A new species of Oecleus Stål (Hemiptera: Fulgoroidea: Cixiidae) from coconut in Brazil.

A new species of cixiid planthopper (Hemiptera: Fulgoroidea) in the genus Oecleus Stål, Oecleus sergipensis n. sp., is described from Sergipe State, Brazil. This new taxon is associated with coconut (Cocos nucifera L.) and date palm (Phoenix L). The species was detected in Auchenorrhyncha surveys to find potential vectors of lethal yellowing type syndrome. This is the first report of the genus Oecleus in Brazil. Sequence data from the mitochondrial cytochrome c oxidase subunit I (COI) barcoding region was obtained and accessioned into GenBank.

Rainfall and Coconut Accession Explain the Composition and Abundance of the Community of Potential Auchenorrhyncha Phytoplasma Vectors in Brazil.

Coconut plantations are attacked by the lethal yellowing (LY), which is spreading rapidly with extremely destructive effects in several countries. The disease is caused by phytoplasmas that occur in the plant phloem and are transmitted by Haplaxius crudus (Van Duzee) (Auchenorrhyncha: Cixiidae). Owing to their phloem-sap feeding habit, other planthopper species possibly act as vectors. Here, we aimed at assessing the seasonal variation in the Auchenorrhyncha community in six dwarf coconut accessions. Also, we assessed the relative contribution of biotic (coconut accession) and abiotic (rainfall, temperature) in explaining Auchenorrhyncha composition and abundance. The Auchenorrhyncha community was monthly evaluated for 1 yr using yellow sticky traps. Among the most abundant species, Oecleus sp., Balclutha sp., Deltocephalinae sp.2, Deltocephalinae sp.3, Cenchreini sp., Omolicna nigripennis Caldwell (Derbidae), and Cedusa sp. are potential phytoplasma vectors. The composition of the Auchenorrhyncha community differed between dwarf coconut accessions and periods, namely, in March and April (transition from dry to rainy season) and August (transition from rainy to dry season). In these months, Oecleus sp. was predominantly found in the accessions Cameroon Red Dwarf, Malayan Red Dwarf, and Brazilian Red Dwarf Gramame, while Cenchreini sp. and Bolbonota sp. were dominant in the accessions Brazilian Yellow Dwarf Gramame, Malayan Yellow Dwarf, and Brazilian Green Dwarf Jequi. We conclude that dwarf coconut host several Auchenorrhyncha species potential phytoplasma vectors. Furthermore, coconut accessions could be exploited in breeding programs aiming at prevention of LY. However, rainfall followed by accessions mostly explained the composition and abundance of the Auchenorrhyncha community.

Presence of 16SrIV phytoplasmas of subgroups A, D and E in planthopper Haplaxius crudus Van Duzee insects in Yucatán, Mexico.

The present study was carried out to determine if group 16SrIV phytoplasmas, causing lethal yellowing (LY) disease, are present in Haplaxius crudus Van Duzee (Hemiptera: Cixiidae) insects associated with palms in Yucatán, Mexico. Haplaxius crudus feral insects were captured from palm foliage at two locations (Chicxulub Puerto and CICY, Mérida, where LY-type diseases are active) and evaluated individually for the presence of phytoplasma DNA by a group 16SrIV-specific nested PCR assay. The results showed positive detection in H. crudus insects in a proportion of 2.7% (of the total 2726 analyzed) during a 3-year period of study. The percentage of detection was different for each site, 5.9% positive of 799 insects from Mérida and 1.7% of 1927 from Chicxulub Puerto. Positive detections were also obtained in extracts from 5.3 to 1.2% of males and females, respectively. Sequencing and in silico RFLP and phylogenetic analyses of PCR-amplified rDNA products indicated that H. crudus insects from Chicxulub Puerto harbored phytoplasma strains of subgroups 16SrIV-A or 16SrIV-D, whereas in insects from Mérida the strains found were 16SrIV-A, 16SrIV-D or 16SrIV-E. The diversity of subgroup strains detected in H. crudus coincided with strains previously identified in palms showing LY-type disease syndromes in Yucatán thereby implicating H. crudus as a candidate vector of 16SrIV phytoplasmas in this region of Mexico.

'Candidatus Phytoplasma palmicola', associated with a lethal yellowing-type disease of coconut (Cocos nucifera L.) in Mozambique.

In this study, the taxonomic position and group classification of the phytoplasma associated with a lethal yellowing-type disease (LYD) of coconut (Cocos nucifera L.) in Mozambique were addressed. Pairwise similarity values based on alignment of nearly full-length 16S rRNA gene sequences (1530 bp) revealed that the Mozambique coconut phytoplasma (LYDM) shared 100% identity with a comparable sequence derived from a phytoplasma strain (LDN) responsible for Awka wilt disease of coconut in Nigeria, and shared 99.0-99.6% identity with 16S rRNA gene sequences from strains associated with Cape St Paul wilt (CSPW) disease of coconut in Ghana and Côte d'Ivoire. Similarity scores further determined that the 16S rRNA gene of the LYDM phytoplasma shared <97.5% sequence identity with all previously described members of 'Candidatus Phytoplasma'. The presence of unique regions in the 16S rRNA gene sequence distinguished the LYDM phytoplasma from all currently described members of 'Candidatus Phytoplasma', justifying its recognition as the reference strain of a novel taxon, 'Candidatus Phytoplasma palmicola'. Virtual RFLP profiles of the F2n/R2 portion (1251 bp) of the 16S rRNA gene and pattern similarity coefficients delineated coconut LYDM phytoplasma strains from Mozambique as novel members of established group 16SrXXII, subgroup A (16SrXXII-A). Similarity coefficients of 0.97 were obtained for comparisons between subgroup 16SrXXII-A strains and CSPW phytoplasmas from Ghana and Côte d'Ivoire. On this basis, the CSPW phytoplasma strains were designated members of a novel subgroup, 16SrXXII-B.

