We continued with work on Objectives 1 and 2, determining the effect of CLas infection on nematode development/populations, and vice versa. The objectives are reiterated for convenience below:1) Determine the effect of CLas infection on subsequent development of citrus parasitic nematodes and their damage to the plants. Hypothesis 1a: CLas alters nematode development and population growth. Hypothesis 1b: Concomitant nematode and CLas infection results in synergistic damage to citrus trees.2) Determine the subsequent effect of citrus parasitic nematodes on CLas infection. Plants will be first attacked by nematodes for 3 months and then graft-inoculated with CLas. Hypothesis 2: CLas symptoms develop more rapidly in nematode damaged plants.Progress on objectivesSince the last report, we sampled leaves from the primary (Exp. 1) study trees (100 trees each Val/SW, 100 trees Val/CZO) inoculated with citrus nematode and HLB to study the interactions between the two pathogens. After testing for HLB by PCR, we found that the infection rate was low (between 5 and 20%), but it is early (6 months), and we anticipate that the rate will be higher at the next check (9 months post-inoculation). Trees that were inoculated with citrus nematodes in late 2019 and early 2020 were check for nematode infection rate in early June. Six, nematode-infected plants were chosen randomly among the treatments and two small soil cores (1.5 cm dia. x 12 cm deep) were taken and combined from each pot. Nematodes were separated from soil in the samples using a sucrose centrifugation technique and counted. The mean number (and standard error) of citrus nematodes per 100 cm3 soil was 681 (175), a level comparable to that of well-infested groves during the summer months. Soil pH of the samples was 7.1, an optimum level for citrus nematode infection. In the upcoming months we plan to begin assessing and comparing root damage among the five treatments, as well as enumerate the nematode populations.In addition to the primary experiment, we added two new experiments:Burrowing nematode (Experiment 2)We initiated a new experiment with 30 trees of Valencia on Kuharske rootstock (nematode tolerant), and 30 trees of Val/Carrizo rootstock (nematode susceptible). 15 of each rootstock were graft-inoculated with HLB+ budwood. All of these trees were inoculated with burrowing nematode (750 nematodes of all life stages per plant) in 10 ml water divided between two holes in soil adjacent to each plant.Rootstock evaluations (Experiment 3)A third experiment was initiated to evaluate rootstocks for their tolerance to burrowing nematode. We planted seedlings of 7 new rootstocks in a soil bed, and inoculated it with the burrowing nematodes. The new rootstocks include: UFR-1, UFR-2, UFR-4, UFR-5, UFR-6, UFR-15 and UFR-17, five replicates each. These rootstocks will be sampled periodically for root damage and nematode infection rate to evaluate their susceptibility to burrowing nematode.