ISTA Method Validation Reports

ISTA Method Validation Reports Published by: The International Seed Testing Association P.O. Box 308 8303 Bassersdorf, CH-Switzerland Volume 2006 Copyright 2006 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 axonopodis pv. phaseoli on bean seeds 1

Remeeus, P.M. and Sheppard, J.W. (2006) ISTA Method Validation Reports 3, 1-11 Proposal for a new method for detecting Xanthomonas axonopodis pv. phaseoli on bean seeds P.M. REMEEUS 1 AND J.W. SHEPPARD 2 1 Naktuinbouw. P.O. Box 40, 2370 AA Roelofarendsveen, The Netherlands; E-mail: p.remeeus@naktuinbouw.nl 2 Canadian Food Inspection Agency. P.O. Box 960 Carling Avenue, Ottawa, Ontario, Canada Approved by ISTA SHC Summary The suitability of the semi-selective agar media MT and XCP1 for the detection of Xanthomonas axonopodis pv. phaseoli in bean seeds was evaluated in a comparative test with 7 laboratories organised by ISTA in collaboration with the International Seed Health Initiative for Vegetables (ISHI-Veg). Four naturally infected bean seed lots were used with five sub-samples of 1000 seeds giving a total of 20 sub-samples per laboratory. 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 pathogenicity testing. It was concluded that MT and XCP1 are both useful mediums for the detection of Xanthomonas axonopodis pv. phaseoli. MT was in this comparative test less selective than XCP1. However, more suspects and confirmed colonies of Xanthomonas axonopodis pv. phaseoli were found on MT versus XCP1. An advantage of MT is that also seed-borne Pseudomonads can be detected. For routine seed testing of bean both complementary semi-selective media, MT and XCP1, are recommended. Introduction Xanthomonas axonopodis pv. phaseoli and Xanthomonas axonopodis pv. phaseoli var. fuscans (Vauterin et al., 1995) (previously Xanthomonas campestris pv. phaseoli) are the causal organisms of common bacterial blight and fuscous blight of bean, respectively. They are important seed-borne diseases occurring world-wide and can cause significant losses in tropical, subtropical and temperate climates. Its wide distribution, capacity to reduce yield and difficult control makes it one of the most economically important pathogens affecting beans (Irigoyen and Garbagnoli, 1997). In the past X. axonopodis pv. phaseoli and X. axonopodis pv. phaseoli var. fuscans were considered as a single pathogen, (Bradbury, 1986). However, X. axonopodis pv. phaseoli var. fuscans isolates are in general more pathogenic than X. axonopodis pv. phaseoli isolates (Crop Protection Compendium, 2005) and several genetic studies, based on reppcr, PFGE and RFLP, revealed that it should be segregated into two subgroups and obtain distinct taxonomic status (Mkandawire et al., 2004). The morphology of fresh fuscans and non-fuscans strains of X. axonopodis pv. phaseoli appear to be similar on agar medium. However after a longer incubation the fuscans colonies are distinguished by a distinct brown pigmentation. On tyrosine medium the production of the diffusible extra cellular brown, melanin-like pigment is particularly prominent (Chan and Goodwin, 1999). Currently there is no standard seed health testing procedure for the detection of X. axonopodis pv. phaseoli and X. axonopodis pv. phaseoli var. fuscans 1 in bean seed lots. Various methods to detect Xap have been described including detection with specific phages and immunoassays but none have been sufficiently specific for accurate routine diagnosis (Sheppard et al., 1989; Crop Protection Compendium, 2005). 1 Hence forth X. axonopodis pv. phaseoli and X. axonopodis pv. phaseoli var. fuscans are collectively referred to as Xap 1

