ISTA Method Validation Reports - International Seed Testing ...

ISTA Method Validation Reports 

Published by: 

The International Seed Testing Association 

P.O. Box 308 

8303 Bassersdorf, CH-Switzerland 

Volume 2005/1 

Copyright © 2005 by the International Seed Testing Association 

All rights reserved. No part of this publication may be reproduced, stored in retrieval system or transmitted in 

any form or by any means, electronic, mechanical, photocopying recording or otherwise, without the prior 

permission of ISTA

Preface 

ISTA Method Validation Reports is an ISTA publication initiated by the Seed Health Committee in 2003. It 

contains reports of method validation studies which support proposals for new or modified methods to be 

included in the International Rules for Seed Testing. Publication will coincide with announcements of rules 

proposals to be voted on by the ISTA Membership at the next Ordinary Meeting.

Contents 

Preface 

Proposal for a new method for detecting Xanthomonas hortorum pv. carotae on carrot seeds 1

Asma, M. (2005) ISTA Method Validation Reports 2, 1-17 

Proposal for a new method for detecting Xanthomonas hortorum pv. carotae on 

carrot seeds 

M. ASMA 

Bejo Zaden B.V. P.O. Box 50, 1749 ZH Warmenhuizen, The Netherlands; E-mail: m.asma@bejo.nl 

International Seed Health Initiative (ISHI): International Technical Group (ITG) Root & Bulbs. 

Approved by ISTA SHC January 2005 

Summary 

The suitability of the semi-selective agar media MD5A, MKM and mTBM for the detection of Xanthomonas 

hortorum pv. carotae in carrot seeds was evaluated in a comparative test with 10 laboratories organised by the 

International Seed Health Initiative (ISHI). Five naturally infected carrot seed lots were used with two to five 

sub samples of 10,000 seeds giving a total of 19 sub samples. For each combination of sub sample and medium, 

the number of suspect and ‘other’ colonies were recorded. The presence of suspect colonies was confirmed by 

PCR or pathogenicity testing. It was concluded that MKM is a more selective medium than MD5A and mTBM. 

For routine testing of carrot seed lots one of the combinations MKM/MD5A or MKM/mTBM is recommended. 

Introduction 

Bacterial leaf blight of carrot (Daucus carota subsp. sativus) caused by Xanthomonas 

hortorum pv. carotae (also known as Xanthomonas campestris pv. carotae) is an important 

seed-borne disease found worldwide. Under conditions favourable to the bacteria this disease 

can cause significant yield losses. Contaminated carrot seeds are a potential primary source 

of inoculum and the use of X. hortorum pv. carotae-free seed is an important disease 

management strategy. Therefore, sensitive detection methods suitable for routine application 

are needed by inspection services, commercial seed testing laboratories and the seed industry 

(Meijerink and van Breukelen, 1995). 

No ISTA Method Sheet is available for the detection of X. hortorum pv. carotae in carrot seed 

lots. The most common method used in seed health testing laboratories is based on a seed 

wash dilution-plating assay. This method involves washing seeds in buffer and plating serial 

dilutions of the extract on the semi-selective modified D5 (MD5) medium (Kuan et al., 1985). 

An improved version of the medium called MD5A (Cubeta and Kuan, 1986) is commonly 

used. The semi-selective XCS medium (Williford and Schaad, 1984) is also used in the seed 

wash dilution-plating assay. However, a poor recovery of X. hortorum pv. carotae on XCS 

medium seems to be a common problem in seed health testing laboratories (Asma, 

unpublished results). Therefore, several other semi-selective media like MKM variants 

(MKMnov, MKMsvs adapted from Kim et al. 1982), mTB and mTBM (adapted from 

McGuire et al. 1986) are currently in use. 

In the past, identification of suspect colonies after dilution plating was difficult with many 

laboratories finding the reproducibility of the pathogenicity assay poor. In addition IF 

(immuno-fluorescence) identification was difficult due to the lack of an antiserum with 

adequate specificity. Recently, identification of suspect colonies has become easier due to the 

availability of X. hortorum pv. carotae-specific primers (Meng et al., 2004). 

In 1999, a first comparative test was organised by the International Seed Health Initiative- 

Vegetables (ISHI-Veg). In this test six semi-selective media (MD5A, XCS, MKMnov, 

MKMsvs, mTB, mTBM) were evaluated for the detection of X. hortorum pv. carotae in two 

1

M. Asma 

carrot seed lots and for the recovery of X. hortorum pv. carotae isolates at seven laboratories. 

The results showed a large variation between laboratories (Asma, 1999) and was due to 

different levels of experience with X. hortorum pv. carotae detection and the semi-selective 

media used. Moreover, there were differences in the chemicals used for the preparation of the 

media and variation in incubation time. Three Dutch laboratories also performed additional 

tests on the effect of agar source, antibiotics and some additional semi-selective media (Asma, 

2000a). 

