Bacteriology e -Xtra*

A New Selective Medium for Isolation of Clavibacter michiganensis

subsp. michiganensis from Tomato Plants and Seed
Radwan M. Ftayeh, Andreas von Tiedemann, and Klaus W. E. Rudolph

Division of Plant Pathology and Crop Protection, Department of Crop Sciences, University of Göttingen, Germany.
Accepted for publication 22 June 2011.

ABSTRACT

Ftayeh, R. M., von Tiedemann, A., and Rudolph, K. W. E. 2011. A new tent of 98 to 100%; and (iii) the remarkable detection sensitivity. Thus,
selective medium for isolation of Clavibacter michiganensis subsp. 8 CFU of Cmm in field plant homogenates containing 12,750 CFU of
michiganensis from tomato plants and seed. Phytopathology 101:1355- accompanying saprophytes were detected on BCT. Under these extreme
1364. conditions, all of the published semiselective media (D2, KBT, D2ANX,
SCM, mSCM, CMM1, mCNS, and EPPO) gave false-negative results.
A new selective and highly sensitive medium was developed for Either some media were rather toxic and Cmm growth was also inhibited
isolation of Clavibacter michiganensis subsp. michiganensis (Cmm), the or the other, less toxic media allowed growth of high numbers of sapro-
causal agent of bacterial canker of tomato, from seed and latently infected phytes, so that Cmm growth was suppressed. Exclusively, BCT also
plants. The new medium (BCT) proved to be superior to all published supported growth of the closely related C. michiganensis subsp. insidiosus,
semiselective media for Cmm and is denoted as selective medium because nebraskensis, and tessellarius. The new medium is recommended for
of (i) its mean plating efficiency, amounting to ≤89% within 7 days for all Cmm detection in tomato seed, and in symptomless tomato plantlets, to
30 Cmm strains from different sources tested; (ii) the high selectivity, improve disease control of bacterial canker of tomato.
because accompanying bacterial species occurring on tomato plants and
seed or bacteria obtained from culture collections were inhibited to an ex- Additional keywords: Solanum lycopersicum.

Clavibacter michiganensis subsp. michiganensis (Cmm) (Smith) medium for routine seed testing and for a reliable isolation and
Davis et al., the causal organism of bacterial canker of tomato detection of Cmm in infested seed and symptomless plants.
(Solanum lycopersicum), has been classified as an A2 quarantine The strategy was, first, to develop an optimum basal medium
organism by the European Plant Protection Organization (EPPO) for Cmm, followed by screening a large group of various anti-
(27). Bacterial canker is a serious emerging disease of tomato biotics and inhibitors, and, finally, testing selected antibiotics and
worldwide wherever tomato plants are grown, and new outbreaks inhibitors in manifold combinations in order to develop a new
of the disease have been reported in several countries recently medium that supports growth of various Cmm strains from differ-
(4,14,15). ent origins but suppresses a wide range of nontarget bacterial
Infected or contaminated seed and transplants are responsible species and strains, such as other phytopathogenic bacterial
for disease transmission into new areas (5,30,32), whereas trans- species affecting tomato plants, and numerous epiphytic or sapro-
mission by soil appears to be of minor importance (12,30). As few phytic bacterial strains occurring on tomato plants and seed.
as 0.01 to 0.05% contaminated seed or transplants can cause an The finally developed new selective medium BCT (bacterial
epidemic in suitable conditions (5). Therefore, indexing of tomato canker of tomato) also showed a remarkable specificity for other
seed lots for Cmm is the key for disease control (3). However, subspecies of C. michiganensis. The underlying mechanisms
some of the recent outbreaks occurred although infected plants could not be explained completely. However, a further elucidation
were raised from tomato seed and transplants that had been of the biochemical interactions involved may open a new avenue
certified as pathogen free when using some of the available for better understanding the host–parasite relationship of these
semiselective media. The most often used detection assay for pathogens and their control.
Cmm is based on dilution plating on semiselective media. Our
study, started in mid-2006, revealed that the main weakness MATERIALS AND METHODS
regarding the EPPO (27) and International Seed Health Initiative
(ISHI) (22) protocols most likely was due to the recommendation Bacterial strains and growth conditions. In all, 72 bacterial
of semiselective media that failed to detect low numbers of Cmm strains and species (Table 1) were used, including 30 Cmm strains
in infected tomato plants and seed, so that false-negative results from different origins and 42 “accompanying” bacterial strains
were obtained in our experiments even when the plant and seed which may occur on tomato plants and seed. These epiphytic or
extracts had been spiked with Cmm. Therefore, the aim of the saproyhytic strains and the phytopathogenic strains were isolated
present work was to develop a new selective and highly sensitive from tomato plants and seed or obtained from bacterial culture
collections. Several saprophytic bacterial strains were antagonists
of Cmm in vitro and taxonomically characterized by their whole
Corresponding author: R. M. Ftayeh; E-mail address: rftayeh@gwdg.de cell fatty acid methyl esters (Table 2).
Working bacterial strains were cultured and stored on nutrient
* The e-Xtra logo stands for “electronic extra” and indicates that Figures 5, 6, and 7 glucose yeast extract (NGY) medium at 4°C while stock cultures
appear in color online.
were maintained in 20% glycerol at –80°C. The NGY medium
(14) contained 0.8% nutrient broth (Roth, Karlsruhe, Germany),
doi:10.1094 / PHYTO-02-11-0045
© 2011 The American Phytopathological Society 1% glucose, and 0.3% yeast extract (Roth); the pH value was

