Identification of Candidatus Liberibacter asiaticus from Foshou (Citrus medica) in China

In China, Foshou or Buddha's hand (Citrus medica var. sarcodactylus), is commonly cultured and valued for its fragrance and used medicinally as a stomach stimulant, expectorant and tonic. We have observed Huanglongbing (HLB) symptoms on Foshou since 1999 (Fig. 1). Yet, the etiological agent, Candidatus Liberibacter sp., a nonculturable, phloem-limited α-proteobacterium, had never been confirmed. Among the three known candidatus species (Ca. L. asiaticus, Ca. L. africanus, and Ca. L. americanus), only Ca. L. asiaticus has been reported in China (Deng and Tang, 1996) but not from Foshou. In January 2006, we found nine Foshou trees showing typical HLB symptoms (leaf mottling and yellowing in young shoots) in Guangning City, Guangdong Province, People's Republic of China.

To identify the pathogen, DNA was extracted from symptomatic leaves with the cetyl trimethylammonium bromide (CTAB) method. Samples were tested for presence of Ca. L. asiaticus by PCR with primer sets OI1/OI2c targeting the 16S rDNA locus (do Carmo Teixeira et al., 2005) and A2/J5 targeting the locus of beta-operon of ribosomal proteins (Hocquellet et al., 1999). DNA samples from all symptomatic leaves were positive with both primer sets. OI1/OI2c generated an amplicon of approximately 1,160 bp. Further digestion of the amplicon by XbaI yielded two DNA fragments of approximately 640 bp and 520 bp and PCR with A2/J5 primer set generated an approximate 703 bp amplicon, excluding Ca. L. africanus as the causal agent . The positive control using a sweet orange (C. sinensis) tree known to have HLB showed the same result. No DNA was amplified from the asymptomatic healthy Foshou tree. Sequences of OI1/OI2c and A2/J5 amplicons were determined and showed 99.6-100% similarity to those of Ca. L. asiaticus in the GenBank database. No DNA was amplified by PCR with primer set GB1/GB2 specific to Ca. L. americanus. These results show that the Foshou HLB etiological agent is Ca. L. asiaticus. This is the first report of molecular identification of Ca. L. asiaticus associated with C. medica HLB in Guangdong (southern) China.