Comparative Host Susceptibility and Sporulation Potential of Phytophthora ramorum on Species, Cultivars, and Hybrids of Camellia

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1 2007. Plant Management Network. This article is in the public domain. Accepted for publication 10 April Published. Comparative Host Susceptibility and Sporulation Potential of Phytophthora ramorum on Species, Cultivars, and Hybrids of Camellia Robert G. Linderman, Research Plant Pathologist, and E. Anne Davis, Biological Laboratory Technician, USDA-ARS Horticultural Crops Research Laboratory, Corvallis, OR Corresponding author: Robert G. Linderman. Linderman, R. G., and Davis, E. A Comparative host susceptibility and sporulation potential of Phytophthora ramorum on species, cultivars, and hybrids of camellia. Online. Plant Health Progress doi: /php rs. Abstract Phytophthora ramorum, causal agent of ramorum blight of woody shrub species, has caused serious damage to cultivars and species of camellia in commercial nurseries. Reports of relative susceptibility of camellia to P. ramorum have indicated a range from high to low susceptibility, both in nurseries and under experimental conditions. We inoculated a series of cultivars of camellia to determine their relative susceptibility to infection, and then compared lesion size to the capacity of the pathogen to produce sporangia on the lesions. We found, as did others, a wide range of susceptibility among cultivars, but lack of correlation between susceptibility (lesion size) and potential to produce sporangia that might spread the pathogen within the nursery. These results indicate that on some cultivars the pathogen might produce small or inconspicuous lesions, yet still produce copious numbers of sporangia that could spread the disease, both within the nursery and from nursery to nursery. Introduction Ramorum blight, caused by the pathogen Phytophthora ramorum (21), has been reported on a wide range of trees, shrubs, and ornamental plants in nurseries and landscapes (20). As a result the disease and the pathogen are regulated by quarantine to prevent geographic dissemination (10). Numerous plant species have been shown to be susceptible (2,20), including camellia (1,13,16,19). Currently, nurseries are inspected to detect infected plants and thereby prevent dissemination of the disease geographically (9). However, the pathogen has been disseminated geographically from nursery to nursery on camellia and other host plants. In the United States, national nursery surveys were performed in 48 states in 2005, and seven states were found to have nurseries with plants that tested positive for P. ramorum (10). In 2004 infected camellia plants were found in nurseries in 22 states, shipped from supply nurseries on the west coast. How that dissemination occurred, in spite of inspections, remains unknown. One explanation, however, is that the pathogen spread within the disseminating nurseries and caused insignificant lesions on camellia leaves that were not detected. Shishkoff (16) described the symptoms and susceptibility of species and cultivars of camellias, and indicated that sometimes lesions were small, and on some cultivars the symptoms were difficult to see on the upper leaf surfaces. Furthermore, infected leaves on camellia often abscised and were no longer on the plants during inspections. If sporangia were produced on leaves that had fallen onto the medium surface, they could have washed down onto the roots, causing root infections that eventually progressed into the shoots, such as was shown for rhododendron (5). The purpose of this study was to test the hypothesis that some camellia cultivars were more susceptible than others, and that variation also occurred in the potential for the pathogen to produce sporangia on lesions on different genotypes of camellia.

