Trees were inoculated in July 2016 and October 2016. P. citrophthora-colonized vermiculite-oat seed inoculum was prepared as described by Matheron and Mircetich with minor modifications. In brief, a volume ratio of 2:4:3 of oat seeds, vermiculite, and 10% regular V8 broth was mixed, 600 ml of the mixture was filled into 1-liter plastic containers, and containers were autoclaved for 45 min each on four consecutive days. Each container was inoculated with ten 10% V8C agar plugs of P. citrophthora cultures , mixed by shaking, and incubated at 25°C for 4 to 6 weeks with weekly shaking and mixing of the contents. Inoculum of the two isolates was mixed in a 1:1 ratio, and 25 ml was buried 10 cm deep at each of two locations at the base of each tree around the root ball. A randomized complete block design with six single-tree replications was used for each fungicide treatment. Fungicides were applied in July 2016 and in May 2017. Fungicides and rates used are listed in Table 2.2, and fungicides were applied as described above. Four replicate trees were treated with water per block and used as controls.Phytophthora root rot incidence and Phytophthora propagule populations in rhizosphere soil were evaluated to assess the efficacy of treatments. In the P. nicotianae trial, feeder roots and rhizosphere soil were sampled in early June and early September 2014 before the second and third fungicide applications, respectively, and in late June 2015 after the last application. Samples were taken from the middle two trees of each four-tree replication and were combined. In the P. citrophthora trial, roots and soil were sampled in December 2016 and July 2017 from each tree. Samples were collected at the dripline of each tree at a depth of ≤ 20 cm, placed in plastic bags, and processed the same day. Feeder roots were carefully separated from soil, rinsed three times with deionized water, and dried on paper towels.

Roots were cut into 1-cm-long sections using a sterilized razor blade, and 20 pieces were plated onto each of two plates of selective Phytophthora isolation medium. When present,cultivo de frambuesas en maceta root pieces with visible lesions were selected. Plates were incubated at 25°C in the dark for 2 to 3 days. P. nicotianae colonies were identified by their typical growth pattern , and representative colonies were sub-cultured and verified for species identity using species-specific TaqMan qPCR. Phytophthora root rot incidence was calculated as the percentage of infected root pieces of 20 pieces plated per plate. Rhizosphere soil was mixed well, and for every treatment replicate, a 10-g aliquot was mixed with 90 ml SDW in a 250 ml flask containing three stainless steel beads on a rotary shaker at 150 rpm for 40 min. Aliquots of 1 ml soil suspension were plated on triplicate plates of PARHFB-V8C medium using a sterilized glass spreader. Plates were rinsed with deionized water after 24 h at 25°C in the dark to remove excess soil, and then further incubated for 1 to 2 days. The number of P. nicotianae colonies on each plate was assessed, and propagule populations in the rhizosphere were calculated as CFU per gram of soil. Tree trunk diameter, canopy size, and fruit production were determined to further evaluate the effectiveness of fungicide treatments in the first field trial. Trunk diameter of every tree at 10 cm above the graft union was measured in February, July, and December 2015 using a caliper. Tree canopy size was measured in April 2015 and 2016 for one representative tree with a visually average canopy size for the four trees per replication. For this, a digital image of each entire canopy was obtained with a blue tarp as background. Pictures were taken at approximately the same distance from each tree, and the tarp was also kept at the same distance. This was done under overcast conditions to minimize shadow effects on tree canopy estimation. Images were analyzed using Assess 1.0: Image Analysis Software for Plant Disease Quantification to calculate the two-dimensional tree canopy. The percentage of tree canopy area in the blue tarp area was computed by defining the two areas by their distinct colors in the software, and the canopy area of each tree was calculated based on the tarp area of 5.96 m2. 

Mature, commercial-grade orange fruit were harvested in December 2016 and 2017 and the number and weight of fruit per tree were determined.Plants were grown from seed in 1-liter pots in UC-C soil mix at 18 to 35°C in the greenhouse, watered using micro drippers, and fertilized with Osmocote® 14-14-14 Slow Release Fertilizer once at transplanting. Six- to nine-month-old plants were inoculated with a chlamydospore suspension of P. nicotianaethat was produced as described above. One milliliter suspension was added into each of four holes around each plant resulting in an equivalent of approximately 20 chlamydospores/g soil. Treatments were applied one week after inoculation. For potassium phosphite, ProPhyt was used. Greenhouse application rates were proportionally reduced from the respective field rates based on the ratio of the average surface of tree basins in the field to the soil surface of a potted plant. A randomized complete block design with four single-pot replications was used for each treatment. Fungicides were applied as aqueous suspensions to the soil around each plant. Plants treated with 50 ml of water were used as controls. The effectiveness of fungicides was evaluated after 6 to 7 months. For each plant, the root ball with soil was carefully removed from the pot. Soil adjacent to roots was collected,maceta larga and root balls were rinsed with water. Soil and feeder roots were plated on duplicate plates of PARHFB-V8C medium as described above for field studies to assess root rot incidence and P. nicotianae propagule soil populations. This experiment was done twice. In another experiment, 10- to 12-month-old plants were inoculated with P. citrophthorathat was grown on long-grain white rice for 4 weeks. For inoculation, two colonized rice grains were buried 3 cm deep at each of four locations around each plant. Fungicides were applied as described above after one week.Plants treated with water were used as controls. After 4 to 5 months, the efficacy of fungicides was evaluated as described above. This experiment was repeated.For field trials, data for Phytophthora root rot incidence and Phytophthora propagule populations in soil, tree trunk diameters, tree canopy sizes, and fruit production at different evaluation times were subjected to a repeated measures univariate analysis of variance to determine the effect of fungicide treatments over the trial periods. Mauchly’s test for sphericity was performed to test the equality of variance of the differences between all combinations of factors at each measurement time when more than two repeated measurements were conducted. When sphericity was violated , valid P values were obtained using the Greenhouse-Geisser correction. Differences between means of treatments at each measurement time were analyzed using Fisher’s least significant difference test, and differences between the means of the control and fungicide treatments were analyzed using pairwise t-tests.

