First Report of Pythium sulcatum Causing Cavity Spot in Processing Carrot Crops in the Columbia Basin of Washington State.

In October 2012, symptoms of cavity spot (1) were observed on roots of two 50 ha, Red Core Chantenay processing carrot (Daucus carota L. subsp. sativus (Hoffm.)) crops in the Columbia Basin of central Washington. Symptoms consisted of sunken, elliptical lesions (3 to 15 mm long) on the root surface. Approximately 6% of the roots in each crop were affected, which was sufficient to present sorting problems for the processor. Symptomatic roots were washed thoroughly in tap water, and then small sections of tissue from the lesion margins were removed aseptically and plated onto water agar (WA) without surface-sterilization. Isolates with morphological characteristics typical of Pythium sulcatum Pratt & Mitchell (2) were obtained consistently from the symptomatic tissue. The genus and species identity of seven isolates was confirmed by sequence analysis of the internal transcribed spacer (ITS) 1-5.8S-ITS2 region of ribosomal DNA (rDNA) using universal eukaryotic primers UN-UP18S42 and UN-LO28S576B with the PCR protocol described by Schroeder et al. (3). The ITS consensus sequences of the seven isolates (Accession Nos. KF509939 to KF509945) were 98 to 99% homologous to ITS sequences of P. sulcatum in GenBank. Pathogenicity of all seven isolates was confirmed by inoculating mature carrot roots of cv. Bolero. Each root was washed with tap water, sprayed to runoff with 70% isopropanol, and dried in a laminar flow hood on sterilized paper toweling. The roots were then placed in plastic bins lined with paper toweling moistened with sterilized, deionized water. Each root was inoculated by placing two 5 mm-diameter agar plugs, taken from the edge of an actively growing WA culture of the appropriate isolate, on the root surface approximately 3 cm apart. Non-colonized agar plugs were used for a non-inoculated control treatment. Four replicate roots were inoculated for each isolate and the control treatment. After inoculation, the roots were misted with sterilized, deionized water, a lid was placed on each bin, and the roots were incubated in the dark at 22°C. Roots were misted daily to maintain high relative humidity. Dark, sunken lesions were first observed 3 days post-inoculation on roots inoculated with the P. sulcatum isolates, and all inoculated roots displayed cavity spot lesions by 7 days. No symptoms were observed on the non-inoculated control roots. Colonies with morphology typical of P. sulcatum were re-isolated from the symptomatic tissue of roots inoculated with the P. sulcatum isolates, and the species identity of the re-isolates was confirmed by ITS rDNA sequence analysis, as described above. Although P. sulcatum is one of several Pythium species that can cause cavity spot of carrot (1), to our knowledge, this is the first report of P. sulcatum causing cavity spot in Washington State, which has the largest acreage of processing carrot crops in the United States (4). References: (1) R. M. Davis and R. N. Raid. Compendium of Umbelliferous Crop Diseases. The American Phytopathological Society, St. Paul, MN, 2002. (2) A. J. van der Plaats-Niterink. Monograph of the Genus Pythium. Stud. Mycol. No. 21. CBS, Baarn, The Netherlands, 1981. (3) K. L. Schroeder et al. Phytopathology 96:637, 2006. (4) E. J. Sorensen. Crop Profile for Carrots in Washington State. U.S. Dept. Agric. National Pest Manage. Centers, 2000.

First Report of Bacterial Blight of Carrot in Indiana Caused by Xanthomonas hortorum pv. carotae.

