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Assays of 77 spinach (Spinacia oleracea) seed lots produced in the United States, Denmark, the Netherlands, or New Zealand in 2000 to 2003 showed that Stemphylium botryosum, causal agent of Stemphylium leaf spot, was present in every lot, at a mean incidence of 29.1% per lot. Either Cladosporium variabile, causal agent of Cladosporium leaf spot, or the morphologically similar species C. macrocarpum, was present in 37 of the 77 lots, at a mean incidence of 1.8% per lot. Some seed isolates of S. botryosum and C. variabile proved pathogenic on spinach. Nonpathogenic isolates resembling C. variabile were identified as C. macrocarpum by the absence of torulose aerial hyphae. Pathogenic isolates of S. botryosum were also detected in each of 12 seed lots stored for up to 11 years at 4.4°C and 60% relative humidity. C. variabile or C. macrocarpum was detected in only 2 of the 11 lots, which had been stored for 3 and 8 years. Component seed assays demonstrated that S. botryosum and C. variabile (or C. macrocarpum) were internal and external in spinach seed. S. botryosum was detected in 5 to 76% of the embryos of five seed lots, but the two Cladosporium species were detected in only 0 to 1% of the embryos of these lots. This suggests greater potential difficulty at eradicating S. botryosum than C. variabile from infected spinach seed using seed treatments. [ABSTRACT FROM AUTHOR]

Cladosporium variabile, Stemphylium botryosum, and Verticillium dahliae are seedborne and seed-transmitted pathogens of spinach. Spinach seed treatments in 1.2% NaOCl for 10 to 60 rain, or hot water (40, 45, 50, 55, and 60°C) for 10 to 40 min, were evaluated for eradication of these fungi from seed. C. variabile and V. dahliae were largely eradicated by chlorine treatment for ≥10 min. Although chlorine treatment reduced the incidence of S. botryosum, this fungus was not eradicated after 60 min in chlorine. Seed germination was not affected adversely by chlorine treatment, even after 60 min. In contrast, germination was reduced significantly by hot water treatment at 50°12 for ≥30 min or 55 or 60°C for ≥10 min. C. variabile was eradicated from seed treated in 40°C water for 10 min. V dahliae was eradicated from seed treated at 55°C for ≥30 rain or 60°C for ≥10 min. S. botryosum was eradicated from a lightly infected seed lot (5% incidence) by hot water treatment at 55 or 60% for ≥10 min, but could not be eradicated from two heavily infected lots (»65% incidence), even at 60°C for 40 min. Using precisely controlled parameters, chlorine or hot water seed treatments can be used to eradicate C. variabile and reduce the incidence of S. botryosum and V. dahliae in spinach seed without damaging germination. [ABSTRACT FROM AUTHOR]

In 2005, Verticillium dahliae was first reported to be pathogenic to spinach seed crops in the Pacific Northwest, with symptoms only de-veloping after initiation of the reproductive stage of plant growth, and to be prevalent on commercial spinach seed lots produced in Denmark, The Netherlands, and the United States. In this study, the genetic diver-sity, pathogenicity, and virulence were examined for a collection of isolates of Verticillium spp. from spinach as well as other hosts (alfalfa, cotton, lettuce, mint, peppermint, potato, radish, and tomato) from various countries and from different vegetative compatibility groups (VCGs). Of a total of 210 isolates of V. dahliae obtained from spinach seed produced in Denmark, the Netherlands, New Zealand, or the United States, 128 were assigned to VCG 4B (89% of 91 U.S. isolates, 86% of 42 isolates from the Netherlands, 19% of 43 Denmark isolates, and 8% of 13 New Zealand isolates), 65 to VCG 2B (92% of the New Zealand isolates, 79% of the Denmark isolates, 14% of the Netherlands isolates, and 9% of the U.S. isolates), and 3 to VCG 2A (2% of each of the Denmark and U.S. isolates, and 0% of the Netherlands and New Zealand isolates); 14 isolates could not be assigned to a VCG. Although little variation in the sequence of the internal transcribed spacer (ITS) region of ribosomal DNA was observed among isolates within each Verticillium sp., the ITS region readily differentiated iso-lates of the species V. dahliae, V. tricorpus, and Gibellulopsis nigres-cens (formerly V. nigrescens) obtained from spinach seed. Greenhouse pathogenicity assays on spinach, cotton, lettuce, and tomato plants using isolates of V. dahliae (n = 29 to 34 isolates), V. tricorpus (n = 3), G. nigrescens (n = 2), and V. albo-atrum (n = 1) originally obtained from these hosts as well as from alfalfa, mint, peppermint, potato, and radish, revealed a wide range in virulence among the isolates. Isolates of V. tricorpus and G. nigrescens recovered from spinach seed and an isolate of V. albo-atrum from alfalfa were not pathogenic on spinach. In addition, isolates of V. dahliae from mint and peppermint were not pathogenic or only weakly virulent on the hosts evaluated. Although there was a wide range in virulence among the isolates of V. dahliae tested, all of the V. dahliae isolates caused Verticillium wilt symptoms on spinach, lettuce, tomato, and cotton. None of the isolates of V. dahl-iae showed host specificity. These results indicate that Verticillium and related species associated with spinach seed display substantial varia-bility in virulence and pathogenicity to spinach and other plants but the V. dahliae isolates were restricted to three VCGs. [ABSTRACT FROM AUTHOR]

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 cirri. 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. [ABSTRACT FROM AUTHOR]

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.

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.