Publications

4707

ABSTRACT. Rickettsia‐like organisms (RLO) are obligate, often highly fastidious, intracellular bacterial parasites associated with a variety of vertebrate and invertebrate hosts. Despite their importance as causative agents of severe mortality outbreaks in farmed aquatic species, little is known about their life cycle and their host range. The present work reports the characterization of “Candidatus Cryptoprodotis polytropus,” a novel Rickettsia‐like bacterium associated with the common ciliate species Pseudomicrothorax dubius by means of the “Full‐Cycle rRNA Approach” and ultrastructural observations. The morphological description by in vivo and scanning electron microscopy and the 18S rRNA gene sequence of the host species is provided as well. Phylogenetic analysis based on the 16S rRNA gene supports the inclusion of “Candidatus Cryptoprodotis polytropus” within the family Rickettsiaceae (cl. Alphaproteobacteria) together with the genera Rickettsia and Orientia. Observations on natural ciliate populations account for the occasional nature of this likely parasitic association. The presence of a previously unknown RLO in ciliates sheds a new light on the possible role of protists as transient hosts, vectors or natural reservoir for some economically important pathogens.

A new disease of glasshouse-grown tomato and pepper in New Zealand has resulted in plant decline and yield loss. Affected plants are characterized by spiky, chlorotic apical growth, curling or cupping of the leaves, and overall stunting. Transmission electron microscopy revealed the presence of phloem-limited bacterium-like organisms in symptomatic plants. The strategy used to identify the bacterium involved using specific prokaryote polymerase chain reaction (PCR) primers in combination with universal 16S rRNA primers. Sequence analysis of the 16S rRNA gene, the 16S/23S rRNA spacer region, and the rplKAJL-rpoBC operon revealed that the bacterium shared high identity with ‘Candidatus Liberibacter’ species. Phylogenetic analysis showed that the bacterium is distinct from the three citrus liberibacter species previously described and has been named ‘Candidatus Liberibacter solanacearum’. This is the first report of a liberibacter naturally infecting a host outside the Rutaceae family. A specific PCR primer pair was developed for its detection.

Citrus huanglongbing (HLB), or greening disease, is strongly associated with any of three nonculturable gram-negative bacteria belonging to ‘Candidatus Liberibacter spp.’ ‘Ca. Liberibacter spp.’ are transmitted by citrus psyllids to all commercial cultivars of citrus. The diseases can be lethal to citrus and have recently become widespread in both São Paulo, Brazil, and Florida, United States, the locations of the largest citrus industries in the world. Asiatic HLB, the form of the disease found in Florida, is associated with ‘Ca. Liberibacter asiaticus’ and is the subject of this report. The nonculturable nature of the pathogen has hampered research and little is known about the distribution of ‘Ca. L. asiaticus’ in infected trees. In this study, we have used a quantitative polymerase chain reaction assay to systematically quantify the distribution of ‘Ca. L. asiaticus’ genomes in tissues of six species of citrus either identified in the field during survey efforts in Florida or propagated in a greenhouse in Beltsville, MD. The populations of ‘Ca. L. asiaticus’ inferred from the distribution of 16S rDNA sequences specific for ‘Ca. L. asiaticus’ in leaf midribs, leaf blades, and bark samples varied by a factor of 1,000 among samples prepared from the six citrus species tested and by a factor of 100 between two sweet orange trees tested. In naturally infected trees, above-ground portions of the tree averaged 1010 ‘Ca. L. asiaticus’ genomes per gram of tissue. Similar levels of ‘Ca. L. asiaticus’ genomes were observed in some but not all root samples from the same plants. In samples taken from greenhouse-inoculated trees, levels of ‘Ca. L. asiaticus’ genomes varied systematically from 104 genomes/g at the graft inoculation site to 1010 genomes/g in some leaf petioles. Root samples from these trees also contained ‘Ca. L. asiaticus’ at 107 genomes/g. In symptomatic fruit tissues, ‘Ca. L. asiaticus’ genomes were also readily detected and quantified. The highest levels of ‘Ca. L. asiaticus’ in fruit tissues were found in the locular membranes and septa (108 genomes/g), with 100-fold lower levels of ‘Ca. L. asiaticus’ in the meso and pericarp of such fruit. Our results demonstrate both the ubiquitous presence of ‘Ca. L. asiaticus’ in symptomatic citrus trees as well as great variation between individual trees and among samples of different tissues from the same trees. Our methods will be useful in both the management and scientific study of citrus HLB, also known as citrus greening disease.