Sawadaea tulasnei Powdery Mildew of Norway Maple (Acer platanoides) in Oregon.

In August 2012, Norway maple 'Crimson Century' trees affected by powdery mildew were observed in Portland, OR. A majority of leaves throughout the crowns on each of the five trees planted in the street terrace on one side of a single city block were affected. White mycelium was present in irregular, discrete, scattered spots or almost continuously on the upper surfaces of leaves and also was present on some petioles. The affected area of the upper surface of leaf blades was estimated to typically be >50%. Chasmothecia were present singly or in groups on the mycelium, and bore simple and bifid appendages (originating from the upper half of the chasmothecia) including some with uncinate or circinate apices that allowed tentative identification to the genus Sawadaea (1). Mean diameter of the chasmothecia was 160 (standard error [SE] = 3.9) µm, and mean appendage length was 72 (SE = 4.9) µm. Asci averaged 77 (SE = 1.2) × 52 (SE = 1.2) µm and ascospores averaged 24 (SE = 0.4) × 12 (SE = 0.3) µm. Braun (1) reported ranges in dimensions of these features as: chasmothecia, (min. 125) 140 to 190 µm; appendages, 40 to 80 µm; asci, 60 to 80 (max. 100) × (min. 30) 35 to 50 (max. 55) µm; and ascospores 15 to 25 (max. 30) × 10 to 15 µm. Nuclear rDNA sequence data was obtained for ITS1, 5.8S, and ITS2 regions using primers PMITS1 and PMITS2. A BLASTn search revealed that the data (430 bp) obtained (GenBank Accession No. KF258718) exactly matched respective sequences of Sawadaea tulasnei (AB193363, AB193385, AB193390, AB193391, and EU247884), and differed from respective sequence data for S. bicornis (AB193380) by 22 nucleotides. S. tulasnei is a European species previously found on maples including Acer platanoides. However, this pathogen has been reported only rarely in North America in the states of New York, Ohio, and Wisconsin, and the province of Quebec (2,3,4). Although not likely to seriously affect the growth or survival of established trees, severe powdery mildew can detract from the aesthetic value of trees. Further, S. tulasnei might seriously damage small seedlings, and detection of this exotic pathogen in nurseries should restrict movement of stock. To our knowledge, this is the first report of S. tulasnei in western North America, and it indicates the distribution of this pathogen is transcontinental. Specimens have been deposited in the U.S. National Fungus Collections (BPI 892675). References (1) U. Braun. The Powdery Mildews (Erysiphales) of Europe. Gustav Fischer Verlag, Jena-Stuttgart-New York, 1995. (2) S. Hirose, et al. Mycol. Res. 109:912, 2005. (3) B. Hudelson, et al. Plant Dis. 92:485, 2008. (4) J. Weiland and G. Stanosz. Plant Dis. 90:830, 2006.

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.

First Report of Erysiphe palczewskii Powdery Mildew of Siberian Pea Tree (Caragana arborescens) in Wisconsin and Quebec.

