RESUMO
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.
Assuntos
Ascomicetos , Resistência à Doença , Lactuca , Doenças das Plantas , Lactuca/microbiologia , California , Doenças das Plantas/microbiologia , Resistência à Doença/genética , Ascomicetos/genética , Ascomicetos/fisiologia , VerticilliumRESUMO
The western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), is an important, worldwide vector of two tospoviruses (Family Bunyaviridae; Genus Tospovirus), Impatiens necrotic spot virus (INSV) and Tomato spotted wilt virus (TSWV). In the Salinas Valley of California, INSV causes severe necrosis, stunting, and crop losses to lettuce (Lactuca sativa L.). Because broccoli (Brassica oleracea var. italica L.) and cauliflower (Brassica oleracea var. botrytis L.) are major rotation crops with lettuce in the Salinas Valley, it is critical to understand if these Brassicas influence the dispersal dynamics of F. occidentalis. In 2013 and 2014, surveys were conducted evaluating yellow sticky cards and plant samples in lettuce, broccoli, and cauliflower fields in three regions of the Salinas Valley. F. occidentalis were collected on sticky cards from April to November and on foliage in all three regions. Two-choice oviposition assays as well as no-choice and two-choice feeding assays were conducted where F. occidentalis adults were exposed to leaves of lettuce, broccoli, and cauliflower. A significantly greater number of eggs was found on lettuce than on broccoli or cauliflower leaves in the choice assay, whereas for the broccoli and cauliflower choice assay, the number of eggs retrieved was low and not significantly different between hosts. The number of F. occidentalis feeding spots and leaf area injury ratings were similar on broccoli and lettuce leaves but were significantly lower on cauliflower than on lettuce and broccoli leaves. The implications of these results on thrips and plant virus management are discussed.
Assuntos
Brassica , Tisanópteros , Tospovirus , Animais , Flores , Lactuca , Óvulo , Doenças das PlantasRESUMO
Downy mildew of spinach is caused by the obligate oomycete pathogen, Peronospora effusa. The disease causes significant economic losses, especially in the organic sector of the industry where the use of synthetic fungicides is not permitted for disease control. New pathotypes of this pathogen are increasingly reported which are capable of breaking resistance. In this study, we took advantage of new spinach genome resources to conduct RNA-seq analyses of transcriptomic changes in leaf tissue of resistant and susceptible spinach cultivars Solomon and Viroflay, respectively, at an early stage of pathogen establishment (48 hours post inoculation, hpi) to a late stage of symptom expression and pathogen sporulation (168 hpi). Fold change differences in gene expression were recorded between the two cultivars to identify candidate genes for resistance. In Solomon, the hypersensitive inducible genes such as pathogenesis-related gene PR-1, glutathione-S-transferase, phospholipid hydroperoxide glutathione peroxidase and peroxidase were significantly up-regulated uniquely at 48 hpi and genes involved in zinc finger CCCH protein, glycosyltransferase, 1-aminocyclopropane-1-carboxylate oxidase homologs, receptor-like protein kinases were expressed at 48 hpi through 168 hpi. The types of genes significantly up-regulated in Solomon in response to the pathogen suggests that salicylic acid and ethylene signaling pathways mediate resistance. Furthermore, many genes involved in the flavonoid and phenylpropanoid pathways were highly expressed in Viroflay compared to Solomon at 168 hpi. As anticipated, an abundance of significantly down-regulated genes was apparent at 168 hpi, reflecting symptom development and sporulation in cultivar Viroflay, but not at 48 hpi. In the pathogen, genes encoding RxLR-type effectors were expressed during early colonization of cultivar Viroflay while crinkler-type effector genes were expressed at the late stage of the colonization. Our results provide insights on gene expression in resistant and susceptible spinach-P. effusa interactions, which can guide future studies to assess candidate genes necessary for downy mildew resistance in spinach.
Assuntos
Resistência à Doença/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Peronospora/fisiologia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Spinacia oleracea/genética , Spinacia oleracea/microbiologia , Suscetibilidade a Doenças , Ontologia Genética , Genoma de Planta , Polimorfismo de Nucleotídeo Único/genética , Propanóis/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcriptoma/genéticaRESUMO
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.
Assuntos
Agricultura , Microbiologia do Ar , Lactuca , Oomicetos , Reação em Cadeia da Polimerase , Esporos Fúngicos , Agricultura/métodos , Ar , Lactuca/microbiologia , Oomicetos/genética , Doenças das Plantas/microbiologia , Esporos Fúngicos/genéticaRESUMO
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 8-10â¯h. When applied in the morning, E. coli populations declined up to 2 log CFU/plant within 2â¯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 (Pâ¯<â¯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.
Assuntos
Escherichia coli O157/crescimento & desenvolvimento , Lactuca/microbiologia , Viabilidade Microbiana , Folhas de Planta/microbiologia , Contagem de Colônia Microbiana , Qualidade de Produtos para o Consumidor , Microbiologia de Alimentos , Umidade , Fatores de TempoRESUMO
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.
Assuntos
Fragaria , Fusarium , Filogenia , California , Fragaria/microbiologia , Fusarium/genética , Fusarium/fisiologia , Genes Fúngicos/genética , Doenças das Plantas/microbiologiaRESUMO
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.
