Symptoms of huanglongbing (HLB) were first reported in Sao Paulo state in Brazil in 2004. Leaves with blotchy mottle symptoms characteristic of HLB were tested using PCR primers specific for the HLB 16S rRNA; however, the majority of samples tested negative for HLB. Using primers, universal for prokaryotic 16S rRNA, evidence of infection by a bacterium was obtained. Sequence analysis and phylogeny studies were then used to determine that the amplified gene possessed the oligonucleotide and secondary loop structures of the alpha-Proteobacteria which includes the liberibacters, the group to which the HLB pathogens belong. Gene sequence similarities of 98.4 and 96.0% to 'Ca. L. asiaticus' and 'Ca. L. africanus', respectively, were obtained. This lower similarity was reflected in the phylogenetic tree where the new bacterium did not cluster with the other HLB liberibacters, but in a separate branch. Sequence similarity of the 16S/23S intergenic region between the new bacterium and 'Ca. L. asiaticus' and 'Ca. L. africanus' were 66.0 and 79.5%, respectively. As sequence similarity with Liberibacter species does not vary greatly, this confirmed that the new bacterium is a novel species for which the name 'Candidatus Liberibacter americanus' is proposed (Texeira et al., 2005a).
The bacteria causing Huanglongbing are restricted to the sieve tubes of the phloem vessels. Electron microscopy studies reveal it possesses the characteristic double membrane cell envelope of the liberibacters (Garnier et al., 1984; Texeira et al., 2005a).
The distribution in this summary table is based on all the information available. When several references are cited, they may give conflicting information on the status. Further details may be available for individual references in the Distribution Table Details section which can be selected by going to Generate Report.
Initial symptoms appear as leaf mottling and chlorosis often occurring in one shoot or sector of the tree. Later leaf symptoms resemble nutritional deficiencies (Zn, Ca and N). Later stages of the disease can cause severe damage to citrus plants including twig dieback, decline and death (Coletto-Filho et al., 2004). The disease frequently produces small, lopsided fruit with aborted seeds (Coletta-Filho et al., 2005).
The American liberibacter can be detected by the PCR amplification of the 16S rRNA gene in Diaphorina citri, the Asian citrus psyllid suggesting that this insect is the vector of the disease. It can also be transmitted by graft inoculations under greenhouse conditions. Inoculated seedlings displayed the typical blotchy mottle symptoms on the foliage. Electron microscopy studies of the organism confirm that it is restricted to the sieve tubes of the phloem tissue and possesses the characteristic double membrane cell envelope of the liberibacters (Garnier et al., 1984; Texeira et al., 2005a). The organism could not be cultured and specific PCR primers for the disease have been developed (Coletta-Filho et al., 2005; Texeira et al., 2005a).
Two PCR methods have been developed to detect the disease, both target the 16S rDNA region of the causal agent. Total DNA is first extracted from leaf midribs.
Primers GB1 (aag tcg agc gag tac gca agt act) and GB3 (cca act taa tga tgg caa ata tag) with reaction conditions of 35 cycles each at 94°C for 45 s, 64°C for 45 s and 72°C for 60 s. These primers lead to a 1027 bp amplicon (Texeira et al., 2005b).
The second lot of primers target what is considered by the authors to be the South American Liberibacter. PCR primers, LSg2f (TTAAGTTAGAGGTGAAATCC) and LSg2r (CAACTTAATGATGGCAAATA) generate a 545 bp amplicon. PCR reactions are performed in 25 µl reaction mixes containing (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2) 2 mM each DNTP, 1 unit of Taq DNA polymerase, 5 µl of DNA extract and 20 ng of each primer. PCR reaction conditions consisted of a first step at 94°C for 3 min followed by 36 cycles of a denaturation step at 94°C for 30 s, an annealing step of 55°C for 45 s, extension at 72°C for 45 s and a final extension of 10 min at 72°C. The amplified DNA was analysed in agarose gel electrophoresis and stained with ethidium bromide. The presence of 545 bp amplicon indicates the presence of the causal agent of the disease (Coletta-Filho et al., 2005).
A diagnostic protocol for Ca. Liberibacter africanus, Ca. Liberibacter americanus and Ca. Liberibacter asiaticus and for their detection in their psyllid vectors Diaphorina citri and Trioza erytreae has been published by EPPO (2014). The protocol involves detection based on the disease symptoms and molecular tests (PCR), and reporting and documentation.
Huanglongbing is difficult to recognize due to symptoms of the disease resembling those of other citrus disorders (see Symptoms). If suspected, the presence of the disease should be confirmed by identifying the bacterium by PCR or electron microscopy.
Disease symptoms are almost identical and can be confused with those of the other strains of Ca. Liberibacter causing HLB. Mixed infections of two of the strains have been reported (Coletta-Filho et al., 2005). Leaf symptoms also resemble nutrient deficiencies, particularly zinc, calcium and nitrogen.
Coletta-Filho H, Takita M, Targon M and Machado M, 2005. Analysis of 16S rDNA sequences from citrus Huanglongbing bacteria reveal a different ’Ca. Liberibacter’ strain associated with citrus disease in São Paulo. Plant Disease, 89:848852
Coletta-Filho H, Targon M, Takita M, De Negri J, Pompeu J, Machado M, 2004. First report of the causal agent of Huanglongbing (’Candidatus Liberibacter asiaticus’) in Brazil. Plant Disease, 88:12
Texeira D, Saillard C, Eveillard S, Danet J, da Costa P, Ayres A, Bove J, 2005. ’Candidatus Liberibacter americanus’, associated with citrus Huanglongbing (greening disease) in São Paulo State, Brazil. International Journal of Systematic and Evolutionary Microbiology, 55:1875-1862