In comparison with the four new fungicides, effectiveness of potassium phosphite in greenhouse studies was high to moderate and was moderate for mefenoxam. In the field study, a significant reduction in disease and soil populations by mefenoxam was only observed after increasing applications to high-label rates. Lower rates were initially used because this fungicide is known to cause phytotoxic effects to young citrus trees as was also observed in preliminary greenhouse studies where a range of rates was evaluated. Thus, the reduced effectiveness of mefenoxam in our study,a treatment that has been used successfully in commercial applications for managing Phytophthora root rot of various tree crops for many years, may have been due to using inadequate rates. Furthermore, trees were inoculated with an isolate of P. nicotianae with an EC50 value for mycelial growth of 0.24 mg/liter that was in the mid-range among 31 isolates from California evaluated previously. Because baseline sensitivities before commercial use of mefenoxam were never established for P. nicotianae from citrus, and the phenylamide class of fungicides has been used since the 1980s in California citriculture, this isolate may be part of a less-sensitive sub-population of the species that cannot be easily managed with mefenoxam applications. Soil populations of untreated control trees in our field and greenhouse studies were often very high considering that >15 propagules/g of soil is considered a threshold level where management is recommended. Still,maceteros fresas disease incidence of feeder roots was mostly low, especially during summer samplings in the field.

We chose ‘Carrizo citrange’ in the field studies because it is commonly used commercially as a rootstock. It is considered of intermediate susceptibility or tolerant to Phytophthora root rot, and this could have accounted for the low disease incidence. In the greenhouse studies, disease incidence may have been increased by pruning feeder roots of seedlings as it was done in studies by others. Root injuries may occur naturally in the field by nematode or root weevil infestations in the soil, and these pests are known to increase the incidence of Phytophthora root rot. Still, although disease incidence was overall low in our studies, fungicide efficacy could be compared and significant differences were observed. The four new Oomycota fungicides are single-site mode of action inhibitors. Their resistance risk currently has not been completely characterized , and resistant field isolates have not yet been detected in Phytophthora species. Resistance, however, has been described for Plasmopara viticola, another Oomycota organism, to mandipropamid. In our previous baseline sensitivity assessments, outliers with higher EC50 values for mycelial growth inhibition of P. syringae by fluopicolide and of P. citrophthora by ethaboxam were identified that were >23-fold less sensitive than the most sensitive isolates of the respective species used in the study, and this was considered to possibly indicate a potential for selecting isolates with reduced sensitivity to these fungicides. Thus, as with any single-site mode of action fungicide, resistance management strategies should be followed from the onset of commercial use. Because two of the new fungicides each have the same registrant in the United States, the commercialization of pre-mixtures will be facilitated. In summary, our study demonstrated that the new Oomycota fungicides ethaboxam, fluopicolide, mandipropamid, and oxathiapiprolin provided highly effective control of Phytophthora root rot of citrus caused by P. nicotianae or P. citrophthora. The efficacy was generally better than for the previously available fungicides mefenoxam and potassium phosphite.

The new compounds promoted the recovery of infected trees andenhanced fruit yield, with fluopicolide and oxathiapiprolin showing the most consistent increases in these measures. Based in part on our studies, fluopicolide recently received a federal and oxathiapiprolin a full registration for use on citrus, whereas registration for ethaboxam and mandipropamid has been requested. Species of Phytophthora cause several diseases on citrus, including root rot, foot rot, brown rot of fruit, and gummosis of tree trunks and larger limbs. Phytophthora root rot is common in orchards in California and other citrus production areas worldwide. The disease can be especially damaging in new citrus plantings where overwatering is conducive for infection, and the limited root system of young trees cannot generate new growth fast enough to replace infected and damaged tissues. This can result in poor tree growth and delayed orchard establishment. In California, the disease is mainly caused by Phytophthora nicotianae Breda de Haan during the warmer months of the year, whereas P. citrophthoraLeonian is active year-round. P. palmivoraE. J. Butler is the major citrus root rot pathogen in Florida. P. cactorumJ. Schröt., P. capsici Leonian, P. cinnamomi Rands, P. drechsleri Tucker, and P. megasperma Drechsler have been occasionally identified in some production areas. Phytophthora root rot is characterized by discoloration and softening of the outer root cortex that becomes water-soaked in appearance and prone to sloughing off, eventually exposing the inner stele. Damage of the root system can lead to tree decline and yield losses from lack of water and nutrient uptake, and if left untreated, to tree death. Phytophthora root rot can be managed by cultural practices such as the use of Phytophthora-tolerant root stocks , irrigation or orchard drainage strategies that avoid overwatering, and fungicide applications. These practices are best used in an integrated approach. Among fungicides,maceta 30l the phenylamides and the phosphonates have been used since the 1980s, and until recently, no alternatives were available.