Vol. 101, No. 11, 2011 1355

adjusted to 7.2 with HCl/NaOH. Pseudomonads were also culti- Development of the basal medium for Cmm. Nine semi-
vated on King’s medium B (KB) (25). For long-term preservation, selective media without addition of antibiotics or other inhibitors
strains were lyophilized and deposited in the Göttingen Collection were tested for growth of Cmm in comparison with growth on the
(Sammlung) of Phytopathogenic Bacteria (GSPB). NGY medium. The nine media were D2 (23); KBT (10); mCNS,
Standardized bacterial suspensions were prepared from 24-h- which was prepared as suggested by Gitaitis et al. (17), based on
old NGY cultures in 0.01M MgSO4. The bacterial concentrations CNS (18) and modified by omission of lithium chloride and
were photometrically adjusted to ≈108 CFU/ml (i.e., an optical Bravo 6F; D2ANX (6); SCM (11); mSCM (31); CMM1 (1,24);
density of 0.06 at 660 nm), followed by 10-fold serial dilutions EPPO, the medium for Cmm recommended by the EPPO (27);
down to the proper concentration. After surface plating onto test and MTNA (20), which was developed for C. michiganensis
media, the agar plates were incubated at 26°C for 48 to 72 h in subsp. sepedonicus. For evaluating the best-suited basal medium,
case of the NGY medium or longer in case of semiselective media three Cmm strains (GSPB 390, GSPB 2973, and Ei-2) differing in
or NGY supplemented with rifampicin and streptomycin. colony morphology and growth rate on NGY were selected.

TABLE 1. Origin of bacterial species and strains used to evaluate semiselective media
GSPB Designation or number Year
Bacterial species numbera in other collectionsb Originc of isolation Isolated byd
Clavibacter michiganensis subsp. michiganensis 3199 Amb-1 Germany, R 2006 R. Ftayeh
C. michiganensis subsp. michiganensis 3200 Ei-1 Germany, NR 2007 R. Ftayeh
C. michiganensis subsp. michiganensis … Ei-2 Germany, NR 2007 R. Ftayeh
C. michiganensis subsp. michiganensis 3201 Lu-1 Germany, KL 2006 R. Ftayeh
C. michiganensis subsp. michiganensis 3202 Mo-1 Germany, R 2006 R. Ftayeh
C. michiganensis subsp. michiganensis … Mo-2 Germany, R 2006 R. Ftayeh
C. michiganensis subsp. michiganensis 3203 Sc-2 Germany, KL 2006 R. Ftayeh
C. michiganensis subsp. michiganensis 3204 BO-RS Germany, NR 2006 R. Ftayeh
C. michiganensis subsp. michiganensis 2972 78-s Germany 1979 E. Griesbach
C. michiganensis subsp. michiganensis 3205 AE-1 Syria, L 2007 R. Ftayeh
C. michiganensis subsp. michiganensis 3206 AH-1 Syria, T 2007 R. Ftayeh
C. michiganensis subsp. michiganensis … ES-1 Syria, T 2007 R. Ftayeh
C. michiganensis subsp. michiganensis 3207 HH-1 Syria, L 2007 R. Ftayeh
C. michiganensis subsp. michiganensis … La-1 Syria, L 2007 R. Ftayeh
C. michiganensis subsp. michiganensis 3208 OS-1 Austria, STM 2007 E. Moltmann
C. michiganensis subsp. michiganensis … OS-2 Austria, STM 2007 E. Moltmann
C. michiganensis subsp. michiganensis … OS-4 Austria, STM 2007 E. Moltmann
C. michiganensis subsp. michiganensis 378 9/79 Greece 1979 A. Mavridis
C. michiganensis subsp. michiganensis 382 24/78 Greece 1978 A. Mavridis
C. michiganensis subsp. michiganensis 390 31/79 Greece 1979 A. Mavridis
C. michiganensis subsp. michiganensis 392 45/78 Greece 1978 A. Mavridis
C. michiganensis subsp. michiganensis … Bulgarian 1 Bulgaria Unknown From E. Griesbach
C. michiganensis subsp. michiganensis 2973 Cm8 Bulgaria Unknown From E. Griesbach
C. michiganensis subsp. michiganensis 2315 KD/1-4 Turkey 1994 Ö. Cinar
C. michiganensis subsp. michiganensis 2221 NCPPB 1573 Hungary 1963 Z. Klement
C. michiganensis subsp. michiganensis 2222 NCPPB Hungary Unknown Unknown
C. michiganensis subsp. michiganensis … 399 unknown Unknown From E. Griesbach
C. michiganensis subsp. michiganensis 3133 NCPPB 3123 United States Unknown E. Echandi
C. michiganensis subsp. michiganensis … 185 United States Unknown From E. Griesbach
C. michiganensis subsp. michiganensis … Leningrad 3 Russia Unknown From E. Griesbach
C. michiganensis subsp. insidiosus 30 NCPPB 1634 United Kingdom 1934 From Lelliott
C. michiganensis subsp. nebraskensis 2223 NCPPB 2581 United States 1971 M. L. Schuster
C. michiganensis subsp. sepedonicus 1522 NCPPB 2140, Cs 1 United States 1942 L.T. Richardson
C. michiganensis subsp. sepedonicus 2823 Solara 3 Germany 1998 A. Mavridis
C. michiganensis subsp. tessellarius 2224 ATCC 33566 United States 1982 R. R. Carlson
Curtobacterium flaccumfaciens pv. flaccumfaciens 2218 NCPPB 559 United States 1958 From Lelliott
Bacillus subtilis 1769 NCPPB 1246 United States 1956 L. S. Bird
B. subtilis … FZB 24 Germany Unknown Unknown
Pectobacterium subsp. carotovorum 436 DSMZ 60442 Germany Unknown Unknown
Pantoea agglomerans 450 NCPPB 651 United Kingdom 1985 E. Billing
Pseudomonas corrugata 2418 PC 1 Germany 1995 A. Mavridis
P. fluorescens 1714 G-1 Germany Unknown Unknown
P. syringae pv. syringae 1142 R-12 Germany 1967 K. Rudolph
P. syringae pv. tomato 1776 14-1 Hungary 1987 S. Süle
P. syringae pv. tomato 2317 Nr.-1 Turkey 1994 A. Mavridis
P. syringae pv. tomato … Syr-1 Syria 2007 R. Ftayeh
Ralstonia solanacearum 2607 180 a Cameroon 1996 A. Mavridis
R. solanacearum 2619 Ps 24 Brazil 1995 O. Martins
Xanthomonas arboricola pv. juglandis 3148 B- 102 Germany 2002 W. Wohanka
X. campestris pv. vesicatoria 2043 S-08 Hungary 1964 Z. Klement
22 isolates of saprophytic bacteriae … S-1, S-2,…, S-23 Germany R, NR, KL 2006–2007 R. Ftayeh
a GSPB = Göttingen Collection (Sammlung) of Phytopathogenic Bacteria.
b NCPPB = National Collection of Plant Pathogenic Bacteria, UK; ATCC = American Type Culture Collection; and DSMZ = German Collection of Micro-
organisms and Cell Cultures.
c R = Reichenau, NR = Niederrhein, KL = Knoblauchsland, Franken, L = Latakia, T = Tartous, and STM = Steiermark.
d “From” indicates obtained from the person named.
e Saprophytes were isolated from tomato seed and tomato plants and differing in color, morphology, Gram’s reaction, or susceptibility to antibiotics, partially

identified by fatty acid analysis.

