Xanthomonas RFLP Pages
GERARD RAYMOND LAZO
May, 1987
Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
Chairman: Dean W. Gabriel, Major Department: Plant Pathology
Gerard R. Lazo, Robin Roffey, and Dean W. Gabriel. 1987. Int. J. Syst. Bacteriol. 37:214-221.
The species Xanthomonas campestris (Pammel 1895) Dowson 1939, a member of the family Pseudomonadaceae which is always found in association with living plants, comprises over 125 different pathovars [6]. The pathovars have been named by "the plant from which first isolated" convention. Unfortunately, strains of some pathovars can be pathogenic on host species in different plant families [157]; thus possible relationships between pathovars may go undetected, since there is no convenient way to determine pathogenicity for all possible host plants, nor is there a useful systematic method for narrowing the possibilities. Further, and despite a widely held view that all xanthomonads are plant pathogens [356], non-pathogenic members of X. campestris are often found as ectoparasitic leaf colonizers (epiphytes) [355] and occasionally as endoparasites [357,88]. Their inability to provoke a pathogenic plant response renders them unclassifiable in this system. The attention given to damaging plant pathogenic strains may not be taxonomically or ecologically warranted. Genes which function to provoke a pathogenic response may be unstable and entirely different from those which confer host selectivity and parasitic ability [120]. Epiphytes and nonpathogenic endoparasites are known to be host selective [223,88]. Host selectivity is thought to be a stable characteristic [359]. Since xanthomonads are always found in association with living plants, host selectivity should result in genetic isolation of a pathovar population. Over time such isolation and random genetic drift should produce distinctive and stable phenotypic characteristics and therefore, establish conserved genetic markers.
Attempts at differentiating pathovars by methods other than pathogenicity have included rRNA-DNA and DNA-DNA hybridization [31,319], serology [220,334,232], phage-typing [332,158], and comparisons of profiles resulting from plasmid and chromosomal DNA restriction enzyme digests [214,360], protein electrophoresis [333], and gas-chromatography of fatty acids [88]. Although all of these methods are useful for specific purposes, few have demonstrated utility for replacing, clarifying, or indicating further pathogenicity tests.
We report here the use of restriction fragment length polymorphism (RFLP) analysis to differentiate pathovars of X. campestris. RFLP analyses have been widely used in the medical field to identify DNA fingerprints specific for inherited diseases or other genetic loci [336,311,252]. These analyses have also been applied to plants [210], bacteria [212], and eukaryotic organelles [320,349]. RFLP analyses allow the observance of genetic variation in organisms within defined regions of the genome due to DNA rearrangements or mutations which affect recognition sites for restriction endonucleases. Variation may be examined over a small (a few base pairs) or large (30-40 kb) stretch of DNA depending on the desired level of polymorphism detection. This variation can be observed for random or selected DNA target sequences within a species against a background of other genomic DNA fragments. In this study, RFLP analyses were applied to investigate the degree of genetic variation among 87 strains of X. campestris, comprising 23 different pathovars. Some of these results have appeared in abstracts [276,274].
Bacterial Strains
The bacterial strains used in this study are shown in Table 6-1. These strains were isolated as plant pathogens, identified as X. campestris, and classified into pathovars according to the susceptible host plants involved. Strains were tested for pathogenicity after single colony purification. For some strains, no pathovar assignments were made due to the lack of known plant pathogenic responses. Broth cultures of bacteria were grown in a peptone-glycerol medium (per L: 10.0 ml glycerol, 20.0 g peptone, and 1.5 g K2HPO4; pH 7.2). The strains were commonly stored and maintained at -80°C in the same medium containing 15% glycerol.
Table 6-1. Strains used in this investigation.
