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Keywords:

  • bacterial culturing;
  • phytosanitary;
  • seed sampling;
  • seedborne;
  • selective media;
  • vacuum extraction

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Xanthomonas axonopodis pv. phaseoli (Xap) is an important seedborne pathogen of Phaseolus vulgaris. Accurate seed health testing methods are critical to protect seed quality and meet phytosanitary requirements. Currently employed selective media-based methods include several variations in extraction procedures. In order to optimize pathogen extraction from seeds, the influence of different extraction steps on the sensitivity of Xap detection was assessed. Seeds were inoculated by vacuum infiltration with Xap to achieve inoculum levels from 101 to 105 CFU per seed; one contaminated seed was mixed into 1000-seed subsamples of uncontaminated P. vulgaris seeds. Thirty subsamples of 1000 seeds were tested using each different extraction procedure. These included soaking whole seeds in sterilized saline phosphate buffer, either overnight at 4°C or for 3 h at room temperature, with or without vacuum extraction, and either with or without concentrating the seed extract by centrifuging. Seed extract dilutions were cultured on semiselective agar media MT and XCP1. The percentages of positive subsamples were compared to measure the effects of each extraction step on detection sensitivity. Vacuum extraction and centrifugation of seed extracts increased sensitivity; the highest sensitivity was obtained with the 3 h vacuum extraction followed by centrifugation. These results were confirmed with naturally infested seeds; Xap was detected in 48 of 70 samples using the 3 h vacuum extraction with centrifugation, whereas only 35 of 70 field samples tested positive using overnight soaking, a significant difference. The results suggest that these steps would be valuable modifications to the current method approved by the International Seed Testing Association (ISTA).


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Xanthomonas axonopodis pv. phaseoli (Xap) and Xanthomonas axonopodis pv. phaseoli var. fuscans (Vauterin et al., 1995) cause common bacterial blight of bean (Phaseolus vulgaris), one of the most destructive diseases in bean production worldwide (Saettler, 1989). Xap var. fuscans can be distinguished by a diffusible brown pigment produced in culture media. The two variants of Xap have also been distinguished by isoenzyme profiling (El-Sharkawy & Huisingh, 1971), plasmid profiling (Lazo & Gabriel, 1987), DNA–DNA hybridization (Hildebrand et al., 1990), and several PCR-based approaches (Birch et al., 1997; Toth et al., 1998; Mahuku et al., 2006). Xap and Xap var. fuscans are both important due to impact on seed quality and phytosanitary concerns. Some studies have indicated that Xap var. fuscans isolates are more aggressive than Xap (Mutlu et al., 2008) but others have found no relationship between virulence on beans and the capacity to produce dark pigment in culture media or the geographical origin of the isolates (Vieira & Souza, 2000; Vieira et al., 2008).

Seedborne bacteria are the primary inoculum sources for outbreaks of common blight (Saettler, 1989), and bacteria in/on the seed can survive longer than the seed itself (Dreo et al., 2003). Infection of approximately 1 in 10 000 seeds (Sutton & Wallen, 1970) or Xap populations as low as 10 CFU per seed (Opio et al., 1993) were capable of outbreaks of common bacterial blight. There is a positive correlation between seed symptoms and the population of Xap per seed (Opio et al., 1993). A range of sample sizes from 4000 to 45 000 is used in seed quality programmes in US seed companies to ensure that the pathogen is not transmitted by commercial seed lots (Maddox, 1997).

Several different approaches have been used for detecting Xap in seeds, including a dome test (Venette et al., 1987), phage test (Kahveci & Maden, 1994), selective media (Mabagala, 1992; Remeeus & Sheppard, 2006; Sheppard et al., 2007), PCR (Audy et al., 1994), and serology such as indirect immunofluorescence microscopy and ELISA (Wong, 1991) or immunostaining and flow cytometry (Tebaldi et al., 2010). Only a few of the published methods have been accepted as routine diagnostic tools. For routine seed testing of bean, two complementary semiselective media, MT and XCP1, are recommended. It has been reported that MT is less selective but more sensitive, and can also be used to detect other seedborne bacterial pathogen pseudomonads (Remeeus & Sheppard, 2006). However, another report found XCP1 more efficient than MT in both quantification and detection of Xap in whole bean seed extracts (Tebaldi et al., 2007).

