Similar to this study, Linde et al. , reported correlations between P. cinnamomi genotypes from Australia and South Africa and the level of virulence in eucalyptus, suggesting that genotype variation may indicate pathogenic variability. On the other hand, Eggers et al. , did not detect differences in pathogenicity or virulence among P. cinnamomi isolates from oak forest soils in Eastern United States Zentmyer did not find significant differences in pathogenicity among twelve A2 California isolates collected from avocado in Northern and Sothern California when inoculated roots of avocado susceptible seedlings.P. cinnamomi isolates less sensitive to phosphite, and more virulent, exhibited slower mycelial growth rate. Based on these results we speculated that a negative correlation might exist between the in vitro growth rates of some P. cinnamomi isolates with the level of virulence and sensitivity to fungicides, which could potentially indicate a fitness cost for some traits over other acquired traits. Our results did not support the standard assumption that increased parasite growth leads to increased virulence but are consistent with an increasing amount of evidence for plant and human pathogens indicating that there is a trade-off between parasite growth and virulence and pathogen survival . Childers et al. showed that many isolates of P. infestans that acquired resistance to mefenoxam exhibited retarded mycelial growth in comparison to the parental isolates that have never been exposed to this fungicide.
Similar to this study, Meyer et al. found that virulence of the necrotrophic fungal pathogen, Pyrenophora semeniperda, procona flower transport containers was significantly negatively correlated with mycelial growth, suggesting a tradeoff between these two traits considering that the production of pathogen toxins necessary to kill the host cells competes with the metabolic processes required for pathogen growth. Similar tradeoff scenarios could be applied for hemibiotrophic pathogens such as P. cinnamomi because at the beginning of the interaction with their host plants, hemibiotrophs need to produce effector proteins to avoid plant recognition and suppress the plant immune system at the biotrophic stages. Furthermore, later in the interaction, these pathogens need to switch to a necrotrophic stage involving the production of another sets of effector proteins to induce host cell death including pathogen toxins . Variability in pathogenicity and virulence among P. cinnamomi isolates has been tested using woody perennial crops such as avocado, eucalyptus, oak, and chestnut . 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 , 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, procona valencia 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 is available 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, 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. A total of 71 P. cinnamomi isolates were obtained from avocado roots and rhizosphere soil from northern and southern avocado growing regions in California . Fifty-three isolates were identified previously, including 32 isolates from an earlier study , and 18 isolates were identified in this study. Isolates were maintained as agar plugs in water during the study, and long-term in liquid nitrogen. The pathogen was cultured on 10% clarified V8 agar . The identity of the 18 recently recovered isolates was confirmed by sequencing the rDNA internal transcribed spacer region . Mycelial DNA was extracted using the Qiagen DNeasy plant mini kit . Universal primers ITS1 and ITS4 were used in PCR reactions. Each 25-µl reaction contained 2 µl of DNA , 2.5 µl of 10× 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, extension at 68°C for 1 min; and a final extension at 68°C for 10 min. PCR products were visualized in ethidium bromide-stained 1% agarose gels. PCR products were treated with Zymo DNA Clean & Concentrator to remove excess primers and nucleotides, and sequenced in both directions .