Centro Internacional de Agricultura Tropical (CIAT), Cali, Colombia, South America

Genotypic Characterization of the Common Bean Bacterial Blight Pathogens,

Xanthomonas axonopodis pv. phaseoli and Xanthomonas axonopodis pv. phaseolivar. fuscans by rep-PCR and PCR–RFLP of the Ribosomal Genes

G. S.G. S. MahukuMahuku,, C.C. JaraJara,, M. A.M. A. HenriquezHenriquez,, G.G. CastellanosCastellanos andand J.J. CuasquerCuasquer

Authors� address: Centro Internacional de Agricultura Tropical (CIAT), A. A. 6713, Cali, Colombia, South America(correspondence to G. S. Mahuku. E-mail: g.mahuku@cgiar.org)

Received April 13, 2005; accepted October 15, 2005

Keywords: common bacterial blight, Phaseolus vulgaris, rep-PCR, restriction fragment length polymorphism, Xanthomonasaxonopodis pv. phaseoli

AbstractCommon bacterial blight (CBB), caused by Xantho-monas axonopodis pv. phaseoli and X. axonopodis pv.phaseoli var. fuscans is one of the most destructive dis-eases of common bean worldwide. The interrelated-ness, genetic diversity and geographical distribution ofthe CBB pathogens was assessed using restriction frag-ment length polymorphism (RFLP) analysis of polym-erase chain reaction amplified 16S ribosomal gene,including the 16S–23S intergenic spacer region andrepetitive element PCR (rep-PCR). RFLP profiles gen-erated by the restriction endonucleases MboI, RsaIand HaeIII differentiated X. axonopodis pv. phaseolifrom X. axonopodis pv. phaseoli var. fuscans and non-pathogenic Xanthomonas species associated with com-mon bean. Cluster analysis of rep-PCR profilesrevealed a high level of genetic differentiation(GST ¼ 0.56) between the two CBB pathogens, show-ing that they are genetically distinct. Significant levelsof genetic diversity were observed within each strain,indicating that the two bacteria are not clonal. Moregenetic diversity was observed in X. axonopodis pv.phaseoli (H ¼ 0.134; I ¼ 0.223) than X. axonopodis pv.phaseoli var. fuscans (H ¼ 0.108; I ¼ 0.184). However,no geographical differentiation was evident for eitherX. axonopodis pv. phaseoli var. fuscans (GST ¼ 0.013)or X. axonopodis pv. phaseoli (GST ¼ 0.017). This lackof geographical differentiation has important practicalimplications, as available host resistance genes arelikely to be effective in controlling the disease indiverse geographical areas.

IntroductionCommon bacterial blight (CBB) of common bean,caused by Xanthomonas axonopodis pv. phaseoli(Smith) Dye, is one of the most destructive diseases in

bean producing areas worldwide (Saettler, 1989). Thepathogen attacks all aerial plant parts, but symptomsare more severe and conspicuous on leaves and pods.The bacteria are seed-borne, this being the primaryinoculum sources (Saettler, 1989), but it can survivefor months in leaves and other plant debris on the soil(Saettler, 1989; Gilbertson et al., 1991; Fourie, 2002).Heavy and early infection, high humidity, tempera-tures fluctuating between 20 and 30�C and alternatedry and wet weather can lead to more than 40% yieldlosses in susceptible cultivars (Saettler, 1989).

Two bacterial strains, X. axonopodis pv. phaseoli, andX. axonopodis pv. phaseoli var. fuscans are associatedwith CBB disease. Both strains cause identical symp-toms, but X. axonopodis pv. phaseoli var. fuscans hasbeen reported to be more aggressive (Ekpo and Saettler,1976; Bozzano-Saguier and Rudolph, 1994; Opio et al.,1996). In addition, X. axonopodis pv. phaseoli var.fuscans produces a brown diffusible pigment in variousculture media (Gilbertson et al., 1991; Goodwin and So-pher, 1994; Fourie, 2002). The pigment results from thesecretion and oxidation of homogenistic acid, an inter-mediate in the tyrosine catabolic pathway (Goodwinand Sopher, 1994). In X. axonopodis pv. phaseoli, a com-plete catabolic pathway that permits the use of tyrosineas a nutrient is present and no pigment is produced(Goodwin and Sopher, 1994). The pigment has not beenassociated with pathogenicity (Fourie, 2002; Gilbertsonet al., 1991).

The interrelatedness of X. axonopodis pv. phaseoliand X. axonopodis pv. phaseoli var. fuscans has been asubject of controversy (Lazo and Gabriel, 1987;Hildebrand et al., 1990; Xue and Goodwin, 1994;Chan and Goodwin, 1999). Several researchers haveproposed that X. axonopodis pv. phaseoli and X. axo-nopodis pv. phaseoli var. fuscans are distinct species ofXanthomonas (Lazo and Gabriel, 1987; Hildebrand

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et al., 1990; Xue and Goodwin, 1994). Pulse-field gelelectrophoresis analysis of whole chromosomes (Chanand Goodwin, 1999) revealed that the average genomesize of X. axonopodis pv. phaseoli (3850.6 + 48.9 kb)was significantly larger than that of X. axonopodis pv.phaseoli var. fuscans (3584.3 + 68.1 kb). Restrictionfragment length polymorphism (RFLP) of XbaI diges-ted genomic DNA using Hrp and pectate lyase genesas probes showed that X. axonopodis pv. phaseoli andX. axonopodis pv. phaseoli var. fuscans were distinct(Chan and Goodwin, 1999). Similarly, informationobtained from RFLP analysis of genomic and plasmidDNA (Gabriel et al., 1989), isoenzyme and plasmidprofiling, RFLP (Lazo and Gabriel, 1987), DNA-DNA hybridizations (Hildebrand et al., 1990) andamplified DNA polymorphisms (Xue and Goodwin,1994; Birch et al., 1997) revealed that X. axonopodispv. phaseoli might be genetically distinct from X. axo-nopodis pv. phaseoli var. fuscans.

