Detection of Erwinia carotovora subsp ... - Wiley Online Library

Download PDF
0 downloads 0 Views 712KB Size Report
Comment
Phalaenopsis sp. Saintpautia .... lished data) and a metalloprotease of Serratia sp. (Nakahama et at, ... M. GC (%)". 1"=. ERWFOR. 5'-ACGCATGAAATCGGCCATGC-3'. 62°C. 55. 2'. ATROREV ..... tubers lo uitecied tubers ratiged Irc^ni 1 to .H^.
Ptant Pathotogy (]995) 44, 1058-1069

Detection of Erwinia carotovora subsp. atroseptica and Erwinia chrysanthemi in potato tubers using polymerase chain reaction E. J. SMID, A. H. J. JANSEN and L. G. M. GORRIS .Agrotechnotogicat Research Institute (ATO-DLO). Bornsesteeg 59. Wageningen NL-6700 AA, The Netherlands Soft rot erwiniae are a group of notorious plant pathogens for which currently available detection methods are inadequate. Based on the polymerase chain reaction, specific and sensitive detection of Erwinia carotovora subsp. atroseptica and E. chrysanthemi in potato tubers has been achieved. The composition of the PCR primers used in two specific detection systems is based on identification of the consensus of sequences of metalloprotease-coding genes present in soft rot erwiniae. Bacterial DNA was extracted from the potato tuber matrix by differential centrifugation in order to avoid interference of potato-derived compounds with the performance of the PCR assay. The PCR assay jjerformed with the E. carotovora subsp. atroseptica specific primer set was found to be capable of distinguishing £. carotovora subsp. atroseptica from all other Erwinia species and the closely related subspecies E. carotovora subsp. carotovora. With the E. chrysanthemi specific primer set, agarose gel electrophoresis is required for unequivocal differentiation between E. chrysanthemi and other erwiniae. Combined with the efficient extraction procedure, the assay allowed specific detection of less than lO' culturable erwiniae per tuber. The specificity and sensitivity of the assay were not reduced in the presence of a 100fold excess of DNA from both related and unrelated bacteria. This PCR-based method for detection of erwiniae in potato tubers provides a relatively fast and sensitive alternative to routinely applied serological methods.

INTRODUCTION Erwinia chrysanthemi, E. carotovora subsp. carotovora and E. carotovora subsp. atroseptica are members of the soft rot erwiniae group. The first two are associated with soft rot diseases on many important horticultural and agricultural crops, whereas E. carotovora subsp. atroseptica is largely restricted to potato. In contrast to the other soft rot erwiniae, E. carotovora subsp. atroseptica is capable of inducing disease symptoms at temperatures below 25 C (Perombelon & Kelman, 1980), and is considered to be the causal agent of potato blackleg, which occurs in cool temperate climates (Bang, 1989). In general, £. f/irv.sa/j/>ifm/' is pathogenic to many different plants in tropical and subtropical regions (Perombelon, 1992), but it is also found in association with potato tubers in cool climates (Janse & Ruissen, 1988). Potato blackleg is initiated by erwiniae from rotting mother tubers and is therefore regarded as a seedborne disease (Perombelon. 1W2). The baeieriii can spread from rotting mother tubers to progeny tubers. The subsequent proliferation

of E. carotovora can be accelerated under hypoxic or anaerobic conditions because of impaired resistance of the tuber to soft rot bacteria (Butler et al., 1990; Vayda et al., 1992) and increased expression of bacterial nitrate reductase activity (Smid et al., 1993). The latter observation also accounts for the increased sensitivity of potato tuber with a high nitrate content to E. carotovora subsp. atroseptica (Kumar et al., 1991; Prokkola, 1994). Measures to reduce the disease level of potato tubers include careful handling dtihng harvest and transit, growing less disease-susceptible cultivars, and the use of certified pathogen-free seed tubers. The level of tuber contamination is regarded as a measure of the blackleg potential of the seed, and reflects the health status of the seed tuber. Therefore, the practice of seed certification benefits from a simple, fast, sensitive and specific method to determine the level of E. carotovora and E. chrysanthemi infection of seed tubers. The mean number of E. carotovora subsp. atroseptica erwiniae per seed tuber necessar>- for the development of blackleg (the infection

