Inflation of 430-parsec bipolar radio bubbles in the Galactic Centre by an energetic event.

The Galactic Centre contains a supermassive black hole with a mass of four million Suns1 within an environment that differs markedly from that of the Galactic disk. Although the black hole is essentially quiescent in the broader context of active galactic nuclei, X-ray observations have provided evidence for energetic outbursts from its surroundings2. Also, although the levels of star formation in the Galactic Centre have been approximately constant over the past few hundred million years, there is evidence of increased short-duration bursts3, strongly influenced by the interaction of the black hole with the enhanced gas density present within the ring-like central molecular zone4 at Galactic longitude |l| < 0.7 degrees and latitude |b| < 0.2 degrees. The inner 200-parsec region is characterized by large amounts of warm molecular gas5, a high cosmic-ray ionization rate6, unusual gas chemistry, enhanced synchrotron emission7,8, and a multitude of radio-emitting magnetized filaments9, the origin of which has not been established. Here we report radio imaging that reveals a bipolar bubble structure, with an overall span of 1 degree by 3 degrees (140 parsecs × 430 parsecs), extending above and below the Galactic plane and apparently associated with the Galactic Centre. The structure is edge-brightened and bounded, with symmetry implying creation by an energetic event in the Galactic Centre. We estimate the age of the bubbles to be a few million years, with a total energy of 7 × 1052 ergs. We postulate that the progenitor event was a major contributor to the increased cosmic-ray density in the Galactic Centre, and is in turn the principal source of the relativistic particles required to power the synchrotron emission of the radio filaments within and in the vicinity of the bubble cavities.

First Report of Bacterial Blight of Crucifers Caused by Pseudomonas cannabina pv. alisalensis in Minnesota on Arugula (Eruca vesicaria subsp. sativa).

In 2011, bacterial blight of arugula (Eruca vesicaria subsp. sativa; cv. Roquette) was observed in organically grown plants under overhead irrigation in a field near Delano, MN. Approximately 80 to 100% of each planting was affected, with greater rates of infection occurring after periods of high humidity. Small, water-soaked, angular spots apparent on both sides of the leaves comprised the initial symptoms, which sometimes expanded and coalesced. Lesions maintained a dark water-soaked appearance or dried and turned a brown/tan color. Additionally, some lesions were outlined by a purple margin. Blue-green fluorescent pseudomonads were isolated consistently on King's Medium B agar (KMB) from symptomatic leaf tissue surface-disinfested with sodium hypochlorite (0.525%). The isolates nucleated ice and produced levan. Isolates were oxidase and arginine dihydrolase negative. They did not rot potato slices but did induce a hypersensitive reaction in tobacco (Nicotiana tabacum cv. Samsun). These data indicated that the bacteria belonged to Lelliott's LOPAT group 1 (2). DNA fragment banding patterns generated by amplifying DNA of the arugula isolates using repetitive extragenic palindromic sequence-polymerase chain reaction (rep-PCR) and the BOX A1R primer were identical and nearly identical to the banding patterns of the Pseudomonas cannabina pv. alisalensis (formerly P. syringae pv. alisalensis) (1) strain (CFBP1637) and the pathotype strain (CFBP 6866PT), respectively. Pathogenicity was confirmed on the arugula cv. My Way in two independent experiments, each with three replicate plants per treatment. Four isolates were grown on KMB for 48 h at 27°C, suspended in 0.01M potassium phosphate buffer (pH 7.0), and adjusted to 0.6 optical density at 600 nm (approximately 1 × 108 CFU/ml). Five- to six-week old plants were spray-inoculated until run-off, incubated in a humidity chamber for 48 h, and then placed in a greenhouse at 20 to 25°C for symptom development. For negative and positive control treatments, a similar number of plants each were sprayed with sterile buffer or P. cannabina pv. alisalensis strains CFBP1637 and CFBP 6866PT, respectively. Water-soaked and brown/tan lesions similar to the original symptoms appeared on plants inoculated with the arugula isolates and P. cannabina pv. alisalensis strains 7 to 14 days postinoculation. No symptoms developed on plants treated with sterile buffer. The bacterial strains re-isolated from surface-disinfested symptomatic tissue were identical by rep-PCR to the isolates used to inoculate the plants, thus, confirming Koch's postulates. Identical replicated experiments conducted on broccoli raab indicated that the arugula isolates were also pathogens of broccoli raab (Brassica rapa subsp. rapa, the original host from which P. cannabina pv. alisalensis was isolated). To our knowledge, this is the first report of bacterial blight of crucifers caused by P. cannabina pv. alisalensis in Minnesota. Arugula germplasm is being evaluated for resistance to this pathogen as an acceptable management method for organic cropping systems. References: (1) C. T. Bull et al. Syst. Appl. Microbiol. 33:105, 2010. (2) R. A. Lelliott. J. Appl. Bacteriol. 29:470, 1966.