There is a lack of conclusive information on thegenetic diversity within and between X. axonopodis pv.phaseoli and X. axonopodis pv. phaseoli var. fuscansfrom large samples collected from different regionswhere CBB is a production constraint. This informa-tion may provide insights into the interrelatedness ofCBB pathogens and the level of genetic diversity pre-sent within each strain, as well as provide the basis forselecting isolates for screening bean genotypes to iden-tify sources of resistance genes for use in breeding pro-grams. The assessment of genetic variation may alsofacilitate investigations on the taxonomy, epidemiologyand detection of the CBB pathogen (Leung et al.,1993) and aid breeding programmes in the develop-ment of CBB resistant cultivars.

The objectives of this study were to assess the inter-relatedness and level of genetic diversity within andbetween X. axonopodis pv. phaseoli and X. axonopodispv. phaseoli var. fuscans and ascertain the level of geo-graphical differentiation for each strain. To avoidpotential bias in genetically characterizing the CBBpathogens, we used primers derived from the entero-bacterial repetitive intergenic consensus (ERIC)sequence, repetitive extragenic palindromic (REP)sequences and the conserved repeated bacterial DNAelement BOX (Louws et al., 1994; Vera Cruz et al.,1996; Scortichini et al., 2001) and RFLP of 16SrDNA, part of the 23 rDNA, including the 16S–23Sintergenic spacer (IGS) region (Manceau and Horvais,1997) to characterize X. axonopodis pv. phaseoli andX. axonopodis pv. phaseoli var. fuscans isolates of adiverse origin.

Materials and MethodsBacterial strains and their identification

A total of 412 bacterial strains consisting of isolatesrandomly obtained from infected bean leaves andseeds, or from the culture collection maintained by thebean pathology laboratory of the Centro Internacionalde Agricultura Tropical (CIAT) were used in thisstudy (Table 1). Pathogenic and non-pathogenic

isolates were identified by their ability to infect thehighly susceptible common bean cultivar BAT 41(Table 2). Pathogenic isolates were inoculated onto sixPhaseolus genotypes that include Phaseolus acutifoliusaccessions, G 40006, OAC and G 40001R and Phaseo-lus vulgaris cultivars, XAN 112, XAN 159 and VAX 6,which were bred for CBB resistance. Inoculations weredone at least twice and using the multiple needlemethod (Zapata et al., 1985). All bred lines have genesfrom different P. acutifolius accessions, which carriesmajor genes for CBB resistance (Singh and Munoz,1999). All pathogenic (343) and a six non-pathogenicisolates were selected for further studies using molecu-lar techniques.

Bacteria DNA extraction

Genomic DNA was prepared using a small-scaleprotocol described by Mahuku (2004). Briefly, bac-teria cells were harvested after 24 h of incubation onYDC medium, collected by centrifugation at5000 · g for 5 min, and washed twice with 1 m NaCland two times with sterile distilled and deionizedwater. The bacterial cell pellet was resuspended inwarm (55�C) extraction buffer (0.2 m Tris–HCl pH8.0; 10 mm EDTA, pH 8.0, 0.5 m NaCl, 1% SDS)containing Proteinase K (10 lg/ml). After 60 min at55�C, 0.5 vol. of 7.5 m ammonium acetate wasadded, gently mixed and left to stand for 10 min atroom temperature. Following centrifugation, thesupernatant was transferred to a fresh tube and theDNA precipitated by adding 1 vol. of ice-cold iso-propanol. The pellet was washed with 70% ethanol,dried and resuspended in 1X TE buffer (10 mm

Tris–HCl, pH 8.0; 1 mm EDTA) containing 10 mg/ml RNase A. Tubes were incubated at 37�C for anhour, and the DNA was precipitated with 1/10 vol.of 3 m NaAc, pH 5.2, and 2 vol. of 95% ethanol.The pellet was dried and finally suspended in 0.1XTE buffer. The quality of extracted DNA was deter-mined by electrophoressis on 0.7% agarose gels. TheDNA concentration was measured with a fluorome-ter (Hoefer� DyNA Quant 2000, Pharmacia Biotech,NJ, USA) and adjusted to 20 ng/ll in 0.1X TEbuffer.