Pathovar Strain Host Location Sourcea
-------------------------------------------------------------------------------------------------------
Xanthomonas campestris pathovar
alfalfae KS Medicago sativa Kansas D.L.Stuteville
FL Medicago sativa Florida R.E.Stall
argemones 084-1052 Argemone mexicana Florida DPI
begoniae 084-155 Begonia sp. Florida DPI
campestris XC1 Brassica o. (cabbage) Oklahoma this study
084-809,084-1136 B. oleracea (cabbage) Florida this study
084-720 B.o. (brussels sprouts) Florida this study
084-1318 B. oleracea (broccoli) Florida DPI
carotae 13 Daucus carota California R.E.Stall
citri X59,X70 Citrus sp Brazil E.L.Civerolo
X62 Citrus sp Japan E.L.Civerolo
X69 Citrus sp Argentina E.L.Civerolo
084-3401 Citrus sp Florida DPI
cyamopsidis 13D5 Cyamopsis tetragonoloba C.I.Kado
X002,X005,X016,X017 Cyamopsis tetragonoloba Arizona J.Mihail
dieffenbachiae 084-729 Anthurium sp Florida DPI
068-1163 Dieffenbachia sp Florida DPI
esculenti 084-1093 Abelmoschus esculentus Florida DPI
glycines B-9-3 Glycine max Brazil W.F.Fett
1717 Glycine max Africa W.F.Fett
17915 Glycine max W.F.Fett
S-9-8 Glycine max Wisconsin W.F.Fett
hederae 084-1789 Hedera helix Florida DPI
holcicola Xh66 Sorghum vulgare Kansas L.Claflin
maculifoliigardeniae 084-6166 Gardenia sp Florida DPI
malvacearum D,M,N,O,U,V,X,Y,Z,TX84 Gossypium hirsutum Texas this study
A,B,E,F,G,H Gossypium hirsutum Oklahoma M.Essenberg
Ch1,Ch2 Gossypium hirsutum Chad L.S.Bird
HV25 Gossypium hirsutum Upper Volta L.S.Bird
Su2,Su3 Gossypium hirsutum Sudan L.S.Bird
FL79 Gossypium hirsutum Florida this study
mangiferaeindicae 084-116 Mangifera indica Florida DPI
nigromaculans 084-1984 Arctium lappa Florida DPI
pelargonii 084-190,084-1370 Geranium sp Florida DPI
phaseoli EK11,Xph25,Xpf11 Phaseolus vulgaris Nebraska M.Schuster
Xpa,Xp11 Phaseolus vulgaris Wisconsin A.W.Saettler
82-1,82-2 Phaseolus vulgaris Florida R.E.Stall
LB-2,SC-3B Phaseolus vulgaris Nebraska A.K.Vidaver
XP2 Phaseolus vulgaris New York J.A.Laurence
XP-JL Phaseolus vulgaris Kansas J.L.Leach
XP-JF Phaseolus vulgaris Missouri this study
XP-DPI Phaseolus vulgaris this study
pisi XP1 Pisum sativum Japan M.Goto
poinsettiicola 083-6248 Euphorbia pulcherrima Florida DPI
pruni 084-1793 Prunus sp Florida DPI
translucens X1105 Hordeum sp Montana D.Sands
vesicatoria E-3 Capsicum annuum Florida R.E.Stall
75-3 Lycopersicon esculentum Florida R.E.Stall
vignicola A81-331,C-1,CB5-1 Vigna ungiuculata Georgia R.D.Gitaitis
Xv19,SN2,432,82-38 Vigna unguiculata Georgia R.D.Gitaitis
vitians ICPB164 Latuca sp R.E.Stall
zinniae 084-1944 Zinnia elegans Florida DPI
unknown 084-1373 Philodendron sp. Florida DPI
084-3928 Fatsia sp. (hederae) Florida DPI
084-4348 Alocasia sp. (vitians) Florida DPI
083-2057 Syngonium sp. (vitians) Florida DPI
084-2848 Cissus sp. Florida DPI
084-1590 Euonymus sp. Florida DPI
251G,084-480 Impatiens sp. Florida DPI
084-6006 Jasminium sp. Florida DPI
X. fragariae Xfra1 Fragaria sp. Florida R.E.Stall
-------------------------------------------------------------------------------------------------------
a DPI = Florida Department of Agricultural and Consumer Services, Division of Plant Industry, Gainesville, Florida.
