12 (P. citricarpa), and water (control) to determine if both isolates are pathogenic to citrus or not. The result showed that only isolate Gc-12 produces symptoms on citrus fruits, demonstrating that. P. citricarpa. appears to be the only plant pathogenic species associated with citrus in Florida and isolates Gc-6 and Gc-7 are non-pathogenic species in citrus. Moreover, a pathogenetic test was performed in. Hymenocallis littoralis. leaves.because .P. hymenocallidicola. was originally described from this host. Leaves were inoculated using two methods (fungal plug and spore suspension), and similar symptoms (brown to reddish spots) were observed as in the only documented report. Koch’s postulates for this disease had never been done. 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.Samples (fruit, twigs, leaves) were collected in the spring of 2021 from groves for mating-type screening using the conventional PCR primers described in Wang et al. (2016) to determine if previously undetected MAT1-1 idiomorph is present or not. Isolates are being purified so the study is ongoing to determine if MAT1-1 is still absent in the Floridian population or if it has entered Florida and the pathogen is reproducing sexually. Thus far, no isolate has been identified with MAT1-1.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.