In summer 2012, bacterial blight symptoms (2) were observed on leaves of carrot plants in 7 out of 70 plots of carrot breeding lines at the Purdue University Meig Horticulture Research Farm, Lafayette, IN. Symptoms included small to large, variably shaped, water-soaked to dry, necrotic lesions, with or without chlorosis, at <5% incidence. Microscopic examination of symptomatic leaf sections revealed bacterial streaming from the cut ends of each leaf piece. For each of the seven plots, symptomatic leaf sections (each 5 to 10 mm2) were surface-sterilized in 1.2% NaOCl for 60 s, triple-rinsed in sterilized, deionized water, dried on sterilized blotter paper, macerated in sterilized water, and a loopful of the suspension was streaked onto yeast dextrose carbonate (YDC) agar medium (1). Colonies with morphology similar to that of strain Car001 of Xanthomonas hortorum pv. carotae from California (3) were obtained consistently from all seven plots, and serial dilutions streaked onto YDC agar medium to obtain pure cultures. One bacterial strain/plot was then subjected to a PCR assay for X. hortorum pv. carotae using the protocol of Meng et al. in (5), except for an annealing temperature of 60°C. All seven Indiana strains and Car001 produced a 355-bp DNA fragment indicative of X. hortorum pv. carotae. The Indiana strains and Car001 were each tested for pathogenicity on five 11-week-old carrot plants of a proprietary Nantes inbred line grown from a seed lot that tested negative for X. hortorum pv. carotae (1,3). Each strain was grown for 16 h in 523 broth (4) on a shaker (200 rpm) at 28°C, and diluted in 0.0125M phosphate buffer to 108 CFU/ml. Approximately 24 h prior to inoculation, the five plants for each strain were enclosed in a large plastic bag to create a moist chamber. The plants were inoculated by atomizing 30 ml of the appropriate bacterial suspension onto the foliage using an airbrush. Five plants inoculated with sterilized phosphate buffer served as a negative control treatment. The plants were re-sealed in plastic bags for 72 h, and placed in a randomized complete block design in a greenhouse set at 25 to 28°C. Symptoms of bacterial blight were first observed 14 days after inoculation, and developed on all inoculated plants by 21 to 28 days after inoculation, with slight variation in severity of symptoms among strains. Symptoms did not develop on negative control plants. Re-isolations were done 32 days after inoculation from symptomatic leaves of three replicate plants/strain and from three plants of the negative control treatment, using the protocol described for the original samples. Bacterial colonies typical of X. hortorum pv. carotae were obtained from symptomatic leaves for all seven Indiana strains and the control strain, but not from the negative control plants. Identity of the re-isolated strains as X. hortorum pv. carotae was confirmed by PCR assay. To our knowledge, this is the first report of bacterial blight of carrot in Indiana. References: (1) M. Asma. Detection of Xanthomonas hortorum pv. carotae on Daucus carota. 7-020. International Rules for Seed Testing, Annex to Chapter 7: Seed Health Testing Methods. Internat. Seed Testing Assoc., Bassersdorf, Switzerland, 2006. (2) R. M. Davis and R. N. Raid. Compendium of Umbelliferous Crop Diseases. The American Phytopathological Society, St. Paul, MN, 2002. (3) L. J. du Toit et al. Plant Dis. 89:896, 2005. (4) E. I. Kado and M. G. Heskett. Phytopathology 60:969, 1970. (5) X. Q. Meng et al. Plant Dis. 88:1226, 2004.

White Rust of Echinacea angustifolia and E. purpurea in North America Caused by a Pustula Species.

