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Stemphylium leaf spot can result in significant losses to spinach seed, processing, and fresh market crops. Stemphylium isolates (n = 1,775) collected from 2000 to 2022 from spinach seed, leaves, and seed crop stem residues were used to assess the diversity of species associated with spinach. Eleven Stemphylium species were identified based on cmdA sequences: S. vesicarium (63.6% of isolates), S. beticola (48.9%), S. amaranthi (5.1%), S. eturmiunum (4.5%), S. astragali (4.0%), S. simmonsii (3.4%), and S. lucomagnoense , S. drummondii , S. gracilariae , S. lycopersici , and S. chrysanthemicola (each 0.6 to 1.7%). Only isolates of S. beticola , S. drummondii , and S. vesicarium were pathogenic to spinach. The incidence of spinach seed on which Stemphylium was observed ranged from 2.5 to 73.5% per seed lot, with S. vesicarium and S. beticola predominant. However, only 60.7 and 62.3% of isolates tested for these two species were pathogenic to spinach, respectively. Therefore, the incidence of Stemphylium species on spinach seed may not reflect accurately the risk of a seed lot carrying pathogenic isolates. Fused MAT1-1 and MAT1-2 genes were detected in isolates of S. vesicarium , but only MAT1-1 was detected in S. beticola isolates, which corroborates previous studies that have proposed the two species to be self-fertile. The duration of ascospore dispersal of S. beticola and S. vesicarium from spinach seed crop stem residues in western Washington, the primary region of spinach seed production in the USA, occurred from mid-winter to late spring or early fall, potentially serving as inoculum for the next season's spinach seed crops. Growers should incorporate residues into the soil after harvest to reduce inoculum production of these pathogens on spinach seed crop residues.

Stemphylium leaf spot of spinach, caused by Stemphylium beticola and S. vesicarium , is a disease of economic importance in fresh market, processing, and seed production. There have been increasing reports of difficulty managing the disease in the southern United States using fungicides in Fungicide Resistance Action Committee (FRAC) group 11. Isolates of S. beticola and S. vesicarium obtained from spinach leaves and seed from 2001 to 2020 were screened for resistance to azoxystrobin and pyraclostrobin in vitro, in vivo, and using PCR assays to detect mutations in cytochrome b associated with resistance in other fungi (F129L, G137R, and G143A). EC 50 values for mycelial growth and conidial germination of S. vesicarium isolates in vitro were significantly less (mean of 0.35 μg/ml) than that of S. vesicarium (mean of 14.17 μg/ml) with both fungicides. All isolates were slightly more sensitive to pyraclostrobin than azoxystrobin in both assays. In vivo assays of plants inoculated with the isolates of S. vesicarium demonstrated poor efficacy of fungicides with each of the two active ingredients. Only the G143A mutation was detected in all spinach isolates of S. vesicarium , including an isolate of S. vesicarium collected in 2003 and 82.9% of isolates from spinach seed lots harvested from crops grown in or after 2017 in Europe, New Zealand, and the United States. The FRAC 11 mutations were not detected in any isolates of S. beticola . The in vitro, in vivo, and DNA mutation assays suggest FRAC group 11 fungicide resistance is widespread in spinach isolates of S. vesicarium but not S. beticola., Competing Interests: The author(s) declare no conflict of interest.

The landscape of scientific publishing is experiencing a transformative shift toward open access, a paradigm that mandates the availability of research outputs such as data, code, materials, and publications. Open access provides increased reproducibility and allows for reuse of these resources. This article provides guidance for best publishing practices of scientific research, data, and associated resources, including code, in The American Phytopathological Society journals. Key areas such as diagnostic assays, experimental design, data sharing, and code deposition are explored in detail. This guidance aligns with that observed by other leading journals. We hope the information assembled in this paper will raise awareness of best practices and enable greater appraisal of the true effects of biological phenomena in plant pathology., Competing Interests: The author(s) declare no conflict of interest.

Arbuscular mycorrhizal fungi (AMF) are ancient and ecologically important symbionts that colonize plant roots. These symbionts assist in the uptake of water and nutrients, particularly phosphorus, from the soil. This important role has led to the development of AMF inoculants for use as biofertilizers in agriculture. Commercial mycorrhizal inoculants are increasingly popular to produce onion and carrot, but their specific effects on native mycorrhizal communities under field conditions are not known. Furthermore, adequate availability of nutrients in soils, specifically phosphorus, can reduce the diversity and abundance of AMF communities in the roots. The type of crop grown can also influence the composition of AMF communities colonizing the plant roots. This study aimed to investigate how AMF inoculants, soil phosphorus levels, and plant species influence the diversity of AMF communities that colonize the roots of onion and carrot plants. Field trials were conducted on high organic matter (muck) soil in the Holland Marsh, Ontario, Canada. The treatments included AMF-coated seeds (three to five propagules of Rhizophagus irregularis per seed) and non-treated onion and carrot seeds grown in soil with low (~46 ppm) and high (~78 ppm) phosphorus levels. The mycorrhizal communities colonizing the onion and carrot roots were identified by Illumina sequencing. Five genera, Diversispora , Claroideoglomus , Funneliformis , Rhizophagus , and Glomus , were identified in roots of both plant species. AMF communities colonizing carrot roots were more diverse and richer than those colonizing onion roots. Diversispora and Funneliformis had a 1.3-fold and 2.9-fold greater abundance, respectively, in onion roots compared to carrots. Claroideoglomus was 1.4-fold more abundant in carrot roots than in onions. Inoculation with R. irregularis increased the abundance and richness of Rhizophagus in AMF communities of onion roots but not in carrot roots. The soil phosphorus level had no effect on the richness and diversity of AMF in the roots of either crop. In summary, AMF inoculant and soil phosphorus levels influenced the composition of AMF communities colonizing the roots of onion and carrot plants, but the effects varied between plant species., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Ilyas, du Toit, Hajibabaei and McDonald.)

