Influence of Nitrogen Fertility on the Susceptibility of Rhododendrons to Phytophthora ramorum

Rita L. Hummel, Marianne Elliott, Gary Chastagner, Robert E. Riley, Kathy Riley, and Annie DeBauw, Washington State University, Puyallup Research and Extension Center, 2606 W. Pioneer, Puyallup, WA 98371; klriley@wsu.edu

Research information demonstrating the effects of various cultural practices on host susceptibility to Phytophthora  ramorum is generally lacking and thus limits the development of an integrated approach to managing diseases caused by this pathogen in irrigated nursery systems.  Rhododendron spp. have accounted for about 78% of the plants associated with P. ramorum-positive nursery finds in Washington State. Nitrogen fertility levels have been reported to influence disease in some Phytophthora disease pathosystems, but data is not available for the P. ramorum-rhododendron pathosystem.

During 2008 we investigated the dynamics between nitrogen application rates and the susceptibility of ‘English Roseum’, ‘Cunningham’s White’ and ‘Compact P.J.M.’ to P. ramorum.  One-gallon plants were potted into 3-gallon containers.  The growing medium was 100% Douglas-fir bark with micromax incorporated at the rate of 1.75 lbs/yd3. Plants were placed on a gravel nursery bed and watered as needed with overhead sprinkler irrigation.  Residual fertilizer in the media was depleted, then ammonium nitrate fertilizer at 100, 300 and 600 ppm N was applied in liquid form twice a week to each of eight plants per cultivar starting on June 2nd.  The same rate of P (potassium phosphate) and K (potassium sulfate), 100 and 200 ppm, respectively, was applied at each fertilization.  Commencing with fertilizer application, the plants were switched to a drip irrigation system.  In early October, plant growth, visual quality and leaf color were measured. At the same time, fully mature, current season leaves from each plant were harvested for determination of leaf tissue nitrogen content and P. ramorum inoculations.

Six detached leaves from each plant were inoculated with suspensions of zoospores from an NA1 lineage rhododendron isolate of P. ramorum by pipetting a 10 ul drop of suspension onto the lower leaf surface. The leaf tissue beneath drops on three leaves was injured using an insect pin, while the tissue beneath each drop on the other leaves was left unwounded. The leaves were then incubated at 19-20 C.

As expected, shoot growth and plant quality indices increased with nitrogen fertility. Based on an overall analysis of lesion size after 10 days, there was a significant difference in the susceptibility of the three cultivars to P. ramorum. “Compact P.J.M.’ had the smallest lesions, while ‘English Roseum’ had the largest. Lesions developed on all the wounded and unwounded inoculation sites on the ‘English Roseum’ and lesion size increased with increasing nitrogen fertility. Nitrogen fertility had no effect on lesion size on the other two cultivars.

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Phenotypic variation in Phytophthora ramorum: wild type vs non-wild type isolates 

M. Elliott, Washington State University, Puyallup Research and Extension Center, Puyallup, WA, USA 98371; melliott2@wsu.edu ; G. Sumampong, S. F. Shamoun, E. Becker, Canadian Forest Service, Pacific Forestry Centre, Victoria, BC, Canada V8Z 1M5; A. Varga, D. James, S. Masri, Sidney Laboratory, Canadian Food Inspection Agency, Sidney, BC, Canada V8L 1H3; and N. J. Grünwald, Horticultural Crops Research Laboratory, USDA ARS, Corvallis, OR, USA 97330

In earlier studies unusual culture morphology and behavior were noticed among some NA1 isolates of Phytophthora ramorum. This “non-wild type” behavior was not observed in our collection of isolates from the EU1 or NA2 lineages, even though the isolates had been in culture for a similar amount of time. It has been suggested that subculturing in vitro cause culture instability and loss of virulence, and passage through the host can revive the isolate back to its original state.  To study this, we compared four less virulent isolates (non-wild type; nwt) with four isolates of normal virulence (wild type; wt) in our culture collection.  One objective of this study was to determine whether wt behavior could be restored to nwt isolates of P. ramorum by successive re-isolation from host material. Detached leaves of rhododendron “Cunningham’s White” were inoculated with each of the isolates, which were re-isolated and tested for pathogenic aggressiveness, growth rate at maximum, optimum, and minimum temperatures, and chlamydospore production in vitro.

In both wt and nwt groups, there were significant differences in lesion size on detached rhododendron leaves between the original culture and the first re-isolation. Successive re-isolations were not different from the original culture and the first re-isolation. After re-isolation from the host, nwt isolates were still less aggressive than wt isolates. Along with lower aggressiveness on rhododendron leaves, nwt isolates produced fewer chlamydospores in V8 agar than did wt isolates.  There was no difference in growth rate between the original culture and the first re-isolation for most isolates.  However, nwt isolates were found to be more sensitive to temperatures below 2°C and above 28°C. The optimum growth temperature was 20°C.

Our preliminary results show that non-wild type isolates were more variable than wild type isolates in all of the characters tested, and were generally lower in aggressiveness, chlamydospore production, and growth rate at all temperatures for both the original culture and when re-isolated from a host. The greater variability suggests that these isolates are unstable or that slightly deleterious mutation(s) have accumulated in accordance with Muller’s ratchet resulting in reduced fitness. Wild type isolates performed better than non-wild type isolates in all of the phenotypic characters examined. Why nwt survives and proliferates is still a mystery. To understand the cause of these phenotypic differences, the role of cytoplasmic elements and differences in mitochondrial and nuclear DNA are being examined.

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Spread of Phytophthora ramorum to water, soil, and vegetation outside a nursery in Pierce County, Washington
G. CHASTAGNER (1), K. Coats (1), M. Elliott (1)
(1) Washington State University, Puyallup, WA, U.S.A.
Phytopathology 101:S32

Volunteer stream monitoring for invasive Phytophthora species in western Washington
M. ELLIOTT (1), G. Chastagner (1), K. P. Coats (1), A. DeBauw (1), K. Riley (1)
(1) Washington State University, Puyallup Research and Extension Center, Puyallup, WA, U.S.A.
Phytopathology 101:S48

Mystery on the Sammamish: What are the sources of Phytophthora ramorum infesting this Washington State waterway?
G. Chastagner (1), K. COATS (1), D. Omdal (2), A. Ramsey-Kroll (2), M. Elliott (3)
(1) Washington State University, Puyallup, WA, U.S.A.; (2) Washington State Department of Natural Resources, Olympia, WA, U.S.A.; (3) Washington State University, Puyallup Research and Extension Center, Puyallup, WA, U.S.A.
Phytopathology 101:S32