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1 Comparative Look at Rust Infection and Resistance in Limber Pine (Pinus flexilis) and Rocky Mountain Bristlecone Pine (P. aristata) Following Artificial Inoculation at Three Inoculum Densities W.R. Jacobi 1, *, H.S.J. Kearns 2, A. Kegley 3, D.P. Savin 3, R. Danchok 3, and R.A. Sniezko 3 1 Colorado State University, Fort Collins, Colorado, USA 2 USDA Forest Service, Forest Health Protection, Sandy, Oregon, USA 3 USDA Forest Service, Dorena Genetic Resource Center, Cottage Grove, Oregon, USA * William.jacobi@colostate.edu INTRODUCTION Limber pine (Pinus flexilis) and Rocky Mountain bristlecone pine (Pinus aristata) are important components of forest ecosystems in the southern Rocky Mountains, United States. Both species are susceptible to white pine blister rust caused by the fungal pathogen Cronartium ribicola. However, very little is known about the relative resistance of the two species. In this study, we used artificial inoculation of seedlings to compare the relative susceptibilities of these two species. The objective of the inoculation test was to compare the performance of seedlings from bulk seedlots of limber pine and bristlecone pine exposed to three inoculum densities (low, medium, and high). This information will provide guidelines for inoculum density levels for future trials of these species and a first look at the extent of resistance present in the general population and its efficacy under exposure to increasing levels of inoculum. METHODS The seedlings were grown by the Colorado State Forest Service Nursery in Fort Collins, Colorado, USA from bulk seedlots collected from parent trees in the Rollinsville, Colorado, area. Limber pine seedlings were 3 years old and bristlecone pine 2 years old when transported by covered truck on August 14, 2003, to the U.S. Department of Agriculture, Forest Service, Dorena Genetic Resource Center (DGRC; Cottage Grove, Oregon, USA). Seedlings were transported and inoculated in 30-cell Styroblocks (Beaver Plastics, Acheson, Alberta, Canada). A randomized complete block design with three replications of three treatments for the two species was used for inoculation and outplanting. Thirty limber pine seedlings were used in each block-by-treatment combination for a total of 90 seedlings per treatment. Twenty-four bristlecone pine seedlings were used per treatment block for a total of 72 seedlings per treatment. The limber pine seedlings were grown in removable containers placed in Styroblocks, and individual seedlings were randomly assigned to the treatment blocks. Seedling numbers were equalized (30 seedlings per treatment block) among the blocks. Limber pine seedlings were also spaced out (15 seedlings per Styroblock) to increase the likelihood of infection. The bristlecone seedlings, however, were directly planted into the Styroblocks, so an entire Styroblock was randomly assigned to a treatment block. Trees were inoculated on September 18, 2003, with naturally infected Ribes leaves placed above the trees (Ribes species are the main alternate host). The In: Schoettle, Anna W.; Sniezko, Richard A.; Kliejunas, John T., eds Proceedings of the IUFRO joint conference: Genetics of five-needle pines, rusts of forest trees, and Strobusphere; 2014 June 15 20; Fort Collins, CO. Proc. RMRS-P-76. Fort Collins, CO: U.S. Department of Agriculture, Forest Service, Rocky Mountain Research Station. 245 p. Papers published in these proceedings were submitted by authors in electronic media. Editing was done for readability and to ensure consistent format and style. Authors are responsible for content and accuracy of their individual papers and the quality of illustrative materials. Opinions expressed may not necessarily reflect the position of the U.S. Department of Agriculture. USDA Forest Service RMRS-P

2 Figure 1 Limber pine seedlings with stem symptoms and mortality following inoculation by C. ribicola at three inoculum spore densities. Figure 2 Bristlecone pine seedlings with stem symptoms and mortality following inoculation by C. ribicola at three inoculum spore densities. infected Ribes leaves were collected from forest stands in Oregon and Washington (USA) and represent nonvcr2 sources. Treatments consisted of three target inoculum densities: low (3,000 basidiospores cm -2 ), medium (6,000 basidiospores cm -2 ), and high (9,000 to 10,000 basidiospores cm -2 ). At DGRC, 3,000 spores cm -2 is the density used in operational inoculations of western white pine (P. monticola); 6,000 spores cm -2 is the density used in inoculation trials of sugar pine (P. lambertiana). When the targeted inoculum 152 USDA Forest Service RMRS-P

