Consensus sequences were generated using BioEdit and submitted to BLASTn searches of the GenBank nucleotide database . The sensitivity of mycelial growth of P. cinnamomi to potassium phosphite was tested using an agar dilution method. Potassium phosphite was amended to 10% V8C agar to obtain final concentrations of 0 , 5, 25, 50, 100, 150, 300, or 600 µg/ml. Each triplicated petri dish for each concentration was inoculated in the center with a mycelial agar plug from 7- to 8-day-old cultures. After incubation in the dark at 22°C for 3 days, radial colony growth was measured. This experiment was done twice. Effective concentrations to inhibit 50% of mycelial growth were determined as described previously . The sensitivity of mycelial growth to ethaboxam, fluopicolide, mandipropamid, mefenoxam, and oxathiapiprolin was evaluated using the spiral gradient dilution method . Isolates were grown on sterile hydrophilic cellophane strips that were placed onto V8C agar. Mycelium-covered agar strips of one-week-old cultures were placed between the cellophane strips. Plates were then incubated in the dark at 22°C for 7 to 9 days until mycelia had grown evenly over the cellophane strips. Aqueous stock solutions of ethaboxam , fluopicolide , mandipropamid , mefenoxam , or oxathiapiprolin , flower bucket or water were plated onto 15-cm V8C agar plates using a spiral plater set at the exponential mode.

Mycelium-covered cellophane strips were radially placed across the fungicide concentration gradient on the SGD plates. For each isolate, two strips were placed on opposite sides of duplicated plates, and the experiment was repeated. SGD plates were incubated in the dark at 22°C for 2 days. The location where mycelial growth was inhibited by 50% was determined, and local fungicide concentrations were calculated using the SGE software .Six- to eight-month-old avocado seedlings and ten-month-old clonal rootstocks, Dusaâ and PS.54 , were obtained from local nurseries. Plants were transplanted into 19-liter pots using UC-A soil mix . The average air temperature was between 25 and 28°C, and the relative humidity was between 40 and 50% during the experiments in the greenhouse. Plants were fertilized once at the beginning of the studies and watered three times a week throughout the study. Plants were inoculated using P. cinnamomi-colonized millet seeds . For this, 175 ml sterile distilled water was added to 144 g of millet seeds in a 500-ml flask, and flasks were autoclaved for 30 min on two consecutive days. Ten plugs of a 4-day-old P. cinnamomi culture were added to each flask, and flasks were incubated in the dark at 25°C for 21 days. Equal proportions of seeds colonized by isolates 2109, 2117, or 2120 were mixed to prepare the final inoculum, and 4.4 g of inoculum was added to the soil around the base of the stem of each plant. Fungicides were applied as a soil drench one week after inoculation. Two rates of fluopicolide, mandipropamid, and oxathiapiprolin were applied to seedlings. Only the high rates of fluopicolide, mandipropamid, oxathiapiprolin, and one rate of ethaboxamwere applied to two rootstocks; and one rate of each mefenoxam and potassium phosphite was applied to seedlings and rootstocks .

Fungicide application rates used for citrus were used to calculate greenhouse rates for avocado based on the ratio of soil surface area of a tree in the field to a potted plant. Seedlings and rootstocks were arranged in a randomized complete block design with ten or six single-pot replicates, respectively. Fungicides were applied to the soil around the plant in each pot. Water was added to each pot immediately after application to move the fungicide into the soil. Inoculated plants treated with water were used as controls. The efficacy of fungicide treatments was evaluated based on PRR incidence and pathogen population sizes in rhizosphere soil 16 to 17 weeks after fungicide applications. Rhizosphere soil was collected, and the root ball of each plant was rinsed with water. Feeder roots were cut into 1-cm-long pieces using a sterilized razor blade, and 20 pieces were placed onto each of two plates of Phytophthora-selective medium PARHFB-V8C . When present, root pieces with discoloration were selected. Plates were incubated at 22°C for 2 to 3 days in the dark. P. cinnamomi colonies were identified by the distinctive coralloid-type mycelium with abundant hyphal swellings , and representative colonies were sub-cultured and verified for species identity using species-specific TaqMan qPCR . PRR incidence was calculated as the percentage of P. cinnamomi infected root pieces of the total pieces plated. For enumeration of soil populations, 10 g of rhizosphere soil from each plant was mixed with 90 ml sterile distilled water in a 250-ml Erlenmeyer flask containing three stainless steel beads on a rotary shaker at 150 rpm for 30 min. Aliquots of 1 ml soil suspension were spread onto two plates of PARHFB-V8C agar per plant. Plates were rinsed with water after 24 h at 22°C in the dark, incubated for another 2 to 3 days in the dark, and P. cinnamomi colonies were enumerated. Phytophthora propagule populations were calculated as CFU per gram of soil. All fungicide treatments evaluated significantly reduced PRR incidence and P. cinnamomi propagule populations in rhizosphere soil compared with the untreated infected control .