In 2012 and 2013, foliar symptoms were observed in certified organic, 2- to 4-ha crops of Echinacea angustifolia and E. purpurea in Grant and Klickitat counties, WA. White pustules were predominant on the abaxial leaf surface, increased in number, and coalesced on E. angustifolia, with 100% infection by the end of the season; in contrast, symptoms remained sparse on E. purpurea. Symptomatic leaves of each species were collected in May 2013 in Grant Co. Sori and sporangia were typical of those of white rust on Asteraceae caused by Pustula obtusata (1), originally named Albugo tragopogonis, then P. tragopogonis (4). Hyaline sporangia (n = 50) averaged 21 ± 2 × 20 ± 2 µm (16 to 25 × 16 to 24 µm) with a 2.6 ± 0.8 µm (1.0 to 4.0 µm) thick wall. Honey-colored to dark brown oospores were embedded in the abaxial leaf surface surrounding sori on older leaves. Oospores (n = 50) averaged 75 ± 7 × 63 ± 6 µm (60 to 96 × 52 to 76 µm) and 52 ± 4 × 51 ± 4 µm (44 to 65 × 44 to 60 µm) with (including protruberances) and without the hyaline outer wall, respectively. Sori were excised and shaken in 100 ml cold (4°C), deionized water at 400 rpm for 15 min on a gyrotory shaker. DNA extracted from the resulting spore suspension was subjected to a PCR assay using oomycete specific primers (2) to amplify the cytochrome oxidase subunit II (cox2) region of mtDNA (3). The 511-nt consensus sequence of the PCR product (GenBank Accession No. KF981439) was 98% identical to a cox2 sequence of A. tragopogonis from sunflower (Helianthus annuus) (AY286221.1), and 96% identical to cox2 sequences of P. tragopogonis (GU292167.1 and GU292168.1) (= P. obtusata) (1,2,4). Pathogenicity of the white rust isolate was confirmed by inoculating 49-day-old plants of E. angustifolia and E. purpurea with a spore suspension prepared as described above. One plant/species was placed in each of six clear plastic bags in a growth chamber at 18°C with a 12-h day/12-h night cycle for 48 h. Five replicate sets of one plant/species were each inoculated with 2.2 × 105 spores/ml on the adaxial and abaxial leaf surfaces using an airbrush (8 psi). One plant/species was sprayed with water as a control treatment. The plants were resealed in the bags for 48 h. After 7 days, white pustules were observed on at least one plant species. The plants were placed in plastic bags again overnight, and re-inoculated with 2.9 × 105 spores/ml. In addition, two sunflower plants at the 4-true-leaf stage were incubated in each of two plastic bags overnight, and inoculated with the spore suspension. Two additional sunflower plants were treated with water as control plants. All plants were removed from the bags after 48 h. White rust sori with sporangia developed on all inoculated Echinacea plants within 10 days, but not on control plants of either species, nor inoculated and non-inoculated sunflower plants, verifying that the pathogen was not P. helianthicola (1,2). Since the cox2 sequence was closest to that of a sunflower white rust isolate, the pathogen appears to be closer to P. helianthicola than P. obtusata, and may be a new Pustula species. To our knowledge, this is the first documentation of white rust on E. angustifolia and E. purpurea in North America. The severity of white rust on E. angustifolia highlights the need for effective management practices. References: (1) C. Rost and M. Thines. Mycol. Progress 11:351, 2012. (2) O. Spring et al. Eur. J. Plant Pathol 131:519, 2011. (3) S. Telle and M. Thines. PloS ONE 3(10):e3584, 2008. (4) M. Thines and O. Spring. Mycotaxon 92:443, 2005.

First Report of Cladosporium Leaf Spot of Spinach Caused by Cladosporium variabile in the Winter Spinach Production Region of California and Arizona.

In December 2011, symptoms typical of Cladosporium leaf spot caused by Cladosporium variabile (4) were observed in organic "baby leaf" spinach (Spinacia oleracea) crops of the cultivars Amazon, Missouri, Tasman, and Tonga in the Imperial Valley (Imperial County, CA and Yuma County, AZ). Leaves had small, circular lesions (1 to 3 mm in diameter), some of which had progressed to necrotic, bleached lesions surrounded by a thin dark margin. The incidence of symptoms in affected crops was ≤20%. Fungal isolates resembling C. variabile were recovered by surfacesterilizing sections (5 mm2) of symptomatic leaf tissue in 0.6% NaOCl, triple-rinsing the sections in sterile water, and plating the sections onto water agar and potato dextrose agar amended with 100 ppm chloramphenicol (cPDA). Single-spore transfers made onto cPDA were maintained at 24 ± 2°C with a natural day/night cycle. Each isolate produced slow growing cultures consisting of dense masses of dark conidiophores (≤350 µm long) with chains of up to three dematiaceous (olive) conidia, and almost no mycelium. Torulose (coiled) aerial hyphae developed from the apices of conidiophores after 5 to 7 days, and distinguished the isolates as C. variabile, not C. macrocarpum (2,4). Pathogenicity was tested for each of six single-spore isolates using 36-dayold plants of the spinach cultivar Carmel. The plants were enclosed in clear plastic bags overnight and inoculated the next day with the isolates of C. variabile by atomizing approximately 30 ml of a spore suspension (1.0 × 106 conidia/ml in sterile water amended with 0.01% Tween 20) of the appropriate isolate onto the upper and lower leaf surfaces of each of five plants/isolate. Five control plants were inoculated similarly with sterile water + 0.01% Tween 20. The plants were resealed in plastic bags for 72 h and then placed on a greenhouse bench. Pinpoint, sunken lesions developed within 4 to 7 days on the leaves of plants inoculated with each of the six test isolates. Lesions developed into dry, circular spots typical of Cladosporium leaf spot. Symptoms were not observed on control plants. After 20 days, C. variabile was reisolated from lesions caused by all six isolates, but not from control plants. Although Cladosporium leaf spot has been reported in the Salinas Valley of California (4), to our knowledge, this is the first report of the disease on spinach crops in the Imperial Valley of California and Arizona, the primary winter, fresh market spinach production region of the United States. Inoculum of C. variabile may have been introduced to this region on spinach seed lots (3), because even seed infestation levels <0.1% could lead to seed transmission (1) under the dense planting populations (≤9 million seeds/ha) and overhead irrigation typical of "baby leaf" spinach crops in this region. Fungicides can be used to manage Cladosporium leaf spot in conventional spinach crops (1), but management in certified organic crops may be more challenging. References: (1) L. J. du Toit et al. Fung. Nemat. Tests 59:V115, 2004. (2) M. B. Ellis. Page 315 in: Dematiaceous Hyphomycetes. Commonwealth Mycological Institute, Surrey, England, 1971. (3) P. Hernandez-Perez. Page 79 in: Management of Seedborne Stemphylium botryosum and Cladosporium variabile Causing Leaf Spot of Spinach Seed Crops in Western Washington, MS thesis, Pullman, WA, 2005. (4) P. Hernandez-Perez and L. J. du Toit. Plant Dis. 90:137, 2006.