2 Inoculum Production, Inoculation, Disease Evaluation, and Sporulation Potential The P. ramorum used in this study, N10-A (N10A), was isolated from rhododendron (Rhododendron sp.) in an Oregon nursery, and is a North American genotype of the A-2 mating type. Cultures were maintained and stored under refrigeration on agar slants until used, and then were transferred frequently on dilute V8 juice agar plates (30 ml/liter of clarified V8 juice instead of the normal 150 to 200 ml/liter) (6). Sporangia to be used as inoculum were produced on dilute V8 juice agar plates (150-mm diameter), starting from a sporangial suspension spread on the plates that were then incubated in a dark incubator at 20 C for 8 days. Sporangia were removed from the plates by flooding with 5 ml of sterile distilled water and scraping the surface of the agar with the edge of a spatula. The aqueous suspension of cauducous sporangia was then poured into a beaker and gently swirled using a magnetic stirrer. Sporangia were used to inoculate leaves from cultivars of Camellia japonica and C. susanqua, and six camellia hybrids (Fig. 1). Three leaves were randomly taken from each of 3 plants per cultivar grown in one-gallon containers. Just prior to inoculation, each leaf was wounded once on the abaxial side, away from the midvein, using a needle probe. A 10-µl drop of sporangial suspension containing approximately 2600 sporangia per ml was pipetted into the wound. The concentration of sporangia (or released zoospores) was determined by plating on PARP medium (4,14) and counting colonies. A single culture plate of N10A was used to inoculate 6 cultivars at a time. Inoculated leaves were placed in plastic box chambers with moist vermiculite in the bottom to maintain high humidity and free water on the leaf surface. Inoculated leaves in boxes were allowed to sit for 5 h in a 20 C incubator, after which they were manually sprayed with distilled water, enough to wet leaves to provide some free-standing droplets. The sealed containers were then placed in a 20 C incubator with a 14-h light regime (average 33 µmol/sec/m) to incubate for 7 days. Leaves were misted daily to maintain free water on the surface for 6 days. Boxes were rotated 180 degrees daily to balance light distribution among replications during the entire incubation time. Quantification of sporulation and lesion size was done on day 8.

3 < Fig. 1. Lesion size and sporangia production by camellia cultivars 7 days after inoculation of detached leaves with Phytophthora ramorum N10A. Data are the means of 3 leaves per plant, 3 plants per cultivar. Means that do not overlap at the 95% confidence interval are considered statistically different. Hybrid cultivar parentage where known: (i) April Remembered : C. japonica Berenice Boddy C. japonica Dr. Tinsley ; (ii) Coral Delight :C. japonica C. saluensis; (iii) Spring's Promise : C. japonica Berenice Boddy C. japonica Kumasaka ; (iv) Taylor's Perfection : C. japonica C. saluensis; (v) Winter's Fire : [C. hiemalis Shishigashira C. oleifera Plain Jane ] C. vernalis Takarazuka ; (vi) Winter's Snowman : C. sasanqua Nurumigata C. hiemalis Shishi-gashira ] C. oleifera Plain Jane.

4 Fig. 2. Lesions and sporangia produced by Phytophthora ramorum N10A on the leaf surface of Camellia japonica Tom Knudsen. Top to bottom: increasing magnification of lesion area. Fig. 3. Variation in lesion size on different camellia genotypes inoculated with sporangia of Phytophthora ramorum N10A. Left to right columns: 3 replicate leaves (rows) of Bob Hope, Coral Delight, and Kanjiro. To quantify sporulation on lesions, each leaf was placed sideways in a deep Petri dish filled with 30 ml of sterile distilled water, so the lesion was submerged. If a lesion area encompassed a large part of a leaf, then the entire leaf was laid flat. The petiole was held out of the water with forceps. With the end of a smooth glass rod, the lesion was rubbed gently on both sides of the leaf to remove sporangia. Pre-trials indicated that these sporangia are not dislodged from the camellia leaf abaxial surface by vortexing or vigorous shaking, and they were almost never seen on the adaxial surface. Each leaf was briefly rinsed with distilled water in a wash bottle to retrieve any adhering sporangia before being replaced in its incubation box. Collected sporangial fluid in the petri dish was decanted through a disposable funnel into a 50-ml polypropylene centrifuge tube, then centrifuged for 3 min at 7500 rpm (4792 g n ) to form a concentrated pellet. The supernatant was drawn off to 5 ml with a pipet and the remaining pellet vortexed for 8 sec to re-suspend the sporangia. Twenty µl were spread evenly over selective PARP medium plates and colonies counted after 2 days incubation at 20 C. Sporangia production was then normalized in relation to lesion size.