Detection oferwiniae in potato tubers threshold value) was found to vary considerably for different cultivars and under different field conditions (Bain et at., 1990). Commonly, between 10^ and 10'' erwiniae per tuber elicit disease symptoms on tubers or plants (Bain et at., 1990). Different methods to assess the microbial contamination of potato tubers have been the subject of various studies. Classical methods based on selective media have proved to be useful tools for the identification and enumeration of soft rot erwiniae (Stewart, 1962; Cuppels & Kelman, 1974; Pierce & McCain, 1992), but are laborious and time consuming. Among the recent enumeration methods are the serological methods such as ELISA (Vernon-Shirley & Burns, 1992; Jones et at., 1993) and slide agglutination tests (McLeod & Perombelon, 1992). However, these methods are generally regarded as being too insensitive. In addition, the serological heterogeneity of soft rot erwiniae (De Boer et at., 1987; Gorris et at., 1994) has hampered the development of their practical application. Recently, DNA probes for detection of E. carotovora (subsp. carotovora and subsp. atroseptica) (Ward & De Boer, 1990) and E. carotovora subsp. atroseptica (Darrasse et al, 1994; Ward & De Boer, 1994) have been isolated. These probes were found to be useful for identification purposes. However, for detection in potato tuber tissue, a 48-h enrichment step is necessary (Ward &DeBoer, 1994), In the polymefase chain reaction (PCR), a targeted DNA fragment is amplified enzymatically. Thus a detection method based on PCR combines a high degree of specificity with a high degree of sensitivity. PCR technology has been successfully developed as a tool for specific and sensitive detection of micro-organisms in clinical samples (Hartskeer et al, 1989), environmental samples (Bej et al, 1991; Atlas et al, 1992), food or dairy samples (Harris & Griffiths, 1992; Niederhauser et al, 1992; Starbuck et al, 1992) and plant samples (Blakemore et at., 1992; Dong et al, 1992), In particular, food, milk and plant samples often contain components that significantly inhibit the polymerase chain reaction (Demeke & Adams, 1992; Rossen et al, 1992; Powell 6/a/., 1994). This paper describes a PCR assay for specific detection of E. carotovora subsp, atroseptica and E. chrysanthemi in potato tubers. In order to overcome the problem of inhibition of the polymerase chain reaction by potato tuberderived compounds, a simple and effective

1059

procedure for extraction of bacteria from tuber tissue has been applied. MATERIALS AND METHODS Bacterial strains and media All bacterial strains listed in Table 1 were routinely cultured in a medium containing 1-6% (w/v) tryptone. 1% (w/v) yeast extract and 0-5% NaCl at 25°C in shaker flasks. Cultures were maintained in the same medium supplemented with 15% (w/v) glycerol at - 7 5 C . A modified double-layer crystal violet pectate (CVP) medium was used for counting Erwinia carotovora in potato peel samples (Stewart, 1962; Cuppels & Kelman, 1974). The number of viable cells in pure cultures was determined by plating on 'Plate Count Agar" (Oxoid. Unipath Ltd. Basingstoke. UK) and CVP medium. Oligonucleotide primers Oligonucleotides were synthesized with a Gene Assembler Plus (Pharmacia-LKB. Uppsala. Sweden), and were released from the matrix of the DNA-synthesizer according to guidelines supplied by the manufacturer. The oligonucleotides were purified on a Sephadex G-20 column (Pharmacia. Uppsala. Sweden) and stored at -20 C in lOmin Tris-HCl. 1 mvi EDTA (pH 8-0). Isolation of bacterial DNA Overnight cultures of volume 1 ml were centrifuged for 5 min at 12 000 x g and resuspended in 1 ml of phosphate-buffered saline (PBS). pH 7-4, containing 0-5% (w/v) Tween-20 (PBS-Tween buffer). The cell suspensions were then boiled for 10 min in 1 ml of PBS-Tween buffer, and subsequently concentrated by centrifugation (5 min, 12 000 X g) and kept on ice. A total of 5 ^l\ of the crude extract was used for PCR. Potato tuber infection Overnight cultures of £. carotovora cells were washed twice in PBS. Potato tubers were incubated for 15 min at 20 C in cell suspensions with concentrations ranging from 10 to lO' viable bacteria/ml. The container in which the incubation was performed was placed on a shaker moving at a speed of 30 rot;itions/min.

E, J. Smid et al.

1060

Table 1 Strains used in this study Species

Strain

Collection

Origin

E. caroiovora subsp. atroseptica E. carotovora subsp. atroseptica E. carotovora subsp. atroseptica E. carotovora subsp. atroseptica E. carotovora subsp. atroseptica E. carotovora subsp. atroseplica E. carotovora subsp. carotovora E. carotovora subsp. carotovora E. carotoYora subsp. carotovora E. carotovora subsp. carotovora E. carotovora subsp. carotovora E carotovora subsp. carotovora E. chrysanihenii E. chrysanihenii E. chrysanthemi E. cypripedii E. herbicola (Panioea agglomerans) E. rhaponlici E. salicis E. siewarlii (Pantoea stewartii) Escherichia coli Laciococcus hciis subsp. lactis

30184 30185 30186 230 739 1025 30168 30169 30170 452 735 1024 4610 30178 30179 3873 4609 4484 30166 30176 K 12 ML3

DSM" DSM" DSM" PD" PD" PD" DSM" DSM" DSM' PD" PD" PD" DSM" DSM" DSM' DSM" DSM" DSM" DSM" DSM" ATO-DLO' RUG"

Potato stem Potato stem Potato tuber Potato tuber Potato tuber Potato tuber Potato (type strain) Brassica oleracea Potato tuber Potato Potato Potato Type strain Phalaenopsis sp. Saintpautia ionantha Type strain Type strain Type strain Type strain Type strain Unknown Unknown

•' German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany. "Culture Collection of The Plant Protection Service, Wageningen, The Netherlands. 'Laboratory collection, Agrotechnological Research Institute (ATO-DLO), Wageningen, The Netherlands. ** Laboratory collection. Department of Microbiology, University of Groningen, Groningen, The Netherlands.