Published seed health testing methods for these pathogens and other seedborne xanthomonads include several variations in the procedure for extracting bacterial cells from seeds. Some methods have involved seed crushing (Maringoni et al., 1998), while most employ soaking whole seeds (Valarini & Menten, 1992; Valarini et al., 1992). The optimal extraction method for Xanthomonas hortorum pv. carotae was suggested as overnight (16–18 h) incubation at 4–7°C (Asma, 2005). The same method was used for Xanthomonas spp. detection from onion seeds (Roumagnac et al., 2000; Sakthivel et al., 2001). The current seed health test method for both variants of Xap, approved by the International Seed Testing Association (ISTA), includes soaking whole seeds overnight in saline buffer at 5°C (±4°C) (Sheppard et al., 2007).

The U.S. National Seed Health System (NSHS) is authorized by the United States Department of Agriculture, Animal and Plant Health Inspection Service (USDA-APHIS) to provide resources to assist seed companies in meeting phytosanitary regulations. As part of its mission, NSHS convenes technical review panels for the evaluation of seed health testing methods. A NSHS review panel evaluated eight published or industry-adopted seed health tests for Xap. The review panel recommended further research on optimization of seed extraction methods for selective media assays. Variations among published assays included seed soaking time and temperature, the use of vacuum extraction, or concentration of the seed extract by centrifugation. The objective of this study was to follow NSHS review panel recommendations by evaluating the effects of incubation time/temperature, vacuum extraction and centrifugation on detection sensitivity for Xap and Xap var. fuscans in bean seeds.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Bacterial strains and growth conditions

The bacterial isolate used in this study to inoculate Phaseolus vulgaris beans was Xap B1, originally from Idaho, USA. Bacterial cultures were grown routinely on nutrient agar (Becton, Dickinson and Company) or YDC medium. Isolation of Xap from naturally infested seed or inoculated seed or plants was conducted on semiselective agar media MT (Goszczynska & Serfontein, 1998; Sheppard et al., 2007) and XCP1 (McGuire et al., 1986; Sheppard et al., 2007). Bacterial cultures were enriched on nutrient agar and YDC agar. Long-term, bacteria were stored on silica beads (Microbank) by shaking beads in a cell suspension, removing excess fluid and freezing at −80°C.

Pathogenicity tests

Seeds of Charlevoix dark red kidney bean (Phaseolus vulgaris), a bean genotype susceptible to common bacterial blight, were planted under greenhouse conditions in pasteurized soil in 20 cm pots. Plants were allowed to grow until the first true leaf was fully established. Plants were watered 2 h before inoculation to ensure sufficient moisture. A sterile needle was contaminated by streaking through colonies from 2-day-old cultures grown on YDC. Seedlings were inoculated by stabbing through the primary node at an angle of about 45° with a contaminated needle. Non-inoculated control plants were wounded with a sterile needle. A culture of a known pathogenic strain (isolate B1) of Xap was used to inoculate plants as a positive control. Plants were covered with transparent plastic bags to maintain humidity at >90%. Symptom development was observed and recorded 10 days after inoculation. The pathogens were reisolated from leaf tissues adjacent to the lesions by culturing on XCP1 semiselective medium. Suspect colonies from infested seed lots were tested using this method and symptoms were recorded and evaluated.