1356 PHYTOPATHOLOGY
Bacterial suspensions containing 25 to 75 CFU were streaked tomato plants which had been previously inoculated with an
with an “L”-shaped glass rod in triplicate per strain on each test antibiotic-resistant Cmm strain and which were highly contami-
medium. The growth area of each Cmm strain was determined in nated with saprophytes were homogenized in sterile water and
square millimeters as the mean of the three replicates on each serially diluted. Aliquots of 100 µl were streaked in triplicate onto
medium at the third and fifth day after plating (i.e., growth area = each test composition. The actual number of Cmm cells contained
number of CFU × πr2). in the plant homogenates from field trials was determined on
Screening and selection of antibiotics and inhibitors. Forty NGY agar supplemented with rifampicin at 50 ppm, streptomycin
antibiotics (13) were initially screened on their inhibitory effect at 200 ppm, and the fungicide Opus Top at 50 µl/liter (BASF,
against two Cmm strains. The screening test was performed Ludwigshafen).
according to the technique of Bauer et al. (2) by means of com- Furthermore, homogenates from healthy field plants (collected
mercially available filter discs containing defined concentrations from different locations in Germany and Syria) were surface
of antibiotics (Oxoid Ltd., England). From these experiments, streaked in triplicate onto NGY agar and test compositions in
eight selected antibiotics (aztreonam, fosfomycin metronidazole, order to estimate selectivity. In parallel, suspensions of two Cmm
mupirocin, nalidixic acid, polymyxin B sulfate, sulfamethoxazole, strains (GSPB 390 and GSPB 2973) differing in growth morphol-
and trimethoprim) were separately added to NGY agar in different ogy and speed were also streaked, each in triplicate, onto agar
concentrations and tested against Cmm and 30 accompanying bac- plates with NGY or test compositions to estimate the growth area
terial strains. The accompanying bacteria had been originally isolated of Cmm. Only those compositions which allowed high selectivity
from tomato seed and plants (Table 2). High concentrations of the concomitantly with large growth areas of Cmm were selected and
bacterial suspensions were streaked by an inoculation loop onto modified repeatedly in further experiments.
the test media, and agar plates were incubated at 25°C. In addi- Naturally infected or spiked plant and seed samples. New
tion, we tested the inhibitors boric acid, lithium chloride, potassium medium compositions were tested with healthy and infected
tellurite, and sodium azide, which are contained in other semiselec- tomato plants and seed harvested from field trials in Göttingen. In
tive media for Cmm. Furthermore, 31 different fungicides (13) addition, samples from naturally infected tomato plants obtained
were tested in order to select those which inhibit a broad range of from different locations in Germany, Austria, and Syria were
fungi occurring on tomato plants without inhibiting Cmm. included to enlarge the diversity of epiphytic microorganisms
Formulation of the optimum concentration of antibiotics. which might interfere with Cmm growth on the test media. For
The goal was to determine the optimum concentration of each inoculation of field plants in 2007 and 2008, Cmm strain BO-RS
component in inhibiting nontarget bacteria while allowing good (GSPB 3204), with resistance against rifampicin and strepto-
growth of various Cmm strains. For testing different medium mycin, was used. Four-month-old field plants were inoculated by
compositions, seed, stems, and side shoots from field-grown stabbing the stem with a fine needle through a drop of a bacterial

TABLE 2. Efficacy of nalidixic acid or trimethoprim toward Clavibacter michiganensis subsp. michiganensis (GSPB 390), 5 phytopathogenic, and 25 epiphytic
or saprophytic bacterial strains isolated from tomato seed and plants on nutrient glucose yeast extract (NGY) medium containing different concentrations of the
two antibiotics
Bacterial growth on NGY amended withc
Nalidixic acid Trimethoprim
Bacterial species or straina Gram reactionb Colony color on NGY 5 10 20 50 100 200 300
C. michiganensis subsp. michiganensis (GSPB 390) G+ Typical + + + ± + + +
Xanthomonas juglandis (GSPB 3148) G– Typical + + – – + – –
X. campestris pv. vesicatoria (GSPB 2043) G– Typical + + – – + – –
Pseudomonas syringae pv. syringae (GSPB 1142) G– Typical – – – – + – –
P. syringae pv. tomato (GSPB 2317) G– Typical – – – – + – –
P. fluorescens (GSPB 1714) G– Typical ± – – – + – –
Pantoea agglomerans (GSPB 450) G– Typical ± – – – + – –
Pectobacterium subsp. carotovorum (GSPB 436) G– Typical + + + – – – –
Bacillus subtilis (GSPB 1769) G+ Typical – – – – – – –
S-1: Pseudomonas putida G– White creamy – – – – + – –
S-2: Microbacterium lacticum G+ Yellow-pink + + + – – – –
S-3: not determined G– Dark yellow – – – – – – –
S-4: Pantoea sp. G– Creamy yellowish + + ± – – – –
S-5: Pantoea sp. G– White yellowish + + – – – – –
S-7: not determined G+ Creamy + + + – – – –
S-8: B. cereus G+ Yellow + + + + – – –
S-9: not determined G– White-creamy + + + + + – –
S-10: Pseudomonas syringae G– White-creamy ± – – – + – –
S-11: B. coagulans G+ Light yellow-pink + + + – ± – –
S-12: Microbacterium sp. G+ Pink-yellowish + + + – – – –
S-13: Pantoea agglomerans G– Light yellow – – – – + – –
S-14: Pseudomonas putida G– White creamy + ± – – + ± ±
S-15: P. putida G– White creamy + – – – + ± ±
S-16: not determined G+ Violet – – – – – – –
S-17: not determined G+ Dark orange + – – – + ± –
S-18: not determined G– Yellow – – – – – – –
S-19: Rahnella aquatilis G– White – – – – + – –
S-20: not determined G+ Dark yellow + – – – + – –
S-21: B. licheniformis G+ Light yellow, creamy + + + – – – –
S-22: not determined G+ White – – – – – – –
S-23: B. pumilus G+ Yellow + + ± – – – –
a GSPB = Göttingen Collection (Sammlung) of Phytopathogenic Bacteria.
b G – = gram negative and G + = gram positive.
c Antibiotic concentrations in milligrams per liter; + = growth, – = no growth, and ± = slight growth.

