AGRICULTURAL RESEARCH FOUNDATION FINAL REPORT FUNDING CYCLE

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1 AGRICULTURAL RESEARCH FOUNDATION FINAL REPORT FUNDING CYCLE TITLE: Efficacy of substrate calcium amendments for control of phytophthora root rot in ornamental crops RESEARCH LEADER: Dr. Luisa Santamaria, Assistant Professor, North Willamette Research & Extension Center, Aurora, OR COOPERATORS: Dr. Jerry Weiland, USDA-ARS-Horticultural Crops Research Laboratory, Corvallis, OR Dr. Carolyn Scagel, USDA-ARS-Horticultural Crops Research Laboratory, Corvallis, OR Gilbert Uribe, North Willamette Research & Extension Center, Aurora, OR SUMMARY: The purpose of this study was to determine whether gypsum substrate amendments are effective in managing phytophthora root rot. The project was timely and important because one of the pathogens used on this project is newly associated with dying nursery plants in the Oregon nursery industry. In addition to the direct loss associated with infected plants, diseased nursery stock may also face export restrictions to other states and/or countries. Recently, two soilborne pathogens (Phytophthora cinnamomi and P. plurivora) have been consistently isolated from diseased plants at several Oregon nurseries and identified as two of the Phytophthora spp. of most concern for crop loss (Parke et al., 2014). Phytophthora cinnamomi is an established pathogen in Oregon, but P. plurivora is a recently discovered species causing severe root rot on many plant hosts in European nurseries (e.g. Prospero et al., 2013). Both pathogens have wide host ranges. However, growers do not know the economic risk associated with these pathogens in their nurseries and there is little information about disease control treatments that are effective for managing these pathogens. Evidence suggests that calcium amendments in plant growth substrates have been effective in controlling P. cinnamomi root rot of avocado (Messenger, et al. 2000) possibly by directly affecting pathogen reproductive structure formation. Other studies suggest that calcium provides a nutritional advantage that allows for greater tolerance to P. cinnamomi in oaks in Spain (Serrano et al., 2013), and Sugimoto et al. (2010) found significant control of soybean stem rot caused by P. sojae in a field study in Japan. OBJECTIVES: Objective 1. Compare disease severity of the novel P. plurivora to that of the more established P. cinnamomi. 1

2 PROCEDURES: Objective 2. Evaluate effectiveness of varying concentrations of calcium amendments, in the form of gypsum, against inoculum of each pathogen to help establish guidelines for developing and implementing disease control strategies. Plant Host Care As the project got underway, it became apparent that it would be difficult to find a sufficient number of clean plants to conduct our trials. Nurseries either did not have enough, or were unwilling to sell rhododendrons for research purposes. We managed to acquire enough, tenmonth old, Rhododendron x Looking glass H-2 liner plants on May 1st, 2015 from an Oregon nursery (Photo 1) to set up our trial. They were placed in a greenhouse under mist for acclimation until they were transferred to gallon-sized pots four weeks later (Photo 2). After transferring them to gallon pots, the plants were fertilized with 50 ppm Jack s Professional Fertilizer approximately once per month until they were inoculated. The plants were transferred from the enclosed greenhouse to an open-roof greenhouse where the trial took place. The plants were hand irrigated twice per week until the plants were inoculated, after which point the plants were irrigated as needed. Potting Medium Preparation Pro-Pell-It, Pellitized Gypsum (Marion Ag Service Inc., Aurora, OR) was used for this trial. Four different gypsum treatments were prepared based on the gypsum-to-substrate volume percentage: 0% control, 1%, 5%, and 10% gypsum. The potting substrate used was a commercial blend of 75% bark, 30% peat moss, and 20% pumice (Pro Gro Mixes, Sherwood, OR). The gypsum-substrate mixture was prepared May 1st, 2015 and thoroughly mixed (Photo 3) in a cement mixer immediately prior to transplanting plants from liners to the gallon pots. The newly potted plants were again placed in the greenhouse and watered by hand as needed. Plants were misted on days with temperatures over 30ºC. Inoculum Preparation Isolates of P. plurivora and P. cinnamomi were obtained from the ARS-Pathology lab and maintained in PARP and PDA media until inoculum preparation. Batches of 50 g of rice were autoclaved in magenta jars twice, for 30 minutes at 24 hour intervals to eliminate any possible source of contamination (Photo 4). After cooling, five, 4mm plugs from a seven-day-old Phytophthora culture was added to the rice, along with 20 ml of sterile water. This process was repeated for both P. plurivora and P. cinnamomi. Inoculum was allowed to grow in rice for ten days before plant inoculation. Inoculation Process Plants were inoculated with P. plurivora on 10/27/15, and P. cinnamomi treatments were inoculated approximately one month later. This difference in time allowed us to avoid cross contamination and account for the slight difference in growth rates of the the two Phytophthora species. We prepared twenty batches of 50 g of inoculated rice. All the prepared inoculum was combined and mixed to ensure a more homogenous inoculum. This was repeated for each pathogen. A total of approximately 10 g of the prepared inoculum were added to each pot. For the inoculation, we made two holes in the substrate of each pot, 9-10 cm deep, placed opposite to 2

