Berthier, Y.

Boher, Bernard

Chevrier, D.

Dongo, P.

Verdier, Valérie

Horizon / Pleins textes

Plant Pathogenic Bacteria, Versailles (France), June 9·12,1992
Ed. IN RA, Paris 1994 (Les CoJloques, nOGG)
Molecular epidemiology of Xanthomonas campestris
pv. manihotis causal agent of cassava bacterial blight
v. VERDIER, Y. BERTHIER", P. DONGO, D. CHEVRIER"" and B. BOHER"""
ORSTOM, Laboratoire de Phytopathologie, Brazzaville, Congo
• INRA, Station de Pathologie vegetale, 78026 Versailles Cedex, France
.. Institut Pasteur, Laboratoire de Predeveloppement des Sondes,
75724 Paris Cedex 15, France
••• ORSTOM, Laboratoire de Phytopathologie, Lome, Togo
Abstract
10 ordtlr to dtlloct and assess gtlndic and tlvolutionary rdationships among strains of Xc pv. ruanihotis a
comparison of strains of distinct geographical origin. rtlpreStlotllg 18 countries. was ptlrformed using a
rangtl of assays including restriction fragmtlnt length polymorphism (RFLP) analysis. Thtl probes usOO
wtlre: 16+23S rRNA gentls from E.coli and three restriction fragments from the chromosomal or plasmid
DNA of Xc pv. mtlnihotis.
Hyhridization with thtl probe correspondmg to thtl rRNA gtlntls allowed thtl distinction of four RFLP
groups. Subgroups were identified based on hybridizatioo profiles with the three others probes.
Genetic variability of Xc. pv. manihotis was exttlnsivtl in strains from thtl area of origin of the host plant
and limited dsewhtlre. ThtlStl results are in agreement with the hypothesis of thtl rocent introduction of the
pathogen to theStl latttlr areas and suggests that the African strains have not yd diversified genetically at the
chromosomal level.
Our results indicate that RNA and DNA prohes are useful tools tor epidemiological studies and in following
the genetic evolution of strains.
Keywords: Xanthomonas campestris pv. f118nihotis. cassava. RFLP. rRNA probe. DNA probtl.
I. INTRODUCTION
Cassava (M3nihot esculenta) , family Euphorbiaceae is a root stock crop native from
South and Central America. Portuguese traders introduced it to West Africa in the
sixteenth century and to East Africa in the eighteenth century (SILVESTRE &
ARRAUDEAU, 1983). It became one of the most important tropical food in countries of
Tropical Africa.
Cassava bacterial blight (C.B.B) caused by Xanthomonas campestris pv. mamhotis is one
of the most important diseases of cassava. The disease was first reported in Brazil in
1912 (BONDAR, 1912) but has also been observed in Colombia and Venezuela
(LOZANO & SEQUEIRA, 1974), as well as in most of African (MARAITE &
MEYER, 1975) and Asian countries (BOOTH & LOZANO, 1978).
Cassava originated from South America and its related bacterial pathogens could have
been propagated to others countries through the cuttings and seeds. To be able to detect
and assess evolutionary relationship among pv. manihotis a comparaison of strains was
developped using a wide range of assays.
n. MATERIAL AND METHODS.
X.C. pv. manmotis coUection.
The bacterial strains used in this study, their geographical origin and their sampling
collecting places are listed in Table I.
Physiological characteristics.
Different phenotypic features were examined: the in vitro susceptibility to 20 antibiotics
was determined, the utilization of carbon sources (19 tested), and the amylase activity
according to described methods (GROUSSON et al, 1990).
Phytopathogenicity test.