Comparison of extraction methods – inoculated seeds

Bean seeds (Phaseolus vulgaris) (cv. Derby, Harris Moran Seed Company), free from contamination by Xap or Xap var. fuscans, were used for these experiments. The seed lot was produced in an area free from Xap, and had been tested by the supplier to confirm absence of the pathogen. The lack of contamination was further confirmed by testing five subsamples of 1000 seeds using the ISTA-approved method (Sheppard et al., 2007). The efficiency of extraction methods was compared for the recovery of Xap cells from artificially inoculated bean seed. The inoculum was made with phosphate-buffered sterile saline solution (0·8% NaCl, 0·02% KCl, 0·17% N2HPO4 +  KH2PO4, pH 7·4) from 2-day-old Xap isolate B1. Original bacterial concentrations were adjusted spectrophotometrically [ODλ600 = 0·6 (≈1 × 109 CFU mL−1)]. Ten-fold dilution series (104–109 CFU mL−1) were made from the original inoculum. Bean seed samples were artificially inoculated by vacuum-infiltration in bacterial suspensions of each concentration for 5 min. Subsequently, seeds were air-dried in a laminar flow hood until excess water had evaporated. In order to confirm inoculation dose, 10 infested seeds from each concentration were soaked individually in 1 mL sterile saline solution (0·85% NaCl, 0·02% Tween 20) at 4°C overnight. One hundred microlitres of seed wash were spread on MT media, two replicates for each seed. After incubation for 2–3 days, colonies were counted and actual infestation level (CFU per seed) was calculated.

Subsamples of 1000 P. vulgaris seeds were used to test extraction method efficiency. One inoculated seed was mixed with 999 clean seeds, using a seed-counting board. The 1000-seed weight was then determined for this seed lot in order to calculate extraction solution volume. Ten replicate subsamples were tested for each treatment and each comparison was repeated three times (total of 30 subsamples per treatment). In one comparison, incubation time/temperature and vacuum extraction were evaluated with inoculum levels of 102, 103 and 105 CFU per seed (inoculum dose per inoculated seed). Incubation treatments were: 4°C overnight, room temperature 3 h and room temperature 3 h with vacuum. Overnight incubation consisted of 18 h duration in a coldroom programmed for 4°C (±2°). Room temperature ranged from 20 to 24°C. Saline volume was 700 mL (2·5 times the 1000-seed weight) (Sheppard et al., 2007). For the vacuum extraction treatment, a vacuum pump aspirator (Nalgene) was applied on flasks containing soaking seeds for 30 min to create low air pressure and the pressure was maintained for 3 h. In another comparison, centrifugation was evaluated using inoculum levels of 101 and 102 CFU per seed. This was a factorial experiment with two incubation conditions (room temperature for 3 h or 4°C overnight), with or without centrifugation of the seed extract. For the centrifugation step, the total seed wash was centrifuged in sealed sterile centrifuge tubes at 5000 g for 10 min immediately after incubation had finished. The pellet was resuspended in 5 mL 0·85% NaCl solution, a dilution series was prepared, and 100 μL aliquots were cultured on selective media (MT and XCP1). A dilution series of the extraction buffer was included to confirm its sterility. Petri dishes were incubated at 28°C (±2°C) for up to 5 days. Initial observations were made after 2 days, and final observations were recorded after 4–5 days. Target colonies were counted to measure detection sensitivity and extraction efficiency. On MT, Xap colonies are yellow, distinguished by two zones of hydrolysis: a large clear zone of casein hydrolysis and a smaller milky zone of Tween 80 lysis. On XCP1, Xap colonies are yellow, glistening and surrounded by a clear zone of starch hydrolysis (Sheppard et al., 2007). The bacterium was not detected in extracts of clean seeds from the original seed lot that were used as controls. Detection sensitivity was recorded as the percentage of subsamples testing positive with each treatment. Treatments (mean percentages for three replications of each comparison) were compared using sas (Statistical Analysis Software) anova procedure GLM and mean separation by Tukey’s method ( 0·05 considered significant). Percentage data were arcsine-square-root transformed in order to meet anova assumptions. Results are presented as untransformed means.