For repeated greenhouse experiments, the homogeneity of variances for Phytophthora root rot incidence and Phytophthora propagule populations in soil were tested using Bartlett’s test of homogeneity. Homogeneous data were combined and analyzed using ANOVA, and the differences between means of treatments were compared using a pairwise t-test and Fisher’s LSD test. All statistical analyses were performed in R using the agricolae and car packages. Results were considered significant at P ≤ 0.05.Variances of data from repeated experiments were homogeneous and therefore, results presented are the mean of two experiments. Similar to the field studies, ethaboxam, fluopicolide, mandipropamid, and oxathiapiprolin effectively reduced the incidence of root rot and soil population sizes of P. nicotianaeto zero or near zero levels. There was no significant difference in efficacy between the two rates of each fungicide evaluated. Mefenoxam, was significantly less effective in reducing soil populations than the other fungicides, but populations were still significantly lower as compared with the control. Potassium phosphite was also highly effective using the label rate for nursery use and significantly reduced root rot incidence and P. nicotianae propagules in the soil. No phytotoxicity was observed on plants with any of the treatments at the rates used, but higher rates of mefenoxam and potassium phosphite used in other studies caused stunting and dieback. In another greenhouse study, orange plants were inoculated with P. citrophthora after soil treatment. Applications with fungicides were done at the lower of the two rates used in the greenhouse study above with P. nicotianae. Although disease incidence in the untreated control was low, significant reductions were obtained using ethaboxam, fluopicolide, mandipropamid, or oxathiapiprolin, and root rot incidence and soil populations were reduced to zero or very low levels. Mefenoxam and potassium phosphite were effective against root rot and showed moderate activity in reducing pathogen soil populations. This is the first study evaluating the new Oomycota fungicides ethaboxam, fluopicolide, mandipropamid, and oxathiapiprolin for managing Phytophthora root rot of citrus. Among soil-borne diseases of citrus, Phytophthora root rot is the most serious one, occurring in most growing regions worldwide, whereas others including Armillaria root rot , dry root rot , and Rosellinia root rot are of only localized importance. Therefore, effective management strategies for Phytophthora root rot need to be available, also in light of increasing restrictions on the use of soil fumigants for preparation of new planting sites.

Moreover, there is some evidence for a positive interaction between infection levels by huanglongbing and Phytophthora root rot and for an enhancement of HLB-induced symptoms in P. nicotianae-infected citrus plants. This further stresses the significance of our study. In comparative studies with the current commercial standards mefenoxam and potassium phosphite, we demonstrated the superior effectiveness of ethaboxam,fluopicolide, mandipropamid, and oxathiapiprolin in reducing Phytophthora spp. soil populations and root rot in field and greenhouse studies as well as increasing crop yield. Our field studies established fungicide effectiveness under California orchard conditions using a common commercial irrigation system that exposes the tree rhizosphere to regular wetness conditions and potential infection periods for the root rot pathogens. In the field study with P. citrophthora, we identified lower highly effective rates for mandipropamid and oxathiapiprolin when results were compared with the first field study on root rot caused by P. nicotianae that used rates originally recommended by the respective registrants. Fungicide rate calculations for plants in the greenhouse were based on pot surface area in comparison with tree trunk basin area in the field. Although the greenhouse-applied rates may still not be equivalent to labeled field rates, these studies demonstrated post-infection activity of the fungicides. This is important because the disease in a newly planted field may be initiated from infected nursery stock. The high efficacy of ethaboxam, fluopicolide, mandipropamid, and oxathiapiprolin in reducing disease and Phytophthora soil populations correlated with their low in vitro effective concentration values against mycelial growth of several Phytophthora species from citrus that we determined previously. The isolates of P. nicotianae and P. citrophthora in the current study were used as representatives of each species with EC50 values for each fungicide within the baseline range of sensitivity. In the Gray et al. study, values for oxathiapiprolin were the lowest among the four compounds for inhibition of five Phytophthora life stages including mycelial growth, sporangium formation, zoospore cyst germination, as well as chlamydospore and oospore formation. This high toxicity was reflected by low effective field rates that we identified in the field study with P. citrophthora. Ethaboxam , fluopicolide and mandipropamid , and oxathiapiprolin were previously shown to provide a high level of control for a range of foliar and root diseases of field and vegetable crops caused by species of Phytophthora. However, our study is the first validation of the effective use of the new compounds on a perennial tree crop, and the results presented are facilitating their registration on citrus. We conducted our studies using orange trees inoculated with P. nicotianae or P. citrophthora, the main Phytophthora root rot pathogens in California. Additional species are major causal agents in other citrus growing areas, for example P. palmivora in Florida , and further studies using these species may be warranted.