Plant Science

Huanglongbing (HLB) is a difficult-to-control and highly destructive citrus disease that, in Brazil, is associated mainly with the bacterium ‘Candidatus Liberibacter asiaticus’ transmitted by the psyllid Diaphorina citri. The aim of this study was to improve our understanding of the ‘Ca. L. asiaticus’ infection process by exposing excised, fully expanded, immature citrus leaves in 50-ml Falcon tubes to one, four, or eight adults from a ‘Ca. L. asiaticus’-exposed colony for 1-, 3-, 7-, or 15-day periods for access to inoculation (IAP). The leaves were incubated at 26°C for 1, 3, 7, 15, and 21 days (incubation period [IP]). Infection frequencies and ‘Ca. L. asiaticus’ titers were assessed by quantitative polymerase chain reaction (qPCR). ‘Ca. L. asiaticus’ infection was a function of leaf age, number of insects, IAP, and IP. In general, higher infection rates were observed on younger leaves inoculated with higher numbers of insects and after longer IAP and IP. The immature excised leaf method allowed determination of 3 to 7 days as the range of time required by ‘Ca. L. asiaticus’ to reach qPCR detectable levels. Even though leaf survival could be prolonged by the maintenance of a branch segment at the base of the leaf petiole, leaf degradation, visible after about 15 days IP, did not allow observation of the entire infection process which, in the intact plant, culminates with the appearance of the blotch mottling symptom on leaf blades.

Although ‘Candidatus Liberibacter asiaticus’ (Las) is a major pathogen associated with citrus huanglongbing (HLB), some characteristics of transmission by the psyllid vector Diaphorina citri are not fully understood. We examined the latent period and persistence of transmission of Las by D. citri in a series of experiments at 25°C, in which third-instar psyllid nymphs and 1-week-old adults were confined on infected citrus for an acquisition access period (AAP), and submitted to sequential inoculation access periods (IAPs) on healthy citrus seedlings. The median latent period (LP50, i.e., acquisition time after which 50% of the individuals can inoculate) of 16.8 and 17.8 days for psyllids that acquired Las as nymphs and adults, respectively, was determined by transferring single individuals in 48-h IAPs. Inoculation events were intermittent and randomly distributed over the IAPs, but were more frequent after acquisition by nymphs. A minimum latent period of 7 to 10 days was observed by transferring groups of 10 psyllids in 48-h IAPs, after a 96-h AAP by nymphs. Psyllids transmitted for up to 5 weeks, when submitted to sequential 1-week IAPs after a 14-day AAP as nymphs. The long latent period and persistence of transmission are indirect evidences of circulative propagation of Las in D. citri.

Premise of the study:The search for resistance/tolerance to the devastating citrus huanglongbing disease (syn. HLB or citrus greening) is generating an increasing number of new plants of diverse genetic makeup. As the increasing number of new plants require more space, resources, and time, the need for faster and more efficient HLB screening tests becomes crucial.Methods and Results:The leaf‐disc grafting system described here consists in replacing a disc of leaf tissue with a similar disc from an infected plant. This can be performed in young seedlings not yet big enough to endure other types of grafting. Graft success and infection rates average approximately 80%.Conclusions:We describe the successful adaptation of leaf‐disc grafting as a powerful screening tool for HLB. The system requires minimal plant material and can be performed in seedlings at a very young age with increased efficiency in terms of time, space, and resources.