Viability of 'Candidatus Liberibacter asiaticus' prolonged by addition of citrus juice to culture medium.

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'.

An HPLC-MS characterization of the changes in sweet orange leaf metabolite profile following infection by the bacterial pathogen Candidatus Liberibacter asiaticus.

Huanglongbing (HLB) presumably caused by Candidatus Liberibacter asiaticus (CLas) threatens the commercial U.S. citrus crop of an annual value of $3 billion. The earliest shift in metabolite profiles of leaves from greenhouse-grown sweet orange trees infected with Clas, and of healthy leaves, was characterized by HPLC-MS concurrently with PCR testing for the presence of Clas bacteria and observation of disease symptoms. Twenty, 8-month-old 'Valencia' and 'Hamlin' trees were grafted with budwood from PCR-positive HLB source trees. Five graft-inoculated trees of each variety and three control trees were sampled biweekly and analyzed by HPLC-MS and PCR. Thirteen weeks after inoculation, Clas was detected in newly growing flushes in 33% and 55% of the inoculated 'Hamlin' and 'Valencia' trees, respectively. Inoculated trees remained asymptomatic in the first 20 weeks, but developed symptoms 30 weeks after grafting. No significant differences in the leaf metabolite profiles were detected in Clas-infected trees 23 weeks after inoculation. However, 27 weeks after inoculation, differences in metabolite profiles between control leaves and those of Clas-infected trees were evident. Affected compounds were identified with authentic standards or structurally classified by their UV and mass spectra. Included among these compounds are flavonoid glycosides, polymethoxylated flavones, and hydroxycinnamates. Four structurally related hydroxycinnamate compounds increased more than 10-fold in leaves from 'Hamlin' and 'Valencia' sweet orange trees in response to Clas infection. Possible roles of these hydroxycinnamates as plant defense compounds against the Clas infection are discussed.

Levels of 2-dodecylcyclobutanone in ground beef patties irradiated by low-energy X-ray and gamma rays.

Food irradiation improves food safety and maintains food quality by controlling microorganisms and extending shelf life. However, acceptance and commercial adoption of food irradiation is still low. Consumer groups such as Public Citizen and the Food and Water Watch have opposed irradiation because of the formation of 2-alkylcyclobutanones (2-ACBs) in irradiated, lipid-containing foods. The objectives of this study were to measure and to compare the level of 2-dodecylcyclobutanone (2-DCB) in ground beef irradiated by low-energy X-rays and gamma rays. Beef patties were irradiated by low-energy X-rays and gamma rays (Cs-137) at 3 targeted absorbed doses of 1.5, 3.0, and 5.0 kGy. The samples were extracted with n-hexane using a Soxhlet apparatus, and the 2-DCB concentration was determined with gas chromatography-mass spectrometry. The 2-DCB concentration increased linearly (P < 0.05) with irradiation dose for gamma-ray and low-energy X-ray irradiated patties. There was no significant difference in 2-DCB concentration between gamma-ray and low-energy X-ray irradiated patties (P > 0.05) at all targeted doses.