Fusarium wilt of spinach, caused by Fusarium oxysporum f. sp. spinaciae , is a significant limitation for producers of vegetative spinach and spinach seed crops during warm temperatures and/or on acid soils. Identification of isolates of F. oxysporum f. sp. spinaciae , and distinction of isolates of the two known races, entails time-intensive pathogenicity tests. In this study, two real-time PCR assays were developed: one for a candidate effector gene common to both races of F. oxysporum f. sp. spinaciae , and another for a candidate effector gene unique to isolates of race 2. The assays were specific to isolates of F. oxysporum f. sp. spinaciae ( n = 44) and isolates of race 2 ( n = 23), respectively. Neither assay amplified DNA from 10 avirulent isolates of F. oxysporum associated with spinach, 57 isolates of other formae speciales and Fusarium spp., or 7 isolates of other spinach pathogens. When the assays were used to detect DNA extracted from spinach plants infected with an isolate of race 1, race 2, or a 1:1 mixture of both races, the amount of target DNA detected increased with increasing severity of wilt. Plants infected with one or both isolates could be distinguished based on the ratio in copy number for each target locus. The real-time PCR assays enable rapid diagnosis of Fusarium wilt of spinach and will facilitate research on the epidemiology and management of this disease, as well as surveys on the prevalence of this understudied pathogen in regions of spinach and/or spinach seed production., Competing Interests: The author(s) declare no conflict of interest.

Two pathogenicity groups of Fusarium oxysporum f. sp. spinaciae , the causal agent of Fusarium wilt of spinach ( Spinacia oleracea ), were described recently based on virulence of isolates on proprietary spinach inbreds. In this study, a wide range in severity of wilt was observed for 68 spinach cultivars inoculated with an isolate of each pathogenicity group, with 22 (32.4%) cultivars displaying differential responses to the isolates. In a second set of trials, seven spinach cultivars were inoculated with five isolates of each pathogenicity group. The cultivars had similar wilt responses to isolates within each group. In both sets of trials, the most severe wilt developed on cultivars inoculated with pathogenicity group 2 isolates when daylength was shorter and light intensity lower. To test whether light intensity exacerbates severity of Fusarium wilt, three spinach cultivars were inoculated with two isolates of each pathogenicity group and grown with or without shading. Shaded plants developed more severe wilt than nonshaded plants. This difference in wilt severity was greatest for plants inoculated with pathogenicity group 2 isolates. We propose naming isolates of pathogenicity groups 1 and 2 as races 1 and 2 of F. oxysporum f. sp. spinaciae , respectively, and recommend the cultivars Kiowa (susceptible to both races) and Magnetic (susceptible to race 2 and highly resistant to race 1) as differentials. Results of this study should help breeders screen spinach germplasm for resistance to both races of F. oxysporum f. sp. spinaciae .

White rust, caused by Albugo occidentalis , is one of the major yield-limiting diseases of spinach ( Spinacia oleracea ) in some major commercial production areas, particularly in southern Texas in the United States. The use of host resistance is the most economical and environment-friendly approach to managing white rust in spinach production. The objectives of this study were to conduct a genome-wide associating study (GWAS), to identify single nucleotide polymorphism (SNP) markers associated with white rust resistance in spinach, and to perform genomic prediction (GP) to estimate the prediction accuracy (PA). A GWAS panel of 346 USDA (US Dept. of Agriculture) germplasm accessions was phenotyped for white rust resistance under field conditions and GWAS was performed using 13 235 whole-genome resequencing (WGR) generated SNPs. Nine SNPs, chr2_53 049 132, chr3_58 479 501, chr3_95 114 909, chr4_9 176 069, chr4_17 807 168, chr4_83 938 338, chr4_87 601 768, chr6_1 877 096, and chr6_31 287 118, located on chromosomes 2, 3, 4, and 6 were associated with white rust resistance in this GWAS panel. Four scenarios were tested for PA using Pearson's correlation coefficient (r) between the genomic estimation breeding value (GEBV) and the observed values: (1) different ratios between the training set and testing set (fold), (2) different GP models, (3) different SNP numbers in three different SNP sets, and (4) the use of GWAS-derived significant SNP markers. The results indicated that a 2- to 10-fold difference in the various GP models had similar, although not identical, averaged r values in each SNP set; using GWAS-derived significant SNP markers would increase PA with a high r-value up to 0.84. The SNP markers and the high PA can provide valuable information for breeders to improve spinach by marker-assisted selection (MAS) and genomic selection (GS)., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nanjing Agricultural University.)

Genotype-by-sequencing (GBS) was used to explore the genetic diversity and structure of Spinacia turkestanica , and the selective sweeps involved in domestication of cultivated spinach, S. oleracea , from S. turkestanica . A total 7,065 single nucleotide polymorphisms (SNPs) generated for 16 Spinacia oleracea and 76 S. turkestanica accessions placed the S. oleracea accessions in one group, Q 1, and the 76 S. turkestanica accessions, which originated from Central Asia, in two distinct groups, Q 2 and Q 3. The Q 2 group shared greater genetic identity with the S. oleracea accessions, Q 1, than the Q 3 S. turkestanica group. Likewise, the S. oleracea Q 1 group had a smaller F st (0.008) with the Q 2 group than with the Q 3 group ( F st = 0.012), and a greater gene flow (Nm = 30.13) with the Q 2 group than with the Q 3 group (Nm = 21.83). The Q 2 accessions originated primarily from Uzbekistan while the Q 3 accessions originated mostly from Tajikistan. The Zarafshan Mountain Range appears to have served as a physical barrier that largely separated members of the Q 2 and Q 3 groups of S. turkestanica . Accessions with admixtures of Q 2 and Q 3 were collected primarily from lower elevations at the southern end of the Zarafshan Mountain Range in Uzbekistan. Selective sweep regions identified at 32, 49, and 52 Mb on chromosomes 1, 2, and 3, respectively, appear to have played a vital role in the domestication of S. oleracea as they are correlated with important domestication traits, including day length sensitivity for bolting (flowering). High XP-CLR scores at the 52 Mb genomic region of chromosome three suggest that a selective sweep at this region was responsible for early differentiation of S. turkestanica into two groups in Central Asia., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Gyawali, Bhattarai, Shi, Kik and du Toit.)

