Candidatus Liberibacter americanus, associated with citrus huanglongbing (greening disease) in São Paulo State, Brazil

Symptoms of huanglongbing (HLB) were reported in São Paulo State (SPS), Brazil, in March 2004. In Asia, HLB is caused by 'Candidatus Liberibacterasiaticus'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 SIPS 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 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 SIPS.

Citrus greening disease investigation using micro synchrotron radiation X-ray fluorescence in association with soft independent modelling of class analogy (SIMCA)

This study describes the use of micro synchrotron radiation X-ray fluorescence (µSR-XRF) to investigate citrus greening disease in sweet orange (Citrus sinensis) plants. An experiment using healthy plants as control and plants of the same variety infected with Candidatus Liberibacter asiaticus (CLas) was performed to verify variations of the mineral composition of citrus leaves. A µSR-XRF system using the D09B X-ray fluorescence beam line at the Brazilian Synchrotron Light Source (LNLS, Campinas, São Paulo State) was employed for this purpose. The data were analyzed using a chemometric tool called soft independent modelling of class analogy (SIMCA). The promising results from SIMCA models reinforce the evidence that plants infected by citrus greening (both asymptomatic and symptomatic) undergo alterations in their micro- and macronutrient compositions.

Spatial distribution of Diaphorina citri Kuwayama (Hemiptera: Psyllidae) in citrus orchards

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

ransmissão de Candidatus Liberibacter asiaticus por Diaphorina citri em folhas destacadas de citros

Pós-graduação em Agronomia (Produção Vegetal) - FCAV

Distribuição espacial e amostragem seqüencial de ninfas e adultos de Diaphorina citri Kuwayama (Hemiptera: Psyllidae) na cultura de citros

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Distribuição espacial de Huanglongbing (Greening) em citros utilizando a geoestatística

Os objetivos do trabalho foram avaliar a distribuição espacial e a expansão da Huanglongbing (greening) em talhões de citros de uma propriedade agrícola localizada no município de Araraquara-SP, utilizando a geoestatística. Para determinar o número de plantas com greening, foram realizadas inspeções periódicas em intervalos de três meses, no período de março de 2005 a julho de 2007, contando-se, em cada talhão, o número de plantas com os sintomas característicos da doença. Realizou-se a análise descritiva dos dados e, para verificar a distribuição espacial do greening, utilizou-se a geoestatística através do ajuste de semivariogramas e da interpolação dos dados por krigagem. A dependência espacial de plantas com greening apresentou raio de agregação de 300 a 560 m, indicando distribuição agregada da doença. Por meio dos mapas de krigagem, observou-se que o foco inicial de plantas doentes ocorreu nos limites da fazenda, com expansão do greening por toda a área. O intervalo de inspeção de três meses não foi adequado para a redução do greening na fazenda.