Multiplex real-time PCR assays for detection of four seedborne spinach pathogens.

AIMS: To develop multiplex TaqMan real-time PCR assays for detection of spinach seedborne pathogens that cause economically important diseases on spinach. METHODS AND RESULTS: Primers and probes were designed from conserved sequences of the internal transcribed spacer (for Peronospora farinosa f. sp. spinaciae and Stemphylium botryosum), the intergenic spacer (for Verticillium dahliae) and the elongation factor 1 alpha (for Cladosporium variabile) regions of DNA. The TaqMan assays were tested on DNA extracted from numerous isolates of the four target pathogens, as well as a wide range of nontarget, related fungi or oomycetes and numerous saprophytes commonly found on spinach seed. Multiplex real-time PCR assays were evaluated by detecting two or three target pathogens simultaneously. Singular and multiplex real-time PCR assays were also applied to DNA extracted from bulked seed and single spinach seed. CONCLUSIONS: The real-time PCR assays were species-specific and sensitive. Singular or multiplex real-time PCR assays could detect target pathogens from both bulked seed samples as well as single spinach seed. SIGNIFICANCE AND IMPACT OF THE STUDY: The freeze-blotter assay that is currently routinely used in the spinach seed industry to detect and quantify three fungal seedborne pathogens of spinach (C. variabile, S. botryosum and V. dahliae) is quite laborious and takes several weeks to process. The real-time PCR assays developed in this study are more sensitive and can be completed in a single day. As the assays can be applied easily for routine seed inspections, these tools could be very useful to the spinach seed industry.

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.

Development and Evaluation of a TaqMan Real-Time PCR Assay for Fusarium oxysporum f. sp. spinaciae.

Fusarium oxysporum f. sp. spinaciae, causal agent of spinach Fusarium wilt, is an important soilborne pathogen in many areas of the world where spinach is grown. The pathogen is persistent in acid soils of maritime western Oregon and Washington, the only region of the United States suitable for commercial spinach seed production. A TaqMan real-time polymerase chain reaction (PCR) assay was developed for rapid identification and quantification of the pathogen, based on sequencing the intergenic spacer (IGS) region of rDNA of isolates of the pathogen. A guanine single-nucleotide polymorphism (G SNP) was detected in the IGS sequences of 36 geographically diverse isolates of F. oxysporum f. sp. spinaciae but not in the sequences of 64 isolates representing other formae speciales and 33 isolates representing other fungal species or genera. The SNP was used to develop a probe for a real-time PCR assay. The real-time PCR assay detected F. oxysporum f. sp. spinaciae at 3-14,056 CFU/g of soil in 82 soil samples collected over 3 years from naturally infested spinach seed production sites in western Washington, although a reliable detection limit of the assay was determined to be 11 CFU/g of soil. A significant (P < 0.05), positive correlation between enumeration of F. oxysporum on Komada's agar and quantification of the pathogen using the TaqMan assay was observed in a comparison of 82 soil samples. Correlations between pathogen DNA levels, Fusarium wilt severity ratings, and spinach biomass were significantly positive for one set of naturally infested soils but not between pathogen DNA levels, wilt incidence ratings, and spinach biomass for other soil samples, suggesting that soilborne pathogen population is not the sole determinant of spinach Fusarium wilt incidence or severity. The presence of the G SNP detected in one isolate of each of F. oxysporum ff. spp. lageneriae, lilii, melongenae, and raphani and reaction of the real-time PCR assay with 16 of 22 nonpathogenic isolates of F. oxysporum associated with spinach plants or soil in which spinach had been grown potentially limits the application of this assay. Nonetheless, because all isolates of F. oxysporum f. sp. spinaciae tested positive with the real-time PCR assay, the assay may provide a valuable means of screening for resistance to Fusarium wilt by quantifying development of the pathogen in spinach plants inoculated with the pathogen.