3 each other, and added 2.5 g of inoculum in each of two holes. The holes were approximately halfway between the edge of the pot and the base of the plant in an attempt to place the pathogen close to the radical system. An additional 5 g were spread and buried around the plant to create conducive pressure for the disease. The P. cinnamomi treatment plants were inoculated on 12/1/15 following the same procedures as above. Each gypsum treatment had 12 control plants, for which uninoculated, autoclaved rice was amended into the substrate following the same procedure as the Phytophthora inoculum. All other pathogen and gypsum treatments also contained 12 plants. Two plants per treatment had been included as backup, so only 10 plants were used for statistical analysis. Each plant served as one repetition. After inoculation, all pots were placed in 4 mil, low-density polyethylene bags to prevent the spread of pathogens within the greenhouse and help maintain higher levels of humidity within the pots (Photo 5). During the first week, the plant substrate was kept saturated, maintaining a water level up to approximately 2-3 inches up the pot, within the bag (Photo 6). Disease Severity Evaluations Visually, it was difficult to discern differences in treatment effect for plants from the different treatments (Photo 7). Due to the objective nature of using visual ratings as a method of disease severity, it was decided to evaluate treatment effects based on root and shoot dry mass. Plants were processed on 1/6/17. Shoots were collected (Photo 8) and placed in a Shel Lab Drying Oven, Model 1685 (Sheldon Manufacturing, Inc. Cornelius, OR) set to 60 C for one week before being weighed. Collecting only root tissue would have been too time consuming and impractical. Therefore, a consistent method of removing as much substrate from the root ball, without losing excessive root tissue, was used to indirectly measure root health. Healthier root systems retained more substrate within them, thereby providing the data used for analysis (Photo 9). The roots were also placed in a Shel Lab Drying Oven, Model 1685 (Sheldon Manufacturing, Inc. Cornelius, OR) and allowed to dry at 60 C for a period of two weeks before being weighed. Statistical Analysis All data was analyzed using R statistical software (R Core Team, 2015). Waller-Duncan K ratios were calculated using the package agricolae as per developer procedures (de Mendiburu, 2015). Shoot data were analyzed independently of root data. SIGNIFICANT ACCOMPLISHMENTS Disease damage among treatments was not evident after the first year and impractical to assess visually (Photo 7). As the trial progressed, it was learned from rhododendron growers that based on their experiences, this cultivar (Rhododendron x Looking glass H-2) was becoming known for being hardy and fairly resistant or tolerant to most diseases related with rhododendrons (personal communication). This same cultivar was used in another trial looking at foliar lesion formation, and in that case, achieving infection was also difficult. For this reason we decided to take advantage of this two-year grant to look at longer term effects. This factor was something we considered for the second year and we set up an additional trial (Photo 10) with a different cultivar (Rhododendron x Percy Wiseman ) known to be susceptible to many diseases. In speaking with other researchers we found that inducing symptoms can be a challenge, so some changes were made to the inoculation protocol in order to provide more conducive conditions, including incorporation of a 48-hour flooding event for all the plants (Photo 11). This trial is still ongoing and in order to compare data with the current trial, the pathogen must be allowed to become more established. An addendum with the results from this 3

4 additional, ongoing trial can be provided once the trial concludes within the next couple of months. Research Results Table 1. Statistical comparison of shoot dry mass for rhododendron plants challenged with different pathogens and amended with different levels of gypsum in their potting substrate x x Values represent mean dry mass (g) for each treatment. y Gypsum percentage calculated by volume of gypsum to total volume of the substrate. z Treatments sharing the same letter are not significantly different based on Waller-Duncan K-ratio t-test at K=100, which approximates to P=0.05. Minimum significant difference between treatments was Table 2. Statistical comparison of root dry mass for rhododendron plants challenged with different pathogens and amended with different levels of gypsum in their potting substrate x x Values represent mean dry mass (g) for each treatment. y Gypsum percentage calculated by volume of gypsum to total volume of the substrate. z Treatments sharing the same letter are not significantly different based on Waller-Duncan K-ratio t-test at K=100, which approximates to P=0.05. Minimum significant difference between treatments was