Main Conclusion: A maize receptor kinase controls defense response to fungal pathogens by regulating jasmonic acid and antimicrobial phytoalexin production. Plants use a range of pattern recognition receptors to detect and respond to biotic threats. Some of these receptors contain leucine-rich repeat (LRR) domains that recognize microbial proteins or peptides. Maize (Zea mays) has 226 LRR-receptor like kinases, making it challenging to identify those important for pathogen recognition. In this study, co-expression analysis with genes for jasmonic acid and phytoalexin biosynthesis was used to identify a fungal induced-receptor like protein kinase (FI-RLPK) likely involved in the response to fungal pathogens. Loss-of-function mutants in fi-rlpk displayed enhanced susceptibility to the necrotrophic fungal pathogen Cochliobolus heterostrophus and reduced accumulation of jasmonic acid and the anti-microbial phytoalexins -kauralexins and zealexins- in infected tissues. In contrast, fi-rlpk mutants displayed increased resistance to stem inoculation with the hemibiotrophic fungal pathogen Fusarium graminearum. These data indicate that FI-RLPK is important for fungal recognition and activation of defenses, and that F. graminearum may be able to exploit FI-RLPK function to increase its virulence., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Spinach (Spinacia oleracea L.) is a member of the Caryophyllales family, a basal eudicot asterid that consists of sugar beet (Beta vulgaris L. subsp. vulgaris), quinoa (Chenopodium quinoa Willd.), and amaranth (Amaranthus hypochondriacus L.). With the introduction of baby leaf types, spinach has become a staple food in many homes. Production issues focus on yield, nitrogen-use efficiency and resistance to downy mildew (Peronospora effusa). Although genomes are available for the above species, a chromosome-level assembly exists only for quinoa, allowing for proper annotation and structural analyses to enhance crop improvement. We independently assembled and annotated genomes of the cultivar Viroflay using short-read strategy (Illumina) and long-read strategies (Pacific Biosciences) to develop a chromosome-level, genetically anchored assembly for spinach. Scaffold N50 for the Illumina assembly was 389 kb, whereas that for Pacific BioSciences was 4.43 Mb, representing 911 Mb (93% of the genome) in 221 scaffolds, 80% of which are anchored and oriented on a sequence-based genetic map, also described within this work. The two assemblies were 99.5% collinear. Independent annotation of the two assemblies with the same comprehensive transcriptome dataset show that the quality of the assembly directly affects the annotation with significantly more genes predicted (26,862 vs. 34,877) in the long-read assembly. Analysis of resistance genes confirms a bias in resistant gene motifs more typical of monocots. Evolutionary analysis indicates that Spinacia is a paleohexaploid with a whole-genome triplication followed by extensive gene rearrangements identified in this work. Diversity analysis of 75 lines indicate that variation in genes is ample for hypothesis-driven, genomic-assisted breeding enabled by this work., (© 2021 The Authors. The Plant Genome published by Wiley Periodicals LLC on behalf of Crop Science Society of America.)

Two fields of coriander ( Coriandrum sativum L.) seed crops of proprietary cultivars were observed in the Columbia Basin of Washington in July 2020 with 40 and 90% incidence of plants showing stunting and leaf and stem discoloration, sometimes with mild leaf curl. Foliar discoloration ranged from yellow to red and purple. Sweep-netting along the field edges collected one beet leafhopper ( Circulifer tenellus Baker; BLH), the known vector of Beet curly top virus (BCTV), Beet leafhopper transmitted virescence agent (BLTVA) phytoplasma, and Spiroplasma citri , all of which affect Solanaceae and Apiaceae crops in Washington (Crosslin et al. 2006; Johnson and Martin 1998; Lee et al. 2006). Nucleic acids extracted from leaves and petioles of 12 coriander plants (8 from Field 1 and 4 from Field 2) using the Dellaporta method, and from the BLH using the CTAB method (Crosslin et al. 2006) were subjected to PCR assays to detect the BLH-transmitted pathogens which cause yellow and purple discoloration in potato ( Solanum tuberosum L.) and carrot ( Daucus carota subsp. sativus (Hoffm.) Arc.) in this region. BLTVA was targeted using a species-specific nested PCR assay with primers P1 and P7, followed by primers FU5 and BLTVA-int (Crosslin et al. 2006); S. citri was targeted using primers P89-F and P89-R (Yokomi et al. 2008); and BCTV was targeted using curtovirus primers BCTV2-F and BCTV2-R (Strausbaugh et al. 2008). BLTVA and S. citri were not detected in the plants, but curtovirus was detected in 10 of the 12 plants. All three pathogens were detected from the single BLH. A 519 bp region of the curtovirus capsid protein gene was amplified from seven plants (5 from Field 1 and 2 from Field 2) and the BLH, and cloned into TOP10 Escherichia coli cells using the pCR-2.1 TOPO vector (Invitrogen, Carlsbad, CA). Three clones were sequenced from each sample. For each of six plant samples and the BLH, the three clones were identical and consensus sequences were generated (GenBank Accessions MW234419 to MW234425). For the seventh plant, two clones were identical in sequence (MW234426) and the third contained 12 single nucleotide polymorphisms (MW234427). All sequences were subjected to an NCBI BLASTn analysis and showed 98.3 to 99.8% identity with BCTV sequences. Additional PCR assays with primers BMCTV-C1 2213F and BMCTV-C1 2609R (Strausbaugh et al. 2008), targeting the C1 gene of the Worland strain of BCTV, detected BCTV-Worland-like strains in all plants and the BLH, confirming that BCTV was present and indicating that the strain-specific primer pair was more sensitive than the universal curtovirus primers. Yield losses in the two fields were approximately 60%, with reduced seed size but not seed quality. BCTV infections in coriander crops have been observed in the Columbia Basin in 2002, 2005, 2008, and 2013, with yield losses ranging from 10 to 100% per field, though official reports were not made following the diagnoses (Crosslin, du Toit, and Frost, unpublished data). BCTV has caused millions of dollars of losses in the U.S. in crops such as sugar beet ( Beta vulgaris subsp. vulgaris L.), tomato ( S. lycopersicum L.), and pepper ( S. annuum L.) (Johnson and Martin 1998). This is the first publication of BCTV affecting seed production of the specialty crop C. sativum . The observation of 90% incidence of symptoms in one field suggests that resistant cultivars and/or insect pest management practices are needed to prevent significant impacts of BCTV on coriander seed production in this semi-arid region.

