M. L. Derie

Verticillium Wilt in Spinach Seed Production

There are no previous reports of Verticillium wilt in fresh and processing spinach (Spinacia oleracea) crops in the United States. In 2002, a hybrid spinach seed crop in the Pacific Northwest developed late-season wilt symptoms. Assays of the harvested seed and stock seed of the male and female parents revealed 59.5, 44.0, and 1.5%, respectively, were infected with Verticillium dahliae. Assays of 13 stock or commercial seed lots grown in 2002 and 62 commercial lots harvested in 2003 in Denmark, Holland, New Zealand, and the United States revealed the prevalence of Verticillium spp. in commercial spinach seed. Sixty-eight lots (89%) were infected with Verticillium spp. at incidences ranging from 0.3 to 84.8%. Five spinach seed isolates of V. dahliae were pathogenic on each of three spinach cultivars by root-dip inoculation. V. dahliae was detected on 26.4% of the seed from 7 of 11 inoculated plants but on none of the seed from 6 control plants, demonstrating systemic movement of V. dahliae. Seed-to-seed transmission was also demonstrated by planting naturally infected seed lots. This is the first report of Verticillium wilt of spinach in the primary region of spinach seed production in the United States.

Bacterial Blight in Carrot Seed Crops in the Pacific Northwest

Carrot (Daucus carota subsp. sativus) seed crops in Oregon and Washington were surveyed in 2001-02 and 2002-03 for development of Xanthomonas campestris pv. carotae, causal agent of bacterial blight. For each state and season, 20 plants were sampled from each of 7 to 12 direct-seeded crops twice in the fall or winter and three times from spring to summer; and from each of 2 to 4 steckling (root-to-seed) crops three times from spring to summer. X. campestris pv. carotae was detected in 1 of 15 and 6 of 32 stock seed lots planted in the fall in Oregon and Washington, respectively, and in 2 of 6 steckling shipments planted in each state in the spring. The pathogen was detected at 102 to 108 CFU/g foliage in 1 of 22 and 10 of 14 direct-seeded crops within 2 months of planting in 2001 and 2002, respectively. The prevalence of X. campestris pv. carotae then increased through the season in most seed crops, although bacterial blight symptoms were not observed until April in Oregon and July in Washington in both seasons. In August 2002 and 2003, X. campestris pv. carotae was detected in all 10 and 13 crops surveyed in Oregon, respectively; and in 11 of 12 and 7 of 10 crops in Washington, respectively. The pathogen was typically less prevalent in steckling versus direct-seeded crops. X. campestris pv. carotae was detected in 20 of 22 and 19 of 23 harvested seed lots in Oregon and Washington, respectively, at populations ranging from 1.3 × 101 to 1.4 × 108 CFU/g seed. Airborne X. campestris pv. carotae, detected ≤1,600 m downwind of crops being threshed in Oregon in September of 2003 and 2004, may provide a source of inoculum for newly planted seed crops between overlapping biennial seasons for carrot seed production. Despite the prevalence of this pathogen in the Pacific Northwest, carrot seed lots free of X. campestris pv. carotae were detected, demonstrating the ability to produce clean seed in this region by adhering to recommended practices for management of bacterial blight.

Prevalence of Botrytis spp. in Onion Seed Crops in the Columbia Basin of Washington

Of 12 onion seed lots harvested in the semi-arid Columbia Basin of Washington in 1999 or 2000, 8 were infected and 10 infested with Botrytis aclada at incidences of 1 to 10% and 2 to 26%, respectively. Twenty to forty plants were sampled from each of nine direct-seeded, biennial seed crops in April, June, and July 2001 and assayed for Botrytis spp. Six direct-seeded crops were sampled in October and November 2001 and April, June, and July 2002. One bulb-to-seed crop was sampled in April, June, and July 2002. The incidence of B. aclada increased through each season, reaching 100% in most fields by July. Infections were primarily asymptomatic, with no apparent relationship between plant infection and infection of harvested seed. B, cinerea, B. squamosa, and B. porri were detected in 16, 4, and 4% of the fields, respectively, at lower incidences than B. aclada. Harvested seed from 15 of the fields were infected with B. aclada at <1 to 28%. B, cinerea, B. porri, and B. squamosa were detected in three, three, and none of the harvested lots, respectively.

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.

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.

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.

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.

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.