First Report of Colletotrichum coccodes Causing Leaf and Neck Anthracnose on Onions (Allium cepa) in Michigan and the United States.

In July of 2010, dry, oval lesions, each with a salmon-colored center and bleached overall appearance, were observed on the leaves and neck of onions plants growing in production fields of Newaygo, Ottawa, Kent, and Ionia counties, Michigan. Acervuli and setae that are characteristic of Colletotrichum spp. were observed with a dissecting microscope, and elliptical conidia (8 to 23 × 3 to 12 µm) with attenuated ends were observed with a compound microscope. Symptomatic tissues were excised and cultured onto potato dextrose agar amended with 30 and 100 ppm of rifampicin and ampicillin, respectively. The cultures produced pale salmon-colored sporulation after growing for 5 days at 22 ± 2°C and black microsclerotia after 2 weeks. Six isolates were confirmed as C. coccodes based on sequence analysis of the internal transcribed (ITS) region of the ribosomal DNA and a 1-kb intron of the glutamine synthase gene (GS) (2). Sequences were submitted to GenBank (Accession Nos. JQ682644 and JQ682645 for ITS and GS, respectively). Pathogenicity tests were conducted on two- to three-leaved 'Stanley' and 'Cortland' onion seedlings. Prior to inoculation, seedlings were enclosed in clear plastic bags overnight to provide high relative humidity. The bags were removed, and seedlings were sprayed inoculated with a C. coccodes conidial suspension (5 × 105 conidia/ml and 25 ml/plant) in sterile double-distilled water. Control seedlings were sprayed with sterile double-distilled water. Tween (0.01%) was added to the conidial suspension and the water. Plants were enclosed in bags for 72 h postinoculation and incubated in growth chambers at 28°C day/23°C night with a 12-h photoperiod. Sunken, oval lesions were observed on the foliage of the onion seedlings inoculated with C. coccodes 4 days postinoculation. Lesions coalesced and foliage collapsed 7 days postinoculation. Control plants remained asymptomatic. When five leaf samples per replication were detached and incubated in a moist chamber for 3 days at 21 ± 2°C, abundant acervuli and setae were observed on the symptomatic tissue but not on control tissue. C. coccodes was consistently recovered from the onion seedling lesions. Six different Colletotrichum spp. have been reported to cause diseases on onions worldwide (1,4). C. circinans, which causes smudge, is an occasional onion pathogen in Michigan, while C. gloeosporioides has only been reported to be infecting onions in Georgia (3). To our knowledge, this is the first report of C. coccodes infecting and causing disease in onions plants. References: (1) D. F. Farr and A. Y. Rossman. Fungal Databases. Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , August 6, 2010. (2) J. C. Guerber et al. Mycologia 95:872. 2003. (3) C. Nischwitz et al. Plant Dis. 92:974. 2008. (4) H. F. Schwartz, and K. S. Mohan. Compendium of Onion and Garlic Diseases and Pests, 2nd ed. The American Phytopathological Society, St. Paul, MN. 1995.

A Leaf Blight of Chive Caused by Botrytis byssoidea in California.

