Distribution of Three Verticillium dahliae Races in Coastal California and Evaluation of Resistance in Lettuce.

Verticillium wilt, caused by Verticillium dahliae, is one of the most devastating soilborne diseases of lettuce (Lactuca sativa L.). There are three races of V. dahliae, and each race has been characterized by markers representing race-specific effectors. Race 1 is differentiated by the presence of the functional secretory Ave1 effector. Similarly, races 2 and 3 are differentiated by effectors VdR2e and VdR3e, respectively. Although the presence of race 1 in coastal California was well established, the presence of effector-based races 2 and 3 was uncertain. This study therefore focused on characterizing 727 isolates collected from 142 ranches of symptomatic lettuce and other crops from coastal California. Based on this evaluation, 523 isolates were designated as race 1, 20 isolates as race 2, 23 isolates as race 3, and 17 as race undefined. Isolates representing other Verticillium species totaled 110, and 34 were non-Verticillium fungal species. Because the use of resistant cultivars is a key strategy to manage this disease, we evaluated 48 lettuce germplasm lines and 1 endive (Cichorium endivia L.) line, comprising commercial cultivars and breeding lines, including the race 1-resistant heirloom cultivar La Brillante and the susceptible cultivar Salinas as controls. Resistance against races 1, 2, and 3 along with VdLs17, a virulent isolate of V. dahliae from lettuce that is currently not assigned to a race, was evaluated in replicated greenhouse experiments. Two crisphead lettuce lines, HL28 and HL29, exhibited resistance against race 1 and a partial resistance against race 2, whereas all other lines were highly susceptible to races 1 and 2 and VdLs17. The majority of lines exhibited higher resistance to race 3 relative to the other two races. This study documents the current distribution of the different races in coastal California. In addition, the sources of resistance currently being developed should be effective or partially effective against these races for targeted deployment as soon as they are available.

Conditions at the time of inoculation influence survival of attenuated Escherichia coli O157:H7 on field-inoculated lettuce.

The impact of plant development, environmental conditions at the time of inoculation, and inoculum concentration on survival of attenuated BSL1 Escherichia coli O157:H7 strain ATCC 700728 on field-grown romaine lettuce was evaluated over 3 years. E. coli 700728 was inoculated onto 4- and 6-week-old romaine lettuce plants in the Salinas Valley, CA, at night or the next morning with either low (5 log) or high (7 log) cell numbers per plant to simulate a single aqueous contamination event. At night, when leaf wetness and humidity levels were high, E. coli cell numbers declined by 0.5 log CFU/plant over the first 8-10 h. When applied in the morning, E. coli populations declined up to 2 log CFU/plant within 2 h. However, similar numbers of E. coli were retrieved from lettuce plants at 2 and 7 days. E. coli cell numbers per plant were significantly lower (P < 0.05) 7 days after application onto 4-week-old compared to 6-week-old plants. E. coli 700728 could be recovered by plating or enrichment from a greater proportion of plants for longer times when inoculated at high compared with low initial concentrations and after inoculation of 6-week-old plants compared with 4-week-old plants, even at the low initial inoculum. A contamination event near harvest or when leaf wetness and humidity levels are high may enhance survivability, even when low numbers of E. coli are introduced.

Measurements of Aerial Spore Load by qPCR Facilitates Lettuce Downy Mildew Risk Advisement.

The lettuce downy mildew pathogen, Bremia lactucae, is an obligate oomycete that causes extensive produce losses. Initial chlorotic symptoms that severely reduce the market value of the produce are followed by the appearance of white, downy sporulation on the abaxial side of the leaves. These spores become airborne and disseminate the pathogen. Controlling lettuce downy mildew has relied on repeated fungicide applications to prevent outbreaks. However, in addition to direct economic costs, heterogeneity and rapid adaptation of this pathogen to repeatedly applied fungicides has led to the development of fungicide-insensitivity in the pathogen. We deployed a quantitative PCR assay-based detection method using a species-specific DNA target for B. lactucae coupled with a spore trap system to measure airborne B. lactucae spore loads within three commercial fields that each contained experimental plots, designated EXP1 to EXP3. Based upon these measurements, when the spore load in the air reached a critical level (8.548 sporangia per m3 air), we advised whether or not to apply fungicides on a weekly basis within EXP1 to EXP3. This approach saved three sprays in EXP1, and one spray each in EXP2 and EXP3 without a significant increase in disease incidence. The reduction in fungicide applications to manage downy mildew can decrease lettuce production costs while slowing the development of fungicide resistance in B. lactucae by eliminating unnecessary fungicide applications.