Comparison of extraction methods – naturally infested seeds

Seed lots known to be naturally infested with common bacterial blight were obtained from various geographic origins within the United States (Table 1). Samples of varying sizes were divided into subsamples of 1000 seeds, using a seed-counting board. Subsamples were then weighed in order to calculate extraction solution volumes. An equal number of subsamples were tested by each of two treatments: the ISTA method (Sheppard et al., 2007) (4°C overnight incubation with neither vacuum nor centrifuge application); or a 3 h incubation at room temperature with vacuum and centrifuge application (modifications to ISTA method based on inoculated-seed experiments). Dilution series of the seed extracts were cultured on the semiselective media MT and XCP1. A dilution series of the extraction buffer was included to confirm its sterility. Petri dishes were incubated at 28°C (±2°C) for up to 5 days. Initial observations were made after 2 days, and final observations were recorded after 4–5 days. Xap colonies were counted and recorded; suspect colonies were confirmed by culturing on YDC medium and pathogenicity testing. The bacterium was not detected in extracts of clean seeds from the original seed lot that were used as controls. Detection sensitivity (proportion of samples testing positive) of each treatment for all subsamples was compared by a chi-square test.

Table 1.   Seed lots from various geographical origins naturally infested with common bacterial blight and used in this study to compare seed extraction methods
Seed lotOriginTypeCultivarSeed lotsSubsamples
  1. NS: not specified; PNW: Pacific North West.

1IARedTrue Red Cranberry112
2IAPolePurple Podded Pole18
3IAGreenBountiful 33716
4PNWGreenThoroughbred110
5–6PNWGreenValentino210, 10
7–8PNWGreenHercules210, 10
9CARed KidneyMontcalm110
10COPintoPoncho120
11–13WIRed KidneyNS34, 6, 8
14NSFrenchNS116

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Inoculated seeds

No significant differences were observed in the percentages of positive subsamples between 3 h room temperature incubation and overnight 4°C incubation (Fig. 1). Vacuum application during the 3 h room temperature extraction procedure typically increased detection sensitivity, but the difference was significant only for the 105 CFU per seed inoculum level (< 0·05) (Fig. 1).

image

Figure 1.  Pathogen detection sensitivity (% of samples testing positive) for seed extraction methods differing in incubation time, temperature and vacuum application for seed samples artificially inoculated with Xanthomonas axonopodis pv. phaseoli B1 (one inoculated seed per 1000-seed subsample). Error bars indicate standard errors. Within each inoculum level, bars with the same letter are not significantly different, according to the Tukey test (< 0·05).

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Centrifugation enhanced sensitivity for both incubation conditions, but the difference was significant (< 0·05) only for the 3 h room temperature incubation. The centrifugation step more than doubled the sensitivity. Room temperature incubation with centrifugation had the highest sensitivity, also significantly higher than the 4°C overnight incubation (< 0·05) (Fig. 2).

image

Figure 2.  Pathogen detection sensitivity (% of subsamples testing positive) for seed extraction at room temperature (20–24°C) and 4°C incubation with or without centrifugation of seed extract for seed samples artificially inoculated with Xanthomonas axonopodis pv. phaseoli B1 (one inoculated seed per 1000-seed subsample). Error bars indicate standard errors. Within each inoculum level, bars with the same letter are not significantly different, according to the Tukey test (< 0·05).

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Naturally infested seeds

The modified extraction treatment resulted in higher detection sensitivity than the original extraction method. For nine of the 14 naturally infested seed lots, the modified method identified a higher proportion of positive subsamples and thus showed improved detection sensitivity. For three of the seed lots (lots 4, 6 and 13), both methods detected equivalent proportions of positive subsamples, and there were two seed lots (lots 9 and 11) for which the ISTA method detected a higher proportion of positive subsamples. Overall, 35 of 70 subsamples were positive by the ISTA-approved method, and 48 of 70 subsamples were positive by the modified method (Table 2). The difference was significant (< 0·05). Both methods correctly detected infestation in at least one subsample from 13 of the 14 seed lots. Suspect colonies were confirmed by culturing on YDC and pathogenicity testing.

