Isolation of “Candidatus Liberibacter” Genes by RAPD and New PCR Detection Technique

The RAPD technique has been extensively applied to reveal polymorphism between closely related genomes. We have used it to isolate genes of the uncultured phloemrestricted bacterium “ Candidatus Liberibacter” sp. of citrus huanglongbing (HLB). Using 102 random 10-mer primers, eight DNA bands were amplified only from HLB-infected plant DNA. These bands were cloned and analyzed. Six of them corresponded to fragments of the liberibacter genome as shown by sequence and hybridization experiments, indicating that the RAPD technique is appropriate to isolate genes from uncultured phloem-restricted plant bacteria. Four genes, nus G, pgm, omp, and an hypothetical protein gene, were identified in the DNA fragments; they will be used for further characterization of the bacterium. A PCR detection method based on the amplification of part of the s operon of the two liberibacter species has been developed. Compared to the previously described PCR detection method in which the 16S ribosomal RNA gene was amplified and which required restriction of the amplified DNA to distinguish between “ Candidatus Liberibacter asiaticus” and “ Candidatus Liberibacter africanus”, the new PCR approach allows direct identification of the two species. In order to characterize the bacterium associated with citrus huanglongbing (HLB), the following genes of the bacterium have been isolated: (i) the highly conserved 16S ribosomal RNA (rRNA) gene obtained by PCR using universal primers (3); (ii) the more variable intergenic 16S/23S region which was also amplified and cloned (5); (iii) three other-HLB DNA fragments were isolated after random cloning of total DNA from infected plants (7). One of these fragments encoded several genes of the wellknown s operon; they corresponded to four ribosomal proteins (L1, L10, L11, and L12) and to the 5’ end of the β subunit of the RNA polymerase gene (8). Sequence analyses of the 16S rDNA and the s operon revealed that the HLB bacterium was a new bacterial genus that we named “ Candidatus Liberibacter” and in which two species could be defined “ Candidatus Liberibacter asiaticus” and “ Candidatus Liberibacter africanus”. This work also resulted in the development of specific and sensitive detection techniques for the liberibacters by DNA/DNA hybridization and PCR (2, 4, 7). However, in order to better characterize the liberibacters, isolation of additional genes is needed. For that purpose, the Random Amplified Polymorphic DNA (RAPD) technique (10) has been carried out on healthy and HLBinfected plant DNA. This method is able to reveal polymorphism in genomic DNA without any prior knowledge on the genome, as this PCR-based technique uses single arbitrary 10-mer primers. Amplification of 16S-rDNA by PCR, allows detection of both liberibacter species. However, in order to distinguish between the two species, digestion of the amplified DNA with restriction enzyme Xba I must be carried out. This step is time consuming and difficult to achieve when little DNA is amplified in a given sample. This is why an alternative PCR method was developed. It is based on the amplification of part of the s operon leading to one size product for “ Candidatus L. asiaticus” and another for “ Candidatus L. africanus”. “ Candidatus Liberibacter” sp. gene isolation using RAPD as well as PCR detection of the two liberibacters with primers A2/J5 selected 364 Fourteenth IOCV Conference, 2000—Short Communications in ribosomal protein genes rpl A and rpl J are presented in this paper. RAPD amplification of DNA from healthy and “ Candidatus Liberibacter”—infected plants . Out of 102 RAPD primers used to amplify DNA extracted from healthy or HLB-infected citrus or periwinkle plants, eight primers revealed additional PCR products on the profiles obtained when DNA from infected plants was used as compared to those obtained with DNA from healthy plants. Figure 1A shows the RAPD profiles obtained with one of these primers (P3). Four bands (P3-Af, P3-As, A17-Af and C13-Af) evidenced with three primers (P3, A17, and C13) were further studied. They were specific of the profiles corresponding to infected plant DNA. Their size were approximately 700 bp, 340 bp, 550 bp and 1 kbp, respectively. Three of them were obtained from plants infected with “ Candidatus L. africanus” and one from plants infected with “ Candidatus L. asiaticus”. Analysis of the selected RAPD products. The selected RAPD fragments were reamplified, radiolabeled and used as probes in dot-blot hybridization against DNA extracted from healthy and infected citrus plants in order to verify that they corresponded to “Candidatus Liberibacter” DNA. In parallel, sequences of the different cloned fragments were determined and compared with those present in data banks. When used as probe under high stringency conditions, fragment P3Af hybridized strongly with DNA from “Candidatus L. africanus”infected citrus plants but not with DNA extracted from healthy or “Candidatus L. asiaticus”-infected plants (Fig. 1B). This indicates that this PCR product is, indeed, DNA of “Candidatus L. africanus”. The sequence of P3 AF (662 bp) had no homology with any of the sequences in the GenBank database. Similarly, P3-As hybridized with DNA from citrus infected with different strains of “Candidatus L. asiaticus” Poona (India), Lipa City (Philippines), Nakom Pathom (Thailand), Taipei (Taiwan) and Fuzhou Fig. 1. A. RAPD profile obtained with primer P3 (A). Amplifications were performed from water (lane 1), DNA from healthy periwinkle (lane 2), and citrus (lane 3); DNA from “Candidatus L. africanus”-infected periwinkle (lane 4) and citrus (lane 5); DNA from “Candidatus L. asiaticus”-infected periwinkle (lane 6) and citrus (lane 7). M: size marker. B. Dot blot hybridization of probe P3Af with DNA extracted from healthy citrus (1), citrus infected with “Candidatus L. africanus” (2), citrus infected with “Candidatus L. asiaticus” from India (3), the Philippines (4), Thailand (5), Taiwan