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Received Date : 11-Feb-2014 Revised Date : 30-Apr-2014 Accepted Date : 07-May-2014 Article type

: Original Article

Corresponding author e-mail id: [email protected]

Multiplex real-time PCR assays for detection of four seedborne spinach pathogens

Chunda Feng (1), Saara Mansouri (1), Burt H. Bluhm (1), Lindsey J. du Toit (2), James C. Correll (1)

(1) University of Arkansas, Fayetteville, AR, U.S.A. (2) Washington State University, Mount Vernon, WA, U.S.A.

Corresponding Author: James C. Correll

ABSTRACT Aim To develop multiplex TaqMan real-time PCR assays for detection of spinach seedborne pathogens that cause economically important diseases on spinach. Materials and Methods Primers and probes were designed from conserved sequences of the internal transcribed spacer (for Peronospora farinosa f. sp. spinaciae and Stemphylium botryosum), the intergenic spacer (for Verticillium dahliae), and the elongation factor 1 alpha (for Cladosporium variabile) regions of DNA. The TaqMan assays were tested on DNA extracted from numerous isolates of the four target pathogens, as well as a wide range of nonThis article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/jam.12541 This article is protected by copyright. All rights reserved.

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target, related fungi or oomycetes, and numerous saprophytes commonly found on spinach seed. Multiplex real-time PCR assays were evaluated by detecting two or three target pathogens simultaneously. Singular and multiplex real-time PCR assays were also applied to DNA extracted from bulked seed and single spinach seed. Conclusions The real-time PCR assays were species-specific and sensitive. Singular or multiplex real-time PCR assays could detect target pathogens from both bulked seed samples as well as single spinach seed. Significance and impact The freeze-blotter assay that is currently routinely used in the spinach seed industry to detect and quantify three fungal seedborne pathogens of spinach (C. variabile, S. botryosum, and V. dahliae) is quite laborious and takes several weeks to process. The real-time PCR assays developed in this study are more sensitive and can be completed in a single day. As the assays can be applied easily for routine seed inspections, these tools could be very useful to the spinach seed industry.

Keywords: downy mildew, Verticillium wilt, Cladosporium leaf spot, Stemphylium leaf spot.

INTRODUCTION Spinach, widely regarded as one of the most nutritious vegetables, contains high levels

of vitamins, minerals, and anti-oxidants (Morelock and Correll 2008). During the last two decades, per capita spinach consumption increased four-fold in the USA (United States Department of Agriculture National Agriculture Statistics Service, USDA NASS, http://www.nass.usda.gov), in part due to the increased availability and popularity of prepackaged baby spinach leaves. Market demand for baby spinach leaves has driven dramatic changes in spinach production practices, including multiple harvests per season and up to a 10fold increase in plant density. The continuous, year-round production of spinach in U.S. production regions, combined with high-density plantings, has created highly favorable conditions for disease development.

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In recent years, seedborne pathogen transmission has become increasingly problematic for spinach production, in part due to high density plantings. Spinach downy mildew, caused by the obligate pathogen Peronospora farinosa f. sp. spinaciae, is the most important disease of spinach in the USA, and the pathogen can be transmitted via seed (Inaba et al. 1983; Inaba 1985). Eleven new races and additional novel strains of P. farinosa f. sp. spinaciae have been identified

in the past two decades (Correll et al. 1990; Irish et al. 2003; Irish et al. 2007; Feng et al. 2014),and the global distribution of some races is consistent with seedborne transmission (Feng

et al. 2014).Verticillium dahliae, a seedborne pathogen of spinach that causes a vascular wilt, has been reported in commercial production fields in the USA, Canada, and the Netherlands (Snyder and Wilhelm 1962; Van der Spek 1972; 1973; Sackston and Sedun 1982). Infections by V. dahliae remain asymptomatic during vegetative growth of spinach and, thus, the pathogen was not considered economically important during crop production (Correll et al. 1994). However, V. dahliae can be highly problematic for spinach seed production(du Toit et al. 2005c), and seed transmission of V. dahliae may introduce the pathogen into new production areas (Atallah et al.

