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, 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.
This is the first study describing the phenotype of P. cinnamomi isolates, collected from PRR infected avocado roots, square pot representing the two A2 mating types groups identified by Paglaccia et al. . The A2 clade I contained P. cinnamomi isolates collected from 1989 to 2010, whereas the A2 clade II only contained a unique set of isolates collected in 2009 and 2010 from Southern California avocado growing regions. This study reported significant differences in vegetative growth, fungicide sensitivity, and virulence among all the isolates tested. Moreover, Southern isolates were less sensitive to potassium phosphite and have slower growth rate at 22℃ whereas Northern isolates were more sensitive to potassium phosphite and have higher growth rates. This trend observed suggests a correlation between mycelial growth rate at 22℃and potassium phosphite sensitivity with specific group of isolates. The group of Northern isolates corresponding to the A2 clade I mating type group exhibited higher mycelial growth rates than the Southern group of isolates at 22°C independent of the nutrient media used . Consistent with the results of Zentmyer et al. , there was a significant effect of nutrient media and my celial growth rate of the P. cinnamomi study isolates, however this effect did not alter the split of the Northern and Southern isolates by their growth rate. The higher sensitivity of the A2 clade I isolates, collected from Northern California, to higher temperatures when compared with the Southern isolates might be explained by the fact that the median temperature is higher in Southern than in Northern California throughout the year.
Previous studies reported phenotypic differences in colony morphology, growth rate, and optimum growth temperature among P. cinnamomi isolates, however the majority of these studies compared isolates from different mating types, origin , and host plants . Only a few studies have included P. cinnamomi associated with avocado when assessing phenotypic variability . In the majority of these studies, the authors could not assign a phenotypic trait to a specific group of isolates. Others have reported varying results on the linkage between genotype groups and colony morphology. Dobrowolski et al. reported that colony morphology of Australian P. cinnamomi isolates grouped with a particularly genotype. In contrast to these results, colony morphology among the twelve isolates tested in this study did not vary significantly. Lopez-Herrera and Perez-Jimenez reported significant differences on colony morphology among P. cinnamomi A2 isolates collected from avocado trees in Spain, but these isolates did not exhibit significant differences on mycelial growth rates. California produces 95% of the avocado crop for the U.S.A and PRR caused by P. cinnamomi is responsible for commercial losses totaling $40 million annually statewide. Mefenoxam and phosphite applications are widely used to prevent and combat this disease , however, there is a notably preference for phosphite products over mefenoxam among the growers and this is particularly true for California growers. This preference could explain why mefenoxam-resistant isolates were not found in this study. Moreover, the range of the EC50 values for mefenoxam was consistent with previous values reported for P. cinnamomi in the U.S.A . Duan et al. reported minor variation in sensitivity to mefenoxam among P. cinnamomi isolates collected from diseased ornamental plants in South Carolina.