In 2000 a second comparative test was organised in which both ISHI-Veg and ISTA 

laboratories participated. Three carrot seed lots were tested, each in three replicates of 10,000 

seeds on three semi-selective media (MD5A, MKM, mTBM) in 10 laboratories. From the 

results it was concluded that the confirmation method (plant pathogenicity assay, ELISA, IF 

or PCR) affected the test results (Asma, 2000b). Moreover, the use of antisera in ELISA and 

IF with insufficient specificity caused false positive confirmation of suspect colonies. 

Therefore, at four laboratories in the Netherlands and USA additional research was performed 

on the identification of X. hortorum pv. carotae with the pathogenicity assay and PCR. 

Results indicated that PCR is a reliable and fast method to confirm the identity of X. hortorum 

pv. carotae isolates (Asma et al., 2002). 

In a final ISHI-Veg comparative test organised in 2003, 10 seed health testing laboratories 

from the Netherlands, France, Israel, USA and United Kingdom participated. In this test, the 

suitability of MD5A, MKM and mTBM medium for the detection of X. hortorum pv. carotae 

in carrot seeds was evaluated. The results of this test are presented in this report. 

Materials and Methods 

Media 

MD5A medium (Modified D5A medium; Cubeta and Kuan, 1986) was prepared by 

dissolving 0.3 g/l MgSO 4 .7H 2 O, 1.0 g/l NaH 2 PO 4 , 1.0 g/l NH 4 Cl, 3.0 g/l K 2 HPO 4 with 17.0 

g/l of agar in distilled water. The pH was adjusted to 6.4 and the medium was autoclaved for 

15 min at 121°C. After cooling to 50°C the following ingredients were added: 20 mg/l 

nystatin, 10 mg/l cephalexin monohydrate, 10 mg/l bacitracin, 5 mg/l L-glutamic acid, 1 mg/l 

L-methionine and 10 g/l filter sterilised cellobiose. 

MKM medium (Modified KM-1 medium; adapted from Kim et al. 1982) was prepared by 

dissolving 1.0 g/l NH 4 Cl, 1.2 g/l K 2 HPO 4 , 1.2 g/l KH 2 PO 4 , 10.0 g/l lactose monohydrate, 4.0 

g/l D(+) trehalose dihydrate, 0.5 g/l yeast extract, 0.2 g/l 2-thiobarbituric acid with 17.0 g/l of 

agar in distilled water. The pH was adjusted to 6.6 and the medium was autoclaved for 15 min 

at 121°C. After cooling to 50°C the following ingredients were added: 20 mg/l nystatin, 

10 mg/l cephalexin monohydrate, 50 mg/l bacitracin and 2 mg/l tobramycin sulphate. 

mTBM medium (modified Tween medium B with milk powder; adapted from McGuire et al. 

1986) was prepared by dissolving 0.3 g/l H 3 BO 3 , 10.0 g/l KBr, 10.0 g/l peptone, with 17.0 g/l 

of agar in distilled water. The pH was adjusted to 7.4 and the medium was autoclaved for 15 

min at 121°C. After it cooling to 50°C the following ingredients were added: 20 mg/l nystatin, 

65 mg/l cephalexin monohydrate, 12 mg/l 5-fluorouracil and 10 ml/l Tween 80 (autoclaved 

separately) and 10.0 g/l skimmed milk (autoclaved separately). 

YDC medium (Yeast Dextrose Chalk (YDC) medium; Schaad, 1988) was formulated 

consisting of 20.0 g/l dextrose, 10.0 g/l yeast extract, 20.0 g/l CaCO 3 with 15.0 g/l of agar in 

one litre of distilled water. YDC medium was autoclaved for 15 min at 121°C. 

2

New method for Xanthomonas hortorum pv. carotae 

Seed samples 

Five carrot seed lots, with variable levels of natural infection were selected by Bejo Zaden 

B.V. Before the comparative test began, the infection level of each seed lot was determined at 

Bejo Zaden B.V. by testing five sub samples of 10,000 seeds from each seed lot. The number 

of infected sub samples for the seed lots P4920, P4925, P4926, P4932 and P4933 was 3, 5, 0, 

1 and 3 respectively. The ‘healthy’ seed lot P4926 was disinfected by hot water treatment for 

30 min at 58°C to ensure that most saprophytic background bacteria were killed. Seed lot 

P4926 could therefore be used as the negative control seed lot. 

For the comparative test each seed lot was divided in sub samples of 10,000 seeds based on 

weight. For the ‘high level infection’ seed lot P4925 (all sub samples infected) and ‘healthy’ 

seed lot P4926 (no infected sub samples) two replicate samples of 10,000 seeds each were 

tested. For the ‘low level infection’ seed lots P4920, P4932, P4933 five replicate samples of 

10,000 seeds each were tested. Paper envelopes containing sub samples of 10,000 seeds were 

coded randomly before sending to participating laboratories ensuring a blind comparative test. 

Seed washing assay 

Sub samples of 10,000 seeds were soaked overnight at 5°C in 100 ml sterile saline (0.85% 

NaCl) with 0.02% Tween 20. A tenfold-dilution series was prepared from each seed soak 

extract and 100 µl of each dilution and the undiluted extract were spread in triplicate on 

MD5A, MKM and mTBM media. Positive control plates were prepared by spreading 100 µl 

of serial tenfold-dilutions of suspensions of a pure culture of a known X. hortorum pv. 

carotae strain (NCPPB 425) on each of the media. After incubation at 27-28°C for 4-8 d the 

numbers of suspect X. hortorum pv. carotae and all ‘other’ colonies present on each plate 

were recorded. Colonies were considered suspect if they appeared similar to colonies of the 

reference strain. 