Evaluation of post-contamination survival and persistence of applied attenuated E. coli O157:H7 and naturally-contaminating E. coli O157:H7 on spinach under field conditions and following postharvest handling.

This study determined the variability in population uniformity of an applied mixture of attenuated E. coli O157:H7 (attEcO157) on spinach leaves as impacted by sampling mass and detection technique over spatial and temporal conditions. Opportunistically, the survival and distribution of naturally contaminating pathogenic E. coli O157:H7 (EcO157), in a single packaged lot following commercial postharvest handling and washing, was also evaluated. From the main study outcomes, differences in the applied inoculum dose of 100-fold, resulted in indistinguishable population densities of approximately Log 1.1 CFU g-1 by 14 days post-inoculation (DPI). Composite leaf samples of 150 g and the inclusion of the spinach petiole resulted in the greatest numerical sensitivity of detection of attEcO157 when compared to 25 and 150 g samples without petioles (P < 0.05). Differences in population density and protected-site survival and potential leaf internalization were observed between growing seasons and locations in California (P < 0.05). A Double Weibull model best described and identified two distinct populations with different inactivation rates of the inoculated attEcO157. Linear die-off rates varied between 0.14 and 0.29 Log/Day irrespective of location. Detection of EcO157- stx1-negative and stx2-positive, resulting from a natural contamination event, was observed in 11 of 26 quarantined commercial units of washed spinach by applying the 150 g sample mass protocol. The capacity to detect EcO157 varied between commercial test kits and non-commercial qPCR. Our findings suggest the need for modifications to routine pathogen sampling protocols employed for lot acceptance of spinach and other leafy greens.

Detection of Fusarium oxysporum f. sp. fragariae from Infected Strawberry Plants.

Isolates of the Fusarium oxysporum species complex have been characterized as plant pathogens that commonly cause vascular wilt, stunting, and yellowing of the leaves in a variety of hosts. F. oxysporum species complex isolates have been grouped into formae speciales based on their ability to cause disease on a specific host. F. oxysporum f. sp. fragariae is the causal agent of Fusarium wilt of strawberry and has become a threat to production as fumigation practices have changed in California. F. oxysporum f. sp. fragariae is polyphyletic and limited genetic markers are available for its detection. In this study, next-generation sequencing and comparative genomics were used to identify a unique genetic locus that can detect all of the somatic compatibility groups of F. oxysporum f. sp. fragariae identified in California. This locus was used to develop a TaqMan quantitative polymerase chain reaction assay and an isothermal recombinase polymerase amplification (RPA) assay that have very high sensitivity and specificity for more than 180 different isolates of the pathogen tested. RPA assay results from multiple field samples were validated with pathogenicity tests of recovered isolates.

Comparative genomics of downy mildews reveals potential adaptations to biotrophy.

BACKGROUND: Spinach downy mildew caused by the oomycete Peronospora effusa is a significant burden on the expanding spinach production industry, especially for organic farms where synthetic fungicides cannot be deployed to control the pathogen. P. effusa is highly variable and 15 new races have been recognized in the past 30 years. RESULTS: We virulence phenotyped, sequenced, and assembled two isolates of P. effusa from the Salinas Valley, California, U.S.A. that were identified as race 13 and 14. These assemblies are high quality in comparison to assemblies of other downy mildews having low total scaffold count (784 & 880), high contig N50s (48 kb & 52 kb), high BUSCO completion and low BUSCO duplication scores and share many syntenic blocks with Phytophthora species. Comparative analysis of four downy mildew and three Phytophthora species revealed parallel absences of genes encoding conserved domains linked to transporters, pathogenesis, and carbohydrate activity in the biotrophic species. Downy mildews surveyed that have lost the ability to produce zoospores have a common loss of flagella/motor and calcium domain encoding genes. Our phylogenomic data support multiple origins of downy mildews from hemibiotrophic progenitors and suggest that common gene losses in these downy mildews may be of genes involved in the necrotrophic stages of Phytophthora spp. CONCLUSIONS: We present a high-quality draft genome of Peronospora effusa that will serve as a reference for Peronospora spp. We identified several Pfam domains as under-represented in the downy mildews consistent with the loss of zoosporegenesis and necrotrophy. Phylogenomics provides further support for a polyphyletic origin of downy mildews.