2010). The latter is potentially problematic because some strains of V. dahliae have a broad host range and spinach is often rotated with high-value vegetables and fruits, including lettuce and strawberry, that can be devastated by V. dahliae (Atallah et al. 2010; Iglesias-Garcia et al. 2013).

Additionally, Cladosporium leaf spot, caused by Cladosporium variabile, and Stemphylium leaf spot, caused by Stemphylium botryosum, have recently emerged as problems for spinach

production as well as spinach seed production (Fuentes-Davila and Gabrielson 1996; du Toit and Derie 2001; Everts and Armentrout 2001; Koike et al. 2001; Koike et al. 2005; Reed et al. 2010;

du Toit and Derie 2012). Both of these pathogens are known to be seedborne (Hernandez-Perez and du Toit 2006).

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The location of seedborne pathogens in or on spinach seed can have practical significance as this affects the potential for seed transmission of the pathogen(s), as well as the potential efficacy of seed treatments such as chlorine, hot water, or fungicides for eradication of the pathogens from seed and/or prevention of seed transmission (du Toit and Hernandez-Perez 2005). Seedborne pathogens can infest seed, whereby the pathogens are limited to the outer structure of the seedor occur superficially on the surface of the seed; or can infect seed, whereby pathogens colonize the seed internally, including the embryo. The terms 'infested seed' vs. 'infected seed' used in this study refer to seed in which infection by a seedborne pathogen is restricted to the outer spinach pericarp (corky outer structure of the seed derived from the maternal parent) or superficially on the seed service vs. infected seed whereby the seed have internal infection of the embryo, respectively. Planting pathogen-free seed can be a critical component of spinach disease management. Seed health assays are routinely performed by the spinach seed industry to determine whether seed lots are contaminated with one or more pathogens. Traditional freezeblotter or selective media assays of spinach seed provide estimates of percent infestation/infection with V. dahliae, C. variabile and S. botryosum (du Toit et al. 2005c; Hernandez-Perez and du Toit 2006). However, these assays are time-consuming and labor intensive, and perhaps most importantly, cannot be used to detect propagules of the obligate pathogen P. farinosa f. sp. spinaciae. Currently, seed assays for P. farinosa f. sp. spinaciae

consist of examining spinach seed washes via light microscopy for sporangia and oospores (Inaba et al. 1983), although viability of these propagules cannot be assessed microscopically. In contrast, a much quicker and more sensitive, quantitative assay could be provided by real-time PCR assays, which have been used to examine diverse seedborne pathogens such as bacteria (Marques et al. 2010; Becker et al. 2011; Cho et al. 2011), fungi (Chilvers et al. 2007; Ha and

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Walcott 2008; Duressa et al. 2012), oomycetes (Montes-Borrego et al. 2011; Ioos et al. 2012) and viruses (Hwang et al. 2007; Ling et al. 2011). Recently, a real-time PCR assay utilizing SYBR Green was developed to detect V. dahliae associated with spinach seed (Duressa et al.

2012), but a real-time PCR assay has not yet been developed for P. farinosa f. sp. spinaciae, C. variabile, or S. botryosum. In this study, we developed specific, sensitive, and robust TaqMan real-time PCR assays to detect and quantify the amount of P. farinosa f. sp. spinaciae, V. dahliae, C. variabile, and S. botryosum in spinach seed, as well as a multiplex approach to detect combinations of these spinach pathogens simultaneously. These assays were effective when tested on batches of spinach seed as well as individual seed, and thus provide useful new tools for spinach seed health assessment.

