Interaction between Diplodia pinea and D. scrobiculata in red and jack pine seedlings.

Sphaeropsis sapinea sensu lato is a conifer fungal pathogen that causes shoot blight and stem cankers. Recently, the former S. sapinea has been divided into two species, Diplodia pinea and D. scrobiculata. The aims of the study were to determine the contribution of each species in disease development on red and jack pines by means of co-inoculations and molecular identifications, and to evaluate how the presence of each species affects the development and aggressiveness of the other. Symptom severity (distance below the inoculation site at which necrotic needles were observed) and identification length (the maximum distance from inoculation site from which either D. pinea or D. scrobiculata was identified using molecular methods) were recorded 4 weeks after inoculating wounded seedlings with agar plugs colonized by these pathogens. The results suggested that D. pinea was much more aggressive on both hosts than D. scrobiculata. When a seedling was co-inoculated with these pathogens, the symptom development appeared to be mainly due to D. pinea. The presence of D. pinea also interfered with the establishment of D. scrobiculata in the plant tissue. However, D. scrobiculata showed antagonism toward D. pinea. When both pathogens co-occurred in a single seedling, symptom severity caused by D. pinea was less than when D. pinea alone was present.

Shoot Blight Caused by Sirococcus tsugae on Eastern Hemlock (Tsuga canadensis) in Georgia.

Eastern hemlock (Tsuga canadensis) is an ecologically and economically important conifer from the north-central United States to the east coast of North America to the southern Appalachian Mountains. In early spring 2010, blighted shoot tips of eastern hemlock were observed at widely separated locations in the Chattahoochee National Forest in north Georgia. Damage did not appear to be directly related to hemlock woolly adelgid (Adelges tsugae) activity, which was sporadic or absent in some areas where symptoms were observed. A preliminary survey in March 2010 revealed that incidence of blighted shoots on individual trees varied, but was as high as 70%. Stems of shoots produced the previous year were frequently necrotic, had lost needles, and bore pycnidia with hyaline, two-celled conidia consistent with those of Sirococcus tsugae (2,3). Later in the spring and summer, shoots of the current year's growth became blighted, with sporulation of S. tsugae also on dead and dying needles. While S. tsugae previously has been reported on T. heterophylla, T. mertensiana, Cedrus atlantica, and C. deodara in western North America, it has only recently been reported on eastern hemlock (1), and its ability to induce shoot blight has not been proven. Pure cultures (2,3) were obtained on streptomycin-amended potato dextrose agar (PDA) and their identity was confirmed by species-specific PCR primers (4). Nuclear rDNA internal transcribed spacer sequence (554 nucleotides) also was obtained for isolate 10-05 and deposited in GenBank (Accession No. HQ256769). This sequence was found to be identical to sequences previously deposited for S. tsugae isolates. Isolate 10-05 and a second isolate (10-06) were used to inoculate potted 2-year-old eastern hemlock seedlings in a growth chamber at 20°C with a 16-h photoperiod. Conidia were collected by flooding 1-month-old colonies on PDA with sterile water. Expanding shoots on one branch of each seedling were wounded using scissors to cut the tips off needles and stems, while another branch remained nonwounded. Ten seedlings per isolate were inoculated by spraying to runoff with a suspension of 5 × 106 conidia ml-1 in sterile water, and five similarly treated control seedlings were sprayed with sterile water. Seedlings were covered with plastic bags to maintain high humidity for 4 days. Germination of conidia of each isolate incubated on water agar in this growth chamber was >80% after 24 h. Symptoms were evaluated and reisolation was attempted on streptomycin-amended PDA 2 months after inoculation. Symptoms of seedlings inoculated with either isolate included chlorotic and necrotic needle spots, browning of cut edges of needles, browning and death of needle tips and entire needles, death of stem tips with retention of dead needles, and needle loss. Symptoms of control seedlings were limited to slight browning of cut edges of needles. The fungus was reisolated from wounded shoots of 17 of 20 inoculated seedlings and nonwounded shoots of 5 of 20 inoculated seedlings and was not cultured from control seedlings. To our knowledge, this is the first report of S. tsugae in Georgia and also the first demonstration of its ability to produce symptoms that have been attributed to it on any tree species. References: (1) M. Miller-Weeks and W. Ostrofsky. USDA. Forest Service. Online publication. NA-PR-01-10, 2010. (2) A. Y. Rossman et al. For. Pathol. 38:47. (3) D. R. Smith et al. For. Pathol. 33:141, 2003. (4) D. R. Smith and G. R. Stanosz. For. Pathol. 38:156, 2008.

