Publications

4470

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.

Summary The anaerobic, mesophilic and moderately halophilic strain L21‐Spi‐D4 T was recently isolated from the suboxic zone of a hypersaline cyanobacterial mat using protein‐rich extracts of Arthrospira (formerly Spirulina ) platensis as substrate. Phylogenetic analyses based on 16S rRNA genes indicated an affiliation of the novel strain with the Bacteroidetes clade MgMjR‐022, which is widely distributed and abundant in hypersaline microbial mats and heretofore comprised only sequences of uncultured bacteria. Analyses of the complete genome sequence of strain L21‐Spi‐D4 T revealed a possible specialization on the degradation of cyanobacterial biomass. Besides genes for enzymes degrading specific cyanobacterial proteins a conspicuous transport complex for the polypeptide cyanophycin could be identified that is homologous to typical polysaccharide utilization loci of Bacteroidetes . A distinct and reproducible co‐occurrence pattern of environmental 16S rRNA gene sequences of the MgMjR‐022 clade and cyanobacteria in the suboxic zone of hypersaline mats points to a specific dependence of members of this clade on decaying cyanobacteria. Based on a comparative analysis of phenotypic, genomic and ecological characteristics we propose to establish the novel taxa Salinivirga cyanobacteriivorans gen. nov., sp. nov., represented by the type strain L21‐Spi‐D4 T , and Salinivirgaceae fam. nov., comprising sequences of the MgMjR‐022 clade.