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Appl Environ Microbiol. 2009 November; 75(21): 6831–6838.
Published online 2009 September 11. doi:  10.1128/AEM.01653-09
PMCID: PMC2772435

Combination of Chromogenic Differential Medium and estA-Specific PCR for Isolation and Detection of Phytopathogenic Xanthomonas spp.[down-pointing small open triangle]

Abstract

A xanthomonad differential medium (designated Xan-D medium) was developed, on which streaks and colonies of xanthomonads, including 13 species of the genus Xanthomonas, turned wet-shining yellow-green and were surrounded with a smaller milky zone and a bigger clear zone in 3 to 4 days. The characteristics could easily be differentiated from those of yellow nonxanthomonads and other bacteria. The mechanism of color change and formation of a milky zone on the medium are mainly due to the Tween 80 hydrolytic capacity of xanthomonads. The gene, estA, responsible for Tween 80 hydrolysis was cloned and expressed in Escherichia coli, which acquired a capacity to hydrolyze Tween 80 and could turn green and form a milky zone on the Xan-D medium. The nucleotide sequence of estA is highly conserved in the xanthomonads, and the sequence was used to design a specific PCR primer set. The PCR amplification using the primer set amplified a 777-bp specific DNA fragment for all xanthomonad strains tested. The Xan-D medium was used to isolate and differentiate Xanthomonas campestris pv. campestris from naturally infected cabbages with black rot symptoms for a rapid diagnosis. All isolated X. campestris pv. campestris strains developed characteristic colonies and were positive in the PCR with the estA primer set. The Xan-D medium was further amended with antibiotics and successfully used for the detection of viable X. campestris pv. campestris cells from plant seeds. Although some yellow nonxanthomonads and other saprophytic bacteria from plant seeds could still grow on the medium, they did not interfere with the color development of X. campestris pv. campestris. However, Stenotrophomonas maltophilia, which is closely related to xanthomonads, existing in a seed lot could also develop yellow-green color but had different colony morphology and was negative in the PCR with the estA primer set. Accordingly, the combination of the Xan-D medium with the estA-specific PCR is a useful and reliable method for the isolation and detection of viable xanthomonad cells from plant materials.

The genus Xanthomonas Dowson 1939 contains phytopathogenic bacteria that are usually yellow pigmented on medium and can cause diseases worldwide. Contaminated plant seeds are an important primary source of inoculum for the bacteria. A very low level of seed infestation (three in 10,000) can give rise to high disease incidence in the field (26). Therefore, planting seed free of bacterial contamination is an important disease management strategy. Several molecular and immunological methods, such as PCR (3, 4, 5, 13, 17, 30), enzyme-linked immunosorbent assay (1, 35), and flow cytometry (7), were developed to test seeds for the presence of these pathogens. However, these methods cannot directly distinguish dead and viable cells and may vary in specificity and sensitivity according to seed and other sample types (plant tissue, soil, and water).

The most commonly used seed test method for recovery of viable bacterial cells is a seed-washing liquid-plating assay. In this assay, bacteria are first extracted from seeds, and the extract is diluted and plated onto semiselective medium. Several semiselective media are recommended in the working sheets in the ISTA Handbook on Seed Health Testing (27). They are considered to be reliable and efficient methods for routine detection (21, 24). Semiselective media permit quantification of viable cells and can be as sensitive as PCR in detecting Xanthomonas albilineans in sugarcane (10, 36) and even more sensitive than immunological techniques for detecting low numbers of bacteria (1, 36). Semiselective medium is easy to use and less costly than molecular and immunological methods and can often be readily used for diverse sample types.

Although the semiselective media for xanthomonads can suppress the overgrowth of most saprophytic microorganisms, the major disadvantage of these media is the presence of yellow nonxanthomonads associated with plant tissues, which may interfere and complicate identification. Therefore, further identification and time-consuming pathogenicity tests must be completed to positively identify pathogenic xanthomonads. Accordingly, the search for a medium to differentiate xanthomonads from yellow nonxanthomonads is a critical task in the progress of isolation, detection, and identification of viable xanthomonads from plant tissues.

In this report we describe a xanthomonad differential medium (called Xan-D medium) on which colonies of xanthomonads were wet-shiny (mucoid), convex, and yellow-green and could easily be differentiated from yellow nonxanthomonads and other bacteria. The medium could be used for the isolation of xanthomonads from naturally diseased plants and infested seeds. The mechanism and the gene responsible for the color development of xanthomonads were determined. The gene was also used to design a specific PCR primer set for the further identification of xanthomonads.

MATERIALS AND METHODS

Bacterial strains and culture conditions.

The bacterial strains used in this study are listed in Table Table1.1. Strains of xanthomonads tested represent 13 species of the genus Xanthomonas. Xanthomonads and other bacteria were routinely cultured on Luria-Bertani agar or in broth medium (22) at 28°C.

TABLE 1.
Bacterial strains testeda

Xan-D medium preparation and testing procedure.