DNA Extraction
DNA was extracted from bacterial cultures at mid- to late-logarithmic growth phase. Extraction of bacterial DNA for cosmid library construction was by a modification of the method of Silhavy et al [335]. A critical modification was to wash cells in 50 mM TRIS, 50 mM Na2EDTA, 150 mM NaCl, resuspend in the same buffer containing 150 µg/ml proteinase K, add SDS (sodium dodecyl sulfate) to 1% (w/v), and
heat for 1 hr at 50 C. The sample was then extracted 2 times with phenol/chloroform/isoamyl alcohol (25:24:1; phenol equilibrated to pH 7.8 with 0.1 M TRIS), and the DNA spooled out from the aqueous phase after adding sodium acetate at 30 mM and 2 volumes 95% ethanol. This DNA was then washed in 70% ethanol. Extracted DNA was resuspended in TE (10 mM Tris(hydroxymethyl)aminomethane [TRIS], 1 mM disodium ethylenediaminetetraacetate [Na2EDTA] containing 20 µg/ml DNase-free pancreatic RNase; pH 7.6).
Agarose Gel Electrophoresis
Approximately 5 µg of DNA was digested with EcoRI or BamHI as specified by the manufacturer (Bethesda Research Laboratories). Digested DNA samples were run in a 20x25 cm agarose gel (0.6%; type II, Sigma) in TRIS-acetate buffer (40 mM TRIS-acetate, 1 mM Na2EDTA; pH 7.6) with electrophoresis of gels set at 35 V for 14-15 hrs. Fragments were visualized by ultraviolet irradiation (302 nm) after staining agarose gels in ethidium bromide (0.5 µg/ml). Photographs were taken using Polaroid Type 55 (or Type 57) film and a yellow filter (Tiffen no. 12). After the gels were photographed, the DNA was transferred to nitrocellulose by the method of Southern as described by Maniatis et al [113]. Restriction fragment sizes were estimated using DNA molecular size standards of lambda phage DNA digested with HindIV.
DNA Probes
The DNA probes used in this study were derived from a genomic library of X. campestris pv. citri strain 3401 constructed into the modified cosmid cloning vector pUCD5B, which was the vector pUCD5 [103] with a 2 kb BamHI fragment deleted from it. DNA of strain 3401 was partially digested with the restriction enzyme MboI and fractionated by size on a 10-40% (w/v) sucrose step gradient [113]. The pUCD5B vector was digested with the restriction enzyme BamHI and treated with calf intestinal alkaline phosphatase (Boehringer-Mannheim). Vector DNA and large fragment size fractions (25-40 kb) from strain 3401 were mixed and treated with T4 DNA ligase (Bethesda Research Laboratories) and recombinant cosmids were packaged in vitro with extracts prepared and utilized according to Scalenghe and Hohn's protocol II as described by Maniatis et al [113]. Transduction with Escherichia coli HB101 was performed using top agar on Luria-Bertani medium containing kanamycin (35 µg/ml). Cloned DNA fragments of strain 3401 in the vector averaged 27-38 kb.
DNA Hybridization
DNA clones used as probes were radiolabeled using 30-60 uCi of 32P-deoxycytidine (Du Pont NEN) with use of a nick translation kit (Bethesda Research Laboratories) and separated from low-molecular weight nucleotides on a mini-column of Sephadex G50-100. Southern blots were pre-hybridized in plastic bags for 4 hr and hybridized with the DNA probe for 16-18 hrs at 68 C. Following hybridization, membranes were washed once in 2X SSC, 0.5% SDS (1X SSC is 0.15 M NaCl, 0.15 M sodium citrate, pH 7.0) and washed once in 2X SSC, 0.1% SDS at ambient temperature, and washed two times in 0.1X SSC, 0.5% SDS at 68°C as described by Maniatis et al [113] for 'stringent' conditions. Nitrocellulose membranes were then air-dried and exposed to Kodak X-Omat AR film at -80°C in cassettes with intensifying screens. Hybridization of the probes to individual strains of X. campestris was repeated at least three times.
Restriction Fragment Patterns and Densitometry
From developed autoradiographs, DNA fragment sizes and profiles were determined and hybridization signals measured using a Gilford Response spectrophotometer equipped with an autoradiograph scanner. Routine scans were done at 600 nm wavelength with 0.5 mm aperture and 0.5 nm bandwidth setting. Scanning data was stored by computer. Using above information, comparison were made among strains of a given pathovar and also strains derived from different pathovars of X. campestris.