3 density was reached, the Ribes leaves were removed from that treatment. The actual inoculation densities achieved were somewhat higher than the target (3,260, 8,227, and 12,053 spores cm -2 ) (table 1). Basidiospore germination was monitored at inoculation and was at least 90 percent for all treatments except the rep one high density, which was 52 percent. Seedlings were transplanted in October 2003 to three standard DGRC outdoor planting boxes (0.9 m wide 1.2 m long 0.3 m high). One box was used per species replication, and there were three contiguous rows of 10 limber pine and 8 to 9 bristlecone pine seedlings per treatment. Seedlings were assessed for needle lesions (spots) twice during the first year and for stem symptoms yearly for 5 years and then at 10 years. 89 percent of seedlings exhibiting needle spots at the low inoculum density, 98 percent at medium density, and 90 percent at high density. In Rocky Mountain bristlecone pine the mean was 35 percent of seedlings with needle spots, with 23 percent at low inoculum density, 42 percent at medium density, and 45 percent at high density. Rocky Mountain bristlecone pine seedlings had a fairly high percentage of primary needles with needle spots (34 to 69 percent over the three densities), whereas limber pine had only 3 to 7 percent of seedlings with primary needle spots. This difference is most likely because the limber pine seedlings were 3 years old and had relatively few primary needles present compared to the 2-year-old bristlecone pine seedlings. RESULTS At the first-year assessments, we noted that inoculum spore density did not affect measures of infection on limber pine as much as Rocky Mountain bristlecone pine (table 2). The effect of inoculum density could be noted in bristlecone pine after 2 years, but limber pine was so susceptible that the low spore density caused as much infection as the high concentration (figs. 1 and 2). We also found that averaged over all spore densities at 1 year post-inoculation, limber pine exhibited a much higher percentage (61 percent) of stems with symptoms than bristlecone pine (26 percent). By 10 years post-inoculation, 83 percent of limber pine and 76 percent of bristlecone pine seedlings exhibited stem symptoms, with little change from the 5-year assessment (figs. 1, 2, 3 5; table 2). There was little or no change in percentage of stems infected after year 2, so we assume there was little to no natural infection from spores in the DGRC area between year 1 and 10. Aecia production occurred by year 3 on many seedlings (figs. 5 and 6). On average, 83 percent of limber pine and 74 percent of bristlecone pine seedlings were dead 10 years post-inoculation, and the temporal dynamics of mortality were similar (figs. 1 and 2). At the first assessment, 10 months post-inoculation, limber pine showed a much higher incidence of needle spots per tree (15 to 23; fig. 6) compared to the 1 to 2 spots per tree on Rocky Mountain bristlecone pine at the three inoculum densities (table 2). The percentage of seedlings with spots on secondary needles was also much higher in limber pine (mean of 92 percent), with SUMMARY AND DISCUSSION The bulk seedlots from the Rollinsville, Colorado, area produced Rocky Mountain bristlecone and limber pine seedlings that were very susceptible to white pine blister rust. The levels of inoculum density utilized influenced infection levels in bristlecone but not limber pine. The lowest inoculum density level used here (~3,260 spores cm -2 ) may be adequate in future trials for limber pine, but Rocky Mountain bristlecone pine is likely to require higher inoculum densities. Over all inoculum densities, limber pine had a higher percentage (61 percent) of stems infected than bristlecone (26 percent) 1 year after inoculation, but by year 5 this was narrowed to 83 and 75 percent, respectively, indicating that latent canker development (a possible partial resistance trait) was more common in bristlecone pine (table 2). Both species had latent infections developing after those present 1 year post-inoculation. The 26-percent survival by bristlecone and 17-percent survival of limber pine at year 10 may indicate a source of resistance in the source population as seen by subsequent tests (Schoettle et al. 2011, 2014). However, only a few inoculated seedlings with stem infections survived the duration of the trial, indicating limited occurrence of bark resistance. The canker-free seedlings in limber pine may be predominantly due to major gene resistance (Schoettle et al. 2014). Major gene resistance has been noted in limber pine (Schoettle et al. 2014) and would not generally be influenced by inoculum density, but this type of USDA Forest Service RMRS-P

