Publications

4852

The aim of this work was to assess the effects of a combined inoculum of a rhizobacterium and an arbuscular mycorrhizal (AM) fungus on plant responses to phytoplasma infection, and on phytoplasma multiplication and viability in Chrysanthemum carinatum plants infected by chrysanthemum yellows phytoplasma (CY). Combined inoculation with Glomus mosseae BEG12 and Pseudomonas putida S1Pf1Rif resulted in some resistance to phytoplasma infection (about 30%), delayed symptom expression in nonresistant plants, improved growth of the aerial part of the infected plants (+68·1%), and altered root morphology (root tip number: +49·9%; branching degree: +82·8%). Combined inoculation with the two beneficial microorganisms did not alter CY multiplication and viability. In inoculated and infected plants, phytoplasma morphology was typical of senescent cells. A more active and efficient root system in double‐inoculated plants probably mediated the effects of the two rhizospheric microorganisms in the infected plants. The practical application of rhizospheric microorganisms for mitigating phytoplasma damage, following evaluation under field conditions, represents an additional tool for the integrated management of phytoplasmosis.

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’.

AbstractWe report the detection of phytoplasmas in Picea abies, Picea glauca and Picea pungens trees with witches’ brooms and other growth abnormalities and also in symptomless trees. Phytoplasmas were detected in c. 25% of the tested plants by polymerase chain reaction using phytoplasma universal P1/P7 followed by R16F2n/R16R2 primer pairs. The phytoplasmas were classified as members of the phylogenetic groups: 16SrI, ‘Ca. Phytoplasma asteris’; 16SrIII, X‐disease phytoplasma group; and 16SrXXI, ‘Ca. Phytoplasma pini’.

The presence of phytoplasmas in seven coniferous plant species ( Abies procera , Pinus banksiana , P. mugo , P. nigra , P. sylvestris , P. tabuliformis and Tsuga canadensis ) was demonstrated using nested PCR with the primer pairs P1/P7 followed by R16F2n/R16R2. The phytoplasmas were detected in pine trees with witches’ broom symptoms growing in natural forest ecosystems and also in plants propagated from witches’ brooms. Identification of phytoplasmas was done using restriction fragment length polymorphism analysis (RFLP) of the 16S rDNA gene fragment with Alu I, Mse I and Rsa I endonucleases. All samples showed RFLP patterns similar to the theoretical pattern of ‘ Candidatus Phytoplasma pini’, based on the sequence of the reference isolate Pin127S. Nested PCR‐amplified products, obtained with primers R16F2n/R16R2, were sequenced. Comparison of the 16S rDNAs obtained revealed high (99·8–100%) nucleotide sequence identity between the phytoplasma isolates. The isolates were also closely related to four other phytoplasma isolates found in pine trees previously. Based on the results of RFLP and sequence analyses, the phytoplasma isolates tested were classified as members of the ‘ Candidatus Phytoplasma pini’, group 16SrXXI.

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.

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 .

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.