Publications

3826

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’.

Huanglongbing (HLB) disease, associated with ‘Candidatus Liberibacter asiaticus’ (CLas), is primarily spread via infected citrus nursery trees and by infective Asian citrus psyllid, the insect vector. Recently, the Texas Department of Agriculture initiated regulations requiring commercial and retail citrus nurseries in Texas to transition from traditional open-field to enclosed facilities with insect-resistant screens to mitigate the risk of nurseries serving as sources of CLas. Although several nursery production facilities have adopted this regulation, non-enclosed nurseries persist and pose a significant threat to the citrus industry as potential sources of CLas. A systematic survey for HLB was embarked on in a semi-open nursery facility in South Texas in April 2014. Leaf tissue samples taken from 94 trees representing 5% of the total number of potted trees in the nursery were tested for CLas by quantitative and conventional PCR assays. Of 94 trees tested, 3.2% (3 trees) were positive for CLas by both assays. The presence of CLas in the PCR-positive samples was confirmed by multi-locus sequence analyses. The results represent the first report of HLB in a nursery facility in Texas, and underscore the need for more intensive surveillance for HLB in citrus nursery stock as an integral component of HLB mitigation efforts in Texas. Accepted for publication 27 August 2014. Published 15 December 2014.

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.’