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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: [email protected] 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

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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 MgSO4.7H2O, 1.0 g/l NaH2PO4, 1.0 g/l NH4Cl, 3.0 g/l K2HPO4 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 NH4Cl, 1.2 g/l K2HPO4, 1.2 g/l KH2PO4, 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 H3BO3, 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 CaCO3 with 15.0 g/l of agar in one litre of distilled water. YDC medium was autoclaved for 15 min at 121°C.

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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

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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 log-link 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.

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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

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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

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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 K2HPO4 and KH2PO4 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.

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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 PCR-based 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.

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Figure 1 Predicted proportions of suspect and PCR confirmed X. hortorum pv. carotae colonies detected on each medium for all laboratories and seed lots1.

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Figure 2 Predicted proportions of suspect X. hortorumpv. carotae colonies detected in carrot seed extract for each laboratory and medium1.

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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.

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

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Figure 6 Predicted counts (CFU/ml) of suspect and PCR confirmed X. hortorum pv. carotae colonies detected in carrot seed extract for each laboratory1.

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Figure 7 Natural logarithm of the predicted counts (CFU/ml) of other colonies detected in carrot seed extract for all laboratories and seed lots.

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Figure 8 Natural logarithm of the predicted counts (CFU/ml) of other colonies detected in carrot seed extract for each laboratory.

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Figure 9 Natural logarithm of the predicted counts (CFU/ml) of other colonies detected in carrot seed extract for each seed lot.

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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*.

Lab MD5A MKM mTBM MD5A MKM mTBM MD5A MKM mTBM MD5A MKM mTBM MD5A MKM mTBM MD5A MKM mTBM1 2 2 2 0 1 0 1 3 3 2 1 1 3 2 1 8 92 2 2 2 0 0 0 0 0 0 2 5 3 4 4 4 8 11 93 2 2 1 0 0 0 0 0 0 3 3 3 3 5 4 8 10 84 2 2 2 0 0 0 1 1 1 3 3 3 4 5 5 10 11 15 2 2 2 0 0 0 2 1 2 5 5 2 4 4 3 13 126 2 2 1 0 0 0 0 1 0 2 2 3 2 2 2 6 77 1 2 2 0 0 0 0 1 0 2 2 2 1 1 2 4 68 2 2 2 0 0 0 1 1 0 3 3 3 3 3 3 9 99 0 2 2 1 0 0 0 0 0 2 5 5 2 4 3 5 11 110 2 2 2 0 0 0 1 1 1 2 3 3 3 4 5 8 10 1total 17 20 18 1 1 0 6 9 7 26 32 28 29 34 32 79 96 85

all seed lotsP4925 (high level) P4932 (low level) P4933 (low level)P4926 (healthy) P4920 (low level)

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

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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.

Labtested PCR+ tested PCR+ tested PCR+ tested PCR+

1 15 15 8 6 14 14 37 32 12 11 12 11 3 3 27 23 6 4 5 5 4 4 154 4 4 4 4 4 4 125 3 2 4 4 2 2 96 3 3 7 5 5 4 157 7 3 5 5 0 0 128 4 4 4 3 4 3 129 1 0 5 4 4 4 1010 17 11 11 9 12 9 40 29all labs 72 57 65 56 52 47 189 160

all mediaP4925P4925

MD5A MKM mTBM

55

13128

128

108


1 1 0 1 1 0 0 22 0 0 0 0 0 0 03 1 0 0 0 0 0 14 0 0 0 0 0 0 05 0 0 0 0 0 0 06 1 0 0 0 0 0 17 0 0 0 0 0 0 08 2 0 4 0 1 0 79 1 1 1 0 0 0 210 3 0 18 0 11 0 32 0all labs 9 1 24 1 12 0 45 2

P4926 P4926MD5A MKM mTBM all media

100000001


1 10 4 29 7 14 6 53 172 0 0 6 0 0 0 63 8 0 19 0 2 0 294 10 1 4 2 11 2 25 55 10 3 2 2 8 2 206 7 0 9 1 4 0 207 16 0 18 1 9 0 438 9 1 10 1 10 0 299 11 0 4 0 10 0 25 010 35 4 47 6 35 2 117 12all labs 116 13 148 20 103 12 367 45


00

7112


1 11 5 6 6 2 2 19 12 18 17 16 16 15 15 49 43 10 10 13 13 8 8 31 34 6 6 12 12 6 6 24 25 10 10 9 9 4 4 236 4 4 4 4 6 6 147 8 8 4 4 4 4 168 6 6 6 6 6 6 189 4 4 12 11 13 13 29 210 19 12 22 14 14 13 55 39all labs 96 82 104 95 78 77 278 254


3814

231416188

13

M. Asma


1 14 11 7 5 2 2 23 12 24 24 23 23 20 20 67 63 9 9 14 14 11 8 34 314 8 8 9 9 12 12 29 25 6 6 7 7 7 6 206 3 3 4 3 4 4 117 3 3 2 2 3 3 88 8 6 6 6 6 6 209 3 3 11 10 6 6 20 110 18 17 28 27 27 27 73 71all labs 96 90 111 106 98 94 305 290


87

919108

189

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 devianceLot 4 230.3 57.6 479.3 119.8Laboratory 8 91.8 11.5 102.7 12.8Lot.Laboratory 32 259.2 8.1 125.5 3.9Lsf 126 638.1 5.1 734.2 5.8Media 2 8.3 4.2 55.5 27.7Lot.Media 8 24.6 3.1 10.4 1.3Laboratory.Media 16 115.0 7.2 110.2 6.9Lot.Laboratory.Media 64 162.9 2.5 116.1 1.8Residual 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 devianceLot 4 38707727 9676932 34350129 8587532Laboratory 8 3333094 416637 3937502 492188Lot.Laboratory 32 2394356 74824 1457889 45559Lsf 126 6653789 52392 6616344 49747Media 2 82134 41067 9415 4707Lot.Media 8 314348 39294 243263 30408Laboratory.Media 16 3254316 203395 3895963 243498Lot.Laboratory.Media 64 1383413 21616 1180496 18445Residual 1218 2342954 1924 2258641 1767

15

M. Asma

Table 6 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 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* * statistical significant differences compared to the F-value with P < 0.05 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 coloniesFactor Df Deviance Mean devianceLot 4 248096079 62024020Laboratory 8 45288068 5661008Lot.Laboratory 32 8436933 263654Lsf 126 10786470 85607Media 2 26767033 13383516Lot.Media 8 2325630 290704Laboratory.Media 16 33098643 2068665Lot.Laboratory.Media 64 4044239 63191Residual 1206 23435397 19432 Table 8 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 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* * statistical significant differences compared to the F-value with P < 0.05

16


17

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

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