5 Lesion area, as a percentage of each total leaf area, was assessed quantitatively on each leaf the same day as sporangia were collected, using digital photographs and ASSESS disease quantification software (American Phytopathological Society, St. Paul, MN). Sporangia counts were correlated to lesion area and are reported as sporangia per square centimeter. Data from two trials, repeated in time, were combined since variance among trials was homogenous by Bartlett s test using Systat 11 (Systat Software Inc., Richmond, CA). Lesion percentage data were transformed to arcsine-square root values and sporangia counts were log-transformed prior to analyses to normalize variances. Transformed data were analyzed by one-way analyses of variance (ANOVA), but real data are presented in our results. Mean comparison tests were not used due to the extreme range of the data. Instead, treatment means of lesion percentages or sporangia production were compared on each cultivar using strict 95% confidence intervals. Means that did not overlap at the 95% confidence interval were considered statistically different. Variation in Camellia Susceptibility and Pathogen Sporulation Generally, camellia leaves developed dark lesions with defined margins, which were more easily seen on the lower than the upper leaf surface. The cultivars within C. japonica and C. sasanqua and several hybrids varied greatly in their susceptibility to inoculation with P. ramorum (Fig. 1). Cultivar effect on variability in lesion size and sporangia production was highly significant (P < 0.001), but comparing camellia species or hybrids by group did not indicate significant differences in sporangia production. Such variability has been reported for several other host groups (10,18), and was shown by Shishkoff (16) to occur within genotypes of camellia. However, what had not been reported previously was the variation in the capacity of the pathogen to produce sporangia on infected camellia tissue. McDonald et al. (8) reported significant variation in sporulation potential on cultivars of rhododendron, however. Tjosvold et al. (17) recently reported that P. ramorum can sporulate on infected camellia flower buds. Also, Parke et al. (12) reported that different plant species had different capacities to support sporulation in an inoculated leaf disk assay. Davidson et al. (3) demonstrated that Bay Laurel in the forests of California produces abundant sporangia from very small lesions on leaves, making that plant a key component in the epidemiology of the sudden oak death syndrome there. Our finding on camellia has significance in the epidemiology of the disease in the nursery as well as in efforts to detect the disease prior to shipping plants. Our findings confirm the hypothesis on which this study was based, that is that different genotypes of camellias have different capacities to produce secondary inoculum that can spread from plant to plant within the nursery, or wash into the pot medium, possibly infecting roots without being detected visually as foliage symptoms. Laboratory studies have shown that chlamydospores and sporangia can survive extended periods of time in growth media (7), and contaminated containers can be shipped, thereby increasing the likelihood of disseminating the pathogen geographically. Since such a high degree of variation occurred in the capacity of the pathogen to produce sporangia on the lesions, with some small lesions producing as many or more sporangia than larger lesions, we conclude that sporulation potential on different host genotypes was independent of lesion size and therefore presumably was related to host genetic factors. Acknowledgements and Disclaimers We acknowledge the excellent assistance from Bryan Beck in this project. Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the USDA and does not imply its approval to the exclusion of other products or vendors that also may be suitable. Literature Cited 1. Beales, P. A., Brodensire, T., Barnes, A. V., Barton, V. C., and Hughes, K. J. D First report of ramorum leaf blight and dieback (Phytophthora ramorum) on Camellia spp. in the UK. Plant Pathol. 53:524.