Materials and Methods Pathogen isolates and DNA extraction A wide range of isolates of spinach seedborne pathogens and saprophytic fungi were

included in this study (Table 1). Twelve isolates of P. farinosa f. sp. spinaciae were used that represented reference strains of races 6, 10, 11, 12, 13, and 14 as well as three novel strains, UA4711, UA1012B, and UA1312(Feng et al. 2014).

Fifteen isolates of V. dahliae were

collected from spinach plants and seed from geographically diverse seed production centers (Denmark, Netherlands, and the USA). Eighteen isolates of C. variabile demonstrated to be

pathogenic on spinach as well as 41 isolates of Cladosporium spp. that were not pathogenic on spinach were examined. The Cladosporium isolates were all from spinach seed. The nonpathogenic isolates included various small-spored Cladosporium spp. readily distinguished from C. variabile with a dissecting microscope, as well as large-spored Cladosporium spp. that closely

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resemble C. vaiabile microscopically but do not produce coiled aerial hyphae and were not pathogenic when inoculated onto spinach plants in a greenhouse using the method previously described (Ellis 1971; Hernandez-Perez and du Toit 2006). Also, 12 isolates of S. botryosum that were pathogenic on spinach were evaluated. All of these isolates were recovered from spinach seed samples from Chile, Denmark, Italy, the Netherlands, and the USA (Table 1). To evaluate specificity of the real-time PCR assays for the target pathogens, five isolates of V. dahliae from other hosts were included, as were strains of Albugo occidentalis (cause of spinach white rust),

Peronospora farinosa f. sp. betae (cause of downy mildew of beet, Beta vulgaris, and Swiss chard, Beta vulgaris subsp. cicla), Peronospora variabilis (cause of downy mildew on quinoa,

Chenopodium quinoa), Verticillium tricorpus and Verticillium nigrescens (saprophytes or weak pathogens of spinach often found associated with spinach seed) (Iglesias-Garcia et al. 2013), V. albo-atrum (from alfalfa, Medicago sativa), V. fungicola (from mushroom), Colletotrichum dematium (cause of spinach anthracnose), Fusarium oxysporum f. sp. spinaciae (cause of Fusarium wilt of spinach), and multiple saprophytic fungal isolates including F. oxysporum, Alternaria spp., Phoma spp., Epicoccum spp.,and Ulocladium spp. that are commonly found on

spinach seed (Correll et al. 1994; du Toit et al. 2005c; du Toit and Hernandez-Perez 2005). For microbial DNA extraction, sporangia of P. farinosa f. sp. spinaciae were washed off

infected leaf tissue with deionized water and collected by centrifugation. Cultures of V. dahliae, C. variabile, and S. botryosum were grown in complete broth medium (Correll et al. 1992) for seven days, and the mycelia were collected by filtration. Sporangia of P. farinosa f. sp. spinaciae, and mycelia of V. dahliae, C. variabile, and S. botryosum were lyophilized. DNA was extracted with a CTAB method (Feng et al. 2011). DNA was quantified with a Nanodrop ND-

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1000 spectrophotometer (Fisher Scientific, Pittsburgh, PA) or Picogreen (Life Technologies, Grand Island, NY) following the manufacturers’ instructions.

Spinach seed samples Spinach seed samples used in the real-time PCR assays were provided by collaborators in

the public and private sectors. The seed lots of spinach cultivars Emu and Raccoon used in this study were confirmed to be contaminated by P. farinosa f. sp. spinaciae using a seed wash assay. Briefly, 5 g seed of each lot were transferred to 25 ml of sterilized deionized water (dH2O) in a

