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Postharvest Biology and Technology 52 (2009) 235–239

Contents lists available at ScienceDirect

Postharvest Biology and Technology

journa l homepage: www.e lsev ier .com/ locate /postharvbio

esearch Note

uantitative and qualitative analysis of Botrytis inoculated on table grapes byPCR and antibodies

ustafa Celika, Tatiana Kalpulovb, Yohanan Zutahyb, Shahar Ish-shalomb, Susan Lurieb,∗, Amnon Lichterb

Adnan Menderes University, Agriculture Faculty, Horticulture Department, Aydın 09100, TurkeyDepartment of Postharvest Science, ARO, The Volcani Center, Bet Dagan, POB 6, 50250, Israel

r t i c l e i n f o

rticle history:eceived 24 July 2008ccepted 26 October 2008

a b s t r a c t

The fungus Botrytis cinerea is the major cause of decay in table grapes during storage, and the severity ofdecay depends in part on contamination with the fungus before storage. The current SO2 technology toprevent decay is robust and independent of the level of contamination by B. cinerea. The introduction ofalternative technologies may however require implementation of means which are proportional to the

eywords:ntibody testotrytis cinereaisease monitoringeal-time PCRtorage

level of contamination. The objectives of this study were to test the feasibility of quantifying B. cinerea inartificially inoculated grapes and to monitor the progress of disease during storage. Two methods werecompared for detection of B. cinerea in grapes; an antibody kit specific for B. cinerea, and quantitative PCRusing fungal specific primers. Antibodies for fast detection of B. cinerea yielded positive results only in thelater stages of decay development. In contrast, the quantitative PCR demonstrated positive identification

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. Introduction

Botrytis cinerea Pers. is the most efficient pathogen of tablerapes during storage. The fungus grows vigorously on harvestedrapes and can spread among berries even at low temperatures−0.5 ◦C). Infections that cause postharvest losses can originaterom conidia present on the surface of the berries or latentnfections before harvest (De Kock and Holz, 1994). If grapesre not treated after harvest or during storage, infection cannitiate on the majority of the berries. Moreover, even if exter-al conidia infection is eliminated, latent infection from singleerries can lead to production of abundant white surface mycelia,hich spread to adjacent berries creating nests of infection. The

porulating nest, which may actually be single berries, can con-aminate and infect an entire package of grapes (Droby and Lichter,004).

The major postharvest technology used to control B. cinereaecay relies on the controlled emission of sulfur dioxide (SO2).

he gas is applied either by weekly fumigation of storage facili-ies (Harvey and Uota, 1978; Luvisi et al., 1992) or by using sachetsontaining the salt sodium metabisulfite (Adaskaveg et al., 2002).or various reasons, alternative technologies to SO2 have been pub-

∗ Corresponding author. Tel.: +972 39683606; fax: +972 39683622.E-mail address: slurie43@agri.gov.il (S. Lurie).

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925-5214/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.postharvbio.2008.10.007

points, and found increasing amounts of the fungus during storage.© 2008 Elsevier B.V. All rights reserved.

ished which can offer different extents of control against decayLichter et al., 2006). For example, dipping grapes in ethanol or

odified atmosphere can offer a sufficient level of control underost conditions but if grapes are highly sensitive or longer storage

uration is required, the combined treatment gives superior resultsLichter et al., 2005).

The major limitation to detection of latent infection within plantaterial is the small quantity of the pathogen relative to the plant

iomass. Recently however, immunological techniques have beeneveloped to monitor the level of B. cinerea contamination of winerapes (Dewey and Yohalem, 2004; Obanor et al., 2004).