Development of Molecular Methods to Detect Macrophomina phaseolina from Strawberry Plants and Soil.

Macrophomina phaseolina is a broad-host-range fungus that shows some degree of host preference on strawberry, and causes symptoms that include crown rot and root rot. Recently, this pathogen has affected strawberry production as fumigation practices have changed, leaving many growers in California and around the world in need of accurate, rapid diagnostic tools for M. phaseolina in soil and infected plants. This study uses next-generation sequencing and comparative genomics to identify a locus that is unique to isolates within a main genotype shared by a majority of isolates that infect strawberry. This locus was used to develop a quantitative single-tube nested TaqMan polymerase chain reaction assay which is able to quantify as little as 2 to 3 microsclerotia/g of soil with 100% genotype specificity. An isothermal assay using recombinase polymerase amplification was developed from the same locus and has been validated on over 200 infected strawberry plants with a diagnostic sensitivity of 93% and a diagnostic specificity of 99%. Together, this work demonstrates the value of using new approaches to identify loci for detection and provides valuable diagnostic tools that can be used to monitor soil and strawberry plant samples for M. phaseolina.

New Races and Novel Strains of the Spinach Downy Mildew Pathogen Peronospora effusa.

Downy mildew disease, caused by Peronospora effusa (=P. farinosa f. sp. spinaciae [Pfs]), is the most economically important disease of spinach. Current high-density fresh-market spinach production provides conducive conditions for disease development, and downy mildew frequently forces growers to harvest early owing to disease development, to cull symptomatic leaves prior to harvest, or to abandon the field if the disease is too severe. The use of resistant cultivars to manage downy mildew, particularly on increasing acreages of organic spinach production, applies strong selection pressure on the pathogen, and many new races of Pfs have been identified in recent years in spinach production areas worldwide. To monitor the virulence diversity in the Pfs population, downy mildew samples were collected from spinach production areas and tested for race identification based on the disease reactions of a standard set of international spinach differentials. Two new races (designated races 15 and 16) and eight novel strains were identified between 2013 and 2017. The disease reaction of Pfs 15 was similar to race 4, except race 4 could not overcome the resistance imparted by the RPF9 locus. Several resistance loci (RPF1, 2, 4, and 6) were effective in preventing disease caused by Pfs 15. The race Pfs 16 could overcome several resistance loci (RPF2, 4, 5, 9, and 10) but not others (RPF1, 3, 6, and 7). One novel strain (UA1014) could overcome the resistance of spinach resistant loci RPF1 to RPF7 but only infected the cotyledons and not the true leaves of certain cultivars. A new set of near-isogenic lines has been developed and evaluated for disease reactions to the new races and novel strains as differentials. None of the 360 U.S. Department of Agriculture spinach germplasm accessions tested were resistant to Pfs 16 or UA1014. A survey of isolates over several years highlighted the dynamic nature of the virulence diversity of the Pfs population. Identification of virulence diversity and evaluation of the genetics of resistance to Pfs will continue to allow for a more effective disease management strategy through resistance gene deployment.

Detection and Quantification of Bremia lactucae by Spore Trapping and Quantitative PCR.