Arizona is an important region of the USA for winter production of baby leaf crops such as spinach ( Spinacia oleracea ), table beet ( Beta vulgaris subsp. vulgaris Condivita Group), and Swiss chard ( B. vulgaris subsp. vulgaris Cicla Group). In the winter of 2019, severe leaf spots were observed at 80% incidence and 40% severity per plant in a 1-ha baby leaf Swiss chard crop of an (unknown cultivar) in Arizona. The lesions were circular to irregular, necrotic, water-soaked, and 1 to 5 mm in diameter. Symptomatic leaf sections (1-cm 2 ) were surface-sterilized with 0.6% NaOCl, rinsed, and macerated in sterilized, deionized water. An aliquot of each macerate was streaked onto King's B (KB) agar medium. Cream-colored, non-fluorescent colonies typical of Pseudomonas were isolated consistently, and all were non-fluorescent. A dozen isolates selected randomly were all negative for potato soft rot, oxidase, and arginine dihydrolase, and positive for levan production and tobacco hypersensitivity, which is typical of fluorescent P. syringae isolates, but can also include non-fluorescent strains (Lelliot et al. 1966). Three isolates were tested for pathogenicity on the table beet cv. Red Ace and Swiss chard cv. Silverado. Strain Pap009 of P. syringae pv. aptata (Psa), demonstrated previously to be pathogenic on Swiss chard and table beet, served as a positive control strain (Derie et al. 2016; Safni et al. 2016). Each isolate was grown inoculated into medium 523 broth and incubated on a shaker at 175 rpm overnight at 25°C. Each bacterial suspension was adjusted to an optical density (OD) of 0.3 at 600 nm (10 8 CFU/ml), and diluted in 0.0125M phosphate buffer to 10 7 CFU/ml. Thirty-day-old seedlings grown in Redi-Earth Plug and Seedling Mix in a greenhouse at 22 to 26°C were inoculated by rubbing the abaxial and adaxial leaf surfaces of each plant with a cotton swab dipped in inoculum to which Carborundum had been added (0.06 g/10 ml). The negative control plants were treated similarly with phosphate buffer with Carborundum. The experiment was set up as a randomized complete block design with 4 replications per treatment and 6 seedlings per experimental unit. In both trials, leaf spots resembling those on the original plants developed on all table beet and Swiss chard plants inoculated with the Arizona isolates and Pap009, but not on negative control plants. Disease severity was greater on Swiss chard (average 39% leaf area with spots) than on table beet (14%). Re-isolates obtained from inoculated seedlings using the same method as the original isolations resembled Psa. Multilocus sequence analysis (MLSA) was carried out for the original three Arizona isolates and the re-isolates using DNA amplified from the housekeeping genes gyrB , rpoD , gapA , and gltA (Hwang et al. 2005; Sarkar and Guttman 2004). Sequence identities of these genes of the Arizona isolates (GenBank accession numbers MW291615 to MW291618 for strain Pap089; MW291619 to MW291622 for Pap095; and MW291623 to MW291626 for Pap096 for gltA , gyrB , rpoD , and gapA , respectively) and the re-isolates ranged from 98 to 100% with those of Psa pathotype strain CFBP 1617 in the PAMDB database (Almeida et al. 2010; Altschul et al. 1997). Based on Koch's postulates, colony characteristics, and MLSA, Psa was the causal agent of leaf spots in the Arizona Swiss chard crop. To our knowledge, this is the first report of bacterial leaf spot on chard in Arizona. The pathogen could have been introduced on infected seed as Psa is readily seedborne and seed transmitted.

Leaf spot diseases of spinach, caused by Colletotrichum spinaciae , has become a major production constraint in several production areas, including Texas, in recent years. Leaf spot symptoms were observed in several fields in Texas in 2016 and 2017, with typical anthracnose-like symptoms and leaves with small, circular, and sunken lesions that appeared similar to injury from windblown sand. The lesions were plated on potato dextrose agar, from which fungal cultures were recovered. The fungi were identified based on morphology and sequence analysis of the introns of glutamate synthetase and glyceraldehyde-3-phosphate dehydrogenase (for isolates determined to be Colletotrichum spp.) and the internal transcribed spacer ribosomal DNA (for isolates determined to be Myrothecium spp.). Based on foliar symptoms, fungal colony and spore morphology, pathogenicity tests of fungal isolates on the spinach cultivar 'Viroflay', and DNA sequence analysis of the isolates, the symptoms on spinach leaves for two sets of samples were caused by Colletotrichum coccodes and Colletotrichum truncatum , and leaf spots resembling damage from windblown sand were caused by Myrothecium verrucaria . This is the first report of spinach leaf spot diseases caused by C. coccodes , C. truncatum , and M. verrucaria in the United States. C. coccodes and C. truncatum caused severe symptoms on the spinach cultivar 'Viroflay', whereas M. verrucaria caused symptoms of intermediate severity. Fungicide efficacy tests demonstrated that chlorothalonil, mancozeb, pyraclostrobin, fluxapyroxad + pyraclostrobin, and penthiopyrad were completely effective at preventing leaf spots caused by any of these pathogens when applied 24 h before inoculation of 'Viroflay' plants in greenhouse trials.

Fusarium wilt of spinach, caused by Fusarium oxysporum f. sp. spinaciae , is an important disease during warm conditions in production regions with acid soils, yet little is known about what confers pathogenicity to spinach in F. oxysporum f. sp. spinaciae genetically. To identify candidate fungal genes that contribute to spinach Fusarium wilt, each of 69 geographically diverse F. oxysporum isolates was tested for pathogenicity on each of three spinach inbreds. Thirty-nine isolates identified as F. oxysporum f. sp. spinaciae caused quantitative differences in disease severity among the inbreds that revealed two distinct pathogenicity groups of F. oxysporum f. sp. spinaciae . Putative effector gene profiles, predicted from whole-genome sequences generated for nine F. oxysporum f. sp. spinaciae isolates and five nonpathogenic, spinach-associated F. oxysporum (NPS) isolates, distinguished the F. oxysporum f. sp. spinaciae isolates from the NPS isolates, and separated the F. oxysporum f. sp. spinaciae isolates into two groups. Five of the putative effector genes appeared to be unique to F. oxysporum f. sp. spinaciae , as they were not found in 222 other publicly available genome assemblies of F. oxysporum , implicating potential involvement of these genes in pathogenicity to spinach. In addition, two combinations of the 14 known Secreted in Xylem ( SIX ) genes that have been affiliated with host pathogenicity in other formae speciales of F. oxysporum were identified in genome assemblies of the nine F. oxysporum f. sp. spinaciae isolates, either SIX8 and SIX9 or SIX4 , SIX8 , and SIX14 . Characterization of these putative effector genes should aid in understanding mechanisms of pathogenicity in F. oxysporum f. sp. spinaciae , developing molecular tools for rapid detection and quantification of F. oxysporum f. sp. spinaciae , and breeding for resistance to Fusarium wilt in spinach.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Leaf spot diseases have become a major concern in spinach production in the United States. Determining the causal agents of leaf spots on spinach, their prevalence and pathogenicity, and fungicide efficacy against these pathogens is vital for effective disease management. Spinach leaves with leaf spots were collected from Texas, California, Arizona, and South Carolina from 2016 to 2018, incubated in a moist chamber, and plated on potato dextrose and tryptic soy agar media. Fungal and bacterial colonies recovered were identified based on morphology and sequence analysis of the internal transcribed spacer rDNA and 16S rRNA, respectively. Two predominant genera were isolated: (i) Colletotrichum spp., which were identified to species based on sequences of both introns of the glutamate synthetase ( GS -I) and glyceraldehyde-3-phosphate dehydrogenase ( gapdh -I) genes; and (ii) Stemphylium spp., identified to species based on sequences of the gapdh and calmodulin ( cmdA ) genes. Anthracnose ( Colletotrichum spinaciae ) and Stemphylium leaf spot ( Stemphylium vesicarium and S. beticola ) were the predominant diseases. Additional fungi recovered at very limited frequencies that were also pathogenic to spinach included Colletotrichum coccodes , C. truncatum , Cercospora beticola , and Myrothecium verrucaria . All of the bacterial isolates were not pathogenic on spinach. Pathogenicity tests showed that C. spinaciae , S. vesicarium , and S. beticola caused significant leaf damage. The fungicides Bravo WeatherStik (chlorothalonil), Dithane F-45 (mancozeb), Cabrio (pyraclostrobin), and Merivon (fluxapyroxad and pyraclostrobin) were highly effective at reducing leaf spot severity caused by an isolate of each of C. spinaciae and S. vesicarium , when inoculated individually and in combination.