50 ml centrifuge tube, which was then shaken at 200 rpm on an orbital shaker for 2 h. Seed were then removed by filtering each suspension through two layers of cheesecloth, followed by centrifugation at 3,000 rpm for 2 min. After decanting the supernatant, the pelleted debris was re-suspended in 1 ml sterilized dH2O, and transferred to microfuge tubes for storage or

examination with a light microscope. Seed of the spinach cultivar Dash was obtained from a spinach crop planted in an isolated region of the Western Cape, South Africa, where P. farinosa f. sp. spinaciae had not previously been documented, yet the baby leaf spinach crop was severely infected with P. farinosa f. sp. spinaciae, suggesting that infested or infected seed was probably

the primary inoculum source Additionally, spinach seed lots 103SN, 201SA and 201MN were produced in the spinach seed production region of Skagit Co., WA, and confirmed to be infested and/or infected naturally with V. dahliae, C. variabile and S. botryosum. The percent infection/infestation of each lot by each of these three pathogens was determined with a freezeblotter assay as described by du Toit et al. (2005c) and Hernandez-Perez and du Toit (2006) (Fig. 4).

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DNA extraction from spinach seed To extract DNA from single spinach seed, individual seed were wrapped with weighing

paper (VWR Cat. #12578-165), and crushed with a hammer. Crushed seed were placed into individual wells of a 96-well microtiter plate, with 100 µl TE buffer added to each well. The plates were each sealed with an EVA cap mat (USA Scientific, Inc., Ocala, FL), incubated in a thermal cycler at 95oC for 5 min or at 65oC in a water bath for 30 min, and centrifuged at 1000

rpm for 3 min in a mini-plate spinner (Labnet, Woodbridge, NJ). Finally, 50 µl of seed extract supernatant from each well was transferred to a well of a new plate containing 50 µl TE buffer. Seed DNA extracts were evaluated immediately via real-time PCR assay or stored at -20oC. To extract DNA from batches of spinach seed, a similar protocol was used with minor

modification. One gram of spinach seed from each seed lot was crushed with a mortar and pestle, transferred to a 15 ml centrifuge tube containing 5 ml TE buffer, and incubated in a 65oC water bath for 30 min. Then 1 ml of the supernatant was transferred to a 1.7 ml tube and centrifuged at 16,000 g for 1 min. Finally, 0.5 ml of the supernatant was transferred to a new 1.7 ml tube containing 0.5 ml TE buffer. The resulting DNA extracts were evaluated immediately via realtime PCR assays or stored at -20oC. Because P. farinosa f. sp. spinaciae is an obligate pathogen, conventional seed assays

such as the freeze-blotter assay cannot be used to determine the percentage of seed infected or infested with P. farinosa f. sp. spinaciae. Therefore, to provide seed samples with known

infection levels for analysis, spinach seeds were inoculated with P. farinosa f. sp. spinaciae. In

summary, 5 g seeds of each of the spinach cultivars Dash and Emu were autoclaved at 13.5 psi and121oC for 20 min, and 10 seeds of each cultivar were then soaked for 5 s in a spore suspension of P. farinosa f. sp. spinaciae (106 spores/ml). DNA was then extracted from

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individual inoculated seed and autoclaved seed of each of the two cultivars following the protocol described above.

Primers and probes for TaqMan real-time PCR assays To identify target sites for primer and probe design, the universal ribosomal DNA (rDNA)

primers ITS1 and ITS4 (White et al. 1990)were used to amplify the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA), including the ITS1-5.8S-ITS2 region, from the isolates of P. farinosa f. sp. spinaciae and S. botryosum. The intergenic spacer (IGS) forward and reverse (F and R) primers previously developed for V. dahliae (Qin et al. 2006)were used to amplify the IGS rDNA from the isolates of V. dahliae. The primers EF1-728F and EF1-986R were used to amplify the translation elongation factor 1α (EF1α) encoding region from the isolates of C. variabile (Carbone and Kohn 1999). The ITS, IGS, and EF1α products were sequenced in the DNA core laboratory at the University of Arkansas, Fayetteville, AR. After sequence alignment and BLASTn searches against GenBank, species-specific and conserved sequences of each pathogen were targeted for primer and probe design using online tools from the Integrated DNA Technology (IDT) website (http://www.idtdna.com). All primers and probes utilized in this study (Table 2) were synthesized by IDT (Coralville, IA). The probes targeting P. farinosa f. sp. spinaciae, V. dahliae, C. variabile and S. botryosum were labeled with Cy5 or FAM, FAM or HEX, HEX, and Cy5 fluorescent dyes, respectively (Table 2).