Bremia lactucae is an obligate, oomycete pathogen of lettuce that causes leaf chlorosis and necrosis and adversely affects marketability. The disease has been managed with a combination of host resistance and fungicide applications with success over the years. Fungicide applications are routinely made under the assumption that inoculum is always present during favorable environmental conditions. This approach often leads to fungicide resistance in B. lactucae populations. Detection and quantification of airborne B. lactucae near lettuce crops provides an estimation of the inoculum load, enabling more judicious timing of fungicide applications. We developed a quantitative polymerase chain reaction (qPCR)-based assay using a target sequence in mitochondrial DNA for specific detection of B. lactucae. Validation using amplicon sequencing of DNA from 83 geographically diverse isolates, representing 14 Bremia spp., confirmed that the primers developed for the TaqMan assays are species specific and only amplify templates from B. lactucae. DNA from a single sporangium could be detected at a quantification cycle (Cq) value of 32, and Cq values >35 were considered to be nonspecific. The coefficient of determination (R2) for regression between sporangial density derived from flow cytometry and Cq values derived from the qPCR was 0.86. The assay was deployed using spore traps in the Salinas Valley, where nearly half of U.S. lettuce is produced. The deployment of this sensitive B. lactucae-specific assay resulted in the detection of the pathogen during the 2-week lettuce-free period as well as during the cropping season. These results demonstrate that this assay will be useful for quantifying inoculum load in and around the lettuce fields for the purpose of timing fungicide applications based on inoculum load.

Plasmolysis and Vital Staining Reveal Viable Oospores of Peronospora effusa in Spinach Seed Lots.

Production of oospores by Peronospora effusa, the causal agent of downy mildew on spinach (Spinacia oleracea), was reported on spinach seed over three decades ago. In view of the rapid proliferation of new races of P. effusa worldwide, seedborne transmission of this pathogen has been suspected but methods to test the viability of seedborne oospores have not been available. Eighty-two seed lots of contemporary spinach cultivars were evaluated for the presence of P. effusa using a seed-wash method and the sediment was examined by microscopy. Of the analyzed seed lots, 16% were positive for oospores and an additional 6% for sporangiophores characteristic of P. effusa. Application of a P. effusa-specific quantitative polymerase chain reaction assay showed that 95% of the 59 tested seed lots were positive for P. effusa. The viability of oospores from five seed lots that were proven to carry the pathogen from the above tests was tested using two independent methods, one involving plasmolysis and the other trypan blue staining. The oospores plasmolyzed in 4 M sodium chloride and were deplasmolyzed in water, demonstrating an active and viable cell membrane. Similarly, viable oospores failed to take up the trypan blue stain. Overall, 59% of the oospores were viable in the plasmolysis test and 45% with the trypan blue test. These results indicate the presence of P. effusa oospores in contemporary spinach seed lots, and suggest that the transmission of viable oospores of P. effusa in spinach seed does occur. Elimination of the pathogen on seed, in addition to other management approaches, will be useful in reducing the extent and severity of downy mildew on spinach crops and diminishing pathogen spread through seed.

Dynamics of verticillium species microsclerotia in field soils in response to fumigation, cropping patterns, and flooding.

Verticillium dahliae is a soilborne, economically significant fungal plant pathogen that persists in the soil for up to 14 years as melanized microsclerotia (ms). Similarly, V. longisporum is a very significant production constraint on members of the family Brassicaceae. Management of Verticillium wilt has relied on methods that reduce ms below crop-specific thresholds at which little or no disease develops. Methyl bromide, a broad-spectrum biocide, has been used as a preplant soil fumigant for over 50 years to reduce V. dahliae ms. However, reductions in the number of ms in the vertical and horizontal soil profiles and the rate at which soil recolonization occurs has not been studied. The dynamics of ms in soil before and after methyl bromide+chloropicrin fumigation were followed over 3 years in six 8-by-8-m sites in two fields. In separate fields, the dynamics of ms in the 60-cm-deep vertical soil profile pre- and postfumigation with methyl bromide+chloropicrin followed by various cropping patterns were studied over 4 years. Finally, ms densities were assessed in six 8-by-8-m sites in a separate field prior to and following a natural 6-week flood. Methyl bromide+chloripicrin significantly reduced but did not eliminate V. dahliae ms in either the vertical or horizontal soil profiles. In field studies, increases in ms were highly dependent upon the crop rotation pattern followed postfumigation. In the vertical soil profile, densities of ms were highest in the top 5 to 20 cm of soil but were consistently detected at 60-cm depths. Six weeks of natural flooding significantly reduced (on average, approximately 65% in the total viable counts of ms) but did not eliminate viable ms of V. longisporum.

