Publications

4511

A novel phytoplasma, designated strain SoyST1c1, associated with a newly emerging disease in soybean (Glycine max), known as soybean stunt (SoyST), was found in 2002 in a soybean plantation in Alajuela Province, Costa Rica. The same phytoplasma, or a very closely related strain, also infected sweet pepper (Capsicum annuum) with purple vein syndrome (SwPPV) and passion fruit vine (Passiflora edulis) with bud proliferation disease (PasFBP) in the same region. Sequence analysis of cloned 16S rRNA gene sequences (GenBank accession nos FJ226068–FJ226073 and HQ225624–HQ225635) indicated that all three affected plants were infected by phytoplasmas that shared <97.5 % sequence similarity with previously described phytoplasmas. The SoyST-causing phytoplasma represents a new taxon, most closely related to phytoplasma group 16SrI and 16SrXII strains. Virtual RFLP analysis indicated that the SoyST-causing phytoplasma and its closely related strains represent a novel 16Sr group, designated 16SrXXXI. Phylogenetic analysis of 16S rRNA gene sequences from the new phytoplasma strains, those previously described as ‘Candidatus Phytoplasma spp.’ and other distinct, as yet unnamed, phytoplasmas indicated that the SoyST-causing phytoplasma represents a distinct lineage within the aster yellows/stolbur branch on the phylogenetic tree. On the basis of its unique 16S rRNA gene sequence and biological properties, strain SoyST1c1 represents a novel taxon, for which the name ‘Candidatus Phytoplasma costaricanum’ is proposed with SoyST1c1 as the reference strain.

Summary In this study, members of a specific group of thin (6–14 µm filament diameter), vacuolated Beggiatoa‐ like filaments from six different hypersaline microbial mats were morphologically and phylogenetically characterized. Therefore, enrichment cultures were established, filaments were stained with fluorochromes to show intracellular structures and 16S rRNA genes were sequenced. Morphological characteristics of Beggiatoa‐ like filaments, in particular the presence of intracellular vacuoles, and the distribution of nucleic acids were visualized. In the intracellular vacuole nitrate reached concentrations of up to 650 mM. Fifteen of the retrieved 16S rRNA gene sequences formed a monophyletic cluster and were phylogenetically closely related (≥ 94.4% sequence identity). Sequences of known filamentous sulfide‐oxidizing genera Beggiatoa and Thioploca that comprise non‐vacuolated and vacuolated filaments from diverse habitats clearly delineated from this cluster. The novel monophyletic cluster was furthermore divided into two sub‐clusters: one contained sequences originating from Guerrero Negro (Mexico) microbial mats and the other comprised sequences from five distinct Spanish hypersaline microbial mats from Ibiza, Formentera and Lake Chiprana. Our data suggest that Beggiatoa ‐like filaments from hypersaline environments displaying a thin filament diameter contain nitrate‐storing vacuoles and are phylogenetically separate from known Beggiatoa . Therefore, we propose a novel genus for these organisms, which we suggest to name ‘ Candidatus Allobeggiatoa’.

AbstractThe bacterium Candidatus Liberibacter asiaticus (Las) has been strongly associated with huanglongbing, or citrus greening, which is one of the most devastating citrus diseases worldwide. Las is transmitted by the Asian citrus psyllid (ACP) Diaphorina citri (Hemiptera: Psyllidae) in a persistent manner, but its interactions with the psyllid vector, particularly at the organ and cellular levels, are poorly understood. We have tested several fluorescence in situ hybridization (FISH) protocols and three nucleic acid probes for the localization of Las in haemolymph smears and dissected organs of ACP adults that fed on Las‐infected plants in the field or laboratory and in sections from Las‐infected citrus leaves. Las was detected by FISH and confocal laser scanning microscopy in the filter chamber, midgut, Malpighian tubules, haemolymph, salivary glands, ovaries and in muscle and fat tissues of ACP that acquired Las from infected plants, as well as in the phloem of infected citrus leaves. Las appeared as pleiomorphic bodies or short thin rods that were much more dispersed and individually distinct in citrus leaf phloem and in ACP haemolymph, but more densely aggregated in cells of the salivary glands and other ACP organs and tissues. Our results provide the first in situ demonstration of Las infection in various psyllid organs and tissues and show the near‐systemic infection of ACP by Las.

