Trypanosomatidae: Phytomonas detection in plants and phytophagous insects by PCR amplification of a genus-specific sequence of the spliced leader gene

In this paper we describe a method for the detection of Phytomonas spp. from plants and phytophagous insects using the PCR technique by targeting a genus-specific sequence of the spliced leader (SL) gene. PCR amplification of DNA from 48 plant and insect isolates previously classified as Phytomonas by morphological, biochemical, and molecular criteria resulted in all cases in a 100-bp fragment that hybridized with the Phytomonas-specific spliced leader-derived probe SL3'. Moreover, this Phytomonas-specific PCR could also detect Phytomonas spp. in crude preparations of naturally infected plants and insects. This method shows no reaction with any other trypanosomatid genera or with plant and insect host DNA, revealing it to be able to detect Phytomonas spp. from fruit, latex, or phloem of various host plants as well as from salivary glands and digestive tubes of several species of insect hosts. Results demonstrated that SLPCR is a simple, fast, specific, and sensitive method that can be applied to the diagnosis of Phytomonas among cultured trypanosomatids and directly in plants and putative vector insects. Therefore, the method was shown to be a very specific and sensitive tool for diagnosis of Phytomonas without the need for isolation, culture, and DNA extraction of flagellates, a feature that is very convenient for practical and epidemiological purposes.     

Monoclonal Antibodies for the Identification of Trypanosomatids of the Genus Phytomonas

Monoclonal antibodies have been produced against culture forms of Phytomonas francai and Phytomonas serpens parasites, respectively, in cassava roots and tomato fruits. These monoclonal antibodies have been tested against 5 other Phytomonas spp. isolated from plants and 14 species of trypanosomatids of various genera. Monoclonal antibodies were found to react exclusively with Phytomonas spp., always giving negative results with other trypanosomatid genera. Thus, these monoclonal antibodies seem to be an effective tool for the identification of phytomonads among insect trypanosomatids.     

Insect trypanosomatids: the need to know more

Of ten recognized trypanosomatid genera, only two - pathogenic Trypanosoma and Leishmania - have been actively investigated for any length of time while the plant flagellates - Phytomonas - have recently begun to attract attention due to their role as agricultural parasites. The remaining genera that comprise parasites associated with insects have been largely neglected except for two or three containing popular isolates. This publication reviews current knowledge of trypanosomatids from insects.     

Trypanosomatids, Other Than Phytomonas spp., Isolated and Cultured from Fruit

Trypanosomatids were isolated from edible fruit. One of the isolates (from tangerine) presented a set of enzymes for the metabolism of arginine-ornithine similar to that of Leptomonas spp., and failed to be recognized by monoclonal antibodies specific for Phytomonas spp. The possibility that trypanosomatids other than Phytomonas spp. could infect fruit was further examined by inoculating tomatoes with species of Crithidia, Leptomonas and Herpetomonas. Some of these flagellates multiplied in tomatoes. Besides, house flies became infected with Crithidia sp. when fed on tomatoes experimentally inoculated with this flagellate. Therefore, isolation of a trypanosomatid from a plant should not constitute an absolute criterion for placing it in the genus Phytomonas.     

A PCR-based survey on Phytomonas (Euglenozoa: Trypanosomatidae) in phytophagous hemipterans of the Amazon region

ABSTRACT. We have surveyed 244 hemipterans from Western Brazilian Amazônia for the presence of trypanosomatids and identification of members of the genus Phytomonas. Examination by phase microscopy of squashes of insect salivary glands (SG) and digestive tubes (DT) revealed that 44% (108/244) of insects from seven families harbored trypanosomatids. Infections were 5 times more frequent in Coreidae than in all other families together. Smears of SG and DT of the dissected insects were fixed on glass slides with methanol and stained with Giemsa for morphological analysis. DNA was recovered from these preparations and submitted to a PCR assay that permitted amplification of all trypanosomatid genera using primers of conserved sequences flanking a segment of the spliced leader (SL) gene. Upon PCR amplification of the recovered DNA, amplicons were hybridized with an oligonucletide probe (SL3′) complementary to a SL intron sequence specific for flagellates of the genus Phytomonas. Among the trypanosomatid‐positive insects, 38.8% harbored Phytomonas spp., corresponding to an overall Phytomonas prevalence of 17.1% among phytophagous bugs, their putative vectors. Since many Phytomonas are pathogenic in plants, this high prevalence in their vectors emphasizes the permanent risk of exposure to disease by native and cultured plants of the Amazon region.     