Vol. 101, No. 11, 2011 1357

suspension (30 to 50 µl containing 30 to 50 CFU of Cmm) placed 0.1 g MgSO4 × 7H2O, 0.015 g MnSO4 × H2O, 0.015 g FeSO4 ×
in the axil of the fourth or fifth true leaf. Two months later, tomato 7H2O, and 0.6 g H3BO3. The resulting pH should be between 7.0
seed were extracted with water, dried, and stored at 4°C for to 7.1. Add 15 g/liter agar (Roth). After autoclaving at 121°C for
maintaining accompanying bacterial populations. In addition, 15 min and cooling at 50 to 55°C under stirring, add per liter:
stems of inoculated and noninoculated (control) plants were cut 20 mg nalidixic acid (AppliChem), 100 mg trimethoprim (Fluka),
and stored at –20°C until use. 20 mg polymyxin B sulfate (8,120 international units/mg) (Appli-
In case of using healthy field plants that were highly contami- Chem), and 50 µl of the fungicide Opus Top (BASF) containing
nated with epiphytic bacteria, defined low amounts of Cmm cells epoxiconazole at 84 g/liter and fenpropimorph at 250 g/liter.
were added to the homogenates (“spiked”) directly before surface Antibiotics and Opus Top should be added as stock solutions,
plating onto test media. freshly prepared, and kept sterile at 4°C. Stock solutions contain:
Criteria for evaluating semiselective media. Three param- nalidixic acid (20 mg/ml of 0.01 N NaOH, filter-sterilized), tri-
eters were determined for evaluation of BCT and the published methoprim (50 mg/ml of dimethyl sulfoxide, kept in the dark),
semiselective media: (i) plating efficiency (recovery rate) (%) = polymyxin B sulfate (10 mg/ml of water, filter-sterilized), and
(CFU of Cmm on test medium/CFU of Cmm on NGY) × 100; Opus Top (50 µl/ml sterile water).
(ii) selectivity (%) = [(Population of nontarget microbes on Effect of boric acid. Depending on the concentration of boric
NGY – population of nontarget microbes on test medium)/popu- acid contained in the NGY medium or the new basal media, the
lation of nontarget microbes on NGY] × 100; and (iii) detection inhibition of saprophytic bacteria was increased but the growth of
sensitivity = lowest number of Cmm CFU occurring in plant Cmm was retarded (data not shown). However, when antibiotics
homogenates which could be detected in the presence of high were added to the new basal media, the effect of boric acid on
concentrations of nontarget bacteria. Cmm was the opposite. By adding boric acid, the recovery rate of
Cmm remained at a high level, even when high amounts of
RESULTS antibiotics were added (Fig. 2). Further experiments revealed that
this effect was due to an interaction between boric acid and
Development of the basal medium for Cmm. The highest polymyxin B sulfate (Fig. 3). Surprisingly, the toxic effect of
growth of Cmm was recorded on the basal medium of MTNA polymyxin B sulfate toward Cmm was significantly reduced by
after 3 and 5 days (Fig. 1). Therefore, this composition was boric acid, whereas this effect was not observed toward the
selected and further adapted to Cmm by several modifications. accompanying bacteria occurring in seed and plant homogenates.
Screening and selection of antibiotics or inhibitors. From the Thus, by discovering this interaction between boric acid and
eight selected antibiotics, only a combination of nalidixic acid polymyxin B sulfate, it was possible to develop the new medium
and trimethoprim suppressed all nontarget bacteria tested (Table BCT that permits a high selectivity and, concomitantly, a high
2). Polymyxin B sulfate was tested separately and also showed a plating efficiency of Cmm.
broad inhibitory efficacy on accompanying bacteria. From the Plating efficiency (recovery rate) of Cmm strains on 10
inhibitors tested, only boric acid was selected, whereas lithium semiselective media. Bacterial suspensions of 30 Cmm strains
chloride, potassium tellurite, and sodium azide did not improve containing 100 to 250 CFU were plated in triplicate for each
the medium. Finally, the selected components were tested in strain onto test media and incubated at 26°C. To avoid counting
multiple combinations in the modified basal medium of MTNA. errors by the possible merging of several joining colonies, count-
After each experimental block, the variants showing the highest ing of Cmm colonies was started as soon as possible on each
potential for Cmm growth speed combined with a good selectivity medium (for example, on NGY after 48 to 72 h). Plating effi-
were chosen and modified repeatedly until the optimal final ciency of each strain after 7, 10, 15, and 20 days was expressed as
composition of the new medium was achieved. percent CFU compared with CFU on NGY agar. Summarized and
Recipe of the new medium BCT. For 1 liter of deionized individual data (Fig. 4; Table 3) show that only four media (D2,
water add: 2.5 g mannitol (Merck, Darmstadt, Germany), 2.0 g SCM, CMM1, and BCT) allowed a plating efficiency similar to
yeast extract (Roth), 1.0 g K2HPO4, 0.1 g KH2PO4, 0.05 g NaCl, that of the nonselective NGY medium. All Cmm strains grew on

Fig. 1. Growth areas in square millimeters of three Clavibacter michiganensis subsp. michiganensis (Cmm) strains on nutrient glucose yeast (NGY) extract
medium and on different basal compositions of semiselective media (without addition of antibiotics) within 3 and 5 days. Growth area (mm2) of each strain was
determined as the mean of three replicates on each medium at the third and fifth day after plating [i.e., growth area = number of CFU × πr2 (r = average radius of
colonies in millimeters)].

