title: 'Candidatus Liberibacter americanus', associated with citrus huanglongbing (greening disease) in São Paulo State, Brazil.
abstract: Symptoms of huanglongbing (HLB) were reported in São Paulo State (SPS), Brazil, in March 2004. In Asia, HLB is caused by 'Candidatus Liberibacter asiaticus' and in Africa by 'Candidatus Liberibacter africanus'. Detection of the liberibacters is based on PCR amplification of their 16S rRNA gene with specific primers. Leaves with blotchy mottle symptoms characteristic of HLB were sampled in several farms of SPS and tested for the presence of liberibacters. 'Ca. L. asiaticus' was detected in a small number of samples but most samples gave negative PCR results. Therefore, a new HLB pathogen was suspected. Evidence for an SPS-HLB bacterium in symptomatic leaves was obtained by PCR amplification with universal primers for prokaryotic 16S rRNA gene sequences. The amplified 16S rRNA gene was cloned and sequenced. Sequence analysis and phylogeny studies showed that the 16S rRNA gene possessed the oligonucleotide signatures and the secondary loop structure characteristic of the alpha-Proteobacteria, including the liberibacters. The 16S rRNA gene sequence phylogenetic tree showed that the SPS-HLB bacterium clustered within the alpha-Proteobacteria, the liberibacters being its closest relatives. For these reasons, the SPS-HLB bacterium is considered a member of the genus 'Ca. Liberibacter'. However, while the 16S rRNA gene sequences of 'Ca. L. asiaticus' and 'Ca. L. africanus' had 98.4% similarity, the 16S rRNA gene sequence of the SPS-HLB liberibacter had only 96.0% similarity with the 16S rRNA gene sequences of 'Ca. L. asiaticus' or 'Ca. L. africanus'. This lower similarity was reflected in the phylogenetic tree, where the SPS-HLB liberibacter did not cluster within the 'Ca. L asiaticus'/'Ca. L. africanus group', but as a separate branch. Within the genus 'Candidatus Liberibacter' and for a given species, the 16S/23S intergenic region does not vary greatly. The intergenic regions of three strains of 'Ca. L. asiaticus', from India, the People's Republic of China and Japan, were found to have identical or almost identical sequences. In contrast, the intergenic regions of the SPS-HLB liberibacter, 'Ca. L. asiaticus' and 'Ca. L. africanus' had quite different sequences, with similarity between 66.0 and 79.5%. These results confirm that the SPS-HLB liberibacter is a novel species for which the name 'Candidatus Liberibacter americanus' is proposed. Like the African and the Asian liberibacters, the 'American' liberibacter is restricted to the sieve tubes of the citrus host. The liberibacter could also be detected by PCR amplification of the 16S rRNA gene in Diaphorina citri, the psyllid vector of 'Ca. L. asiaticus', suggesting that this psyllid is also a vector of 'Ca. L. americanus' in SPS. 'Ca. L. americanus' was detected in 216 of 218 symptomatic leaf samples from 47 farms in 35 municipalities, while 'Ca. L. asiaticus' was detected in only 4 of the 218 samples, indicating that 'Ca. L. americanus' is the major cause of HLB in SPS.
authors.affiliation: Instituto de Química - UNESP, R. Francisco Degni, s/n, CEP 14800-900, Araraquara, SP, Brazil.|Fundecitrus, Av. Dr. Adhemar Pereira de Barros, 201, CEP 14807-040, Araraquara, SP, Brazil.
authors.forename: Diva do Carmo
authors.initials: DDC
authors.lastname: Teixeira
authors.affiliation: UMR 1090, Institut National de la Recherche Agronomique et Université Victor Segalen Bordeaux 2, BP 81, 33883 Villenave d'Ornon cedex, France.
authors.forename: Colette
authors.initials: C
authors.lastname: Saillard
authors.affiliation: UMR 1090, Institut National de la Recherche Agronomique et Université Victor Segalen Bordeaux 2, BP 81, 33883 Villenave d'Ornon cedex, France.
authors.forename: Sandrine
authors.initials: S
authors.lastname: Eveillard
authors.affiliation: UMR 1090, Institut National de la Recherche Agronomique et Université Victor Segalen Bordeaux 2, BP 81, 33883 Villenave d'Ornon cedex, France.
authors.forename: Jean Luc
authors.initials: JL
authors.lastname: Danet
authors.affiliation: Instituto de Química - UNESP, R. Francisco Degni, s/n, CEP 14800-900, Araraquara, SP, Brazil.
authors.forename: Paulo Inácio da
authors.initials: PID
authors.lastname: Costa
authors.affiliation: Fundecitrus, Av. Dr. Adhemar Pereira de Barros, 201, CEP 14807-040, Araraquara, SP, Brazil.
authors.forename: Antonio Juliano
authors.initials: AJ
authors.lastname: Ayres
authors.affiliation: UMR 1090, Institut National de la Recherche Agronomique et Université Victor Segalen Bordeaux 2, BP 81, 33883 Villenave d'Ornon cedex, France.
authors.forename: Joseph
authors.initials: J
authors.lastname: Bové
mesh_terms: D001938:Brazil; D002957:Citrus / Q000382:microbiology*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000032:analysis; D021903:DNA, Ribosomal Spacer / Q000032:analysis; D008969:Molecular Sequence Data; D010802:Phylogeny; D010935:Plant Diseases / Q000382:microbiology*; D018515:Plant Leaves / Q000382:microbiology; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012338:RNA, Ribosomal, 23S / Q000235:genetics; D012230:Rhizobiaceae / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification / Q000472:pathogenicity; D017422:Sequence Analysis, DNA
chemical_list: D004269:DNA, Bacterial; D021903:DNA, Ribosomal Spacer; D012336:RNA, Ribosomal, 16S; D012338:RNA, Ribosomal, 23S