Stunting of Onion in the Columbia Basin of Oregon and Washington Caused by Rhizoctonia spp.

During 2009 and 2010, 45 isolates of Rhizoctonia spp. were recovered from onion bulb crops in the semiarid Columbia Basin of Oregon and Washington, in which patches of severely stunted onion plants developed following rotation with winter cereal cover crops. Characterization of isolates recovered from naturally infested soil and roots was performed by sequence analysis of the ribosomal DNA (rDNA) internal transcribed spacer region, with the majority of isolates (64%) identified as Rhizoctonia solani. In steam-pasteurized field soil, stunting of onion was caused by isolates of R. solani anastamosis groups (AGs) 2-1, 3, 4, and 8, as well as Waitea circinata var. circinata and binucleate Rhizoctonia AG E evaluated at 13 and 8 or 15 and 15°C day and night temperatures, respectively, typical of spring planting conditions in the Columbia Basin. Isolates of R. solani AG 5 as well as binucleate AG A and I were nonpathogenic. The most virulent isolates belonged to AG 8, although an AG 3 and an AG E isolate were also highly virulent. Isolates of AG 2-1 and 3 caused moderate levels of disease, while isolates of AG 4 and W. circinata var. circinata caused low levels of disease. Emergence was reduced by isolates of AG 2-1, 3, and E. When the various AGs were grown at temperatures of 5 to 30°C, the relative growth rate of the Rhizoctonia isolates was not positively correlated with virulence on onion within an AG.

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.

Effects of Postharvest Onion Curing Parameters on the Development of Sour Skin and Slippery Skin in Storage.

The influence of postharvest curing temperature and duration on development of slippery skin (caused by Burkholderia gladioli pv. alliicola) and sour skin (caused by B. cepacia) in onion (Allium cepa) bulbs during storage was evaluated by inoculating bulbs of the storage cultivars 'Redwing' and 'Vaquero' with each of the pathogens after harvest, curing the bulbs at 25, 30, 35, or 40°C for 2 or 14 days, and storing the bulbs at 5°C for 1, 2, or 3 months. Noninoculated bulbs and bulbs injected with sterile water served as control treatments. The onion bulbs were from drip-irrigated, commercial onion crops grown in the semiarid Columbia Basin of central Washington in 2009 and 2010. Each bulb was cut through the point of inoculation from the neck to the basal plate to assess severity of bulb rot (percentage of cut bulb surface area with bacterial rot symptoms) after 1, 2, or 3 months of storage. Bulb rot severity in the 2009-10 and 2010-11 trials was negligible for noninoculated bulbs (mean of 4.0 and 4.5%, respectively) and bulbs injected with water (6.2 and 10.1%, respectively) compared with bulbs inoculated with B. cepacia (34.6 and 39.8%, respectively) and B. gladioli pv. alliicola (20.7 and 27.4%, respectively). Bulbs inoculated with B. cepacia developed significantly more severe rot than those inoculated with B. gladioli pv. alliicola, even though a 10-fold greater inoculum concentration was used for B. gladioli pv. alliicola, demonstrating the more aggressive nature of B. cepacia compared with B. gladioli pv. alliicola. Severity of bulb decay caused by B. cepacia or B. gladioli pv. alliicola was affected significantly (P < 0.05) by season (trial), cultivar, curing temperature, curing duration, and storage duration, with significant interactions among these factors. In both trials and for both pathogens, bulb rot was significantly more severe the greater the curing temperature and the severity of bulb rot was significantly greater when bulbs were cured for 14 versus 2 days prior to cold storage. Overall, the severity of bulb rot was greater with a longer duration of storage after curing. This increase in bulb rot severity, which resulted from an increase in curing temperature and duration, was significantly greater for Vaquero than Redwing and significantly greater for bulbs inoculated with B. cepacia than B. gladioli pv. alliicola. The results suggest that postharvest curing at temperatures <35°C for a limited duration can significantly reduce the severity of sour skin or slippery skin in storage.