On the MD5A medium, colonies of X. hortorum pv. carotae after 7-8 d are typically straw 

yellow, glistening, round, smooth, convex with entire margins, and 2-3 mm in diameter. 

On the MKM medium, colonies of X. hortorum pv. carotae after 4-6 d are typically light 

yellow-cream, light brown to peach yellow, glistening, round and 2-4 mm in diameter. 

On the mTBM medium, colonies of X. hortorum pv. carotae after 7-8 d are white or yellow or 

white-yellow, glistening, round, smooth, convex with entire margins, 1-2 mm in diameter and 

surrounded by a large clear zone of casein hydrolysis. 

If present, up to six suspect colonies from each sub sample were sub-cultured to sectored 

plates of YDC, which were then incubated at 28°C for 3-4 d. Sub-cultured isolates were 

compared visually with the reference strain and were confirmed by PCR with the S3 primer 

pair (Meng et al., 2004) or pathogenicity testing (Kuan, 1985) 

Data analysis 

For each combination of laboratory, medium, seed lot, sub-sample and dilution the number of 

suspect (X. hortorum pv. carotae) and ‘other’ colonies were recorded. As lab#8 did not count 

the number of colonies on the plates and instead made estimations of colony numbers, its 

results were not included in the raw data for the statistical analysis. 

An estimation of the number of suspect and ‘other’ colonies was made according to the 

results of the four dilutions for each combination of laboratory, medium, seed lot and sub 

sample. The results of the confirmation tests were used to calculate the number of confirmed 

3

M. Asma 

positive colonies as the number of suspect colonies multiplied by the proportion of PCR 

positive colonies. For example, if 100 suspect colonies are recorded on MKM medium and 

five are run in a PCR, four positive in the confirmation test gives 80 (100 * 4/5) confirmed 

positives. The non-confirmed colonies (i.e. 100-80=20) are added to the ‘other’ colonies. 

The number of suspect and the total number of confirmed positives were analysed in two 

different generalised linear modelling facilities of Genstat (Payne et al., 2003), a binomial 

model (i.e. data in terms of either a positive or negative result), and a Poisson model (i.e. 

count data for the number of X. hortorum pv. carotae and ‘other’ colonies detected). 

The binomial model was specified as having a binomial error distribution with a 

complementary log-log link function. The effects of both seed lot and laboratory were tested 

against the mean deviance of samples within laboratory under the assumption that the mean 

deviance ratio by approximation follows an F-distribution. The predictions (based on the 

model) and standard errors were calculated taking the mean deviance of the samples within 

laboratory as the dispersion factor. For the binomial data no over-dispersion occurred at the 

level of the residuals. So the effect of media and interaction with media were tested according 

to the model assumption that the deviance (of these effects) follows a Chi-squared 

distribution. The standard errors are based on the binomial distribution with dispersion factor 

equals one. 

The model for the count data was specified as having a Poisson error distribution with a loglink 

function and the dilution was accounted for by an offset term (the natural log of the 

dilution). The effects of both seed lot and laboratory were tested against the mean deviance of 

the samples within laboratory. The effect of medium was tested against the lot x laboratory x 

medium term in the model. The predictions (based on the model) and corresponding standard 

errors were calculated. The standard errors are based on the dispersion factor that was set to 

the mean deviance of the sample within laboratory or the lot x laboratory x medium 

respectively. 

The repeatability (within laboratory variability) is equivalent to the mean deviance of the 

samples within laboratories (Lsf) value. The reproducibility dispersion (between laboratory 

variability) is based on the between laboratory dispersion plus the within laboratory 

dispersion. In practice this is equivalent to the deviances of the laboratory, lsf, lot x 

laboratory, in total, divided by the degrees of freedom of all three. 

Results 

In all three media, nystatin (20 mg/l) was included to inhibit the growth of fungi on the plates. 

However, several laboratories noted the growth of fungi on the media in the laboratory notes 

accompanying their test results. 

The number of positive sub samples for each combination of seed lot, laboratory and medium 

was determined (table 1). Laboratory 5 detected the highest number of positive sub samples 

(34) and laboratory 7 the lowest number (16). Two laboratories detected some X. hortorum 

pv. carotae in the ‘healthy’ seed lot P4926. Not all laboratories were able to detect and 

identify X. hortorum pv. carotae in both sub samples on all media from the ‘high level 

infection’ seed lot P4925. In addition, three laboratories were unable to detect any X. 

hortorum pv. carotae in the ‘low level infection’ seed lot P4920. Most laboratories recorded 

the highest number of positive sub samples on MKM. All laboratories recorded the highest 

number of positive sub samples on a combination of MKM with MD5A or mTBM. 