From 1998 through 2002, commercial chives (Allium schoenoprasum) in coastal California (Monterey County) were damaged by an undescribed disease. Initial symptoms were chlorosis and tan-colored necrosis at the leaf tips; as the disease progressed, extensive tan-to-light brown discoloration extended down affected leaves, resulting in their death. The damage prevented growers from harvesting affected crops. Stems of the chive plants were unaffected. Diseased plants continued to grow new leaves that subsequently became infected. A fungus was consistently isolated from symptomatic leaves. Isolates grown on potato dextrose agar (PDA) in petri plates incubated at 24°C under fluorescent lights produced extensive mycelial growth without conidia. However, on onion leaf straw agar (2), the isolates produced abundantly sporulating colonies with conidiophores and conidia typical of a Botrytis species. Conidiophores rarely exceeded 1 mm long. Ellipsoidal conidia measured 11 to 17 × 5 to 8 µm. On green bean pod agar (4), the isolates produced a few, black, irregularly shaped sclerotia measuring 1 to 2 mm in diameter. Morphological comparisons were made on PDA between five chive isolates and isolates of the following Botrytis species known to infect Allium species (1): B. aclada BA5, B. allii BA3, B. byssoidea ATCC 60837, B. cinerea from an onion seed crop, B. porri 749, B. squamosa 392, and B. tulipae GC-1. B. elliptica strain MARLI-3 was also compared with the chive isolates. Chive isolates produced floccose, off white-to-light tan mycelium, lacked sporulation (except where mycelium contacted the edge of the plastic petri dish), and did not form sclerotia on PDA, thereby resembling B. byssoidea. Identification of the chive isolates as B. byssoidea was confirmed by ApoI restriction fragment length polymorphism digests of a 423-bp PCR amplicon obtained from each of the five chive isolates and the eight known Botrytis species (1,3). Pathogenicity of the chive isolates of B. byssoidea was confirmed by spraying a conidial suspension (1 × 105 conidia/ml) of each of 12 isolates onto chive (cv. Fine Leaved) and onion (A. cepa cv. Southport White) plants until runoff, incubating the plants in a humidity chamber at 24 to 26°C for 48 h and then maintaining the plants under ambient light in a greenhouse. After 6 to 8 days, inoculated chives and onions developed symptoms similar to those observed in the field and B. byssoidea was reisolated. Noninoculated control chives and onions sprayed with distilled water did not develop symptoms. The experiment was conducted three times and the results were the same. To our knowledge, this is the first report of a leaf blight of chive caused by B. byssoidea in North America. After 2002, the commercial chive plantings were placed on farms further east in Monterey County away from the coast. The disease has not been observed since this move to a drier climate. References: (1) M. I. Chilvers and L. J. du Toit. Online publication. doi:10.1094/PHP-2006-1127-01-DG. Plant Health Progress, 2006. (2) L. A. Ellerbrock and J. W. Lorbeer. Phytopathology 67:219, 1977. (3) K. Nielsen et al. Plant Dis. 86:682, 2002. (4) A. H. C. van Bruggen and P. A. Arneson. Plant Dis. 69:966, 1985.

Botrytis porri in Onion Seed Crops and Onion Seed.

