‘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 a-Proteobacteria, including the liberibacters. The 16S rRNA gene sequence phylogenetic tree showed that the SPS-HLB bacterium clustered within the a-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.Abbreviations: HLB, huanglongbing; RIR, ribosomal intergenic region; SPS, Sã o Paulo State.The GenBan...

High Incidence of Preharvest Colonization of Huanglongbing-Symptomatic Citrus sinensis Fruit by Lasiodiplodia theobromae (Diplodia natalensis) and Exacerbation of Postharvest Fruit Decay by That Fungus

“…HLB is associated with "Candidatus Liberibacter" spp., which are Gram-negative, phloem-limited bacteria (2,3). The Asian form of HLB is currently present in the United States, and the causal pathogen, bacterium "Candidatus Liberibacter asiaticus," was first confirmed in Southeast Florida in 2005 (1) and now is prevalent in all Florida citrus-growing areas in the state.…”

A New Huanglongbing Species, “ Candidatus Liberibacter psyllaurous,” Found To Infect Tomato and Potato, Is Vectored by the Psyllid Bactericera cockerelli (Sulc)

“…HLB is caused by a phloem-restricted, nonculturable bacterium that belongs to the genus "Candidatus Liberibacter," which belongs to the Alphaproteobacteria (12). Currently, the genus "Candidatus Liberibacter" is composed of three known species that cause disease, primarily in citrus (3): "Candidatus Liberibacter asiaticus" (12,13), "Candidatus Liberibacter africanus" (9,12,13,20), and "Candidatus Liberibacter americanus" (26,27). In this study, we report a new HLB "Candidatus Liberibacter" species which is unique from the other three species because it causes disease in solanaceous plants, such as tomato and potato, and is vectored by the psyllid species Bactericera cockerelli (Sulc).…”

Natural parasitism of Diaphorina citri Kuwayama (Hemiptera, Psyllidae) nymphs by Tamarixia radiata Waterston (Hymenoptera, Eulophidae) in São Paulo orange groves

“…The citrus psyllid, Diaphorina citri Kuwayama 1908 (Hemiptera, Psyllidae), has become the main citrus pest in the state of São Paulo in the past few years, after the introduction of the bacteria associated with huanglongbing (HLB, citrus greening), Candidatus Liberibacter asiaticus and Candidatus Liberibacter americanus (Coletta-Filho et al 2004;Teixeira et al 2005), both transmitted by this insect (Aubert 1987b;Bové 2006). Aggressive chemical control of the vector with the aim of excluding it from citrus groves has been recommended as part of programs to reduce HLB damage.…”