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.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, 10 liter drainage pot 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 Dusaâ rootstock that is considered more tolerant to PRR is likely due to our selection of discolored root pieces for plating of all samples. The four new fungicides were moderately to highly effective in reducing PRR and P. cinnamomi populations in rhizosphere soil of the avocado seedlings and rootstocks used. Overall, oxathiapiprolin was the most effective among fungicides evaluated. In experiments with Zutano seedlings, the efficacy of oxathiapiprolin at the low rate of 70 g/Ha was 2- to 33-times higher than that of the other fungicides and 2- to 4-times higher than that of mandipropamid, a CAA fungicide. In a study on managing P. capsici on peppers , the difference in effectiveness of oxathiapiprolin at 30 g/Ha as compared to the CAA dimethomorph at 262.5 g/Ha was similar to our study using the same FRAC codes of fungicides. In response to reducing PRR, avocado plants treated with oxathiapiprolin generally developed more shoot and root growth as compared with untreated plants. On the avocado seedlings and rootstocks used, fluopicolide, mandipropamid, and ethaboxam treatments also effectively reduced the incidence of PRR compared with the control. P. cinnamomi propagules in the rhizosphere soil were only significantly reduced on the Zutano seedlings and the Dusa rootstock. These latter treatments were often significantly more effective than potassium phosphite or mefenoxam; whereas fluopicolide often performed statistically similar to oxathiapiprolin. Still, the efficacy of potassium phosphite was demonstrated with significant reductions in PRR on the seedlings and rootstocks although its overall performance may have been compromisedby the use of three P. cinnamomi isolates with reduced sensitivities to the fungicide in our soil inoculations. These results also could explain why potassium phosphite is still effectively used in managing PRR in California since many growers cultivate avocado trees grafted on the Dusaâ rootstock. Thus, highly effective alternatives to mefenoxam and the phosphonates were identified by us for the management of avocado PRR. Oxathiapiprolin, fluopicolide, mandipropamid, and ethaboxam previously demonstrated high efficacy against selected foliar and root diseases of vegetable and tree crops caused by Oomycota organisms in greenhouse and field studies. Thus, the four fungicides were highly efficacious in reducing Phytophthora root rot of citrus caused by P. nicotianae and P. citrophthora . Oxathiapiprolin, fluopicolide, and mandipropamid were more effective in managing P. capsici on watermelon than mefenoxam or potassium phosphite . In other studies, oxathiapiprolin was shown to be highly effective in managing diseases of vegetable crops caused by Phytophthora species including P. capsici and P. infestans and controlled black shank of tobacco caused by P. nicotianae . Ethaboxam was shown to be an effective treatment for tomato late blight , as well as Phytophthora blight of pepper .

Based on our studies, 25 liter pot registration of oxathiapiprolin for use on avocado has been initiated through the Inter-regional Research Project No. 4 , and ethaboxam,fluopicolide, and mandipropamid are proposed for further development on avocado. Additional evaluations will have to be done under field conditions using rootstocks with different growth characteristics and susceptibilities to PRR. The availability of fungicides with new modes of action and options for rotation and mixture programs using previously registered and new fungicides will help reduce the risk of development and spread of resistance in P. cinnamomi populations in California avocado production. Growers currently rely heavily on the use of phosphonate-based fungicides, and as we demonstrated, pathogen populations are shifting towards reduced sensitivity to this fungicide class. Thus, there is an urgent need to register fungicides with new modes of action. In our greenhouse studies, overall treatment efficacy in reducing PRR and soil inoculum levels of the pathogen on the susceptible PS.54 was reduced as compared with the more tolerant Dusaâ rootstock, indicating additive effects of fungicide use and rootstock selection. In an integrated approach for a durable and effective management of PRR that allows the continued economical production of avocados in P. cinnamomi infested soils, the use of tolerant rootstocks is critical along with irrigation management and cultural practices such as using mulching and planting in areas with good soil drainage.Plant pathogenic oomycetes fall into two general categories when it comes to pathogenicity. There are Phytophthora species that can infect only one, or a few different hosts like Phytophthora infestans de Bary, and then there are species that can infect hundreds or even thousands of different plant species such as P. cinnamomi Rands . P. cinnamomi is of particular interest in California because it causes Phytophthora root rot of avocado, in fact, PRR is the most destructive disease of avocado production worldwide . PRR limits production of avocado by killing feeder roots which reduces fruit yield and can cause tree death . P. cinnamomi impacts other fruit crops such as peach, pineapple, and highbush blueberry, as well as affecting natural stands of eucalyptus, pine, and oak . Areas that have become infested with P. cinnamomi will never completely remove this pathogen from the soil. Current chemical treatments are being challenged by the emergence of isolates that are more virulent and less sensitive to potassium phosphite . The current challenges of PRR treatment of avocado necessitates a better understanding of the molecular and genetic basis of plant-P. cinnamomi interactions. Taking advantage of the wide host range of P. cinnamomi, we developed a detached leaf assay in Nicotiana benthamiana to elucidate the molecular and genetic basis of plant immunity against P. cinnamomi . The hemibiotrophic lifestyle of P. cinnamomi was confirmed in this model system through differential staining and quantitative PCR pathogen DNA quantification. The model plant, N. benthamiana , has been widely used to study the pathogenicity and virulence of similar broad range and root Phytophthora pathogens such as P. capsici , P. palmivora , and P. parasitica . Furthermore, several studies using model plants, crops, and tree crops to study pathogenicity, virulence, and fungicide efficacy of root rot pathogens such as P. sojae, P. capsici, P. parasitica, P. palmivora, P. cinnamomi, and P. ramorum have been performed using detached-leaf assays . Using the tools developed in previous studies and combining them with RNAseq analysis as well as functional assays using this model plant it becomes possible to gain a better understanding of plant defense responses against P. cinnamomi infection. Previous transcriptomic studies on avocado and model systems provides important information on plant gene expression in response to infection by P. cinnamomi. Avocado defense gene expression has been analyzed three separate times over the last eight years . Mahomed and Van den Berg used the tolerant avocado rootstock Dusaâ to study the gene expression changes after P. cinnamomi inoculation. By comparing expressed sequence tags and 454 pyrosequencing they were able to identify six defense related genes. The defense genes identified encoded: cytochrome P450-like TBP , thaumatin, PR10 , metallothionein-like protein, MLO transmembrane protein encoding gene, and a gene encoding a universal stress protein . In a follow up study, again on the resistant avocado rootstock Dusaâ , 16 additional defense genes encoding: WRKY transcription factors, phenylalanine ammonia-lyase , beta-glucanase, allene oxide synthase, allene oxide cyclase, oxophytodienoate reductase, 3-ketoacyl CoA thiolase, Fbox proteins, ethylene biosynthesis, isoflavone reductase, glutathione s-transferase, cinnamyl alcohol dehydrogenase, cinnamoyl-CoA reductase, cysteine synthase, quinone reductase, and NPR1 were differentially expressed after P. cinnamomi infection. Reeksting et al. found up-regulated transcripts corresponding to cytochrome P450, a germin-like protein , and chitinase genes after P. cinnamomi infection using microarray technology. It has been stated , that an important difference between gene expression in avocado and model systems is that the salicylic acid response is only seen in infected avocado, which is associated with a defense response to biotrophic and hemibiotrophic pathogens. It has been further asserted that P. cinnamomi infection of model plants initiates the jasmonic acid and ethylene pathways associated with necrotrophic pathogens. Although there are differences between expression patterns in avocado and the numerous model plants that have been studied to better understand plant defense to P. cinnamomi, there are also many similarities.