Agarose Gel Electrophoresis
For a given pathovar, the banding patterns appeared very similar and polymorphism in these strains was apparently limited (Fig. 6-1). In all, DNA from 87 strains of X. campestris, representing 23 different pathovars and including some with no known pathovar status were digested with restriction enzymes EcoRI and BamHI and separated by size on agarose gels. Based on restriction fragment patterns alone, it was possible to visualize DNA variability among the strains, but recognition of a particular DNA fragment banding patterns was difficult due to the numerous DNA fragments involved (Fig. 6-2). Some of the apparent variation was due to the presence of plasmid bands, which appear brighter against the chromosomal bands due to their relatively higher copy number within each cell.
Figure 6-1. Genomic DNA of strains of X. campestris digested with the restriction endonuclease EcoRI. Lane A, probe XCT1; lanes B-K, X. c. pv. phaseoli; lane B, JL; lane C, LB-2; lane D, Xph25; lane E, EK11; lane F, 82-1; lane G, 82-2; lane H, Xpa; lane I, XP2; lane J, XP2-1; lane K, JF; lanes L-M, X. c. pv. phaseoli var. fuscans; lane L, SC-3B; lane M, Xpf11; lanes N-O, X. c. pv. alfalfae; lane N, KS; lane O, FL;lanes P-R, X. c. pv. campestris; lane P, XC1 (cabbage); lane Q, 084-1318 (broccoli); lane R, 084-720 (brussels sprouts); lane S, unknown X. c. G65; lane T, probe XCT11.
Figure 6-2. Genomic DNA of strains of X. campestris digested with the restriction endonuclease EcoRI. Lane A, probe XCT1; lane B, X. c. pv. alfalfae FL; lane C, X. c. pv. begoniae 084-155; lane D, X. c. pv. campestris 084-809; lane E, X. c. pv. carotae 13; lane F, X. c. pv. cyamopsidis X002; lane G, X. c. pv. dieffenbachiae 068-1163; lane H, X. c. pv. glycines S-9-3; lane I, X. c. pv. holcicola Xh66; lane J, X. c. pv. maculifoliigardeniae 084-6166; lane K, X. c. pv. malvacearum FL79; lane L, X. c. pv. pelargonii 084-190; lane M, X. c. pv. phaseoli var. fuscans SC-3B; lane N, X. c. pv. pisi XP1; lane O, X. c. pv. translucens X1105; lane P, X. c. pv. vesicatoria 75-3; lane Q, X. c. pv. vignicola SN2; lane R, X. c. pv. vitians ICPB164; lane S, X. c. pv. zinniae 084-1944; lane T, probe XCT11.
DNA Probes
Two cosmid DNA clones, XCT1 and XCT11 were randomly selected from a genomic library of strain 3401 for use as DNA probes. XCT1 carried a 30 kb insert, and XCT11 carried a 37 kb insert. There were no detectable plasmids in strain 3401, thus the cosmid clones are assumed to contain chromosomal fragments. Assuming a 3333 kb genome for X. campestris [245], each cosmid probe represents approximately 1% of the total genome.
DNA Hybridization
Autoradiographs of Southern blots hybridized against either of the two DNA probes revealed conserved DNA fragments within each pathovar. For example, in figure 6-3 (lanes B - K) are shown the RFLP pattern from 10 different strains of X. campestris pv. phaseoli from different geographic locations. At least five EcoRI cut DNA fragments which hybridized to XCT1 appeared to be conserved in this pathovar. In lanes L and M are the RFLP patterns of two strains of X. campestris pv. phaseoli var. fuscans. These X. campestris pv. phaseoli var. fuscans strains also attack beans (and hence have the pv. phaseoli designation), but are biochemically distinct [6]. They produce a dark, melanin-like pigment in nutrient media and in these tests are clearly genetically distinguishable from the other X. campestris pv. phaseoli strains. Conserved DNA fragments were also seen for X. campestris pv. alfalfae (Fig. 6-3, lanes N and O) in which six of the up to 15 fragments appeared identical. Likewise, in four different strains of X. campestris pv. campestris (Fig. 6-3, lanes P - Q, Fig. 6-5, lane D) isolated from three different crucifer sources (cabbage, broccoli, and
brussels sprouts) at least four of over 10 fragments appeared to be identical. Sizes of hybridizing DNA fragments are given in Table 6-2. In Figure 6-4 is shown the RFLP patterns revealed using clone XCT11. In each case, a different RFLP pattern is shown, but the same genetic relationship is revealed. Other sets of conserved DNA fragments within a pathovar were seen among seven strains of X. campestris pv. vignicola (identical pattern), seven strains of X. campestris pv. cyamopsidis (nearly identical), and at least three strains each for X. campestris pvs. citri, glycines, malvacearum, pelargonii, and vesicatoria (not shown).