The xanthomonad differential medium (Xan-D medium) was adapted from Tween medium (16) and milk-Tween medium (12). The Xan-D medium was made by mixing skim milk and bromothymol blue solutions to the basal medium as detailed below. The basal medium consisted of 10 g of Bacto-Soytone (Becton Dickinson and Company, Sparks, MD), 10 ml of Tween 80 (Sigma, St. Louis, MO), 10 g of potassium bromide (KBr; J. T. Baker, Phillipsburg, NJ), and 15 g of Bacto agar (Becton Dickinson) in 500 ml of water. Skim milk solution was prepared by adding 10 g of skim milk (Becton Dickinson) to 500 ml of distilled water. The basal medium and skim milk solution were autoclaved separately and mixed when still hot to a final volume of 1 liter. After being cooled to 50 to 60°C, the above medium was supplemented with 24 mg of bromothymol blue (1.5 ml of 1.6% [wt/vol] bromothymol blue in ethanol) per liter under sterile conditions and poured into petri dishes. The resulting Xan-D medium had a pH of 6.5 and a light orange-yellow color. Bromothymol blue is a pH indicator which changes color from yellow to green to blue when pH transitions from 6.0 to 7.6. Cycloheximide (75 μg/ml; Sigma), cephalexin (cefalexin) (65 μg/ml; Sigma), and 5-fluorouracil (12 μg/ml; Sigma) were added as necessary to prevent the overgrowth of contaminating fungi and bacteria in plant materials. The Xan-D medium amended with the three antibiotics was called Xan-D(CCF) hereafter.

To test the color development of Xanthomonas spp. and other bacteria on the Xan-D medium, four to six bacterial cultures were streaked per plate and incubated for 3 to 4 days at 28°C. For observation of colony morphology, 100 μl of bacterial suspension (103 CFU/ml) was pipetted onto the surface of the Xan-D medium and spread evenly with an L-shaped rod. The plate was incubated for 3 to 4 days at 28°C prior to observation.

Gene cloning and analysis of a gene for Tween 80 hydrolysis.

To clone a gene for Tween 80 hydrolysis from Xanthomonas campestris pv. campestris XCC1-1, a data search found that ApeE of Salmonella enterica serovar Typhimurium (accession no. AF047014) has a capacity to hydrolyze Tween 80 (6) and shares similarity to EstA of X. campestris pv. campestris ATCC 33913 (accession no. AE008922). The DNA fragment containing the full coding region of estA was amplified from X. campestris pv. campestris XCC1-1 total DNA by a PCR using primer pair Xc-lip-F (5′-TTGAGCAGGCATTCCCATGGCTTCAAC-3′)-Xc-lip-R (5′-GCCTTGGGCGCAATGCGTGGCGCACATGAC-3′) designed from the estA nucleotide sequence. The PCR amplification was performed using the protocol described below. The 1.9-kb PCR-amplified DNA fragment was ligated into pGEM-T Easy vectors (Promega Corp., Madison, WI) and transferred into Escherichia coli DH5α. Sequence analysis and comparison were performed by Vector NTI software (Invitrogen Corp., Carlsbad, CA) and the computer programs of GCG (Genetics Computer Group, Madison, WI). Based on the sequence analysis, a primer pair specific to estA of xanthomonads was designed, Xc-lip-F2 (5′-TATGTGATGGTGCCGACCATTC-3′)-Xc-lip-R2 (5′-GGACTTCGCGGTCCACGTCGTAGC-3′), which yielded a 777-bp DNA fragment in PCR amplification.

EstA protein expression and assay.

The plasmid for expression of estA of X. campestris pv. campestris XCC1-1 under the control of the T7 RNA polymerase system was constructed as follows. The estA gene was PCR amplified with Vent DNA polymerase (2 U/μl; New England Biolabs, Ipswich, MA) with primers Xc-lip-BamHI-F3m (5′-GCGGATCCATGGCCGTGGCCATCGCGCT-3′) and Xc-lip-XhoI-R3m (5′-GCCTCGAGGAAGTTGCCGCTGAAGT-3′). The amplified fragment was digested with BamHI and XhoI and ligated between the BamHI and XhoI sites of plasmid pET29a (Novagen, Madison, WI), and the resulting plasmid, called pET29a-estA, was transformed into E. coli Rosetta(DE3)pLysS. The expression of estA was performed as described by the pET29a supplier. Tween 80 hydrolysis activity (esterase) was assayed simply by streaking the recombinant clone E. coli Rosetta(DE3)(pLysS, pET29a-estA) on the Xan-D medium and incubating it for 3 to 4 days at 37°C. The development of green color on a bacterial streak and formation of a milky zone around the streak indicate the Tween 80 hydrolysis.

PCR analysis with estA primer pairs.