4


In the comparative test PCR was used for the identification of suspect colonies. In the ‘high 

level infection’ seed lot P4925 most laboratories were able to retrieve suspect colonies from 

all media and identify as X. hortorum pv. carotae by PCR (table 2). 

It should be noted that two laboratories detected and confirmed the identity of colonies of X. 

hortorum pv. carotae in the putatively X. hortorum pv. carotae – free (‘healthy’) seed lot 

P4926. 

For the three ‘low level infection’ seed lots P4920, P4932, P4933 the number of colonies on 

YDC, considered suspect and subsequently confirmed with PCR, was strongly dependent on 

the laboratory. For seed lot P4920 only 45 colonies of the 367 tested colonies were PCR 

positive (12,3%). 

From the ten laboratories, only laboratory 7 tested a small number of colonies with a plant 

pathogenicity assay also. From a total of 9 PCR positive colonies, 8 colonies were positive in 

the pathogenicity assay. Nine PCR negative colonies did not cause any reaction in the plant 

pathogenicity assay (data not shown). 

Binomial model 

The analysis of deviances for the number of suspect and PCR confirmed colonies indicated 

that the differences between seed lots and laboratories were significant. A significant lot x 

laboratory interaction for the number of suspect colonies was also detected. The differences 

between media in the number of PCR confirmed colonies were also significant. However, a 

significant laboratory x media interaction was also detected (tables 3 and 4). 

The predicted proportions of suspect and PCR confirmed colonies for each medium are 

summarised in figure 1. The highest predicted proportion of suspect and PCR confirmed 

colonies were detected on MKM. 

In figure 2 the predicted proportions of suspect colonies for each laboratory and medium are 

presented. There was no association between the detection of the highest proportion of 

suspect colonies on an individual medium and laboratories. 

Poisson model 

The analysis of deviances for the number of suspect and PCR confirmed colonies indicated 

that there were significant differences between seed lots and laboratories. However, a 

significant laboratory x media interaction was also detected (tables 5 and 6). 

The predicted counts of suspect and PCR confirmed colonies for each medium are 

summarised in figure 3. The highest number of suspect colonies was detected on MD5A. The 

medium affects the proportion of false positive suspect colonies scored. On MKM the lowest 

number of false positive suspect colonies was scored. 

A large effect of the laboratory on the predicted counts of suspect colonies per medium was 

found (figure 4). 

In figure 5 the natural logarithm of the predicted counts of the number of suspect and PCR 

confirmed positives for each seed lot is presented. Not all suspect colonies in the seed lots 

were confirmed positive with PCR. In the healthy seed lot P4926 nearly all recorded suspect 

colonies were not confirmed positive. 

The predicted counts of the number of suspect and PCR confirmed colonies for each 

laboratory is presented in figure 6. A large effect of the laboratory is seen. Laboratory 1 

5

M. Asma 

recorded the lowest number of suspect colonies and laboratory 2 the highest number. 

Moreover, nearly all suspect colonies recorded at laboratory 2 were confirmed positive with 

PCR. 

The analysis of deviances (tables 7 and 8) for the number of all ‘other’ colonies indicated that 

there were significant differences between seed lots, laboratories and media. All interactions 

are significant as well. The natural logarithm of the predicted counts of the number of ‘other’ 

colonies for each medium is presented in figure 7. On MKM the lowest number of ‘other’ 

colonies was recorded. 

In figures 8 and 9, the natural logarithm of the predicted counts for laboratories and seed lots 

on each medium respectively is shown. The five seed lots had a different level of ‘other’ 

colonies with most ‘other’ colonies recorded on seed lot P4920 and P4925. In all seed lots, the 

lowest number of ‘other’ colonies was detected on MKM. At seven laboratories the lowest 

number of ‘other’ colonies was recorded on MKM and the highest on MD5A. 

In tables 9 and 10 the reproducibility dispersion (between laboratory variability) and the 

repeatability dispersion (within laboratory variability) for the confirmed positive colonies 

based on the binomial data and count data respectively are presented. Despite the differences 

between laboratories and seed lots all media performed the same in all laboratories for 

detecting presence or absence of X. hortorum pv. carotae regarding reproducibility and 

repeatability. 

Discussion 

Most participants confirmed the infection levels of the four infected seed lots P4920, P4925, 

P4932 and P4933. This result agreed with that obtained at Bejo Zaden B.V. where the 

infection level was determined before the comparative test started. Laboratory 2, 3 and 9 did 

not detect any pathogen in seed lot P4920 and a high proportion of false positive suspect 

colonies were recorded. For seed lot P4932 and P4933 nearly all suspect colonies were 

confirmed positive. This indicates that the ‘other’ colonies on seed lot P4920 did resemble X. 

hortorum pv. carotae colonies, which made identification difficult. 

The seed lot P4926 was severely disinfected by a hot water treatment for 30 minutes at 58°C 

after it had already been found free of contamination. However, two laboratories detected 

some confirmed positive colonies in this ‘healthy’ seed lot. It seems more likely that this was 

caused by cross-contamination or a false-positive PCR reaction then by a very low infection 

level of this seed lot. 

