A New Liberibacter Species, Candidatus Liberibacter americanus sp. nov., is Associated with Citrus Huanglongbing (Greening Disease) in São Paulo State, Brazil

Abstract

Candidatus Liberibacter africanus and Ca. L. asiaticus are α-Proteobacteria , responsible for citrus huanglongbing (HLB) in Africa and Asia, respectively. Until 2004, the disease had never been reported on the American continent. However, South, Central, and North America (Argentina, Brazil, Venezuela, the Caribbean basin, Florida, Texas, Yucatan) harbour Diaphorina citri , the Asian psyllid vector of Ca. L. asiaticus. In early 2004, leaf and fruit symptoms resembling those of HLB were observed in several sweet orange orchards in the Araraquara area of Sao Paulo State (SPS). Shoots with affected leaves were yellowish. Leaves showed characteristic blotchy mottle symptoms. Fruits were small and lopsided, had strong color inversion and contained many aborted seeds. Symptomatic and symptomless sweet orange leaves were analyzed for the presence of the HLB-liberibacters by PCR using the HLB-specific primers OA1+OI1/OI2c. None of the symptomatic leaf samples gave a positive PCR amplification, while liberibacter-infected citrus leaves from the Bordeaux HLB collection, tested positive. These results suggested the occurrence of a new bacterial HLB-agent in SPS. When universal primers fD1/rP1 for the amplification of the bacterial 16S rDNA were used, all symptomatic leaf samples, but none of the symptomless ones, yielded the same 16S rDNA amplification product, indicating the presence of a bacterium in the symptomatic leaves. This 16S rDNA product was cloned and sequenced, and its sequence compared with those of Ca. L. africanus and Ca. L. asiaticus, and other bacteria. The 16S rDNA sequence of the new bacterium (SPS-HLB bacterium) possessed all the oligonucleotide signatures characteristic of the liberibacters, but on the phylogeny tree, it did not cluster within the Ca. L. africanus/ Ca. L. asiaticus group, and formed instead a separate branch. The 16S/23S ribosomal intergenic region (RIR) of the SPS-HLB was also amplified, cloned and sequenced, and compared to the RIRs of other liberibacters. All tested strains of Ca. L. asiaticus had identical or almost identical RIR sequences (99% to 100% sequence identity). However, the SPS-HLB bacterium and Ca. L. asiaticus or Ca. L. africanus had less than 80% sequence identity. For these reasons , we propose that the SPS-HLB bacterium is a member of the genus Candidatus Liberibacter, but sufficiently different from known liberibacter species to warrant a new species designation as Candidatus Liberibacter americanus sp. nov.