5 Figure 1. Mean, dry shoot and root mass for each gypsum and pathogen treatment. BENEFITS & IMPACT: When comparing the dry masses of the rhododendrons challenged with P. cinnamomi and the different gypsum treatments, shoot and root masses were significantly higher for plants treated with 10% gypsum, while the plants treated with 0% and 1% gypsum had the significantly lowest mass. For rhododendrons challenged with P. plurivora, those with the 5% and 10% gypsum amendment had significantly higher mass, while those with the 0% and 1% gypsum amendment had significantly lower mass for both roots and shoots. Finally, the rhododendrons with no pathogen also had significantly higher root mass for those with the 10% gypsum amendment, and significantly higher shoot mass for those with the 5% and 10% gypsum amendment, while those with the 0% and 1% gypsum amendment had significantly lower shoot and root mass. All means referred to as being the highest or the lowest were not significantly different from one another, regardless of the pathogen used. Conclusions In short, gypsum had a significant effect on a plant's shoot and root mass with 5% and 10% amendments being most favorable. We saw no evidence of differences in severity between P. cinnamomi and P. plurivora (Figure 1). Objective 1. Compare disease severity of the novel P. plurivora to that of the more established P. cinnamomi. Root and shoot values were not significantly different for plants challenged with each of the pathogen and no gypsum amendment, indicating that there is no difference in virulence or severity, from either pathogen, on this rhododendron cultivar (Table 1 & Table 2). Objective 2. Evaluate effectiveness of varying concentrations of calcium amendments, in the form of gypsum, against inoculum of each pathogen to help establish guidelines for developing and implementing disease control strategies. Although the plants challenged with P. plurivora and P. cinnamomi treated with 0% and 1% gypsum had lower means for their shoot mass than the control plants inoculated with sterile 5

6 rice, the differences were not significantly different (Table 1). However, across all pathogen treatments, it was very clear and significant that incorporating 5-10% gypsum into the substrate had a significantly improved effect on both root and shoot mass (Table 1 & Table 2). This was an unexpected result since Messenger et al. (2000) had reported no significant root mass differences among their avocado treatments with different amounts of gypsum. We plan to share our findings through either a peer reviewed publication or trade journal, to help growers make management decisions. ADDITIONAL FUNDING RECEIVED DURING PROJECT TERM: Additional, supplemental funding for this project was received as part of a larger, collaborative Floriculture and Nursery Research Initiative (FNRI) grant titled Improving plant health for nursery production in the Pacific Northwest Integration of detection tool development, fungal soil metagenome analysis, and improved soil health to manage Pythium and Phytophthora in PNW nurseries. FUTURE FUNDING POSSIBILITIES: We would like to seek additional funds to expand the gypsum amendment trials to other Rhododendron cultivars and simultaneously evaluate resistance to P. plurivora, as this could be of additional interest and benefit to the industry. Photo 1. Experimental plants, Rhododendron x Looking glass H-2 liners approximately ten months old. 6

7 Photo 2. Rhododendron x Looking glass H-2 liner plants transferred to 1 gallon containers after four weeks of acclimation under greenhouse conditions. Photo 3. Preparation of the substrate mix with different gypsum amendments. 7

8 Photo 4. Batches of 50 g of rice were inoculated to prepare inoculum for each of the pathogens tested in this trial. The batches of inoculated rice were mixed to prepare homogeneous inoculum Photo 5. Inoculated plants in the greenhouse, pots were placed in plastic bags to prevent leaching and potential contamination in the experimental area. 8

9 Photo 6. Excess water was provided to ensure substrate saturation during the first week. Photo 7. Plants challenged with P. cinnamomi with 1% gypsum amendment (left) compared to plants challenged with P. cinnamomi with 10% gypsum amendment (right). 9

10 Photo 8. Dr. Santamaria processing the shoots and roots before placing them in the drying oven. Photo 9. Processed roots and shoots prior to being placed in the drying oven. 10

11 Photo 10. Plants from the second trial, awaiting symptom development. Photo 11. Plants from the second trial during flooding. 11

12 REFERENCES Felipe de Mendiburu agricolae: Statistical Procedures for Agricultural Research. R package version Messenger, B.J., Menge, J.A., and E. Pond Effects of gypsum soil amendments on avocado growth, soil drainage, and resistance to Phytophthora cinnamomi. Plant Disease, 84(60): Parke, J.L., Knaus, B.J., Fieland, V.J., Lewis, C., and N.J. Grunwald Phytophthora community structure analyses in Oregon nurseries inform systems approaches to disease management. Phytopathology, 104(10): Prospero, S., Vercauteren, A., Heungens, K., Belbahri, L., and D. Rigling Phytophthora diversity and the population structure of Phytophthora ramorum in Swiss ornamental nurseries. R Core Team (2015). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL: Serrano, M.S., Fernández-Rebollo, P., De Vita, P., and M.E. Sánchez Calcium mineral nutrition increases the tolerance of Quercus ilex to Phytophthora root disease affecting oak rangeland ecosystems in Spain. Agroforestry Systems, 87: Sugimoto, T., Watanabe, K., Yoshida, S., Aino, M., Furiki, M., Shiono, M., Matoh, T., and A.R. Biggs Field application of calcium to reduce Phytophthora stem rot of soybean, and calcium distribution in plants. Plant Disease, 94(7):