The first use of molecular detection of B. cinerea used conven-ional PCR from a unique PCR fragment obtained by RAPD analysisRigotti et al., 2002). The detection limit of this method was 2 pgn 1 �g of strawberry DNA. The introduction of real-time or quan-itative PCR (qPCR) greatly improved quantitative analysis, andhe very wide dynamic range of the method enabled disease pro-ression to be monitored in many pathogen–plant model systemsrom infection to symptom expression (Schaad and Frederick, 2002;

cCartney et al., 2003; Schena et al., 2004).In the present study our objective was to follow the infection

f grapes by B. cinerea both at optimal growth temperature (20 ◦C)or the fungus for 4 d and during storage at 0 ◦C for 4 weeks. Therogress of infection was monitored with a rapid immunologicalssay and qPCR. The results showed that qPCR detected the fungusithin the infected grapes from the initial stage of infection, while

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36 M. Celik et al. / Postharvest Biolo

he immunological assay detected the fungus during the late stagesf infection at both 20 ◦C and 0 ◦C.

. Material and methods

‘Superior Seedless’ (‘Sugarone’) table grapes at physiologicalaturity, i.e. TSS 15.8% and acidity 0.57% in tartaric acid equivalents,ere harvested from a commercial vineyard on July 15. The clustersere dipped in 70% ethanol and dried on paper towels under a bio-

ogical hood. The berries were removed from the cluster with theedicel attached and positioned in 24-well multidishes (Nunclonurface, Nunc, Roskilde, Denmark). The berries were divided intoour groups. The berries in the first three groups were wounded bynserting a 21-gauge needle to a depth of 1 mm. Those of the firstroup were only wounded, those of the second group had a 10 �Lrop containing 103 conidia (105 mL−1) of B. cinerea placed in theound, and the third group was inoculated with a drop contain-

ng 104 (106 mL−1) conidia. The berries of the fourth group wereeither wounded, nor inoculated.

The inoculations used B. cinerea Pers.: Fr. Botryotinia fuckelianade Bary) Whetz., strain BO5.10. The fungus was maintained on PDAishes. Conidia were collected by adding 10 mL of sterile waterontaining 0.03% Tween 20 and 0.7% NaCl. The solution was fil-ered through four layers of gauze, and the concentration of conidiaas determined with a hemocytometer and adjusted to 105 or 106

onidia mL−1.Following the inoculation treatments, the trays containing the

erries were placed into two covered plastic boxes. One box wasncubated at 20 ◦C for 4 d and the other was incubated at 20 ◦C for4 h and then at 0 ◦C for 4 weeks. The berries held at 20 ◦C were sam-led immediately after wounding and inoculation, and then after4, 48 and 96 h at 20 ◦C. The berries in cold storage were sampledollowing the 24 h incubation at 20 ◦C, and after 1, 2, 3 and 4 weeksf storage. The sample from each treatment and time point con-isted of three berries, which were observed microscopically andhen wrapped in aluminum foil and frozen, as described below.

After microscopic observation, the 3-berry samples wereubmerged in liquid nitrogen, crushed with a mortar and pes-le, and lyophilized for 48 h. The original fresh weight of theerries, 20–25 g, was reduced to approximately one-seventh after

yophilization. The samples were ground to fine powder under liq-id nitrogen, and the powder transferred to 1.5 mL tubes that wereept at −32 ◦C pending analysis.

For immunological analysis, 100 mg (dry weight) samples wereuspended in 1 mL of distilled water in 1.5 mL tubes and vortexedor 2–3 min. The suspension was added to 19 mL of 0.1 M phosphateuffer (pH 7.2) included in the BOTR00083, Pocket Diagnostic Lat-ral Flow Antibody Kit (Pocket Diagnostic, http://pdiag.csl.gov.uk).fter mixing as recommended by the manufacturer, 75 �L aliquotsere applied to the well of the lateral flow device. Appearance of theositive line indicating presence of B. cinerea antigens was recordednd photographed after 10 min of incubation.