Table 2.   Pathogen detection sensitivity following seed extraction using the ISTA-approved method and modified method on seed lots naturally infested with Xanthomonas axonopodis pv. phaseoli, evaluated by culturing on selective media. Each entry is the proportion of positive 1000-seed subsamples over the total number of subsamples
Seed lotExtraction method
ISTA methodaModified methodb
  1. aOvernight incubation at 4°C without vacuum or centrifugation.

  2. bThree-hour incubation at room temperature with vacuum extraction and centrifugation of seed extract.

  3. cMethods were significantly different (chi-square test, < 0·05).

 12/64/6
 21/44/4
 31/32/3
 44/54/5
 53/55/5
 63/53/5
 72/53/5
 84/55/5
 94/53/5
106/108/10
111/20/2
120/31/3
132/42/4
142/84/8
Total35/70c48/70c

Pathogenicity tests

Suspect colonies from seed lots 2, 3, 5, 6, 7, 11 and 14 were inoculated into plants. There were 2–6 colonies picked from each seed lot. After 1 week, most plants inoculated with colonies recovered from naturally infested bean seeds had symptoms of leaf spots surrounded by a yellow halo; two or more colonies from each seed lot were pathogenic. Leaf washes with sterile distilled water were cultured on semiselective XCP1 medium and Xap colonies were recovered from the leaf lesions. Plants inoculated with Xap isolate B1 all showed typical symptoms and the bacterium was recovered. Negative controls that were wounded but not inoculated did not show common blight symptoms, nor did they yield any colonies of Xap following the 1 week incubation of the plants.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Short soaking times at room temperature for seed extraction are attractive because results can be obtained faster than with overnight soaking. According to the results here, seed soaking for a shorter period of time (i.e. 3 h) at room temperature with vacuum applied was as effective as overnight soaking at 4°C. Vacuum extraction is not usually practical for overnight extraction because of the low temperature requirement. With centrifugation of the seed extract, the 3 h room temperature extraction was the most effective method tested in this study. Most commercial labs use overnight incubation. Longer extraction times may be favoured because they allow the bacteria more time either to enter into suspension or to resuscitate. In this study, these potential advantages may have been counteracted by using vacuum extraction along with the shorter incubation time. Because healthy seeds were used, which were kept in storage for more than 6 months, the inoculated seed samples did not display heavy natural contamination of other microorganisms which can be a problem for some seed lots, even on these semiselective media. However, most fungal and bacterial species are inhibited on the semiselective MT and XCP1 media. Some of the naturally infested seed lots tested were recently harvested and displayed a substantial amount of contamination with saprophytic organisms; however, the abundance of these organisms did not noticeably differ between the two methods compared on naturally infested seed lots. Vacuum application (Gitaitis & Walcott, 2007) on seed soaking could force air from beneath the seed coat and also bring bacterial cells out of the seed. It may also force the seed coat to loosen and fall apart, allowing deeper extraction. When it was used during 3 h incubation at room temperature, the results showed that it significantly increased recovery. Centrifugation (Hadas et al., 2005) has been used to enhance test sensitivity when a low infestation dose was expected. It enriches the concentration of target bacteria, but also concentrates other microorganisms and debris/chemicals from seeds. Interestingly, the centrifuge step added much more to the sensitivity for short incubation at room temperature than for cold overnight incubation. This may have occurred because the overnight seed extract contained more debris particles, soil and seed constituents that may have interfered with growth of pathogen cells. Significant effects of vacuum extraction or centrifugation at the lowest inoculum levels tested on inoculated seeds were not always detected; however, the levels indicated in Figures 1 and 2 are for the inoculated seeds, which represented only 1/1000 seeds in the sample. Therefore, the inoculum levels in the whole sample were three orders of magnitude lower. Significant effects for vacuum extraction and centrifugation were detected for (sample mean) inoculum levels as low as 102 and 10−1 CFU per seed, respectively. Although vacuum extraction and centrifugation represent additional steps in the procedure compared to the ISTA method, these steps facilitate shortening the time required to obtain results by as much as a day, with equal or better sensitivity. This can represent significant value for commercial seed exporters.