The characteristic colonies of the bacterium isolated from the Xan-D medium were suspended in 50 μl of sterile distilled water. The bacterial suspension was incubated for 10 min at 100°C for cell lysis and was maintained at 4°C before use. The PCR assay was carried out using the Xc-lip-F2-Xc-lip-R2 primer set for estA. The Xc-lip-F2-Xc-lip-R2 primer set yielded a 777-bp PCR product. The PCR amplification was performed with a 2720 thermal cycler (Applied Biosystems, Foster City, CA) in a 30-μl reaction mixture containing 1 μl of cell lysate, 0.35 to 0.5 μM of each primer, 0.25 mM of each deoxynucleoside triphosphate, 1× reaction buffer (1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 10 mM Tris-HCl; pH 8.8), 6% dimethyl sulfoxide (J. T. Baker, Phillipsburg, NJ), and 2.0 U of Taq DNA polymerase (DyNAzyme II; Finnzymes Oy, Finland) by using the following program: one cycle of denaturation for 1 min at 94°C and 35 cycles consisting of denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 1 min. Reaction mixtures were stored at 15°C until they were used for analysis. Amplified DNA was detected by electrophoresis in 0.8% agarose (agarose I; Amresco Inc., Solon, OH) in 1× TAE buffer (40 mM Tris-acetate, 1 mM EDTA, pH 8.0).

Isolation of X. campestris pv. campestris from naturally diseased cabbage plants.

Cabbage plants with symptoms of black rot were collected during the growing season from three cultivation areas in Taiwan. The diseased leaves were washed in running water and air dried in a laminar flow hood. Small pieces (about 0.2 by 0.2 cm2) from the leading edge of black rot lesions were excised and further cut into smaller pieces in 100 μl of sterile water. One loopfu1 of the suspension was streaked onto the Xan-D medium, and plates were incubated at 28°C and observed for the presence of typical pathogen colonies daily for 4 days.

Recovery of X. campestris pv. campestris from artificially and naturally infested cabbage seeds.

To determine whether saprophytic bacteria or fungi on seeds might affect the growth and color development of xanthomonads on the Xan-D medium, the recovery of X. campestris pv. campestris on the Xan-D medium was first determined from seed wash to which X. campestris pv. campestris cells were added. Three grams of cabbage seeds was incubated in 10 ml of washing buffer (0.85% NaCl, 0.02% Tween 80) in a 120-ml flask. The flasks were placed on a rotary shaker (125 rpm) at 28°C for 3 h. X. campestris pv. campestris XCC1-1 cell suspension was added to the flasks to a final concentration of 103 CFU/ml. The 100-μl seed wash and its 10- and 100-fold dilutions were spread onto the plates of Xan-D and Xan-D(CCF) media, respectively. Plates were incubated at 28°C and observed for the presence of typical colonies of xanthomonads daily for 3 to 5 days. For isolation of X. campestris pv. campestris from naturally infested cabbage seeds, the seeds were washed and seed washes were diluted and spread onto the media as described above.

Identification and pathogenicity testing of yellow nonxanthomonads and xanthomonads.

Some yellow bacterial colonies from plant seeds tested could grow on the Xan-D medium. Their colony color and morphology were different from those of xanthomonads, and they were considered yellow nonxanthomonads. For determination of their identities, the colonies were taken for a PCR assay to obtain their partial 16S rRNA gene fragments using 16S-F3 (5′-CCAGACTCCTACGGGAGGCAGC-3′) and 16S-R1 (5′-GCTGACGACAGCCATGCAGCACC-3′) as primers, which yield an approximately 730-bp DNA fragment. The DNA fragments were ligated into pGEM-T Easy vectors (Promega Corp., Madison, WI), and double-stranded DNA sequencing was performed with standard universal T7 and SP6 primers. Sequence data were analyzed using sequence analysis tools and the database of the National Center for Biotechnology Information. The API 20NE system (BioMerieux Vitek Inc., Hazelwood, MO) was used for physiological and biochemical tests. The pathogenicity testing was done for the isolated xanthomonads and yellow nonxanthomonads by inoculating cabbage seedlings (Brassica oleraceae L. var. capitata) by cutting leaf margins with a pair of sterilized scissors that had been dipped in a suspension of bacteria containing 107 to 108 CFU/ml. The inoculated plants were covered with a plastic bag in a growth chamber at 28°C under conditions of 16 h of light and 8 h of darkness. After 24 h, the plastic bag was removed, plants were maintained in the growth chamber, and symptoms were evaluated after 7 to 14 days.

RESULTS

Growth, colony morphology, and color development of xanthomonads on the Xan-D medium.

The strains of xanthomonads, including 13 species of Xanthomonas (Table (Table1),1), were tested. All the bacterial streaks of Xanthomonas spp. grew well, turned wet-shining yellow-green, and were surrounded with a smaller milky and a bigger clear zone on the Xan-D medium in 3 to 4 days. Ten isolates of X. campestris pv. campestris (XCCN1 to -N10 in Table Table1)1) from cabbage leaves with black rot symptoms in three cultivation areas of Taiwan were tested. All showed the same characteristics as those described above on the Xan-D medium. This indicated that the phenotypic properties on the medium were stable in the xanthomonads tested and in the population of X. campestris pv. campestris. Other plant-pathogenic bacteria listed in Table Table11 did not develop a green color on the Xan-D medium. The developed color of xanthomonads on the Xan-D medium did not disappear with age.