An Ornamental Swiss Stone Pine (Pinus cembra) in Wisconsin is a Host of the Shoot Blight Pathogen Diplodia pinea.

Swiss stone pine (Pinus cembra) is a slow-growing, hardy tree native to high-mountain regions of Europe from the Alps to the Carpathians. It also is planted as an ornamental in North America. Shoot blight and branch dieback were observed in the fall of 2008 on a single, 25- to 30-year-old Swiss stone pine growing on the University of Wisconsin-Madison campus. This tree is located between two mature Austrian pines (P. nigra) that exhibit symptoms of Diplodia blight and show signs of the conifer pathogen Diplodia pinea. Approximately 20% of the Swiss stone pine shoots were affected with needles and stems killed before full elongation. Symptom development appeared to have progressed from tips into older portions of branches with several years' growth often heavily resinous and necrotic. Five samples each of needles, stems, and cones bearing erumpent, black pycnidia were collected for microscopic examination. Each sample yielded conidia consistent with those of D. pinea (2). Using tannic acid agar (TAA) (1) on which autoclaved pine needles were placed to induce sporulation, this fungus was cultured from all 15 samples. The identity of the pathogen was confirmed as D. pinea with species-specific PCR primers (4) that allow differentiation from the similar fungus D. scrobiculata. Single-conidial isolate 09-03 from the affected Swiss stone pine was used to inoculate potted seedlings of this species in a greenhouse. Growing shoots of 12 seedlings were wounded by removing a needle fascicle and then were inoculated by placing on the wound a 5-mm-diameter plug cut from an actively growing colony on water agar (WA). Noncolonized WA plugs were placed on five wounded control seedlings, and five nonwounded control seedlings were used. Seedlings were covered with plastic bags to maintain high humidity for 2 weeks and then the bags were removed. The initial symptom, present 1 week after inoculation, was chlorosis of the bases of current-year needles near the point of inoculation. Affected needles became necrotic and pycnidia were visible on some by 10 days after inoculation. Needle chlorosis, necrosis, and dark discoloration of vascular tissue had developed on 11 of 12 inoculated seedlings by 6 weeks after inoculation, but not on wounded or nonwounded control seedlings. At that time, one or more symptomatic needles and a stem segment from each inoculated seedling and comparable material from control seedlings were surface disinfested and placed on TAA. The pathogen was cultured from needles of 10 of 12 inoculated seedlings and from stems of all inoculated seedlings. The fungus was not cultured from needles of control seedlings, but was cultured from stems of 2 of 10 control seedlings, one wounded and one nonwounded. D. pinea often severely damages species in the Pinus subgenus Diploxylon (two- and three-needle pines), but it is much less frequently reported as a cause of damage to hosts in the subgenus Haploxylon (five-needle pines), which includes Swiss stone pine. Although an unidentified Diplodia species was listed among fungi cultured from a healthy shoot of P. cembra (3), to our knowledge this is the first report of D. pinea as a pathogen of Swiss stone pine. References: (1) J. T. Blodgett et al. For. Pathol. 33:395, 2003. (2) E. Punithalingam and J. M. Waterston. No. 273 in: Descriptions of Pathogenic Fungi and Bacteria. Commonwealth Mycological Institute, Kew, Surrey, England, 1970. (3) G. R. Schnell. Eur. J. For. Pathol. 17:19, 1987. (4) D. R. Smith and G. R. Stanosz. Plant Dis. 90:307, 2006.

Diplodia pinea, the Cause of Diplodia Blight of Pines, Confirmed in Alabama, Louisiana, and Mississippi.