In all three media, nystatin (20 mg/l) was included as an alternative for the very toxic and 

expensive cycloheximide to inhibit the growth of fungi on the plates. From this test it was 

concluded that nystatin at this concentration is not sufficient for inhibition of fungal growth. 

The use of nystatin at a higher concentration or cycloheximide, which is more potent than 

nystatin, is an improvement. In several laboratories, amongst others the Naktuinbouw (NL), 

nystatin is routinely used at 35 mg/l in several semi-selective media for detection of bacteria. 

This concentration controls fungal contaminations in media such as MD5A and MKM 

(Koenraadt, pers. comm.) 

In the past the identification of X. hortorum pv. carotae by pathogenicity assay was a major 

limitation in the testing of carrot seed lots. These tests are laborious, require greenhouse or 

growth chamber facilities and take 3-4 weeks to complete. Although one PCR positive isolate 

did not give a positive reaction in the pathogenicity assay this comparative test showed that 

PCR identification was possible in all laboratories. The results from laboratory 7 showed that 

6


the PCR results correlated well with the plant pathogenicity assay. This also corresponds with 

earlier results obtained by Asma et al. (2002). Therefore, the author believes that PCR could 

reduce time and resources required to identify X. hortorum pv. carotae. 

The semi-selective XCS medium is based on a modification of KM-1 medium (Kim et al., 

1982). The final concentration of tobramycin in XCS medium is 8 mg/l. It is not clear from 

reference literature which chemical structure of tobramycin should be used in the preparation 

of XCS. From earlier results it was concluded that some strains of X. hortorum pv. carotae 

are sensitive to tobramycin sulphate when used at concentrations higher than 4 mg/l (M. 

Asma, unpublished results). Therefore, a concentration of 8 mg/l tobramycin in XCS might 

negatively influence the recovery of X. hortorum pv. carotae, particularly when using a free 

base structure. This phenomenon probably explains the problems with the poor growth of X. 

hortorum pv. carotae on XCS medium. 

The semi-selective MKM medium is a modification of XCS medium. The modifications refer 

to increasing the amount of both K 2 HPO 4 and KH 2 PO 4 from 0.8 g/l to 1.2 g/l, the use of 

cephalexin and bacitracin instead of ampicillin and vancomycin, and lowering the 

concentration of tobramycin from 8 mg/l to 2 mg/l. 

The lowest number of false positive suspect colonies was scored on MKM. This implies that 

suspect colonies were more easily recognised on MKM than on MD5A or mTBM. Moreover, 

for all seed lots, the number of ‘other’ colonies on MKM was lower compared to MD5A and 

mTBM. The results of the Binomial data analysis and the Poisson data analysis correspond 

with respect to the significance of the effect of the lot and the laboratory on the suspect 

colonies and confirmed positive colonies. With respect to the significance of the effect of the 

medium on the confirmed positive colonies the results do not correspond. However, on MKM 

the highest number of positive sub samples was detected, a result supported by the binomial 

data analysis. 

The dispersions based on the count data showed that MKM performed better in different 

laboratories and within laboratories due to the lower reproducibility dispersion and 

repeatability dispersion. This suggests that all laboratories detected differences in the 

infection level of seed on the three media but that the number of colonies detected on the three 

media differed. 

Conclusions and Recommendations 

All three semi-selective media are suitable for the detection of X. hortorum pv. carotae on 

carrot seed. However, the highest number of positive sub samples was detected and the lowest 

number of false positive suspect colonies was recorded on MKM. The number of ‘other’ 

colonies on MKM was lower compared to MD5A and mTBM. 

Most laboratories detected the highest number of positive sub samples on a combination of 

MKM with MD5A or on a combination of MKM with mTBM. 

Nystatin (20 mg/l) is not sufficient to inhibit fungal growth. The use of nystatin at a higher 

concentration (35 mg/l), is an improvement. The use of cycloheximide, which is more potent 

than nystatin, could be considered as an alternative. 

PCR with the S3 primer pair can be used to identify X. hortorum pv. carotae. 

Most labs were able to detect X. hortorum pv. carotae – contaminated seed lots. For routine 

testing of carrot seed lots it is recommended to use a combination of two semi-selective 

media, MKM/MD5A or MKM/mTBM. 

7

M. Asma 

Acknowledgements 

The input of the participating laboratories, ISHI-Veg ITG Root & Bulbs, Petra Remeeus 

(NAKtuinbouw) and Steve Roberts (HRI) for their help in the statistical analysis of the results 

is greatly acknowledged. 

References 

Asma, M. (1999). Test Report Comparative Test Xanthomonas campestris pv. carotae in 

carrot seed. ISHI Report, Bejo Zaden BV, Research Report P9411 

Asma, M. (2000a) Additioneel onderzoek Xanthomonas campestris pv. carotae.ISHI Report, 

Bejo Zaden BV, Research Report P9317-15 / P9416 

Asma, M. (2000b). Report of the ISHI-ISTA comparative test Xanthomonas campestris pv. 

carotae in carrot seed. ISHI Report, Bejo Zaden BV, Research Report P9417 

Asma, M., de Vogel, R., Woudt, B. and Krause, D. (2002). Evaluation of pathogenicity 

testing, rep-fingerprinting and PCR for the identification of Xanthomonas campestris pv. 

carotae, ISHI Report Bejo Zaden BV, Research Report P9317-16. 