After incubation, the tubers were dried for 30 min in the air and were then stored in closed plastic containers (dimensions 45 30 • 16 cm) for 4 to 7 days at 20 C. Recovery of bacterial DNA from potato tubers Using a sterile knife, 1-lOg of potato peel was collected and transferred to a sterile Stomacher 400 bag (Colworth Stomacher, A.J. Seward & Co. Ltd, London, UK) containing 25 ml of sterile PBS. The mixture was homogenized in a blender (Stomacher) for 2 min. The supernatant from the peel L-xtract was transferred to a sterile tube and centrifuged for 5 min at 70 x g, and the supernatant was then subjected to high-speed centrifugation for 20 min at 1000 \g. Finally, the pellet was resuspended in 1 ml of PBS supplemented with 0 5% Tween-20, and DNA was isolated as described above. Again, 5/il of the crude preparation were used in the PCR assay.

PCR assay PCR was performed in a total volume of 50/J in 0 5-ml GeneAmp reaction tubes (PerkinElmer Cetus Corp., Norwalk, CT, USA) under a layer of paraffin oil. The mixture contained lOmM Tris-HCl, pH 8 3, 1 5mM MgCh, 50mM KCl, lOO^g/ml gelatine, 200^M of each of the deoxynucleoside triphosphates, 20/IM of each oligonucleotide primer, 0-5% Tween-20, and 1 25U of Taq DNA polymerase (Perkin-Ehner Cetus Corp., Norwalk, CT, USA). When sensitive detection of the amplified DNA fragments was required, 2 ^ digoxigenin-lldUTP (Boehringer, Mannheim, Germany) was added to the PCR-assay mixture. Template DNA was denatured at 94°C for 4min before it was added to the PCR mixture. The PCR was run on a DNA thermal cycler (PerkinElmer Cetus Corp.) for 30 cycles at 9 4 X for I min, 55 C for I min, and 72°C for 2 min. The PCR was terminated with one cycle at

Detection oferwiniae in potato tubers for lmin, 55°C for lOmin.

1 min, and 72°C for

DNA electrophoresis Amplified DNA fragments were run on a 1 % (w/ v) agarose gel. Electrophoresis was performed under standard conditions in a 40 mM Trisacetate buffer (pH 7-8-8 0) supplemented with 2mM EDTA and 0-2 mg/1 ethidium bromide. Samples were loaded onto the gel in a buffer containing 0-05% (w/v) brome-phenol blue, 0-05% (w/v) xylene cyanol and 3% (w/v) Ficoll, type 400. Detection of Erwinia carotovora DNA in potato peel extracts Bacteria were recovered from the peel extract and lysed as described above. After amplification, the digoxigenin-labelled DNA was transferred directly to a positively charged nylon membrane (Boehringer, Mannheim, Germany) by pressure-slot blotting, or it was separated by agarose gel electrophoresis first, and subsequently transferred to a membrane by Southernpressure blotting. The relative intensity of the dots or bands on the nylon membrane was analysed and quantified by reading grey values from video images captured with a high-sensitivity CCD monochrome camera (Vilbert Lourmat, Marne La Vallee, France) coupled to a computer running a video-image analysis program (BIO PROFIL version 4-6, Vilbert Lourmat, Marne La Vallee, France). The values were transferred to a scale of 0-100 units. The signal obtained with the highest number of bacteria (10^ per reaction tube) was set at 100 units, and a negative control containing no target DNA was set at 0 units.

1061

soaked in 2 x SSC (saline sodium citrate buffer; l5mM Na-citrate, pH 7-0, supplemented with 0-15 M NaCl) for 1 min. After the excess bufTer had been removed with tissue, cross-linking of DNA to the membrane was carried out using a UV Stratalinker^'^ 1800 (Stratagene Cloning Systems, La Jolla, CA, USA). Slot-blotting of the digoxigenin-l 1-dUTPlabelled DNA was performed with a vacuum slot-blot system (The Convertible^^' Filtration Manifold System, Gibco BRL, Life Technologies Inc. Gaithersburgh, USA). Aliquots of 25^1 of PCR product were diluted in an equal volume of denaturation buffer (1 M NaOH and 3 M NaCl). A total volume of 180/il of TE buffer (10 mM Tris-HCI, pH 8-0, and 1 mM EDTA) was then added to the denatured DNA sample. A positively charged nylon membrane was soaked in TE buffer and mounted in the slot-blot apparatus. After the slots had been rinsed with TE buffer, 0-2 ml ofthe DNA sample was transferred into the slots, a vacuum was applied and the slots were washed again with 0-2 ml of TE buffer. Subsequently, the membrane was incubated for 1 min in a 2 X SSC solution. After cross-linking DNA to the membrane, the membrane was washed twice in 2 x SSC supplemented with 0-1% (w/v) sodium dodecyl sulphate (SDS) and twice in 0-1 < SSC supplemented with 0-1 % SDS (w/v). Digoxigenin-ll-dUTP-labelled DNA was detected using the DIG System (Boehringer, Mannheim, Germany) with anti-digoxigenin antibodies conjugated with alkaline phosphatase, 4-nitroblue tetrazolium chloride and 5bromo-4-chloro-3-indolyl-phosphate. RESULTS Development of specific PCR primers

