We carried out a quantitative detection of Candidatus Liberibacter asiaticus, the bacterium associated with the disease of huanglongbing, in the vector psyllid Diaphorina citri by using a TaqMan real-time PCR assay. The concentration of the bacterium was monitored at 5-day intervals for a period of 20 days after psyllids were exposed as fifth instar nymphs or adults to a Ca. L. asiaticus-infected plant for an acquisition access period of 24 h. When adults fed on Ca. L. asiaticus-infected plant, the concentration of the bacterium did not increase significantly and the pathogen was not transmitted to any citrus seedlings. In contrast, when psyllids fed on infected plant as nymphs, the concentration of the pathogen significantly increased by 25-, 360-and 130-fold from the initial acquisition day to 10, 15 and 20 days, respectively. Additionally, the pathogen was successfully transmitted to 67% of citrus seedlings by emerging adults. Our data suggested that multiplication of the bacterium into the psyllids is essential for an efficient transmission and show that it is difficult for adults to transmit the pathogen unless they acquire it as nymphs.

Huanglongbing (HLB) is one of the most devastating diseases of citrus worldwide, and is caused by a phloem-limited fastidious prokaryotic alpha-proteobacterium that is yet to be cultured. In this study, a combination of traditional polymerase chain reaction (PCR) and real-time PCR targeting the putative DNA polymerase and 16S rDNA sequence of 'Candidatus Liberibacter asiaticus,' respectively, were used to examine the distribution and movement of the HLB pathogen in the infected citrus tree. We found that 'Ca. Liberibacter asiaticus' was distributed in bark tissue, leaf midrib, roots, and different floral and fruit parts, but not in endosperm and embryo, of infected citrus trees. Quantification analysis of the HLB bacterium indicated that it was distributed unevenly in planta and ranged from 14 to 137,031 cells/mug of total DNA in different tissues. A relatively high concentration of 'Ca. Liberibacter asiaticus' was observed in fruit peduncles. Our data from greenhouse-infected plants also indicated that 'Ca. Liberibacter asiaticus' was transmitted systemically from infection site to different parts of the plant. Understanding the distribution and movement of the HLB bacterium inside an individual citrus tree is critical for discerning its virulence mechanism and to develop management strategies for HLB.

Members of the family Secoviridae are non-enveloped viruses with mono- or bipartite (RNA-1 and RNA-2) linear positive-sense ssRNA genomes with the size of the RNAs combined ranging from 9 to 13.7 kb. They are related to picornaviruses and are classified in the order Picornavirales. The majority of known members infect dicotyledonous plants and many are important plant pathogens (e.g. grapevine fanleaf virus and rice tungro spherical virus). This is a summary of the current International Committee on Taxonomy of Viruses (ICTV) report on the taxonomy of the family Secoviridae available at www.ictv.global/report/secoviridae.

Thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) was performed to amplify the uncharacterized regions adjacent to the nusG-rplKAJL-rpoB gene cluster of citrus greening organism (GO) isolates from different locations in Japan and Indonesia. Conventional PCR was used to amplify the internal nusG-rplKAJL-rpoB gene cluster of these isolates, and the complete sequence of this 6.1-kb fragment was determined. Comparisons with other bacterial sequences showed that the fragment is the tufB-secE-nusG-rplKAJL-rpoB gene cluster. The organization of this gene cluster is similar to that of the homologous cluster found in Escherichia coli. Except for three nucleotide changes, the sequence was identical among Japanese and Indonesian isolates. A loop-mediated isothermal amplification (LAMP) assay based on the conserved sequence of the nusG-rplKAJL-rpoB gene cluster was developed for the detection of the GO. The LAMP product was rapidly detected on nylon membranes by staining with AzurB. LAMP could detect as low as about 300 copies of the nusG-rplKAJL-rpoB fragment of the Japanese and Indonesian isolates of GO. The LAMP-based detection method, which does not depend upon a thermal cycler and electrophoresis apparatus, will be useful for under-equipped laboratories, including those found in extension centers and quarantine offices.

The aim of this study was to investigate the genetic diversity and relationships among 'Candidatus Liberibacter asiaticus' isolates from different hosts and distinct geographical areas in Southeast Asia. Genetic diversity among 'Ca. Liberibacter asiaticus' was estimated by sequencing four well-characterized DNA fragments: the 16S ribosomal DNA (rDNA) and 16S/23S intergenic spacer regions; the outer membrane protein (omp) gene region; the trmU-tufB-secE-nusG-rplKAJL-rpoB region (gene cluster region); and the bacteriophage-type DNA polymerase region. The sequences of the 16S rDNA and 16S/23S intergenic spacer regions were identical among all 'Ca. Liberibacter asiaticus' isolates. In contrast, nucleotide substitutions were observed in both the omp gene and the gene cluster regions. However, extended bacteriophage-type DNA polymerase sequences acquired by thermal asymmetric interlaced polymerase chain reaction provided the most sequence diversity among isolates. Phylogenetic analysis of the bacteriophage-type DNA polymerase sequences revealed three clusters in the Southeast Asian 'Ca. Liberibacter asiaticus' population. All Indonesian 'Ca. Liberibacter asiaticus' isolates clustered in one group. The other clusters were not correlated with geographic distribution. The differences in genetic sequences did not reflect differences in the original citrus host (mandarin or pummelo). These results suggest that the bacteriophage-type DNA polymerase region would be useful for molecular differentiation between different Southeast Asian 'Ca. Liberibacter asiaticus' isolates.