A test run was conducted on 10 October 2013 to determine the optimal runoff sampling time intervals and the number of samples needed to capture the peak and total loadings. For the test run, the water soluble fertilizer was applied to both of the sites at a rate of 2.24 g m−2 . Grab samples were collected at a 10-min frequency over a two hour irrigation period from the control site. Water samples were collected immediately before the runoff was directed to the underground tank. The water samples were coarse filtered during sample collection with coffee filters to remove large tree leaves, grass clippings and large soil particles. Based on the results of the test run, the water sampling frequency for subsequent trials was extended to six hours with a variable sampling interval to better characterize the runoff pollutant pattern. The composite water sample from the treatment site was used for calculating the total loading of the treatment site where little surface runoff occurred in this study. A 5.1 cm diameter and 0.9 m long PVC drainage pipe was vertically installed into the middle of the treatment bioswale to collect a representative water sample for monitoring pollutants concentration dynamics in the bioswale. The treatment site water samples were not affected by successive flow from the control site because the treatment site was located upslope of the control site. The water sample collected from the control site was surface runoff, which was not affected by subsurface flow of the treatment site because of the site’s relatively flat surface. The oomycete pathogen Phytophthora cinnamomi Rands, procona buckets causal agent of Phytophthora root rot , is the most destructive disease of avocado worldwide .
In California, avocado PRR affects 60-75% of avocado growers who lose approximately $40 million annually . This globally distributed oomycete is called “the biological bulldozer” for its capacity to infect over 3000 plant species causing devastating impacts in natural ecosystems, forestry, agriculture, and the nursery industry . The economic impact due to P. cinnamomi infestation is evident in the forest and food industry, affecting eucalyptus, pine, oak , and other fruit crops such as pineapple, peach, and high bush blueberry . Losses include not only decreases in crop yield and product value, but also large amounts of money spent annually on control measures. There are no effective means to eradicate P. cinnamomi from infested areas as it survives in moist soil or dead plant material as chlamydospores for long periods under adverse conditions . Several PRR control strategies have been found to reduce the impact of this invasive pathogen including the use of chemical treatment , tolerant plants, and management practices . P. cinnamomi is a hemibiotrophic pathogen feeding initially from living host cells and then switching to necrotrophy by killing the host cells and feeding from the nutrients released by them . The entry into the plant is achieved by the adhesion of the motile zoospores to the host tissue, encystment, and germ tube formation. The germ tubes usually grow and penetrate the root surface via appressorium like swelling structures and then plant tissue is rapidly colonized . During its biotrophic stage, P. cinnamomi projects haustoria into the plant cells for the acquisition of nutrients and release of pathogen proteins to aid the infection process in the host . This is followed by a necrotrophic stage characterized by host cell death, hyphal proliferation, and production of numerous sporangia .
Currently, the molecular and genetic basis of P. cinnamomi pathogenicity, virulence, and plant immunity against this pathogen are largely unknown due to limitations associated with tree crop biology and the lack of tools available for functional studies in tree crops such as avocado . The model plant, Nicotiana 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 . Moreover, several studies using model plants, crops, and tree crops to study pathogenicity, virulence, and fungicide efficacy of PRR pathogens such as P. sojae, P. capsici, P. parasitica, P. palmivora, P. cinnamomi, and P. ramorum have been done using detached-leaf assays . Phosphite is the most widely used chemical control method for managing PRR caused by several Phytophthora spp. including P. cinnamomi . Phosphorous acid dissociates to form the phosphonate ion , also called phosphite. Phosphorous acid and its ionized compounds are often referred to as phosphonate or phosphonite. The specific mode of action of potassium phosphite is largely unknown, however appears to involve both a direct and an indirect effect on the pathogen . Several studies have assessed the in vitro sensitivity of P. cinnamomi to phosphite using mycelial radial growth inhibition in solid and liquid media to identify sensitive and tolerant isolates . In California, avocado growers heavily rely on the use of phosphite products to control P. cinnamomi, however the phosphite sensitivity of California avocado isolates is largely unknown. In addition to phosphite, phenylamide fungicides such as metalaxyl and mefenoxam are also used for managing diseases caused by oomycetes including P. cinnamomi . Resistance to metalaxyl has developed in P. capsici, P. infestans, and P. nicotianae . Phenylamides usually do not inhibit germination of sporangia or encysted zoospores as effectively as they do mycelial growth . Consequently, inhibition of mycelial growth in vitro has been used as the primary method of determining the sensitivity to these fungicides among isolates of Phytophthora spp. .