Southern-pressure hlotting and vacuum-slot hlotting Following electrophoresis, gels were incubated twice for 10 min in a solution of 0-5 M NaOH and 1-5M NaCI. The gels were then transferred to a 1M ammonium acetate buffer and incubated twice for 10 min. Subsequently, the neutralized gel was mounted in a Stratagene Posiblot^'*^ pressure blotter (Stratagene Cloning Systems, La Jolla, CA, USA). The DNA was transferred to a positively charged nylon membrane (Boehringer, Mannheim, Germany) within I h, at 75 mmHg pressure. Subsequently, the membrane was

Based on sequence comparison of a gene coding for a metalloprotease (proB) of Erwinia chrysanthemi B374 (Delepelaire & Wandersman, 1989), a metalloprotease (prtC) of £. chrysanthemi EC 16 (Dahler ei at,, 1990), a metalloprotease of Erwinia carotovora subsp. atroseptica DSM 30186 (E.J. Smid and C.J Tuijn, unpublished data) and a metalloprotease of Serratia sp. (Nakahama et at,, 1986), a PCR-primer tentatively termed 'ERWFOR' annealing to all four metulloprotease genes was designed. In addition, two reverse PCR-primers termed ATROREV and CHRREV were designed specifically for detection of £. carotovora subsp. atroseptica and

1062

E. J. Smid tt al. Table 2 Sequences and melting temperatures of PCR primers Sequence (5' - 3')

Primer 1"=. ERWFOR 2'. ATROREV 3^CHRREV

5'-ACGCATGAAATCGGCCATGC-3' 5'-ATCGATAATTTGATTGTCCT-3' 5'-AGTGCTGCCGTACAGCACGT-3'

• M

GC (%)"

62°C 52''C 64°C

55 30 60

' Melting temperature was calculated according to the method of Itakura et al. (1984), ''Percentage of guanine and cytosine present in primer, M -t- 2, Eca-primer set; 1 + 3, Ech-primer set; 1 -t- 2 -t- 3, triple PCR set. E. chrysanthemi, respectively (Table 2). The sequence of these primers was taken from a region of low similarity between the metalloprotease genes from E. chrysanthemi B374 (Delepelaire & Wandersman, 1989) and E. carotovora subsp, atroseptica DSM 30186 (E.J, Smid and CJ. Tuijn, unpublished data)

In a standard PCR protocol performed with primers ERWFOR and ATROREV and using purified chromosomal DNA of E. carotovora subsp, atroseptica DSM 30186 as a template, only one band of 389 bp was obtained. Combination of the primers ERWFOR and CHRREV, using E. chrysanthemi DSM 30178 DNA as a

Table 3 Polymerase chain reaction for 20 different Erwinia strains and two non-Erwinia strains with different primer sets" Oligonucleotide primer set

Species

Strain

E. carotovora subsp. atroseptica £. carotovora subsp. atroseptica E. carotovora subsp. atroseptica £. carotovora subsp. atroseptica £. carotovora subsp. atroseplica £. carotovora subsp. atroseptica £. carorovora subsp. carorovora £. carotovora subsp. carotovora £. carotovora subsp. carolovora £. carotovora subsp. carotovora £. carolovora subsp. carotovora £. carotovora subsp. carotovora £ chrysanthemi £. chrysanthemi £. chrysanthemi £. cypripedii £. herbicota £. rhapontici £. 5a/icu £. stewartii Escherichia coti Lactococcus tactis subsp, /actu

DSM 30184 DSM 30185 DSM 30186 PD 230 PD 739 PD 1025 DSM 30168 DSM 30169 DSM 30170 PD 452 PD 735 PD 1024 DSM 4610 DSM 30178 DSM 30179 DSM 3873 DSM 4609 DSM 4484 DSM 30166 DSM 30176 KI2 ML3

Eca-specific (389 bp)

Ech-specific (450 bp)

Combination (389/450 bp) -I-I-I-

X X

+ +

nd* nd

nd nd

' Crude extracts of pure cultures of the bacterial strains were used in the PCR assay. Tlie assay was performed under standard PCR conditions and the results were analysed by agaroaegd - I

