A rapid multiplication system for'Candidatus Liberibacter asiaticus'through regeneration of axillary buds in vitro

‘Candidatus Liberibacter asiaticus(CLas)’,which causes citrus Huanglongbing(HLB)disease,has not been successfully cultured in vitro to date.Here,a rapid multiplication system for CLas was established through in vitro regeneration of axillary buds from CLas-infected‘Changyecheng’sweet orange(Citrus sinensis Osbeck).Stem segments with a single axillary bud were cultured in vitro to allow CLas to multiply in the regenerating axillary buds.A high CLas titer was detected in the regenerated shoots on an optimized medium at 30 days after germination(DAG).This titer was 28.2-fold higher than in the midribs from CLas-infected trees growing in the greenhouse.To minimize contamination during in vitro regeneration,CLas-infected axillary buds were micrografted onto seedlings of‘Changyecheng’sweet orange and cultured in a liquid medium.In this culture,the titers of CLas in regenerated shoots rapidly increased from 7.5×10^(4)to 1.4×10^(8)cellsμg^(-1)of citrus DNA during the first 40 DAG.The percentages of shoots with>1×10^(8)CLas cellsμg^(-1)DNA were 30 and 40%at 30 and 40 DAG,respectively.Direct tissue blot immunoassay(DTBIA)indicated that the distribution of CLas was much more uniform in regenerated plantlets than in CLas-infected trees growing in the greenhouse.The disease symptoms in the plantlets were die-back,stunted growth,leaf necrosis/yellowing,and defoliation.The death rate of the plantlets was 82.0%at 60 DAG.Our results show that CLas can effectively multiply in citrus plantlests in vitro.This method will be useful for studying plant-HLB interactions and for rapid screening of therapeutic compounds against CLas in citrus.

Limited infection by ‘Candidatus Liberibacter asiaticus’ in ‘Valencia’ sweet orange trees in the presence of Citrus tristeza virus

Huanglongbing (HLB) is the most destructive disease of citrus and is associated with ‘Candidatus Liberibacter asiaticus’(CLas), a member of the α-proteobacteria. Citrus tristeza virus (CTV) is another pathogen of citrus with very great historic as well as current importance. Both CLas and CTV are phloem-restricted pathogens. A severe CTV isolate, CTV-B6, and CLas-B232 induce a group of symptoms of phloem dysfunction that overlap, but the mild isolate CTV-B2 does not cause any loss to commercial trees. Prior inoculation and establishment of CLas-B232 did not affect subsequent establishment of either CTV-B2 or CTV-B6, while super infection by CLas-B232 was reduced by prior establishment of CTV-B2 and to a lesser extent by prior infection with CTV-B6. Trees co-infected with CTV-B6 and CLas-B232 developed more severe symptoms, typical of CTV-B6, than either of the two pathogens co-infected with CTV-B2. In this study, we confirmed that CLas established in the rootlets earlier and with higher concentration than in leaves. The distribution of CLas in the plant infected by CLas-B438 alone and with CTV-B2 fits a previously proposed model but CLas was more sporadically distributed in a plant co-infected by CLas and CTV-B2 than in a plant infected by CLas alone. These biological phenomena are aligned with previously analyzed transcriptome data and the study provides a novel idea that mild CTV strains may provide some protection against CLas by limiting its multiplication and spread. The protective effect may be due to opposite regulation of key host defense pathways in response to CTV-B2 and CLas-B438.