P.M. Remeeus and J.W. Sheppard A number of semi-selective media have been reported for the isolation of Xap including; MXP (Claflin et al., 1987); M-SSM (Mabagala and Saettler,1992); and MT (Goszczynska and Serfontein, 1998). These media depend on the ability of Xanthomonads to degrade starch, casein or Tween leaving clear zones of hydrolysis around the colony. A number of other agar media have been used in seed health testing laboratories to detect Xap including PTSA (peptone, tyrosine starch agar) (Van Vuurde et al., 1983); and XCP1 (McGuire et al., 1986). Some strains of Xap do not grow on some semi-selective media. For routine testing by laboratories either a nonselective media in addition to a selective media or a combination of two selective media have been commonly used. Previous ISTA/ISHI tests evaluated several semi-selective media for use in the detection of Xap in bean seed. XCP1 was consistently found to have good selection for both fuscans and non-fuscans types and was preferred by participants for ease of detection. Other media evaluated included BBD, MT, YSSM, MXP and PTSA and no significant difference was found in the detection of Xap between media. However, characteristics of some media proved more suitable for routine testing of bean seed than others: YSSM - was difficult to prepare consistently, gave lower recovery, and there was difficulty in detecting zones of hydrolysis; MXP - although recovery was good colonies were in general difficult to read and heavy saprophytic growth was noted by some participants; PTSA - provided good recovery and identification of fuscans strains but it was difficult to detect non fuscans strains; BBD - was considered to show promise and it was thought that through its modification to MT as described by Goszczynska and Serfontein, 1998 it had the additional benefit of showing promise for the detection of other major bean seed pathogens (Pseudomonas syringae pv. phaseolicola and Pseudomonas syringae pv. syringae); and XCP1 was easy to use and selective for Xap. As a result of these previous tests MT and XCP1 were chosen for this validation study. In this validation study the use of XCP1 and MT media for the detection of Xap was evaluated by seven seed-health testing laboratories, with practical experience in detecting this pathogen, from the Netherlands, France, Canada and South Africa. Materials and Methods Seed samples A range of seed lots with varying levels of natural infection were tested by the Canadian Food Inspection Agency. Four bean seed lots were chosen B, E, D, and A. The number of infected sub-samples from five sub-samples of 1000 seeds tested for each seed lot was 0, 1, 3 and 5 respectively. For the comparative test each seed lot was divided in to sub-samples of 1000 seeds based on weight. Plastic Ziploc bags containing the sub-samples of 1000 seeds were coded randomly to ensure a blind comparative test. For each seed lot and media five replicates (sub-samples) of 1000 seeds were sent to each of the seven laboratories for testing. Media MT medium (Milk Tween agar) (Goszczynska and Serfontein, 1998) was prepared by dissolving 10.0 g/l Proteose peptone #3, 0.25 g/l CaCl 2, 0.5 g/l tyrosine, 15.0 g/l of agar in one litre of distilled water. The medium was autoclaved for 15 min at 121 C. After cooling to 50 C the following ingredients were added: 40 mg/l nystatin, 80 mg/l cephalexin, 10.0 mg/l vancomycin and 10 ml/l Tween 80 (autoclaved separately) and 10.0 g/l skimmed milk (autoclaved separately). 2

New method for Xanthomonas axonopodis pv. phaseoli XCP1 medium (Xanthomonas campestris pv. phaseoli medium) (adapted from McGuire et al., 1986) was prepared by dissolving 10.0 g/l KBr, 0.25 g/l CaCl 2, 10.0 g/l soluble potato starch, 10.0 g/l peptone, with 15.0 g/l of agar in one litre of distilled water. The medium was autoclaved for 15 min at 121 C. After cooling to 50 C the following ingredients were added: 0.15 ml crystal violet (1% aqueous), 40 mg/l nystatin, 10 mg/l cephalexin, 3 mg/l fluorouracil, 0.16 mg/l tobramycin and 10 ml/l Tween 80 (autoclaved separately). To prepare YDC medium (Yeast Dextrose Chalk medium) (Wilson et al., 1967), 20.0 g dextrose, 10.0 g yeast extract, 20.0 g CaCO 3 and 15.0 g of agar were dissolved in one litre of distilled water and autoclaved for 15 min at 121 C. In order to reduce the chance of introducing variability in the comparative test media, antibiotics and other critical materials, like skim milk powder, were prepared and distributed by Canadian Food Inspection Agency in Canada and Naktuinbouw in the Netherlands. Seed washing assay Sub-samples of 1000 seeds were soaked overnight at 5 C in 600 ml sterile distilled water. A tenfold-dilution series (100, 10-1, 10-2, 10-3) in nutrient broth was prepared from each seed soak extract and 100 μl of each dilution and the undiluted extract were spread in duplicate on MT and XCP1 media. A sterility check was done by preparing a dilution series from a sample of the extraction medium, containing no seeds, and plated on each of the media as for samples. Positive control plates were prepared by spreading 100 μl of serial tenfold-dilutions of suspensions of pure cultures of two known X. axonopodis pv. phaseoli strains, fuscans and nonfuscans (XAPh.f.1; IPO 482 and XAPh.n.1; IPO 103 2 ) on each of the media. All plates were incubated at 28 C for 3-5 d and then examined for yellow mucoid, convex colonies surrounded by zones of hydrolysis. On the MT medium, colonies of X. axonopodis pv. phaseoli after 4 d are surrounded by a clear zone of starch hydrolysis and on the XCP1 medium, colonies of X. axonopodis pv. phaseoli after 4 d can be distinguished by two zones of hydrolysis; a large clear zone of casein hydrolysis and a smaller milky zone of Tween 80 lysis. After more than 5 days of incubation of colonies of X. axonopodis pv. phaseoli fuscans develops a brown pigment. The numbers of suspect X. axonopodis pv. phaseoli and all other colonies present on each plate were recorded. Colonies were considered suspect if their appearance was similar to colonies of the reference strains. If present, up to two suspect colonies from each sub-sample were sub-cultured to sectored plates of YDC, which were then incubated at 27 C for 2-3 d. Sub-cultured isolates were compared visually with the reference strain and were confirmed by pathogenicity testing (Saettler, 1971). Data analysis For each combination of laboratory, medium, seed lot, sub-sample and dilution the number of suspect XAP and other colonies were recorded. 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 pathogenicity test were used to calculate the number of confirmed positive colo- 2 Isolates can be ordered at the Plant Research International, Wageningen, The Netherlands. 3