Ribosomal DNA restriction analysis and synthetic oligonucleotide probing in the identification of genera of lower trypanosomatids.

Fifty-four species or isolates of insect trypanosomatids were examined for the presence of selected restriction enzyme sites in the small (SSU) and large (LSU) rRNA coding units of ribosomal genes. In the SSU, sites for Eco RI, Bgl II, Pst I, and Hind III were found to occur at the same location for all species examined, thus displaying a universal distribution among trypanosomatids. In the LSU, a site for Bgl II in the 24S-alpha sequence and sites for Hind III and Pst I in the 24S-beta sequence were found in all species examined. In contrast, a site for Pvu II in the SSU exhibited a genus-related distribution, being present in Crithidia and Herpetomonas but absent in Phytomonas. A site for Hind III in the 24S-alpha sequence of the LSU also exhibited genus-restricted distribution. The site was present in Crithidia but absent in Phytomonas and Herpetomonas. These findings were confirmed by dot hybridization with a synthetic oligonucleotide complementary to the 18S rRNA sequence containing the Pvu II site. Results point to the usefulness of restriction markers as diagnostic tools for distinguishing the lower trypanosomatid genera Crithidia, Herpetomonas, and Phytomonas at the same time revealing a marked complexity within the genus Leptomonas.     

5S ribosomal RNA gene repeat sequences define at least eight groups of plant trypanosomatids (Phytomonas spp.): phloem-restricted pathogens form a distinct section

Trypanosomatids isolated from plants have been assigned typically into the genus Phytomonas. Such designations do not reflect the biology of the diverse isolates; confusion may arise due to the transient presence in plants of monogenetic (insect) trypanosomatids deposited by phytophagous bugs. To develop further molecular markers for the plant kinetoplastids, we have obtained the DNA sequence of the 5S ribosomal RNA gene from 24 isolates harvested from phloem, latex, and fruit. Small, distinct sequence differences were found at the 3'-ends of the transcribed regions; substantial sequence and size differences were found in the non-transcribed regions. Alignment of the gene sequences from all the isolates suggested the presence of eight groupings. While six groups contained isolates from single plant tissues, groups C and A contained isolates from both fruit and latex. The DNA sequences of the 10 phloem-restricted pathogenic isolates from South America and the Carribean were highly conserved and thus comprised a single group (H). The conserved nature of the 5S ribosomal RNA genes in these plant pathogens supports the proposal that they be considered as a distinct section, the phloemicola.     

The more insect trypanosomatids under study-the more diverse Trypanosomatidae appears

From 10 trypanosomatids genera six comprise monogenetic parasites of insects and for the rest of four genera insects may serve as vectors. The invertebrate host is an essential element of trypanosomatids life cycle, but from more than 900 recognised vertebrate hosts only about 500 species of insects have been discovered to be the hosts of homoxenous trypanosomatids. Nothing or very little is known about insect trypanosomatids in many extensive areas such as South East Asia, Australia, Japan and some others. Each new region explored brings many new findings. Recently flagellates were found in new insect species and families. The border of parasites distribution was expanded till Central Asia, Far East and North over the Polar Circle. As paleogeographical events are now under contemplating in trypanosomatids phylogeny researches so northern insect trypanosomatids may attract some attention as the elements of postglacial fauna which is definitely young. Very broad host specificity of insect trypanosomatids and high probability to isolate non-specific parasite show causes that only the investigation of a culture may solve the question 'what parasite was really isolated?'. Examination of cell morphotypes in the host has clearly demonstrated that they are not sufficient for classification and may lead us to be mistaken. The number of insect trypanosomatid cultures is inadequate for characterisation of the diversity of insects trypanosomatids. Trypanosoma is actually the only trypanosomatid genus which is out of questions. Insect trypanosomatids comprise the most diversified part of trypanosomatids evolutionary tree. Recent ssrRNA phylogenetic analysis and morphological data show that three insect isolates represent new lineages on trypanosomatid evolutionary tree, as well as dendrograms derived from PCR data demonstrated some new groups of isolates. Therefore, the more insect trypanosomatids are involved in laboratory investigations--the more new clusters or/and new lineages are appearing on the tree.     