1358 PHYTOPATHOLOGY
NGY within 3 days, and most Cmm strains tested started to grow could be recovered. Thus, 25 to 100% of the existing Cmm cells
on the new selective medium BCT on the fourth or fifth day after added to or contained in plant and seed homogenates were de-
plating. Finally, 29 of the 30 strains tested grew on the new tected on BCT. In contrast, none of the earlier published semi-
medium within the first 7 days. One Cmm strain took 7 to 10 days selective media resulted in the detection of Cmm under these
to grow on BCT and did not grow on BCT-2. On BCT, colony extreme conditions.
diameter of Cmm was 2.0 to 5.0 mm within 7 days. In other experiments when the differences in population densi-
Selectivity of the new medium BCT. All 42 nontarget ties between saprophytes and Cmm were lower, by increasing the
bacterial species and pathovars tested were unable to grow on amounts of Cmm in plant homogenates or by reducing amounts of
BCT but grew very well on NGY or KB. These bacterial strains saprophytic bacteria in dilutions, detection of Cmm was possible
included Bacillus subtilis, Pantoea agglomerans, Pectobacterium on some of the published media; however, distinguishing between
carotovorum subsp. carotovorum, Pseudomonas corrugata, P. Cmm and saprophytes was often difficult. In fact, on these
fluorescens, P. syringae pv. syringae, P. syringae pv. tomato, published semiselective media, Cmm colonies could not always
Ralstonia solanacearum, Xanthomonas arboricola pv. juglandis, be distinguished from contaminants, particularly (i) if certain
X. campestris pv. vesicatoria, and 22 different saprophytic bac- Cmm strains did not show the typical colony morphology as
terial isolates from tomato plants (Table 2, S-1 to S-23). In known from those media, or (ii) when the morphology of some
contrast to the nontarget bacterial strains, all 30 Cmm strains saprophytic bacteria was similar to Cmm, resulting in confusions
tested grew on BCT and 29 of 30 strains grew on BCT-2. with some saprophytes. In contrast, on BCT, Cmm colonies were
Further experiments with homogenates from field tomato plants clearly distinguishable from saprophytes once they had increased
and seed containing extremely high levels of naturally occurring in colony size with time, whereas saprophytic bacteria remained
accompanying bacteria and which were spiked with low Cmm smaller due to selective inhibition and were mostly white in color
populations showed a high selectivity of BCT, because an average (Fig. 5). In contrast, Cmm colonies were shining, convex, slimy,
of 98.5% of nontarget bacterial populations did not grow on and more or less circular and the color varied from white creamy
BCT (Table 4) and the remaining colonies of nontarget bacteria to yellow (Fig. 6).
were rather small and easily distinguishable from the larger Modifications of BCT. BCT was modified into BCT-2 by
Cmm colonies (Fig. 5). All eight published semiselective media increasing the phosphate concentration (K2HPO4 at 2.0 g/liter and
for Cmm revealed false-negative results under these extreme KH2PO4 at 0.5 g/liter). The resulting pH value of BCT-2 should
conditions (i.e., the added Cmm bacteria could not be visually be 7.2. While BCT supported growth of all 30 Cmm strains tested,
detected, even after restreaking and microscopic tests of and most of the Cmm strains started to grow after 4 to 5 days,
suspected colonies). BCT-2 supported growth of 29 Cmm strains and growth was 1 day
Detection sensitivity of the semiselective media. Stem sec- delayed (Table 3). However, the detection sensitivity of Cmm
tions of 1 to 2 cm from field tomato plants were crushed in sterile cells spiked into seed and plant extracts was greater on BCT than
mortars with 5 to 8 ml of sterile water, or 10 to 25 seeds from on BCT-2, apparent by the larger colony size of Cmm on BCT
field plants were crushed in 1 to 2 ml of sterile water. Serial (Table 4).
dilutions in 0.01M MgSO4 were surface plated on test media and Opus Top was selected out of 31 fungicides tested because of
incubated at 26°C. Counting nontarget bacteria was started as its high efficacy toward a wide range of fungi and its antimi-
soon as colonies became visible, and the final counts recorded at crobial effect against some saprophytic bacteria (data not shown).
7 to 10 days for semiselective media were compared with bac- From these fungicides, only cycloheximde or nystatin are con-
terial populations recovered on NGY medium within 1 to 4 days. tained in the published semiselective media for Cmm. However,
These results were confirmed by repeated streaking of suspected both fungicides did not suppress fungal growth on agar media as
Cmm colonies on NGY agar plates, a microscopic check, and effectively as Opus Top. Opus Top also showed an antimicrobial
Gram reaction. Even as few as 8 CFU of Cmm among 12,750
saprophytes or 58 CFU of Cmm among 18,000 CFU of sapro-
phytes from plant homogenates streaked onto one petri plate
revealed positive results (Table 4), although not every single CFU

Fig. 3. Interactive effects of boric acid with different inhibitors in the basal
medium of BCT on the growth of Clavibacter michiganensis subsp.
Fig. 2. Mean number of CFU per plate recovered from pure cultures of 13 michiganensis (Cmm) (data represent the mean of 11 Cmm strains, each in three
Clavibacter michiganensis subsp. michiganensis (Cmm) strains (each in three replicates), 100 OT = Opus Top at 100 µl/liter, 30 NA = nalidixic acid at 30
replicates) on the new medium (BCT) with and without boric acid, when ≈90 ppm, 200 Tr = trimethoprim at 200 ppm, and 30 PB = polymyxin B sulfate at
CFU were streaked on each petri dish. 30 ppm (8,120 IU/mg).

