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

Abstract

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 (SL3P) 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 palms and fruit. ß

5S Ribosomal RNA Gene Repeat Sequences Define at Least Eight Groups of Plant Trypanosomatids (Phytomonas spp.): Phloem‐Restricted Pathogens Form a Distinct Section

“…For example, the Spliced Leader (SL) RNA gene, also known as the mini-exon gene, has emerged as a pivotal marker for diverse groups of protozoa in the kinetoplastideuglenoid lineage (Campbell et al 1997). The transcribed region may be used as a marker to distinguish Phytomonas from insect trypanosomatids (Serrano et al 1999b;Ser-ran0 et al 1999c;Teixeira et al 1996) and the DNA sequence of the non-transcribed spacer region may be used to distinguish among phloem, latex, and fruit isolates (Nunes et al 1995;Sturm et al 1995). Here we extend our studies on the tandemlyrepeated genes of kinetoplastid protozoa by investigating the 5s ribosomal RNA (rRNA) gene as a molecular marker.…”

“…Here, we focus on the latter problem, which is being resolved by the characterization of biochemical and genetic markers. Originally distinguished by morphological criteria, monogenetic insect kinetoplastids (Crithidia, Herpetomonas or Leptomonas) that are accidental visitors to plants may be identified now by molecular features (Serrano et al 1999b;Serrano et al 1999c;Teixeira et al 1996). The plant-obligate parasites can be classified by their cytological preferences and pathological profiles (Vickerman and Dollet 1992) in the latex of lactiferous plants where they behave as symbionts; in the phloem (sieve tubes) of non-lactiferous plants where they apparently cause the wilt of the host (Dollet et al 1977;Parthasarathy et al 1976), or restricted to the fruit with an inability to survive in other parts of the plant.…”

Classification of trypanosomatids from fruits and seeds using morphological, biochemical and molecular markers revealed several genera among fruit isolates

“…Several studies have shown that plants can harbor trypanosomatids of various genera besides Phytomonas [7,14,15,20,26]. In contrast to Phytomonas, for which spe-ci¢c molecular taxonomic markers are available [7,14,15], classi¢cation of members of Herpetomonas, Leptomonas and Crithidia still requires the development of speci¢c methods. However, despite the lack of genus-speci¢c markers, these £agellates can be classi¢ed at the genus level using a combination of criteria which, taken together, indicate a reliable taxonomic position.…”

“…Various approaches have been proposed to classify plant trypanosomatids, such as reactivity with monoclonal antibodies (mAbs) [6,7], enzymatic activities [8^10] and molecular markers based on ribosomal genes [11^13]. In addition, we developed two methods based on splicedleader (SL) genes for generic identi¢cation of Phytomonas: (a) hybridization of genomic DNA with a SL (SL3P)-based probe [7], and (b) PCR ampli¢cation (Phy-SLPCR) of an SL sequence able to detect Phytomonas from di¡erent plant tissues and phytophagous insects, using either culture £agellates [14] or crude preparations of ¢eld-collected plants and insects [15,16].…”

A PCR‐Based Survey on Phytomonas (Euglenozoa+Trypanosomatidae) in Phytophagous Hemipterans of the Amazon Region

“…Distinct PCR assays were then developed to amplify SL sequences of all trypanosomatid genera (Try-SLPCR) or exclusively of Phytomonas (Phy-SLPCR) (Serrano et al 1999b). Finally, these PCR assays were adapted to amplify SL genes from methanol-fixed flagellates in glass smears of insect and plant parts and juices (Serrano et al 1999a;Teixeira, Serrano, and Camargo 2000).…”