Figure 6-3. Genomic DNA of strains of X. campestris digested with the restriction endonuclease EcoRI and probed with cosmid clone XCT1. Lane A, probe XCT1; lanes B-K, X. c. pv. phaseoli; lane B, JL; lane C, LB-2; lane D, Xph25; lane E, EK11; lane F, 82-1; lane G, 82-2; lane H, Xpa; lane I, XP2; lane J, XP2-1; lane K, JF; lanes L-M, X. c. pv. phaseoli var. fuscans; lane L, SC-3B; lane M, Xpf11; lanes N-O, X. c. pv. alfalfae; lane N, KS; lane O, FL;lanes P-R, X. c. pv. campestris; lane P, XC1 (cabbage); lane Q, 084-1318 (broccoli); lane R, 084-720 (brussels sprouts); lane S, unknown X. c. G65; lane T, probe XCT11.
Table 6-2. Sizes of DNA fragments from Xanthomonas campestris genomic digests (EcoRI) which hybridized to the XCT1 DNA probe. See Figure 6-3.
A 12.2, 11.2, 7.9, 5.6, 5.1, 3.2, 2.2, 1.6, 1.0
B 14.1, 13.3, 10.4, 7.6, 6.4, 4.6, 4.3, 4.0, 3.7, 3.3, 3.1
C 14.4, 13.3, 10.4, 7.6, 6.4, 4.6, 4.3, 4.0, 3.7, 3.3, 3.1
D 14.1, 13.1, 10.4, 7.5, 6.4, 5.2, 4.6, 4.3, 4.0, 3.7, 3.1
E 13.9, 11.6, 10.4, 8.9, 5.2, 5.0, 4.6, 4.3, 4.0, 3.7, 3.1
F 13.7, 13.1, 11.6, 10.4, 8.9, 5.2, 5.0, 4.6, 4.3, 4.0, 3.7, 3.1, 1.9
G 13.7, 13.1, 11.6, 10.4, 8.9, 5.2, 5.0, 4.6, 4.3, 4.0, 3.7, 3.1, 1.9
H 13.1, 10.4, 7.5, 5.2, 5.0, 4.6, 4.3, 4.0, 3.7, 3.1, 1.9
I 13.1, 10.4, 7.5, 5.2, 5.0, 4.6, 4.3, 4.0, 3.7, 3.1, 1.9
J 13.1, 10.4, 7.5, 5.2, 5.0, 4.6, 4.3, 4.0, 3.7, 3.1, 1.9
K 13.1, 11.6, 10.4, 8.9, 5.2, 5.0, 4.6, 4.3, 4.0, 3.7, 3.1
L 11.9, 8.4, 7.1, 6.7, 4.5
M 17.0, 15.3, 10.0, 8.4, 4.5
N 19.7, 19.1, 15.5, 14.9, 12.9, 12.1, 8.0, 4.6, 3.8, 3.1, 2.7, 1.7
O 19.4, 18.0, 14.7, 13.7, 13.1, 11.7, 9.7, 7.3, 6.2, 5.2, 4.6, 3.7, 3.4, 3.1
P 20.3, 15.3, 10.9, 10.5, 10.2, 9.3, 8.4, 6.9, 5.9, 5.2, 4.4, 3.9, 2.6, 2.2, 1.9
Q 12.9, 10.8, 10.5, 9.1, 8.4, 7.7, 7.2, 6.3, 4.6, 4.4, 3.9, 3.3, 2.4, 2.2, 1.9
R 18.6, 15.8, 12.7, 10.8, 10.2, 9.1, 8.4, 7.7, 6.7, 5.6, 4.4, 3.3, 2.2, 1.9
S 11.3, 8.0, 6.9, 3.4, 2.5, 1.6
T 17.2, 15.3, 14.3, 10.5, 8.6, 7.2, 6.8, 5.8
Figure 6-4. Genomic DNA of strains of X. campestris digested with the restriction endonuclease EcoRI and probed with cosmid clone XCT11. Lane A, probe XCT1; lanes B-K, X. c. pv. phaseoli; lane B, JL; lane C, LB-2; lane D, Xph25; lane E, EK11; lane F, 82-1; lane G, 82-2; lane H, Xpa; lane I, XP2; lane J, XP2-1; lane K, JF; lanes L-M, X. c. pv. phaseoli var. fuscans; lane L, SC-3B; lane M, Xpf11; lanes N-O, X. c. pv. alfalfae; lane N, KS; lane O, FL;lanes P-R, X. c. pv. campestris; lane P, XC1 (cabbage); lane Q, 084-1318 (broccoli); lane R, 084-720 (brussels sprouts); lane S, unknown X. c. G65; lane T, probe XCT11.