The colonies of xanthomonads tested on the Xan-D medium were also yellow-green, mucoid, circular, convex, and surrounded with two zones, a bigger clear zone of skim milk hydrolysis and a smaller milky zone of Tween 80 lipolysis. Most yellow nonxanthomonads isolated from plant seeds, such as Arthrobacter sp., Pseudomonas sp., and Pantoea sp., were flat without a milky zone, remained yellow, and did not turn green on the medium. Representative results are shown in Fig. Fig.1.1. Plating efficiency, defined as average number of CFU on the Xan-D plate/average number of CFU on the Luria-Bertani agar plate, of the xanthomonad strains tested in Table Table11 ranged from 100 to 124% with a mean of 106%. Accordingly, the Xan-D medium could be used as a differential medium for differentiation of xanthomonads from other bacteria by colony color and morphology.

FIG. 1.
Colony color and morphology of xanthomonads and yellow nonxanthomonads on the Xan-D medium incubated at 28°C for 4 days. (A) X. campestris pv. campestris XCC1-1; (B) X. campestris pv. coriandri NPCCB 1457; (C) X. axonopodis pv. citri XW134; (D) ...

On the Xan-D medium without Tween 80, the xanthomonads remained yellow, did not turn yellow-green, and were also not surrounded with a milky zone. Addition of Tween 80 solution could decrease the pH and turn the color of bromothymol blue in the Xan-D medium to yellow. It was reasoned that the xanthomonads have a gene product for hydrolyzing Tween 80, which could cause a milky zone (the precipitation of fatty acid of Tween 80) and raise the pH, leading to the color change of bromothymol blue from yellow to green and making the xanthomonads become yellow-green.

Gene cloning and expression of esterase gene of X. campestris pv. campestris.

The estA gene of X. campestris pv. campestris XCC1-1, whose product may have a capacity to hydrolyze Tween 80, was cloned by a homology search as described in Materials and Methods. The nucleotide sequence of estA from XCC1-1 was determined and is 100% identical to that of estA from X. campestris pv. campestris ATCC 33913 (accession no. AE008922). The coding region of estA is 1,785 bp, encoding an esterase/lipase of 594 amino acids. The coding region of estA was further ligated to pET29a expression vector (called pET29a-estA) and could be expressed in E. coli Rosetta(DE3)pLysS. The expressed protein has 635 amino acids (66 kDa) and was found mostly in the insoluble (pellet) fraction of cell lysates (Fig. (Fig.2A).2A). Nevertheless, the bacterial streak of E. coli Rosetta(DE3)(pLysS, pET29a-estA) could still develop green color and was surrounded with a smaller milky zone on the Xan-D medium after 3 days of incubation at 37°C and then 4°C for 2 more days (Fig. (Fig.2B).2B). E. coli Rosetta(DE3)pLysS containing only pET29a did not develop green color, and no milky zone was formed. The results indicated that EstA of X. campestris pv. campestris XCC1-1 expressed in E. coli had Tween 80 hydrolytic ability and could cause the color change and milky zone formation on the Xan-D medium.

FIG. 2.
Analysis of expression of the estA gene in Escherichia coli Rosetta(DE3)(pLysS, pET29a-estA) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (A) and on the Xan-D medium (B). (A) Separation in a polyacrylamide gel of proteins from the pellet ...

Sequence analysis of estA and specific PCR primers for xanthomonads.

Comparison of the nucleotide sequence of estA of X. campestris pv. campestris XCC1-1 with those in the GenBank database indicated that estA exists in all complete genome sequences of xanthomonads, such as Xanthomonas oryzae pv. oryzae (accession no. AE013598 and AP008229), Xanthomonas axonopodis pv. citri (AE011975), X. campestris pv. campestris (AE012430, CP000050, and AM920689), and X. campestris pv. vesicatoria (AM039952), and the coding region of estA is highly conserved in the xanthomonads. The extent of overall nucleotide sequence identity between these xanthomonads ranges between 87 and 100%. The estE gene of Xanthomonas vesicatoria (AF536208) (32) shares 98% identity with estA and could be considered a homolog of estA. For nonxanthomonads, the highest nucleotide sequence identity was found to be those with a putative lipase/esterase from Stenotrophomonas maltophilia (70% identity; CP001111) and Xylella fastidiosa (69% identity; CP001011), which are closely related to xanthomonads (20, 29, 31, 37). The lipase/esterase genes from other nonxanthomonads, including apeE of S. enterica serovar Typhimurium, share only about less than 38% identity with that of X. campestris pv. campestris XCC1-1. An S. maltophilia strain (ATCC 17806) was found to grow on the Xan-D medium and could also develop a yellow-green color.

Since the nucleotide sequence of estA is highly conserved in xanthomonads, the sequence was used to design a specific PCR primer set, Xc-lip-F2-Xc-lip-R2. To determine the specificity of the primer set, PCRs were carried out with colonies of all of the strains listed in Table Table1.1. The PCR amplification using Xc-lip-F2-Xc-lip-R2 primers amplified a 777-bp DNA fragment for all xanthomonad strains tested. No amplification was observed with yellow nonxanthomonads and other plant-pathogenic bacteria tested, such as Erwinia, Pseudomonas, or Ralstonia strains (Table (Table1).1). The S. maltophilia strain (ATCC 17806) was also tested and did not produce the 777-bp DNA fragment by the PCR primer set (Fig. (Fig.3).3). Thus, the primer set designed is specific to xanthomonads. The developed PCR assay was used for the further identification of xanthomonads after the isolation of viable yellow-green bacterial colonies on the Xan-D medium from plant materials.