Light leaf spot, caused by the ascomycete Pyrenopeziza brassicae , is an established disease of Brassicaceae in the United Kingdom (UK), continental Europe, and Oceania (OC, including New Zealand and Australia). The disease was reported in North America (NA) for the first time in 2014 on Brassica spp. in the Willamette Valley of western Oregon, followed by detection in Brassica juncea cover crops and on Brassica rapa weeds in northwestern Washington in 2016. Preliminary DNA sequence data and field observations suggest that isolates of the pathogen present in NA might be distinct from those in the UK, continental Europe, and OC. Comparisons of isolates from these regions using genetic (multilocus sequence analysis, MAT gene sequences, and rep-PCR DNA fingerprinting), pathogenic ( B. rapa inoculation studies), biological (sexual compatibility), and morphological (colony and conidial morphology) analyses demonstrated two genetically distinct evolutionary lineages. Lineage 1 comprised isolates from the UK, continental Europe, and OC, and included the P. brassicae type specimen. Lineage 2 contained the NA isolates associated with recent disease outbreaks in the Pacific Northwest region of the USA. Symptoms caused by isolates of the two lineages on B. rapa and B. juncea differed, and therefore "chlorotic leaf spot" is proposed for the disease caused by Lineage 2 isolates of P. brassicae . Isolates of the two lineages differed in genetic diversity as well as sensitivity to the fungicides carbendazim and prothioconazole., Competing Interests: The authors have no conflicts of interest to declare., (© 2019 The Authors. Plant Pathology published by John Wiley & Sons Ltd on behalf of British Society for Plant Pathology.)

The Galactic Centre contains a supermassive black hole with a mass of four million Suns 1 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 surroundings 2 . 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 bursts 3 , strongly influenced by the interaction of the black hole with the enhanced gas density present within the ring-like central molecular zone 4 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 gas 5 , a high cosmic-ray ionization rate 6 , unusual gas chemistry, enhanced synchrotron emission 7,8 , and a multitude of radio-emitting magnetized filaments 9 , 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 × 10 52  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.

Phoma betae is an important seedborne pathogen of table beet worldwide that is capable of causing foliar, root, and damping-off diseases. Ten microsatellite and mating type markers were developed to investigate the genetics of P. betae populations in table beet root crops in New York and in table beet seed crops in Washington, from where table beet seed is predominantly sourced. The markers were used to characterize 175 isolates comprising five P. betae populations (two from New York and three from Washington), and they were highly polymorphic with an allelic range of 4 to 33 and an average of 11.7 alleles per locus. All populations had high genotypic diversity (Simpson's complement index = 0.857 to 0.924) and moderate allelic diversity (Nei's unbiased gene diversity = 0.582 to 0.653). Greater differentiation observed between populations from the two states compared with populations within the same state suggested that an external inoculum source, such as windblown ascospores, may be homogenizing the populations. However, most genetic diversity (87%) was among individual isolates within populations (pairwise index of population differentiation = 0.127; P = 0.001), suggesting that local within-field inoculum source(s), such as infested field debris or infected weeds, may also be important in initiating disease outbreaks. Standardized index of association, proportion of compatible pairs of loci, and mating type ratio calculations showed evidence for a mixed reproduction mode in all populations. These findings could be useful in designing more effective management strategies for diseases caused by P. betae in table beet production.

Blackleg (Phoma stem canker) of crucifers is a globally important disease caused by the ascomycete species complex comprising of Leptosphaeria maculans and Leptosphaeria biglobosa . Six blackleg isolates recovered from Brassica rapa cv. Mizspoona in the Willamette Valley of Oregon were characterized as L. biglobosa based on standard pathogenicity tests and molecular phylogenetic analysis. These isolates were compared to 88 characterized L. biglobosa isolates from western Canada, 22 isolates from Australia, and 6 L. maculans isolates from Idaho, USA using maximum parsimony and distance analysis of phylogenetic trees generated from the ITS rDNA (internal transcribed spacer rDNA) sequence, and the actin and β-tubulin gene sequences. The L. biglobosa isolates derived from B. rapa collected in Oregon formed a separate subclade based on concatenated gene sequences or a single gene sequence, regardless of the analyses. Pathogenicity tests showed that these isolates failed to infect either resistant or susceptible B. napus cultivars, but caused severe symptoms on three B. rapa cultivars (Accession number: UM1113, UM1112, and UM1161), a B. oleracea var. capitata (cabbage) cultivar (Copenhagen Market), and two B. juncea cultivars (CBM, a common brown Mustard, and Forge). These findings demonstrated that the L. biglobosa isolates derived from a B. rapa crop in Oregon were genetically distinct from existing species of L. biglobosa , and constitute a new subclade, herein proposed as L. biglobosa 'americensis'.

