Quantitative determination of selective alternative hosts of candidatus liberibacter asiaticus and potential for transmission to citrus.

摘要

Citrus huanglongbing (HLB) is a devastating citrus disease. HLB in Florida is associated with bacterial pathogen Candidatus Liberibacter asiaticus (Las) and transmitted by Asian citrus psyllid ( Diaphorina citri). Some citrus relatives have been listed as hosts of the psyllid and/or the associated bacterium based on field surveys or PCR tests on field samples, but their status has never been systematically studied. In this work, 8 citrus relatives, i.e., Severinia buxifolia, x Citrofortunella microcarpa, Citropsis gilletiana , Esenbeckia runyonii, Zanthoxylum fagara , Choisya aztec 'Pearl', Choisya ternata 'Sundance', and Amyris texana, were studied to investigate their alternative host status. Possible transmission pathways for each plant were tested with repeated psyllid transmission experiments as well as grafting where compatible. After inoculation, plants were monitored for symptom development and tested by real-time PCR (qPCR). The results showed that all plants studied except A. texana were infected by Las although their transmissibility varied a lot based on bacterial persistency and psyllid activities.;Although estimating live bacterial genome (LBG) is critical for HLB research, PCR has limitations on differentiating live and dead cells. Propidium monoazide (PMA), a novel DNA-binding dye, has already been successfully used on many bacterial plant pathogens to effectively remove DNA from dead cells, but no applications on uncultured bacteria like Las were reported. In this study, PMA-qPCR protocols were first optimized to work with plant and psyllid materials, respectively. Then, they were used to determine LBG in various studies, such as establishing correlation between LBG and microscopic counting, checking the reactions of different citrus plants to Las infection, and checking the connection between LBG and leaf symptom expression. Lastly, the LBG dynamics inside HLB positive citrus and non-citrus hosts was monitored monthly through a 20-month period, and a seasonal development pattern was observed in both hosts.;This study experimentally demonstrated the alternative host status of 7 plant species, of which 6 plants were 1st time reported in the world. The optimized PMA-qPCR provides an accurate way to determine LBG in hosts of Las, which should benefit various HLB research and serve as a crucial component in HLB management.