Loblolly pine (Pinus taeda) is the major commercial pine species cultivated in the Gulf Coast Region of the southern United States. Symptoms of Diplodia shoot blight (including yellow and brown needles and resin-soaked, dead, small twigs), pycnidia with conidia typical of Diplodia pinea on blighted shoots, and damaged, immature seed cones were observed during the summer of 2007 in loblolly pine seed orchards near Ward, AL, Winn Parish, LA, and Moselle, MS. Similar conidia also were obtained from pycnidia on opened seed cones of longleaf pine (P. palustris) collected on the campus of Mississipi State University, Starkville. Pure cultures obtained from specimens collected at each location were confirmed as D. pinea using species-specific PCR primers (3) that allow differentiation of D. pinea from the similar pine shoot blight pathogen D. scrobiculata. Isolates from loblolly pines in Alabama (07-58), Louisiana (07-38), and Mississippi (06-45) were used individually to inoculate potted 6- to 7-month-old loblolly pine seedlings grown from seed in a greenhouse in each of two independent trials. Elongating terminal shoots of seedlings to be inoculated were wounded by removing a needle fascicle ∼2 cm below the shoot apex. A 4-mm-diameter plug cut from an actively growing colony on water agar (WA) was placed on the wound, mycelium side toward the stem. Noncolonized WA plugs were placed in the same manner on similarly wounded control seedlings and nonwounded control seedlings also were used. Parafilm was wrapped around the shoots to hold the agar plugs in place and was removed after 1 week. Each of the five isolate-treatment combinations was applied to seven (trial 1) or eight (trial 2) seedlings (35 and 40 seedlings per trial, respectively). One week after inoculation, small, brown lesions were visible at the point of inoculation on stems of most of the inoculated seedlings. At 25 days after inoculation, all inoculated seedlings exhibited needle browning and stem cankers ranging from 0.6 cm to 9.0 cm long (mean 2.5 cm) that girdled and killed distal portions of the shoots of ∼25% of the inoculated seedlings in each trial. Wounded control and nonwounded control seedlings did not develop symptoms. Stem segments including the point of inoculation (or comparable segments of wounded and nonwounded control seedlings) were excised, surface disinfested, and incubated on tannic acid agar with sterile red pine needles. D. pinea was cultured from all inoculated seedlings and also from one wounded control seedling. Although occurrence of D. pinea on Cedrus spp. is included in an index (1), to our knowledge this is the first confirmed report of D. pinea on pines in Alabama, Louisiana, and Mississippi. The degree of risk presented by D. pinea to loblolly pine, longleaf pine, and other pine species native to the southern United States when grown in their native ranges is unknown. Reports of Diplodia shoot blight of southern U.S. pines when grown as exotics in the southern hemisphere (4) and the potential for epidemics to develop suddenly under severe weather conditions (2,4) justify additional studies to evaluate the potential for damage to these hosts in their native ranges. References: (1) Anonymous. Page 333 in: Index of Plant Diseases in the United States. Agric. Handb. 165, U.S. Dep. Agric. Washington, DC, 1960. (2) T. H. Nicholls and M. E. Ostry. Plant Dis. 74:54, 1990. (3) D. R. Smith and G. R. Stanosz. Plant Dis. 90:307, 2006. (4) W. J. Swart and M. J. Wingfield. Plant Dis. 75:761, 1991.

Shoot Blight Caused by Diplodia pinea on Afghan and Austrian Pines in Texas.

Shoot blight was observed on ornamental Afghan (Pinus eldarica) and Austrian pines (P. nigra) at several sites in metropolitan Dallas, TX in the summer of 2005. Shoots were stunted, cankered, often resinous, sometimes curled or crooked at the tips, and bore brown needles that often had been killed before full elongation. Pycnidia on necrotic needles, stems, and cones of each host species yielded conidia characteristic of the fungus Diplodia pinea. Individual conidia and hyphal tip transfers produced pure cultures confirmed as D. pinea using a species-specific PCR assay (1), which allows differentiation from the similar pine shoot blight pathogen D. scrobiculata. Five isolates (three from Afghan pine and two from Austrian pine) were tested for pathogenicity by inoculation of potted 1-year-old Afghan pine seedlings obtained from the Texas Forest Service Nursery. Elongating terminal shoots were wounded by removing a needle pair approximately 2 cm below the shoot apex. A 4-mm-diameter plug cut from an actively growing culture on water agar (WA) was placed fungus side down on the wound. Noncolonized WA plugs were placed on similarly wounded control seedlings. Nonwounded control seedlings also were used. Parafilm was wrapped around the shoots to hold the agar plugs in place and was removed 2 weeks later. Each treatment was applied to four seedlings. Five weeks after inoculation, 9 of the 20 inoculated seedlings (including at least one inoculated with each isolate) exhibited dieback of shoot tips. One wounded control seedling exhibited slight tip dieback, no other nonwounded or wounded control seedlings developed symptoms. Segments of shoots were harvested, surface disinfested, and incubated on WA to determine the presence of the pathogen. The pathogen was reisolated from 11 of the 20 inoculated seedlings but not from any control seedlings. To our knowledge, this is the first report of D. pinea as a cause of shoot blight of Afghan pine and the first substantiated report of the occurrence of D. pinea in Texas. Although widely distributed in much of eastern North America, reports of the presence of D. pinea in the other southern gulf coast states of Alabama, Louisiana, and Mississippi, as well as the western states of Colorado, New Mexico, and Utah, are lacking. Reference: (1) D. R. Smith and G. R. Stanosz. Plant Dis. 90:307, 2006.

