There are several limitations in using tree crops to study and characterize pathogenicity and virulence for a large number of P. cinnamomi isolates including: the requirement for using genetically uniform clonal material, only a small number of experiments can be completed annually, the large greenhouse space required to conduct the experiments, and the long time to score the disease. Model plants including Arabidopsis , Lupinus , and Medicago 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. N. benthamiana, is a model plant that has been widely used by the oomycete community to understand Phytophthora pathogenicity and the molecular interaction with their host plants . Recently, this model plant has been used to study other hemibiotrophic Phytophthora root pathogens such as P. palmivora , P. capsici , and the citrus pathogen P. parasitica . Taking advantage of the extremely wide host range of P. cinnamomi, we assessed and found that P. cinnamomicould infect and colonize N. benthamiana. While P. cinnamomi has been reported on other Nicotiana specie in Russia , 10 liter pot this study is the first report showing that N. benthamiana is also a host for this pathogen.

Similar to the detached leaf P. cinnamomi inoculation method developed in Arabidopsis, we used this detached leaf assay to inoculate N. benthamiana leaves with this oomycete. Detached leaf assays offer several advantages over whole plant inoculations including: i) greater reproducibility due to similar size and age of leaves, ii) increased replication, iii) more consisted delivery and localization of the inoculum in the leaves, iv) uniform incubation conditions, v) more accurately quantification of disease, and vi) reduction of space require for inoculations. Robin and Guest used detached leaf assays to characterize the pathogenicity of P. parasitica isolates in four tobacco cultivars. Detached leaf assays have been also used in tree crops to study Phytophthora root pathogens pathogenicity and virulence. Denman et al. used detached leaf inoculations using different tree species to study the response of several isolates of P. ramorum. Helliwel et al. used detached leaf assays using Theobroma cacao to study P. palmivora. In agreement with previous studies using other host plants including avocado, we found that P cinnamomi as expected exhibited a hemibiotrophic lifestyle when inoculated on N. benthamiana leaves as supported by our microscopic studies and DNA pathogen quantification using TaqMan real-time PCR. P. cinnamomi haustoria-like structure were detected in N. benthamiana leaves as was found in infected roots of other host plants such as avocado , Medicago; and Quercus .

Furthermore, the trend of P. cinnamomi growth in N. benthamiana leaves, determined by DNA quantification using TaqMan quantitative PCR, resembled the pathogen growth in avocado roots monitored using quantitative PCR . In avocado infected roots, P. cinnamomi continuously growth until 7 days post inoculation, then the pathogen growth decreased due to the complete necrosis of the avocado root system. Similarly, P. cinnamomi in N. benthamiana infected leaves continuously growth until 48 hpi and then decreased due to a complete necrosis of the inoculated area sampled for TaqMan DNA quantification . Finally, this N. benthamiana detached leaf P. cinnamomi inoculation method was validated by successfully detecting significant differences in virulence between the S-2109 and the S-2113 isolates, which was consistent with our avocado inoculation results, indicating that this method could be used as an alternative inoculation method to circumvent the difficulties of using whole avocado root inoculation methods to assess the virulence of large number of isolates. All the experiments conducted in this work provide initial evidence of variability in growth rate, optimal growth temperature, fungicide sensitivity, and virulence among isolates representing the two A2 clonal populations identified by Paglaccia et al. . More importantly, the existence of P. cinnamomi isolates collected from PRR diseased avocado roots that are more virulent and less sensitive to the current chemical control methods used by avocado growers in California will greatly influence the development of resistant avocado rootstocks and help the implementation of more effective cultural practices for managing avocado PRR in California including for example the registration of new fungicides for avocado that can be used in combination with mefenoxam and phosphonates.