Pathogenicity of all strains was tested on cassava plants, Congo's cultivar PMB,
multiplied from cuttings. The stem inoculation was done according to previously
described methods (MARAITE et al, 1981).
709
Table 1 :Xanthomonas campestris pv. manihotis strains used and information on tbeir origin and isolation.
Strain no' and in other collection Place and year of isolation Isolated y Strain no • and in other collection Place and year of isolation Isolated y
.....
o
CFBP1851,CIAT1111 Colombia 1974 Lozano.J
LMG 776, NCPPB2443,HMB72,CFBP2603 1970 Lozano.J
ORSTl CIATl060 CFBPI849 1970 Lozano.J
ORST2, CIATl06I. CFBPI850 Venezuela 1971 LOZ800
ATCC 23380, HMB68.NCPPBI159 Brazil 1941 Burkholder
HMB 70, NCPBB 1160, LMG5273 1941 Drurnond Hipolito
HMB 55a,NCPBBI834*. LMF784 1965 Robbs C.
ORST7, CFBP1854 1973 Neto JR.
HMB23, LMG770 1973 Pereira A.
ORST3, CIAT 1120, CFBP1852 1974 Lozano.J
ORST5. CFBP1855 1974 Neto J.R.
ORST6, CFBPI856 1976 Neto JR.
HMB79. LMG778 1978 Takatsu.A.
LMG777. HMB78 1978 Takatsu.A.
LMG779 HMB80 1978 Takatsu.A.
HMB25, NCPBB3060. LMG 771 Nigeria 1976 Maraite H.
ORST42 1978 DanielJF
ORST43 1978 «
CFBPI857, ORSTOM Al02.1 1978 «
CFBPI858, ORSTOM Al03.1 1978 «
CFBPI859. ORSTOM Al05.1 1978 «
CFBPI860. ORSTOM Al07 1978 «
ORST34 Benin 1982 Daniel J.F
ORST35 1982 «
ORST36 \ 1982 «ORST37 1982 «
ORST38 I 1982 «
CFBPI944 Ivory Coast 1979 RideM.
LMG5249,HMB203 1981 Maraite H.
ORST55 1984 Daniel J.F
ORST56 1984 «
ORST (198 strains) Congo 1977-1991 Daniel JF
Boher B.
Verdier V.
ORST (29 strains) TOl(o 1987-1991 Boher B.
HMB6, LMG 767 zaire 1973 Maraite H.
HMB9, LMG 768. NCPBB3058 » »
LMG 769. NCPPB3059. HMBIO » »
LMG 766, HMB3 » »
ORST44 1979 Daniel JF
ORST45 » »
ORST46 » »
ORST47 » »
ORST48 » »
ORST49 » »
ORST50 » »
ORST51 » »
ORST52 » »
ORST53 » »
ORST54 » »
ORSTI86 1987 Boher B
ORSTI87 » »
ORST39 RCA 1977 Daniel J.F
ORST40 » »
ORST41 » »
LMG 5287, NCPPB 3161 Cameroon 1976 PersleyG.
HMB27 LMG629 1977 Maraite H.
LMG780, HMB81 Uganda 1979 Maraite H.
LMG782 HMs93 » »
LMG783, H,MB148 Kenva 1979 Maraite H.
LMG5288 NCPPB 3194 Nill:er 1978 Bradburv.J
LMG765 Malaysia 1980 LanG.
LMG774, HMB60 TaIwan 1978 Maraite H.
HMB71, NCPBBI161, LMG775 Mauritius 1946 Orian G.
CFBP2624 Reunion 1986 Pruvost O.
CFBP2635 1987 Girard JC
ATCC : Americain Type Culture, Rockville. Maryland, USA. CFBP : Collection Franyaise de Bactl!ries Phytopathogenes. Angers, France.
NCPPB: National Collection of Plant Pathogenic Bacteria, Harpenden, U.K. HMB: H. Maraite's Bacterial Collection, LOUVAlN La Neuve, Belgium.
LMG: Laboratorium voor Microbiologie Gent culture Collection. Gent, Belgium. ORST : Collection du Laboratoire de Phytopathologie, ORSTOM, Brazzaville. Congo.
CIAT : Centro International de Agricultura Tropical, Cali, Colombia. * : Pathovar reference strain.
1 Brasil
1 Taiwan
\/
1
. eE)
Table 2: Representat Ion of RFLP groups of X.C. pv manfhot/s straIns.
pBS8
pBS6
...,
RFLP analysis.
Total genomic DNA isolation, endonuclease digestion, electrophoresis and Southern blot
were done according to previously described methods (BERTHIER et aI, 1992).
~ybridization was made with different probes. Acetyl Amino Fluorene labeled
n~somal 16+~3S RNA genes ,from E.coli (Eurogentec, Liege, Belgium) hybridized
WIth the genomIc DNA of bactena. The rRNA - rDNA duplexes were detected using the
anti-AAF monoclonal antibody (GR1MONT et al. 1989).