FIG. 3.
Agarose gel electrophoresis of PCR products from Xanthomonas spp., yellow nonxanthomonads, and other plant-pathogenic bacteria with the estA (Xc-lip-F2-Xc-lip-R2) primer set. (A) Lanes: 1, X. campestris pv. campestris XCC1-1; 2, Xanthomonas arboricola ...

Isolation of Xanthomonas spp. from naturally infected plants.

Most bacterial streaks or colonies isolated from the leading edge of cabbage leaves with black rot lesions became visible as yellow first, then turned yellow-green, and were surrounded with clear and milky zones on the Xan-D medium after incubation for 3 to 4 days at 28°C, indicating that the pathogenic bacterium was a xanthomonad. Because the Xan-D medium is not a selective medium, a few saprophytes grew out but did not interfere with the development of characteristic colonies of the xanthomonad. One characteristic colony on the Xan-D medium was selected from a separate diseased cabbage. Three to four colonies were chosen from each of three cultivation areas. A total of 10 characteristic colonies (XCCN1 to -N10 in Table Table1)1) were further assayed by PCR using the Xc-lip-F2-Xc-lip-R2 primer set, and all amplified a 777-bp DNA fragment. The isolated bacterial colonies were also tested for their pathogenicity, and all caused symptoms of black rot.

Recovery of X. campestris pv. campestris from artificially infested cabbage seeds.

The Xan-D medium was tested to recover X. campestris pv. campestris from artificially infested cabbage seeds. The overgrowth of saprophytic fungi and bacteria present in some seed lots interfered with the growth of X. campestris pv. campestris and also turned the medium green. To prevent the overgrowth, seed wash was diluted 10- or 100-fold before being plated to the Xan-D or Xan-D(CCF) media, respectively. Although the growth of saprophytic fungi and bacteria was not totally inhibited, X. campestris pv. campestris still formed a characteristic colony, which was easily differentiated from saprophytes. The number of colonies of X. campestris pv. campestris recovered from artificially infested seeds ranged from 80 to 90% of the original number on the Xan-D medium and 75 to 85% of the original number on Xan-D(CCF) medium, indicating that some added X. campestris pv. campestris cells were absorbed by seed constituents and that the antibiotics in the Xan-D(CCF) medium reduced the colony recovery of X. campestris pv. campestris by 5%.

In addition to X. campestris pv. campestris, yellow and flat bacterial colonies that grow on the Xan-D medium were also isolated from cabbage seeds. These bacteria were identified as Arthrobacter sp., Pantoea sp., or Pseudomonas sp. (Table (Table11 and Fig. Fig.1),1), based on analysis of their 16S rRNA gene nucleotide sequences and physiological and biochemical properties. These yellow nonxanthomonads did not turn yellow-green on the Xan-D medium and were easily differentiated from X. campestris pv. campestris by their colony color and morphology.

Recovery of X. campestris pv. campestris from naturally infested seeds.

The Xan-D(CCF) medium was used to detect X. campestris pv. campestris from five cabbage and three cauliflower seed samples offered by seed producers in Taiwan. Xanthomonads were not detected in cabbage seeds tested but were found in one of three cauliflower seed lots. Populations recovered from the cauliflower seed lots on Xan-D(CCF) medium were about 3 × 102 CFU per milliliter of seed wash. Four characteristic colonies (XCCP1 to -P4 in Table Table1)1) were isolated and further identified by the PCR assay using the estA (Xc-lip-F2-Xc-lip-R2) primer set, and all amplified a 777-bp DNA fragment. The bacterial colonies were also tested for their pathogenicity, and all caused symptoms of black rot.

It was found that the cauliflower seed lot which was infested with X. campestris pv. campestris was also contaminated with another yellow bacterium capable of turning yellow-green on the Xan-D(CCF) medium. The yellow bacteria were isolated and identified as S. maltophilia. Although it could turn yellow-green, S. maltophilia had a different colony morphology on the medium. The S. maltophilia colonies are raised with an undulate margin and turn yellow-green first and then green-blue when the colonies grow bigger, in contrast to the X. campestris pv. campestris colonies, which are mucoid and convex with an entirely circular margin (Fig. (Fig.4).4). The S. maltophilia isolates (Clb1 to -b4 in Table Table1)1) did not produce the 777-bp DNA fragment by PCR with the estA primer set and did not cause any disease on cabbage plants.

FIG. 4.
The colony morphology of X. campestris pv. campestris and S. maltophilia from cauliflower seeds on Xan-D(CCF) medium. The colonies of X. campestris pv. campestris are yellow-green and convex with an entirely circular margin (1), and those of S. maltophilia ...