P.M. Remeeus and J.W. Sheppard nies as the number of suspect colonies multiplied by the proportion of pathogenicity positive colonies. For example, if 100 suspect colonies are recorded on the MT medium and five are tested in the pathogenicity test, four positive in the pathogenicity test are equivalent to 80 (100 * 4/5) confirmed positives. 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. axonopodis pv. phaseoli 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 a dispersion factor of 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 laboratories. 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 All laboratories were able to detect X. axonopodis pv. phaseoli in the positive seed lots A, D and E with the exception of laboratory 1, seed lot E. Colonies of X. axonopodis pv. phaseoli were also detected and confirmed in the previously negative seed lot B. No X. axonopodis pv. phaseoli var. fuscans were recorded. The two positive isolates were recovered on both media in most laboratoriess, however some laboratories did not see good recovery of isolate XAPh.f.1; IPO 482 fuscans type on MT media. Binomial model The analysis of deviances for the number of suspect and the confirmed colonies indicated that the differences between seed lots were significant. The differences between the laboratories and media in the number of suspect colonies were also significant. Significant media x seed lot and media x laboratories interactions for the number of confirmed colonies were also detected (Tables 1 and 2). 4

New method for Xanthomonas axonopodis pv. phaseoli Table 1. Analysis of deviance for the binomial data of the number of suspect and confirmed X. axonopodis pv. phaseoli colonies detected in bean seed extract for all media, laboratories and seed lots. Suspect colonies Confirmed colonies Factor Df Deviance Mean deviance Deviance Mean deviance Lot 3 246.3 82.1 257.8 85.9 Laboratory 6 81.8 13.6 50.3 8.4 Lot.Laboratory 18 43.4 2.4 45.5 2.5 Lsf 112 277.7 2.5 329.6 2.9 Media 1 60.0 60.0 3.4 3.4 Lot.Media 3 4.0 1.3 21.5 7.2 Laboratory.Media 6 13.0 2.2 36.1 6.0 Lot.Laboratory.Media 18 0.0 0.0 0.0 0.0 Residual 392 42.5 0.1 11.1 0.03 Table 2. Determination of statistical significant differences for lot-, laboratory- and media-effects and their interaction. Effect of lot and lab 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 (2a). For the binomial data no over-dispersion occurred so the effect of media and interaction with media was tested with the deviance (of these effects) which follows a Chi-squared distribution (2b). Table 2a Factor Df Binomial Suspect colonies Binomial Confirmed colonies Lot effect 3/112 32.8* 29.6* Laboratory effect 6/112 5.4* 2.9* Lot.Laboratory effect 18/112 1.0* 0.9* * statistical significant differences compared to the F-value (p < 0.05 ) Table 2b Factor Df Binomial Suspects Deviance Binomial Confirmed Deviance Media effect 1 60.0* 3.4* Lot.Media 3 4.0* 21.5* Laboratory.Media 6 13.0* 36.1* * statistical significant differences compared to the Chi-value (p < 0.05) The predicted proportions of suspect and confirmed colonies for each medium are summarised in Figure 1. The highest predicted proportion of suspect and confirmed colonies were detected on XCP1. Figure 1 Predicted proportions of suspect and confirmed X. axonopodis pv. phaseoli colonies detected on each medium for all laboratories and seed lots 3. 3 Note error bars are equivalent to standard errors. 5

