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 Dusaâ 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 compromised by 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, flower buckets wholesale highly effective alternatives to mefenoxam and the phosphonates were identified by us for the management of avocado PRR.

Oxathiapiprolin used at low rates provided similar or better efficacy than the other fungicides. 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, 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 Dusa 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. cinnamomiinfested 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.

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 high bush 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 Dusa 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 , flower harvest buckets 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. Model plants used to better understand plant defense gene response to P. cinnamomi infection include; Zea mays, Arabidopsis thaliana, Lupinus angustifolius, Castanea sativa , Eucalyptus nitens, Lomandra longifolia, and most recently N. benthamiana . The gene expression in susceptible model hosts such as L. angustifolius and N. benthamiana can be compared to tolerant hosts like A. thaliana and L. longifolia to identify differences that may be associated with resistance to P. cinnamomi. Santos et al compared the gene expression between a susceptible and resistant variety of chestnut. They found that genes encoding for proteins involved in pathogen recognition proteins , were significantly upregulated in the resistant variety especially before inoculation. Six out of eight defense related genes including; WRKY31 and LRR-RLK’s were more highly expressed in the uninoculated C. crenata when compared to the uninoculated C. sativa. This increased basal defense to P. cinnamomi may contribute to this variety’s resistance. Gene expression in E. nitens in response to P. cinnamomi infection included up-regulated overrepresented gene ontology terms related to JA and ET signaling . Interestingly, pathogenesis-related gene 9 was down-regulated and represents a cross-species effector target during P. cinnamomi infection. Functional genomics and validation of these defense genes has only been performed in one study in A. thaliana. Eshraghi et al. reported that an auxin Arabidopsis mutant was more susceptible to P. cinnamomi infection than the wild type indicating the role of auxin pathways in P. cinnamomi defenses. The main challenges for the identification of P. cinnamomi resistance genes in avocado are the lack of tools available for functional genomic studies and limitations associated with tree crop biology. Next-generation sequencing has provided some information on the expression of defense-related genes in avocado infected with P. cinnamomi. However, the lack of the genome sequence and absence of functional genomic tools for avocado makes it difficult to determine and confirm their contributions to resistance against P. cinnamomi.

The N. benthamiana model plant provides the opportunity to conduct functional genomic studies to determine the role of defense response genes to P. cinnamomi resistance that is not yet available in the avocado system or other tree hosts. Model plants including A. thaliana , L. angustifolius , and Medicago truncatula have been previously reported as susceptible hosts for this oomycete pathogen and have been used to study P. cinnamomi pathogenesis and plant responses to this pathogen. Although whole genome sequencing was available for these pathosystems, functional assays were not conducted with the exception of one study in Arabidopsis implicating the auxin signaling pathway with defense response against P. cinnamomi . Conducting RNAseq studies in N. benthamiana system at different time points during the infection process will provide a foot-hold into the defense gene expression pattern during P. cinnamomi infection and will allow us to conduct functional studies of selected defense genes using this N. benthamiana–P. cinnamomi pathosystem. Differentially expressed pathways and genes can be then validated by RT-qPCR in N. benthamiana and in avocado inoculated with P. cinnamomi using a detached leaf assay. Functional validation of the most promising genes can be done in N. benthamiana by transient over expression or silencing to determine their contribution to P. cinnamomi resistance. If similar expression patterns are found in avocado it is reasonable to consider this gene a good candidate for marker assisted breeding or biotechnology in avocado. As genomic tools for avocado quickly become more available the methods developed in this system will become more applicable to this fruit tree crop. RNAseq analysis of infected N. benthamiana roots can complement this system by identifying what genes are universally expressed in the plant in response to P. cinnamomi infection and what gene expression is unique to the roots. Functional genomics are lacking in avocado; therefore, the objectives of this study were i) to establish a model system to look at defense gene expression in response to P. cinnamomi infection, ii) validate differentially expressed defense genes using overexpression in the same N. benthamiana model system, and iii) establishing connections to similarly expressed defense genes in avocado in response to P. cinnamomi infection. This information will help to select candidate defense genes in avocado for marker assisted breeding or biotechnology. The average total million reads for the sequenced mock-inoculated N. benthamiana samples were 66, 75, 75, 81, and 70 million for the 6, 12, 24, 36, and 48 hpi time points respectively. The average total reads for the inoculated N. benthamiana samples were 145, 123, 80, 62, and 70 million reads for the 6, 12, 24, 36, and 48 hpi time points respectively. The average percentage of reads mapped for the five-mock inoculated time points was between 86 and 88%.