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.

The internal transcribed spacer of ribosomal RNA genes in plant trypanosomes (Phytomonas spp.) resolves 10 groups.

The distinction between plant trypanosomatids and opportunistic monoxenous insect trypanosomatids has not been demarcated clearly due to the mass placement of all trypanosomatids isolated from plants into the arbitrary genus Phytomonas spp. The advent of molecular markers has been useful in distinguishing plant trypanosomatids from the rest of the Trypanosomatidae family. Here we have examined the internal transcribed spacer (ITS) region of the ribosomal RNA (rRNA) locus for classification purposes. This region contains two distinct ITSs flanked by the small subunit and large subunit of ribosomal RNA genes and separated by the 5.8S ribosomal RNA gene. Sequences within the 5.8S ribosomal RNA gene and in the ITS sequences can serve as specific markers for several of the Phytomonas groups. Microsatellite sequences were identified in Phytomonas spp. in both ITS regions. Several classes of microsatellites were seen, with inter-isolate variation that has potential for future use. Maximum Likelihood analysis of the ITS sequences of 20 Phytomonas isolates representing the eight defined groups and a few unclassified isolates revealed a total of 10 distinct subgroups within our collection, of which two are new. The ITS region, which includes the 5.8S sequence, is a robust marker for the subdivisions within the genus Phytomonas spp.

Isolation of a protozoan parasite genetically related to the insect trypanosomatid Herpetomonas samuelpessoai from a human immunodeficiency virus-positive patient.

Severely immunocompromised human immunodeficiency virus (HIV) patients can develop various opportunistic infections due to bacteria, viruses, fungi, or protozoa. Here we report the first isolation of a flagellated protozoan genetically closely related to Herpetomonas samuelpessoai, which is usually a parasite of insects, from the blood of an HIV-infected patient.

First complete chromosomal organization of a protozoan plant parasite (Phytomonas spp.).

Phytomonas spp. are members of the family Trypanosomatidae that parasitize plants and may cause lethal diseases in crops such as Coffee Phloem necrosis, Hartrot in coconut, and Marchitez sorpresiva in oil palm. In this study, the molecular karyotype of 6 isolates from latex plants has been entirely elucidated by pulsed-field gel electrophoresis and DNA hybridization. Twenty-one chromosomal linkage groups constituting heterologous chromosomes and sizing between 0.3 and 3 Mb could be physically defined by the use of 75 DNA markers (sequence-tagged sites and genes). From these data, the genome size can be estimated at 25.5 (+/-2) Mb. The physical linkage groups were consistently conserved in all strains examined. Moreover, the finding of several pairs of different-sized homologous chromosomes strongly suggest diploidy for this organism. The definition of the complete molecular karyotype of Phytomonas represents an essential primary step toward sequencing the genome of this parasite of economical importance.

Rational sub-division of plant trypanosomes (Phytomonas spp.) based on minicircle conserved region analysis.

The sequences of minicircle conserved regions from various plant trypanosomatids have been determined and analyzed. The goal of this study was to add another tool to the arsenal of molecular probes for distinguishing between the different trypanosomatids occurring in plants: systemic trypanosomatids multiplying in the sap, those from the laticiferous tubes, and those developing in fruits, seeds or flowers but not in the plant itself and that are frequently considered as opportunistic insect trypanosomatids. As some plant intraphloemic trypanosomatids are the causative agents of important diseases, a clear definition of the different types of trypanosomatids is critical. The conserved region of the mitochondrial minicircle provides several specific features in a small sequence region containing three functionally elements required for minicircle replication. Trees generated from the analysis recapitulated trees drawn from analyses of isoenzymes, RAPD, and particular gene sequences, supporting the validity of the small region used in this work. Three groups of isolates were significant and in accordance with previous work. The peculiarity of phloem-restricted trypanosomatids associated with wilts of coconut and oil palm in Latin America - group H - is confirmed. In agreement with previous studies on their biological and serological properties the results highlighted this group called 'phloemicola'. It always differentiated from all other latex and fruit isolates or opportunistic trypanosomatids, like insect trypanosomatids. We can assert that phloemicola is the only well-defined taxon among all plant trypanosomatids. A group of non-pathogenic latex isolates from South American euphorbs (G), and a heterogenous group (A) including one fruit, one possible latex and one insect isolate are clearly distinct groups. The group of Mediterranean isolates from latex (D), even with a low boostrap, stood out well from other groups. The remainder of the isolates fell into a heterogeneous cluster. At least eight different groups in the plant trypanosomatids were identified.

Herpetomonas spp. isolated from tomato fruits (Lycopersicon esculentum) in southern Spain.

A flagellate 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. Amplification 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.

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.

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.