Cubeta, M.S. and Kuan, T.L. (1986). Comparison of MD5 and XCS media and development 

of MD5A medium for detection of Xanthomonas campestris pv. carotae in carrot seed, 

Phytopathology 76,1109. (Abstr.) 

Kim, H.K., Sasser, M. and Sands, D.C. (1982). Selective medium for Xanthomonas 

campestris pv. translucens. Phytopathology 72, 936 (Abstr.) 

Kuan, T.L., (1985). Detection of Xanthomonas campestris pv. carotae in carrot. In Detection 

of Bacteria in Seed and Other Planting Material (eds. A.W. Saettler, N.W. Schaad and D.A. 

Roth), p 63. APS Press, St. Paul, MN, USA. 

Kuan, T.L., Minsavage, G.V. and Gabrielson, R.L. (1985). Detection of Xanthomonas 

campestris pv. carotae in carrot seed. Plant Disease 69, pp. 758-760 

McGuire, R.G., Jones, J.B. and Sasser, M. (1986). Tween media for the semiselective 

isolation of Xanthomonas campestris pv. vesicatoria from soil and plant material. Plant 

Disease 70:887-891. 

Meijerink, G.A.A.M. and van Breukelen, E.W.M. (1995). International initiative standardizes 

test protocols. Prophyta annual 49, pp. 58-65. 

Meng, X.Q., Umesh, K.C., Davis, R.M. and Gilbertson, R.L. (2004). Development of PCRbased 

assays for detecting the carrot bacterial leaf blight pathogen (Xanthomonas campestris 

pv. carotae) from different substrates. Plant Disease 88, pp. 1226-1234 

Payne, R.W., Baird, D.B., Cherry, M., Gilmour, A.R., Harding, S.A., Kane, A.F., Lane, P.W., 

Murray, D.A., Soutar, D.M., Thompson, R., Todd, A.D., Tunnicliffe Wilson, G., Webster, R., 

Welham, S.J. (2003), GenStat Release 7.1 Reference Manual. VSN International, Wilkinson 

House, Jordan Hill Road, Oxford, UK. 

Williford, R.E. and Schaad, N.W. (1984). Agar medium for selective isolation of 

Xanthomonas campestris pv. carotae from carrot seeds. Phytopathology 74, 1142 (Abstr.) 

Schaad, N.W. (1988). Initial Identification of Common Genera. In Laboratory Guide for 

Identification of Plant Pathogenic Bacteria 2nd Edition (ed. N.W. Schaad) p. 3. APS Press, 

St.Paul, MN, USA. 

8


0,8 

Prediction 

0,6 

0,4 

0,2 

suspects 

confirmed 

0 

MD5A MKM mTBM 

Media 

Figure 1 Predicted proportions of suspect and PCR confirmed X. hortorum pv. carotae colonies detected on 

each medium for all laboratories and seed lots 1 . 

1 

Prediction 

0,8 

0,6 

0,4 

0,2 

MD5A 

MKM 

mTBM 

0 

1 2 3 4 5 6 7 9 10 

Laboratories 

Figure 2 Predicted proportions of suspect X. hortorumpv. carotae colonies detected in carrot seed extract for 

each laboratory and medium 1 . 

10000 

8000 

Prediction 

6000 

4000 

2000 

suspects 

confirmed 

0 

MD5A MKM mTBM 

Media 

Figure 3 Predicted counts (CFU/ml) of suspect and PCR confirmed X. hortorum pv. carotae colonies detected 

on each medium for all laboratories and seed lots. 

1 Note error bars are equivalent to standard errors 

9

M. Asma 

20000 

Prediction 

15000 

10000 

5000 

MD5A 

MKM 

mTBM 

0 

1 2 3 4 5 6 7 9 10 

Laboratories 

Figure 4 Predicted counts (CFU/ml) of suspect X. hortorum pv. carotae colonies detected in carrot seed extract 

for each laboratory. 

Prediction (ln) 

12 

10 

8 

6 

4 

2 

0 

P4920 P4925 P4926 P4932 P4933 

Seed lots 

suspects 

confirmed 

Figure 5 Natural logarithm of the predicted counts (CFU/ml) of suspect and PCR confirmed X. hortorum pv. 

carotae colonies detected in carrot seed extract for each seed lot. 

Prediction 

16000 

14000 

12000 

10000 

8000 

6000 

4000 

2000 

0 

1 2 3 4 5 6 7 9 10 

Laboratories 

suspects 

confirmed 

Figure 6 Predicted counts (CFU/ml) of suspect and PCR confirmed X. hortorum pv. carotae colonies detected 

in carrot seed extract for each laboratory 1 . 

10



14 

12 

10 

8 

6 

4 

2 

0 

MD5A MKM mTBM 

Media 

other 

Figure 7 Natural logarithm of the predicted counts (CFU/ml) of other colonies detected in carrot seed extract 

for all laboratories and seed lots. 