6 2. Davidson, J. M., Werres, S., Garbelotto, M., Hansen, E. M., and Rizzo, D. M Sudden oak death and associated diseases caused by Phytophthora ramorum. Online. Plant Health Progress doi: /php dg. 3. Davidson, J. M., Wickland, A. C., Patterson, H. A., Falk, K. R., and Rizzo, D. M Transmission of Phytophthora ramorum in mixed-evergreen forest in California. Phytopathology 95: Kannwischer, M. E., and Mitchell, D. J The influence of a fungicide on the epidemiology of black shank of tobacco. Phytopathology 68: Lewis, C. D., Roth, M. L., Choquette, C. J., and Parke, J. L Root infection of rhododendron by Phytophthora ramorum. (Abstr.) Phytopathology 94:S Linderman, R. G., and Davis, E. A Response of selected nursery crop plants to inoculation with isolates of Phytophthora ramorum and other Phytophthora species. HortTechnology 16: Linderman, R. G. and Davis, E. A Survival of Phytophthora ramorum compared to other species of Phytophthora in potting media components, compost, and soil. HortTechnology 16: McDonald, V., Grunwald, N. J., and Linderman, R. G Evaluation of infection potential and sporulation of Phytophthora ramorum on five Rhododendron cultivars. Online. (Abstr.) Abstr. of the Joint Meet. of the Amer. Phytopathol. Soc., the Can. Phytopathol. Soc., and the Mycolog. Soc. of Amer., July 29-Aug. 2, 2006, Québec City, Canada. American Phytopatholocical Society, St. Paul, MN. 9. Osterbauer, N. K., Griesbach, J. A., and Hedberg, J Surveying for and eradicating Phytophthora ramorum in agricultural commodities. Online. Plant Health Progress doi: /php rs. 10. Palmieri, K., and Frankel, S. J Sudden Oak Death and Phytophthora ramorum: A Compilation of the 2005 COMTF Monthly Newsletters. California Oak Mortality Task Force, Univ. of Calif., Berkely, CA. 11. Parke, J. L., Linderman, R. G., and Hansen, E. M Susceptibility of Vaccinium species and cultivars to Phytophthora ramorum, cause of sudden oak death. Phytopathology 92:S Parke, J. L., Hansen, E. M., and Linderman, R. G Sporulation potential of Phytophthora ramorum on leaf disks from selected hosts. Proc. of Sudden Oak Death Sci. Symp., Monterey, CA. Integ. Hardwood Range Manage. Prog., Agric. and Natur. Resources, Univ. of Calif. 13. Parke, J. L., Linderman, R. G., Osterbauer, N. K., and Griesbach, J. A Detection of Phytophthora ramorum blight in Oregon nurseries and completion of Koch s Postulates on Pieris, Rhododendron, Viburnum, and Camellia. Plant Dis. 88: Ribeiro, O. K A Source Book of the Genus Phytophthora. J. Cramer, Vaduz, Fla. 15. Rizvi, R., and Inman, A Phytophthora ramorum: Susceptibility and sporulation potential of some British heathland plants, especially Vaccinium species, in relation to risk. Rep. of the Central Sci. Lab., Dept. for Environ., Food and Rural Affairs, UK. 16. Shishkoff, N Susceptibility of Camellia to Phytophthora ramorum. Online. Plant Health Progress doi: /php rs. 17. Tjosvold, S. A., Chambers, D. L., Thomas, S. L., and Blomquist, C. L First Report of Phytophthora ramorum infecting Camellia flower buds in North America. Online. Plant Health Progress doi: /php br. 18. Tooley, P. W., Kyde, K. L., and Englander, L Susceptibility of selected ericaceous ornamental host species to Phytophthora ramorum. Plant Dis. 88: Tubajika, K. M., Bulluck, R., Shiel, P. J., Scott, S. E., and Sawyer, A. J The occurrence of Phytophthora ramorum in nursery stock in California, Oregon, and Washington states. Online. Plant Health Progress doi: /php RS. 20. USDA-APHIS List of regulated hosts and plants associated with Phytophthora ramorum. Online. USDA-APHIS, Washington, DC. 21. Werres, S., Marwitz, R., Man In t Veld, W. A., De Cock, W. A. M., Bonants, P. J. M., DeWeerdt, M., Themann, K., Ilieva, E., and Baayen, R. P Phytophthora ramorum sp. nov: A new pathogen on Rhododendron and Viburnum. Mycol. Res. 105: