April 20241. Please state project objectives and what work was done this quarter to address them:The objectives are to 2) determine if the flush cycle and infection period for Z. citri-griseum have changed due to the influence of HLB on citrus physiology or changing environmental factors; 4) evaluate the potential promotion of greasy-green symptoms related to nutrition programs or to peel reactions like a chemical burn from different pesticide and combinations of pesticide tank mixes; and 5) evaluate if postharvest degreening treatments might be modified to adequately remove the green coloration while mitigating poor shelf life from anticipated longer degreening times. Objective 2: To determine whether the flush cycle and infection period for Zasmidium citri-griseum have changed due to the influence of HLB on citrus physiology and other factors such as the changing environment, a site located in Fort Pierce was selected based on feedback from growers. Last year, two blocks with different grapefruit varieties were selected at the site for monitoring. Within each block, two groups of twenty mature grapefruit trees with similar canopy health status were selected and ten flush per tree was tagged. The same blocks were used for our second year of the field trial, which has been layed out. Fruit and leaf assessments and collection for epiphytic growth continued until early November when the fruit were harvested. The data are being entered and evaluated but the flush and fruit growth trends mirrored the previous season so we will not need a third year of citrus growth data. We are working to match the growth data with environmental data. Evaluation of epiphytic growth on fruit and leaves is still ongoing. Previously, we had subsampled the leaf and fruit samples to understand the trends and we are working on examining all the leaf disks to ensure our conclusions from the subsamples were correct. For the epiphytic growth on fruit, the results suggest that epiphytic growth started in September on both white and red grapefruit which was unexpected. We had hoped to collect earlier samples in 2023, but the grower hedged severely in the block, removing the majority of fruit. We decided to sample again starting in September because we would otherwise run out of fruit to sample. Initial results show spores on the fruit in 2023 but no epiphytic growth until part way through September in a similar fashion to 2022. In 2022, the epiphytic growth stayed at a about 10% surface coverage, once we were able to detect it in September to the end of the season at harvest. When we evaluated the leaves collected in 2022 for density of epiphytic growth, we found tht there was denser mycelial growth on the adaxial side than the abaxial side which is different from what was observed in previous studies and is not where the majority of stomates are located. The growth started in early June and accelerated by the beginning of July with leaf disks showing nearly 50% coverage by mycelium on the adaxial side. The growth remained present on the leaves until we finished sampling in December 2022. We have the beginning of 2023 epiphytic data on the leaves. We started sampling the epiphytic growth in April rather than June. In this early season, there was epiphytic growth observed in the early season, but at much lower density, mostly scattered hyphae. Again the trend of more epiphytic growth on the adaxial rather than abaxial surfaces was observed. We will continue to record the data for the remaining 2023 season. When the trees were sampled, there were very few symptomatic leaves present in the grove in both seasons, however greasy green symptoms were present on the fruit. We performed DNA sanger sequencing of the transcription elongation factor (TEF1), long ribosomal subunit (LSU), and the internal transcribed spacer (ITS) regions for a set of putative fungal isolates collected in Florida from leaf litter from grapefruit trees exhibiting greasy spot symptoms. We successfully identified Zasmidum citri-griseum with a high sequence identity percentage (>95%) from a blast search against the NR database at NCBI. From the blast results, we also discarded a few isolates (6 in total) that corresponded to Cercospora loranthi co-occurring fungus which is not considered a citrus pathogen (Huang et al., 2015). Objective 4: We are also trying to meet with growers to compare programs from greasy-green affected and non- or less-affected blocks. The response is that there is not much difference between blocks. Some have indicated a willing to meet, but the actual dates for those meetings are still pending.The Co-PI has indicated that it has been very challenging to get grower participation, which is unfortunate. The responses would be very helpful in pinpointing what has caused the greasy green challenges. Objective 5: Fruit were collected for a second season of degreening trials. They were completed and data analysis should be completed soon. 2. Please state what work is anticipated for next quarter: Continued observation of the epiphytic growth on leaves and fruit. Work on data analysis. Primer design. Collect new fruit for epiphytic growth observations, particularly earlier in the season. Data analysis of post-harvest experiments. 3. Please state budget status (underspend or overspend, and why): No over or underspend on budget currently 4. Please show all potential commercialization products resulting from this research, and the status of each: None at this date.