June 2020Objective 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 as of March 13. We evaluated 50 fruit each for disease severity on approximately 125 trees in 32 rows. The data entered for analysis but analysis has not yet begun. The treatments were in a randomized split-plot design with skirting as the main plot and fungicide timings (early, standard, late) along with an untreated control were the minor plots. However, all the treatments appear to have had significantly better disease management than the untreated control. 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). The delay was caused by the travel and work restrictions cause by COVID-19. Several subsequent sprays have been applied. During the 2019 -2020 growing season, 6 applications of fungicide were made. There was no major delay or issue with any of our treatments. In our trials, we had nine fungicides along with a water control total of 10 treatments. On March 9-13, 2020, we completed our first trial’s evaluations. We inspected 50 fruit per tree in all treated trees using a 0 (no disease) to 5 (high severity) rating scale. The data are entered and a very preliminary analysis shows all products or product rotations had significantly greater odds of no black spot compared to the untreated control. We were unable to set up the second planned fungicide trial this year because of the pre-treatment data collection needed but we plan to conduct the trial next year if a no cost extension is granted. New fungicide products are being tested for the management of CBS in South Africa. Products being evaluated include Kocide (copper hydroxide) sprayed in alternation with Amistar Top, Luna Sensation or Headline. The final monthly application of fungicide has been completed. The fungicides are being tested in a ‘Valencia’ orange orchard with a history of CBS. The trial evaluation is planned for the end of August. Despite this, 23 isolates were sequenced (6 from South Africa and 17 from the USA) using the Ion Torrent System. The genomes of all the isolates have been successfully assembled and analysed using a customised bioinformatics pipeline. Previous genotypes obtained with SSR primers were confirmed and new SSR primers were developed in silico. To date, mapping and SNP variant statistics as well as in silico genotyping data revealed significantly less variation between the USA isolates than between the isolates from South Africa. To investigate the fine-scale genetic differences within the USA P. citricarpa population, the assembled genomes were annotated by mapping the reference genes to the assembled contig sets, using GMAP. The variant calling results together with the annotations were further analysed using SNPeff, to detect putative variable genes. In silico detection of mating types were also performed, and confirmed that only one mating type is present in the USA.Eight of the 12 South African isolates send for 200bp sequencing passed the quality control checks and were sequenced. Quality and completeness of the genome assemblies will be assessed, as well as number of SSRs that can be detected, to determine whether 200bp sequencing is a viable and more cost-effective sequencing approach. Our research facilities were closed from mid-March to end of May due to the Covid-19 pandemic, causing the delay in evaluating the 200bp sequencing. The isolates that failed quality control, as well as additional South African isolates, are in the process of being cultured and DNA will by re-extracted and sequenced. A manuscript was submitted describing the analysis and results from the USA isolates.In total, 11 South African isolates have been sequenced and analysed in the same manner as the USA isolates, to investigate the population structure of P. citricarpa in South Africa. Twelve more South African isolates have been cultured, DNA extracted, and are in the process of being sequenced. A more cost effective sequencing approach (200bp rather than 600bp sequencing) are currently investigated.Objective 3 (Survey for the MAT-1-1 mating type and two closely related Phyllosticta spp.). Studies on the diversity of Phyllosticta species in Florida is ongoing to determine which species (pathogenic and endophyte) are associated with citrus. High quality genomic DNA has been extracted and purified for fifty-nine of the 125 single-spored Phyllosticta isolates in our collection. Based on tef1 sequences, two isolates (Gc-6 and Gc-7) demonstrated polymorphism distinct from P. capitalensis and P. citricarpa. Greatest sequence identity was found with P. hymenocallidicola. Additional multi-locus phylogenetic analysis of ITS, LSU, and actA sequence of these two isolates was conducted. Analysese of these loci against a larger diversity of Phyllosticta species confirmed that the isolates Gc-6 and Gc-7 uniquely clade with P. hymenocallidicola. This finding strongly suggests that these two isolates from citrus are identical to one another and are P. hymenocallidicola. Collaboration with Dr. Hector Urbina and colleagues at the Division of Plant Industry is ongoing to assess the mating type of isolates collected at the expanding margins of the quarantine zone. Fifteen isolates from 2019 -2020 have been single spored and are being prepped for DNA extraction to confirm species identification and to determine mating type. Isolates from additional sites in Southwest Florida were collected and under going single spore isolates. To better understand the diversity of P. citricarpa in the region our partnership with Cuban researchers has been strengthened. Permits for the acquisition of genomic DNA from Cuban P. citricarpa isolates has been approved. We are, amidst current travel restriction, devising plans to have the DNA shipped from Cuba. DNA representing these isolates will be screened for mating type and used within a larger analysis of the global P. citricarpa population structure. Additional efforts have been focused on determining the role of fruit developmental etiology on susceptibility to CBS. Our established quarantine greenhouse experiment with fruit-bearing Myer lemon trees is ongoing. Eighty-six fruit of varying developmental stages, as well as controls, continue to be monitored following inoculation in December. Data is being collected on temperature, relative humidity and light intensity in addition to monitoring for symptom development to determine developmental and environmental parameters of symptom development. Little progress has been made other than isolate maintenance and monitoring for fruit symptoms since the last report due to COVID-19 restrictions. We hope to be able to accomplish more very soon. A poster of the phylogenetic results is being prepared for the virtual annual meeting of APS this August.