Shoots affected by powdery mildew were collected from Siberian pea trees in July 2009 on the University of Wisconsin-Madison campus and on the campus of Université Laval, Quebec City, Quebec. This exotic shrub or small tree is infrequently planted in Wisconsin and three shrubs in a group that were affected are the only examples known on the UW-Madison campus. In Quebec City, Siberian pea tree is more commonly used as an ornamental, often in hedges (as is the case of the affected plants on the Université Laval campus). In both locations, <10% of foliage was visibly affected, but incidence was greater on shoots closer to the ground than on higher shoots. White-to-grayish mycelium was present on leaves and young stems and sometimes completely covered both upper and lower leaf surfaces. Dark brown-to-black chasmothecia were numerous on leaf blades, petioles, and young stems, but were most abundant on lower surfaces of leaves. Morphology of chasmothecia, including appendages with distinctive terminal dichotomous branching, (1) was consistent with descriptions and illustrations of the fungus Erysiphe palczewskii Jacz. (synonym Microsphaera palczewskii) (1-4) thought to be native to Asia, but also known as an invader of Europe where it occurs on the same host. For a sample from Université Laval, mean diameter of chasmothecia was 113 µm, mean appendage length was 185 µm, and barrel-shaped conidia that lacked fibrosin bodies averaged 30 × 14 µm. Asci contained oval, yellow ascospores with mean dimensions of 20 × 12 µm. DNA was extracted from chasmothecia, and nuclear rDNA sequences (633 nucleotides) of the Wisconsin (GenBank Accession No. GQ497277) and Quebec (GenBank Accession No. GQ497276) specimens differed by only one nucleotide. The sequences that were obtained most closely matched GenBank sequences for Oidium spp. (98%) and Erysiphe spp. (97%). Further observations indicated that the same pathogen affected Siberian pea trees planted as ornamentals at several locations separated by ≥15 km in the metropolitan Quebec area. This report extends the eastern known limit of E. palczewskii in the United States, previously known from collections in Alaska (2), Washington (4), Idaho (4), North Dakota (3), and Minnesota (3). To our knowledge, this is the first report of this disease in Canada, and it indicates that the distribution of E. palczewskii is transcontinental. Specimens from Madison, WI and Quebec, QC have been deposited in the U.S. National Fungus Collections (BPI 879152) and the Rene Pomerleau Herbarium of the Canadian Forest Service Laurentian Forestry Centre (QFB-22601). References: (1) U. Braun. Beih. Nova Hedwigia 89:1, 1987. (2) D. A. Glawe and G. A. Laursen. Online publication. doi:10:1094/PHP-2005-1017-01-BR. Plant Health Progress, 2005. (3) D. A. Glawe et al. Online publication. doi:10.1094/PHP-2006-0117-01-BR. Plant Health Progress, 2006. (4) C. Nischwitz and G. Newcombe. Plant Dis. 87:451, 2003.

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.

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.

First Report of Sawadaea tulasnei Powdery Mildew of Norway Maple (Acer platanoides) in Wisconsin.

Leaves affected by powdery mildew were collected from a Norway maple tree in early October 2007 in Beaver Dam, WI (Dodge County). Diseased leaves were present throughout the crown of this tree, with white mycelium in irregular and often vein-associated spots and often covering as much as 50% of the upper surfaces of leaves. Examination of the lower surfaces revealed necrosis of the areas underlying mycelium. Blades of samaras also bore white mycelium. Chasmothecia were present singly or in groups on the mycelium. Morphology of chasmothecia, including simple and bifid appendages with uncinate to circinate apices, was sufficient to identify the pathogen to the genus Sawadaea (1). Data for nuclear rDNA ITS sequence (546 bp) obtained for a specimen (GenBank Accession No. EU247884) exactly matched sequences for Sawadea tulasnei (GenBank Accession Nos. AB 193363, 478 bp; AB193385, 490 bp; AB193390 and AB193391, 546 bp). This data was 96% similar (528 of 552 nucleotides) to that of another European powdery mildew pathogen, S. bicornis (GenBank Accession No. AB193380), which is also reported to occur on maples in Idaho, Washington, and Wisconsin (2,3). A further survey revealed the same fungus on several additional nearby Norway maples along streets and in yards (including varieties with both darkly colored and variegated leaves), but on these trees very few leaves were affected and usually less than 5% of the upper leaf surfaces bore mycelium. This pathogen was not observed on leaves of either red (A. rubrum) or silver maples (A. saccharinum) examined in the same area. S. tulasnei was previously known in North America only by collections in New York, Ohio, and Montreal, Canada (4), but our observation indicates that the geographic distribution of this pathogen is probably much broader and overlaps with that of S. bicornis. Specimens from Beaver Dam, WI have been deposited in the U.S. National Fungus Collections (BPI 878273). References: (1) U. Braun. The Powdery Mildews (Erysiphales) of Europe. Gustav Fischer Verlag, Jena-Stuttgart, New York, 1995. (2) C. Nischwitz and G. Newcombe. Plant Dis. 87:451, 2003. (3) G. Stanosz et al. Plant Dis. 91:636, 2007. (4) J. Weiland and G. Stanosz. Plant Dis. 90:830, 2006.

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.

Sawadaea Powdery Mildew of Box Elder (Acer negundo) in Wisconsin.