Identification of excreted iron superoxide dismutase for the diagnosis of Phtytomonas

An excreted iron superoxide dismutase (FeSODe) of pI 3.6 with a molecular weight of 28-30 kDa was detected in the in vitro culture of Phytomonas isolated from Euphorbia characias (SODeCHA) and from Lycopersicon esculentum (SODeTOM), in Grace's medium without serum. These FeSODe excreted into the medium had immunogenic capacity: the positivity of the anti-SODeCHA serum persisted to a dilution of 1/30,000, and for the anti-SODeTOM to 1/10,000 by Western blot. In addition, cross reaction was detected between the anti-SODe serum of Phytomonas isolated from E. characias against SODeTOM, and the anti-SODe serum from L. esculentum with SODeCHA. This characteristic offers the possibility of its use to diagnose plant trypanosomatids. The validation of the test was confirmed by experimental inoculation of tomato fruits with Phytomonas isolated from L. esculentum. At 7, 10, 15, and 21 days post infection, it was possible to detect the presence of the parasites with the anti-SODe serum of Phytomonas isolated from L. esculentum at a dilution of 1/250. These serological results were confirmed by visualization of the parasites by optical microscopy. The data of this study confirm that the SOD is sufficient to identify a trypanosomatid isolated from plants as belonging to the genus Phytomonas.     

Characterization of Phytomonas isolated from fruits by electrophoretic isoenzymes and kinetoplast-DNA analysis

Abstract Two flagellates of the family trypanosomatidae were isolated from the fruits of Lycopersicon esculentum (tomato) and Annona cherimolia (cherimoya) in the southeastern region of Spain. The isolates were characterized by isoenzyme analysis using nine different isoenzymes and by analysis of kinetoplast DNA (kDNA) restriction fragment length polymorphism using four different restriction endonucleases. Most of the isoenzymes were unable to distinguish between the two fruit isolates, while they were all able to distinguish these two from four other Phytomonas isolates, three of which were from laticiferous plants i.e. Euphorbia characias E. hirta and E. hyssopifolia , and one was a phloem-restricted isolate associated with Hartrot disease. Only the enzyme Superoxide dismutase was able to differentiate between the two fruit isolates. Electrophoretic and restriction endonuclease analysis of kDNA minicircles, using four restriction enzymes, showed similar if not identical restriction cleavage patterns of the minicircles of the two isolates from fruits, while the patterns were different for the other isolates. These results confirm the hypothesis that the two isolates from fruits constitute a group of trypanosomatids that are the same or closely related and that this group can parasitize more than one host plant.     

Genetic variability of trypanosomatids isolated from phytophagous hemiptera defined by morphological, biochemical, and molecular taxonomic markers.

In the present study, we investigated the genetic variability among 49 new isolates of trypanosomatids from phytophagous Hemiptera by means of morphological characters, growth features, and biochemical (enzymes of ornithine-arginine cycle) and molecular markers (based on spliced-leader, and ribosomal genes). From 402 phytophagous insects dissected and examined for the presence of trypanosomatids, 228 species belonging to Pyrrhocoridae, Coreidae, Lygaeidae, and Pentatomidae families harbored trypanosomatids in their salivary glands, or digestive tubes. Among these insects, 211 carried promastigotes and only 17 had choanomastigote forms. The results show a strong association among morphology, growth features, and biochemical and molecular markers and reveal the genetic diversity of the isolates, which were assigned to Crithidia, Phytomonas, and Leptomonas; we found genetic polymorphism within all these genera, thus indicating high genetic variability among trypanosomatids from phytophagous insects.     