Vol. 101, No. 11, 2011 1359

effect against some saprophytic bacteria without inhibiting Cmm. DISCUSSION
Thus, by replacing Opus Top with cycloheximide or nystatin in
BCT or BCT-2, growth and recovery rates of Cmm were increased Recently, bacterial canker of tomato has emerged on tomato
but the selectivity was reduced, even when antibiotic concen- crops growing on sterilized artificial substrate in some European
trations were increased (13). Therefore, it is not recommended to countries for the first time (personal observation). These disease
replace Opus Top with other fungicides. outbreaks suggest seed as the source of inoculum even though the
Selectivity of BCT for other pathovars or species of coryne- seed had been certified as healthy according to the EPPO (27) and
form bacteria. In additional experiments, we tested the suitability ISHI (22) detection protocols. Thus, questions arose about the
of BCT for detection of other coryneform phytopathogenic reliability of the currently applied protocols for seed health
bacteria (Table 5). The results revealed that only those bacteria testing, especially the protocols that are based on plating assays of
which are very closely related to Cmm according to Davis et al. seed extracts on semiselective media recommended by the EPPO
(8) (i.e., the C. michiganensis subsp. tessellarius, insidiosus, and (27) or by the ISHI (22). The earlier reported semiselective media
nebraskensis) grew exclusively on the new selective medium BCT for Cmm do not permit an acceptable balance between high
(Table 5). plating efficiency and high selectivity. Thus, several earlier
The colony appearance of the three subspecies on BCT was published semiselective media for Cmm proved to allow high
quite distinct within 7 days. C. michiganensis subsp. tessellarius plating efficiencies (D2, KBT, SCM, CMM1, and D2ANX) in our
(Fig. 7A) had light pink, shining, and slimy colonies with a experiments, but these media failed to strongly suppress nontarget
diameter of 1.5 to 2.5 mm; C. michiganensis subsp. insidiosus bacteria that inhibited growth and detection of Cmm. In case of
(Fig. 7B) had small, pink colonies with violet internal flecks and a high inhibition of the accompanying bacteria by semiselective
diameter of 1.0 to 1.8 mm; and C. michiganensis subsp. nebras- media (mSCM, EPPO, and mCNS), this feature was due to a
kensis: colonies were similar to Cmm (i.e., large, yellow, shining, general toxicity, so that many Cmm strains also failed to grow on
slimy colonies with a diameter of 2.0 to 3.0 mm). these media. Hadas and co-workers (19) obtained similar results,

Fig. 4. Recovery of Clavibacter michiganensis subsp. michiganensis (Cmm) on nine semiselective media and on the nonselective medium nutrient glucose yeast
extract (NGY). Results were obtained from single experiments with 30 Cmm strains. Each column represents the mean of 30 Cmm strains, each in triplicate.
Starting inocula contained 100 to 250 CFU per strain and plate. The same letter (a) above columns indicates no significant difference to the standard nonselective
NGY medium. Statistical analysis was performed by Fisher’s least significant difference test. P ≤ 0.05, n = 900.

1360 PHYTOPATHOLOGY
because some of their Cmm strains tested were not able to grow However, mannitol is more selective than glucose and sucrose, the
on D2ANX, CNS, or mSCM, and other Cmm strains grew with growth of Cmm is well supported, and mannitol is more heat
very low plating efficiency. Thus, a sensitive detection with a very stable than glucose. Mannose is also selective but it does not
low threshold was impossible to reach with any of these earlier reliably support growth of Cmm. These results are similar to those
published semiselective media. Therefore, an increased effort to of De la Cruz (9) and Jansing and Rudolph (20) in the case of the
improve the detection sensitivity of the pathogen in seed and closely related pathogen C. michiganensis subsp. sepedonicus.
transplants has been suggested (21,28). The main problem in designing a selective medium for Cmm
Development of an absolute synthetic selective medium for Cmm was to find the optimum combination and dosage of antibiotics
was impossible because of its partial fastidious nature. Therefore, and inhibitors, because (i) great differences among Cmm strains in
we included yeast extract serving as nitrogen and carbon source. sensitivity toward antibiotics and inhibitors, and (ii) the finding
The main carbon source of BCT is D (–) mannitol, whereas that some nontarget bacteria occurring on tomato plants and seed
previous semiselective media used glucose, sucrose, or mannose. were more tolerant toward antibiotics and inhibitors than Cmm.