Phytophthora root rot caused by Phytophthora cinnamomi Rands is the most important disease of avocado and limits production in California, Florida, and other locations worldwide. P. cinnamomi kills feeder roots and can also cause trunk cankers, resulting in reduced fruit yield and often tree death . PRR historically affected 60 to 75% of California avocado growers, causing losses of $40 million annually . Major expenditures for managing PRR also include cost and application of fungicides. Favorable conditions for spread and proliferation of the pathogen are wet, poorly drained soils at a wide range of temperatures . The main infection propagules of P. cinnamomi are zoospores that are chemotactically attracted to the roots of plants . Chlamydospores, long-term resting structures, enable P. cinnamomi to persist in the soil for many years making it nearly impossible to completely eliminate the pathogen once the soil is infested . Avocado PRR management includes the use of resistant rootstocks, proper irrigation practices, and chemical treatments . Commercially available, moderately resistant rootstocks include Dusaâ, Toro Canyon, Duke 7, Steddom, Uzi, and Zentmyer . Among them, Dusaâ is the current California industry standard enabling growers to cultivate avocado in P. cinnamomic-infested soil and maintain production. However, resistance of this rootstock is challenged by a new clonal group of more virulent P. cinnamomi isolates recently identified in California . Cultural management practices include mulching,gypsum application, and proper irrigation using water sources not contaminated with propagules of P. cinnamomi . The pathogen has a very broad host range and is capable of infecting more than 5,000 plant species . Thus, even with the best management program, the pathogen can be re-introduced into an orchard from other plants or with irrigation and run-off water. At present, the only fungicides available to control PRR of avocado are phosphonate-based  and phenylamide compounds . Mefenoxam is an R-enantiomer of metalaxyl that was introduced in 1977. It has been effectively used for managing diseases caused by Phytophthora spp. and other Oomycota organisms . It is strongly inhibitory to mycelial growth and sporulation of these organisms due to interfering with RNA polymerases and blocking RNA synthesis to these organisms. The risk of phenylamide resistance development is considered high due to a single-site mode of action , and resistance has developed in P. infestans, P. citricola, P. megasperma, and P. nicotianae only few years after metalaxyl and mefenoxam became available for use . Little information is available on the sensitivity of P. cinnamomi populations to mefenoxam , and no information isavailable for isolates from avocado in California where this fungicide is mostly used by the nursery industry. Phosphorous acid and its ionized compounds belong to the phosphonate group of fungicides. The specific mode of action of phosphite is largely unknown, but direct inhibition of pathogen growth and induction of the host plant defense system appear to be involved . Reduced in vitro sensitivity to potassium phosphite in several Phytophthora spp., including P. capsici, P. cinnamomi, P. citrophthora, P. infestans, and P. syringae has been reported . Potassium phosphite is the preferred PRR control treatment by avocado growers because it is less expensive than mefenoxam. Its optimal application by trunk injection, however, can be labor-intensive and costly, and furthermore, injection sites provide entry points for insect pests. New Oomycota fungicides with different modes of action from mefenoxam and phosphonate fungicides have become available in recent years. Ethaboxam, 10 liter drainage collection pot a thiazole carboxamide , disrupts microtubule organization in Oomycota . Fluopicolide is a pyridinylmethyl-benzamide that disrupts cell division and mitosis by acting on spectrin-like proteins . Mandipropamid is a carboxylic acid amide fungicide targeting the pathogen cellulose synthase gene that is involved in cell wall biosynthesis . Oxathiapiprolin, a piperidinyl-thiazole-isoxazoline , targets the oxysterol-binding protein of Oomycota organisms .

The goal of this study was to determine whether the new Oomycota fungicides could be used to manage PRR of avocado. Thus, the objectives were to establish baseline sensitivities of a large number of isolates of P. cinnamomi representing the current pathogen population on avocado in California, compare these sensitivities to those of mefenoxam and potassium phosphite, and evaluate the efficacy of the four new fungicides as compared to the two registered ones for the management of PRR of avocado seedlings and clonal rootstocks in greenhouse studies.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. Dry weights of shoots of Zutano seedlings and roots of all plants were determined after drying at 50°C for 5 days. The experiment was conducted twice using avocado seedlings and repeated on two clonal rootstocks, Dusaâ and PS.54.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 veryeffective 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 . In both studies, 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.