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.

Effects of Postharvest Onion Curing Parameters on Enterobacter Bulb Decay in Storage.

Enterobacter bulb decay is a recently described storage disease of onion (Allium cepa) bulbs caused by Enterobacter cloacae. The disease is generally considered minor but, on occasion, can cause significant losses for onion producers. The impact of postharvest curing temperature and duration on Enterobacter bulb decay of onion was evaluated by inoculating bulbs of the cultivars Redwing and Vaquero with E. cloacae after harvest, curing the bulbs at 25, 30, 35, or 40°C for 2 or 14 days, and storing the bulbs at 5°C for 1, 2, or 3 months. Noninoculated bulbs and bulbs injected with sterile water served as control treatments. The trial was completed using bulbs harvested from commercial onion crops grown in the semi-arid Columbia Basin of central Washington in each of 2008-09 (center-pivot irrigated crop) and 2009-10 (drip irrigated crop). Severity of bulb rot was assessed by cutting each bulb down the center from the neck to the basal plate, and rating the percentage of cut surface area with bacterial rot symptoms. Bulb rot severity was negligible for noninoculated bulbs (mean of 0.3% in the 2008-09 storage trial and 1.0% in the 2009-10 storage trial) and bulbs injected with water (0.8% in the 2008-09 trial and 1.3% in the 2009-10 trial) compared to bulbs inoculated with E. cloacae (15.3% in 2008-09 and 23.3% in 2009-10). Severity of Enterobacter bulb decay was affected significantly (P < 0.05) by season (trial), cultivar, curing temperature, curing duration, and storage duration, with significant interactions among these factors. Enterobacter bulb decay was significantly more severe for bulbs cured at 40°C than for bulbs cured at 25, 30, or 35°C. This effect was even greater when bulbs were cured for 14 days versus 2 days prior to cold storage, and in bulbs stored for 2 or 3 months after curing compared to bulbs stored for 1 month. The increase in bulb rot severity caused by curing bulbs at 40°C for 14 days compared to lower temperatures and shorter durations was greater for Vaquero than Redwing, particularly in the 2008-09 trial. The results suggest that curing temperatures ≤35°C should significantly reduce the risk of Enterobacter bulb decay in storage for these cultivars. If higher curing temperatures are used in order to dry onion necks for long-term storage and reduce the risk of fungal diseases such as neck rot (caused by Botrytis spp.), a shorter curing duration may be necessary to minimize the risk of Enterobacter bulb decay in storage.

Evaluation of Onion Cultivars for Resistance to Enterobacter cloacae in Storage.

Sixty-nine storage onion (Allium cepa) cultivars (seven white, five red, and 57 yellow cultivars) were evaluated in the Washington State University Onion Cultivar Trials in the semiarid Columbia Basin of central Washington in 2007-08 and/or 2008-09. Each cultivar was inoculated with Enterobacter cloacae, cured, stored under commercial storage conditions, and evaluated for bacterial storage rot symptoms approximately 4.5 months after storage. Noninoculated bulbs of each cultivar served as a control treatment in each experiment. In addition, bulbs injected with water served as a second control treatment in the 2008-09 experiment. Inoculation of onion bulbs with E. cloacae resulted in significantly higher incidence and severity of Enterobacter bulb decay compared to noninoculated bulbs and bulbs injected with sterile water. For bulbs inoculated with E. cloacae, mean severity of bacterial storage rot per cultivar ranged from 5 to 19% of the cross-section evaluated for each onion bulb in 2007-08 and from 9 to 29% in 2008-09. For noninoculated bulbs, mean severity ranged from 0 to 1% in 2007-08 and 0 to 3% in 2008-09. For bulbs injected with water in the 2008-09 experiment, severity of bulb rot ranged from 0 to 10% per cultivar, with four cultivars (OLYX05-26, RE-E, Redwing, and Talon) displaying bulb rot ratings significantly greater than 0%. For the 33 cultivars included in both experiments, a significant correlation in bulb rot severity ratings was detected for the 2007-08 versus 2008-09 experiments (r = 0.43 at P = 0.013). Redwing, Red Bull, T-433, Centerstone, and Salsa had low severity ratings in both experiments; whereas Montero, OLYS05N5, Caveat, and Granero had severe bulb rot ratings in both experiments. The results demonstrate that it should be possible to select for increased resistance to Enterobacter bulb decay in storage onion cultivars.