Real-time PCR assay conditions Real-time PCR assays were performed in 20 µl reactions containing: 1X Taq DNA

polymerase buffer, 2mM Mg2+, 80 µM each dNTP, 1 U Taq DNA polymerase, 0.4 µM primers,

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0.2 µM probe, 0.5 µM ROX reference dye, 1 µl target DNA at the appropriate concentration, and sterilized dH2O to make a final volume of 20 µl. Reactions were also performed with sterilized

dH2O and DNA extracted from autoclaved spinach seed as the template to serve as negative control treatments. The PCR reactions were carried out at 94oC for 5 min, followed by 40 cycles of 94oC for 10 sec and 60oC for 30 sec. PCR assays were performed primarily with a Stratagene

Mx 3000P thermal cycler (Stratagene Corporation, La Jolla, CA), but some reactions were performed with a Bio-rad C1000 thermal cycler (Hercules, CA) or a StepOne thermal cycler (Life Technologies, Grand Island, NY) to test the assays. For multiplex reactions, the PCR parameters described above were used with

combinations of two or three sets of primers/probes (each at 0.2 µM). Ten-fold serial dilutions of pathogen genomic DNA were prepared and tested to evaluate the sensitivity of each primer/probe set, with a minimum of three replications performed for each DNA template, in at least three repeated experiments per combination. For each template, standard curves calculated based on regression of the Ct values versus the target DNA concentrations, were used to evaluate reaction performance and to quantify target pathogen DNA in samples tested with the real-time PCR assays. For the serial dilutions analyzed, DNA concentrations ranged from 1 fg/µl to 1 ng/µl. To evaluate specificity of each real-time PCR assay, each set of primers and probe was

tested on taxonomically related pathogens and other unrelated fungi commonly associated with spinach seed (Table 1).The threshold for detection was placed automatically by the analysis software of the real-time PCR thermal cyclers, with minimal modification as needed based on the negative control samples in each run. Reactions with threshold cycle (Ct) values ≤35 were

regarded as positive for the presence of the target DNA.

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Real-time PCR assays for detection of pathogens from spinach seed To test and/or confirm whether selected spinach seed lots were infested and/or infected

by one or more of the four target pathogens, 1 g of seed was weighed and used for DNA extraction for each seed lot tested. To estimate the percentage of seed infected, 88 seeds from each seed lot were selected randomly and used separately for DNA extraction. Thus, each 96well PCR plate contained 88 wells for seed assays, plus 8 control samples consisting of DNA from spores or mycelia and inoculated seed as positive control treatments, and sterilized dH2O

and autoclaved seed as negative control treatments.

RESULTS Specificity and sensitivity of real-time PCR assays for four spinach seedborne pathogens Neither false positive nor false negative reactions were observed in the real-time PCR

assays. The real-time PCR assay for P. farinosa f. sp. spinaciae detected all 12 isolates of that pathogen utilized in the study, which represented races 6, 10, 11, 12, 13, and 14, as well as three novel strains (UA4711, UA1012B and UA1312) (Feng et al. 2014). The assay also did not cross react with DNA from a variety of other organisms, including the spinach white rust pathogen A.

occidentalis, beet and Swiss chard downy mildew pathogen, P. farinosa f. sp. betae, and quinoa downy mildew pathogen, P. variabilis, as well as the spinach Verticillium wilt pathogen, V. dahliae, and other spinach pathogenic or saprophytic fungal isolates. Likewise, the real-time

PCR assay for the downy mildew pathogen produced negative results for the two negative control treatments, dH2O, and DNA extracted from autoclaved seed. The real-time PCR assay for V. dahliae detected all 20 isolates of V. dahliae, regardless

of the host or geographic origin, and did not amplify DNA from a wide range of saprophytic