Sera of chagasic patients react with antigens from the tomato parasite Phytomonas serpens

The genus Phytomonas comprises trypanosomatids that can parasitize a broad range of plant species. These flagellates can cause diseases in some plant families with a wide geographic distribution, which can result in great economic losses. We have demonstrated previously that Phytomonas serpens 15T, a tomato trypanosomatid, shares antigens with Trypanosoma cruzi, the agent of human Chagas disease. Herein, two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) were used to identify proteins of P. serpens 15T that are recognized by sera from patients with Chagas disease. After 2D-electrophoresis of whole-cell lysates, 31 peptides were selected and analyzed by tandem mass spectrometry. Twenty-eight polypeptides were identified, resulting in 22 different putative proteins. The identified proteins were classified into 8 groups according to biological process, most of which were clustered into a cellular metabolic process category. These results generated a collection of proteins that can provide a starting point to obtain insights into antigenic cross reactivity among trypanosomatids and to explore P. serpens antigens as candidates for vaccine and immunologic diagnosis studies.     

Phylogeny of the kinetoplastida: taxonomic problems and insights into the evolution of parasitism

To further investigate phylogeny of kinetoplastid protozoa, the sequences of small subunit (18S) ribosomal RNA of nine bodonid isolates and ten isolates of insect trypanosomatids have been determined. The root of the kinetoplastid tree was attached to the branch of Bodo designis and/or Cruzella marina. The suborder Trypanosomatina appeared as a monophyletic group, while the suborder Bodonina was paraphyletic. Among bodonid lineages, parasitic organisms were intermingled with free-living ones, implying multiple transitions to parasitism and supporting the 'vertebrate-first hypothesis'. The tree indicated that the genera Cryptobia and Bodo are artificial taxa. Separation of fish cryptobias and Trypanoplasma borreli as different genera was not supported. In trypanosomatids, the genera Leptomonas and Blastocrithidia were polyphyletic, similar to the genera Herpetomonas and Crithidia and in contrast to the monophyletic genera Trypanosoma and Phytomonas. This analysis has shown that the morphological classification of kinetoplastids does not in general reflect their genetic affinities and needs a revision.     

Use of Monoclonal Antibodies for Differentiation of Different Isolates of Phytomonas (Plant Trypanosomatids)

The Phytomonas genus was created arbitrarily to designate plant trypanosomes. A serological study with polyclonal and monoclonal antibodies was carried out to situate these trypanosomatids with respect to other trypanosomatids - Herpetomonas, Crithidia, Trypanosoma - and to compare different plant trypanosome strains with each other. The use of monoclonal antibodies directed against two different isolates makes it possible to distinguish plant trypanosomatids according to their geographical origin and to separate clearly the plant trypanosomatids from the South of France from other lower trypanosomatids, which seems to justify creating the Phytomonas genus.     

A Gene Encoding the Plant-Like Alternative Oxidase is Present in Phytomonas but Absent in Leishmania spp.

The constituents of the respiratory chain are believed to differ among the trypanosomatids; bloodstream stages of African trypanosomes and Phytomonas promastigotes oxidize ubiquinol by a ubiquinol:oxygen oxidoreductase, also known as alternative oxidase, whereas Leishmania spp. oxidize ubiquinol via a classic cytochrome-containing respiratory chain. The molecular basis for this elementary difference in ubiquinol oxidation by the mitochondrial electron-transport chain in distinct trypanosomatids was investigated. The presence of a gene encoding the plant-like alternative oxidase could be demonstrated in Phytomonas and Trypanosoma brucei , trypanosomatids that are known to contain alternative oxidase activity. Our results further demonstrated that Leishmania spp. lack a gene encoding the plant-like alternative oxidase, and therefore, all stages of Leishmania spp. will lack the alternative oxidase protein. The observed fundamental differences between the respiratory chains of distinct members of the trypanosomatid family are thus caused by the presence or absence of a gene encoding the plant-like alternative oxidase.     

PCR amplification of the spliced leader gene for the diagnosis of trypanosomatid parasites of plants and insects in methanol-fixed smears

A PCR-based method was adapted for the amplification of DNA from methanol-fixed smears of insects and plants parasitized by trypanosomatids. The PCR target was the multicopy spliced leader (SL) gene. Amplicons were hybridized with an oligonucleotide probe (SL3') specific for Phytomonas. The method has the advantage of dispensing with the cultivation of parasites, many of which are very fastidious or non-cultivable. The technique was applied to archival glass slides and to newly collected material. It proved to specific for Phytomonas spp., enabling their detection in plants and insects. Sequence comparison of the amplicons obtained revealed the existence of different strains/species of Phytomonas circulating among diseased palsms and fruit.     