TABLE 3. Plating efficiency of 30 Clavibacter michiganensis subsp. michiganensis (Cmm) strains on 10 semiselective media, expressed as percent CFU compared
with the nonselective nutrient glucose yeast extract (NGY) mediuma
Plating efficiency (%) within 7/10/15/20 days, respectively, on
Strain D2 KBT mCNS D2ANX SCM mSCM CMM1 EPPO BCT BCT-2
Amb-1 117/117/117/117 120/120/120/120 0/0/0/0 61/61/61/61 125/125/125/125 0/0/86/113 109/109/109/109 0/0/0/0 67/90/90/90 76/76/90/90
Ei-1 121/121/121/121 123/123/123/123 0/0/1/1 104/104/104/104 119/119/119/119 0/0/101/103 106/106/106/106 0/0/19/43 87/109/109/109 119/119/119/119
Ei-2 110/110/110/110 95/95/95/95 0/0/0/0 13/13/13/13 94/94/94/94 0/84/84/96 89/89/89/89 0/0/0/0 73/73/73/73 98/98/98/98
Lu-1 127/127/127/127 92/92/92/92 0/0/0/0 98/98/98/98 94/94/94/94 0/69/69/98 105/105/105/105 0/0/0/0 102/102/102/102 114/114/114/114
Mo-1 112/112/112/112 89/89/89/89 47/47/51/51 91/91/91/91 105/105/105/105 0/0 /113/113 93/93/93/93 0/0/104/104 96/96/96/96 97/97/97/97
Mo-2 103/103/103/103 103/103/103/103 0/0/3/6 90/90/90/90 104/104/104/104 0/51/85/102 111/111/111/111 0/0/0/2 95/95/95/95 87/87/87/87
Sc-1 112/112/112/112 106/106/106/106 0/0/3/6 90/90/90/90 107/107/107/107 0/35/86/95 108/108/108/108 0/0/0/0 112/112/112/112 110/110/110/110
BO-RS 91/91/91/91 68/68/68/68 0/0/0/0 36/36/36/36 25/35/35/35 0/0/0/0 67/67/67/67 0/0/0/0 76/76/76/76 71/71/71/71
GSPB 2972 84/84/84/84 61/61/61/61 0/0/0/0 107/107/107/107 80/80/80/80 0/0/0/0 97/97/97/97 0/0/0/57 92/92/92/92 81/81/88/88
AE-1 112/112/112/112 87/87/87/87 0/0/0/0 56/56/56/56 102/102/102/102 0/10/24/38 94/94/94/94 0/12/12/21 106/106/106/106 94/94/94/94
AH-1 117/117/117/117 114/114/114/114 0/0/0/0 89/89/89/89 107/107/107/107 0/44/77/89 118/118/118/118 0/111/111/122 100/100/100/100 68/68/68/68
ES-1 98/98/98/98 90/90/90/90 0/0/0/0 95/95/95/95 100/100/100/100 0/0/99/99 90/90/90/90 0/0/92/92 93/93/93/93 79/79/79/79
HH-1 109/109/109/109 99/99/99/99 0/0/4/5 75/75/75/75 104/104/104/104 0/76/77/79 86/86/86/86 0/0/0/0 45/45/45/45 107/107/107/107
La-1 96/96/96/96 64/64/64/64 0/0/0/0 43/43/43/43 89/89/89/89 71/79/87/88 89/89/89/89 0/87/87/91 87/87/87/87 73/73/73/73
OS-1 93/93/93/93 74/74/74/74 0/0/4/4 93/93/93/93 101/101/101/101 0/0/99/99 94/94/94/94 0/0/79/84 96/96/96/96 93/93/93/93
OS-2 91/91/91/91 84/84/84/84 0/0/0/0 81/81/81/81 129/129/129/129 0/0/103/105 91/91/91/91 0/0/65/73 91/91/91/91 93/93/93/93
OS-4 91/91/91/91 87/87/87/87 0/0/0/0 47/47/47/47 120/120/120/120 0/89/89/101 91/91/91/91 0/0/0/10 109/109/109/109 83/83/83/83
GSPB 378 88/88/88/88 88/88/88/88 0/0/0/0 43/43/43/43 93/93/93/93 0/76/98/98 75/75/75/75 0/79/87/88 66/66/66/66 81/81/81/81
GSPB 382 83/83/83/83 55/55/55/55 0/0/0/0 0/0/0/0 65/65/65/65 48/67/67/67 49/49/49/49 0/65/65/66 45/45/45/45 20/42/46/46
GSPB 390 111/111/111/111 102/102/102/102 0/0/0/0 85/85/85/85 88/88/88/88 0/76/76/93 103/103/103/103 0/113/113/116 108/108/108/108 111/111/111/111
GSPB 392 106/106/106/106 91/91/91/91 0/0/0/1 72/72/72/72 92/92/92/92 0/0/115/115 96/96/96/96 0/0/113/113 102/102/102/102 107/107/107/107
Bulgarian 1 97/97/97/97 104/104/104/104 0/0/0/0 79/79/79/79 103/103/103/103 0/85/102/102 87/87/87/87 0/0/96/96 0/23/58/64 0/0/0/0
GSPB 2973 94/94/94/94 99/99/99/99 0/7/9/9 94/94/94/94 82/82/82/82 0/0/0/46 100/100/100/100 0/0/55/73 84/84/84/84 85/85/85/85
GSPB 2315 128/128/128/128 78/78/78/78 0/0/0/4 79/79/79/79 91/91/91/91 0/14/14/101 106/106/106/106 0/0/0/38 102/102/102/102 82/82/82/82
GSPB 2221 103/103/103/103 79/79/79/79 7/7/13/13 86/86/86/86 99/99/99/99 0/0/95/97 98/98/98/98 0/0/95/97 91/91/91/91 90/90/90/90
GSPB 2222 105/105/105/105 61/61/61/61 0/0/0/0 66/66/66/66 90/90/90/90 0/0/0/81 95/95/95/95 0/0/0/22 103/103/103/103 74/74/74/74
399 102/102/102/102 47/47/47/47 0/0/2/7 103/103/103/103 75/75/75/75 0/0/87/87 100/100/100/100 0/0/99/101 97/97/97/97 87/87/90/90
GSPB 3133 103/103/103/103 88/88/88/88 0/0/0/0 23/23/23/23 87/87/87/87 0/59/59/66 92/92/92/92 0/0/34/47 85/85/85/85 94/94/94/94
185 98/98/98/98 81/81/81/81 0/0/0/0 29/29/29/29 98/98/98/98 86/98/98/98 80/80/80/80 0/114/115/115 100/100/100/100 89/89/89/89
Leningrad 3 107/107/107/107 61/61/61/61 0/0/42/43 81/81/81/81 110/110/110/110 0/55/80/81 98/98/98/98 0/0/83/85 84/85/85/85 106/106/106/106
All strains 104/104/104/104 85/85/85/85 2/2/5/5 74/74/74/74 95/96/96/96 6/29/70/84 95/95/95/95 0/17/53/62 89/91/92/92 88/88/89/89
a In all, 100 to 250 CFU were plated in triplicate onto each medium. Plating efficiency percent = (CFU of Cmm on test medium/CFU of Cmm on NGY) × 100.
Each value for each strain and test medium was derived from triplicate.