First Report of Enterobacter cloacae Causing Onion Bulb Rot in the Columbia Basin of Washington State.

In August of 2006, onion plants of cv. Redwing exhibiting premature dieback and bulb rot were obtained from a commercial onion crop under center pivot irrigation in the Columbia Basin of Washington State. High temperatures during the summer were similar to those in 2004, which preceded significant outbreaks of Enterobacter rot of onion bulbs in storage. Fungal pathogens of onion were not observed. Bacteria from infected bulb tissue were isolated and purified on nutrient broth yeast extract (NBY) agar, and 537 isolates were evaluated for the ability to ferment glucose anaerobically. Of the facultative anaerobes (~50% of all isolates), 48 isolates were arginine dihydrolase positive, indole negative, and unable to degrade pectin, i.e., characteristics typical of the genus Enterobacter (2), which includes Enterobacter cloacae, a bacterial pathogen reported to cause onion bulb rot in California and Colorado (1,3). Sixteen of the putative Enterobacter isolates, along with four strains of E. cloacae known to be pathogenic on onion (1) (ATCC 23355 and ATCC 13047, 310 (H. F. Schwartz, Colorado State University), and E6 (J. Loper, USDA ARS), were tested for pathogenicity on onion bulbs (8 to 10 cm in diameter; cv. Tamara). The isolates were grown overnight in NBY broth at 28°C, harvested by centrifugation and resuspended to an OD600 = 0.3 (~108 CFU/ml) in sterile distilled water. After the outermost fleshy scale of each bulb was removed, each bulb was surface disinfected in 0.6% NaOCl for 2 min, dipped in sterile distilled water, and then dipped in 95% ethanol. Each bulb was air dried before a 0.5-ml aliquot of bacterial suspension was injected into the shoulder of the bulb with a 20-gauge needle. Three bulbs were inoculated for each isolate, placed in individual plastic bags, sealed, and incubated at 30°C in the dark. Three bulbs injected with water and three noninjected bulbs served as controls. After 14 days, each bulb was sliced through the center and rated for rot. Thirteen isolates induced rot symptoms on the inner fleshy scales of all inoculated bulbs. Of these, seven also caused tan-to-brown discoloration of the inner fleshy scales; similar symptoms were caused by the four pathogenic reference strains of E. cloacae (1). No symptoms were observed in any of the controls. Symptoms were not observed when the bacteria, prepared as described above, were infiltrated into onion leaves. Bacteria were reisolated from the symptomatic inoculated bulb tissue and confirmed to be Enterobacter spp. by the above physiological tests. In addition, an isolate designated ECWSU2 and the corresponding strain recovered from one of the inoculated symptomatic bulbs, along with the four reference strains, were evaluated for anaerobic growth on a variety of carbon sources by using API 50 CHE test strips (bio Mérieux Vitek, Inc., Hazlewood, MO). The physiological test data along with sequence analysis of a portion of the 16S rRNA gene of each isolate confirmed all of these isolates to be E. cloacae (4; Ribosomal Database Project [ http://rdp.cme.msu.edu/ ]). To our knowledge, this is the first report of E. cloacae causing a bulb rot of onion in Washington State. References: (1) A. L. Bishop and R. M. Davis. Plant Dis. 74:692, 1990. (2) J. G. Holt et al. Bergey's Manual of Determinative Bacteriology. Williams and Wilkins, Baltimore, MD, 1994. (3) H. F. Schwartz and K. Otto. Plant Dis. 84:808, 2000. (4) L. Verdonck et al. Int. J. Syst. Bacteriol. 37:4, 1987.

Helleborus net necrosis virus: A New Carlavirus Associated with 'Black Death' of Helleborus spp.

