A2 67
Large molecular karyotype divergences between
two groups of the genus ”Phytomonas”
Particularity of the phloem-restricted group
C. Marín1, B. Alberge2, M. Pagés2, P. Bastien2 and M. Dollet3
1. Instituto de Biotecnología, Departamento de Parasitología,
Facultad de Ciencias, Universidad de Granada, Spain
2. UMR 5093 CNRS/Université Montpellier 1, Laboratoire de
Parasitologie-Mycologie, France
3. CIRAD, Tree Crops Department, Coconut Lethal Yellowing and
Citrus Greening unit, TA 80/F, 34398 Montpellier Cedex 5, France
[email protected]
A
lthough a single genus name, Phytomonas, has been proposed for all plant
trypanosomes, they can differ from one another in terms of their ecological and
biological properties, and of their effects. Phloem-restricted trypanosomatids
(group H) are specifically associated with lethal diseases: Hartrot of coconut
(Fig. 1), Marchitez Sorpresiva of oil palm (Fig. 2) and yellow wilt of Alpinia purpurata [1]. They have been established
as a homogeneous group, due to its specific features, as well as being confined to a single continent: Latin America and the Caribbean.
On the other hand, in general, trypanosomatids located in the latex tubes of latex-bearing plants do not induce any defined pathology.
Introduction
(B)
Due to the absence of chromosome condensation during mitotic division, the
chromosomal structure and organization of the genome was not studied until
the advent of techniques enabling the separation of large DNA molecules, such
as Pulsed Field Gel Electrophoresis (PFGE). This technique was used to define
molecular karyotypes, which can be so polymorphic as to be almost strainspecific [2].
In our study, we started verifying the homogeneity of the genus Phytomonas by
establishing molecular karyotypes of different groups by PFGE.
(A)
Fig 1.
(A) Hartrot in coconut palm.
(B) Hartrot in coconut flowers.
(C) Phytomonas isolates from
coconut phloem.
Fig 2. Phytomonas isolates
from oil palm phloem.
Material and Methods
1- The Phytomonas isolates from phloem and latex plants (E.M.1) used in this
study (Table 1) were routinely cultured in Grace’s Insect Medium
supplemented with 10% heat-inactivated foetal bovine serum at 28°C.
Leishmania major “Friedlin” (LmF) (MHOM/IL/81/FRIEDLIN) was grown
as in [3].
2- For the preparation of chromosomal DNA, all the parasites were cultured
up to a final concentration of 7 x 108 cells.ml-1. DNA agarose blocks were
prepared and processed as described [4]. PFGE was carried out on homemade devices according to [5]. As chromosome size markers, we used the
chromosomes of LmF, as determined by [6]. Different PFGE conditions were
used in order to resolve all chromosomal size classes.
After migration, the gels were stained with ethidium-bromide, photographed
under UV transillumination and transferred onto nylon filters (Hybond N+,
Amersham) by alkaline transfer, according to the manufacturer’s instructions.
Results
The molecular karyotypes were completely resolved (including the compression
zone at 2.5-3 Mb) by using different PFGE conditions (Fig.3).
The molecular karyotype of Phytomonas isolates from latex (E.M.1) showed 18
non stoichiometrically-staining bands with 1 band having a “double” staining
intensity (Fig.3, lane 1). If each single and “double” band represented 1 and 2
chromosomes respectively, the number of chromosomes would be 19: 5 large
chromosomes of ~3-1.6 Mb, 8 “intermediate” chromosomes of 1.3-0.75 Mb and
6 small chromosomes of 0.68-0.38 Mb. Thus, the minimum size of the haploid
genome of these isolates appeared to be 22 Mb. By contrast, the molecular
karyotype of phloem-restricted trypanosomatids showed 4-7 chromosomal
bands ranging from 0.3 to 3 Mb. A similar analysis to the one above was used
to determine a total haploid genome size of around 13 Mb (Fig. 3, lanes 2-5).
The karyotypes of the three groups of phloem-restricted isolates (Hartrot,
Marchitez and Alpinia) appeared similar.
2.8
1.6
1.2
1.1
A
L
1
2 3
.
.
.
.
.
..
.
4
Mb
5
Isol ate
Pla nt
Tissue
Country /Year
E.M .1
Hart 4
Hart 1
Mar 1
Alp 3
Euphorbia pinea
Cocos nucifera
C. nucifera
Elaeis guineensis
Alpinia purpurata
latex
phloem
phloem
phloem
phloem
Mo ntpel lier , Fr ance/ 1980
Fr. Guiana /1987
Fr. Guiana /1986
Colom bia /1989
Grenada , C aribbean W indward Islands /1992
Table 1. Phytomonas spp. isolates used.
Conclusion
The karyotypic divergences observed between the phloem-restricted group
and Phytomonas isolated from laticiferous plants, strongly suggested that these
two groups are evolutionarily very distant, and might belong to different
genera.
Reference
[1] Dollet M. et al., 2001. Int. J. Parasitol. 31: 459-467.
[2] Schwartz D.C. and Cantor C.R., 1984. Cell 37: 67-75.
[3] Blaineau et al., 1992. Mol. Biochem. Parasitol. 50(2):
197-204.
[4]Bastien et al., 1992. In Avila, J.L. and Harris, J.R. (eds),
Subcellular Biochemistry. Plenum Press, New York, NY,
Vol 18, pp. 131-187.
[5] Ravel C. et al., 1996. Genomics 35(3): 509-516.
[6] Ravel C. et al., 1998. Parasitol. Today 14(8): 301-303.
B
L
1
2 3
4
5
<
<
2.8
<
1.6
1.2
<
C.Z.
<
<
0.83
0.28
C.Z.
.
.
.
.
.
.
..
.
.
.
Fig 3.
CIRAD
Complete resolution of the karyotype of Phytomonas isolates from latex and phloem. The
<
sizes of L. major “Friedlin” (LmF) are indicated on the left in megabase pairs (Mb) and the
position of the chromosomal bands are shown by dots for E.M.1 and squares for Hart 4. C.Z.
compression zone. The C.Z. was resolved on other gels using different conditions (not
<
shown). (L) LmF, (1) E.M.1, (2) Hart 4, (3) Hart 1, (4) Mar 1 and (5) Alp 3. (A) Separation of the
<
intermediate chromosomes was achieved using pulse times of 220s/36h, 180s/36h and
120s/36h in a 1.5% agararose gel at 7.5 V/cm. (B) Separation above 2 Mb was achieved using
pulse times of 500s/36h, 400s/48h and 300s/36h in a 1.5% agararose gel at 5 V/cm.
French
Agricultural
Research
Centre
for International
Development
Design and production: CIRAD -June 2006
Mb
(C)