Box elder leaves affected by powdery mildew were collected from a single tree in an urban yard in the fall of 2006 in Madison, WI. As much as half of the area of the leaf blades, when viewed from above or below, was pale green-to-tan and necrotic. Mycelium was not visible to the eye on either the upper or lower leaf surfaces, but very sparse white mycelium was observed on the lower surface of leaves with the aid of a dissecting microscope. Chasmothecia were present singly or in groups of a few on the lower surface of leaves. Morphology of the chasmothecia, including simple and bifid appendages with uncinate to circinate apices, was sufficient to identify the pathogen to the genus Sawadaea (1). Other characters were not sufficiently distinct to make an identification of the species. However, DNA was extracted from the chasmothecia, and analysis of a 542-bp sequence of nuclear rDNA ITS (GenBank Accession No. EF122238) revealed a 100% match with the respective sequence obtained from Genbank for S. bicornis (Accession No. AB193380). A specimen from which these chasmothecia were obtained has been deposited in the U.S. National Fungus Collections (BPI 877328). S. bicornis is a European pathogen of maples, including box elder when grown there (1). Knowledge of the geographic distribution and hosts of Sawadaea powdery mildews in North America is extremely limited. S. bicornis was first reported in North America only recently, occurring in the states of Idaho and Washington on Norway maple (Acer platanoides) (3). The morphologically very similar European powdery mildew pathogen of maples, S. tulasnei, is known only from New York, Ohio, and Montreal, Canada (4), and an unidentified Sawadaea species on box elder has been reported from California (2). References (1) U. Braun. The Powdery Mildews (Erysiphales) of Europe. Gustav Fischer Verlag, Jena-Stuttgart-New York, 1995. (2) S. Hirose et al. Mycol. Res. 109:912, 2005. (3) C. Nischwitz and G. Newcombe. Plant Dis. 87:451, 2003. (4) J. Weiland and G. Stanosz. Plant Dis. 90:830, 2006.

Sawadaea tulasnei Powdery Mildew of Norway Maple (Acer platanoides) in North America.

Norway maple leaves bearing powdery mildew were collected from one location in the fall of 2003 and four locations (as much as 1.5 km apart) in the fall of 2005 in Buffalo, NY. No powdery mildew was observed on leaves collected from sugar maples (Acer saccharum) that were present in the vicinity of affected Norway maples at two locations. Trees were located along streets and in yards. Diseased leaves were present throughout tree crowns but lower leaves were more commonly affected. White mycelium was present in irregular, discrete, scattered spots only on the upper surface of leaves and on both sides of wings of samaras. Typically, <10% of the upper leaf area bore visible mycelium. Cleistothecia were present singly or in groups on the mycelium. Morphology of cleistothecia on leaves collected each year, including simple and bifid appendages with uncinate to circinate apices, was sufficient to identify the pathogen to the genus Sawadaea (1). Other characteristics were not sufficiently distinct to make an identification of S. bicornis or S. tulasnei (1), each a European species found on Acer spp. However, a sample from 2003 was supplied by the authors for use in a study of phylogeny of the genus (2) that served as a first report of the species in the United States. Analysis of nuclear rDNA ITS sequence of this specimen (GenBank Accession No. AB193390) placed the sample in a clade with S. tulasnei specimens from Europe. In the same study, powdery mildew samples from Acer spp. in Ohio and Montreal, Canada also were placed in this clade. Thus, occurrence of S. tulasnei in North America is confirmed. S. bicornis was recently identified (based on morphology) on Norway maple in the western United States (3). Specimens from Buffalo, NY have been deposited in the U.S. National Fungus Collections (BPI 871210). References: (1) U. Braun. The Powdery Mildews (Erysiphales) of Europe. Gustav Fischer Verlag, Jena-Stuttgart-New York, 1995. (2) S. Hirose et al. Mycol. Res. 109:912, 2005. (3) C. Nischwitz and G. Newcombe. Plant Dis. 87:451, 2003.

First Report of Diplodia Canker of Cypress Caused by Diplodia pinea f. sp. cupressi on Mediterranean Cypress in Tunisia.

