Raiol-Junior, Laudecir Lemos

Huanglongbing (HLB) is a devastating disease of citrus plants caused by the non-culturable phloem-inhabiting bacterium Candidatus Liberibacter ssp., being Ca. Liberibacter asiaticus (CLas) the most aggressive species. CLas is vectored by the psyllid Diaphorina citri and introduced into sieve cells, establishing a successful infection in all Citrus species. Partial or complete resistance has been documented in the distant relatives Murraya paniculata and Bergera koenigii, respectively, providing excellent systems to investigate the molecular basis of HLB-resistance. It has been shown previously that the first weeks after bacterial release into the phloem are critical for the establishment of the bacterium. In this study, a thorough transcriptomic analysis of young flushes exposed to CLas-positive and negative psyllids has been performed in Citrus × sinensis, as well as in the aforementioned resistant species, along the first eight weeks after exposure. Our results indicate that the resistant species do not deploy a classical immunity response upon CLas recognition. Instead, transcriptome changes are scarce and only a few genes are differentially expressed when flushes exposed to CLas-positive and negative psyllid are compared. Functional analysis suggests that primary metabolism and other basic cellular functions could be rewired in the resistant species to limit infection. Transcriptomes of young flushes of the three species are very different, supporting the existence of distinct biochemical niches for the bacterium. These findings suggest that both intrinsic metabolic inadequacies to CLas survival, as well as inducible reprogramming of physiological functions upon CLas recognition, could orchestrate together restriction of bacterial multiplication in these resistant hosts.

Huanglongbing is a highly destructive citrus disease associated with “Candidatus Liberibacter asiaticus” (Las), a phloem−limited and non-culturable bacterium, naturally transmitted by the psyllid Diaphorina citri. Although diverse approaches have been used to understand the molecular mechanisms involved in the pathogen–host interaction, such approaches have focused on already infected and/or symptomatic plants, missing early events in the initial days post-inoculation. This study aimed to identify the time course of Las multiplication and whole-plant colonization immediately following inoculation by infected psyllids feeding for 2 days. Thus, the experimental approach was to track Las titers after psyllid inoculation in new shoots (NS) of Citrus × sinensis (susceptible), Murraya paniculata (partially resistant), and Bergera koenigii (fully resistant). Soon after psyllid removal, Las titers dropped until the 10–12th days in all three species. Following this, Las titers increased exponentially only in C. × sinensis and M. paniculata, indicating active bacterial multiplication. In C. × sinensis, Las reached a stationary phase at ∼5 log Las cells/g of tissue from the 40th day onward, while in M. paniculata, Las increased at a lower rate of up to ∼3 log Las cells/g of tissue between the 40th and 60th days, decreasing gradually thereafter and becoming undetectable from the 160th day onward. In B. koenigii, Las titers decreased from the start and remained undetectable. In C. × sinensis, an average of 2.6 log of Las cells/g of tissue was necessary for Las to move out of 50% of the NS in 23.6 days and to colonize the rest of the plant, causing a successful infection. Conversely, the probability of Las moving out of the NS remained below 50% in M. paniculata and zero in B. koenigii. To our knowledge, this is the first study on Las dynamics and whole-plant colonization during the earliest stages of infection. Identification of critical time-points for either successful multiplication or Las resistance may help to elucidate initial events of Las–host interactions that may be missed due to longer sampling intervals and at later stages of infection.

‘Candidatus Liberibacter asiaticus’ (Las) is an unculturable, phloem-limited, insect-transmitted bacterium associated with the Asiatic form of huanglongbing (HLB), the most destructive citrus disease. In Asia and the Americas, it is transmitted by the Asian citrus psyllid (Diaphorina citri Kuwavama). Despite considerable research, little is known about the processes involved in plant infection and colonization by Las. This study was conducted to determine whether the basal portion (below girdling) of the plant is an important route for Las to move laterally from a point of inoculation on a branch to pathogen-free branches elsewhere in the canopy, and to quantify the influence of actively growing tissues on vertical upward (acropetally) or downward (basipetally) movement of Las. Nongirdled and fully or partially girdled stems of potted plants of ‘Pera’ sweet orange, graft-inoculated above or below girdling, were sampled in distinct regions and assessed by qPCR, 6 months postinoculation. Las invaded all regions of partially and nongirdled plants but remained restricted to the inoculated regions of fully girdled plants, evidence that in planta bacterium movement is limited to the phloem. In fully girdled plants, starch accumulated above the girdling site, probably because of changes in flow of phloem sap. To study the influence of actively growing tissues, inoculated ‘Valencia’ sweet orange plants were kept intact or were top- or root-pruned to force production of new tissues, and sampled at 15-day intervals. Las migrated rapidly and most predominantly toward newly developing root and leaf tissues. The rapid and predominant movement of Las to newly developed shoots and roots would explain failures of canopy heat treatments and pruning to cure HLB-affected trees, and reinforces the need to protect rapidly growing new shoots from feeding by D. citri in order to minimize transmission and spread of the pathogen by the vector within and between orchards.