Figure 6-5. Genomic DNA of strains of X. campestris digested with the restriction endonuclease EcoRI and probed with cosmid clone XCT1. Lane A, probe XCT1; lane B, X. c. pv. alfalfae FL; lane C, X. c. pv. begoniae 084-155; lane D, X. c. pv. campestris 084-809; lane E, X. c. pv. carotae 13; lane F, X. c. pv. cyamopsidis X002; lane G, X. c. pv. dieffenbachiae 068-1163; lane H, X. c. pv. glycines S-9-3; lane I, X. c. pv. holcicola Xh66; lane J, X. c. pv. maculifoliigardeniae 084-6166; lane K, X. c. pv. malvacearum FL79; lane L, X. c. pv. pelargonii 084-190; lane M, X. c. pv. phaseoli var. fuscans SC-3B; lane N, X. c. pv. pisi XP1; lane O, X. c. pv. translucens X1105; lane P, X. c. pv. vesicatoria 75-3; lane Q, X. c. pv. vignicola SN2; lane R, X. c. pv. vitians ICPB164; lane S, X. c. pv. zinniae 084-1944; lane T, probe XCT11.
Table 6-3. Sizes of DNA fragments from Xanthomonas campestris genomic digests (EcoRI) which hybridized to the XCT1 DNA probe. See Figure 6-5.
A 12.2, 11.2, 7.9, 5.6, 5.1, 3.2, 2.2, 1.6, 1.0
B 20.5, 18.8, 15.3, 14.2, 13.5, 12.0, 7.3, 6.2, 5.1, 4.6, 3.7, 3.4, 3.1
C 18.7, 12.1, 9.5, 8.6, 8.2, 7.7, 6.9, 6.7, 6.2, 6.0, 4.8, 4.2, 4.0, 3.4, 3.0, 2.9, 2.5, 2.3
D 19.4, 16.4, 14.4, 13.1, 11.1, 10.5, 9.2, 8.5, 7.7, 6.7, 5.6, 4.3, 4.0, 2.2, 1.9
E 17.9, 12.9, 4.1, 3.5, 3.0, 2.3, 1.5
F 16.2, 11.5, 9.5, 8.7, 5.7, 4.0, 3.6, 3.1, 3.0, 1.4
G 14.2, 11.1, 4.5, 3.9, 3.5, 3.0
H 12.2, 10.2, 8.3, 7.9, 6.5, 5.5, 4.2, 3.1
I 12.9, 12.0, 7.6, 7.1, 6.5, 4.7, 3.0, 1.7
J 15.6, 14.2, 9.4, 7.9, 6.3, 5.0, 4.6, 4.0, 3.1, 2.5, 2.2
K 20.9, 17.1, 16.2, 15.5, 14.2, 11.0, 10.4, 9.2, 8.2, 7.5, 6.7, 4.2, 4.0, 3.3, 3.1, 3.0
L 19.4, 12.7, 10.4, 9.0, 7.9, 7.7, 6.8, 5.7, 4.2, 3.9, 3.0, 2.7, 2.3, 1.9, 1.5, 1.4
M 12.0, 8.4, 7.0, 6.8, 4.5
N 15.9, 8.4, 4.9, 3.4, 2.1, 1.9
O 9.2, 4.4
P 13.3, 9.2, 7.2, 6.9, 6.2, 4.6, 3.8, 3.1, 2.1
Q 11.0, 8.5, 7.0, 6.3, 4.2, 3.2
R 16.7, 14.2, 12.9, 9.9, 7.4, 6.9, 6.3, 4.8, 3.8, 3.5, 3.2, 2.6, 2.4, 2.2, 1.6
S 12.9, 12.5, 9.5, 7.7, 6.5, 4.4, 4.0, 3.8
T 17.6, 15.6, 14.7, 10.5, 8.6, 7.2, 1.4
DNA of strains representing other pathovars of X. campestris were also digested with either EcoRI or BamHI, and hybridized with either XCT1 or XCT11. The pathovars of X. campestris included in these experiments were X. campestris pvs. alfalfae, begoniae, campestris, carotae, citri, cyamopsidis, dieffenbachiae, esculenti, glycines, hederae, holcicola, maculifoliigardeniae, malvacearum, mangiferaeindicae, nigromaculans, pelargonii, phaseoli, pisi, translucens, vesicatoria, vignicola, vitians, and zinniae. Also included were some strains isolated as pathogens of an Alocasia sp., Cissus sp., Impatiens sp., Fatsia sp., Jasmine sp., Argemone sp., and a Euonymus species for which no pathovar designations were available.
The results of hybridization of probe XCT1 to DNA of different pathovars of X. campestris digested with EcoRI are shown in Figure 6-5. The clone XCT1 appeared to contain DNA fragments of strain 3401 which were able to demonstrate variability of restriction fragment patterns among different pathovars of X. campestris. Variability in