Photosynthetic consequences of Marssonina leaf spot differ between two poplar hybrids.

• In foliage of two hybrid poplars, clone DN-34 (Populus deltoides × P. nigra) and clone NM-6 (P. nigra × P. maximowiczii), we examined relationships between photosynthesis and severity of leaf spot induced by Marssonina brunnea f. sp. brunnea, a common disease of many tree species in the Populus genus with the potential to affect growth. • Gas exchange was measured on asymptomatic and diseased foliage in monoculture stands of each clone. The equation Y = (1 - x)ß was used to characterize the relationship between relative photosynthesis (Y) and percent leaf spot (x), where ß represents the ratio between functional impairment and measured lesion area. • Leaf photosynthesis was strongly and negatively correlated with leaf spot severity in both hybrids, and ß-values indicated that photosynthetic impairment extended beyond visibly damaged leaf tissue. However, large differences in ß between hybrids indicated differential photosynthetic consequences for a given leaf spot severity. For each hybrid, values of ß were positively related to photosynthetic photon flux density incident upon the leaf during gas exchange measurement. • Declines in leaf photosynthesis appeared to result from a disruption of the photosynthetic apparatus by the invading pathogen. However, specific causes for the differential photosynthetic responses of the two hybrids to disease remained elusive.

Water stress and Sphaeropsissapinea as a latent pathogen of red pine seedlings.

• The role of water stress in the initiation of collar rot by Sphaeropsis sapinea in asymptomatically colonized Pinus resinosa seedlings is reported. • Mortality and frequency of identification of the pathogen was quantified for seedlings subjected to different water regimes or watering regime-fungicide (benomyl) combinations in glasshouse experiments. • In experiment 1, seedling mortality ranged from 8% of repeatedly watered seedlings to 50% of those in the driest regime; data analysis indicated a high probability that mortality was not independent of watering regime. Seedlings developed symptoms resembling those of Sphaeropsis collar rot, and S. sapinea was identified from living (42%) and dead (92%) seedlings. In experiment 2, mortality of repeatedly watered seedlings was low, irrespective of fungicide application. For nonwatered seedlings, however, mortality was greater among seedlings not treated with fungicide (61%) than among benomyl-treated seedlings (37%); data analysis indicated a high probability that mortality was not independent of fungicide treatment. • Sphaeropsis sapinea can act as a latent pathogen; physiological alteration, through water stress, can effect release from the quiescent condition to result in rapid disease development.

In vitro inhibition of Sphaeropsis sapinea by natural stilbenes.

The effects of pinosylvin, pinosylvin monomethyl ether, pinosylvin dimethyl ether, and resveratrol on the fungal shoot blight and canker pathogen of conifers Sphaeropsis sapinea were examined in vitro. Effects of compounds, isolates, and concentrations on both conidial germination and mycelial growth were significant (values of P < 0.001), indicating inhibitory activity of these compounds.

Monoterpene and Phenolic Compound Concentrations in Water-Stressed Red Pine Inoculated with Sphaeropsis sapinea.

ABSTRACT Changes in monoterpene and phenolic compounds resulting from water stress and colonization by Sphaeropsis sapinea were examined for 9- and 11-year-old red pine trees in a plantation and 3-year-old seedlings in a growth chamber. Four treatments were assigned at random to individual trees in the field: no treatment, herbicide to kill surrounding weeds, supplemental water, and both herbicide and supplemental water. In the growth chamber, seedlings were either not watered (water stressed) or watered daily (nonstressed). Shoots were inoculated with agar plugs colonized with either S. sapinea isolates of morphotype A and B (field) or only isolates of morphotype A (growth chamber). Nine monoterpenes were detected in tissue extracts; the most common were alpha-pinene (59 to 74% of the total), beta-pinene (13 to 33% of the total), and delta-3-carene (1 to 5% of the total). Shoots inoculated with isolates of morphotype A had more severe symptoms and produced higher concentrations of monoterpenes in both experiments compared with the controls. In the growth chamber, inoculations with isolates of morphotype A caused higher concentrations of phenolics compared with the controls. In the field experiment, monoterpenes increased in quantity only in shoots of stressed trees inoculated with isolates of morphotype A. Isolates of morphotype B caused few symptoms and did not alter monoterpene concentrations. Increases in monoterpenes do not appear to be involved in the response to infection by morphotype A in nonstressed trees, and the role of phenolics is unclear. However, these results are consistent with previous observations that monoterpenes may be involved in the differences in aggressiveness between morphotypes on red pine.