TABLE 4. Selectivity and detection sensitivity of the media BCT and BCT-2 for Clavibacter michiganensis subsp. michiganensis (Cmm) occurring in tomato seed
and plant homogenates which contained high concentrations of saprophytic bacteria, compared with eight earlier published semiselective media
Inhibition of saprophytes (Inhib.) and visible detection (Det.) of Cmm (%) ina
A B C D E F Average
Medium Inhib. Det. Inhib. Det. Inhib. Det. Inhib. Det. Inhib. Det. Inhib. Det. Inhib. Det.
D2 70.0 0.0 91.2 0.0 93.0 0.0 93.9 0.0 90.8 0.0 84.9 0.0 87.3 0.0
KBT 84.9 0.0 81.0 0.0 80.9 0.0 98.6 0.0 72.5 0.0 74.8 0.0 82.1 0.0
mCNS 95.1 0.0 98.6 0.0 98.4 0.0 100.0 0.0 99.2 0.0 98.3 0.0 98.3 0.0
EPPO 95.6 0.0 98.3 0.0 97.8 0.0 100.0 0.0 94.7 0.0 97.9 0.0 97.4 0.0
CMM1 88.0 0.0 87.8 0.0 98.6 0.0 98.1 0.0 98.8 0.0 77.4 0.0 91.5 0.0
D2ANX 82.0 0.0 89.1 0.0 99.0 0.0 86.9 0.0 91.6 0.0 91.5 0.0 90.0 0.0
SCM 79.9 0.0 99.3 0.0 95.1 0.0 95.8 0.0 99.6 0.0 94.1 0.0 94.0 0.0
mSCM 88.4 0.0 95.5 0.0 95.8 0.0 97.6 0.0 99.7 0.0 95.2 0.0 95.4 0.0
BCT 97.8 67.3 98.0 39.7 98.7 100.0 99.4 66.7 98.6 100.0 98.2 25.0 98.5 66.4
BCT-2 99.8 63.6 98.0 50.0 99.3 98.4 99.6 0.0 98.1 0.0 100.0 0.0 99.1 35.3
a A, B, C, D, E, and F = different seed or plant homogenates plated onto selective media and containing different cell-numbers of saprophytes (S) and Cmm, A,
field seed homogenate (11,500 S + 110 Cmm BO-RS/agar plate); B, field plant homogenate (18,000 S + 58 Cmm 382/agar plate); C, homogenate from
greenhouse plants infested (15,000 S + 250 Cmm BO-RS/agar plate); D, field seed homogenate (1,150 + 21 Cmm BO-RS/agar plate); E, homogenate of field
plants (1,200 S + 3 Cmm OS-2/agar plate), and F, homogenate of field plants (12,750 S + 8 Cmm 382/agar plate).

Vol. 101, No. 11, 2011 1361

Fig. 5. Detection of Clavibacter michiganensis subsp. michiganensis (Cmm) in asymptomatic plant samples on different media. When plant samples were only
slightly infected with Cmm and highly contaminated with saprophytic bacteria, Cmm was detected only on the new medium (BCT). On BCT, Cmm colonies were
easily recognized (creamy to yellow in color, convex, shining, and had increased size with time), whereas colonies of saprophytes were depressed (small, faint, and
mostly white in color).

Fig. 6. Growth of Clavibacter michiganensis subsp. michiganensis on the new medium (BCT): colonies are shining, convex, slimy, and circular, and the color
varies from white creamy at the beginning to yellow later.

Therefore, initially 40 different antibiotics, several inhibitors concentration of 20 mg/liter (8,120 IU/mg) was actually toxic
(boric acid, lithium chloride, potassium tellurite, and sodium toward Cmm when applied in the basal medium. However,
azide), and 31 fungicides were tested. Finally, three antibiotics this toxic effect on Cmm was prevented when boric acid was
(polymyxin B sulfate, nalidixic acid, and trimethoprim), the also added to the medium. In contrast, the toxic effect of
inhibitor boric acid, and the fungicide Opus Top were selected polymyxin B sulfate against a large variety of accompanying
and tested in manifold combinations (13). saprophytes tested so far was not prevented by addition of boric
Polymyxin B sulfate has a very broad inhibiting spectrum on acid. Thus, polymyxin B sulfate at 20 mg/liter and boric acid at
nontarget organisms and considerably enhances the selectivity. A 600 mg/liter together supported the medium with higher selec-

1362 PHYTOPATHOLOGY
TABLE 5. Growth of other coryneform phytopathogenic bacterial species on nutrient glucose yeast extract (NGY) medium and the new medium (BCT) and
colony diameter after 7 days
Colony diameter (mm) on Plating efficiency (%) on
Bacterial species NGY BCT BCT
Clavibacter michiganensis subsp. insidiosus (GSPB 30) 2.0–5.0 1.0–1.8 68.0
C. michiganensis subsp. nebraskensis (GSPB 2223) 2.0–5.0 2.0–3.0 99.0
C. michiganensis subsp. tessellarius (GSPB 2224) 2.0–5.0 1.5–2.5 98.0
C. michiganensis subsp. sepedonicus (GSPB 1522) 0.5 0.0 0.0
C. michiganensis subsp. sepedonicus (GSPB 2823) 0.2 0.0 0.0
Curtobacterium flaccumfaciens pv. flaccumfaciens (GSPB 2218) 2.0–4.0 0.0 0.0

BCT, even when the pathogen was present in 1,000-fold lower

concentration than accompanying nontarget bacteria. Such highly
sensitive detection was impossible with any of the earlier pub-
lished semiselective media for Cmm. Therefore, BCT should be
very helpful in improving the detection sensitivity of the pathogen
in tomato seed and transplants and, thus, allow an effective
control of bacterial canker of tomato.

ACKNOWLEDGMENTS
Fig. 7. A, Clavibacter michiganensis subsp. tessellarius GSPB 2224 and B, C.
michiganensis subsp. insidiosus GSPB 30 on the new medium (BCT).
This study was supported, in part, by the Georg-August University of
Göttingen, the German Academic Exchange Service, and the Hanns-
Seidel-Foundation. Fatty acid methyl ester analyses were kindly carried
out under the supervision of D. Felgentreu, Institute for Ecological
tivity and allowed a very good growth of all Cmm strains tested,
Chemistry, Plant Analysis and Stored Products Protection, Julius-Kühn-
whereas very low or no growth of Cmm was recorded without Institut, Berlin. We thank H. Eiffert, Institute of Medical Microbiology,
boric acid. University of Göttingen, for supplying us with different antibiotics
The underlying mechanism for this effect, which was never needed for the initial screening tests; and C. Nordmann for her excellent
observed against any of the other bacterial species tested, is still technical assistance.
unknown and can only be speculated upon. Because the toxicity
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