For extraction of B. cinerea DNA, 105 conidia were inoculatednto a 250 mL Erlenmeyer flask containing 60 mL of Gambourg B5

edium including vitamins (DUCHEFA, Haarlem, the Netherlands;at No. GO210.0025). The culture was grown for 7 d at 25 ◦C withentle shaking at 120 rpm. After 7 d the mycelia were collectedy vacuum filtration through 3 mm paper (Whatman, Maidstone,K) and were washed three times with 20 mL of sterile water. The

ashed mycelia were collected into a 15 mL polypropylene tube,

rozen in liquid nitrogen and lyophilized for 48 h.B. cinerea DNA was extracted from lyophilized mycelia according

o Hofman and Winston (1987) with some modifications. Sixty-fiveg of mycelia were placed into a 2 mL polypropylene tube together

3

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Technology 52 (2009) 235–239

ith the equivalent of 100 �L of glass beads (212–300 �m; Sigma),00 �L of breaking buffer, and 500 �L of extraction solution. Thereaking buffer consisted of Triton-X (2%), sodium dodecylsulfate1%), sodium chloride (100 mM), Tris–Cl (10 mM, pH 8), and EDTA1 mM). The extraction buffer consisted of phenol buffered to pH 8Biolabs, Jerusalem, Israel), chloroform (Biolabs), and isoamyl alco-ol (Sigma) in proportions of 24:24:1 (v/v/v). The tubes were sealednd vortexed for 5 min at maximum speed and centrifuged formin at maximum speed, after which 500 �L of the supernatantere removed to a new tube. Sodium acetate (50 �L, 3 M, pH 5.2)

nd 1 mL of cold ethanol (96%) were added, and the tubes werencubated for 30 min at −20 ◦C. The tubes were centrifuged for0 min at maximum speed, and the pellet was washed with 70%thanol and dried under a microbiological hood for 30 min. Sixty-ve microliters of distilled water were added to the tube, whichas then incubated overnight at 4 ◦C. The yield of B. cinerea DNA,hich was used as reference stock for the study, was 163 ng �L−1,

s measured with a NanoDrop instrument (NanoDrop Technologies,ilmington, DE, USA).DNA from inoculated grape berries was extracted from 50 mg of

yophilized tissue. The extraction was carried out with the G-SpinIp Genomic DNA Extraction Kit for Plants (Cat. No. 17271) (INtRONitechnology, Kyungki-Do, Korea), according to the manufacturer’s

nstructions. The extracted DNA was stored at −32 ◦C pending anal-sis. The average yield of DNA was 20 ng �L−1, as measured bybsorbance at 260 nm using a NanoDrop instrument.

B. cinerea-specific primers used in this study were designedrom the sequence of an RNA helicase gene (Accession num-er: BC1G 13792). The forward and reverse primers were:Hel81-ACAGACTTTGGGCACATTCC and RHel82-CGGTACGTCCAA-ACGCTTT. The primers were synthesized by Syntezza (Jerusalem,srael). The grape-specific primers used in this study were basedn the sequence of the grape catalase gene (Accession number:F236127) (Or et al., 2002). The primers were kindly provided by T.alleli and E. Or; they were: forward: FCat141-TCAAGCAGCCTGGT-AGAGATAC; reverse: RCat141-CAGCCTGAGACCAATA-TGAAATCC.

Real-time PCR was carried out with the Rotor-Gene 3000 instru-ent (Corbett Research, Mortlake, NSW, Australia). The reactionsere carried out in 0.1 mL tubes, and in a volume of 10 �L. The reac-

ion mixture comprised AmpliqonIII Real-Time quantitative PCRqPCR) Master Mix (Cat No. 250506) (Vestre Teglgade, Copenhagen,enmark) with MgCl2, at a final concentration of 3 mM. Primer con-entrations were 0.625 �M for primers FHel81 and RHel82, and.30 �M for FCat141 and RCat141. The cycling conditions were: ini-ial incubation of 15 min at 95 ◦C, followed by 40 cycles of 5 s at5 ◦C, 15 s at 60 or 61 ◦C for the grape and the B. cinerea primer,espectively, and 20 s at 72 ◦C. Fluorescence data were collected at2 ◦C after completion of cycling, and a melting curve was producedy ramping the temperature from 72 to 99 ◦C.

