June 2019Objective 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. In the fungicide and skirting control trial, three applications of fungicides have been completed. They are applied approximately every 28 days. Skirting was planned for early June. We expect to continue with the applications through the summer and into the fall. The two potential groves for the fungicide spray trial were scouted for CBS incidence and symptom severities in in March 2019. The selected grovewas chosen for the trial based on the higher incidence of CBS. To keep treatments consistent, only mature trees were rated for CBS. Twenty-fve fruit per tree were rated and three trees were grouped based on the average rating values into a replication. There are five replications per treatment and each treatment consists of at least one replicate at each CBS incidence level. The trial is spread out over eight rows, each consisting of 117 trees, plus guard rows on both sides of the rows containing the treatments. A total of 10 treatments were selected and treatments are applied with a handgun at 200 psi. The first application was done over a two-day period starting on May 16th, this was a two-week delay from the original plan due to unforeseen grove activities. The second application was done over a two-day period beginning on June 3rd. The first two applications were completed as planned without any weather delays; although there was significant rainfall five hours after the last treatment of the second application. The third application is planned to begin on June 24th. Whilst the CRDF-funded project has already officially started, the sub-contract between UF and CRI must still be concluded. Funding has therefore not been allocated yet. Nonetheless, a postdoctoral researcher has been appointed from 1 March 2019, and preparations for genotyping-by-sequencing of a collection of Phyllosticta citricarpa isolates from USA has been initiated. Eighteen isolates (4 from South Africa and 14 from USA) were sequenced (600nt read length) using the Ion Torrent System. Thus far, the genomes of eight isolates ( 3 from SA and 5 from the USA) have been successfully assembled and analysed using a customised bioinformatics pipeline. The following available reference sequences were used: GC12: 5748 contigs (Florida, USA) and GCMC3: 6716 contigs (Zhejiang, China). The rest of the isolates will be sequenced before comparative analysis commences. Jeff Rollins has been able to secure the most of the necessary permits for him to be able to travel to Cuba and collect isolates to determine the mating type and species identifications. As the travel rules change based on Federal government policy, a few more details need to be worked out and final trip arrangement. We recieved MAT 1-1 DNA from South Africa to use as positive controls for our experiments. Trees are fruiting for some on tree experiments.