Distinction of CBB pathogens

Xanthomonas axonopodis pv. phaseoli var. fuscans iso-lates were distinguished from X. axonopodis pv. phase-oli by their ability to produce a brown diffusiblepigment on yeast extract-dextrose CaCO3 (YDC) med-ium and by the presence of an 820 bp (X. axonopodispv. phaseoli var. fuscans) or 900 bp (X. axonopodis pv.phaseoli) fragment obtained using the RAPD primerOPG-11 (Birch et al., 1997). RAPD–PCR amplifica-tions were conducted in 12.5 ll reaction volumes con-taining 20 ng of genomic DNA, 1 U Taq DNApolymerase (Promega, Madison, WI, USA), 0.6 lmprimer, 100 lm of each dNTP, 1X PCR reactionbuffer, and 0.75 mm MgCl2 using cycling conditionsdescribed by Birch et al. (1997).

36 Mahuku et al.

PCR–RFLP of bacterial ribosomal genes

The 16S gene including the IGS spacer region wasamplified using the conserved primers Phill-1

[5¢-TGGCTCAGATTGAACGCTGGCGGC-3¢ and P322-Anti [5¢-GGTTCTTTTCACCTTTCCCTC-3¢ thatanneals at the 5¢ end of the 16S ribosomal genes and 23Sribosomal genes, respectively. PCR amplifications werecarried out in a 50 ll reaction volume that contained 1XPCR buffer (pH 8.3), 30 ng of genomic DNA, 0.5 lmeach primer, 0.2 mm dNTP, 2 mm MgCl2 and 2 U Taqpolymerase (Promega). Reactions were performed in anautomated MJ Research PTC 100 programmable ther-mal cycler (Watertown, MS, USA), programmed for 35cycles of 95�C 1 min; 65�C 1 min; 72�C 3 min; and afinal extension at 72�C for 10 min. An aliquot of thereaction mixture (6 ll) was analysed by agarose gel elec-trophoresis (1.0% agarose). Subsequently, aliquots(8 ll) of PCR products were digested with the tetramer-ic restriction endonucleases MboI, RsaI, HinfI, TaqI,CfoI, HpaII, AluI and HaeIII as recommended by themanufacturer (New England Biolabs, MA, USA).Restricted DNA was visualized by gel electrophoresis in1.5% agarose gels and 100 bp ladder used as a molecu-lar marker. Gel images were captured using the EagleEye II gel documentation system (Stratagene Inc., LaJolla, CA, USA) and band position was determinedusing the Quantity One scientific Software, Version 4(Bio-Rad, Hercules, CA, USA).

Table 2Response of 412 Xanthomonas species isolated from common beanfollowing inoculation on six Phaseolus sp. genotypes carrying differ-ent CBB resistance genes

Phaseolus sp.genotypea

Isolates with acompatible interaction

Isolates with anincompatible interaction

XAN 112 118 225G 40006 320 23XAN 159 117 226OAC 166 177G 40001R 6 337VAX 6 0 343BAT 41 343b 0 (69)c

aXAN 112, Xan 159 and VAX 6 are bred lines that contain CBBresistance genes from P. acutifolius. G 40006, OAC and G 40001Rare P. acutifolius genotypes carrying different major genes for CBBresistance. BAT 41 is a P. vulgaris bred line that has no known CBBresistance.bOnly isolates that were pathogenic on BAT 41 were inoculated onXAN 112, G40006, XAN 159, OAC, G40001R and VAX 6.cThe number in brackets signifies non-pathogenic Xanthomonas iso-lates.

Table 1Origin of CBB pathogens(X. axonopodis pv. phaseoli andX. axonopodis pv. phaseolivar. fuscans) of common beanused in this study

Geographical region Country

CBB pathogen strains

X. axonopodispv. phaseoli var. fuscans

X. axonopodispv. phaseoli Total

South America Argentina 9 3 12Brazil 12 26 38Colombia 25 36 61Paraguay 6 20 26Peru 1 0 1Uruguay 0 5 5Sub-total 53 90 143

Central America Costa Rica 8 17 25El Salvador 5 4 9Guatemala 9 6 15Honduras 6 9 15Nicaragua 3 2 5Sub-total 31 38 69

Caribbean Cuba 3 10 13Dominican Republic 0 14 14Haiti 4 16 20Jamaica 0 1 1Puerto Rico 7 9 16Sub-total 14 50 64

Africa Burundi 3 2 5Malawi 6 0 6South Africa 1 0 1Uganda 0 1 1Zambia 2 0 2Sub-total 12 3 15

North America Canada 1 2 3Mexico 7 6 13USA 6 9 15Sub-total 14 17 31

Middle East Iran 0 7 7Turkey 4 4 8Sub-total 4 11 15

Europe Spain 0 2 2Australia Australia 4 0 4

Total 132 211 343

CBB, common bacterial blight.

37Genetic Diversity of Bean Bacterial Blight Pathogen

rep-PCR amplification

rep-PCR protocols were carried out as described byLouws et al. (1994) in 12.5 ll reaction volumes con-taining 20 ng of template DNA and 2 U Taq polym-erase (Promega) and using primers corresponding toERIC, REP and BOX elements (Martin et al., 1992).Primers were synthesized by Integrated DNA Technol-ogies Inc. (IDT, Coralville, IA, USA). PCR amplifica-tions were performed in an automated MJ ResearchPTC 100 programmable thermal cycler using the pro-gramme described by Vera Cruz et al. (1996). A 8 llportion of amplified PCR product was separated byelectrophoresis on 1.5% agarose gels in 0.75X Tris–acetate-EDTA buffer (40 mm Tris–HCL and 1 mm

EDTA, adjusted to pH 7.6 with glacial acetic acid) at45 V for 15 h. Gel images were captured using theEagle Eye II gel documentation system (StratageneInc.), and band position was determined using theQuantity One scientific Software, Version 4 (Bio-Rad).Amplifications were done at least twice for each isolate.