Lea and I cl l ) \

. /» •.,iiiili,nii

ir.iin /

,nii|T|ilK.i

D S M I

. ,iiii


D S M ?01fiX on the with the Eca

s i \ li

that

P( R-ci)mhina(i

h.ieieii.i

w e t e .iit i h e k i l l v

,,;;,,/,.,,./,;

u i ' i n t f ' l It ,1 111 .1 s e n i i - L | n . i n 1 1 1 . 1 1 1 \ e n i . i n n e i

Ktfects of forcicn DN V on l'( R The

eltects n i | ) N\

elosel\

lel.iied

a m i

I III \ s i i n i l i i III! o n l ' ( R . i n i p h l i e . i l i o n

I tiiiiltiviiiii s u b s p primer

,,(/.I/-M IM,, s u b s p

isubi-

s u c h

/

. i s /

I r o m

s p e c i e s

. ..-i , r ; , M , i ; , ;

, i i n ' \ i I ' l l , ,: I > \ \ w i t h

setw,is e \ . m m i e d

i ndel.nl

ol t h e,^S')-hp li.iijnieni l i o n i / i l l i i n c p n i ll W A S n o l .illeiled

o l/

t h e 1 e . i

\niplilk.ition

>,;/r^;, ii, i,,; s u b s p b \

| ) \\

lioiii

E. J. Stuict e{ al.

3

4

5

6

8

9

10

389 b -

Fig. 4 D e l e d I on o l i rile tubers ic\

lur.ih'unu

s u h s p uirascpliiu

in s a m p l e s c o m p o s e d o f i n f e c t e d a n d n o n - i n l e c t e d p o t a t o t u b e r s ,

B i n t i e I w e r e i n f e e t e d « i l h A" i uiciiivni,!

s u h s p u in m pin a D S M 3(11.S6 P C R u a s p e r t o r m e d u i t h t h e

l e a p r i m e r set a n d t h e r e s u l t s \ s e i e a n a l y s e d b \ S o u l h e r n - p r e s s u r e h l u t t i n g infected tubers to non-infeeled uihers « e r e a n a l \ s e d 111. I 2 ( l i l . i n e ^ ) . I .'II ( l a n e S ) . II I ( l a n e ' - ' I iiiiiiscpiuu

L a n e I r e p r e s e n t s t h e positive ci>ntrol ( p u n l i e d f

i l i r \ \ ( i i l l l i c i i i i o r //

(iiioiinnici subsp

, iirnlinoiLi

DNA (l-ig : ) In order to assess the ellects .if noti-target DNA on the etliciencv o\ aniplitication o\ E : Liinrinniii

subsp

iiim^cpiuii

D N A 111 t h e P C R

Detection in mixed samples

assav based on the hca primer set. PC R was perlornied m the presence o\ DN X from unrelated bacteria originating from the same habitat Crude DN \ preparations were made ol strain

ol Ciir\>ul\H h n u n i

l\ciuli'iiuiihi\

ihi\

spp

and

s p p. l o u r

two

Hiiiillif-

The sensitiMtv of the PC R assa\ with the Eca primer set was investigated bv mixing E air,'hum,I subsp c///(nc/'Hi w-intcVted potato tubers with dillerent numbers ol nc^n-inlected tubers I or ditVerent lots, consisting of inteeted and non-mlected tubers, mixed samples vxere prepared in which the ratio of non-intected tubers lo uitecied tubers ratiged Irc^ni 1 to .H^ (lur results show that a satnple ot.^d tubers. onl>

strains

s p p . t w ostrains ol

were isiilated t r o m

XLIIIIIU'IIIO-

s t r a i n s , all o l

t h e s t u l a c e ol p o t . i t o

which tubers

W h e n a d d e d \\\ a d .•< • I d - f v i k i e x c e s s t o a PC R with

Ica

primers

and

/;

1

2

ti'iiiiiie aiialvsis o l / , w a s siihieiUd 1, 111 r

^ub^p

ciiin\cpiuii target DN.A. a preparation ot" DNA from (.'iirvnthiuicnuDi spp. had no etTeel on the ethciencv ol aniplitication (lane 4. Fig ?•). Onh in the presence ol a 6 3 lll'-fold excess of Ci'r\ lU'hiu ifniiiii D N A was a significant inhibition of the PCR obser\ed (lane .>. Fig. ?). Comparable results were obtained with DNA from the other isolates from the tuber surtace

fold excess ( F i g 21 .\s e x p e c t e d , n o a m p h h c a tion w a s o b s e r v e d in the a b s e n c e ol E nirnim :'iu stibsp iiirincpihii DN.A but Itl the p r e s e n c e of £

of

laroniuirn

D N A l a n d l a n e ID r e p r e s e n t s i h e n e g a t i v e c o n l r o l ( n o t a r g e t D N A a d d e d I