Host Range of Verticillium isaacii and Verticillium klebahnii from Artichoke, Spinach, and Lettuce.

Verticillium is a genus that includes major vascular wilt pathogens. Recently, multilocus phylogenetic analyses of the genus identified five new species, including Verticillium isaacii and V. klebahnii, both of which occur in agricultural soils in coastal California and have been isolated from asymptomatic and diseased spinach and lettuce plants. Little data are available regarding their pathogenicity and virulence on a broader range of crops important to the region. Four isolates each of V. isaacii and V. klebahnii along with two reference isolates of V. dahliae races 1 and 2 were inoculated on eight crops (artichoke, cauliflower, eggplant, lettuce, pepper, tomato, spinach, and strawberry) in a greenhouse experiment. After 8 weeks, plants were assessed for disease severity to determine the relative host ranges of Verticillium isolates. Additionally, 13 lettuce lines resistant to race 1 and partially resistant to race 2 of V. dahliae were screened against V. isaacii and V. klebahnii to evaluate their responses. Three of four V. isaacii and four of four V. klebahnii isolates tested were nonpathogenic on all crops tested except those indicated below. One V. isaacii isolate caused wilt on artichoke and 'Salinas' lettuce and most isolates of both species caused varying degrees of Verticillium wilt on strawberry. Lettuce lines resistant to V. dahliae race 1 and partially resistant to V. dahliae race 2 also exhibited resistance to all of the isolates of V. isaacii and V. klebahnii. Thus, at least some isolates in the populations of V. isaacii and V. klebahnii have the potential to become significant pathogens of coastal California crops. However, resistance developed against V. dahliae also offers resistance to the pathogenic isolates of both species, at least in lettuce.

Coupling Spore Traps and Quantitative PCR Assays for Detection of the Downy Mildew Pathogens of Spinach (Peronospora effusa) and Beet (P. schachtii).

Downy mildew of spinach (Spinacia oleracea), caused by Peronospora effusa, is a production constraint on production worldwide, including in California, where the majority of U.S. spinach is grown. The aim of this study was to develop a real-time quantitative polymerase chain reaction (qPCR) assay for detection of airborne inoculum of P. effusa in California. Among oomycete ribosomal DNA (rDNA) sequences examined for assay development, the highest nucleotide sequence identity was observed between rDNA sequences of P. effusa and P. schachtii, the cause of downy mildew on sugar beet and Swiss chard in the leaf beet group (Beta vulgaris subsp. vulgaris). Single-nucleotide polymorphisms were detected between P. effusa and P. schachtii in the 18S rDNA regions for design of P. effusa- and P. schachtii-specific TaqMan probes and reverse primers. An allele-specific probe and primer amplification method was applied to determine the frequency of both P. effusa and P. schachtii rDNA target sequences in pooled DNA samples, enabling quantification of rDNA of P. effusa from impaction spore trap samples collected from spinach production fields. The rDNA copy numbers of P. effusa were, on average, ≈3,300-fold higher from trap samples collected near an infected field compared with those levels recorded at a site without a nearby spinach field. In combination with disease-conducive weather forecasting, application of the assays may be helpful to time fungicide applications for disease management.

Identification of New Races and Deviating Strains of the Spinach Downy Mildew Pathogen Peronospora farinosa f. sp. spinaciae.