Nine fields direct-seeded with onion (Allium cepa L.) were surveyed in central Washington in the spring and summer of 2001 for Botrytis species associated with onion seed crops produced in this semiarid region. Forty plants were sampled from each field in a 'W' pattern in April, and 20 plants were similarly sampled from each field in June and July. Each plant was placed in a separate plastic bag, stored at 4 ± 2°C for 3 to 5 weeks, sliced lengthwise using a knife sterilized with 70% ethyl alcohol, incubated in a moist chamber for 5 days, and examined under a dissecting microscope. Fungal growth resembling Botrytis spp. was transferred to acidified potato dextrose agar (PDA) for species identification based on colony morphology, rate of growth, and spore and sclerotium characteristics (3). Cultures were incubated on a laboratory bench at 24 ± 4°C with 8 to 16 h of daylight. A species resembling B. porri (3) was detected in 3 fields in April at an incidence ranging from 3 to 28%, and in 2 of the same 3 fields in each of June and July at incidences ranging from 5 to 10%. Infected plants were asymptomatic at the time of sampling. The isolates formed brown, cerebriform sclerotia and sporulated sparsely. Subsamples of seed harvested from each field were assayed for Botrytis spp. To detect internal infection, 400 seeds from each of the nine fields were soaked in 0.525% NaOCl for 60 s, triple-rinsed in sterile deionized water, air dried, placed on a selective agar medium (2) with 20 seed per 9-cm-diameter petri plate, and incubated at 24°C (12 h day/night) for 14 days. Seeds were examined 5, 10, and 14 days after plating, and fungi resembling Botrytis spp. were transferred to acidified PDA for species determination. Isolates resembling B. porri were detected in 0.75% of seed from two of the three fields in which this species was isolated from plant samples. The internal transcribed spacer 1 region of ribosomal DNA of four isolates of the putative B. porri (two from plant samples and two from seed) were sequenced, and all four sequences matched that of B. porri registered in GenBank (Accession No. Z99666) most closely. Botrytis porri is a pathogen of garlic (A. sativum L.), leek (A. porrum L.), and wild garlic (A. vineale L.), but can infect onion and shallot (A. ascalonicum L.) when inoculated on these hosts (1). To our knowledge, this is the first report of natural infection of onion by B. porri, and the first report of seedborne B. porri on onion. References: (1) W. R. Jarvis. Pathology. Page 62 in: Botryotinia and Botrytis Species: Taxonomy, Physiology, and Pathogenicity. Canada Department of Agriculture, Monograph No. 15, 1977. (2) G. Kritzman and D. Netzer. Phytoparasitica 6:3, 1978. (3) A. H. Presly. Plant Pathol. 34:422, 1985.

Persistence of Complex Virulences in Populations of Phytophthora infestans in Western Washington.

ABSTRACT Isolates of Phytophthora infestans, collected from bittersweet, hairy nightshade, petunia, potato, potato vine, and tomato in western Washington, 1998 to 1999, were evaluated for virulence complexity as well as mating type, metalaxyl insensitivity, allozymes of glucose-6-phosphate isomerase and peptidase, and DNA fingerprint with the RG57 probe. Results were compared with those from similar collections made in the same region during the 1990s. Generally, virulence complexity was high for most of the isolates regardless of year, genotype, or host. No marked shift in virulence complexity was evident for the populations studied, and unnecessary virulences were maintained. During 1998 and 1999, isolates of the US-8 and US-11 genotypes had 4 or more virulence factors. US-8 isolates averaged 8.2 and 9.3, whereas US-11 isolates averaged 5.4 and 6.3 virulence factors. The frequency of US-8 isolates that were sensitive to metalaxyl ranged from 5% in 1998 to 72% in 1999. All of the US-11 isolates tested in 1998 and 1999 were insensitive to metalaxyl. From 1996 to 1999 on potato, the recovery of US-8 increased, whereas the recovery of US-11 decreased. No evidence of new genotypes or sexual recombination was found. Western Washington was a desirable location for screening germ plasm for durable resistance to late blight due to the high frequency and persistence of complex virulences.

Stemphylium botryosum Pathogenic on Spinach Seed Crops in Washington.

In September 2000, symptoms typical of leaf spot caused by Cladosporium variabile were observed on a spinach (Spinacea oleracea L.) seed crop in western Washington. Dry, bleached spots (1 to 20 mm) were most abundant on lower leaves. Two isolates of C. variabile and three isolates of Stemphylium were recovered by plating surface-sterilized (0.1% sodium hypochlorite) sections of symptomatic leaf tissue onto water agar and acidified potato dextrose agar (PDA). Transfers of each isolate were made to PDA, and cultures were kept at 24 ± 2°C on a lab bench (natural day/night cycle) for 10 to 14 days. Spore suspensions (105/ml) of the isolates of C. variabile were prepared in a 0.01% solution of Tween 80. Isolates of Stemphylium produced few spores, so mycelial suspensions (105 fragments/ml) were prepared. Five 8-week-old seedlings of each of the cultivars Winter Bloomsdale and Ozarka II were inoculated per fungal isolate by atomizing the inoculum onto each seedling until all leaves were covered with a thin film of droplets (4 to 5 ml of inoculum per seedling). Plants were enclosed in plastic bags on a greenhouse bench (24 ± 3°C) for 72 h (8 h/16 h day/night). Symptoms developed within 80 h of inoculation for both isolates of C. variabile and two isolates of Stemphylium. Small (1 to 2 mm) sunken spots turned white 24 to 48 h later and became dry and bleached. Lesions caused by isolates of Stemphylium enlarged and coalesced more rapidly than lesions caused by C. variabile, and were more irregular and usually not delimited by the thin brown margin typical of lesions caused by C. variabile. The differences in symptoms were consistent on both spinach cultivars. Symptoms were not observed on non-inoculated control plants nor on plants inoculated with the third isolate of Stemphylium. C. variabile and Stemphylium were reisolated from symptomatic leaf tissue. Colony morphology, conidiophores, and conidia of the pathogenic Stemphylium isolates were similar to those of pathogenic isolates of Stemphylium botryosum obtained from spinach plants in California (2). This is the first report of S. botryosum as a foliar pathogen of spinach seed crops in Washington. Although Correll et al. (1) noted Stemphylium to be damaging on mature spinach plants grown for seed production, S. botryosum may not have been diagnosed previously on spinach seed crops in Washington because of the similarity of symptoms caused by S. botryosum and C. variabile. S. botryosum was recently reported as a foliar pathogen of spinach in California (2). References: (1) J. C. Correll et al. Plant Dis. 78:653, 1994. (2) S. T. Koike et al. Plant Dis. 85:126, 2001.