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fungi from spinach seed including V. tricorpus, V. nigrescens and other Verticillium species or other fungal genera. The V. dahliae primers did not amplify DNA from other spinach seedborne pathogens, including C. variabile, S. botryosum, and C. dematium. The real-time PCR assay for S. botryosum detected all 12 isolates of S. botryosum tested, but did not amplify DNA of P. farinosa, V. dahliae, C. variabile or other spinach pathogenic and saprophytic isolates. The realtime PCR assay for C. variabile amplified DNA from the 20 pathogenic isolates of C. variabile

tested, but not from the 41 isolates of Cladosporium spp. that were not pathogenic on spinach or the isolates of non-target fungi tested and the spinach downy mildew pathogen. Thus, the primers and TaqMan probes developed in this study appeared to be specific to the four target spinach pathogens among the diverse fungal and oomycete isolates tested (Table 1). Serial dilutions of 1 ng to 1 fg DNA of each of the target spinach pathogens were used to

test sensitivity of the real-time PCR assays. The Ct values for 1 ng DNA from each of the four target pathogens were approximately 17 to 22.6 cycles (with standard deviations of 0.15 to 0.40). However, due to different amplification efficiencies, the Ct values for 10 fg template DNA from each of P. farinosa f. sp. spinaciae, V. dahliae, and S. botryosum were 35, 30 and 34, respectively; and the Ct value for 100 fg of C. variabile DNA was 37. Therefore, the detection threshold for the four pathogens was set at 10 fg for P. farinosa f. sp. spinaciae, V. dahliae, and S. botryosum; and 1 pg for C. variabile with a Ct cut-off of 35 (Table 3). The stability of the four real-time PCR assays was evaluated with three replications of a serial dilution of DNA from each of the four target pathogens in three or more experiments. When evaluating the relationship between Ct value and DNA template concentration, standard curves indicated that amplification efficiencies ranged from 84.6 to 125.5%, with the coefficient of determination, R2, ranging from

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0.953 for C. variabile to 0.995 for P. farinosa f. sp. spinaciae (Fig. 1), indicating that the Ct values were solely determined by the concentration of target DNA in each assay.

Multiplex real-time PCR assays for simultaneous detection of multiple spinach seedborne pathogens Multiplex real-time PCR assays for simultaneous detection of multiple spinach pathogens

were evaluated by combining DNA templates from various combinations of two or three of the target pathogens. All the multiplex real-time PCR assays completed using DNA extracted from the four spinach seedborne pathogens were successful. However, the sensitivity of each multiplex assay was lower than that of the single-pathogen real-time PCR assays. The duplex real-time PCR assays simultaneously detected 100 fg DNA from each of any two pathogen combinations of P. farinosa f. sp. spinaciae, V. dahliae, and S. botryosum, but only 1 pg DNA of each for any combination of C. variabile with P. farinosa f. sp. spinaciae, V. dahlia or S.

botryosum. Only three fluorescent dyes could be detected simultaneously with the Mx 3000p real-time PCR thermal cycler. All four combinations of triplex real-time PCR assays were able to detect the three target pathogens concurrently. The triplex real-time PCR assay for P. farinosa f. sp. spinaciae + V. dahliae + S. botryosum detected as little as 1 pg DNA of each pathogen. However, for the other triplex combinations (P. farinosa f. sp. spinaciae + V. dahliae + C.

variabile, P. farinosa f. sp. spinaciae + S. botryosum + C. variabile, and V. dahliae + S.

botryosum+ C. variabile), the detection thresholds were only 10 pg for each pathogen. The

triplex real-time PCR sensitivity for each of these three pathogens was equivalent to one-tenth that of the duplex assays for each combination of two of the three target pathogens. The standard curves (Fig. 2) showed the relationship between Ct value and template DNA concentration for

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three duplex and one triplex real-time PCR assays. The R2 for each of these assays was >0.95, indicating that the Ct value for each pathogen was determined mainly by DNA concentration of the target pathogens. The amplification efficiencies for the multiplex assays were close to or >80% (Fig. 2). Assays for P. farinosa f. sp. spinaciae and V. dahliae were more sensitive

than those for C. variabile and S. botryosum. Thus the latter two appear to be the limiting pathogens regarding sensitivity of the multiplex real-time PCR assays.