RFLP pattern appeared as qualitative and quantitative differences among hybridization profiles of various sized DNA fragments in the strains. For instance, the weak hybridization signal seen from X. campestris pv. translucens (fig. 6-5, lane O) suggests that this strain is distinct from the other pathovars compared, at least over the 1% of the genome contained on the clone XCT1. In other pathovars such as X. campestris pv. alfalfae (fig. 6-5, lane B), X. campestris pv. begoniae (lane C), X. campestris pv. cyamopsidis (lane F), X. campestris pv. malvacearum (lane K), X. campestris pv. vignicola (lane Q), and X. campestris pv. vitians (lane R) where some DNA fragments had strong hybridization signals, a close relatedness with portions of DNA in the cosmid clone is suggested. Some of the DNA fragments from different pathovars which hybridized most strongly to the probe were also of identical size with the EcoRI fragments of the XCT1 clone. As expected, DNA of X. campestris pv. citri strain 3401 digested with EcoRI, or BamHI [276,274] contained fragments which corresponded to DNA fragments of the XCT1 probe (Fig. 6-5, lane A). Sizes of hybridizing DNA fragments are given in Table 6-3.
From observations using different enzymes and different probes, the same patterns emerged, indicating that the basic chromosome structure of X. campestris can be used to differentiate strains into specific RFLP types. Although a pathovar may contain more than one type of variant (e.g. X. campestris pv. phaseoli), all strains of a given type appear to exhibit the same host selectivity. The RFLP pattern is highly distinctive for each type, and can be used to unambiguously assign an unknown sample to a pathovar, using simple
visual comparisons of pattern against a set of known samples, such as those given in Figure 6-5.
The only widely accepted and most practical method for differentiating pathovars of X. campestris is to inoculate a plant suspected as the host for that pathovar. This practice can sometimes be tedious, time consuming, subjective and subject to a surprising number of artifactual influences. It is not known whether host selectivity is unstable as suggested by Dye [358]; stable as suggested by Schnathorst [359] or even taxonomically significant. Although the classification is thought to be useful, it can be highly misleading since emphasis is placed upon one characteristic - pathogenicity. If only one or few gene differences were involved in host selection, then differentiation by pathovar could be highly misleading, at least in the sense that a given group of strains might be capable of attacking more than one host in some cases. Alternatively, two or more strains of relatively unrelated groups could be cataloged together because they happen to attack the same host. This latter situation appears to be the case with X. campestris pv. phaseoli and X. campestris pv. phaseoli var. fuscans (see Fig. 6-3), and with Florida strains of X. campestris pv. citri [276].