Data analysis

Each unique banding pattern generated by rep-PCRprimers or PCR–RFLP was regarded as a haplotype.The presence of a band was coded as 1, and itsabsence as 0. The genetic distance between two isolateswas calculated based on Dice’s coefficient using SASprogramme (SAS Version 6, SAS Institute Inc., Cary,NC, USA). Multiple correspondence analysis (MCA)was used to assign isolates to clusters to avoid definingevery clonally related pair of haplotyes as a separategroup. Each MCA defined group of isolates was con-sidered as a separate genetic lineage. A genetic diver-sity index (HT) was calculated for each cluster basedon the band presence or absence. HT represents theprobability that two isolates randomly chosen andbelonging to the same cluster are different.

Genetic diversity

Diversity statistics for each population, including thepercentage of polymorphic loci (Lin et al., 1997); gen-etic diversity (H) at each locus in each population(Nei, 1973), and the average diversity (Ho) over lociusing both the unbiased Nei’s gene diversity (Nei,1987) and Shannon’s information index (Shannon andWeaver, 1949), were calculated using PopGene Version1.31 (Yeh et al., 1999). The degree of differentiationamong populations was quantified using the unbiasedgenetic identity (I) and genetic divergence among pop-ulations was estimated by calculating Nei’s GST value(Nei, 1987). Divergence statistics were estimated usingthe analysis of molecular variance (amova, Excoffieret al., 1992). The total variance was partitioned intothat ascribed to differences within and between X. axo-nopodis pv. phaseoli and X. axonopodis pv. phaseolivar. fuscans, and to geographical origin of the isolates.Genetic distances between populations were calculatedusing Nei’s unbiased genetic distance (Nei, 1978) andused to infer the unweighted pair grouping by mathe-matical averaging (UPGMA) topology using TFPGA

(Miller, 1997). The genotypic diversity within X. axo-nopodis pv. phaseoli and X. axonopodis pv. phaseolivar. fuscans was evaluated using Shannon diversityindex (Shannon and Weaver, 1949).

ResultsIdentification of CBB pathogens

Of the 412 isolates tested, 343 invoked symptoms onthe common bean cultivar BAT 41, while the rest (69)did not cause disease symptoms on this cultivar andwere considered non-pathogenic to common bean(Table 2). No X. axonopodis pv. phaseoli or X. axono-podis pv. phaseoli var. fuscans isolate caused disease onVAX 6, a line that was developed at CIAT throughinterspecific hybridization of P. vulgaris · P. acutifo-lious, and embryo rescue techniques, followed byresistance gene pyramiding (Singh and Munoz, 1999).All pathogenic (343) and six non-pathogenic isolateswere included for further analysis using moleculartechniques.

Distinction of CBB pathogens

Of the 343 CBB pathogen isolates, 211 did not pro-duce a brown pigment in culture and were confirmedas X. axonopodis pv. phaseoli by the presence of a900 bp fragment amplified using the RAPD primerOPG-11 (Fig. 1). Xanthomonas axonopodis pv. phaseolivar. fuscans isolates (132) produced a brown pigmentin culture and an 820 bp fragment was amplified fromall isolates using the OPG-11 primer (Fig. 1). Moreisolates (61%) were identified as X. axonopodis pv.phaseoli, but both strains were detected from all coun-tries and regions that were represented in this study,except Australia. There was a tendency of one strainto predominate in some regions. For example, 12 of

1 2 3 4 5 6 7 8

0.9 kb0.8 kb

0.6 kb

Fig. 1 Agarose gel showing PCR products amplified from genomicDNA of X. axonopodis pv. phaseoli and X. axonopodis pv. phaseolivar. fuscans strains using the RAPD primer OPG11. Lanes 1–4 areX. axonopodis pv. phaseoli isolates, lanes 4–6 are X. axonopodispv. phaseoli var. fuscans isolates, lane 7 is a non-pathogenicXanthomonas isolate and lane 8 is a 100 bp molecular size marker

38 Mahuku et al.

the 15 isolates from Africa were X. axonopodis pv.phaseoli var. fuscans, whereas in the Caribbean,X. axonopodis pv. phaseoli predominated, accountingfor 78% of the isolates from this region (Table 1). InCentral America, Mexico, USA and Canada, there wasan almost even distribution of the two forms (Table 1).