Spinach downy mildew disease, caused by the obligate pathogen Peronospora farinosa f. sp. spinaciae, is the most economically important spinach (Spinacia oleracea) disease. New races of this pathogen have been emerging at a rapid rate over the last 15 years. This is likely due to production changes, particularly in California, such as high-density plantings and year-round spinach production. As of 2004, 10 races of P. farinosa f. sp. spinaciae had been identified, and the spinach resistance locus RPF2 provided resistance to races 1 to 10. Based on disease reactions on a set of spinach differentials containing six hypothesized resistance loci (RPF1-RPF6), races 11, 12, 13, and 14 of P. farinosa f. sp. spinaciae were characterized based on samples collected in the past 5 years as part of this study. Race 11, identified in 2008, could overcome the resistance of spinach cultivars resistant to races 1 to 10. Spinach resistance loci RPF1, RPF3, and RPF6 provided resistance to race 11. Race 12 was identified in 2009 and could overcome the resistances of the RPF1 and RPF2 loci. The RPF3 locus was effective against race 12. Race 13 was identified in 2010 and could overcome the resistance imparted by the RPF2 and RPF3 loci, whereas the RPF1 locus was effective against race 13. Race 14 was similar to race 12 and caused identical disease responses on the standard differentials but could be distinguished from race 12 by its ability to cause disease on a number of newly released cultivars, including 'Pigeon', 'Cello', and 'Celesta'. Five novel strains of P. farinosa f. sp. spinaciae were also identified. For example, isolate UA4711 of the pathogen, collected from Spain in 2011, was able to overcome the resistance imparted by the RPF1 and RPF3 loci, while RPF2 and RPF4 were effective against this strain. A total of 116 spinach cultivars, including 103 commercial lines and 13 differential cultivars, were evaluated for resistance to race 10 and the newly designated races 11, 12, 13, and 14.

Characterization and Epidemiology of Outbreaks of Impatiens necrotic spot virus on Lettuce in Coastal California.

California is the leading producer of lettuce (Lactuca sativa) for the United States and grows 77% of the country's supply. Prior to 2006, coastal California lettuce was only periodically and incidentally infected by a single tospoviruses species: Tomato spotted wilt virus (TSWV). However, beginning in 2006 and continuing through 2012, severe outbreaks of disease caused by Impatiens necrotic spot virus (INSV) have affected the coastal lettuce crop, though TSWV was also present. In contrast, TSWV was the only tospovirus associated with disease outbreaks in Central Valley lettuce during this period. Disease surveys conducted over two seasons (2008 and 2009) in 10 commercial fields (acreage of 6 to 20 ha) indicated that INSV was the only tospovirus associated with economically damaging disease outbreaks in lettuce in the coastal region, with incidences of 0.5 to 27% (mean = 5.7%). Molecular characterization of INSV isolates associated with these disease outbreaks revealed little genetic diversity and indicated that lettuce-infecting INSV isolates were nearly identical to those previously characterized from ornamental or other hosts from different locations in the United States and the world. Monitoring of thrips revealed moderate to large populations in all surveyed lettuce fields, and the majority of thrips identified from these fields were western flower thrips, Frankliniella occidentalis. There was significant positive correlation (r2 = 0.91, P = 0.003) between thrips populations and INSV incidence in the most commonly encountered type of commercial lettuce (romaine, direct seeded, conventional) included in this study. A reverse-transcription polymerase chain reaction assay developed for detection of INSV in thrips showed promise as a monitoring tool in the field. Surveys for INSV reservoir hosts in the coastal production area revealed that the weeds little mallow (Malva parvifolia) and shepherd's purse (Capsella bursa-pastoris) were commonly infected. M. parvifolia plants infected in the field did not show obvious symptoms, whereas plants of this species inoculated in the laboratory with INSV by sap transmission developed necrotic spots and chlorosis. Eleven other weed species growing in the lettuce production areas were found to be hosts of INSV. Coastal crops found to be infected with INSV included basil (Ocimum basilicum), bell pepper (Capsicum annuum), calla lily (Zantedeschia aethiopica), faba bean (Vicia faba), radicchio (Cichorium intybus), and spinach (Spinacia oleracea). Thus, it is likely that INSV was introduced into coastal California lettuce fields via viruliferous thrips that initially acquired the virus from other local susceptible plant species. Results of this study provide a better understanding of INSV epidemiology in coastal California and may help growers devise appropriate disease management strategies.

Stemphylium Leaf Spot of Parsley in California Caused by Stemphylium vesicarium.