In order to address these problems, we evaluated the potential of using DNA sequence variation of X. campestris for strain classification purposes. DNA sequence variation was first examined by digesting DNA with restriction enzymes and visualizing directly the resulting fragments on ethidium bromide stained gels. These stained gels were useful for side by side comparisons of restriction fragments for samples run on the same gel, but comparisons between different gels were more difficult. Subsequently, visualizing and comparing variation in bacterial genomes with cosmid clones carrying 30-38 kb cloned X. campestris genome fragments was simplified. In both cases variation was revealed by alterations in the sizes of visualized DNA restriction fragments (RFLPs). The use of DNA probes derived from chromosomal DNA fragments also alleviated some of the difficulty in comparing stained gels with the sometimes present polymorphism of the higher copy number plasmid DNA fragments which can occur in the background.
Some of the selected clones tested as DNA probes appeared to be useful for identifying DNA sequences conserved within a given pathovar, while others appeared to identify DNA sequences which were highly conserved at the species level. The DNA which was considered conserved at the species level was represented as banding patterns which were nearly identical among all strains over the several pathovars tested. Examples of DNA sequences which are known to be highly conserved are rRNA encoding genes [212,213]. There are undoubtedly others. If smaller DNA probes containing known genetic loci such as those associated with genes for pectate lyase and protease [29], or avirulence activity [106,234] were tested, these smaller, more defined DNA probes could be used to determine if such genetic loci were highly conserved or variable. With larger, randomly selected probes, the likelihood was increased for detecting strain- or pathovar-specific variation. The large (greater than 30 kb) size of the probes used in this study allowed detection of both.
Using RFLP genomic blots, it appeared that phylogenetic relationships between the full spectrum of described pathovars of X. campestris might be determinable. Some mathematical approaches toward determining phylogeny based on restriction cleavage sites has been proposed [342,346]. DNA probes have been used to make phylogenetic comparisons among the relatively conserved mitochondrial [320] and chloroplast [349] genomes. The variation seen among the different pathovars of X. campestris appeared within the range that is usefully distinguishable with this test. Only limited amounts of variation can be usefully distinguished, such as that occurring within a species. Significantly, the RFLP groupings closely corresponded with the pathovar groupings, strengthening the taxonomic significance of this classification. All strains tested were readily grouped by RFLP phenotypes, and the classification based on RFLP patterns correlated very well with the classification based on pathogenicity. This technique may provide a more convenient means of classifying these bacteria. In addition, unexpected taxonomic relationships between pathovars might be revealed. The potential pathogenic range of each RFLP group would be of obvious value.
By comparing observed RFLPs among strains of X. campestris using selected DNA probes, it was possible to identify unknown strains when known standards were included. Additionally, strains previously undescribed could be classified as being related to known pathovars. For instance, similarities were found between undescribed strains isolated from a Alocasia sp. and Argemone sp.; and a Cissus sp. and Jasmine specie. The strains isolated from a Philodendron sp. and Dieffenbachia sp. both corresponded to that of X. campestris pv. dieffenbachiae. The other unknown strains tested appeared different from one another, and did not appear similar to any of the other X. campestris pathovars as characterized by the DNA probes. Finally, this technique may provide a basis for the classification of nonpathogenic and epiphytic xanthomonads. Experiments can now be conducted to determine whether epiphytes are all basically similar or form diverse groups, whether they are restricted in host range or if they are nonpathogenic, but closely related to described pathovars. Although this approach provides another classification scheme for strains, it appears to be basically compatible with the currently recognized and useful pathovar naming scheme, which relies primarily on plant-specificity. Although certain pathovars may need to be redefined, this work supports and helps validate the natural taxonomic groupings provided by the pathovar naming system.