Naturally infested seeds were from various origins and of different types. Infestation level of common bacterial blight varied widely and so did the accompanying microflora. However, Xap colonies were distinguishable from other Gram-negative bacteria on semiselective media by their glossy and transparent appearance, yellow pigment and smooth edge. Although each seed lot was infested with Xap, the results suggest that most of the seed lots had <1% incidence of infection (Remund et al., 2007), except for seed lots 2, 5 and 8. In some seed lots, Xap was the most common organism and only a few other colonies appeared on plates; in other cases, plates had numerous Pseudomonas-like colonies and Xap colonies were scarce but detectable. In some subsamples, Xap was non-detectable. Both methods detected contamination of 13 of the 14 naturally infested seed lots, with one false negative for each method (seed lot 11, modified method; seed lot 12, ISTA method). However, each subsample from a given seed lot had the same probability of containing infested seeds, and the modified method demonstrated superior sensitivity according to the percentage of positive subsamples. Assuming there were no false positives, the average incidence of infested seeds (excluding seed lots 2, 5 and 8) can be estimated as 0·103%; therefore, each 1000-seed subsample had an average probability of 0·6422 of containing an infested seed (Remund et al., 2007). This corresponds to an expectation that 36 of the 56 subsamples would contain an infested seed. Calculating the sensitivity (Agarwal, 2006) of both methods based on this expectation, sensitivity of the ISTA method was 75%, and the modified method had a sensitivity of 94·4%. Seed lots 2, 5 and 8 were not included in this calculation because all subsamples of these seed lots tested positive by the modified method, and the incidence of infection cannot be estimated for these samples. However, the modified method was more sensitive for these samples as well (14/14 positive subsamples compared to 8/14 for the ISTA method). This improved sensitivity demonstrates that the modified method is more likely to detect infested subsamples (and therefore infested seed lots) compared to the ISTA method.

No Xap var. fuscans was found in the naturally infested seed lots, and no comparison was made with the extraction steps with Xap var. fuscans-inoculated samples. However, there is no evidence that var. fuscans behaves differently in terms of extraction from seed, association with seed tissues or detectability, compared to Xap. Seed health tests currently used for both variants are identical (Sheppard et al., 2007).