P.M. Remeeus and J.W. Sheppard 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. The standard errors were almost zero except for laboratory 7. The predicted proportions of suspect and confirmed colonies for each seed lot are summarised in Figure 3. The highest number of suspect colonies was detected on seed lot A. In Table 3 the reproducibility dispersion (between laboratory variability) and the repeatability dispersion (within laboratory variability) for the confirmed positive colonies based on the binomial data are presented. Repeatability and reproducibility were higher for MT than XCP1. Figure 2. Predicted proportions of suspect X. axonopodis pv. phaseoli colonies detected in bean seed extract for each laboratory and medium 3. Figure 3. Predicted proportions of suspect and confirmed X. axonopodis pv. phaseoli colonies detected in bean seed extract for each seed lot 3. Table 3. Reproducibility dispersion and repeatability dispersion for each medium (based on the binomial data) of the confirmed positive X. axonopodis pv. phaseoli colonies detected in bean seed extract for each medium and all laboratories and seed lots. Medium Reproducibility dispersion Repeatability dispersion MT 1.70 1.61 XCP1 1.93 1.77 6

New method for Xanthomonas axonopodis pv. phaseoli Poisson model The analysis of deviances for the number of suspect and confirmed colonies indicated that there were significant differences between seed lots, media and laboratories. However, a significant seed lot x media interaction was also detected (Tables 4 and 5). Table 4. Analysis of deviance for the counts of the number of suspect and total confirmed X. axonopodis pv phaseoli colonies detected in bean seed extract for all media, laboratories and seed lots. Suspect colonies Total confirmed colonies Factor Df Deviance Mean deviance Deviance Mean deviance Lot 3 17271898 5757299 17042375 5680792 Laboratory 6 23442800 3907133 24052884 4008814 Lot.Laboratory 18 2434244 135236 2475610 137534 Lsf 112 20301208 181260 19695339 175851 Media 1 476346 476346 470225 470225 Lot.Media 3 2112056 704019 2053171 684390 Laboratory.Media 6 85620 14270 43023 7170 Lot.Laboratory.Media 18 128175 7121 167596 9311 Residual 392 680760 1737 885631 2259 Table 5. Determination of statistical significant differences for lot-, lab- and media-effects and their interaction. Effect of lot and lab 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.laboratory.med for the count-data. Factor Df Counts suspect colonies Counts total confirmed Lot effect 3/112 31.8* 32.3* Laboratory effect 6/112 21.6* 22.8* Lot.Laboratory effect 18/112 0.7* 0.8* Media effect 1/18 66.9* 50.5* Lot.Media 3/18 98.9* 73.5* Laboratory.Media 6/18 2.0* 0.8* * statistical significant differences compared to the F-value (p < 0.05) In Figure 4, the natural logarithm of the predicted counts for laboratories on each medium is shown. Most colonies were found by laboratory 3 on MT and XCP1. In Figure 5, the predicted counts of suspect, confirmed and other colonies for media is shown. Most suspect and confirmed colonies were recorded on MT. In total the lowest number of other colonies was recorded on XCP1 and the highest on MT. In Table 6 the reproducibility dispersion (between laboratory variability) and the repeatability dispersion (within laboratory variability) for the count data are presented. Repeatability and reproducibility were higher for XCP1 than MT. 7

P.M. Remeeus and J.W. Sheppard Figure 4 Natural logarithm of the predicted counts (CFU/ml) of suspect X. axonopodis pv. phaseoli colonies detected in bean seed extract for each laboratory. Figure 5. Predicted counts (CFU/ml) of suspect and confirmed X. axonopodis pv. phaseoli colonies detected in bean seed extract for each medium 3. Table 6. Reproducibility dispersion and repeatability dispersion for each medium (based on the count data) of the confirmed positive X. axonopodis pv. phaseoli colonies detected in bean seed extract for each medium and all laboratories and seed lots. Medium Reproducibility dispersion Repeatability dispersion MT 250558 2073 XCP1 95138 526 Discussion All participants (with the exception of lab 1, sample E) detected Xap in the three infected bean seed lots A, D and E. Three laboratories detected and confirmed the presence of Xap in the negative seed lot B, of the 70 sub-samples tested overall four were found to be positive. In the pre-test a relatively small number of seeds (5 000) were tested. In the more extensive comparative test many more seeds (35 000) from the negative seed lot were tested. It is possible that a low level of infection existed in the lot and that the number of contaminated seeds is lower than 1 out of 5 000 but higher than 1 out of 35 000. The results of the Binomial data analysis and the Poisson data analysis both show a significant effect of the lot on suspect and confirmed positive colonies. The effect of laboratory and media was significant in both data analyses for suspect colonies but was only significant in the Poisson analysis for confirmed positive colonies. However a significant lot x media and lab x media 8