PCR–RFLP analysis

PCR amplification of the ribosomal gene cassetteresulted in a fragment of approximately 3.5 kb forX. axonopodis pv. phaseoli, X. axonopodis pv. phaseolivar. fuscans and non-pathogenic Xanthomonas isolates.No polymorphisms were apparent in the size of theamplified ribosomal genes, but size estimations follow-ing restriction digestion revealed that X. axonopodispv. phaseoli var. fuscans had a smaller fragment(3.2 kb) than X. axonopodis pv. phaseoli (approxi-mately 3.5 kb). Informative RFLP profiles wereobtained using the restriction endonuclease MboI, RsaIand HaeIII, allowing the differentiation of X. axonopo-dis pv. phaseoli from X. axonopodis pv. phaseoli var.fuscans strains and non-pathogenic Xanthomonas iso-lates (Fig. 2a–c). RFLP analysis using RsaI revealedtwo genotypes with six well-resolved bands (Fig. 2a).Two fragments of approximately 1.1 and 1.2 kb werepresent in X. axonopodis pv. phaseoli var. fuscans iso-lates only, while three fragments of approximately 500,600 and 750 bp were present only in X. axonopodis pv.phaseoli isolates (Fig. 2a). Digestion with HaeIIIyielded five fragments in X. axonopodis pv. phaseolivar. fuscans and six in X. axonopodis pv. phaseoli iso-lates (Fig. 2b). Two fragments of approximately 300and 400 bp were present only in X. axonopodis pv.phaseoli, while a 700 bp fragment was only present inX. axonopodis pv. phaseoli var. fuscans isolates. MboIgenerated the most polymorphic bands and severalfragments that were present in X. axonopodis pv.phaseoli var. fuscans were absent from X. axonopodispv. phaseoli and vice versa (Fig. 2c). For all enzymes,the pattern from non-pathogenic isolates was morecomplex, and distinctly different from patternsobserved for X. axonopodis pv. phaseoli and X. axo-nopodis pv. phaseoli var. fuscans (Fig. 2a–c).

Cluster and MCA of data resulting from profilesgenerated by the three restriction endonucleases sep-arated X. axonopodis pv. phaseoli isolates into agroup that was distinct from that occupied byX. axonopodis pv. phaseoli var. fuscans, or non-pathogenic Xanthomonas isolates (Fig. 3). Analysis ofcombined data showed little within strain variationin RFLP profiles, except for non-pathogenic Xantho-monas isolates, where profiles for the six isolateswere distinct.

rep-PCR analysis

Complex genomic fingerprints were generated with allthree-primer sets. Figure 4 gives examples of profilesgenerated by ERIC and REP–PCR primers. X. axo-nopodis pv. phaseoli isolates could be clearly differenti-ated from X. axonopodis pv. phaseoli var. fuscans and

non-pathogenic Xanthomonas isolates. A total of 63polymorphic fragments were evaluated, 95% of whichwere polymorphic in X. axonopodis pv. phaseoli com-pared to 72% in X. axonopodis pv. phaseoli var.fuscans (Table 3). The average gene (H) and genetic(Ho) diversity were higher for X. axonopodis pv.

(a)M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

(b)1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 M

(c)1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 M

Fig. 2 Agarose gel showing RFLP profiles for X. axonopodis pv.phaseoli, X. axonopodis pv. phaseoli var. fuscans and non-pathogenicXanthomoas strains that were generated by digesting products ofPCR amplified ribosomal genes with the restriction endonucleaseenzymes RsaI (a), HaeIII (b) and MboI (c). (a)RsaI: lane 1 is negat-ive control, lanes 2–4, 7, 10–14, 16–18, 20–21 and 23–24 are X. axo-nopodis pv. phaseoli; lanes 5, 6, 8, 15, 19 and 22 are X. axonopodispv. phaseoli var. fuscans; lane 9 is a non-pathogenic Xanthomonasstrain. Lanes 4 and 13 did not contain sufficient bacteria DNA to bevisualized on an agarose gel. Lane M is the 100 bp molecular sizemarker. (b) HaeIII: lanes 1, 2, 4, 6, 7, 11–15, 18 and 20–27 representisolates of X. axonopodis pv. phaseoli; lanes 3, 5, 8–10, 16, 19 28 and29 are X. axonopodis pv. phaseoli var. fuscans; lane 17 is a non-pathogenic Xanthomonas strain. Lane M is the 100 bp molecular sizemarker. (c) MboI: lane 1, 3, 6, 9–11, 13, 15–17 and 19 are X. axonop-odis pv. phaseoli; Lanes 2, 4, 5, 7, 14 and 18 are X. axonopodis pv.phaseoli var. fuscans; lane 8 is a non-pathogenic Xanthomonas strain.No DNA was added to lane 12. Lane M is the 100 bp molecular sizemarker

39Genetic Diversity of Bean Bacterial Blight Pathogen

phaseoli (H ¼ 0.134; Ho ¼ 0.223) than for X. axonopo-dis pv. phaseoli var. fuscans (H ¼ 0.108; Ho ¼ 0.184),revealing that X. axonopodis pv. phaseoli is more gen-etically diverse than X. axonopodis pv. phaseoli var.fuscans (Table 3). Non-pathogenic Xanthomonas iso-lates were the most diverse (H ¼ 0.215; Ho ¼ 0.319).

Cluster analysis of data resulting from profiles gen-erated by rep-PCR revealed that X. axonopodis pv.phaseoli and X. axonopodis pv. phaseoli var. fuscansare genetically different (Fig. 5). Isolates clustered intotwo broad groups and the average genetic similaritybetween the two groups was 0.45. However, more vari-ation was found within X. axonopodis pv. phaseoli(average similarity coefficient 0.80) than in X. axonopo-dis pv. phaseoli var. fuscans (0.89). Both strains weresignificantly distinct from non-pathogenic Xanthomon-as isolates included in this study (Fig. 5).