A chr]Miiuhcmi DN.A or E iciniiavcru subsp iiiiiiunoid D N . \ . e\en when present in a HHi-

one

S a m p l e s \Mth t h e tollovMng raticts of

I u i U i n e Z ) . 1 l l l a n e ? ) . 1 ? l l a n e 4 ) . 1 5 l l a n e 5). 1 10 i l j n e

t iii numn

\i

subsp

3

,,//, W.M . u . i s u b s p

lo pussiiu

sloi

4

doi 1 S

l(i\ells

d o i4 S

6

7

,;/;, •!,/'-;, , i b v I ' l R \v i ( h t h e K . i p i u i i e i s e t A m p h t i e \ l O N A m t h e

bl.'iiiuL' . u u l MilisequenI

11 I I n l . . | | . . « i n i ' . i b s . i l u i e n i i m l i e i s o l v i . i b l e c e l l s iclls

5

Id'lells

detection

usuig

the digoxiseiiui

svstcm

lXit>

d e U i m m e i l b v p i . i t m e o n P i " A d o l I . S > t(l'~ c t l K . d o l ^

d.'i s s . l(l\ells. dot h. S • Id'eclK. .iiiddot

'. S •

ItV'eflb

Detection of erwiniae in potato tubers one of which is infected with approximately lO** erwiniae/tuber, generates a positive signal in the PCR assay based on the Eca primer set (Fig. 4). Rapid analysis by pressure-slot blotting

For practical application of the PCR assay, the procedure should be as simple as possible. Since the PCR assay based on the Eca primer set produces a single DNA band, separation of the amplified DNA by gel electrophoresis can be omitted. Therefore the sample containing the amplified digoxigenin-labelled target DNA was directly spotted onto a nylon membrane mounted in a pressure slot-blot apparatus. After blotting, the labelled DNA was visualized using anti-digoxigenin antibodies conjugated with alkaline phosphatase, 4-nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolylphosphate. Figure 5 shows that this procedure allows the specific detection of 100 to 1000 bacteria in a potato tuber sample. DISCUSSION This study describes the development of a PCR assay for the specific detection of E. carotovora subsp. atroseptica and E. chrysanthemi in potato tubers. A major obstacle to the application of PCR using crude plant extracts and more specifically potato tuber extracts is the presence of plant-derived compounds which inhibit amplification of DNA in the assay (Demeke & Adams, 1992; Rossen et at., 1992). Therefore we applied a simple procedure to allow the more specific extraction of bacterial DNA from the potato tuber matrix. The procedure includes low-sjjeed centrifugation of the potato extract, high-speed centrifugation of the low-speed supernatant, and subsequent lysis of the extracted bacteria. In this way a DNA preparation is obtained which is free from plant-derived compounds that would interfere with the assay. Our results show that enzymatic amplification of bacterial DNA extracted from potato peel samples via the twostep procedure is not suppressed by any plantderived factor. Therefore, time-consuming purification procedures can be omitted. Since inhibitory compounds from rotting tuber tissue also reduce the affinity of antibodies for their targets (McLeod & Perombelon, 1992), a similar extraction procedure is applied in serological tests for bacterial ring rot of potato (De Boer eta/., 1989). The PCR assay described in this paper is based

1067

on a combination of an oligonucleotide primer with a specific sequence and a primer that anneals to homologous DNA sequences from different related species. This approach allows the highly specific detection of E. carotovora subsp. atroseptica and E. ctirysanthemi. By combining the two specific primers with the Erninia-spedfic primer (ERWFOR), simultaneous detection of both species can be achieved. However, the appearance of faint bands with non-target DNA from two of the tested E, carotovora subsp. carotovora strains demonstrates that the specificity of this shared-primer PCR assay is less stringent than the PCR performed with the Eca or Ech primer sets. The successful use of shared-primer PCR for simultaneous detection of the closely related species Bordetella pertussis and B, parapertussis (Li et al, 1994), shows that this technique can be further optimized and has potential for practical application. The PCR assay with the Eca primer set yields the amplification of a single DNA band in all cases tested. The amplification of this specific band is not hindered by DNA from related or unrelated bacteria. Hence, for routine application, the time-consuming analysis of the amphfied DNA by agarose gel electrophoresis and Southern-pressure blotting can be omitted, and PCR samples can be directly transferred to a nylon membrane and visualized by a procedure similar to ELISA (Fig. 5). The actual number of erwiniae required for induction of disease symptoms on potato tubers determines the minimum required sensitivity of the detection method. Different studies have demonstrated that between 10" and 10'' erwiniae/tuber elicit disease symptoms on tubers or plants (Bain et at., 1990). The detection limit of ELISA in potato peel extracts is approximately 10' cells/ml (van der Wolf & Gussenhoven. 1992; Gorris et at,, 1994). Thus the sensitivity of ELISA performed without additional enrichment is too low for accurate evaluation of the health status of seed tubers. Furthermore, when digoxigenin-labelled DNA probes are applied for detection, an additional 48-h enrichment step has to be included (Ward & De Boer. 1994). Our results show that the sensitivity of PCR performed with a specific primer set and in combination with the two-step extraction procedure is sufficient for the detection of less than lO' target bacteria per seed tuber. Consequently, no time-consuming enrichment procedures with selective or general growth medui are needed. A