In summary, this study found that a 3 h vacuum extraction followed by centrifugation and culturing on MT or XCP1 media resulted in the most sensitive detection of Xap from contaminated P. vulgaris seed. This extraction procedure should be considered as a modification to the ISTA-approved method.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The authors are grateful to several people who assisted with locating or providing seed for this research: Elisabetta Vivoda (Harris Moran Seed Co., Davis, CA), Laurel Carter (Monsanto Vegetable Seeds, Inc., Woodland, CA), Aaron Whaley (Seed Savers Exchange, Decorah, IA), Mark Brick and Howard Schwarz (Colorado St. Univ.), Robert Rand (Univ. of WI – Madison) and Wayne Wiebe (Syngenta Seeds, Inc., Nampa, ID). Thanks to Elizabeth Vavricka (ID Dept. of Agric.) for providing bacterial cultures. This research was funded and supported by the Vegetable Seed Research Committee of the American Seed Trade Assoc.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • Agarwal VK, 2006. Seed Health. Lucknow, India: International Book Distributing Co.
  • Asma M, 2005. Proposal for a new method for detecting Xanthomonas hortorum pv. carotae on carrot seeds. ISTA Method Validation Reports 2, 117.
  • Audy P, Laroche A, Saindon G, Huang HC, Gilbertson RL, 1994. Detection of the bean common blight bacteria, Xanthomonas campestris pv. phaseoli and X. c. phaseoli var. fuscans, using the polymerase chain reaction. Phytopathology 84, 118592.
  • Birch PRJ, Hyman LJ, Taylor R, Opio AF, Bragard C, Toth IK, 1997. RAPD PCR-based differentiation of Xanthomonas campestris pv. phaseoli and Xanthomonas campestris pv. phaseoli var. fuscans. European Journal of Plant Pathology 103, 80914.
  • Dreo T, Demsar T, Ravnikar M, 2003. Quarantine of Xanthomonas campestris pv. phaseoli in beans. Zbornik predavanj in referatov 6. Slovenskega Posvetovanje o Varstvu Rastlin, Zrece, Slovenije, 1237.
  • El-Sharkawy TA, Huisingh D, 1971. Differentiation among Xanthomonas species by polyacrylamide gel electrophoresis of soluble proteins. Journal of General Microbiology 68, 15565.
  • Gitaitis R, Walcott R, 2007. The epidemiology and management of seedborne bacterial diseases. Annual Review of Phytopathology 45, 37197.
  • Goszczynska T, Serfontein JJ, 1998. Milk–Tween agar, a semiselective medium for isolation and differentiation of Pseudomonas syringae pv. syringae, Pseudomonas syringae pv. phaseolicola and Xanthomonas axonopodis pv. phaseoli. Journal of Microbiological Methods 32, 6572.
  • Hadas R, Kritzman G, Klietman F, Gefen T, Manulis S, 2005. Comparison of extraction procedures and determination of the detection threshold for Clavibacter michiganensis ssp. michiganensis in tomato seeds. Plant Pathology 54, 6439.
  • Hildebrand DC, Palleroni NJ, Schroth MN, 1990. Deoxyribonucleic acid relatedness of 24 xanthomonad strains representing 23 Xanthomonas campestris pathovars and Xanthomonas fragariae. Journal of Applied Bacteriology 68, 2639.
  • Kahveci E, Maden S, 1994. Detection of Xanthomonas campestris pv. phaseoli and Pseudomonas syringae pv. phaseolicola by bacteriophages. Journal of Turkish Phytopathology 23, 7985.
  • Lazo GR, Gabriel DW, 1987. Conservation of plasmid DNA-sequences and pathovar identification of strains of Xanthomonas campestris. Phytopathology 77, 44853.
  • Mabagala RB, 1992. An improved semiselective medium for recovery of Xanthomonas campestris pv. phaseoli. Plant Disease 76, 4436.
  • Maddox DA, 1997. Regulatory needs for standardized seed health tests. In: McGee DC, ed. Plant Pathogens and the Worldwide Movement of Seeds. St. Paul, MN, USA: APS, 8192.
  • Mahuku GS, Jara C, Henriquez MA, Castellanos G, Cuasquer J, 2006. Genotypic characterization of the common bean bacterial blight pathogens, Xanthomonas axonopodis pv. phaseoli and Xanthomonas axonopodis pv. phaseoli var. fuscans by rep-PCR and PCR-RFLP of the ribosomal genes. Journal of Phytopathology 154, 3544.