4 Table 1 Mean inoculation time, inoculum density, and basidiospore germination percentage (+ 1 standard error) for three treatments of bristlecone and limber pine. Treatment Duration (h) Mean inoculum density % basidiospore germination Low Medium High Table 2 Average number of spots and stem symptoms per tree and percentages of pine seedlings with stem symptoms and killed by white pine blister rust infections. Inoculum density Year one needle spots/tree Year one stem symptoms/tree % seedlings with stem symptoms / % mortality year one year five year ten limber bristlecone limber bristlecone limber bristlecone limber bristlecone limber bristlecone low / 0 11/ 0 84/ 78 62/ 53 84/ 83 64/ 58 Medium / 0 31/ 0 89/ 84 77/ 73 89/ 88 78/ 77 High / 0 36/ 0 77/ 76 87/ 84 77/ 77 87/ USDA Forest Service RMRS-P

5 Figure 3 Limber pine seedling 1 year after inoculation with needle spots. Figure 4 Limber pine seedling 1 year after inoculation with stem symptoms and needle spots. USDA Forest Service RMRS-P

6 Figure 5 Limber pine with aecia 3 years after inoculation. Figure 6 Bristlecone pine 3 years after inoculation with aecia on the stem. resistance has not been confirmed in bristlecone pine. The increasing level of infection in bristlecone pine with increasing inoculum density may suggest that partial resistance is present and can be at least somewhat eroded at very high inoculum densities. Future trials should investigate even higher levels of inoculum density to ascertain whether some level of resistance is maintained in Rocky Mountain bristlecone pine under extreme pathogen pressure. The inoculum levels used at DGRC may need adjustment depending on the seedling culture (seedling physiological state) and local environment pre- and post-inoculation. Primary needles are more susceptible than secondary needles. Nursery protocols that provide young seedlings with predominantly secondary needles, which are likely to give the best correspondence with field resistance, should be developed. Artificial inoculation trials to examine blister rust resistance using seed from individual parent tree collections (half-sib seedlots) has begun for seedlings of both limber pine and Rocky Mountain bristlecone pine (Schoettle et al. 2011, 2014; Sniezko et al. 2011, 2016). Such trials will help delineate the level and type of partial resistance, as has been noted in other species (Sniezko et al. 2014). Field trials of these two species to validate resistance from seedling trials, like those established for P. monticola, P. lambertiana, and whitebark pine (P. albicaulis), are needed. Several small trials have recently been established (see Schoettle et al., this proceedings, Southern Rockies Rust Resistance Trial). If confirmed in other tests, the level of resistance found in the bulk seedlots used in this study is encouraging, and the information can be used by land managers for restoration efforts. 156 USDA Forest Service RMRS-P

7 REFERENCES Schoettle, Anna W.; Sniezko, Richard A.; Kegley, Angelia; [et al.] Preliminary overview of the first extensive rust resistance screening tests of Pinus flexilis and Pinus aristata. In: Keane, R.E.; Tomback, D.F.; Murray, M.P.; [et al.], eds. The future of high-elevation, five-needle white pines in western North America: Proceedings of the high five symposium. Proceedings RMRS-P-63. Fort Collins, CO: U.S. Department of Agriculture, Forest Service, Rocky Mountain Research Station: Schoettle, Anna W.; Sniezko, Richard A.; Kegley, Angelia; [et al.] White pine blister rust resistance in limber pine: Evidence for a major gene. Phytopathology. 104(2): Sniezko, Richard A.; Danchok, Robert; Savin, Douglas P.; [et al.] Genetic resistance to white pine blister rust in limber pine (Pinus flexilis): Major gene resistance in a northern population. Canadian Journal of Forest Research. 46 (9): doi: / cjfr Sniezko, Richard A.; Mahalovich, Mary Francis; Schoettle, Anna W.; [et al.] Past and current investigations of the genetic resistance to Cronartium ribicola in high-elevation five-needle pines. In: Keane, R.E.; Tomback, D.F.; Murray, M.P.; [et al.], eds. The future of high-elevation, five-needle white pines in western North America: Proceedings of the high five symposium. Proceedings RMRS-P-63. Fort Collins, CO: U.S. Department of Agriculture, Forest Service, Rocky Mountain Research Station: USDA Forest Service RMRS-P