1068

E. J. Smid et al.

definite advantage of the high sensitivity of the PCR assay is that a single infected tuber can be detected in a sample of 30 tubers. Thus a smaller number of assays is required for accurate analysis of a given batch of seed tubers. A detection method based on PCR always provides semi-quantitative information about the degree of infection of tubers. This means that the actual number of bacteria present in a sample cannot be directly determined by this assay. In fact, the number of specific target sequences in the sample correlates with the number of specifically amplified DNA fragments present in the sample after PCR. Our results show that this correlation can be used if between 10 and 10^ amplifiable targets are present in the sample (Fig, 1). Detection of E. carotovora subsp. atroseptica in potato peel extracts by means of the PCR assay is calibrated on the basis of viable counts in pure cultures. The validity of this calibration procedure is confirmed by the observation that a given number of viable E. carotovora subsp. atroseptica cells extracted from tuber tissue generate a signal in the assay which matches the signal obtained with pure cultures (Fig. 1). This indicates that the contribution of inactive or dead bacteria to the signal obtained in potato peel extracts and pure cultures is approximately the same in both cases. This paper shows that PCR based on the Eca primer set allows specific, sensitive and rapid indexing of potato tubers for the important plant pathogenic bacterium E. carotovora subsp. atroseptica. Stable performance of the PCR assay is achieved by using a simple and practical procedure for the extraction of bacterial DNA from the tuber matrix. The implementation of pressure-slot blotting allows rapid detection of the digoxigenin-labelled amplified DNA fragments. ACKNOWLEDGEMENTS We thank J. Springer, E.J.H. Wolbert and G.J.A. Rouwendal for valuable suggestions throughout this work. REFERENCES Atlas RM, Sayler G, Burlagc RS, Bej AK, 1992. Molecular approaches for environmental monitoring of microorganisms. Biottittniques 12. 706. Bain RA, Perombelon MCM, 1 sror L, Nachmias A, 1990. Blackleg development and tuber yield in relation to numbers of Erwiniu carolovora subsp

atroseptica on seed potatoes. Ptant Pathotogy 39, 125-33. Bang HO, 1989, Prevalence of Erwinia species in basic seed lots of potatoes grown in northern Sweden. Acta Agricutturae Scandinavica 39, 373-9. Bej AK, Mahbubani MH, Dicesare JL, Atlas RM, 1991. Polymerase chain reaction-gene probe detection of microorganisms by using filter-concentrated samples. Apptied and Environmentat Microbiotogy 57, 3529-34. Blakemore EJA, Reeves JC, Ball SFL, 1992. Polymerase chain reaction used in the development of a DNA probe to identify Erwinia stewartii, a bacterial pathogen of maize. Seed Science and Technology 20, 331-5. Butler W, Cook L, Vayda ME, 1990. Hypoxic stress inhibits multiple aspects of the potato tuber wound response. Ptant Physiotogy 93, 264-70. Cuppels D, Kelman A, 1974. Evaluation of selective media for isolation of soft-rot bacteria from soil and plant tissue. Phytopathotogy 64, 468-75. Dahler GS, Barras F, Keen NT, 1990. Cloning of genes encoding extracellular metalloproteases from Erwinia chrvsanlhemi EC16. Journat of Bacleriotogy 172, 5803-15. Darrasse A, Kotoujansky A, Bertheau Y, 1994. Isolation by genomic subtraction of DNA probes specific for Erwinia carotovora subsp. atroseptica. Apptied and Environmenlat Microbiotogy 60, 298306. De Boer SH, Verdonck L, Vruggink H, Harju P, Bang HO, de Ley J, 1987. Serological and biochemical variation among potato strains of Erwinia carotovora subsp. atroseptica and their taxonomic relationship to other £. carotovora strains. Journal of Apptied Bacteriotogy 63, 487-95. De Boer SH, De Haan T-L, Mawhinney J, 1989, Predictive value of post-harvest serological tests for bacterial ring rot of potato. Canadian Joumai of Ptant Pathotogy II, 317-21. Delepelaire P, Wandersman C, 1989. Protease secretion by Erwinia chrysanthemi. Proteases B and C are synthesized and secreted as zymogens without a signal peptide. Journal of Biological Chemistrv 264, 9083-9. Demeke T, Adams RP, 1992. The effects of plant polysaccharides and buffer additives on PCR. Biotechniques 12, 332-4 Dong LC, Sun CW, Thies KL, Luthe DS, Graves CH, 1992. Use of polymerase cham reaction to detect pathogenic strains of .Agrobacterium. Phytopathotogy %Z, 4i4-9. Gorris MT, Alarcon B. Lopez MM, Cambra M, l^M Characterization of monoclonal antibodies specific for Erwinia carotovora subsp. atroseptica and comparison of serological nMthods for its sensitive detection on potato tubers. Apptied and Envinmmcntat Murobiohgv 60, 2076-85, Harris U , Grittiths MW, 1992 The detection of foodbome pathogens by the polymerase chain