Fusarium wilt of spinach is caused by the soilborne fungus Fusarium oxysporum f. sp. spinaciae and occurs in most regions of spinach production. The disease is favored by acid soils and warm temperatures, and the fungus can survive extended periods as chlamydospores or by asymptomatic colonization of the roots of nonhost plant species. The 10- to 15-year rotation required to minimize losses to Fusarium wilt is the primary constraint on spinach seed production in the maritime Pacific Northwest, the only region of the United States suitable for this cool-season, daylength-sensitive crop. Raising soil pH with agricultural limestone (97% CaCO 3 ) results in a transitory, partially suppressive effect on spinach Fusarium wilt. A field trial was completed from 2009 to 2012 to assess the potential for annual applications of agricultural limestone at 0, 2.24, and 4.48 tons/ha for 3 years prior to a spinach seed crop to improve Fusarium wilt suppression compared with the level of suppression attained from a single limestone amendment at 4.48 tons/ha. Three proprietary female spinach lines were planted that ranged from highly susceptible to partially resistant to Fusarium wilt. Three successive annual applications of limestone at 4.48 tons/ha reduced midseason wilt incidence by an average of 20%, increased spinach biomass by 33%, and increased marketable spinach seed yield by 45% compared with plots amended once with the same rate of limestone in the spring of planting. The suppressive effect increased with increasing rate of limestone amendment, with the greatest difference observed when limestone was applied at between 0 and 2.24 tons/ha annually for 3 years. The effects on seed yield were greatest for the partially resistant female line, followed by the moderately susceptible and highly susceptible female lines. Overall, the results demonstrate that annual applications of agricultural limestone on acid soils of the maritime Pacific Northwest of the United States can enhance suppression of spinach Fusarium wilt, potentially reducing the required rotation interval by as much as 50%, thereby doubling the capacity for spinach seed production in the United States.

Stunting caused by Rhizoctonia spp. is economically important in irrigated onion bulb crops in the semiarid Columbia Basin of Oregon and Washington, where cereal winter cover crops commonly are planted the previous fall to prevent wind erosion of soil. The cover crop is killed with herbicide application just before or shortly after onion seeding, so that the dead rows of cereal plants provide a physical barrier tall enough to protect onion seedlings against wind and sand blasting but not tall enough to shade onion seedlings. However, the cover crop also serves as a green bridge for Rhizoctonia spp. on cereal roots to colonize the onion roots, potentially resulting in severe stunting of onion seedlings. To determine the effect of timing of application of the herbicide glyphosate to reduce this green bridge effect and, subsequently, onion stunting, three herbicide application intervals preceding onion planting were evaluated in a grower's onion field in each of 2012 and 2014 in the Columbia Basin. The wheat cover crop was killed with a glyphosate application 27, 17, and 3 days before onion seeding in 2012 and 19, 10, and 3 days before seeding in 2014. As the interval between herbicide application and onion planting increased from 3 days to 19 and 27 days, the number of patches of stunted onion plants decreased by ≥55%, total area of stunted patches decreased by 54 to 63%, and patch severity index decreased by 59 to 65%. Similarly, the Rhizoctonia solani AG 8 DNA concentration in soil sampled from the dead cover crop rows declined as the interval between glyphosate application and onion seeding increased in the 2012 trial but not in the 2014 trial. R. solani AG 3 and AG 8 DNA concentrations in soil sampled from the cover crop rows were significantly positively correlated with the number of patches of stunted onion plants (r = 0.490 and 0.607 at P = 0.039 and 0.008, respectively), total area of stunted patches (r = 0.496 and 0.659 at P = 0.035 and 0.003, respectively), and patch severity index (r = 0.492 and 0.635 at P = 0.038 and 0.005, respectively) in the 2012 trial; however, these variables were only correlated significantly with R. solani AG 3 DNA concentration in the 2014 trial. Increasing the interval between herbicide application to the cover crop and onion planting provides a practical management tool for stunting in onion bulb crops.

Organic vegetable production accounted for 19% of the total organic acreage in Washington State in 2013, with 1,700 ha of certified organic vegetable pea. However, production is challenged constantly with the threat of poor emergence after planting due to damping-off caused by Pythium spp. A survey of Pythium spp. in organic vegetable production areas of the semiarid Columbia Basin of central Washington was carried out in fall 2009 to identify species associated with damping-off during early spring planting. Of 305 isolates baited from soil sampled from 37 certified organic fields, 264 were identified to 16 Pythium spp. by sequencing the internal transcribed spacer region of ribosomal DNA. A soil DNA-CFU regression curve was developed using real-time quantitative polymerase chain reaction assays for each of the three predominant pathogenic species (Pythium abappressorium, the P. irregulare complex, and P. ultimum var. ultimum) found in soil sampled from the 37 fields. The P. irregulare complex, P. abappressorium, and P. ultimum var. ultimum were detected in 57, 78, and 100% of the fields sampled, respectively. A regression analysis was used to determine that P. ultimum var. ultimum ranged from 14 to 332 CFU/g of soil in the 37 fields, the P. irregulare complex ranged from 25 to 228 CFU/g of soil, and P. abappressorium DNA was below the quantifiable limit. In summary, P. ultimum var. ultimum was the most prevalent pathogenic Pythium sp. detected in certified organic fields in the semiarid Columbia Basin of central Washington but multiple Pythium spp. may be associated with damping-off in cool and wet, early spring planting conditions.

Onion stunting caused by Rhizoctonia spp. is an important soilborne disease on very sandy soils in the Columbia Basin of Oregon and Washington. From 2010 to 2013, 251 isolates of Rhizoctonia or Rhizoctonia-like spp. were obtained from soil and onion plant samples collected from inside and outside patches of stunted plants in 29 onion fields in the Columbia Basin. Sequence analysis of the internal transcribed spacer (ITS) region was used to identify the isolates, with 13 anastomosis groups (AGs) or subspecies detected. The most frequent was Waitea circinata var. circinata (25%), followed by Rhizoctonia solani AG 3 (17%), R. solani AG 4 (14%), Ceratobasidium sp. AG A (10%), R. solani AG 8 (7%), Ceratobasidium sp. AG K (6%), R. solani AG 2-1 (6%), W. circinata var. zeae (6%), R. solani AG 5 (4%), Ceratobasidium sp. AG G (2%), R. solani AG 11 (2%), and R. solani AG 1-1B and AG 10 (each <1%). However, the distribution of AGs and subspecies varied depending on whether soil or onion plants samples were collected within or adjacent to patches of stunted onion plants. In an attempt to predict the risk of onion stunting for a field prior to planting, DNA concentrations of AG 2-1, AG 3, AG 4, and AG 8 were quantified from bulk soil samples collected from each of nine growers' fields approximately 1 month before onion sowing in 2012. The preplant DNA concentrations did not show a significant association with the amount of stunting observed in the fields during the growing season. In contrast, the frequency of isolation and DNA concentration of R. solani AG 8 detected in soil samples collected during the growing season were greater from inside patches of stunted onion plants than from adjacent healthy areas of an onion crop sampled in 2012, but not for soil samples collected similarly from an onion crop in 2013. AG 2-1, AG 3, and AG 4 DNA concentrations did not differ significantly in soil sampled inside versus outside stunted patches in the fields sampled in 2012 and 2013. Relationships between the number of bulbs harvested or bulb weight versus severity of stunting were defined using correlation and regression analyses for six onion cultivars grown in seven fields surveyed in 2012 and 2013. Onion stunting reduced the average marketable bulb yield by 25 to 60% within stunted patches of the six cultivars. Stunting did not reduce onion plant stand but consistently reduced the size of bulbs, and yield reduction increased with increasing disease severity.

