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 only very preliminarly analysis has 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). Applications have been made on time in the spray trial We were unable to set up the second planned fungicide trial this year because of the COVID-19 shut down. It occured just as the pre-treatment data collection should have done but we plan to conduct the trial next year if a no cost extension is granted. The trial in which different fungicides were tested for their efficacy to protect Valencia orange fruit from CBS infection was evaluated in August 2020. All the fungicides tested were effective in protecting fruit as they achieved more than 97% CBS free fruit, except for Luna Sensation (applied 6x on its own), which achieved 67.5% clean fruit. More than 99% CBS free fruit were yielded by applying Enable (Indar) 6 times on its own and also applying Amistar Top in alternation with copper hydroxide. The highest percentage (100%) of CBS free fruit was achieved with standard program consisting of copper oxychloride followed by the application of two sprays of azoxystrobin + copper oxychloride + mineral oil and lastly copper oxychloride. Trees that were sprayed with copper hydroxide in alternation with Cabrio (Headline) yielded 97.3% CBS fruit. The trial site was, however, characterised by a low incidence of citrus black spot during the 2019-20 season with the untreated trees yielding 60.2% fruit without CBS lesions. With the exception of programs alternating copper hydroxide with either Cabrio (92% fruit showing no phytotoxicity) or Amistar Top (97.8% fruit showing no phytotoxicity), the experimental fungicides did not produce any phytotoxicity on fruit. Due to the unavailability in South Africa, Miravis, Miravis Top, PhD, Priaxor and Luna Experience were not tested in this trial. Twenty-three 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.A manuscript was submitted describing the analysis and results from the USA isolates and we are still waiting for a decision by the journal of Molecular Plant Pathology.Eight of the 12 South African isolates sent 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. NGS data from the eight samples were received and are in the process of being analyzed. Eight more samples were submitted for NGS. 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.). A study on the diversity of Phyllosticta species is ongoing to determine which species (pathogenic and endophyte) are associated with citrus in Florida. Currently, fifty-nine isolates have been extracted and high-quality DNA purified. Based on tef1, ITS and actA sequences, two isolates (Gc-6 and Gc-7) demonstrated polymorphism with P. capitalensis and P. citricarpa, and the highest sequence identity was found with P. hymenocallidicola. This finding strongly suggests that the two isolates are identical to P. hymenocallidicola. Additional multi-locus phylogenetic analysis of GPDH sequence of these two isolates is underway to support our preliminary result and reject any possibility of misidentification. Sexual and vegetative compatibility test of isolate Gc-6 and Gc-7 is underway to determine if both isolates are the same clone or not. Moreover, isolates Gc-6 and Gc-7 failed to produce symptoms in citrus when tested on lemons in quarantine. Leaf inoculation of Amaryllis species is underway for a pathogenicity test of both isolates. Hymenocallis littoralis will be tested as well since P. hymenocallidicola was originally described from this host. To obtain robust information on the diversity of species of Phyllosticta in Florida. Further Phyllosticta isolates are being prepared for screening using tef1 primers. To date, no P. paracitricarpa or P. paracapitalensis have been identified but using the tef1 primers will identify any isolates that maybe part of our remaining collection. Out of 88 samples, 63 isolates are from our Phyllosticta collection, 14 isolates were isolated from fruit lesions collected from different locations under quarantine in Florida (samples provided by Dr. Hector Urbina from the Division of Plant Industry), and 11 isolates isolated from fruit lesions collected in groves, in the La Belle area on the leading northern edge of the spring 2020 quarantine zones. 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. The final data are expected to be collected in October. Data are 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. A poster of the phylogenetic results was prepared and presented at the virtual annual meeting of APS in August.