Symptoms of 'black death' were observed on Helleborus spp. in each of three independent nurseries from across the United States. A new virus of the genus Carlavirus was identified in association with this disease. Symptomatic plants contained curved, rod-shaped particles averaging 800 by 17 nm, and yielded predominant bands of double-stranded (ds)RNA corresponding to approximately 9.0, 2.6, and 1.7 kbp. Amplification with degenerate primers for carlaviruses yielded a product of approximately 3,000 bp from diseased plants. Complete genomic sequences of two virus isolates were determined. Particle size, dsRNA patterns, genome organization, and sequence were consistent with members of the family Flexiviridae, genus Carlavirus. The name Helleborus net necrosis virus (HeNNV) is proposed for the virus associated with black death of Helleborus spp. in the United States. The sequence of the 3' terminus of Helleborus mosaic virus (HeMV) (genus Carlavirus) was also determined. Nucleotide sequences of HeNNV and HeMV were only 49% identical, revealing the distinct nature of these viruses. Assays for other viruses failed to reveal a consistent association of any other virus with black death symptoms. Cucumber mosaic virus was detected in hellebore specimens both with and without distinct black death symptoms.

First Report of Bacterial Blight on Conventionally and Organically Grown Arugula in Nevada Caused by Pseudomonas syringae pv. alisalensis.

In 2007, leaf spots were observed on arugula (Eruca vesicaria subsp. sativa cv. My Way) grown under sprinkler irrigation for fresh market in conventional and organic production fields located above 1,200 m (4,000 feet) in Nevada (NV). Approximately 30% of each planting was affected. Initially, symptoms consisted of small (<2 mm in diameter), angular, water-soaked spots visible from both sides of the leaf, some of which developed a shot-hole appearance. The spots enlarged and coalesced, remaining angular. Lesions ranged from black to tan, occasionally developing a chlorotic or purple margin. Some lesions resembled symptoms of downy mildew on arugula, but microscopic examination revealed no sporangiophores associated with the lesions. Bacterial ooze was observed when sections of symptomatic leaves were examined microscopically. Blue-green fluorescent pseudomonads were isolated from lesions on King's medium B agar from three arugula plantings. Twelve strains (at least three from each planting) were evaluated along with known strains of Pseudomonas syringae pv. alisalensis and P. syringae pv. maculicola in all assays. Bacterial strains were levan positive, oxidase negative, and arginine dihydrolase negative. Strains did not rot potato slices but induced a hypersensitive reaction on tobacco (Nicotiana tabacum L. cv. Sansun) indicating that the bacteria belonged to Lelliot's LOPAT group 1 (2). This was confirmed by analysis of fatty acid methyl esters (MIS-TSBA version 4.10; MIDI, Inc., Newark, DE), which indicated that the strains were highly similar (similarity >0.80) to P. syringae. Amplification of DNA between repetitive bacterial sequences (rep-PCR) using the BOXA1R primer resulted in identical banding patterns for the NV arugula strains and P. syringae pv. alisalensis from arugula in California (1). Koch's postulates were completed by confirming pathogenicity of the isolated strains on the arugula cvs. Italian and Astro. Strains were grown on nutrient agar for 48 h at 27°C, adjusted to 108 CFU/ml in sterile 0.01 M phosphate buffer (pH 7.0), and spray inoculated until runoff onto 2- to 3-week-old plants. Control plants were similarly sprayed with sterile phosphate buffer. Plants were held for 2 days in a mist chamber and 7 days on a greenhouse bench (24 to 26°C). Angular lesions similar to symptoms observed on the original plants developed on leaves of all inoculated arugula plants. In addition, some plants developed blackening of the smaller veins accompanied by chlorosis of the surrounding interveinal tissue in 10- to 20-mm diameter areas of the leaves. Small black lesions (as much as 10 mm long) were also observed on the petioles. Bacterial strains reisolated from the symptomatic tissue were identical to P. syringae pv. alisalensis by rep-PCR. Control plants remained symptomless. Similar inoculation and incubation methods confirmed that the host range of the NV arugula isolates was identical to that of known strains of P. syringae pv. alisalensis. The arugula and P. syringae pv. alisalensis isolates caused leaf spots on broccoli raab (Brassica rapa subsp. rapa cv. Sorento) and oats (Avena sativa cv. Montezuma). Pathogenicity tests were repeated. This confirms that the leaf spot observed on conventionally and organically grown arugula in NV was caused by P. syringae pv. alisalensis. To our knowledge, this is the first report of this disease on arugula in NV. References: (1) C. T. Bull et al. Plant Dis. 88:1384, 2004. (2) R. A. Lelliott. J. Appl. Bacteriol. 29:470, 1966.