Differential Inhibition of Sphaeropsis sapinea Morphotypes by a Phenolic Compound and Several Monoterpenes of Red Pine.

ABSTRACT The in vitro effects of a red pine phenolic compound (pinosylvin), a phenolic compound common to other species (tannic acid), and the major red pine monoterpenes (alpha-pinene, beta-pinene, and delta-3-carene) on spore germination and mycelial growth of Sphaeropsis sapinea were examined. Two A and two B morphotype isolates were used. At 88 mug/mm(2), pinosylvin inhibited spore germination of all four isolates (98 to 100%). At 8.8mug/mm(2), spore germination of B isolates was inhibited more than that of A isolates (73 versus 30%). Pinosylvin also inhibited mycelial growth of B isolates more than that of A isolates (84 versus 13% at 88 mug/mm(2)). Tannic acid stimulated or had little affect on spore germination and had little affect on mycelial growth of either morphotype. Spore germination of B isolates was inhibited more than that of A isolates by beta-pinene at saturation (79 versus 37%). Spore germination of B isolates was inhibited and germination of A isolates was stimulated by delta-3-carene below saturation (49 versus -7%). Mycelial growth of B isolates was inhibited more than that of A isolates by all monoterpenes at saturation. Differences observed between morphotypes below saturation were significant only for beta-pinene. These results demonstrate the biological activity of a phenolic compound and monoterpenes that occur in red pine. The differential responses might provide means of distinguishing morphotypes and offer a potential explanation for ecological specialization.

Effects of Water Stress on Colonization of Poplar Stems and Excised Leaf Disks by Septoria musiva.

ABSTRACT Septoria musiva causes leaf spot and canker diseases of trees in the genus Populus, and is one of the most damaging fungal pathogens of hybrid poplar in eastern North America. The effect of host water stress on Septoria canker development was studied in two separate greenhouse experiments. Hybrid poplar clones NM6, NC11396, and NE308 were stressed by withholding water until predawn water potential fell below -1.0 MPa. Stems were treated by removing a leaf and applying agar plugs that were either colonized by S. musiva (inoculated) or sterile (control) to the wound. Cankers on inoculated water-stressed trees were significantly larger than those on nonstressed trees. A leaf disk assay also was conducted three times with the NM6 and NE308 trees. We cut two disks from each of 120 stressed and 120 well-watered trees, placing them on water agar in 24-well tissue culture plates. A conidial suspension was applied to one disk in each pair and sterile water to the other. Inoculated disks from water-stressed trees developed less necrosis than those from well-watered trees. These results demonstrate that environmental influences on host condition must be considered in evaluating resistance of clones proposed for widespread culture of hybrid poplar.

Effects of Moderate Water Stress on Disease Development by Sphaeropsis sapinea on Red Pine.

ABSTRACT The aggressiveness of Sphaeropsis sapinea isolates was compared on water-stressed and nonstressed 3-year-old red pines (Pinus resinosa) in greenhouse and growth chamber experiments. Water was withheld from stressed seedlings to achieve mean predawn needle water potentials (psi(PD)) above -1.9 MPa. The lowest mean psi(PD) of well-watered seedlings was maintained at or above -0.8 MPa. Young shoots were inoculated by placing colonized agar plugs on wounds made by removing a needle fascicle. Two isolates of each recognized morphotype (A and B) were used in the greenhouse experiment and two isolates of morphotype A were used in the growth chamber experiment. After 4 weeks, isolates of morphotype A caused more severe symptoms and could be recovered farther from the inoculation site on water-stressed than on nonstressed trees in both experiments. In the greenhouse experiment, isolates of mor-photype A also caused more severe symptoms and could be recovered farther from the inoculation site than isolates of morphotype B, regardless of watering regime. These results indicate that water stress at levels observed typically in the field can result in increased disease development by isolates of S. sapinea morphotype A on red pine. The reduction of water stress of red pines in the field may reduce losses due to Sphaeropsis shoot blight.

Sphaeropsis sapinea and Water Stress in a Red Pine Plantation in Central Wisconsin.

