Experimental Parasitology 116 (2007) 88–90
www.elsevier.com/locate/yexpr
Research brief
Herpetomonas spp. isolated from tomato fruits
(Lycopersicon esculentum) in southern Spain
Clotilde Marín a, Sandrine Fabre b, Manuel Sánchez-Moreno a, Michel Dollet b,¤
a
Instituto de Biotecnología, Departamento de Parasitología, Facultad de Ciencias, Universidad de Granada, C/Severo Ochoa s/n. 18071 Granada, Spain
b
CIRAD, UPR 29, TA 80/F 34398 Montpellier Cedex 5, France
Received 2 June 2006; received in revised form 13 October 2006; accepted 8 November 2006
Available online 30 January 2007
Abstract
A Xagellate of the family Trypanosomatidae was isolated from fruits of Lycopersicon esculentum (tomato) in southeastern Spain. The
isolate was successfully adapted to in vitro culture in monophasic media. The morphology showed the kinetoplast to be positioned
towards the middle of the body, and the typical opistomastigote form characteristic of members of the genus Herpetomonas. AmpliWcation of the mini-exon gene was negative, whilst for the 5S ribosomal rRNA gene the result was positive. The DNA sequence was obtained
and its alignment with other trypasomatids, obtained using the BLAST algorithm, suggested it was closely related to Herpetomonas
samuelpessoai.
© 2006 Elsevier Inc. All rights reserved.
Index Descriptors and Abbreviations: Phytomonas spp.; Fruit; 5S rRNA; SL RNA; Herpetomonas
The genus Phytomonas was proposed by Donovan
(1909) to diVerentiate between plant and animal trypanosomatids. Phytomonas spp. have been described as digenetic
trypanosomatids found in plants with a typical promastigote appearance (Vickerman, 1976). But that deWnition is
ambiguous, because promastigote stages also occur in Leptomonas, Herpetomonas, Crithidia, and Blastocrithidia, in
insects. As Phytomonas are transmitted by insects, they can
be confused with these other genera which also occur in
plant tissues (Conchon et al., 1989; Wallace et al., 1992;
Camargo and Wallace, 1994; Fiorini et al., 2001). Plant trypanosomatids have been isolated from latex tubes of laticiferous plants, phloem of trees, and mature fruits and seeds
of many plant species, with a wide geographical distribution (Dollet, 1984; Camargo, 1999; Dollet, 2001). Phloemrestricted isolates, all from South America, are speciWcally
associated with pathological syndromes in plants: Hartrot
of coconut, Marchitez Sorpresiva of oil palm, yellow wilt of
*
Corresponding author. Fax: +33 4 67 59 38 14.
E-mail address: [email protected] (M. Dollet).
0014-4894/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.exppara.2006.11.003
Alpinia purpurata, and phloem necrosis of coVee trees (Dollet, 2001). Conversely, trypanosomatids found in fruits or
seeds do not cause any disease, only local damage, such as
yellow spots or necrotic spots.
Consequently, there are no well-deWned criteria for
attributing the genus Phytomonas to any plant-isolated trypanosome. In this study we show that a trypanosomatid
isolated from tomato fruits in southeastern Spain was a
member of the genus Herpetomonas.
Fruit samples of Lycopersicon esculentum were collected
from greenhouses along the southern coast of Spain (provinces of Almeria and Murcia). For experimental purposes,
we selected plants with fruits showing deformations and
pigment alterations in the pericarp (necrosis or yellow spots
caused by insect bites). The fruit surface was cleaned with
70% ethanol, then small sections of the fruits were homogenized in phosphate buVered saline and examined by phasecontrast microscopy. Sample smears were also Wxed in
methanol and stained with buVered Giemsa (pH 7.0) for
light microscopy. The samples found to be infected with
Xagellates were inoculated into 3 ml of Grace’s Insect
medium (Sigma®) supplemented with 10% heat-inactivated
C. Marín et al. / Experimental Parasitology 116 (2007) 88–90
Fig. 1. Light microscopy of Giemsa-stained (A–F). (! D kinetoplast,
foetal calf serum, as previously described (Sánchez-Moreno
et al., 1995). Growth was assessed by cell counting in a Neubauer hemocytometer. Once the Xagellates had adapted to
in vitro culture, they could reach a density of 108 cells/ml
after 72 h at 26 °C. This fast growth did not match the diYculties generally encountered with the culture of phloemrestricted or even latex isolates (Dollet, 2001).