14 

12 

10 

8 

6 

4 

2 

0 

1 2 3 4 5 6 7 9 10 

Laboratories 

MD5A 

MKM 

mTBM 


for each laboratory. 


14 

12 

10 

8 

6 

4 

2 

0 

P4920 P4925 P4926 P4932 P4933 

Seed lots 

MD5A 

MKM 

mTBM 


for each seed lot. 

11

M. Asma 

Table 1 Number of sub samples of 10,000 seeds with PCR confirmed X. hortorum pv. carotae colonies 

detected in carrot seed extract and diluted on MD5A, MKM and mTBM media for each laboratory and seed lot * . 

P4925 (high level) P4926 (healthy) P4920 (low level) P4932 (low level) P4933 (low level) all seed lots 

Lab MD5A MKM mTBM MD5A MKM mTBM MD5A MKM mTBM MD5A MKM mTBM MD5A MKM mTBM MD5A MKM mTBM 

1 2 2 2 0 1 0 1 3 3 2 1 1 3 2 1 8 9 7 

2 2 2 2 0 0 0 0 0 0 2 5 3 4 4 4 8 11 9 

3 2 2 1 0 0 0 0 0 0 3 3 3 3 5 4 8 10 8 

4 2 2 2 0 0 0 1 1 1 3 3 3 4 5 5 10 11 11 

5 2 2 2 0 0 0 2 1 2 5 5 2 4 4 3 13 12 9 

6 2 2 1 0 0 0 0 1 0 2 2 3 2 2 2 6 7 6 

7 1 2 2 0 0 0 0 1 0 2 2 2 1 1 2 4 6 6 

8 2 2 2 0 0 0 1 1 0 3 3 3 3 3 3 9 9 8 

9 0 2 2 1 0 0 0 0 0 2 5 5 2 4 3 5 11 10 

10 2 2 2 0 0 0 1 1 1 2 3 3 3 4 5 8 10 11 

total 17 20 18 1 1 0 6 9 7 26 32 28 29 34 32 79 96 85 

* The number of sub samples tested for the seed lots P4925, P4926, P4920, P4932 and P4933 was 2, 2, 5, 5 and 

5 respectively 

12


Table 2 Number of PCR tested and PCR confirmed X. hortorum pv. carotae colonies detected in carrot seed 

extract for each seed lot, medium and laboratory. 

P4925 

P4925 

Lab MD5A MKM mTBM all media 

tested PCR+ tested PCR+ tested PCR+ tested PCR+ 

1 15 15 8 6 14 14 37 35 

2 12 11 12 11 3 3 27 25 

3 6 4 5 5 4 4 15 13 

4 4 4 4 4 4 4 12 12 

5 3 2 4 4 2 2 9 8 

6 3 3 7 5 5 4 15 12 

7 7 3 5 5 0 0 12 8 

8 4 4 4 3 4 3 12 10 

9 1 0 5 4 4 4 10 8 

10 17 11 11 9 12 9 40 29 

all labs 72 57 65 56 52 47 189 160 

P4926 

P4926 



1 1 0 1 1 0 0 2 1 

2 0 0 0 0 0 0 0 0 

3 1 0 0 0 0 0 1 0 

4 0 0 0 0 0 0 0 0 

5 0 0 0 0 0 0 0 0 

6 1 0 0 0 0 0 1 0 

7 0 0 0 0 0 0 0 0 

8 2 0 4 0 1 0 7 0 

9 1 1 1 0 0 0 2 1 

10 3 0 18 0 11 0 32 0 

all labs 9 1 24 1 12 0 45 2 

P4920 

P4920 



1 10 4 29 7 14 6 53 17 

2 0 0 6 0 0 0 6 0 

3 8 0 19 0 2 0 29 0 

4 10 1 4 2 11 2 25 5 

5 10 3 2 2 8 2 20 7 

6 7 0 9 1 4 0 20 1 

7 16 0 18 1 9 0 43 1 

8 9 1 10 1 10 0 29 2 

9 11 0 4 0 10 0 25 0 

10 35 4 47 6 35 2 117 12 

all labs 116 13 148 20 103 12 367 45 

P4932 

P4932 



1 11 5 6 6 2 2 19 13 

2 18 17 16 16 15 15 49 48 

3 10 10 13 13 8 8 31 31 

4 6 6 12 12 6 6 24 24 

5 10 10 9 9 4 4 23 23 

6 4 4 4 4 6 6 14 14 

7 8 8 4 4 4 4 16 16 

8 6 6 6 6 6 6 18 18 

9 4 4 12 11 13 13 29 28 

10 19 12 22 14 14 13 55 39 

all labs 96 82 104 95 78 77 278 254 

13

M. Asma 

P4933 

P4933 



1 14 11 7 5 2 2 23 18 

2 24 24 23 23 20 20 67 67 

3 9 9 14 14 11 8 34 31 

4 8 8 9 9 12 12 29 29 

5 6 6 7 7 7 6 20 19 

6 3 3 4 3 4 4 11 10 

7 3 3 2 2 3 3 8 8 

8 8 6 6 6 6 6 20 18 

9 3 3 11 10 6 6 20 19 

10 18 17 28 27 27 27 73 71 

all labs 96 90 111 106 98 94 305 290 

14


Table 3 Analysis of deviance for the binomial data of the number suspect and PCR confirmed X. hortorum pv. 

carotae colonies detected in carrot seed extract for all media, laboratories and seed lots. 