ABSTRACT A study was conducted to determine the effects of water stress resulting from competing vegetation on disease development of Sphaeropsis sapinea in red pine plantations. A 9-year-old plantation was selected in 1992 and experiments were conducted for three consecutive years. Four treatments were assigned at random to individual trees: no treatment, herbicide to kill surrounding weeds, supplemental water, and both herbicide and supplemental water. Two isolates of each S. sapinea morphotype (A and B) were used to inoculate wounded lateral shoots. Disease development was measured as the maximum distance below the inoculation site at which necrotic needles were observed. Nonwatered trees with competing vegetation (nontreated condition) had significantly lower predawn needle water potentials (more water stress) and more severe disease development than trees that received the herbicide, water, or combined herbicide and water treatments. The most severe disease occurred in the driest year and the least in the wettest year. Competing vegetation indirectly affected disease development by inducing water stress, even in relatively moist years, on trees previously considered well established. Isolates of morphotype A were more aggressive than isolates of morphotype B. Conclusions from this research have implications for sustainable management of the region's conifer forests.

Effect of ozone on growth of mosses on disturbed forest soil.

Mixed forest floor organic matter and upper mineral soil from a 1580 m elevation conifer-hardwood stand in the Pisgah National Forest, NC, was placed in 4-cm diameter x 21-cm deep tubes and exposed to 0.00, 0.08, 0.16, 0.24 or 0.32 microl O3/liter air (ppm). Twelve tubes in each of three replications/treatment were fumigated in continuously-stirred tank reactors in a greenhouse for 6 h/day on 4 consecutive days/week. Soil was watered 3 days/week with deionized water amended with ions and adjusted to pH 4.3 with H2SO4 + HNO3 (70 meq SO4(2-): 30 meq NO3(-)). After 10 weeks the amount of soil surface covered by moss (predominantly Ditrichum pusillum, but also D. lineare, and Pohlia nutans) was estimated visually and assigned a rating on a scale of: 1 = 0-25%; 2 = 26-50%; 3 = 51-75%; 4 = 76-100%. Linear regression analysis revealed a significant (p < 0.001) negative relationship between coverage ratings and O3 concentration. Surface coverage in tubes exposed to 0.32 or 0.24 ppm was about half of that for 0.00 ppm (mean ratings of 1.1, 1.4 and 2.6, respectively). Coverage differences appeared to be due in part to O3 suppression of plant numbers. Linear regression analysis also revealed a significant (p < 0.001) negative relationship between heights of D. pusillum plants (measured after 12 weeks treatment) and O3 concentration.

Shoot Blight of Lodgepole Pine Seedlings in Nebraska Caused by Sphaeropsis sapinea.

Shoot blight was observed on lodgepole pine (Pinus contorta Douglas ex Loud.) seedlings in fall 1994 at the USDA Forest Service Bessey Tree Nursery, Halsey, NE. Shoots were stunted, cankered, and sometimes curled at the tips, and bore brown needles that often had been killed before full elongation. Pycnidia were present on necrotic needles and stems. Conidia from these pycnidia produced pure cultures of Sphaeropsis sapinea (Fr.:Fr.) Dyko & Sutton in Sutton. The fungus was identified based on cultural, pycnidial, and conidial characteristics and comparison with known isolates. Also, random amplified polymorphic DNA markers of isolates were consistent with those of the A morphotype of S. sapinea (1). The ability of each of the two recognized morphotypes of S. sapinea to cause shoot blight of lodgepole pine was tested by inoculation of potted, 2-year-old seedlings in a greenhouse. Elongating terminal shoots were wounded by removing a needle pair approximately 1 cm below the shoot apex. A plug cut from an actively growing culture on water agar (WA) was placed fungus-side-down on the wound. The two isolates used (128, A morphotype; and 124, B morphotype) are representative of larger collections of these two morphotypes for which aggressiveness has been compared (2). Noncolonized WA plugs were placed on similarly wounded control seedlings. Nonwounded control seedlings also were used. Parafilm was wrapped around the shoots to hold the agar plugs in place and was removed 1 week later. Each treatment was applied to 10 seedlings in each of five completely randomized replicates. After 4 weeks, the condition (living or dead) of shoot tips was recorded, and the length of necrotic stem (canker) below the point of inoculation was measured to the nearest 0.25 cm. Segments of shoots were harvested, surface sterilized, and incubated to determine the presence of the pathogen. Analyses of variance were performed with Minitab for Windows version 10.1 software (Minitab Inc., State College, PA). Seedlings inoculated with either morphotype developed symptoms resembling those observed in the nursery. Neither incidence of shoot tip death nor severity (as indicated by canker length) differed greatly according to isolate morphotype. The means for number of shoot tips killed by the A and B isolates were 8.8 and 6.4 (out of 10), respectively (P = 0.07). The means for lengths of cankers produced by the A and B isolates were 4.9 and 3.8 cm, respectively (P = 0.10). Among controls, only one wounded seedling developed any symptoms. The pathogen was identified on incubated shoot segments from inoculated seedlings, but not those of control seedlings. This is the first report of S. sapinea as a cause of shoot blight of lodgepole pine in the U.S., and it associates the A morphotype with damage observed in the Bessey nursery. Further, the ability of the B morphotype to cause disease of lodgepole pine is established. Results of inoculations contrast with those obtained with red pine (Pinus resinosa Aiton) seedlings, on which the B morphotype was not aggressive (2). Susceptibility of lodgepole pine seedlings to both A and B morphotypes of S. sapinea, however, is similar to that exhibited by the related species, jack pine (P. banksiana Lamb.) (2). These species are classified in subsection "contortae" of the genus Pinus. Both morphotypes of S. sapinea should be considered as potential causes of shoot blight encountered on lodgepole pine. References: (1) G. R. Stanosz et al. Plant Dis. 80:1175, 1996. (2) J. T. Blodgett and G. R. Stanosz. Plant Dis. 81:143, 1997.