Diversity within and between CBB pathogens

Analysis of gene diversity, using rep-PCR datarevealed significant differences between X. axonopodispv. phaseoli, X. axonopodis pv. phaseoli var. fuscansand non-pathogenic Xanthomonas isolates. The totalgene diversity (HT) among the CBB pathogens was0.317, indicating that 31.7% of the isolates wereunique. The mean gene diversity within population(HS) was 0.139. An analysis of the partitioning ofgenetic diversity for all polymorphic loci showed arelatively high level of genetic differentiation betweenthe two CBB pathogens (GST ¼ 0.56). This indicatesthat 56% of the genetic variation observedin this study was due to differentiation between

X. axonopodis pv. phaseoli and X. axonopodis pv.phaseoli var. fuscans populations when comparedwith 44% within-population variation. The geneidentity index between X. axonopodis pv. phaseolivar. fuscans and X. axonopodis pv. phaseoli was0.648, while the genetic distances between them was0.434 (Table 4). Taken together, these results revealthat X. axonopodis pv. phaseoli and X. axonopodispv. phaseoli var. fuscans are genetically distinct. Bothstrains were almost equally distant from non-patho-genic Xanthomonas isolates, with an average geneidentity of 0.650 and 0.643 for X. axonopodis pv.phaseoli var. fuscans and X. axonopodis pv. phaseoli,respectively (Table 4). Analysis of molecular varianceof rep-PCR data revealed no geographical differenti-ation for either X. axonopodis pv. phaseoli var. fu-scans (GST ¼ 0.013) or X. axonopodis pv. phaseoli(GST ¼ 0.017), revealing the presence of similar geno-types in distinct geographical areas.

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Fig. 3 Three-dimensional graph based on MCA of RFLP data ofpolymerase chain reaction amplified 16S ribosomal gene, includingthe 16S–23S IGS region of X. axonopodis pv. phaseoli var. fuscans,X. axonopodis pv. phaseoli and non-pathogenic Xanthomonas isolatesand plotted using the spin platform of the JMP programme in SAS.Symbols indicate the positions of isolates within each cluster

M(a)

M1 2 3 4 5 6 7 8 9 10

(b)M M1 2 3 4 5 6 7 8 9 10

Fig. 4 Agarose gel showing rep-PCR profiles generated by amplify-ing genomic DNA with (a) ERIC and (b) REP primers. Lanes 1–4are X. axonopodis pv. phaseoli var. fuscans isolates; lanes 5–9 X. axo-nopodis pv. phaseoli and lane 10 is a non-pathogenic Xanthomonasstrain. Lane M is the 100 bp molecular size marker

40 Mahuku et al.

Table 3Overall polymorphism, mean gene diversity (H), and genetic diversity (Ho) estimated by Shannon’s information index using rep-PCR data forcommon bean bacterial blight pathogens and non-pathogenic Xanthomonas strains

Population No. of isolates Polymorphic bands (%) Average gene diversity (H) Genetic diversity (Ho)

X. c. pv. phaseoli 211 95 0.134 ± 0.165 0.223 ± 0.226X. c. pv. phaseoli var. fuscans 132 72 0.108 ± 0.146 0.184 ± 0.209Non-pathogenic isolates 6 79 0.215 ± 0.205 0.319 ± 0.260All isolates 349 97 0.278 ± 0.209 0.414 ± 0.272

Genetic Distances

Non-pathogenic

X. a

xono

podi

s pv

. pha

seol

iX

.axo

nopo

dis

pv. p

hase

oli v

ar. f

usca

ns

011.5 0.5

Fig. 5 Dendrogram fromUPGMA of similarities betweenrep-PCR profiles of Xanthomonasaxonopodis pv. phaseoli,X. axonopodis pv. phaseoli var.fuscans and non-pathogenicXanthomonas strains

41Genetic Diversity of Bean Bacterial Blight Pathogen

DiscussionThere are two forms of the CBB pathogen of commonbean, the yellow-pigmented X. axonopodis pv. phaseoli,and the brown-pigmented X. axonopodis pv. phaseolivar. fuscans (Gilbertson et al., 1991; Fourie, 2002;Mkandawire et al., 2004). Several molecular assays havebeen developed that distinguish X. axonopodis pv.phaseoli from X. axonopodis pv. phaseoli var. fuscans(Birch et al., 1997) and non-pathogenic Xanthomonasspecies (Audy et al., 1994). We have shown that amplifi-cation of the 16S ribosomal gene, including the 16S–23Sintergenic spacer region, followed by digestion with thetetrameric restriction endonuclease MboI, RsaI andHaeIII (PCR–RFLP) is another simple and reliable wayto distinguish and differentiate X. axonopodis pv. phase-oli from X. axonopodis pv. phaseoli var. fuscans andnon-pathogenic Xanthomonas isolates. Results obtainedfrom PCR–RFLP analysis concurred with morphologi-cal classification obtained following growth on YDCmedia and amplification with the OPG 11 RAPD primer(Birch et al., 1997). As the PCR–RFLP method usesprimers developed from conserved regions of the ribo-somal genes to amplify the target region (Louws et al.,1999) followed by restriction analysis, it is a simple diag-nostic tool that is more reliable than the use of theOPG11 RAPD primer. It is not affected by the problemsof reproducibility that are normally encountered withthe use of RAPD primers (Welsh and McClelland,1990). Furthermore, primers targeting differences in therecognition sequences for informative restrictionenzymes can be designed to develop specific PCR-baseddiagnostic probes for routine identification and differen-tiation of the CBB pathogens.