New method for Xanthomonas axonopodis pv. phaseoli effect was found with the binomial analysis of the confirmed positive colonies whereas only the lot x media was significant for both suspect and confirmed colonies with Poisson analysis. A lot x media interaction is not unexpected as the micro flora associated with bean seed will vary from one lot to another and the different media will control this micro flora in a different way. A few laboratories commented that it was more difficult to recognise Xap colonies on MT medium compared with XCP1 medium. Where laboratories are unfamiliar with a new medium it is not unusual for them to have difficulties in identifying how the organism grows on the new medium. However, overall MT medium had the highest count of confirmed positives in this comparative test. XCP1 medium on the other hand had fewer other colonies than MT medium, suggesting it was more selective for Xap than MT medium. Despite being more selective XCP1 medium provides better recovery of the fuscans type of X. axonopodis pv. phaseoli than MT medium (pers. comm. H. Koenraadt). This was demonstrated in the results for the positive control plates where recovery of the fuscans strain (XAPh.f.1:IPO 482) was higher on XCP1 medium than MT medium. Unfortunately no seed lots with the fuscans type of X. axonopodis pv. phaseoli were available for this comparative test. Although the dispersions based on the binomial analysis showed that MT performed better in different laboratories and within laboratories due to the lower reproducibility dispersion and repeatability dispersion. Dispersion based on the count data showed that XCP1 performed better in different laboratories and within laboratories due to a lower reproducibility dispersion and repeatability dispersion. XCP1 showed benefits in selectivity and its ability to pick up the fuscans strain more readily than MT whereas MT has the ability to also detect Pseudomonas savastanoi pv. phaseolicola. It is therefore recommended that the two complementary semiselective media are used for routine testing of Xap. Media preparation To prevent introducing variability in the comparative test due to the ingredients of the test media, two laboratories prepared and distributed the individual ingredients. The source of milk powder is critical for MT medium. The quality of milk powder is vital to develop the hydrolysis of starch in MT medium. Two milk sources that work well are Oxoid and Sigma brands. Cycloheximide was widely used to control fungal contamination in semi-selective media for the detection of bacteria. However in recent years the availability of cycloheximide has been in question. It is also expensive and very toxic. Nystatin was identified by seed health testing laboratories as a suitable alternative for controlling fungal contamination (Asma, 2005). In this comparative test nystatin was used effectively in place of cycloheximide for MT medium at 40 ppm. However one laboratory did not agree and commented that with nystatin fungi were present on some plates whereas when they used cyclohexamide in repeat tests no fungi were present. Dilution buffer Nutrient broth was selected as the dilution buffer for this comparative test. Since this experiment, comparative tests for other bacterial bean pathogens have been completed, particularly for the detection of P. savastanoi pv. phaseolicola in bean seed. For P. savastanoi pv. phaseolicola saline (0.85%) is used in preference to nutrient broth. In order to assist laboratories to combine the tests for X. axonopodis pv. phaseoli and P. savastanoi pv. phaseolicola and therefore reduce costs. An additional experiment using 2 infected seed samples and 10 sub-samples was conducted in one laboratory to compare the number of suspect colonies found when 0.85% 9