Detection oferwiniae in potato tubers reaction (PCR). Food Research International 25, 457-69. Hartskeer RA, De Vit MYL, KJatser PR, 1989. Polymerase chain reaction for the detection of Mycobacterium leprae. Journal of General Microbiology 135, 2357-64. Itakura K, Rossi JJ, Wallace RB, 1984. Synthesis and use of synthetic oligonucleotides. Annual Review of Biochemistry 53, 323. Janse JD, Ruissen MA, 1988. Characterization and classification of Erwinia chrysanthemi strains from several hosts in The Netherlands. Phytopathology 78, 800-8. Jones DAC, McLeod A, Hyman LJ, Perombelon MCM, 1993. Specificity of an antiserum against Erwinia carotovora subsp. atroseptica in indirect ELISA. Journal of Applied Bacteriology 74, 620-4. Kumar A, Pundhir VS, Gupta KC, 1991. The role of phenols in potato tuber resistance against soft rot by Erwinia carotovora ssp. carotovora. Potato Research 34,9-16. Li ZM, Jansen DL, Finn TM, Halperin SA, Kasina A, O'Connor SP, Aoyama T, Manclark CR, Brennan MJ, 1994. Identification of Bordetella pertussis infection by shared-primer PCR. Journal of Clinical Microbiology 32, 783-9. McLeod A, Perombelon MCM, 1992. Rapid detection and identification of Erwinia carotovora subsp. airoseptica by a conjugated Staphylococcus aureus slide agglutination test. Journal of Applied Bacteriology 72, 274-80. Nakahama K, Yoshimura K, Marumoto R, Kikuchi M, Lee IS, Hase T, Matsubara H, 1986. Cloning and sequencing of Serratia protease gene. Nucleic Acid Research 14, 5843-55. Niederhauser C, Candrian U, Hofelein C, Jermini M. Buhler HP, Luthy J, 1992. Use of polymerase chain reaction for detection of Listeria monocytogenes in food. Applied and Environmental Microbiology 58, 1564-8. Perombelon MCM, 1992. Potato blackleg: epidemiology, host-pathogen interaction and control. Netherlands Journal of Plant Pathology 98 (Suppl. 2), 135-46. Perombelon MCM, Kelman A, 1980. Ecology of the soft rot envinias. Annual Review of Phytopathology 18,361-87.

1069

Pierce L, McCain AH, 1992. Selective medium for isolation of pectolytic Erwinia sp. Plant Disease 76, 382-4. Powell HA, Gooding CM, Garrett SD. Lund BM, McKee RA, 1994. Proteinase inhibition of the detection of Liiteria monocytogenes in milk using the polymerase chain reaction. Letters of Applied Microbiology 18, 59-61. Prokkola S, 1994. Effect of applying nitrogen fertilizer to a potato seed crop on the susceptibility of the daughter plants to Erwinia carotovora subsp. airoseptica. Potato Research 37, 103-11. Rossen L, Norskov P. Holmstrom K. Rasmussen OF, 1992. Inhibition of PCR by components of food samples, microbial diagnostic assays and DNAextraction solutions. Internaiional Journal of Food Microbiology 17, 37—15. Smid EJ, Jansen AHJ. Tuijn CJ. 1993. Anaerobic nitrate respiration by Erwinia carotovora subsp. atroseptica during potato tuber invasion. Applied and Environmental Microbiology 59, 3648-53. Starbuck MAB, Hill PJ. Stewart GSAB, 1992. Ultrasensitive detection of Listeria monocytogenes in milk by the polymerase chain reaction (PCR). Letters of Applied Microbiology 15, 248 52. Stewart DJ, 1962. A selective-diagnostic medium for the isolation of pectinolytic organisms in the Enterobacteriaceae. Suiiirc 195, 1023. Van der Wolf JM. Gussenhoven GC, 1992. Reaction of saprophytic bacteria from potato peel extracts and plant pathogenic bacteria in ELISA with antisera to Erwinia chrysanthemi (serogroup OlHa). Netherlands Journal of Plant Pathology 98, 33- 44 Vayda ME. Antonov LS, Yang Z, Butler WO, Lacy GH. 1992. Hypoxic stress inhibits aerobic wound-induced resistance and activates hypoxic resistance to bacterial soft rot. American Potato Journal 69, 239 53. Vernon-Shirley M, Burns R, 1992. The development and use of monoclonal antibodies for detection of Erwinia. Journal of Applied Bacteriology 72, 97-102. Ward LJ. De Boer SH, 1990. A DNA probe specific for serologically diverse strains of Erwinia carotovora. Phytopathology 80, 665-9. Ward LJ, De Boer SH, 1994. Specific detection of Erwinia caroiovora s u b s p . atroseplica with a d i g o x igenin-labeled DNA probe. Phytopathology 84, 18086.