June 2017 The objectives of this proposal are 1) To determine the temperature and relative humidity optima for Guignardia citricarpa pycnidiospore infection and production on citrus twigs, leaf litter, and fruit; 2) To determine the relative potential of Guignardia citricarpa to form pycnidiospores on citrus twigs, leaf litter, and fruit; 3) To determine whether Guignardia citricarpa can survive and reproduce on citrus debris on grove equipment. During this time, the focus was on extraction of fungal DNA from plant tissues that had previous been enriched and then stored at -20�C. 1,056 extractions were carried out using the Qiagen MoBio PowerSoil kit. Another 576 samples were taken from the field biweekly, 384 of these samples were processed for incubation. After incubation 288 of these samples were excised of their bark and stored for the DNA extraction process. Salts have been selected and will be used in the future for the RH temperature treatments. Experiments were started to look at the effect of temperature on the level of sporulation of P. citricarpa. It can be quite difficult to get consistent sporulation even under controlled conditions. The temperatures that are being tested 15, 20, 24, 28, 32, and 36C. After incubation in complete darkness to avoid the confounding effects of light, it was found for 5 isolates that 24C was the best temperature for sporulation (P < 0.05) followed by 28C. The repetition of the experiment is not yet completed. Work on the effect of FDACS recommended disinfectants (200 ppm bleach or 2000 ppm quaternary ammonium) on conidia germination was conducted. Effective concentrations to inhibit either 50% or 90% of conidia germination for 2 quat products, Canker Solve and C-Quat, and bleach were found to be well below 5 ppm for all products. Bleach was about ten times more effective but is not as stable as quat. The disinfectants have been preliminarily evaluated in the presence of finely ground plant debris (twigs and leaves as would be found on mowers or hedgers). Citrus debris itself had no significant effect on conidia germination but there was a significant effect on the efficacy of the disinfectants. Disinfectant treatment in the presence of citrus tissue debris has a much lower efficacy than determined from previous experiments lacking citrus tissue debris. This loss of efficacy can be attributed to two factors. The first is a reduction in potency due to the presence of tissue debris within the liquid treatment. The second and more profound factor is the availability of disinfectant as a free liquid. Testing of quaternary ammonium was completed during this quarter. Results showed that at the lowest ratio of disinfectant to debris, which is 100�l of disinfectant, 500 ppm a.i. was required to reduce the percentage spore germination to zero. This is still well below the recommended rate of 2000 ppm. At the highest volume (1500 �l), only 20-50 ppm a.i. was required to kill 100% of the spores. In the absence of debris, only 20 ppm a.i. of quaternary ammonium was required to reduce spore germination to zero. Results of our work demonstrate that the presence of debris significantly reduces the efficacy of disinfectant, but this can be mitigated by using a large volume, relative to the amount of debris. Therefore, the results of this study illustrate that when decontaminating equipment with disinfectant, it should be applied to the point of run-off, rather than lightly sprayed on.