Phytomonas iron superoxide dismutase: a possible molecular marker

We have isolated and biochemically characterized two iron superoxide dismutases activities (SODI and SODII) from a plant trypanosomatid isolated from Euphorbia characias. The isoenzyme FeSODII has immunogenic capacity, and the positivity of the anti-SODII serum persists to a dilution of 1/40,000, by Western blot. In addition, Western blot has been used to test the positivity of the anti-SODII serum against antigen fractions (SOD) from 17 isolates belonging to the family Trypanosomatidae and for which we had previously determined the isoenzymatic profile. The reaction proved positive only with those plant isolates considered to belong to the genus Phytomonas, whereas there was no reaction of the anti-SODII serum, against the antigen fractions from the species Trypanosoma cruzi, Leishmania donovani, Herpetomonas samuelpessoai, Herpetomonas davidi, Crithidia luciliae and Leptomonas collosoma. FeSODII is located mainly over the entire surface of the parasite, as well as in the nucleus, glycosomes and membranes. The above makes FeSODII promising as a molecular tool for diagnosis and identification, and as a potential chemotherapeutic target for designing drugs aimed at controlling not only of the diseases caused by Phytomonas species, but also for the great metabolic similarity to other trypanosomatids of animals and humans, it may be possible for these results to be extrapolated. Moreover, the sequencing of the amino-terminal end of the FeSODII enables the design of primers that in the near future will make it possible to sequence the gene of this isoenzyme.     

The Streamlined Genome of Phytomonas spp. Relative to Human Pathogenic Kinetoplastids Reveals a Parasite Tailored for Plants

Members of the family Trypanosomatidae infect many organisms, including animals, plants and humans. Plant-infecting trypanosomes are grouped under the single genus Phytomonas, failing to reflect the wide biological and pathological diversity of these protists. While some Phytomonas spp. multiply in the latex of plants, or in fruit or seeds without apparent pathogenicity, others colonize the phloem sap and afflict plants of substantial economic value, including the coffee tree, coconut and oil palms. Plant trypanosomes have not been studied extensively at the genome level, a major gap in understanding and controlling pathogenesis. We describe the genome sequences of two plant trypanosomatids, one pathogenic isolate from a Guianan coconut and one non-symptomatic isolate from Euphorbia collected in France. Although these parasites have extremely distinct pathogenic impacts, very few genes are unique to either, with the vast majority of genes shared by both isolates. Significantly, both Phytomonas spp. genomes consist essentially of single copy genes for the bulk of their metabolic enzymes, whereas other trypanosomatids e.g. Leishmania and Trypanosoma possess multiple paralogous genes or families. Indeed, comparison with other trypanosomatid genomes revealed a highly streamlined genome, encoding for a minimized metabolic system while conserving the major pathways, and with retention of a full complement of endomembrane organelles, but with no evidence for functional complexity. Identification of the metabolic genes of Phytomonas provides opportunities for establishing in vitro culturing of these fastidious parasites and new tools for the control of agricultural plant disease.     

Diversity of insect trypanosomatids assessed from the spliced leader RNA and 5S rRNA genes and intergenic regions

We have determined the sequences of 5S rRNA and spliced leader (SL) RNA genes, and adjacent intergenic regions for representatives of all known trypanosomatid genera parasitizing insects. The genetic loci have been analyzed separately as well as by a combined approach. Several isolates, assigned by morphology to different genera (Leptomonas spp., Blastocrithidia spp.), seem to belong to a single species with an unexpectedly wide host and geographical range. An unnamed trypanosomatid isolated from rats in Egypt was found to belong to the genus Herpetomonas, so far associated with insect hosts only. It is closely related to Herpetomonas ztiplika, a parasite of a blood-sucking biting midge. Apparently several different trypanosomatid species can infect one insect species, as exemplified by Leptomonas sp. PL and Wallaceina sp. Wsd, which were isolated from different specimens of Salda littoralis on the same locality and day. However, since the same species of Leptomonas was obtained from insect hosts belonging to different genera, some insect trypanosomatids may have low host specificity. Our data revealed additional discrepancies between molecular phylogenetic data and cell morphology, rendering current trypanosomatid taxonomy unreliable.