For quantitative PCR analysis of the amount of B. cinerea DNAn grape DNA extractions, 40 ng of DNA were used, together withHel81 and RHel82 primers, in triplicate. For normalization of themount of grape DNA in each sample, primers FCat141 and RCat141ere used in separate reactions in triplicate. The amount of B.

inerea or grape DNA in each sample was calculated by using thetandard curves generated by the qPCR software version 6.0.19 (Cor-ett Research). The normalized results were obtained by dividinghe amount of B. cinerea DNA by the amount of grape DNA in valuesf �g per �g.

. Results and discussion

Standard curves of B. cinerea and grape DNA were generated withPCR to be used in determination of the amount of B. cinerea or

M. Celik et al. / Postharvest Biology and Technology 52 (2009) 235–239 237

Table 1Quantitative analysis by qPCR of B. cinerea in grapes at 20 ◦C and 0 ◦C after inoculation and qualitative analysis with antibodies in lateral flow device. The berries were onlywounded, or wounded and inoculated with 103 or 104 conidia of B. cinerea. Unwounded, non-inoculated berries are designated as ‘intact’. Pools of three berries were sampledimmediately after inoculation (0) or 1 and 4 d after inoculation at 20 ◦C and 1 and 4 weeks at 0 ◦C. In addition, mixed samples containing 9 symptomless and 1 infected berrywere sampled after 4 d at 20 ◦C or 4 weeks at 0 ◦C and are designated “9 + 1”. The DNA was assayed by qPCR and the amounts of B. cinerea and grape DNA are presented asB�g (�g Botrytis DNA) G�g−1 (�g grape DNA). Standard errors correspond to triplicates in the qPCR analysis. Positive or negative antibody assays are designated as P or N.

B�g G�g−1 Ab B�g G�g−1 (20 ◦C) Ab (20 ◦C) B�g G�g−1 (0 ◦C) Ab (0 ◦C)

0 d 1 d 1 week

Wounded 0.00 N 0.00 N 0.00 N103 0.45 ± 0.45 N 0.45 ± 0.03 N 4.24 ± 0.26 N104 4.23 ± 0.18 N 2.87 ± 0.98 N 36.77 ± 11.51 NIntact 0.00 N 0.00 N 0.05 ± 0.01 N

4 d 4 weeksWounded 0.14 ± 0.14 N 0.12 ± 0.04 N103 1540.54 ± 57.71 P 1268.36 ± 287.76 P1 4 5I“

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rape DNA in the unknown samples. The R2 values for the standardurves were 0.99 and 0.98 for B. cinerea and grape DNA, respectively.he M values, which represent the slopes of the curves and the effi-iency of the PCR reaction, were −3.26 and −3.50 for B. cinereand grape DNA, respectively. These values differ slightly from theheoretically ideal M value of −3.32, but during optimization ofhe reaction near-ideal curves were generated, which suggests thathese primer sets are suitable for quantitative analysis and thateviations from this ideal amplification rate are within the range ofhe experimental error. In the samples, the calculated amount of B.inerea DNA was normalized against grape DNA and expressed asicrograms of B. cinerea DNA per grape DNA.Unwounded, wounded and inoculated berries were sampled on

he day of inoculation and after 1 and 4 d at 20 ◦C. Results presentedn Table 1 show that B. cinerea DNA could be identified immediatelyfter inoculation in berries containing either 103 or 104 conidia,nd that the proportions of B. cinerea to grape DNA detected were.45 and 4.23 �g �g−1, respectively. One day after inoculation thereas no dramatic change in the amount of B. cinerea DNA in the

noculated berries. After 4 d at 20 ◦C there was a sharp increasen the amounts of B. cinerea DNA amounting to 1.54 × 103 and.07 × 103 �g �g−1 in samples inoculated with 103 or 104 conidia,espectively.