Oxathiapiprolin at the high rate of 0.028 g/pot resulted in significantly the lowest incidence of PRR with a 95% reduction from the untreated control where 82% of the plated root pieces were found to be colonized by P. cinnamomi . The lower rate of this fungicide was also very effective and was statistically similar to either rate of fluopicolide. The low rate of fluopicolide performed similar to either rate of mandipropamid, whereas mefenoxam and potassium phosphite were the least effective with reductions in PRR incidence from the control of 51 and 43%, respectively. Oxathiapiprolin at either rate most effectively reduced the number of viable P. cinnamomi propagules in the soil compared with all other treatments . All other treatments significantly reduced pathogen populations as compared with the control, and there were no significant differences among these latter fungicides . Variances of shoot and root dry weights were not homogenous between experiments according to Bartlett’s test , thus, data are presented for each experiment . Similar trends were observed for most of the treatments in the two experiments. In both studies, plastic flower bucket all treatments significantly increased shoot dry weight as compared with the untreated control. Oxathiapiprolin at both rates and fluopicolide at the high rate in both experiments, and mandipropamid at the high rate in the second experiment had significantly the highest shoot growth. Shoot dry weight of these treatments was increased between 192% and 265.5% as compared with the untreated control. Root dry weight of inoculated plants was highest after using oxathiapiprolin at either rate or fluopicolide at the high rate in both experiments, mandipropamid at the high rate in the second experiment, or fluopicolide at the low rate in the first experiment. Increases as compared to the control ranged from 192.8% to 306.5% . Root dry weight was not significantly different as compared with the control after potassium phosphite treatment in the first experiment. Only the higher of the two concentrations of fluopicolide, mandipropamid, and oxathiapiprolin from the seedling studies, and ethaboxam were evaluated in experiments with clonal rootstocks and compared with mefenoxam and potassium phosphite. Untreated plants of both rootstocks developed a similar incidence of PRR . All treatments significantly reduced the incidence of PRR compared with the untreated controls on both rootstocks . On Dusaâ, a significant difference among treatments was only observed between oxathiapiprolin with 2.1% incidence and potassium phosphite with 16.3% incidence. The other fungicides were all intermediate in efficacy. On PS.54, oxathiapiprolin resulted in the lowest incidence of PRR , and this was statistically similar to fluopicolide . Ethaboxam at the rate used was the least effective of the fungicides with 51.7% incidence. Pathogen propagules were effectively reduced by all treatments on the Dusa rootstock with no significant difference among treatments . Still, mandipropamid, oxathiapiprolin, mefenoxam, and potassium phosphite reduced soil populations to zero levels. In contrast, on the PS.54 rootstock, only oxathiapiprolin significantly reduced P. cinnamomi populations in the soil . In this study, the four new Oomycota-targeting fungicides ethaboxam, fluopicolide, mandipropamid, and oxathiapiprolin demonstrated high in vitro toxicity with relatively low mean EC50 values to the avocado root rot pathogen P. cinnamomi. The in vitro sensitivities for each of these compounds displayed a unimodal distribution and a narrow range of EC50 values for mycelial growth inhibition of 71 isolates representing the current P. cinnamomi population in major avocado growing areas in California. The narrow ranges in sensitivities among isolates with no distinct less sensitive outliers in the distribution may suggest a reduced potential for selection of resistance with the proper use of these fungicides.

Because P. cinnamomi isolates were never previously exposed to ethaboxam, fluopicolide, mandipropamid, and oxathiapiprolin, the sensitivity ranges reported herein can be referred to as baseline distributions that can be used as references in future monitoring for fungicide resistance in populations of the pathogen.In our study, oxathiapiprolin had the lowest EC50 values for all isolates among the new fungicides evaluated ranging from 0.0002 to 0.0007 µg/ml. This fungicide also was shown to be highly inhibitory to other Phytophthora spp. from a wide range of hosts by others with mean EC50 values of less than 0.001 µg/ml . Similarly, Gray et al. found that oxathiapiprolin had the lowest range of EC50 values of 0.0002 to 0.0015, 0.0002 to 0.0003, 0.0003 to 0.001, and <0.0003 µg/ml for P. citrophthora, P. syringae, P. nicotianae, and P. hibernalis, respectively, as compared with the other three compounds. Together, reported inhibitory values for oxathiapiprolin are generally 10- to 1000-fold lower than those for ethaboxam, fluopicolide, mandipropamid, and mefenoxam, depending on the fungicide-species combination. Thus, the in vitro toxicity of oxathiapiprolin to P. cinnamomi from avocado reported in our study is lower than for any previous fungicide evaluated against this pathogen. EC50 values for fluopicolide, mandipropamid, and ethaboxam for P. cinnamomi in our study were also within the range of values previously determined for several other Phytophthora spp. . The range of EC50 values for mefenoxam in our study was similar to that previously reported for P. cinnamomi from avocado , Fraser fir , and woody ornamentals in the United States. Thus, the current usage pattern for this fungicide to control avocado PRR in California nurseries and orchards has not resulted in mefenoxam resistance in P. cinnamomi populations. In contrast to the other fungicides, a wide range of in vitro sensitivities was detected for potassium phosphite, and there was a significant difference in mean EC50 values between isolates from the two geographical regions, confirming a previous report . The higher value for isolates from southern California production areas may be due to higher field rates or more frequent applications of potassium phosphite to manage PRR in avocado orchards. The bimodal distribution for the 71 isolates in this study separates the current pathogen population into two sensitivity groups indicating a shift in population sensitivity. A baseline for this compound, however, was never established before commercial field usage. Still, prolonged use of phosphite caused a shift toward reduced sensitivity of P. cinnamomi isolates from avocado orchards in Australia and South Africa . Phosphonate resistance has also been reported for P. cinnamomi from Chamaecyparis lawsoniana in nurseries , downy mildew of lettuce , and recently in P. citrophthora, P. nicotianae, and P. syringae from citrus in California . With direct and indirect effects on the pathogen, the resistance potential of potassium phosphite is considered relatively low . The extensive and often sole use of this FRAC group in California avocado orchards to combat PRR , however, is expected to eventually lead to resistance. In our greenhouse studies, avocado seedlings and rootstocks were inoculated with P. cinnamomi isolates from southern avocado production areas that have been described as more virulent . A high incidence of PRR developed on untreated control plants of seedlings and both rootstocks with more than 75% of plated root pieces colonized by the pathogen. The high incidence on the Dusa rootstock that is considered more tolerant to PRR is likely due to our selection of discolored root pieces for plating of all samples.