Real-time PCR assay for detection of P. farinosa f. sp. spinaciae in spinach seed The real-time PCR assay was applied to batches of spinach seeds for detection of P.

farinosa f. sp. spinaciae. Spores of this pathogen were observed from the seed washes of spinach cultivars Dash, Emu and Raccoon (data not shown). The real-time PCR assays using DNA extracted from 1 g of seed from each seed lot also confirmed that all three samples tested positive for P. farinose f. sp. spinaciae (data not shown). To evaluate reliability of the real-time PCR assay for P. farinosa f. sp. spinaciae on

single seed, DNA was extracted from 10 autoclaved seeds as well as 10 autoclaved seeds that had been inoculated (spiked) with P. farinosa f. sp. spinaciae spores, for each of the spinach cultivars Dash and Emu. The real-time PCR assay detected P. farinosa f. sp. spinaciae from all 20 spiked seeds, but the 20 autoclaved seeds tested negative for the pathogen (Fig. 3). This suggested that the real-time PCR assay for the downy mildew pathogen could be used for single seed assays for detection of this important seedborne pathogen of spinach. When 88 seeds were randomly selected from each seed lot, the single seed assays showed

that 93.2% of the Dash seeds, 70% of the Emu seeds, and 69.3% of Raccoon seeds were infested and/or infected with P. farinosa f. sp. spinaciae. To test reliability of the assay with naturally

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infested and/or infected seed lots, 2 µl DNA was taken from 6, 8, and 8 wells that had tested negative with the real-time PCR assays for the Dash, Emu, and Raccoon seed DNA plates, respectively. A 1 µl aliquot of DNA of each sample was used directly for repeating the real-time PCR assay. These samples tested negative for P. farinosa f. sp. spinaciae. The remaining 1 µl

DNA aliquot was spiked with 0.1 ng DNA of isolate INT-1 of P. farinosa f. sp. spinaciae, and

then tested with the real-time PCR assay. All 22 spiked seeds tested positive for the downy mildew pathogen, and the average amount of DNA detected for the 22 samples was estimated as 0.085 ng, suggesting that efficiency of DNA quantification of the test was approximately 85%.

Real-time PCR assays for detection of V. dahliae, C. variabile, and S. botryosum in spinach seed When using DNA extracted from 1g of seed as a template, V. dahliae, C. variabile and S.

botryosum were detected from all three spinach seed lots evaluated (103SN, 201SA, and 201MN) using the single real-time PCR assay designed for each pathogen. Duplex and triplex real-time PCR assays confirmed these results when primers/probes for two or three pathogens were combined (data not shown). Real-time PCR assays were also run for DNA extracted from individual seeds to evaluate the percentage of seed infested and/or infected with each pathogen. The estimated percentage of infection for each of the three pathogens in each seed lot using the real-time PCR assays was equivalent to or greater than results with the freeze-blotter assays (Fig. 4). Compared to the percentage of V. dahliae infection estimated using the freeze-blotter assay for these three lots, the percentage infested/infected seed estimated with the real-time PCR assay for this pathogen was 2 to 17% greater, but only the percentage of infection or infestation for seed lot 201MN was significantly different (P

Multiplex real-time PCR assays for detection of four seedborne spinach pathogens.

To develop multiplex TaqMan real-time PCR assays for detection of spinach seedborne pathogens that cause economically important diseases on spinach...
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