Open Access and Reproducibility in Plant Pathology Research: Guidelines and Best Practices.

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.

Prevalence of FRAC Group 11 Fungicide Resistance in Stemphylium vesicarium Isolates, but Not S. beticola Isolates, Causing Stemphylium Leaf Spot of Spinach (Spinacia oleracea).

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). EC50 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.

Characterization of Stemphylium species associated with Stemphylium leaf spot of spinach (Spinacia oleracea).

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.

Real-Time PCR Assays for Races of the Spinach Fusarium Wilt Pathogen, Fusarium oxysporum f. sp. spinaciae.

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.

Shedding Light on Races of the Spinach Fusarium Wilt Pathogen, Fusarium oxysporum f. sp. spinaciae.

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.

Putative Effector Genes Distinguish Two Pathogenicity Groups of Fusarium oxysporum f. sp. spinaciae.

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.

A maize leucine-rich repeat receptor-like protein kinase mediates responses to fungal attack.

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.

First Report of Bacterial Leaf Spot Caused by Pseudomonas syringae pv. aptata on Swiss Chard, Beta vulgaris subsp. vulgaris, in Arizona.

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-cm2) 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 (108 CFU/ml), and diluted in 0.0125M phosphate buffer to 107 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.

Three New Fungal Leaf Spot Diseases of Spinach in the United States and the Evaluation of Fungicide Efficacy for Disease Management.

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.

First Report of Curly Top of Coriandrum sativum Caused by Beet curly top virus in the Columbia Basin of Washington State.

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.

Characterization of Leaf Spot Pathogens from Several Spinach Production Areas in the United States.

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.

Genetic Diversity and Differentiation in Phoma betae Populations on Table Beet in New York and Washington States.

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.

A New Subclade of Leptosphaeria biglobosa Identified from Brassica rapa.

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 ß-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'.

Limestone-Mediated Suppression of Fusarium Wilt in Spinach Seed Crops.

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% CaCO3) 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.

Timing of Glyphosate Applications to Wheat Cover Crops to Reduce Onion Stunting Caused by Rhizoctonia solani.

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.

Pythium Species Associated with Damping-off of Pea in Certified Organic Fields in the Columbia Basin of Central Washington.

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.

A Soil Bioassay for Predicting the Risk of Spinach Fusarium wilt.

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.

Stunted Patches in Onion Bulb Crops in Oregon and Washington: Etiology and Yield Loss.

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.

Characterization and Pathogenicity of Rhizoctonia and Rhizoctonia-Like spp. From Pea Crops in the Columbia Basin of Oregon and Washington.

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.

Quantitative Molecular Detection of Xanthomonas hortorum pv. carotae in Carrot Seed Before and After Hot-Water Treatment.

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 101 CFU for pure cultures of X. hortorum pv. carotae, and 102 to 103 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 (R2 = 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.