DISCUSSION

The Xan-D medium can be widely used for most xanthomonads regardless of their species and pathovars. Several other semiselective media were developed before for certain pathovars of X. campestris based on starch hydrolysis (8, 9, 15, 18, 25). However, not all strains of xanthomonads are starch hydrolyzing. Many isolates of X. campestris pv. manihotis, X. campestris pv. vesicatoria, and X. campestris pv. vitians do not hydrolyze starch (11, 14, 16, 33). Unlike starch hydrolysis, Tween 80 hydrolysis (lipolysis) is a more constant characteristic of xanthomonads. All species, pathovars, and strains of the genus Xanthomonas in Table Table11 grew well on the Xan-D medium and hydrolyzed Tween 80, leading to the formation of a milky zone (the precipitation of fatty acid of Tween 80) and the development of wet-shiny yellow-green color. The xanthomonads also hydrolyzed skim milk in the medium and resulted in a clear zone. In addition, the Xan-D medium contains KBr, which can enhance production of yellow dibromomethoxyphenyl polyene pigment of xanthomonads (2). Although Tween 80 hydrolysis has been used before to develop semiselective media for X. campestris pv. vesicatoria (16) and X. axonopodis pv. phaseoli (12), these media do not make xanthomonads develop easily recognizable yellow-green color as the Xan-D medium does. Most yellow nonxanthomonads isolated in this study did not develop yellow-green color on the medium. Thus, the unique color development makes the Xan-D medium more useful for the detection and identification of xanthomonads.

The estA gene of X. campestris pv. campestris was cloned by the homology search and PCR amplification. The Tween 80 hydrolytic ability of the EstA expressed by E. coli Rosetta(DE3)pLysS was tested by the plate assay using the Xan-D medium. After 3 days of incubation at 37°C, a milky zone was not visible. The plates were then incubated at 4°C for two more days, and a milky zone became apparent around E. coli. The color of E. coli also turned green on the Xan-D medium. The result suggested that the EstA expressed by E. coli could indeed cause the cleavage of Tween 80, but the amount of fatty acid precipitation was so small that the milky zone became visible only after incubation at a low temperature. This could be due to the fact that most of the EstA proteins expressed from E. coli were membrane bound and only a few were secreted into the Xan-D medium and that the EstA expressed by E. coli was not as active as that expressed by X. campestris pv. campestris.

Since estA is highly conserved in xanthomonads, one option for reliable identification of xanthomonads is the detection of estA by a PCR assay. Using the specific primer set derived from estA, a specific DNA fragment was amplified from all xanthomonads listed in Table Table1,1, consistent with the capacity of Tween 80 hydrolysis on Xan-D medium. The result indicated that Tween 80 hydrolysis is not only a phenotypic but also a genotypic property for xanthomonads. The primer set also worked well in distinguishing xanthomonads from yellow nonxanthomonads commonly isolated from plant seeds and leaves, and from other plant-pathogenic bacteria tested, such as Erwinia, Pseudomonas, or Ralstonia strains. The PCR assay developed was used for the further identification of xanthomonads after the isolation of viable wet-shiny yellow-green bacterial cells from diseased plant tissues or seeds on the Xan-D medium.

Although the Xan-D medium was developed primarily for its differentiation feature for the identification of xanthomonads, the medium was also suitable for the isolation of xanthomonads from low-level-contaminated plant materials, such as the leading edge of lesions in naturally diseased plant tissues. The medium was successfully used to isolate the causal agents of cabbage black rot. Bacteria from the diseased plant tissues grew well and became characteristic wet-shiny yellow-green, indicating that the bacteria were xanthomonads. Only a few saprophytic bacteria could grow, but they did not interfere with the color development of xanthomonads on the medium. Therefore, the medium is useful for a rapid diagnosis to determine whether plant diseases are caused by xanthomonads and for the further studies of characterization and epidemiology of the isolated xanthomonad strains.

Since plant seeds are primary sources of pathogenic xanthomonads, effective plant seed quarantine and detection measures are needed to prevent the introduction and disease spread of xanthomonads from seeds. Plant seeds tested were contaminated with saprophytic fungi and bacteria, and the overgrowth of these fungi and bacteria would turn the Xan-D medium green and make xanthomonads indistinguishable. To prevent such overgrowth, the Xan-D medium was amended with cycloheximide, cephalexin, and 5-fluorouracil, and a serial dilution of seed wash was made before being plated to the medium. Cycloheximide can inhibit fungal growth, and cephalexin and 5-fluorouracil suppress most gram-positive bacteria and fluorescent pseudomonads, which have been used in some selective media for xanthomonads (9, 19, 23, 28, 34). Although saprophytic fungi and bacteria were not totally inhibited on the developed Xan-D(CCF) medium, they did not interfere with the growth and color development of xanthomonads. The Xan-D(CCF) medium was used to successfully isolate X. campestris pv. campestris from artificially and naturally infested seeds in this study.