The need for new oomycete-targeted fungicides to control diseases caused by these pathogens especially those that have developed resistance to phenylamide fungicides has resulted in the development of several new chemicals with varying modes of action such as fluopicolide and oxathiapiprolin . Fluopicolide is a pyridinylmethyl-benzamide fungicide that disrupts cell division and mitosis by acting on spectrin-like proteins . This fungicide is effective to control diseases caused by P. capsici and P. infestans . Oxathiapiprolin is the first of the new piperidinyl thiazole isoxazoline class fungicides discovered and developed by DuPont Co. in 2007. The molecular target of oxathiapiprolin is the oxysterol binding protein . This new fungicide exhibits strong inhibitory activity against a range of agriculturally important plant-pathogenic oomycetes including P. capsici, P. infestans, P. sojae, Peronospora belbahrii, and Pythium ultimun . However, its inhibitory activity against P. cinnamomi has not been tested. P. cinnamomi is a heterothallic species that requires the presence of both A1 and A2 mating types to undergo sexual reproduction. Despite that both mating types arepathogenic , avocado PRR disease in California is mainly associated with A2 mating type isolates . Previous P. cinnamomi population studies have revealed low levels of genotypic and phenotypic variation among isolates from different mating types, origin, isolation source, and host plants, however, only a few were conducted or have included isolates from avocado . These studies described the existence of three clonal lineages for P. cinnamomi, one corresponding to the A1 mating type isolates and two different clonal lineages for the A2 mating type isolates . Pagliaccia et al. conducted the first study to assess the genetic diversity of P. cinnamomi isolates from avocado in California and also found two genetically distinct clades of A2 mating type isolates . The A2 clade II consisted of isolates with unique genotypes collected only in Southern California. Interestingly, the authors in this study included isolates from P. cinnamomi previously identified as belonging to the A2 type 1 and A2 type 2 described by Dobrowolski et al. , however these isolates clustered within the A2 clade I group, suggesting that the A2 clade II group identified in Paglaccia et al. in California could be another clonal lineage. No studies have been conducted to assess the phenotype of avocado isolates corresponding to these genetically distinct A2 groups identified in California by Paglaccia et al. , therefore, procona florida container the objectives of this study were to i) assess the phenotype of several avocado isolates corresponding to these A2 clades regarding in vitro mycelial growth rate, optimal growth temperature, sensitivity to potassium phosphite and mefenoxam, and virulence, ii) test the sensitivity of avocado isolates to fluopicolide and oxathiapiprolin as alternative chemistries for controlling avocado PRR in California, and iii) develop and validate a detached leaf assay inoculation method using N. benthamiana to circumvent the difficulties associated with the avocado whole plant root inoculation method to assess the virulence of P. cinnamomi isolates. This information will help to design appropriate measures for managing avocado PRR in California and implement efficient and reliable screening methods towards the selection and development of new P. cinnamomi resistant avocado rootstocks effective against a more diverse pathogen population.A total of twelve P. cinnamomi isolates associated with avocado PRR were used in this study, including six isolates from Northern California corresponding to the A2 clade I group, four isolates corresponding to the unique A2 clade II subpopulation from Southern California, and two isolates collected from the same Southern California region from where the A2 clade II isolates were collected .
A2 clade I isolates from this study represent five of the ten total genotypes identified by Paglaccia et al. . Isolates representing the A2 clade II group covered three from the six unique genotypes identified in Southern California. All the isolates in this study were obtained from a P. cinnamomi small collection at the UCR Avocado Rootstock Breeding Program. Isolates were maintained as water agar plugs . Agar plugs were removed one at the time as needed for each experiment and plated on 10% clarified cV8A agar to resume growth. To ensure that isolates in the collection are free of contamination and avoid any problems with mix samples or not appropriate maintenance of the collection, we sequenced all the isolates using ITS and COX1 spacer regions. Mycelial DNA was extracted using the Qiagen DNeasy plant mini kit . Universal primers ITS1 and ITS4 were used for the ITS region, and primers OOM and FM85 were used for the COX1 spacer region, both sets of primers are described in Kroon et al. . Each 25-µl PCR reaction contained 2 µl of DNA , 2.5 µl of 10x PCR buffer , 200 µM dNTPs, 0.4 µM of each primer, and 1.25 units of Taq DNA polymerase . PCR reactions were performed using a Programmed Thermal Controller with conditions as follows: 95°C for 5 min; followed by 35 cycles at 95°C for 1 min, 50°C for 1 min, and 68°C for 1 min; and a final extension at 68°C for 10 min. PCR products were confirmed on 1% certified molecular biology agarose gel , stained with ethidium bromide , and visualized under UV light using a Universal Hood UV Light Table . PCR products were treated with Zymo DNA Clean and Concentrator according to the manufacturer’s instructions to remove excess primers and nucleotides before submitting the samples for Sanger sequencing. DNA sequencing was conducted in both directions with the same primers used for amplification at the UCR Genomics Core facility. Contiguous sequences were generated which were then subjected to a BLASTn search to determine the highest maximum identity to the sequence of the type isolates in GenBank.Two avocado rootstocks were selected based on their P. cinnamomi resistance phenotype. Clonal rootstocks corresponding to the moderate resistant Dusaâ and the susceptible PS.54 were obtained from Brokaw Nursery. The 6-month-old clonally propagated plants were removed from their bags and transplanted into pots after the nurse seed was removed. Plants were grown in a greenhouse with an average maximum temperature range of 25-28°C at 40-50% relative humidity. Plants were fertilized twice a week and watered every day. Nicotiana benthamiana seeds were germinated in trays and transferred to individual pots 2 weeks post germination. Plants were grown at 22°C with 16 h of light and 8 h dark cycles at 40-50% relative humidity. The effective concentration of potassium phosphite at which 50% of the mycelial growth of each isolate was inhibited was determined using the traditional agar dilution method as described in Adaskaveg et al. . Potassium phosphite was added to 10% cV8A to obtain final concentrations of 5, 25, and 100 µg/ml. Mycelial agar plugs from 6-day old cultures of P cinnamomi were placed at the center of the plates containing the corresponding chemical concentration. Three replicates were done per each treatment. After incubation for 3 days in the dark at 22°C, radial colony growth was measured. Colony growth inhibition of each isolate in the presence of potassium phosphite was then calculated as compared with their corresponding control plates without potassium phosphite.