The berries were stored at 0 ◦C after 1 d of incubation at 20 ◦C andere sampled after 1 and 4 weeks of storage at 0 ◦C (Table 1). After 1eek at 0 ◦C, the concentrations of B. cinerea DNA in the wounded

nd intact berries were 0 and 0.05 �g �g−1, respectively. After 4eeks at 0 ◦C, the concentrations of B. cinerea DNA in these con-

rols were 0.12 and 0.14 �g �g−1, respectively. After 1 week at 0 ◦C,he concentrations of B. cinerea DNA in samples inoculated with03 or 104 conidia were 4.24 and 36.77 �g �g−1, respectively. Afterweeks at 0 ◦C the corresponding values in these treatments were

.26 × 103 and 0.99 × 103 �g �g−1. The presence of fungal DNA afterweeks at 0 ◦C in the control (137 �g �g−1) may be due to inocula-

ion after surface sterilization and development during storage at◦C. However, the amount in the control after 4 weeks is 5 orders

ower than in the inoculated berries.The feasibility of detecting B. cinerea DNA in grape samples

y qPCR was tested by combining nine symptomless berries withne inoculated grape berry. The amount of B. cinerea DNA in suchmixed sample taken from berries incubated at 20 ◦C for 4 d

as 41.88 �g �g−1. Another set of samples taken from inoculatederries that had been stored at 0 ◦C for 4 weeks contained an aver-ge B. cinerea DNA concentration of 137.27 �g �g−1 (Table 1).

PCR-based detection of B. cinerea in various biological samplesas reported for several systems (Rigotti et al., 2002; Gachon and

ateo2

P 994.61 ± 97.35 PN 0.14 ± 0.02 NN 137.28 ± 21.30 P

aindrenan, 2004; Brouwer et al., 2003; Mehli et al., 2005; Suarez etl., 2005; Chilvers et al., 2007). The present study does not offer newechnological advances compared to some of the previous work,ut rather focuses on the potential of monitoring infection of B.inerea in inoculated grape samples. It showed that B. cinerea can beetected on the day of inoculation in berry samples containing 1000onidia. The calculated amount of conidia that would be present inn assay of this sample would be one to two conidia. In addition,t showed that there is no significant increase in the amount ofNA during the first 24 h after inoculation, which is the expectedermination time at 20 ◦C. However by 2 d after inoculation thereas a tenfold increase in the amount of fungal DNA (Fig. 1a), and byd at 20 ◦C the amount of fungal DNA increased by more than threerders of magnitude (Table 1). Such a sharp increase in inoculumontent was also found by Mehli et al. (2005).

A qualitative immunoassay of the amount of B. cinerea antigensn the samples used for qPCR analysis was performed using lateralow devices. Unwounded, wounded and inoculated berries wereampled at different time points during incubation at 20 ◦C or stor-ge at 0 ◦C. The results show positive detection of B. cinerea by thentibodies in inoculated berries incubated at 20 ◦C at 4 d after inoc-lation (Table 1) and a strong positive signal after 4 weeks at 0 ◦C.ne of the inoculated samples was also detected as positive afterweeks at 0 ◦C (data not shown) while other times of sampling

ad negative results with this detection method. B. cinerea anti-ens could be detected in samples containing one inoculated berryith visible decay symptoms pooled together with nine symptom-

ree healthy berries, after 4 weeks of storage at 0 ◦C followingnoculation (Table 1). However, when a similar sample was pre-ared with berries after 4 d of incubation at 20 ◦C, the results wereegative.