The existence of yellow nonxanthomonads on plant seeds was considered a major problem for using the selective media developed previously for the isolation of xanthomonads from plant seeds, because yellow nonxanthomonads could not be totally inhibited and would interfere with the identification of xanthomonads on the media. In this study, contaminating yellow nonxanthomonads on cabbage and cauliflower seeds which grew on the Xan-D or Xan-D(CCF) medium were isolated and identified. These bacteria included strains of Arthrobacter species, Pantoea species, Pseudomonas species, and S. maltophilia. Among these bacteria, only S. maltophilia grew and turned yellow-green as did X. campestris pv. campestris on the Xan-D(CCF) medium. Although the colony morphology of S. maltophilia is different from that of X. campestris pv. campestris, S. maltophilia strains could still be misidentified as xanthomonads. Therefore, the yellow-green colonies should be chosen for the further PCR assay using the estA (Xc-lip-F2-Xc-lip-R2) primer set, and only xanthomonads can produce a specific PCR product. Accordingly, the combination of the Xan-D or Xan-D(CCF) medium and the specific PCR for estA is practically useful in plant disease diagnosis and in seed detection and certification programs for the isolation and identification of phytopathogenic Xanthomonas spp.

Acknowledgments

We thank Ya-Chun Chang (Department of Plant Pathology and Microbiology, National Taiwan University, Taipei, Taiwan) for helpful discussions and critical reading of the manuscript.

This research was supported by grants from the Council of Agriculture (96AS-14.4.1-BQ-B3), Taiwan, Republic of China.

Footnotes

[down-pointing small open triangle]Published ahead of print on 11 September 2009.