Although X. axonopodis pv. phaseoli and X. axonop-odis pv. phaseoli var. fuscans cause identical symptomson common bean and have the same host range (Saet-tler, 1989; Opio et al., 1996; Fourie, 2002), all avail-able molecular analysis reveal that they are distinctgenetically (Hildebrand et al., 1990; Gilbertson et al.,1991; Vauterin et al., 1991; Xue and Goodwin, 1994;Chan and Goodwin, 1999). Differences between thetwo types have been noted in protein profiles (Vauterinet al., 1991), reassociation values in DNA–DNAhybridization studies (Hildebrand et al., 1990), analysisof the two component regulatory system (Xue andGoodwin, 1994), RFLP analysis with repetitive DNAprobes (Lazo and Gabriel, 1987; Gilbertson et al.,1991) and pulse-field analysis of whole chromosomes(Chan and Goodwin, 1999). Analysis of a large popu-lation of the CBB pathogens of a diverse origin

generated distinct rep-PCR and PCR–RFLP profilesthat clearly distinguished X. axonopodis pv. phaseolifrom X. axonopodis pv. phaseoli var. fuscans. Clusterand multiple correspondence analyses of both PCR–RFLP and rep-PCR data confirmed this distinctive-ness. Of the total genetic diversity observed in thisstudy, 56% was due to differences among X. axonopo-dis pv. phaseoli and X. axonopodis pv. phaseoli var.fuscans populations, compared to 44% within them.The observed levels of genetic distances between thetwo CBB pathogens (D ¼ 0.434) and the level of gen-etic differentiation (GST ¼ 0.56) supports and confirmsprevious reports (Hildebrand et al., 1990; Gilbertsonet al., 1991; Vauterin et al., 1991; Xue and Goodwin,1994; Chan and Goodwin, 1999; Mkandawire et al.,2004), that X. axonopodis pv. phaseoli and X. axonopo-dis pv. phaseoli var. fuscans are genetically distinct.The ability of X. axonopodis pv. phaseoli and

X. axonopodis pv. phaseoli var. fuscans to have identi-cal host range and cause similar symptoms may bereflective of convergent evolution, or the presence ofsimilar types of plasmids with conserved homologousDNA regions that may encode virulence and hostrange specific functions. Such functions have beendescribed on plasmids within Rhizobiaceae (Longet al., 1982) and Agrobacterium tumefaciens (Loperand Kado, 1979), and need to be tested for the CBBpathogens. However, previous plasmid analysis (Lazoand Gabriel, 1987) revealed distinct differences indigestion patterns of plasmids from X. axonopodis pv.phaseoli var. fuscans and from X. axonopodis pv.phaseoli strain that might be indicative of differenttypes of plasmids in the two bacteria. There is a needto test the hypothesis of the presence of homologousplasmid DNA that encodes host virulence factors inboth X. axonopodis pv. phaseoli and X. axonopodispv. phaseoli var. fuscans, and elucidate why two bac-teria that are genetically distinct have the same hostrange and cause similar symptoms.Considerable levels of genetic diversity were

observed in both X. axonopodis pv. phaseoli andX. axonopodis pv. phaseoli var. fuscans. More geneticdiversity was observed in X. axonopodis pv. phaseolithan in X. axonopodis pv. phaseoli var. fuscans. Theseresults are in agreement with those of Mkandawireet al. (2004) who described three distinct X. axonopodispv. phaseoli genotypes, two that were found exclusivelyin East Africa and a third that included isolates fromeast Africa and the rest of the world. However, in ourcase we observed only one genetically diverse genotype

Table 4Nei’s (1978) unbiased measure of genetic identity (I) (above diagonal) and genetic distance (D) (below diagonal) for three Xanthomonas popu-lations

PopulationX. axonopodispv. phaseoli

X. axonopodispv. phaseoli var. fuscans

Non-pathogenicXanthomonas strains

X. axonopodis pv. phaseoli – 0.648 0.650X. axonopodis pv. phaseoli var. fuscans 0.434 – 0.643Non-pathogenic Xanthomonas strains 0.430 0.442 –

42 Mahuku et al.

for X. axonopodis pv. phaseoli, irrespective of the ori-gin of the isolates. The small number of X. axonopodispv. phaseoli isolates from Africa (three) analysed inthis study preclude us from drawing strong conclusionson the observations that Mkandawire et al. (2004)made in Africa. Like Mkandawire et al. (2004), weobserved one large group for X. axonopodis pv. phase-oli var. fuscans that was composed of isolates fromAfrica and other parts of the world. Furthermore, nogeographical differentiation was observed for X. axo-nopodis pv. phaseoli var. fuscans (GST ¼ 0.013) or forX. axonopodis pv. phaseoli (GST ¼ 0.017). As the CBBpathogens are seed borne (Saettler, 1989; Gilbertsonet al., 1991; Fourie, 2002), the lack of geographicaldifferentiation could be a result of continuous intro-duction and movement of new bacterium genotypesbetween regions.