Sphaeropsis sapinea Morphotypes Differ in Aggressiveness, but Both Infect Nonwounded Red or Jack Pines.

Two morphotypes of Sphaeropsis sapinea, designated A and B, are recognized in the north central United States. Nonwounded seedlings of red (Pinus resinosa) and jack pine (P. banksiana) were inoculated with conidial suspensions of isolates of each morphotype obtained from hosts in Michigan, Minnesota, and Wisconsin. Wounded seedlings were inoculated with water agar plugs colonized by these isolates. Both morphotypes penetrated both hosts without wounding. On nonwounded seedlings the frequency of symptoms was 97% for A isolates and 18% for B isolates on red pine and 42% for A isolates and 6% for B isolates on jack pine. On average, isolates of the A morphotype also were more aggressive than B isolates on wounded seedlings of both pine species (A isolates causing needle necrosis 7.0 cm from the inoculation sites and B isolates 1.4 cm). Based on symptom severity (distance from the inoculation site at which necrotic needles were observed), A isolates could be distinguished from all B isolates on red pine, but not on jack pine. These observations indicate the potential importance of distinguishing between S. sapinea morphotypes encountered in nurseries, plantations, and natural stands.

Branch Dieback of Savin Juniper in Pennsylvania Caused by Diplodia mutila.

Branch dieback of savin juniper (Juniperus sabina L.) was observed on a tree in Dauphin County, PA, in May 1996. The symptomatic tree was in an ornamental planting that had been established approximately 10 years previously. Branches were cankered and girdled, causing yellowing and death of foliage beyond the cankers. Black pycnidia occurred in necrotic bark of cankers. Dark, two-celled conidia obtained from these pycnidia produced pure cultures of Diplodia mutila (Fr.:Fr.) Mont., the anamorph of Botryosphaeria stevensii Shoemaker. The fungus was identified based on pycnidial, conidial, and cultural characteristics, and comparison with known isolates provided by N. A. Tisserat (2). Pathogenicity of a single conidial isolate from Pennsylvania was tested in a greenhouse by wounding and inoculating twigs of potted eastern red cedar (Juniperus virginiana L.) with water agar plugs colonized by mycelium. Cankers formed and enlarged to girdle and kill the inoculated shoots, from which the pathogen was reisolated. No symptoms developed on, nor was the pathogen isolated from, control twigs. B. stevensii has been reported more frequently on angiosperms, such as apple (Malus Mill.) and oak (Quercus L.), than on gymnosperms. However, a canker disease caused by B. stevensii previously has been reported to affect J. scopulorum Sarg. and J. virginiana in Kansas, Missouri, and Iowa (1,2). References: (1) P. H. Flynn and M. L. Gleason. Plant Dis. 77:210, 1993. (2) N. A. Tisserat et al. Plant Dis. 72:699, 1988.

Differences in Aggressiveness of Sphaeropsis sapinea RAPD Marker Group Isolates on Several Conifers.

