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2007 Plant Management Network.
Accepted for publication 20 December 2007. Published 19 April 2007.


Detection of Candidatus Liberibacter asiaticus from Wampee (Clausena lansium Skeels) by Nested PCR


Xiaoling Deng, Gen Zhou, and Huaping Li, Laboratory of Citrus Huanglongbing Research, Department of Plant Pathology, South China Agricultural University, Guangzhou, Guangdong 510642, People's Republic of China; and Jianchi Chen and Edwin L. Civerolo, San Joaquin Valley Agricultural Sciences Center, Crop Diseases, Pests, and Genetics Research Unit, USDA-ARS, Parlier, CA 93648


Corresponding author: Jianchi Chen.  jichen@fresno.ars.usda.gov


Deng, X., Zhou, G., Li, H., Chen, J., and Civerolo, E. L. 2007. Detection of Candidatus Liberibacter asiaticus from wampee (Clausena lansium Skeels) by nested PCR. Online. Plant Health Progress doi:10.1094/PHP-2007-0419-01-BR.


Wampee (Clausena lansium Skeels) is native to southern China and Southeast Asia. Wampee trees are attractive, with grape-like fruits and a muscat taste and are popular in home gardens. Like other members of Rutaceae, wampee has long been suspected to have "yellow shoot" disease or Huanglongbing (HLB) and Diaphorina citri, the disease vector, was capable of a long-term survival on Wampee (2). The importance of wampee HLB is its potential to serve as a source of inoculum of the HLB pathogen, Candidatus Liberibacter spp., for sweet orange, mandarin, and other economically important citrus crops. Yet, the presence of Ca. Liberibacter spp. in wampee has not been confirmed with the 16S rDNA signature sequence that define this unculturable bacterium (1,3).

In August of 2006, we identified two wampee trees showing "yellow shoots" symptoms (Fig. 1A) adjacent to a mandarin orchard with HLB in Luoding City, Guangdong Province, Peoples Republic of China. Symptomatic leaves ranged from mottling and to yellowing (Fig. 1B). Leaf samples were collected, and DNA was extracted using the CTAB (cetyltrimethylammoniumbromide) method (4). DNA samples were assayed by nested-PCR. The general bacterial 16S rDNA primer set fDl/rD1 (AGA GTT TGA TCC TGG CTC AG / AAG GAG GTG ATC CAG CC) (1) was used for the outer round amplification. The PCR reaction (25 l) mixture contained:10 mM Tris-HCl, pH 8.3; 50 mM KCl; 1.5 mM MgCl2; 100 M each dNTPs; 400 mM each primer, 1 U of Taq DNA polymerase and 1 L of template DNA. Amplification was conducted with an initial denature at 96C for 10 min, followed by 10 cycles consisting of: denaturing at 96C for 30 s, annealing at 55C for 30 s, and extension at 72C for 30 s. Two l of the amplicon were then used for inner round amplification using the same procedure but 35 PCR cycles with primer set OI1/OI2c (5' GCG CGT ATG CAA TAC GAG CGG CA 3' / 5' GCC TCG CGA CTT CGC AAC CCA T 3') specific for Ca. L. asiaticus (1,3). The amplified DNAs were resolved through agarose gel electrophoresis.


 

Fig. 1. Huanglongbing symptoms in Clausena lansium: (A) "yellow shoots"; (B) comparison of symptomatic and asymptomatic leaves.


As shown in Fig. 2, a 1.1 kb amplicon was obtained from symptomatic but not asymptomatic leaf samples. Non-nested PCR using primer set OI1/OI2c-only did not yield any detectable DNA bands from either symptomatic or asymptomatic leaves. XbaI digestion yielded two fragments of 520 bp and 640 bp, characteristic to Ca. L. asiaticus. PCR amplicons were further sequenced to be 1,095 bp and shared a > 98% similarity to sequences of Ca. L. asiaticus in the GenBank database. No DNA was amplified with primer set GB1/GB2 (AAG TCG AGC GAG TAC GCA AGT ACT / CCA ACT TAA TGA TGG CAA ATA TAG) specific to Ca. L. americanus (5). We note that nested-PCR is necessary for Ca. L. asiaticus detection in wampee, suggesting low bacterial titer in this host. We recommend that eradication of wampee trees surrounding citrus orchards should be part of the overall management of citrus HLB.


 

Fig. 2. Detection of Candidatus Liberibacter asiaticus by non-nested (using primer set OI1/OI2c-only) and nested PCR (using the general 16S rDNA primer set fDl/rD1 and then primer set OI1/OI2c) and results from Xba I digestion of PCR amplicons.

 

Literature Cited

1. Jagoueix, S., Bove, J. M., and Garnier, M. 1994. The phloem-limited bacterium of greening disease of citrus is a member of the alpha subdivision of the Proteobacteria. Int. J. Syst. Bacteriol. 44:379-386.

2. Koizumi, M., Prommintara, M., and Ohtsu, Y. 1996. Wood apple, Limonia acidissima: A new host for the huanglongbing (greening) vector, Diaphorina citri. Pages 271-275 in: Proc. 13th Conf. of the Intl. Organization of Citrus Virologists. J. V. da Graa, P. Moreno, and R. K.Yokomi, eds. Univ. of California, Riverside.

3. Murray, R. G. E., and Stackebrandt, E. 1995. Taxonomic Note: Implementation of the provisional status Candidatus for incompletely described procaryotes. Int. J. Syst. Bacteriol. 45:186-187.

4. Murray, M. G., and Thompson, W. F. 1980. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res. 8:4321-4324.

5. Teixeira, Ddo C., Luc Danet, J., Eveillard, S., Cristina Martins, E., de Jesus Junior, W. C., Takao Yamamoto, P., Aparecido Lopes, S., Beozzo Bassanezi, R., Juliano Ayres, A., Saillard, C., and Bove, J. M. 2005. Citrus huanglongbing in Sao Paulo State, Brazil: PCR detection of the 'Candidatus' Liberibacter species associated with the disease. Mol. Cell. Probes. 19:173-179.