The ,DNA probes used in this study were: BS6 (7kb-EcoRI) and BS8 (8kb-EcoRI), two
restncted fragments from the chromosomal DNA (Xc. pv manihotis strain CNBPI851-
CIATIIII) and pBsF2 derivated from the 13kb-HindlII fragment of plasmid DNA
cloned in the bluescript vector plasmid. DNA probes were labeled in vitro by using a
random priming kit with 32 P deoxycytidine triphosphate (Multiprime Amersham).
m. RESULTS
RFLP patterns.
Using the rRNA probe, the distinction of 4 RFLP groups among the 290 strains tested
could be possible. Strains from South America were heterogenous and gave different
patterns, on the contrary no polymorphism was noticed in African strains (Table 2).
Hybridization profiles with DNA probes could differentiate 6 groups with BS8 probe and
8 groups with BS6 probe, each group representing strains with identical RFLP pattern
(Table 2). Polymorphism could be noticed in South American strains which are
represented in groups mentioned above. In contrast, no polymorphism was observed in
African strain with BS8 and BS6 probes suggesting that these regions are well conserved
into the genome.
Variability among RFLP patterns of African strains was only noticed with the plasmid
DNA probe pSF2.
Pathogenic characteristics.
Variability among pathogenic characteristics exists but was not related with the
geographical origin of strains.
Pbeootypic features.
Same results were obtained for two of the three phenotypic features tested (sensitivity to
antibiotics and utilization of carbon sources). Starch hydrolysis was observed for all
strains but two groups were differentiated. All African Reunion and Malaysian strains
showed a low amylase activity similar to that found in 3 Brasilian and Colombian strains.
IV. DISCUSSION
Based on numerical analysis of protein gel eJectrophoregrams and, 267 phenotypic
features, VAN DEN MOOTER et aI, (1987) and VAUTERlN et aI, (1991) indicate that
the pv manihotis strains constitute a phenotypically and genetically homogeneous group.
In this study, using the RFLP analysis, small changes in DNA organization were
observed. Genetic variability of pv manihotis was more extensive in strains from the area
of origin of the host plant and more limited in those coming from elsewhere. Among
African strains homogeneity was observed with the probe corresponding to the rRNA
genes and thus was confirmed with genomic probes used in this study. In our previous
data based on plasmid DNA study we have indicated the hypothesis of one common
geographic origin within strains of Xc. pv. manihotis (VERDIER, 1988). The results
presented here agree with the hypothesis of the recent introduction of this pathogen from
South America to the other countries, and suggest that African strains are not already
diversified at chromosomal level. Using the DNA plasmid fragment as a probe, this
study revealed that DNA polymorphisms exist in African strains. Plasmids are mobile
elements which easily perform genetic exchange in bacterial strains (COPLlN, 1989;
EBERHARD, 1990). Presence of essential pathogenicity genes on these plasmid
fragment was previously demonstrated (VERDIER et aI, 1989).
RNA and DNA probes used here were particularly useful in our epidemiological studies,
providing information on the genetic population structure of these pathogens and its
ability to identify clonally related individuals.
712
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