Symptoms of decline were observed on Mediterranean cypress trees (Cupressus sempervirens L.) in Tunisia in 2003 and 2004; disease specimens were vouchered as FIAF 38649. The declining, windbreak trees in the Cap Bon Region were 25 to 30 years old. Severity of symptoms varied among trees. Thus, areas of reddish, withered foliage alternated with areas that were still green. Other trees were completely withered. The bases of withered branches and tree trunks bore slightly sunken cankers with longitudinal bark cracks that oozed drops of resin. When the outer layer of a cankered area was scraped away, dark brown inner bark tissue was seen to extend up to several centimeters around the canker. Cross sections through cankers on trunks and branches revealed extensive darkened, wedge-shaped sectors in the wood. The affected bark bore numerous black pycnidia. Conidia were mostly smooth, ovoid, hyaline, and aseptate; a few were brown with a medial septum. The mean conidial dimensions (n = 100) were 27 × 11 µm; the extreme range was 19 to 31 × 8 to 13 µm. Isolates obtained from affected host tissue and conidia developed mainly floccose colonies that were white, then greyish green, and finally dark gray black on potato dextrose agar (PDA) at 25°C. Blackish pycnidial fructifications containing typical conidia were produced after 5 weeks on autoclaved cypress seeds placed on the colonies under light. Pathogenicity was tested using five 3-year-old potted Mediterranean cypress trees. These were inoculated by placing 3-mm-diameter plugs of mycelium of isolate DF IMG86 (DAOM 234788) from the edge of a 15-day-old colony on PDA on 5-mm-diameter wounds made in the bark. The wounds were covered with cotton wool moistened with sterile water and wrapped in adhesive tape. Similar wounds on five control trees received a plug of sterile PDA. Symptoms occurred as early as the third week after inoculation. The leaves first became yellow and then turned amaranth red, after which they progressively withered. Two months after inoculation, cankers were clearly visible at the inoculation site. Isolates from these cankers were morphologically similar to those used for inoculation. The control plants did not show any disease symptoms and their wounds healed normally. Morphological, cultural, and pathological characteristics of the fungus isolated from cypress with decline symptoms were similar to those of the fungus referred to as Diplodia pinea f. sp. cupressi or Sphaeropsis sapinea f. sp. cupressi (1). Identification was confirmed by marker analysis by using intersimple sequence repeat polymerase chain reaction (2). Banding patterns for isolate DF IMG86 were produced using primers HYH(GY)7 and (CAG)5 and were identical to those for Diplodia pinea f. sp. cupressi isolates 94-3 (DAOM 229437) and 95-158 (DAOM 229439) and differed from those obtained for isolates of Diplodia pinea (S. sapinea A group), D. scrobiculata (S. sapinea B group), Botryosphaeria obtusa, and B. stevensii. To our knowledge, this is the first report of this pathogen in Tunisia. The development of D. pinea f. sp. cupressi on cypress windbreaks in the Cap Bon Region may be related to a drought that has afflicted Tunisia for the past 5 years. Reference: (1) Z. Solel et al. Can. J. Plant Pathol. 9:115, 1987. (2) S. Zhou et al. Mycol. Res. 105:919, 2001.

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.

First Report of Sirococcus conigenus on Deodar Cedar in Oregon.

Cedrus deodara is a highly valued conifer widely grown as an ornamental in the Pacific Northwest and southern United States. C. deodara in the Pacific Northwest is normally problem free but occasionally is damaged by dieback of shoot tips, which has been associated with a fungus resembling Sirococcus conigenus. In February 2002, bleeding cankers were observed on 2- to 4-year-old stems of potted nursery stock of C. deodara cv. Karl Fuchs from Clackamas County, OR. Cankers were dark with indistinct margins, shallow, and up to 30 cm long. Infection appeared to have originated with small twigs that had died. Cultures isolated from discolored bark on streptomycin-amended potato dextrose agar (PDA) produced conidiomata with hyaline, fusiform, two-celled conidia typical of S. conigenus (1,3). Inter-simple sequence repeat-polymerase chain reaction fingerprints of an isolate from one of these trees were consistent with the P group of S. conigenus (mostly from hosts in Picea and Pinus spp.) (2). This isolate (02-04, ATCC MYA-2969) was used to inoculate two shoots on each of 12 3-year-old potted deodar cedars in each of two trials. Removing a needle wounded each shoot, and an agar plug colonized with mycelium was placed over the wound and held in place for 2 weeks with Parafilm. Sterile agar plugs were applied to two wounded control shoots on each tree in each trial. After 10 weeks, 25 of 48 inoculated shoots were blighted and drooped with yellow to brown needles that eventually dropped. The pathogen was reisolated from 24 of 25 symptomatic shoots but not from asymptomatic or control shoots. To our knowledge, this is the first confirmed report of S. conigenus as a pathogen of C. deodara. References: (1) P. F. Cannon and D. W. Minter. Taxon 32:572, 1983. (2) D. R. Smith et al. For. Pathol. 33:141, 2003. (3) B. Sutton. The Coelomycetes. Commonw. Mycol. Inst., Kew, Surrey, England, 1980.

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.

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.

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.

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.

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.