From 2009 through 2011, a previously undescribed disease occurred on commercial parsley in coastal (Ventura County) California. Symptoms of the disease consisted of circular to oval, tan to brown leaf spots and resulted in loss of crop quality and, hence, reduced yields. A fungus was consistently isolated from symptomatic parsley. Morphological and molecular data identified the fungus as Stemphylium vesicarium. When inoculated onto parsley leaves, the isolates caused symptoms that were identical to those seen in the field; the same fungus was recovered from test plants, thus completing Koch's postulates. Additional inoculation experiments demonstrated that 10 of 11 tested flat leaf and curly parsley cultivars were susceptible. The parsley isolates also caused small leaf spots on other Apiaceae family plants (carrot and celery) but not on leek, onion, spinach, and tomato. Isolates caused brown lesions to form when inoculated onto pear fruit but only when the fruit tissue was wounded. Using a freeze-blotter seedborne pathogen assay, parsley seed was found to have a low incidence (0.25%) of S. vesicarium. When inoculated onto parsley leaves, three of four isolates from seed caused the same leaf spot disease. This is the first documentation of a foliar parsley disease caused by S. vesicarium. The occurrence of S. vesicarium on parsley seed indicates that infested seed may be one source of initial inoculum. Based on the negative results in the host range experiments, it appears that this parsley pathogen differs from the S. vesicarium that causes disease on leek, garlic, onion, and pear fruit.

Development of an assay for rapid detection and quantification of Verticillium dahliae in soil.

ABSTRACT Verticillium dahliae is responsible for Verticillium wilt on a wide range of hosts, including strawberry, on which low soil inoculum densities can cause significant crop loss. Determination of inoculum density is currently done by soil plating but this can take 6 to 8 weeks to complete and delay the grower's ability to make planting decisions. To provide a faster means for estimating pathogen populations in the soil, a multiplexed TaqMan real-time polymerase chain reaction (PCR) assay based on the ribosomal DNA (rDNA) intergenic spacer (IGS) was developed for V. dahliae. The assay was specific for V. dahliae and included an internal control for evaluation of inhibition due to the presence of PCR inhibitors in DNA extracted from soil samples. An excellent correlation was observed in regression analysis (R(2) = 0.96) between real-time PCR results and inoculum densities determined by soil plating in a range of field soils with pathogen densities as low as 1 to 2 microsclerotia/g of soil. Variation in copy number of the rDNA was also evaluated among isolates by SYBR Green real-time PCR amplification of the V. dahliae-specific amplicon compared with amplification of several single-copy genes and was estimated to range from ≈24 to 73 copies per haploid genome, which translated into possible differences in results among isolates of ≈1.8 cycle thresholds. Analysis of the variation in results of V. dahliae quantification among extractions of the same soil sample indicated that assaying four replicate DNA extractions for each field sample would provide accurate results. A TaqMan assay also was developed to help identify colonies of V. tricorpus on soil plates.

A real-time PCR assay for detection and quantification of Verticillium dahliae in spinach seed.

Verticillium dahliae is a soilborne fungus that causes Verticillium wilt on multiple crops in central coastal California. Although spinach crops grown in this region for fresh and processing commercial production do not display Verticillium wilt symptoms, spinach seeds produced in the United States or Europe are commonly infected with V. dahliae. Planting of the infected seed increases the soil inoculum density and may introduce exotic strains that contribute to Verticillium wilt epidemics on lettuce and other crops grown in rotation with spinach. A sensitive, rapid, and reliable method for quantification of V. dahliae in spinach seed may help identify highly infected lots, curtail their planting, and minimize the spread of exotic strains via spinach seed. In this study, a quantitative real-time polymerase chain reaction (qPCR) assay was optimized and employed for detection and quantification of V. dahliae in spinach germplasm and 15 commercial spinach seed lots. The assay used a previously reported V. dahliae-specific primer pair (VertBt-F and VertBt-R) and an analytical mill for grinding tough spinach seed for DNA extraction. The assay enabled reliable quantification of V. dahliae in spinach seed, with a sensitivity limit of ≈1 infected seed per 100 (1.3% infection in a seed lot). The quantification was highly reproducible between replicate samples of a seed lot and in different real-time PCR instruments. When tested on commercial seed lots, a pathogen DNA content corresponding to a quantification cycle value of ≥31 corresponded with a percent seed infection of ≤1.3%. The assay is useful in qualitatively assessing seed lots for V. dahliae infection levels, and the results of the assay can be helpful to guide decisions on whether to apply seed treatments.