Seedlings of Scot's pine varieties East Anglia and Austrian Hills, red pine, mugho pine variety Pumileo, Colorado blue spruce, Douglas-fir, and balsam fir were wounded and inoculated with water agar plugs colonized by isolates of the two random amplified polymorphic DNA (RAPD) marker groups (A and B) of Sphaeropsis sapinea. Isolates were obtained from hosts in Michi-gan, Minnesota, and Wisconsin. Symptom severity (distance from the inoculation site at which necrotic needles were present) resulting from inoculations with each group A isolate exceeded that from inoculations with each group B isolate on all hosts except Colorado blue spruce. Hosts varied considerably in their responses to group A isolates. Based on symptom severity, East Anglia Scot's pine was most susceptible and balsam fir was least susceptible when inoculated with group A isolates. The pathogen was recovered from both symptomatic and asymptomatic seedlings inoculated with isolates of either group. Results emphasize the importance of characterizing a RAPD marker group(s) of S. sapinea encountered in the field or used in research; the need for comparative evaluations of resistance among coniferous genera, species, and varieties to S. sapinea of both groups; and the potential for asymptomatic persistence of S. sapinea from both groups in or on several coniferous hosts.

Chrysomyxa weirii on Colorado Blue Spruce in Wisconsin.

In early June 2002, yellow spots and bands with erumpent telia on previous year's needles of Colorado blue spruce (Picea pungens) were noted in landscape tree nurseries in both northern (Sawyer County) and southern (Dane County) Wisconsin. Many 1 to 2 m tall trees were symptomatic at each location. Based on the age of affected needles, time of year of telium development, and telial characteristics including the size and shape of teliospores, the pathogen was identified as Chrysomyxa weirii, the cause of Weir's cushion rust (1,2). Identification of the pathogen was confirmed by Dale Bergdahl, (School of Natural Resources, University of Vermont), who also observed basidiospores. C. weirii is an autoecious microcyclic rust pathogen known to affect P. englemanii, P. glauca, P. mariana, P. pungens, and P. sitchensis. Although this fungus has been reported in the western United States from the Black Hills of South Dakota to Washington State, in the eastern United States from the southern Appalachian Mountains (Tennessee and West Virginia) to Vermont, and in most Canadian provinces and territories (1,2), to our knowledge, this is the first report from the Great Lakes Region of the United States. The occurrence of Weir's cushion rust in Wisconsin has direct implications for the economically important nursery and Christmas tree industry in this region. References: (1) D. Bergdahl and D. Smeltzer. Plant Dis. 67:918, 1983. (2) W. Ziller. The Tree Rusts of Western Canada. Canadian Forestry Service, Victoria, BC, 1974.

Purple Coneflower Is a Host of the Aster Yellows Phytoplasma.

The perennial plant purple coneflower (Echinacea purpurea (L.) Moench, Asteraceae) is native to open woods and prairies of the eastern U.S. and is gaining popularity as a garden and roadside ornamental. It is propagated both by seed and by division of established plant crowns. During late summer 1996, three of seven purple coneflower plants located in a residential garden in Dane County, WI, exhibited symptoms associated with diseases caused by phytoplasmas. These plants had been established from seed in spring 1995. Stems were thickened and brittle. Leaves were slightly twisted and drooped, and flower parts lacked normal pigmentation. Petals of ligulate flowers were narrow. Clusters of short, thin stems bearing sterile, dwarfed, distorted, green heads had grown from the receptacles of severely affected flower heads. A presumptive fluorescence test with 4',6-diamidino-2-phenylindole staining was positive for phytoplasmalike bodies in phloem sieve elements in the midveins of leaves from a symptomatic plant. Nested polymerase chain reaction (PCR) amplification of extracted DNA with phytoplasma-specific oligo-nucleotide primer pairs (1,2) confirmed the presence of phytoplasmas in this plant. Based on restriction fragment length polymorphism analysis of the PCR products, the associated phytoplasma was identified as phytoplasma 16S rRNA group I (aster yellows [AY] and related phytoplasmas), subgroup A. This is the first report of a host of the AY phytoplasma in the genus Echinacea. Because no attempt was made to transfer the phytoplasma via leafhoppers, the potential for purple coneflower to be a source of inoculum is unknown. References: (1) D. E. Gunderson and I.-M. Lee. Phytopathol. Mediterr. 35:144, 1996. (2) I.-M. Lee et al. Phytopathology 84:559, 1994.