Suspect colonies 

Confirmed 

colonies 

Factor Df Deviance Mean deviance Deviance Mean deviance 

Lot 4 230.3 57.6 479.3 119.8 

Laboratory 8 91.8 11.5 102.7 12.8 

Lot.Laboratory 32 259.2 8.1 125.5 3.9 

Lsf 126 638.1 5.1 734.2 5.8 

Media 2 8.3 4.2 55.5 27.7 

Lot.Media 8 24.6 3.1 10.4 1.3 

Laboratory.Media 16 115.0 7.2 110.2 6.9 

Lot.Laboratory.Media 64 162.9 2.5 116.1 1.8 

Residual 1218 490.0 0.4 283.3 0.2 

Table 4 Determination of statistical significant differences for lot-, laboratory- and media-effects and their 

interaction. Effect of lot and laboratory is tested against the mean deviance of samples within laboratories (Lsf) 

under the assumption that the mean deviance ratio by approximation follows an F-distribution. Media and 

interaction with media was tested against the mean deviance of lot x laboratory x medium for the binomial data. 

Factor Df Bin. Suspect colonies Bin. Confirmed 

Lot effect 4/126 11.3* 20.7* 

Laboratory effect 8/126 2.3* 2.2* 

Lot.Laboratory effect 32/126 1.6* 0.7 

Media effect 2/64 1.7 15.4* 

Lot.Media 8/64 1.2 0.7 

Laboratory.Media 16/64 2.9* 3.8* 

* statistical significant differences compared to the F-value with P < 0.05 

Table 5 Analysis of deviance for the counts of the number of suspect and PCR confirmed X. hortorum pv. 

carotae colonies detected in carrot seed extract for all media, laboratories and seed lots. 

Suspect colonies 

Total confirmed 

colonies 

Factor Df Deviance Mean deviance Deviance Mean deviance 

Lot 4 38707727 9676932 34350129 8587532 

Laboratory 8 3333094 416637 3937502 492188 

Lot.Laboratory 32 2394356 74824 1457889 45559 

Lsf 126 6653789 52392 6616344 49747 

Media 2 82134 41067 9415 4707 

Lot.Media 8 314348 39294 243263 30408 

Laboratory.Media 16 3254316 203395 3895963 243498 

Lot.Laboratory.Media 64 1383413 21616 1180496 18445 

Residual 1218 2342954 1924 2258641 1767 

15

M. Asma 



under the assumption that the mean deviance ratio by approximation follows a F-distribution. Media and 

interaction with media is tested against the mean deviance of lot x laboratory x media for the count-data (Table 3 

and 5). 

Factor Df Counts suspect colonies Counts total confirmed 

Lot effect 4/126 184.7* 172.6* 

Laboratory effect 8/126 8.0* 9.9* 

Lot.Laboratory effect 32/126 1.4 0.9 

Media effect 2/64 1.9 0.2 

Lot.Media 8/64 1.8 1.6 

Laboratory.Media 16/64 9.4* 13.2* 


Table 7 Analysis of deviance for the counts of the number of other colonies detected in carrot seed extract for 

all laboratories, media and seed lots. 

Other colonies 

Factor Df Deviance Mean deviance 

Lot 4 248096079 62024020 

Laboratory 8 45288068 5661008 

Lot.Laboratory 32 8436933 263654 

Lsf 126 10786470 85607 

Media 2 26767033 13383516 

Lot.Media 8 2325630 290704 

Laboratory.Media 16 33098643 2068665 

Lot.Laboratory.Media 64 4044239 63191 

Residual 1206 23435397 19432 



under the assumption that the mean deviance ratio by approximation follows a F-distribution. Media and 

interaction with media is tested against the mean deviance of lot x laboratory x med for the count-data (Table 3 

and 5). 

Factor Df Other colonies 

Lot effect 4/126 724.5* 

Laboratory effect 8/126 66.1* 

Lot.Laboratory effect 32/126 3.1* 

Media effect 2/64 211.8* 

Lot.Media 8/64 4.6* 

Laboratory.Media 16/64 32.7* 


16


Table 9 Reproducibility dispersion and repeatability dispersion (based on the binomial data) of the PCR 

confirmed positive X. hortorum pv. carotae colonies detected in carrot seed extract for each medium and all 

laboratories and seed lots. 

Medium Reproducibility dispersion Repeatability dispersion 

MD5A 2.76 2.73 

MKM 2.73 2.56 

mTBM 2.72 2.65 

Table 10 Reproducibility dispersion and repeatability dispersion (based on the count data) of the PCR 

confirmed positive X. hortorum pv. carotae colonies detected in carrot seed extract for each medium and all 

laboratories and seed lots. 

Medium Reproducibility dispersion Repeatability dispersion 

MD5A 34000 20423 

MKM 31658 15355 

mTBM 41218 24548 

17

ISTA Method Validation Reports - International Seed Testing ...

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