Distribution, Cultivar Susceptibility, and Epidemiology of Apium virus Y on Celery in Coastal California.

Apium virus Y (ApVY) is a potyvirus that was recently found to cause crop loss to celery (Apium graveolens) in California. Symptoms on leaves exhibit varying forms of chlorosis and necrosis. Depending on the cultivar, celery petioles could also exhibit extensive necrotic, sunken, elongated lesions. Severely affected plants were unmarketable. Disease incidence surveys found that a susceptible celery (cv. 414) showed 55% (2007) and 71% (2008) disease. Because it was noted that the Apiaceae weed poison hemlock (Conium maculatum) was present in almost all areas where ApVY affected celery, a 4-year survey collected overwintered hemlock from six coastal county regions and tested composite samples for ApVY using reverse transcription-polymerase chain reaction (RT-PCR) and ApVY-specific primers. These plants were consistently positive for ApVY. Seeds collected from these plants were also positive when tested with the same RTPCR method. However, when ApVY-positive hemlock seeds were germinated and the resulting seedlings tested, all results were negative. The failure of ApVY to be transmitted from hemlock seeds to seedlings was further documented by collecting newly germinated hemlock seedlings from the field and testing them with RT-PCR. All such seedlings were negative for ApVY even though large, adjacent, overwintered hemlock plants tested positive. Two crops of celery seed were produced from ApVY-positive mother plants; celery seed from these infected plants likewise tested positive for ApVY, but seedlings grown from the seed lots were negative for ApVY. Twenty-one celery and celeriac cultivars were inoculated with ApVY using viruliferous aphids, planted in a replicated field trial, and then grown to maturity. Seven cultivars remained symptomless, tested negative for ApVY, and showed signs of possible resistance. The epidemiology of disease caused by ApVY in California evidently involves poison hemlock as a common overwintering host with subsequent vectoring of the virus from hemlock to celery via aphids. ApVY was not seedborne in this weed host or in celery in our experiments. Our data suggest that growers can manage this disease by controlling poison hemlock weed populations and by planting celery cultivars that are not susceptible to ApVY.

Multilocus sequence typing of Pseudomonas syringae sensu lato confirms previously described genomospecies and permits rapid identification of P. syringae pv. coriandricola and P. syringae pv. apii causing bacterial leaf spot on parsley.

Since 2002, severe leaf spotting on parsley (Petroselinum crispum) has occurred in Monterey County, CA. Either of two different pathovars of Pseudomonas syringae sensu lato were isolated from diseased leaves from eight distinct outbreaks and once from the same outbreak. Fragment analysis of DNA amplified between repetitive sequence polymerase chain reaction; 16S rDNA sequence analysis; and biochemical, physiological, and host range tests identified the pathogens as Pseudomonas syringae pv. apii and P. syringae pv. coriandricola. Koch's postulates were completed for the isolates from parsley, and host range tests with parsley isolates and pathotype strains demonstrated that P. syringae pv. apii and P. syringae pv. coriandricola cause leaf spot diseases on parsley, celery, and coriander or cilantro. In a multilocus sequence typing (MLST) approach, four housekeeping gene fragments were sequenced from 10 strains isolated from parsley and 56 pathotype strains of P. syringae. Allele sequences were uploaded to the Plant-Associated Microbes Database and a phylogenetic tree was built based on concatenated sequences. Tree topology directly corresponded to P. syringae genomospecies and P. syringae pv. apii was allocated appropriately to genomospecies 3. This is the first demonstration that MLST can accurately allocate new pathogens directly to P. syringae sensu lato genomospecies. According to MLST, P. syringae pv. coriandricola is a member of genomospecies 9, P. cannabina. In a blind test, both P. syringae pv. coriandricola and P. syringae pv. apii isolates from parsley were correctly identified to pathovar. In both cases, MLST described diversity within each pathovar that was previously unknown.