  • Maringoni AC, Kimati H, Kurozawa C, 1998. Comparison between two extraction methods of Xanthomonas campestris pv. phaseoli from bean seeds. Pesquisa Agropecuaria Brasileira 33, 65964.
  • McGuire RG, Jones JB, Sasser M, 1986. Tween media for semi selective isolation of Xanthomonas campestris pv. vesicatoria from soil and plant material. Plant Disease 70, 88791.
  • Mutlu N, Vidaver AK, Coyne DP, Steadman JR, Lambrecht PA, Reiser J, 2008. Differential pathogenicity of Xanthomonas campestris pv. phaseoli and X. fuscans subsp. fuscans strains on bean genotypes with common blight resistance. Plant Disease 92, 54654.
  • Opio AF, Teri JM, Allen DJ, 1993. Studies on seed transmission of Xanthomonas campestris pv. phaseoli in common beans in Uganda. African Crop Science Journal 1, 5967.
  • Remeeus PM, Sheppard JW, 2006. Proposal for a new method for detecting Xanthomonas axonopodis pv. phaseoli on bean seeds. ISTA Method Validation Reports 3, 111.
  • Remund K, Simpson R, Laffont J-L, Wright D, Gregoire S, 2007. SeedCalc8, ver. 8.1.0. http://www.seedtest.org/en/stats-tool-box-_content---1--1143.html .
  • Roumagnac P, Gagnevin L, Pruvost O, 2000. Detection of Xanthomonas sp., the causal agent of onion bacterial blight, in onion seeds using a newly developed semi-selective isolation medium. European Journal of Plant Pathology 106, 86777.
  • Saettler AW, 1989. Common bacterial blight. In: Schwartz HF, Pastor-Corrales MA, eds. Bean Production Problems in the Tropics. Cali, Colombia: CIAT, 26184.
  • Sakthivel N, Mortensen CN, Mathur SB, 2001. Detection of Xanthomonas oryzae pv.oryzae in artificially inoculated and naturally infected rice seeds and plants by molecular techniques. Applied Microbiology and Biotechnology 56, 43541.
  • Sheppard J, Kurowski C, Remeeus PM, 2007. Detection of Xanthomonas axonopodis pv.phaseoli and Xanthomonas axonopodis pv. phaseoli var.fuscans on Phaseolus vulgaris. Bassersdorf, Switzerland: ISTA: International Rules for Seed Testing 7-021.
  • Sutton MD, Wallen VR, 1970. Epidemiological and ecological relationship of Xanthomonas phaseoli and X. c. var. fuscans on bean in Southern Ontario, 1961–1968. Canadian Journal of Botany 48, 132934.
  • Tebaldi ND, Souza RM, Machado JC, 2007. Detection of Xanthomonas axonopodis pv. phaseoli in seeds of common bean on semi-selective medium. Fitopatologia Brasileira 32, 568.
  • Tebaldi ND, Peters J, Souza RM et al. , 2010. Detection of Xanthomonas axonopodis pv. phaseoli in bean seeds by flow cytometry, immunostaining and direct viable counting. Tropical Plant Pathology 35, 21322.
  • Toth IK, Hyman LJ, Taylor R, Birch PRJ, 1998. PCR-based detection of Xanthomonas campestris pv. phaseoli var. fuscans in plant material and its differentiation from X. c. pv. phaseoli. Journal of Applied Microbiology 85, 32736.
  • Valarini PJ, Menten JOM, 1992. Methodology for detection of Xanthomonas campestris pv. phaseoli in bean seeds. Fitopatologia Brasileira 17, 37383.
  • Valarini PJ, Menten JOM, Lollato MA, 1992. Incidence of the common bacterial blight in the field and transmission of Xanthomonas campestris pv. phaseoli through bean seeds, evaluated by different methods. Summa Phytopathologica 18, 1606.
  • Vauterin L, Hoste B, Kersters K, Swings J, 1995. Reclassification of Xanthomonas. International Journal of Systematic Bacteriology 45, 47289.
  • Venette JR, Lamppa RS, Albaugh DA, Nayes JB, 1987. Presumptive procedure (dome test) for detection of seed-borne bacterial pathogens in dry beans. Plant Disease 71, 98490.
  • Vieira B de AH, Souza RM de, 2000. Virulence of isolates of Xanthomonas axonopodis pv. phaseoli and its variant fuscans. Ciencia e Agrotecnologia 24, 94102.
  • Vieira RF, Carneiro JES, Lynch JP, 2008. Root traits of common bean genotypes used in breeding programs for disease resistance. Pesquisa Agropecuaria Brasileira 43, 70712.
  • Wong WC, 1991. Methods for recovery and immunodetection of Xanthomonas campestris pv. phaseoli in navy bean seed. Journal of Applied Bacteriology 71, 1249.