Isolates of Rhizoctonia and Rhizoctonia-like spp. (n = 179) were baited selectively from soil and plant samples collected from irrigated pea crops in the semiarid Columbia Basin of Oregon and Washington from 2011 to 2013, and characterized to species, subspecies, and anastomosis groups (AG) based on sequences of the internal transcribed spacer region of ribosomal DNA. Rhizoctonia solani comprised 76% of all isolates, and included isolates of AG 4 (31% of all isolates), AG 2-1 (18%), AG 3 (10%), AG 8 (8%), AG 5 (5%), AG 10 (3%), and AG 9 (1%). The isolates of Ceratobasidium spp. (20%) comprised four AGs: AG K (11%), AG A (6%), AG I (2%), and AG I-like (1%). Waitea circinata isolates (4%) comprised two subspecies: W. circinata var. circinata (approximately 4%) and W. circinata var. zeae (<1%). Repeated pathogenicity tests of isolates of the 10 most frequently detected AGs and subspecies on 'Serge' pea at 15°C revealed that R. solani AG 2-1 caused the greatest reduction in pea emergence, followed by R. solani AG 4. R. solani AG 4 caused the most severe root rot, stunting, and reduction in pea seedling biomass, followed by isolates of AG 2-1. R. solani AG 8 did not affect emergence, plant height, and total biomass compared with noninoculated control plants; however, root rot caused by isolates of AG 8 was ranked the third most severe among isolates of the 10 Rhizoctonia subgroups, after that caused by isolates of AG 4 and AG 2-1. Isolates of other AGs and subspecies were either weakly virulent or nonpathogenic on pea. The most common AGs (AG 4 and AG 2-1) detected in pea fields in the Columbia Basin were also the most virulent. In a growers' pea crop grown for seed ('Prevail') planted 5 days after herbicide application and incorporation of a preceding winter wheat crop, severe stunting caused by Rhizoctonia spp. resulted in an average 75% yield loss within patches of stunted plants. In contrast, the yield of processing pea from a green pea crop of Serge did not differ significantly for plants sampled within versus outside patches of stunted plants; however, plants within patches were significantly more mature. In the Prevail seed crop, a greater frequency of R. solani AG 8 was detected than AG 2-1 or AG 4 from within patches of stunted plants, indicating that isolates of AG 8 may be associated with the root rot complex in some pea crops in the Columbia Basin.

The maritime Pacific Northwest is the only region of the United States suitable for production of spinach seed, a cool-season, daylength-sensitive crop. However, the acidic soils of this region are highly conducive to spinach Fusarium wilt, caused by Fusarium oxysporum f. sp. spinaciae. Rotations of at least 10 to 15 years between spinach seed crops are necessary to reduce the high risk of losses to this disease. The objectives of this study were to develop a greenhouse soil bioassay to assess the relative risk of Fusarium wilt in fields intended for spinach seed production, and to identify soil chemical and physical properties associated with conduciveness to this disease. Preliminary bioassays established a protocol for growing spinach plants in a greenhouse environment and inducing Fusarium wilt symptoms so that the bioassay can be completed in <2 months. Test soils with a range of Fusarium wilt inoculum potentials, and three spinach inbred parent lines (highly susceptible, moderately susceptible, and moderately resistant to Fusarium wilt) were used to evaluate sensitivity of the bioassay to different levels of risk of Fusarium wilt. Then, from 2010 to 2013, spinach seed growers and stakeholders submitted soil samples from 147 fields for evaluation with the bioassay. The fields were each under consideration for planting a spinach seed crop, yet the bioassay revealed a wide range in Fusarium wilt inoculum potential among soil samples. Differences in susceptibility to Fusarium wilt of the three inbred lines were key to detecting differences in wilt risk among soils. Visits to spinach seed crops planted in fields evaluated in the bioassay, as well as test plots of the three inbred lines planted in growers' seed crops, confirmed the predictive value of the bioassay for Fusarium wilt risk. Correlation analyses for 23 soil properties revealed significant relationships of 15 soil properties with the Fusarium wilt potential of a soil, but the correlations were influenced significantly by susceptibility of the inbred line to Fusarium wilt (13, 10, and 8 soil properties correlated significantly with Fusarium wilt risk for the susceptible, moderate, and partially resistant inbreds, respectively). Multiple regression analyses identified different statistical models for prediction of Fusarium wilt risk depending on the spinach inbred line, but the best fitting model explained <34% of the variability in Fusarium wilt risk among 121 fields evaluated in the soil bioassay. Thus, no model was robust enough to replace the bioassay for the purpose of predicting Fusarium wilt risk.

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 6866 PT ), 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 × 10 8 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 6866 PT , 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.

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., (© 2014 The Society for Applied Microbiology.)

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 × 10 5 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 × 10 5 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.

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 mm 2 ) 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 10 8 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.

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.