AbstractBackground"CandidatusPhytoplasma aurantifolia", is the causative agent of witches' broom disease in Mexican lime trees (Citrus aurantifoliaL.), and is responsible for major losses of Mexican lime trees in Southern Iran and Oman. The pathogen is strictly biotrophic, and thus is completely dependent on living host cells for its survival. The molecular basis of compatibility and disease development in this system is poorly understood. Therefore, we have applied a cDNA- amplified fragment length polymorphism (AFLP) approach to analyze gene expression in Mexican lime trees infected by "Ca. Phytoplasma aurantifolia".ResultsWe carried out cDNA-AFLP analysis on grafted infected Mexican lime trees of the susceptible cultivar at the representative symptoms stage. Selective amplifications with 43 primer combinations allowed the visualisation of 55 transcript-derived fragments that were expressed differentially between infected and non-infected leaves. We sequenced 51 fragments, 36 of which were identified as lime tree transcripts after homology searching. Of the 36 genes, 70.5% were down-regulated during infection and could be classified into various functional groups. We showed that Mexican lime tree genes that were homologous to known resistance genes tended to be repressed in response to infection. These included the genes for modifier of snc1 and autophagy protein 5. Furthermore, down-regulation of genes involved in metabolism, transcription, transport and cytoskeleton was observed, which included the genes for formin, importin β 3, transducin, L-asparaginase, glycerophosphoryl diester phosphodiesterase, and RNA polymerase β. In contrast, genes that encoded a proline-rich protein, ubiquitin-protein ligase, phosphatidyl glycerol specific phospholipase C-like, and serine/threonine-protein kinase were up-regulated during the infection.ConclusionThe present study identifies a number of candidate genes that might be involved in the interaction of Mexican lime trees with "CandidatusPhytoplasma aurantifolia". These results should help to elucidate the molecular basis of the infection process and to identify genes that could be targeted to increase plant resistance and inhibit the growth and reproduction of the pathogen.

Abstract Previously, we reported that Candidatus Liberibacter asiaticus (Las)‐infected Diaphorina citri are characterized by lower levels of cytochrome P450 monooxygenases than uninfected counterparts. In the present study, we investigated expression levels of family 4 cytochrome P450 ( CYP4 ) genes in Las‐infected and uninfected D. citri adults. Five novel CYP4 genes ( CYP4C67 , CYP4DA1 , CYP4C68 , CYP4DB1 and CYP4G70 ) were identified. Four of the five CYP4 genes were expressed at significantly higher levels in uninfected than Las‐infected males, whereas only one was expressed at significantly higher levels in uninfected than Las‐infected females. These results suggest that levels of cytochrome P450 monooxygenases in D. citri may be linked to expression levels of these CYP4 genes. Expression of all five CYP4 genes was induced by exposure of D. citri to imidacloprid, suggesting their possible involvement in metabolism of this toxin. Higher expression of the five CYP4 genes was found in nymphs than adults, which is congruent with previous results indicating higher levels of cytochrome P450 monooxygenases in nymphs than adults. These five CYP4 genes may be promising candidates for RNA‐interference to silence overexpression of genes associated with insecticide resistance in D. citri . These newly identified genes may also serve as DNA‐based screening markers for cytochrome P450‐mediated insecticide resistance in field populations of D. citri .

Citrus Huanglongbing (HLB) has been threatening citrus production worldwide. In this study, a comparative proteomic approach was applied to understand the pathogenic process of HLB in affected sweet orange leaves. Using the isobaric tags for relative and absolute quantification (iTRAQ) technique, we identified 686 unique proteins in the mature leaves of both mock‐inoculated and diseased ‘Madam Vinous' sweet orange plants. Of the identified proteins, 20 and 10 were differentially expressed in leaves with and without symptoms of HLB (fold change > 2.5), respectively, compared with mock‐inoculated controls. Most significantly, upregulated proteins were involved in stress/defense response, such as four miraculin‐like proteins, chitinase, Cu/Zn superoxide dismutase and lipoxygenase. Microarray analysis also showed that stress‐related genes were significantly upregulated at the transcriptional level. For example, remarkable upregulations of miraculin‐like proteins and Cu/Zn superoxide dismutase transcripts were observed. Moreover, the transcriptional patterns of miraculin‐like protein 1 and Cu/Zn superoxide dismutase were examined at different stages of HLB disease development. Combined with the transcriptomic data, the proteomic data can provide an enhanced understanding of citrus stress/defense responses to HLB.