The EC50 values of the majority of these isolates were less than 0.1 µg/ml. Hu et al. found that there were more variations in mefenoxam sensitivity among P. cinnamomi isolates collected from different host species than from the same host. In this study, the authors classified isolates as sensitive to mefenoxam when their EC50 values ranged from 0.01 to 0.02 µg/ml and as intermediate when EC50 values ranged from 0.03 to 0.08 µg/ml. The EC50 values for mefenoxam in this study were also less than 0.1 µg/ml suggesting that the repeated use of this fungicide to control PRR in nurseries and avocado orchards does not appear to have reduced the mefenoxam sensitivity of P. cinnamomi isolates. In contrast to mefenoxam, we detected a significant variability among the isolates in potassium phosphite sensitivity but we did not find potassium phosphite-resistant isolates. The Northern isolates corresponding to the A2 clade I group had EC50 values of < 34.1 µg/ml, whereas the Southern isolates had EC50 values of > 98.9 µg/ml. It is worrisome that the Southern group of isolates including the A2 clade II isolates that exhibited more virulence in the moderate resistant Dusa, which is the current industry standard rootstock among California avocado growers, were less sensitive to potassium phosphite. Their higher EC50 values could represent a selection from higher doses of potassium phosphite being necessary to suppress and control avocado root rot where these isolates are present. A more detailed study with a larger number of isolates and history of phosphonate applications in the field are needed it to test this hypothesis. Dobrowolski et al. demonstrated that P. cinnamomi isolates exposed to long periods of phosphite treatment in avocado orchards in Australia exhibited reduced sensitivity to phosphite when evaluated on avocado, lupin, and eucalyptus suggesting the onset of resistance to this fungicide. Similar results have been reported for P. cinnamomi isolates from avocado orchards in South Africa . This study reports for the first time, the presence of P. cinnamomi isolates, collected from PRR diseased avocado roots in California, that are less sensitive to potassium phosphate. To help delay the development of phosphite-resistant P cinnamomi isolates, care should be taken to alternate or mix phosphite products with other effective fungicides with different mode of action to control avocado PRR. Phosphite and mefenoxam rotation with alternative fungicides is commonly used to prevent or reduce the emergence of Phytophthora resistant isolates , drainage collection pot however there are no other fungicides tested or registered to control P. cinnamomi in avocado. Fluopicolide and oxathiapiprolin are two new oomycete-targeted fungicides that have been tested for several Phytophthora spp., but not for P. cinnamomi . In this study, we report for the first time that the EC50 values for mycelial growth inhibition of P. cinnamomi avocado isolates are within the range of the EC50 values reported for other Phytophthora spp. using these two fungicides . Gray et al. recently reported the Fluopicolide and oxathiapiprolin EC50 values for severalPhytophthora species associated with citrus on California. Fluopicolide EC50 values for P. citrophthora, P. syringae, P. nicotianae, and P. hibernalis ranged from 0.031 to 0.087, 0.02 to 0.0461, 0.039 to 0.095, and 0.017 to 0.018 µg/ml, respectively. Of the four fungicides evaluated in this study, oxathiapiprolin had the lowest EC50 values . This range is similar to EC50 values reported for other Phytophthora spp. including P. sojae, another member of the phylogenetic clade 7, which includes P. cinnamomi . In agreement to our study, Gray et al. , also found that this fungicide had the lowest EC50 values among five different fungicides tested. The authors reported oxathiapiprolin EC50 values for P. citrophthora, P. syringae, P. nicotianae, and P. hibernalis ranged from 0.0002 to 0.0015, 0.0002 to 0.0003, 0.0003 to 0.001, and < 0.0003 µg/ml, respectively. Interestingly, in contrast to potassium phosphite sensitivity, the Southern isolates in this study exhibited lower EC50 values than the Northern A2 clade I isolates.
These results suggest the potential to rotate oxathiapiprolin with phosphonates and mefenoxam for controlling avocado PRR in California reducing the risk of the emergence of phosphonate-resistant P. cinnamomi isolates. Significant variation in pathogenicity and virulence among P. cinnamomi isolates collected from different host plants have been previously reported . Few cases have reported no differences in virulence among P. cinnamomi isolates collected from different hosts and unique host including one study in avocado . In this study we found significant differences in virulence among three representative P. cinnamomi isolates from the two A2 clonal groups identified by Paglaccia et al. when we inoculated a moderate resistant avocado rootstock but not the most susceptible one. The use of highly susceptible plants to distinguish between isolates with different levels of virulence is challenging and could explain why we did not detect significant differences in virulence among our isolates when we used a susceptible avocado rootstock . Although the mixture of the A2 clade II isolates were more virulent than the mixture of the A2 clade I isolates , we observed that the mixture of all the isolates together exhibited the same disease severity as the mixture of the less aggressive ones . This observation might be explained by the level of competition between these two groups of might reflect the induction of specific plant defense responses triggered by these two distinct groups of isolates, a more comprehensive study is required to test these two possibilities. 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, 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 .