Assuntos
Ascomicetos/isolamento & purificação , Fragaria/microbiologia , Doenças das Plantas/microbiologia , Reação em Cadeia da Polimerase/métodos , Ascomicetos/genética , California , SoloRESUMO
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.
Assuntos
Lactuca/parasitologia , Oomicetos/isolamento & purificação , Doenças das Plantas/parasitologia , Microbiologia do Ar , Primers do DNA/genética , Fungicidas Industriais , Geografia , Oomicetos/genética , Folhas de Planta/parasitologia , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , EsporosRESUMO
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.
RESUMO
Knowing the identity of bacterial plant pathogens is essential to strategic and sustainable disease management in agricultural systems. This knowledge is critical for growers, diagnosticians, extension agents, and others dealing with crops. However, such identifications are linked to bacterial taxonomy, a complicated and changing discipline that depends on methods and information that are often not used by those who are diagnosing field problems. Modern molecular tools for fingerprinting and sequencing allow for pathogen identification in the absence of distinguishing or conveniently tested phenotypic characteristics. These methods are also useful in studying the etiology and epidemiology of phytopathogenic bacteria from epidemics, as was done in numerous studies conducted in California's Salinas Valley. Multilocus and whole-genome sequence analyses are becoming the cornerstones of studies of microbial diversity and bacterial taxonomy. Whole-genome sequence analysis needs to become adequately accessible, automated, and affordable in order to be used routinely for identification and epidemiology. The power of molecular tools in accurately identifying bacterial pathogenesis is therefore of value to the farmer, diagnostician, phytobacteriologist, and taxonomist.
Assuntos
Bactérias/classificação , Produtos Agrícolas/microbiologia , Biologia Molecular/métodos , Doenças das Plantas/microbiologia , Patologia Vegetal , Bactérias/genética , California , Patologia Vegetal/educaçãoRESUMO
A field trial in Salinas Valley, California, was conducted during July 2011 to quantify the microbial load that transfers from wildlife feces onto nearby lettuce during foliar irrigation. Romaine lettuce was grown using standard commercial practices and irrigated using an impact sprinkler design. Five grams of rabbit feces was spiked with 1.29 × 10(8) CFU of Escherichia coli O157:H7 and placed - 3, - 2, and - 1 days and immediately before a 2-h irrigation event. Immediately after irrigation, 168 heads of lettuce ranging from ca. 23 to 69 cm (from 9 to 27 in.) from the fecal deposits were collected, and the concentration of E. coli O157:H7 was determined. Thirty-eight percent of the collected lettuce heads had detectable E. coli O157:H7, ranging from 1 MPN to 2.30 × 10(5) MPN per head and a mean concentration of 7.37 × 10(3) MPN per head. Based on this weighted arithmetic mean concentration of 7.37 × 10(3) MPN of bacteria per positive head, only 0.00573% of the original 5 g of scat with its mean load of 1.29 × 10(8) CFU was transferred to the positive heads of lettuce. Bacterial contamination was limited to the outer leaves of lettuce. In addition, factors associated with the transfer of E. coli O157:H7 from scat to lettuce were distance between the scat and lettuce, age of scat before irrigation, and mean distance between scat and the irrigation sprinkler heads. This study quantified the transfer coefficient between scat and adjacent heads of lettuce as a function of irrigation. The data can be used to populate a quantitative produce risk assessment model for E. coli O157:H7 in romaine lettuce to inform risk management and food safety policies.
Assuntos
Escherichia coli O157/isolamento & purificação , Fezes/microbiologia , Contaminação de Alimentos/análise , Lactuca/microbiologia , Animais , Animais Selvagens , California , Contagem de Colônia Microbiana , Qualidade de Produtos para o Consumidor , Microbiologia de Alimentos , Inocuidade dos Alimentos , Folhas de Planta/microbiologiaRESUMO
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.
Assuntos
Brassicaceae/microbiologia , Microbiologia do Solo , Verticillium/fisiologia , Fumigação , Hifas/fisiologia , Doenças das Plantas/microbiologia , Solo , Água/fisiologiaRESUMO
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.
RESUMO
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.
Assuntos
Beta vulgaris/microbiologia , Peronospora/isolamento & purificação , Doenças das Plantas/microbiologia , Spinacia oleracea/microbiologia , Esporos/isolamento & purificação , Sequência de Bases , Primers do DNA/genética , DNA Ribossômico/genética , Limite de Detecção , Dados de Sequência Molecular , Peronospora/classificação , Peronospora/genética , Reação em Cadeia da Polimerase em Tempo Real , Alinhamento de Sequência , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
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.
RESUMO
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.
RESUMO
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.
RESUMO
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.
Assuntos
Doenças das Plantas/microbiologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sementes/microbiologia , Spinacia oleracea/microbiologia , Verticillium/isolamento & purificação , DNA Fúngico/análise , DNA Fúngico/genética , Reação em Cadeia da Polimerase em Tempo Real/instrumentação , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Verticillium/genéticaRESUMO
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.
RESUMO
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.