The limited number of fungicides registered resulted in their over-use, and in subsequent resistance development. Resistance to the phenylamide class of fungicides has been reported in Oomycota pathogens of numerous crops including Phytophthora spp. that are known to be pathogenic to citrus such as P. citricola and P. nicotianae. Phenylamide-resistant populations of P. nicotianae are established in Florida orchards and nurseries. Phosphonate resistance is less common but has been identified in isolates of P. cinnamomi and P. infestans , and more recently in isolates of P. citrophthora, P. syringae, and P. nicotianae from California citrus orchards. With the need for alternative chemical treatments to manage Phytophthora diseases of citrus, new fungicides have recently become available for evaluation. They include the thiazole carboxamide ethaboxam, the benzamide fluopicolide, the carboxylic acid amide mandipropamid, and the piperidinyl thiazole isoxazoline oxathiapiprolin. Each compound has a unique mode of action that is different from those of the previously registered compounds. Among these, oxathiapiprolin was recently registered on citrus for foliar and soil treatments against Phytophthora diseases. This compound was found to be toxic in vitroat very low concentrations against several life stages of the pathogens and was shown to be highly effective in managing root rot and brown rot. Belonging to the Fungicide Resistance Action Committee code 49, its mode of action is the inhibition of an oxysterol binding protein, resulting in the inhibition of multiple cellular processes. Uptake of oxathiapiprolin into citrus plants after soil application is unknown. Previous work has been conducted on annual crops , however, there is currently no information on the mobility and activity of oxathiapiprolin within perennial tree crops. This information may provide a better understanding of its protective and eradicative capabilities in controlling Phytophthora root rot of citrus and have implications on its field use in managing the disease. Therefore, the objectives of this research were to determine if oxathiapiprolin can be detected inside roots and above ground portions of citrus seedlings after soil application as compared to mefenoxam and if concentrations of the fungicides inside the plants can be effective against P. citrophthora. For this, bio-assays and analytical residue analyses were performed at selected time periods after treatment of plants. Sweet orange Osbeck) cv. ‘Madam Vinous’ seedlings in 15 cm x 15 cm x 15 cm pots were grown from seeds in the greenhouse at 24°C to 30°C for 6 to 7 months. At this time, plants were between 25 cm and 30 cm tall. Prior to treatment, plants were moved to an incubator set for a 12-h photoperiod with 34˚C during the light cycle and 26.7˚C during the dark cycle. There were three single-plant replicates for each fungicide treatment and each of the four sample timings. Plants were arranged in a randomized complete block design with all four sampling times in each block. Additionally, three replications of untreated control plants were used.

Solutions of oxathiapiprolin and mefenoxam were prepared in distilled water, and 50 ml was added to each pot, resulting in final applications amounts of 50 mg of oxathiapiprolin and 130 mg of mefenoxam per pot. These amounts are comparable to labeled chemigation rate ranges based on the total basin area for 288 trees/Ha and considering that the treatment area of a potted plant is approximately 1/9 of the basin of a newly planted citrus tree. Solutions were added to each pot without wetting the stem, and distilled water was used for the controls. Each pot was then placed in a plastic bag that was tied around the bottom of the stem to reduce evaporation. Plants were watered once nine days after treatment. Plants were harvested 7, 10, 13, or 16 days after treatment. The root ball was shaken to remove most of the soil, washed using tap water, and allowed to air-dry briefly at ambient temperature. Roots were sampled randomly. The stems were cut 1.5 cm above the soil line to avoid fungicide contamination from the soil application. Another cut was done 10 cm above the first cut, and stem and leaf tissues within this stem segment were separated.One gram of each tissue was placed into glass scintillation vials. The vials were covered with a single layer of cheesecloth and frozen at -80°C for 24 h, lyophilized for 24 h , and then capped and stored at -20°C. A standard procedure was followed for extraction of plant tissues for both fungicides. For this, the lyophilized tissues were transferred to 2-ml impact resistant tubes containing two stainless-steel grinding balls and pulverized for 60 s using the FastPrep-24 set at 6.0 m/s. The contents of each tube were transferred to a 15-ml conical polypropylene plastic tube for a single-phase extraction. For this, 1 ml of sterile ultrapure water was added to each tube and the tube was incubated for 5 min to allow soaking of the sample. An additional 800 µl of sterile ddiH2O, 2.4 ml of acetonitrile, and 20 µl of formic acid were added, and the tubes were placed on an orbital shaker at 300 rpm for 5 min. The tubes were centrifuged at 1,380 g for 10 min. The supernatant was transferred to a 15-ml plastic tube, stored at -20ºC, and used for determining fungicide activity in a bio-assay within 7 days. For analytical residue analyses using HPLC-MS/MS , 500 µl of each tissue extract was transferred into a scintillation glass vial, 2 ml of methanol , and 4.5 ml of 1% formic acid were added, 0.6 ml of the resulting solution was aliquoted into a 2-ml low absorption vial , and vials were stored at -20°C until analyses. The experiment was done twice. Analytical grade oxathiapiprolin and mefenoxam were dissolved in acetonitrile and serially diluted. The samples were analyzed for oxathiapiprolin and mefenoxam using a standard curve method. The concentration of standards used for quantitation were 0.1, 0.5, 1.0, and 10 ng/ml. Each dilution was transferred to a 2-ml low-absorption vial, and aliquots were transferred to auto-sampler vials for analysis that was performed by Environmental Micro Analysis Inc.. The autosampler vials were analyzed using high-pressure liquid chromatography coupled with tandem electrospray mass spectrometer. The chromatographic separation was achieved on a XB-C18 HPLC column. The samples were analyzed with standard concentration levels indicated above. For bio-assays using plant extracts with unknown amounts of fungicides, the dependent variable was the log10-transformed mean inhibition zone; whereas for plant extracts analyzed using HPLC-MS/MS, the dependent variable was the log10-transformed mean amount of fungicide calculated per g of tissue. These data were analyzed using generalized linear mixed models with the GLIMMIX procedure of SAS. For this, root, stem, and leaf extracts or days after treatment were treated as fixed effects, and experiment, replication , and the overall error term were treated as random effects.