In this study, X. axonopodis pv. phaseoli and X. axo-nopodis pv. phaseoli var. fuscans isolates from the samegeographical region often had different rep-PCR pro-files, revealing that the CBB pathogen is not clonal.Several molecular mechanisms, such as spontaneousmutations arising from DNA replication infidelity, therepair of DNA mismatches, transposition, site-specificrecombination and horizontal DNA acquisition pro-mote genetic variation that can account for geneticdiversity within bacterial populations (Scortichiniet al., 2001). It is possible that any one of these mech-anisms could be occurring in the CBB pathogens, lead-ing to the levels of genetic diversity observed in thisstudy. The introduction of the CBB pathogens throughcontaminated seed (Saettler, 1989; Gilbertson et al.,1991; Fourie, 2002), coupled with survival and possibleselection on symptomless hosts (Opio et al., 1994;Gent et al., 2005), may also contribute to generatinghigh genetic diversity. Both CBB pathogens have beenreported to survive epiphytically on other non-hostspecies (Opio et al., 1994; Gent et al., 2005). Further-more, reports of non-clonality in the CBB pathogenshave been made (Gilbertson et al., 1991; Vauterinet al., 1991; Xue and Goodwin, 1994; Mkandawireet al., 2004), and variation in aggressiveness, phagetypes and in vitro growth rates both within andbetween X. axonopodis pv. phaseoli and X. axonopodispv. phaseoli var. fuscans (Opio et al., 1996) have beennoted. Our results concur and support those reportedelsewhere (Gilbertson et al., 1991; Xue and Goodwin,1994; Opio et al., 1996; Chan and Goodwin, 1999), toconfirm that X. axonopodis pv. phaseoli and X. axo-nopodis pv. phaseoli var. fuscans are not clonal.

As the majority of bean growers in Africa and LatinAmerica are small subsistence resource limited farmers,the most appropriate and feasible approach for con-trolling CBB is through host resistance (Opio et al.,1996; Dursun et al., 2002). It has been shown thatresistance to CBB in P. vulgaris is quantitatively inher-ited with predominance of additive gene action andmoderate to high heritability (Beebe, 1989). However,qualitative host-specific resistance to the bean bacterialblight pathogens that fit the gene for – gene relation-

ship has been observed in P. acutifolius (Urrea et al.,1999). A set of P. acutifolius differential cultivars wasassembled and used to reveal races of X. axonopodispv. phaseoli and X. axonopodis pv. phaseoli var.fuscans (Opio et al., 1996). To exploit the P. acutifoliusresistance genes in developing bean cultivars with last-ing resistance to the bean bacterial blight pathogen,breeders need to harness the high levels of resistancein P. acutifolius and pyramid the major genes inappropriate backgrounds of P. vulgaris. Following thisstrategy, CIAT developed the VAX 1–6 lines, throughinterspecific hybridization and embryo rescue, followedby resistance gene stacking and classical selection pro-cedures (Singh and Munoz, 1999). In this study, theusefulness of this strategy in developing CBB resistantbean cultivars was revealed as none of the isolates tes-ted had a compatible interaction with VAX 6. Becausethe seed type of VAX 6 is not acceptable commer-cially, the challenge is to transfer all the resistancegenes in VAX 6 into preferred commercial type culti-vars, while maintaining the same level of disease resist-ance. Molecular markers tightly linked to these geneswill play a crucial part in assuring the transfer of theseresistance genes into susceptible but preferred beancultivars (Miklas et al., 2003).

This study has provided further data to show thatX. axonopodis pv. phaseoli and X. axonopodis pv.phaseoli var. fuscans are genetically distinct. This con-clusion is supported by studies from many otherresearchers (Gabriel et al., 1989; Hildebrand et al.,1990; Xue and Goodwin, 1994; Chan and Goodwin,1999; Mkandawire et al., 2004). Xanthomonas axonop-odis pv. phaseoli was observed to be more geneticallydiverse than X. axonopodis pv. phaseoli var. fuscans,but this diversity was not geographically structured foreither of the two forms of the CBB pathogens. Thelack of geographical differentiation has importantpractical implications in breeding for CBB resistance,as available resistance genes are likely to be effective inmanaging the CBB disease in diverse geographicalareas. Furthermore, routine screening of bean geno-types for resistance to CBB disease should includeboth X. axonopodis pv. phaseoli and X. axonopodis pv.phaseoli var. fuscans isolates. The usefulness of theresistance genes in VAX 6 to control CBB pathogenswas confirmed, showing the effectiveness of pyramid-ing major and minor resistance genes. Restriction ana-lysis of the PCR amplified ribosomal genes isdiagnostic of the CBB pathogens, distinguishingbetween X. axonopodis pv. phaseoli, X. axonopodis pv.phaseoli var. fuscans and non-pathogenic Xanthomonasspecies associated with common bean. This approachoffers a reliable diagnostic tool that potentially can bedeveloped into a simple PCR-based test to assay seedfor these bacteria.

Acknowledgements

We are grateful to Jorge Fory and Marıa del Carmen Hernadez for

technical assistance, and to Drs Fernando Correa and Steve Beebe

for their suggestions and critical reading of this manuscript.

43Genetic Diversity of Bean Bacterial Blight Pathogen

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