Huanglongbing (HLB), caused by phloem-limited Candidatus Liberibacter asiaticus (CLas) is the primary limiting factor of production in most citrus regions of the world. After infection CLas is transported systemically throughout the phloem tissues following the source-sink movement. Split-root rhizoboxes and one-side graft inoculation above the split trunk was used to understand if vertical distance of the inoculum source and different anatomical structures (grafted or seedlings trees) can affect the speed of the CLas movement as well as the effects of the seasonality on these movements. The time for CLas to reach the roots was not affected by either distance of the inoculum source or tree type. The season infection period appears to have an important effect on CLas movement. Trees inoculated in the summer had fast and uniform movement (first detection at 4 weeks after inoculation). Plants inoculated in the winter had a slow and uneven movement (first CLas detection at 14 weeks after inoculation). Our results indicate that summer and spring are the seasons of CLas down and lateral movement but is independent of vertical distance of inoculum source or anatomical structures of the plants. The findings from this study aid in the management of HLB in the field as well as improve the methods for CLas detection.
Candidatus Phytoplasma pruni is the causative agent of X-disease on peach (Prunus persica) trees. Infected trees exhibit premature yellowing, leaf necrosis causing a shot-hole appearance, limb dieback, and eventual death. How pathogen infection leads to these symptoms is unknown. This study undertook a modern characterization of the disease by assessing the physiological and transcriptomic consequences of phytoplasma infection. Phytoplasma titer was high in the symptomatic tissues and undetected or at low titer in asymptomatic tissues. Symptomatic leaves had a significant decrease in chlorophyll a, chlorophyll b, and carotenoids. Transcriptomic analysis showed alterations in genes related to phytohormone synthesis and signaling, circadian rhythms, lignification, and sugar synthesis and transport. Several transcripts that may be related to symptom development were identified. Collectively these data give a much clearer picture of symptom development in Ca. P. pruni infected P. persica and provide several avenues of further research in determining how Ca. P. pruni interacts with its host to elicit the observed symptoms.
An updated real-time multiplex quantitative polymerase chain reaction (qPCR) assay was designed and validated for the simultaneous detection of three ‘Candidatus Liberibacter species’ (CLsp), Ca. Liberibacter asiaticus (CLas), africanus (CLaf) and americanus (CLam), associated with the Huanglongbing (HLB) disease of citrus. The multiplex assay was designed based on the previously published qPCR assay by Li et al., 2006, taking into consideration all available CLsp 16S rRNA gene sequences in the GenBank and the MIQE guidelines and workflow for qPCR optimization, which became available after 2006. When using the updated multiplex CLsp qPCR assay compared to singleplex qPCR, no significant increase in Cq values was detected. The specificity and sensitivity of the updated qPCR assay was optimal and measuring the intra and inter assay variations confirmed the reproducibility and repeatability of the assay. The assay was also successfully used with a large number of diverse samples, at independent laboratories in four countries, thus demonstrating its transferability, applicability, practicability, and robustness as different qPCR reaction conditions or instruments had a minor effect on Cq values. This updated multiplex CLsp qPCR assay can be used in a variety of citrus surveys, germplasm, or nursery stock programs that require different pathogen detection tools for their successful operation. Keywords: Citrus greening disease, COX internal DNA control; validation; citrus germplasm; budwood; citrus nursery, citrus survey, regulatory diagnostics, Citrus Clonal Protection Program (CCPP), National Clean Plant Network (NCPN)