The Xagellates appeared with diVerent morphological
forms (Fig. 1). Generally, the kinetoplast position was
located towards the middle of the body and in exponential
cultures a low percentage of organisms showed a typical
opistomastigote form (Fig. 1F) characteristic of the Herpetomonas species.
Genomic DNA was obtained by phenol/chloroform
extraction and ethanol precipitation from 108 to 109 cells.
We tried to amplify the 5S Ribosomal RNA gene and the
tandemly repeated spliced leader RNA gene (SL RNA or
mini-exon) from about 10 ng of genomic DNA following
the protocols described elsewhere (Dollet et al., 2000, 2001).
The ampliWcation of the SL RNA gene and the neighbouring intergenic region using primers ME-L and ME-R was
89
D nucleus).
negative. However, the 5S rRNA gene repeat was successfully ampliWed using primers 5S-L and 5S-R, giving a product with a size of around 650 bp. The amplicon was cloned
into pCR2.1 TOPO TA (Invitrogen) and both strands were
sequenced. The nucleotide sequence has been deposited in
the GenBank database with accession number DQ441589.
The 630 bp sequenced region was composed of the complete 5S rRNA gene, and the adjacent intergenic region
including the mini-exon gene sequence, as it appears in
Herpetomonas samuelpessoai (Aksoy, 1992).
The BLAST algorithm was used to Wnd sequence readings
displaying similarity. The complete sequence had 97%
homology with the H. samuelpessoai gene 5SrRNA
(X62331). The 5S rRNA gene sequence itself showed 99%
homology with the H. samuelpessoai 5SrRNA gene
(X62331), and 98% homology with the trypanosomatid registered in the genebank as “Trypanosomatidae sp. Alexandria_1983” (AY547487.1), a Herpetomonas isolated from rats
and dogs in Egypt (Podlipaev et al., 2004a,b), and Herpetomonas ztiplika, a herpetomonad from a blood-sucking midge
(AY308759) (Podlipaev et al., 2004a,b). The Exon sequence
90
C. Marín et al. / Experimental Parasitology 116 (2007) 88–90
was a 100% match with the Exon sequence of several Herpetomonas spp. (H. mariadeanei, AY547489.1; Trypanosomatidae sp. Alexandria and H. samuelpessoai) plus Leptomonas
sp. Nfm (AY547485.1), which should be transferred into the
genus Herpetomonas as H. sp. Nfm (Podlipaev, 2000). The
Intron sequence showed 100% homology with H. samuelpessoai and 94% with Trypanosomatidae sp. Alexandria.
It has thus been proven once again that trypanosomatids
other than Phytomonas can colonize and multiply in plants
(Camargo, 1999), and consequently we can imagine that
infected tomatoes in the Weld can serve as an infection
source for insects, as was shown experimentally in the laboratory (Conchon et al., 1989).
In this work is stated a recurrent fact, concerning that the
generic identiWcation of new isolates of trypanosomatids is
somehow confuse when it is only based on host-speciWcity
criterion. As there is no clear limit between the taxa Leptomonas, Herpetomonas, and Phytomonas, due to the facts that: (1)
insects may carry Phytomonas, as vectors; (2) insect vectors
can carry diVerent “insect trypanosomatids”; (3) plants can
be parasitized with trypanosomatids called “insect tryopanosomatids” such as Herpetomonas spp., Leptomonas spp. or
Crithidia spp., and “plant trypanosomatids” called Phytomonas spp. This taxonomic question can be answered by the
hypothesis that the ancestors of phytomonads were of insect
origin (Podlipaev et al., 2004a,b).
To conclude, the tomato Xagellate isolated in southern
Spain is possibly a strain of H. samuelpessoai, another
“insect trypanosome” isolated from plants (and, not a Phytomonas sp.). Thus, plant origin cannot be used as an exclusive taxonomic parameter for classifying a trypanosomatid
as a Phytomonas, and the concept of “monoxenus insect
trypanosomatids” or “plant trypanosomatids” needs to be
revised.
Acknowledgments
We thank Valérie Gil (CIRAD, Montpellier), Peter Biggins (CIRAD, Paris) for her kind help.
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