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CLas Genomics

The complete genome sequence of CLas has been obtained by using DNA extracted from a single psyllid carrying a high titer of CLas (Duan et al., 2009). Metagenomic analysis of DNA from phloem of CLas-infected citrus revealed more than one CLas genome per phloem cell and only trace representation of any other bacterial cell (Tyler et al., 2009). Although the analysis was not capable of revealing viruses, the result suggests very strongly that CLas alone is the causal agent of HLB in Florida. Recent work by Wulff et al. (2009) and Duan et al. (2009) have shown that the genomes of both Clam and CLas appear to be circular with 3 ribosomal ribonucleic acid (rRNA) operons and similar size. Analysis of the Clas genome has revealed many other properties of the organism, particularly the absence of pathogenicity systems involving toxins, enzymes or specialized secretion systems. The absence of such specialized secretion systems (referred to as Type III secretion systems or T3SS), which are common in Gram-negative bacteria that are pathogenic to humans, animals, insects, and plants, had led to speculation that other mechanisms of pathogenicity may be involved (Bové and Garnier, 2003). The gene for a bacteriophage DNA polymerase, discovered in 1993, has now been shown to be part of a bacteriophage DNA genome associated with HLB on citrus (Gabriel and Zhang, 2009). These results raise the question of a possible role of liberibacter phages in HLB. Sequencing also has revealed genetic diversity among CLas isolates collected in Southeast Asia (Tomimura et al., 2009).

Obtaining the CLas genome sequence has added to our knowledge of HLB, but how this information can contribute to HLB mitigation remains to be demonstrated. The genomes of several other bacterial plant pathogens have been sequenced. These include the genomes of Xylella fastidiosa, causal organism of citrus variegated chlorosis (and Pierce’s disease of grapevine), Xanthomonas axonopodis pv. citri, causal organism of citrus canker, and Spiroplasma citri, causal agent of citrus stubborn disease. These bacteria have been cultured on synthetic media. The genomes of several phytoplasmas, which have not been cultured, have also been completed. In each case new candidate pathogenicity genes have been discovered and in some cases those genes have been functionally confirmed to contribute to pathogenicity. An interesting example is the onion yellows phytoplasma. Its genome sequence revealed that it possesses specific genes that allow it to send protein molecules out of the sieve tubes to carry out functions beneficial to the phytoplasma. This protein, called Tengu, is believed to inhibit an auxin-related pathway which affects plant development (Hoshi et al., 2009).

CLas Culture

When HLB was first detected in Florida in 2005, the three citrus liberibacters, CLas, Candidatus Liberibacter africanus (CLaf), and Candidatus Liberibacter americanus (Clam), were known, but no evidence indicates any agent for HLB in Florida other than CLas (Tyler et al., 2009). In 2008, co-cultivation of the Asian HLB-associated bacterium (CLas) with Gram-positive actinobacteria was reported by a research group in Florida, but a pure culture of CLas could not be obtained (Davis et al., 2008). In 2009, a report on cultivation of all three HLB-associated Liberibacters and fulfillment of Koch’s postulates was published by Sechler et al. (2009). However, neither the Sechler et al. (2009) result or previous reports of successful axenic



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