Immunological detection of pathogens in biological samples ishe method of choice in many applications, because of its relativeimplicity, accuracy, and the possibility for high-throughput anal-ses. Antibodies and methodologies for B. cinerea were developedor diverse uses, including detection of B. cinerea contamination inrape juice-contamination that could interfere with wine makingDewey and Meyer, 2004). The use of antibodies for this application

akes good sense, because the detection level is below the thresh-ld of interference by the contaminant, and the decision is made athe end-point of decay. In contrast, for decision making prior to stor-

ge, it is unlikely that the sensitivity of antibodies will be sufficiento detect the extremely low fungal biomass within the berries. Forxample, B. cinerea antibodies were able to detect the effect of 1–3%f decayed berries, as manifested in their juice (Dewey and Meyer,004). In contrast, in this study the lateral flow device was able to

238 M. Celik et al. / Postharvest Biology and Technology 52 (2009) 235–239

Fig. 1. The kinetics of accumulation of Botrytis cinerea DNA in grape berries incubated at 20 ◦C or 0 ◦C. Berries were wounded and inoculated with conidia of B. cinerea. Poolso at 20 ◦

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f three berries were sampled immediately after inoculation (0) and after 1, 2 or 4 dhe amounts of B. cinerea and grape DNA were calculated from standard curves andith 103 or 104 conidia per berry.

dentify B. cinerea only in the late stages of infection. Therefore, thisethod was less sensitive than the qPCR assay.A logarithmic model describes the kinetics of accumulation of

. cinarea DNA in grapes held at 20 ◦C or at 0 ◦C, with correlationoefficients of 0.90 (Fig. 1a) and 0.96 (Fig. 1b), respectively. Theegree of difference between incubation at 20 ◦C and that at 0 ◦C is

ndicated by the difference between the exponent constants, whichere 2.23 and 0.25, respectively. An important aspect of the present

tudy concerns the kinetics of development of B. cinerea at lowemperature. The data show that at 0 ◦C the amount of fungal DNAccumulating after 4 weeks was equivalent to the amount detectedfter 4 d at 20 ◦C. However, when the kinetics of DNA accumula-ion were calculated from the slopes in Fig. 1, the ratio betweenhe constants of the exponent was approximately 9, which sug-ests that 1 d at 20 ◦C is equivalent to 9 d of development at 0 ◦C,nder our experimental conditions. However, this number is ques-ionable, because the model of DNA accumulation at 20 ◦C seemso be more complex than that presented here. This may be partlyue to the germination rate or percentage, and better resolution isequired to improve the model. There is also significant uncertaintyegarding what to use as a reference in such studies, especially withespect to degradation of DNA during infection. One approach is tose plant biomass as a reference parameter (e.g., Brouwer et al.,003) and the other is to use a plant gene as a reference (Gachonnd Saindrenan, 2004). The advantage of the latter approach, whichas used in the present study, is that it takes into account the dif-

erences in DNA extraction efficiency between samples, and alsohe issue of PCR inhibition, which may have significant implica-ions for quantification. Also, it was assumed that DNA degradationad similar effects on the pathogen and the host, which led to an

ntegrative view of the interaction (Gachon and Saindrenan, 2004).The molecular diagnostic tools for B. cinerea, as well as for other

athogens, could be significantly improved by optimization of var-ous parameters. One of the obvious problematic areas is sampleandling and DNA extraction which, in the present study, were notptimal in terms of throughput and yield, respectively. The mainandicap to future use of the quantitative diagnostic approach wille found in the sampling methodology.

In summary, in our hands antibodies in lateral flow devicesere not suitable for following infection in samples containing

ne infected berry and nine symptomless berries prepared for DNAxtraction. In contrast, qPCR was capable of efficiently following

isease progress from the inoculation stage to a late infection stage,t both ambient and at low temperature. It is predicted that in theuture qPCR technology can be incorporated into decision-supportystems that will specify which technologies should be used fortorage of grapes.

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C (A) or after 1, 2, 3 or 4 weeks of storage at 0 ◦C. The DNA was assayed by qPCR andesented as �g �g−1 B. cinerea to grape DNA. The results are averages of inoculation

cknowledgments

This paper is a contribution no. 505–08 from the Agriculturalesearch Organization, the Volcani Center, Bet Dagan, Israel. Thetudy was partly funded by research grant award no. IS-3947-06o A.L. from BARD, the United States–Israel Binational Agriculturalesearch and Development Fund.

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