Evidence that Cercospora carotae Causes Leaf Spot on Carrot in Western Washington.

During 1999, a leaf spot on carrot (Daucus carota L. subsp. sativus [Hoffm.] Arcang.) was observed on nearly every plant in a 20-ha field of carrots (cv. Red Chantenay) grown for processing in western Washington. Circular to elongate, light brown lesions surrounded by chlorosis were present on leaflet margins and petioles of affected plants. Conidia of Cercospora carotae (Pass.) Solheim were present in the lesions. Small pieces of surface-sterilized leaf tissue were placed onto potato dextrose agar plates and incubated at room temperature to obtain fungal isolates. Koch's postulates were completed by atomizing the upper and lower leaves of carrot seedlings at the three to four leaf stage with sterile water or C. carotae at 1.0 × 104 conidia/ml in sterile 0.01% Tween 80. Treatments were replicated five times using single plants. The plants were bagged in clear plastic and placed in a greenhouse at 25°C for 72 h. Disease symptoms developed within 10 days as light brown lesions on leaflet margins and petioles, and were similar to those found in the field. The fungus was reisolated as described above. Symptoms did not develop in control plants sprayed with water. Farr et al. (1) report that C. carotae occurs in several states but not Washington, and Shaw (2) lists C. carotae only from British Columbia and Oregon. To our knowledge, this is the first report of Cercospora leaf spot on carrot in Washington. References: (1) D. F. Farr et al. 1989. Fungi on Plants and Plant Products in the United States. American Phytopathological Society, St. Paul, MN. (2) C. G. Shaw. W.S.U. Agric. Exp. Sta. Bull. 765, 1969.

Characterization of Phytophthora infestans Populations in Western Washington.

The first detection in the United States of isolates of Phytophthora infestans having metalaxyl insensitivity and complex pathotypes occurred in western Washington during the early 1990s. To determine the genetic structure of the current population in western Washington, a total of 115 isolates of P. infestans were obtained during 1996 from infected tubers or foliage of potato, tomato, nightshade, and bittersweet throughout the region. An additional 45 isolates were collected from a single field. Based on mating type, metalaxyl-insensitivity, and molecular markers (allozymes of glucose-6-phosphate isomerase, peptidase, and RG57 DNA fingerprint), all of the isolates were A1 mating type and had the US-11 multilocus genotype. Analyses of an additional 120 isolates collected during 1997 from potato, tomato, and nightshade were performed. As in 1996, US-11 was the predominant genotype detected on all three hosts. However, three additional A2 mating type genotypes were also detected: US-7, US-8, and US-14. These three genotypes represent the first A2 mating type isolates detected in western Washington. Most of a subset of 60 isolates infected 4 to 7 of the 10 potato differentials tested. This included 90% of the isolates collected in 1996 (all US-11), plus 72% of the US-11 and 100% of the US-8 and US-14 isolates collected during 1997. Virulence phenotypes in this region are complex even without the selection pressure of R-genes in the local commercial cultivars. The apparent increase in genetic variation observed in populations of P. infestans in western Washington from 1996 to 1997 most likely occurred by migration rather than by sexual recombination.