Publications

4857

Huanglongbing, or citrus greening disease, is associated with infection by the phloem-limited bacterium ‘Candidatus Liberibacter asiaticus’. Infection with ‘Ca. L. asiaticus’ is incurable; therefore, knowledge regarding ‘Ca. L. asiaticus’ biology and pathogenesis is essential to develop a treatment. However, ‘Ca. L. asiaticus’ cannot currently be successfully cultured, limiting its study. To gain insight into the conditions conducive for growth of ‘Ca. L. asiaticus’ in vitro, ‘Ca. L. asiaticus’ inoculum obtained from seed of fruit from infected pomelo trees (Citrus maxima ‘Mato Buntan’) was added to different media, and cell viability was monitored for up to 2 months using quantitative polymerase chain reaction in conjunction with ethidium monoazide. Media tested included one-third King's B (K), K with 50% juice from the infected fruit, K with 50% commercially available grapefruit juice, and 100% commercially available grapefruit juice. Results show that juice-containing media dramatically prolong viability compared with K in experiments reproduced during 2 years using different juice sources. Furthermore, biofilm formed at the air–liquid interface of juice cultures contained ‘Ca. L. asiaticus’ cells, though next-generation sequencing indicated that other bacterial genera were predominant. Chemical characterization of the media was conducted to discuss possible factors sustaining ‘Ca. L. asiaticus’ viability in vitro, which will contribute to future development of a culture medium for ‘Ca. L. asiaticus’.

Analysis of the completely determined genomes of the plant-derived Acholeplasma brassicae strain O502 and A. palmae strain J233 revealed that the circular chromosomes are 1,877,792 and 1,554,229 bp in size, have a G + C content of 36 and 29%, and encode 1,690 and 1,439 proteins, respectively. Comparative analysis of these sequences and previously published genomes of A. laidlawii strain PG-8, ‘Candidatus Phytoplasma asteris' strains, ‘Ca. P. australiense' and ‘Ca. P. mali' show a limited shared basic genetic repertoire. The acholeplasma genomes are characterized by a low number of rearrangements, duplication and integration events. Exceptions are the unusual duplication of rRNA operons in A. brassicae and an independently introduced second gene for a single-stranded binding protein in both genera. In contrast to phytoplasmas, the acholeplasma genomes differ by encoding the cell division protein FtsZ, a wide variety of ABC transporters, the F₀F1 ATP synthase, the Rnf-complex, SecG of the Sec-dependent secretion system, a richly equipped repertoire for carbohydrate metabolism, fatty acid, isoprenoid and partial amino acid metabolism. Conserved metabolic proteins encoded in phytoplasma genomes such as the malate dehydrogenase SfcA, several transporters and proteins involved in host-interaction, and virulence-associated effectors were not predicted for the acholeplasmas.

The Asian citrus psyllid, Diaphorina citri Kuwayama, one of the known vectors for citrus greening disease or Huanglongbing (HLB) pathogens, has been present in Texas for over a decade, but the detection of the disease is recent. HLB has been confirmed in only two adjacent commercial citrus groves of grapefruit and sweet orange. A study was conducted to compare the population of Candidatus Liberibacter asiaticus (CLas) cells in different plant parts including peduncle, columella, leaves, seeds, young shoots, flower buds, flowers, and bark of 6-year-old known infected grapefruit and sweet orange trees. The bacterial population was estimated using a previously described grand universal regression equation Y = 13.82 – 0.2866X, where Y is the log of the target copy number and X is the Ct (threshold cycle) of the assay. Except for bark tissue, there was no significant difference in the concentration of CLas cells in other plant parts between the two cultivars. Within the cultivar, the bacterial concentration also varied with the plant part, with peduncle, columella, midrib having significantly higher titer of CLas compared with other plant parts. The obtained results here are in agreement with previous studies conducted on Florida samples, but the consistently lowest bacterial titer recorded in young shoots, leaf blade, and especially leaf margins relative to the midrib has never been previously reported.

Abstract ‘Candidatus Liberibacter solanacearum’ is a pathogen of solanaceous crops (Solanales: Solanaceae) that causes zebra chip disease of potato (Solanum tuberosum L.) and plant dieback in tomato (S. lycopersicum L.) and pepper (Capsicum spp.). This pathogen is vectored by the potato/ tomato psyllid Bactericera cockerelli (Šulc) (Hemiptera: Triozidae), but little is known about the interactions between B. cockerelli and ‘Ca. Liberibacter solanacearum.’ Fluorescence in situ hybridization was used to assess the incidence of ‘Ca. Liberibacter solanacearum’ in the hemolymph, bacteriomes, alimentary canals, and salivary glands of B. cockerelli. Liberibacter was observed in 66% of alimentary canals, 39% of salivary glands, and 40% of bacteriomes dissected from adult psyllids. Compared with adults, the organs of fifth instars appeared less likely to harbor Liberibacter, which was observed in 52% of alimentary canals, 10% of salivary glands, and 6% of bacteriomes dissected from the nymphs. Results of real-time polymerase chain reaction confirmed that fewer fifth instars were infected with Liberibacter compared with adults and indicated that fifth instars were less likely to transmit the pathogen to noninfected host plants. These observations of the localization of ‘Ca. Liberibacter solanacearum’ in the organs and tissues of B. cockerelli adults and nymphs will aid the study of Liberibacter-psyllid interactions and the epidemiology of ‘Ca. Liberibacter solanacearum.’