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.

Sequence diversity of the nucleoprotein gene of iris yellow spot virus (genus Tospovirus, family Bunyaviridae) isolates from the western region of the United States.

Iris yellow spot virus (IYSV), a tentative virus species in the genus Tospovirus and family Bunyaviridae, is considered a rapidly emerging threat to onion production in the western United States (US). The present study was undertaken to determine the sequence diversity of IYSV isolates from infected onion plants grown in California, Colorado, Idaho, Oregon, Utah and Washington. Using primers derived from the small RNA of IYSV, the complete sequence of the nucleoprotein (NP) gene of each isolate was determined and the sequences compared. In addition, a shallot isolate of IYSV from Washington was included in the study. The US isolates of IYSV shared a high degree of sequence identity (95 to 99%) with one another and to previously reported isolates. Phylogenetic analyses showed that with the exception of one isolate from central Oregon and one isolate from California, all the onion and shallot isolates from the western US clustered together. This cluster also included onion and lisianthus isolates from Japan. A second distinct cluster consisted of isolates from Australia (onion), Brazil (onion), Israel (lisianthus), Japan (alstroemeria), The Netherlands (iris) and Slovenia (leek). The IYSV isolates evaluated in this study appear to represent two distinct groups, one of which largely represents isolates from the western US. Understanding of the population structure of IYSV would potentially provide insights into the molecular epidemiology of this virus.

First Report of Leaf Spot of Spinach Caused by Stemphylium botryosum in Arizona.

During the winter (December through February) of 2003-2004, and again during 2004-2005, spinach (Spinacia oleracea) crops in the Yuma region of Arizona developed a foliar disease that previously had not been diagnosed in this geographic area. The problem was found on only a few acres and severity was low. The first symptoms consisted of round to oval leaf spots that were gray to olive green and visible from both adaxial and abaxial leaf surfaces. The spots were 3 to 6 mm in diameter but expanded up to as much as 10 mm. As disease progressed, leaf spots became tan and dry and papery in texture. Fungal growth was not observed on the spots. Isolations from the edges of surface-sterilized lesions onto V8 juice agar consistently resulted in fungal colonies. The fungus was identified as Stemphylium botryosum based on the following morphological characteristics of isolates incubated under fluorescent lights: dark green-to-brown mycelial growth, unbranched conidiophores with distinctly swollen apical cells that had dark bands, and dictyoconidia. The conidia were brown, ellipsoidal to ovoid, verrucose, borne singly, and measured 17 to 28 × 13 to 19 µm. To test pathogenicity, inoculum of each of five isolates (approximately 1 × 105 conidia/ml) was sprayed separately onto 20 to 25 plants each of spinach cvs. Whitney, Rushmore, Lion, Springfield, Nordic IV, and Unipak 144. Inoculated plants were incubated in a humidity chamber for 48 h and then maintained in a greenhouse (24 to 26°C). After 10 to 14 days, leaf spots resembling those seen in the field developed on all inoculated plants, and S. botryosum was reisolated from the spots. Control plants were similarly inoculated with water but did not develop symptoms. To our knowledge, this is the first report of leaf spot of spinach caused by S. botryosum in Arizona. The possibility of seedborne S. botryosum (3) may account for the development of this disease in winter spinach crops in this arid region. Leaf spot could be damaging to spinach grown in this region if rainfall is higher than normal, such as in 2004-2005. This disease has been reported in production spinach crops in California, Delaware, Florida, and Maryland (2,4) and in spinach seed crops in Washington (1). References: (1) L. J. du Toit and M. L. Derie. Plant Dis. 85:920, 2001. (2) K. L. Everts and D. K. Armentrout. Plant Dis. 85:1209, 2001. (3) P. Hernandez-Perez and L. J. du Toit. (Abstr.) Phytopathology 95:S41, 2005. (4) R. N. Raid and T. Kucharek. 2003 Florida Plant Disease Management Guide: Spinach. University of Florida, Gainesville, 2003.