Molecular assays to detect and quantify DNA from viable cells of the seedborne pathogen Xanthomonas hortorum pv. carotae in carrot seed were developed and evaluated for use on nontreated and hot-water-treated seed lots. Both a TaqMan real-time polymerase chain reaction (PCR) assay and a loop-mediated isothermal amplification (LAMP) dilution endpoint assay detected and quantified DNA from viable pathogen cells after treatment of carrot seed washes with the live-dead discriminating dye propidium monoazide (PMA). The detection limits of the assays were approximately 10 1 CFU for pure cultures of X. hortorum pv. carotae, and 10 2 to 10 3 CFU/g seed from naturally infested carrot seed lots. X. hortorum pv. carotae in and on carrot seed was killed by soaking the seed in hot water (52°C for 25 min), and a subsequent PMA treatment of these hot-water-treated seed washes suppressed detection of the pathogen with both the real-time PCR and LAMP assays. For 36 commercial seed lots treated with PMA but not hot water, regression of colony counts of X. hortorum pv. carotae measured by dilution plating on a semiselective agar medium versus estimates of pathogen CFU determined by the molecular assays resulted in significant (P ≤ 0.05) linear relationships (R 2 = 0.68 for the real-time PCR assay and 0.79 for the LAMP assay). The molecular assays provided quantitative estimates of X. hortorum pv. carotae infestations in carrot seed lots in <24 h, which is a significant improvement over the 7 to 14 days required to obtain results from the traditional dilution-plating assay.

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.

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.

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.

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 mm 2 ) 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 × 10 6 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.

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 × 10 5 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 commercial skullcap (Scutellaria lateriflora, family Lamiaceae) crop with wilted and necrotic plants was examined in Washington State in 2008. Three fungal isolates were obtained and identified as Verticillium dahliae based on morphology and sequencing of the internal transcribed spacer DNA region. All three skullcap isolates caused typical Verticillium wilt symptoms on skullcap and two peppermint cultivars. Inoculations of skullcap with the V. dahliae isolates from skullcap and an isolate from peppermint resulted in severe symptoms and a 21 to 78% reduction in aboveground biomass. Isolates from skullcap caused severe symptoms on the susceptible peppermint 'Black Mitcham' and reduced yield by up to 82%. One skullcap isolate caused severe symptoms on the moderately resistant 'Redefined Murray' in three of four trials and reduced biomass up to 71% compared with noninoculated control plants. The pathogen was recovered from 43 to 69% of skullcap stems from plants inoculated with skullcap or peppermint isolates, and was isolated from 2.5% of seed harvested from skullcap plants inoculated with the peppermint isolate of V. dahliae. This is the first report of V. dahliae infecting skullcap, and the first demonstration of V. dahliae isolates from skullcap and peppermint causing symptoms on both hosts, as well as the seedborne nature of V. dahliae in skullcap.

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.

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.

The efficacy of 14 seed and drench treatments for control of soilborne damping-off pathogens in organic production of spinach was evaluated in a greenhouse study. The efficacy of each treatment was compared with nontreated seed and seed treated with a conventional fungicide for control of Fusarium oxysporum f. sp. spinaciae, Pythium ultimum, and Rhizoctonia solani. Two experimental seed treatments, GTG I and GTG II (each comprised of a proprietary organic disinfectant and the latter also containing Trichoderma harzianum T22), provided equivalent control to the conventional fungicide, mefenoxam, against P. ultimum in one trial and significant reduction of damping-off in the second trial. Natural II and Natural X (Streptomycete products), and Subtilex (Bacillus subtilis) seed treatments each suppressed damping-off significantly in one of the two trials. For R. solani, GTG I and Natural II seed treatments reduced damping-off as effectively as a drench with the fungicide Terraclor (pentachloronitrobenzene). A soil drench with Prestop (Gliocladium catenulatum) suppressed postemergence wilt caused by F. oxysporum in both trials; a compost tea drench and seed treatment with Yield Shield (Bacillus pumilis) each suppressed postemergence wilt in only one of two trials. GTG I and GTG II significantly increased seed germination compared to nontreated seed. No treatment was effective against all three pathogens, and some treatments exacerbated damping-off.

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 × 10 5 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.

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.

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 OD 600 = 0.3 (~10 8 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.

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 10 8 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 real-time fluorescent polymerase chain reaction (PCR) assay was developed using SYBR Green chemistry to quantify the Botrytis spp. associated with onion (Allium cepa) seed that are also able to induce neck rot of onion bulbs, i.e., B. aclada, B. allii, and B. byssoidea. The nuclear ribosomal intergenic spacer (IGS) regions of target and nontarget Botrytis spp. were sequenced, aligned, and used to design a primer pair specific to B. aclada, B. allii, and B. byssoidea. Primers and amplification parameters were optimized to avoid amplifying the related species B. cinerea, B. porri, and B. squamosa, as well as Sclerotinia sclerotiorum and isolates of 15 other fungal species commonly found associated with onion seed. The primers reliably detected 10 fg of genomic DNA per PCR reaction extracted from pure cultures of B. aclada and B. allii. Conventional assays of surface-disinfested and nondisinfested seed on an agar medium were used to determine the incidence of neck rot Botrytis spp. associated with each of 23 commercial onion seed lots, and the real-time PCR assay was used to determine the quantity of DNA of neck rot Botrytis spp. in each seed lot. A linear relationship could not be found between the incidence of seed infected with the neck rot Botrytis spp. using the conventional agar seed assays and the quantity of DNA of the neck rot Botrytis spp. detected by the real-time PCR assay. However, the real-time PCR assay appeared to be more sensitive than the conventional agar assay, allowing detection of neck rot Botrytis spp. in 5 of the 23 seed lots that tested negative using the conventional agar seed assay.

During the growing seasons of 2003 and 2004, a disease occurred in several carrot crops in south central Washington with symptoms suggestive of infection by phytopathogenic mollicutes (phytoplasmas and spiroplasmas). In the fall, many affected carrot plants exhibited extensive purple or yellow-purple leaf discoloration, general stunting of shoots and taproots, and formation of bunchy, fibrous secondary roots. For detection of the putative causal agents, polymerase chain reaction (PCR) assays were performed using primers specific to phytoplasmas as well as primers specific to plant-pathogenic spiroplasmas. Restriction fragment length polymorphism (RFLP) analyses of PCR-amplified 16S rDNA sequences revealed that about 81% of affected plants showing dark purple or yellow-purple leaf symptoms tested positive for Spiroplasma citri. Of affected plants showing mild purple discoloration of leaf margins, 18% tested positive for a phytoplasma strain belonging to the clover proliferation group (16SrVI), subgroup 16SrVI-A, and 11% for another phytoplasma strain belonging to the aster yellows group (16SrI), subgroup 16SrI-A. Nucleotide sequence analysis of cloned 16S rDNA confirmed the phytoplasma group affiliations. Some symptomatic plants were co-infected with S. citri and either aster yellows phytoplasma or clover proliferation group phytoplasma. To our knowledge, this is the first documentation of spiroplasma infection of carrot in 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.