REFERENCES

1. Alvarez, A. M., and K. Lou. 1985. Rapid identification of Xanthomonas campestris pv. campestris by ELISA. Plant Dis. 69:1082-1086.
2. Andrewes, A. G., S. Hertzberg, S. Liaaen-Jensen, and M. P. Starr. 1973. The Xanthomonas ‘carotenoids’—non-carotenoid, brominated, aryl-polyene esters. Acta Chem. Scand. 27:2382-2395. [PubMed]
3. Audy, P., C. E. Braat, G. Saindon, H. C. Huong, and A. Laroche. 1996. A rapid and sensitive PCR-based assay for concurrent detection of bacteria causing common and halo blights in bean seed. Phytopathology 86:361-366.
4. Audy, P., A. Laroche, G. Saindon, H. C. Huang, and R. L. Gilbertson. 1994. Detection of the bean common blight bacteria, Xanthomonas campestris pv. phaseoli and X. c. phaseoli var. fuscans, using the polymerase chain reaction. Phytopathology 84:1185-1192.
5. Berg, T., L. Tesoriero, and D. L. Hailstones. 2005. PCR-based detection of Xanthomonas campestris pathovars in Brassica seed. Plant Pathol. 54:416-427.
6. Carinato, M. E., P. Collin-Osdoby, X. Yang, T. M. Knox, C. A. Conlin, and C. G. Miller. 1998. The apeE gene of Salmonella typhimurium encodes an outer membrane esterase not present in E. coli. J. Bacteriol. 180:3517-3521. [PMC free article] [PubMed]
7. Chitarra, L. G., C. J. Langerak, J. H. W. Bergervoet, and R. W. van den Bulk. 2002. Detection of the plant pathogenic bacterium Xanthomonas campestris pv. campestris in seed extracts of Brassica sp. applying fluorescent antibodies and flow cytometry. Cytometry 47:118-126. [PubMed]
8. Chun, W. W. C., and A. M. Alvarez. 1983. A starch-methionine medium for isolation of Xanthomonas campestris pv. campestris from plant debris in soil. Plant Dis. 67:632-635.
9. Claflin, L. E., A. K. Vidaver, and M. Sasser. 1987. MXP, a semi-selective medium for Xanthomonas campestris pv. phaseoli. Phytopathology 77:730-734.
10. Davis, M. J., P. Rott, P. Baudin, and J. L. Dean. 1994. Evaluation of selective media and immunoassays for detection of Xanthomonas albilineans, causal agent of sugarcane leaf scald disease. Plant Dis. 78:78-82.
11. Fessehaie, A., K. Wydra, and K. Rudolph. 1999. Development of a new semiselective medium for isolating Xanthomonas campestris pv. manihotis from plant material and soil. Phytopathology 89:591-597. [PubMed]
12. Goszczynska, T., and J. J. Serfontein. 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. J. Microbiol. Methods 32:65-72.
13. Hartung, J. S., J. F. Daniel, and O. P. Pruvost. 1993. Detection of Xanthomonas campestris pv. citri by the polymerase chain reaction method. Appl. Environ. Microbiol. 59:1143-1148. [PMC free article] [PubMed]
14. Jones, J. B., R. E. Stall, and H. Bouzar. 1998. Diversity among xanthomonads pathogenic on pepper and tomato. Annu. Rev. Phytopathol. 36:41-58. [PubMed]
15. Kado, C. I., and M. G. Heskett. 1970. Selective media for isolation of Agrobacterium, Corynebacterium, Erwinia, Pseudomonas, and Xanthomonas. Phytopathology 60:969-976. [PubMed]
16. McGuire, R. G., J. B. Jones, and M. Sasser. 1986. Tween media for semiselective isolation of Xanthomonas campestris pv. vesicatoria from soil and plant material. Plant Dis. 70:887-891.
17. Meng, X. Q., K. C. Umesh, R. M. Davis, and R. L. Gilbertson. 2004. Development of PCR-based assays for detecting Xanthomonas campestris pv. carotae, the carrot bacterial leaf blight pathogen, from different substrates. Plant Dis. 88:1226-1234.
18. Mulrean, E. N., and M. N. Schroth. 1981. A semiselective medium for the isolation of Xanthomonas campestris pv. juglandis from walnut buds and catkins. Phytopathology 71:336-339.
19. Mwangi, M., M. Mwebaze, R. Bandyopadhyay, V. Aritua, S. Eden-Green, W. Tushemereirwe, and J. Smith. 2007. Development of a semiselective medium for isolating Xanthomonas campestris pv. musacearum from insect vectors, infected plant material and soil. Plant Pathol. 56:383-390.
20. Palleroni, N. J., and J. F. Bradbury. 1993. Strenotrophomonas, a new bacterial genus for Xanthomonas maltophilia (Hugh 1980) Swings et al. 1983. Int. J. Syst. Bacteriol. 43:606-609. [PubMed]
21. Randhawa, P. S., and N. W. Schaad. 1984. Selective isolation of Xanthomonas campestris pv. campestris from crucifer seeds. Phytopathology 74:268-272.
22. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
23. Schaad, N. W., J. B. Jones, and W. Chun. 2001. Laboratory guide for identification of plant pathogenic bacteria, 3rd ed. American Phytopathological Society, St. Paul, MN.
24. Schaad, N. W. 1989. Detection of Xanthomonas campestris pv. campestris in crucifers, p. 68-75. In A. W. Saettler, N. W. Schaad, and D. A. Roth (ed.), Detection of bacteria in seed and other planting material. American Phytopathological Society, St. Paul, MN.
25. Schaad, N. W., and W. C. White. 1974. A selective medium for soil isolation and enumeration of Xanthomonas campestris. Phytopathology 64:876-880.
26. Schaad, N. W., W. R. Sitterly, and H. Humaydan. 1980. Relationship of incidence of seedborne Xanthomonas campestris to black rot of crucifers. Plant Dis. 64:91-92.
27. Schaad, N. W., and A. A. J. M. Franken. 1996. ISTA handbook on seed health testing. Working sheet no. 50 (2nd ed.): Xanthomonas campestris pv. campestris. International Seed Testing Association, Zurich, Switzerland.
28. Sijam, K., C. J. Chang, and R. D. Gitaitis. 1991. An agar medium for isolation and identification of Xanthomonas campestris pv. vesicatoria from seed. Phytopathology 81:831-834.
29. Simpson, A. J., et al. 2000. The genome sequence of the plant pathogen Xylella fastidiosa. Nature 406:151-157. [PubMed]
30. Sulzinski, M. A., G. W. Moorman, B. Schlagnhaufer, and C. P. Romaine. 1998. PCR based detection of artificial latent infections of geranium by Xanthomonas campestris pv. pelargonii. J. Phytopathol. 146:111-147.
31. Swings, J., P. De Vos, M. Van den Mooter, and J. De Ley. 1983. Transfer of Pseudomonas maltophilia Hugh 1981 to the genus Xanthomonas as Xanthomonas maltophilia (Hugh 1981) comb. nov. Int. J. Syst. Bacteriol. 33:409-413.
32. Talker-Huiber, D., J. Jose, A. Glieder, M. Pressnig, G. Stubenrauch, and H. Schwab. 2003. Esterase EstE from Xanthomonas vesicatoria (Xv_EstE) is an outer membrane protein capable of hydrolyzing long-chain polar esters. Appl. Microbiol. Biotechnol. 61:479-487. [PubMed]
33. Toussaint, V., C. E. Morris, and O. Carisse. 2001. A new semi-selective medium for Xanthomonas campestris pv. vitians, the causal agent of bacterial leaf spot of lettuce. Plant Dis. 85:131-136.
34. Tripathi, L., J. N. Tripathi, W. K. Tushemereirwe, and R. Bandyopadhyay. 2007. Development of a semi-selective medium for isolation of Xanthomonas campestris pv. musacearum from banana plants. Eur. J. Plant Pathol. 117:177-186.
35. Van Vuurde, J. W. L., L. Van den Bovenkamp, and G. W. Birnbaum. 1983. Immunofluorescence microscopy and enzyme-linked immunosorbent assay as potential routine tests for the detection of Pseudomonas syringae pv. phaseolicola and Xanthomonas campestris pv. phaseoli in bean seed. Seed Sci. Technol. 11:547-559.
36. Wang, Z. K., J. C. Comstock, E. Hatzoloukas, and N. W. Schaad. 1999. Comparison of PCR, BIO-PCR, DIA, ELISA, and isolation on semiselective medium for detection of Xanthomonas albilineans, the causal agent of leaf scald of sugarcane. Plant Pathol. 48:245-252.
37. Wells, J. M., B. C. Raju, H.-Y. Hung, W. G. Weisburg, L. Mandelco-Paul, and D. J. Brenner. 1987. Xylella fastidiosa gen. nov., sp. nov: gram-negative, xylem-limited, fastidious plant bacteria related to Xanthomonas spp. Int. J. Syst. Bacteriol. 37:136-143.

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