P.M. Remeeus and J.W. Sheppard saline was used instead of nutrient broth as the dilution buffer. There is no significant difference between the number of suspects found using either 0.85% saline or nutrient broth (Figure 6). Therefore, saline is recommended as a dilution buffer for this method (Olivier and Remeeus, 2004). Figure 6 Predicted proportions of suspect X. axonopodis pv. phaseoli colonies detected in bean seed extract for each dilution buffer 3. Conclusions and Recommendations Both semi-selective media are suitable for the detection of X. axonopodis pv. phaseoli on bean seed. The highest number of confirmed positive colonies was recorded on MT. However, the number of other colonies on XCP1 was lower compared to MT and growth of X. axonopodis pv. phaseoli var fuscans is better on XCP1. With regard to analysis of repeatability and reproducibility, dispersion values from count data (Poisson) and binomial data do not correspond, with one analysis favouring XCP1 and the other favouring MT. So for routine testing of bean seed lots it is recommended to use the two semi-selective media, MT and XCP1. To combine the detection of Xap and P. savastanoi pv. phaseolicola in one test saline must be used as the extraction buffer. Acknowledgements The input of the participating laboratories, ISHI-Veg ITG Bean and Steve Roberts (HRI) for their help in the statistical analysis of the results is greatly acknowledged. References Asma, M. 2005. Proposal for a new method for detecting Xanthomonas hortorum pv. carotae on carrot seeds. ISTA Method Validation reports 2, 1-17. Bradbury, J.F. 1986. Guide to Plant Pathogenic bacteria, Wallingford, UK, CAB International. Chan, J.W.Y.F. and P.H. Goodwin. 1999. Differentiation of Xanthomonas campestris pv. phaseoli from Xanthomonas campestris pv. phaseoli var. fuscans by PFGE and RFLP. European Journal of Plant Pathology 105: 867-878. Claflin, L.E., Vidaver, A.K. and M. Sasser. 1987. MXP, a semi-selctive medium for Xanthomonas campestris pv. phaseoli. Phytopathology 77(5): 730-734. Crop Protection Compendium. 2005. Crop Protection Compendium 2005 Edition (Web-based). Wallingford, UK, CAB International. Goszczynska, T. and J.J. Serfontein. 1998. Milk-Tween agar, a semiselective medium for 10

New method for Xanthomonas axonopodis pv. phaseoli isolation and differentiation of Pseudomonas syringae pv. syringae, Pseudomonas syringae pv. phaseolicola and Xanthomonas axonopodis pv. phaseoli. Journal of Microbiological Methods 32: 65-72. Irigoyen, E.D. and C. Garbagnoli. 1997. Common bacteriosis in bean (Xanthomonas campestris pv. phaseoli [E.F. Smith] Dowson): detection, infection and transmission through seeds. Fitopatologa. Mabagala, R.B. and A.W. Saettler. 1992. An improved semiselective medium for recovery of Xanthomonas campestris pv. phaseoli. Plant Disease 76(5): 443-446. McGuire, R.G., Jones, J.B., Sasser, M. 1986. Tween media for semi selective isolation of Xanthomonas campestris pv. vesicatoria from soil and plant material. Plant Disease 70: 887-891. Mkandawire, A.B.C, Mabagala, R.B., Guzmán, P. and R.L. Gilbertson. 2004. Genetic diversity and pathogenic variation of common blight bacteria (Xanthomonas campestris pv. phaseoli and X. campestris pv. phaseoli var. fuscans) suggests pathogen co evolution with the common bean. Phytopathology 94: 593-603. Olivier, V. and P.M. Remeeus. 2004. Additional experiment for the extraction and dilution buffer for the detection of Xanthomonas axonopodis pv. phaseoli in bean seeds. ISHI Report, Naktuinbouw, Research report 0305XAP. 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. Saettler, A.W. 1971. Seedling injection as an aid to identifying bean blight bacteria. Plant Disease Reporter 55: 703-706. Sheppard JW, Roth DA, Saettler AW, 1989. Detection of Xanthomonas campestris pv. phaseoli. In: Saettler AW, Schaad NW, Roth DA, eds. Detection of bacteria in seed and other planting material. St. Paul, USA: APS Press, 17-29. Van Vuurde, J.W.L., Van den Bovenkamp, G.W. and Birnbaum, Y. 1983. Immunofluorescence microscopy and enzyme-linked immunosorbent assay as a potential routine tests for the detection of Pseudomonas syringae pv. phaseolicola and Xanthomonas campestris pv. phaseoli in bean seeds. Seed Science and Technology 11: 547-559. Vauterin, L., Hoste, B., Kersters, K. and J. Swings. 1995. Reclassification of Xanthomonas. International Journal of Systematic Bacteriology 45(3): 472-489. Wilson, E.E., Zeitoun, F.M. and Fredrickson, D.L. (1967) Bacterial phloem canker, a new disease in Persian walnut trees. Phytopathology 57: 618-621. 11