Citrus plants affected by Huanglongbing host multiple populations of Candidatus Liberibacter asiaticus

Huanglongbing (HLB) is an infectious disease of citrus trees. The phloem-limited Candidatus Liberibacter asiaticus is associated with HLB. We studied microbial populations in bark and leaf midrib tissues of infected citron and mandarin trees in China. The presence of Ca. Liberibacter was quantitatively assessed via qPCR. The sample DNA was used for whole-community analysis using pyrosequencing of the 16S rDNA region. Contrary to the expectations, qPCR indicated that barks were more heavily colonized that leaf midribs. Whole-community analysis indicated that citrus and mandarin trees shared a large portion of their bacterial populations. Bacterial communities associated with bark and midrib were also highly similar. Despite the similarities, samples from the different species and tissues could be grouped in a Principal Coordinates Analysis plot generated using UniFrac distances. Using pyrosequencing we deteced Ca. Liberibacter sequences in all samples. We noted that the relative frequency of sequences under the order Rhizobiales was constant and unaffected by the variables considered in this work. Increased numbers of Ca. Liberibacter corresponded to fewer sequences assigned to other families in the Rhizobiales (chiefly Methylocystaceae and Methylobacterium). Four separate OTUs were classified as Ca. Liberibacter. The majority of samples hosted multiple (2-3) OTUs of Ca. Liberibacter, and the control sample (whose bacterial microfauna was dominated by Ca. Liberibacter) hosted all four OTUs of the pathogen. Our findings show that during development of HLB disease, citrus trees harbor multiple populations of Ca. Liberibacter, and raise new questions on their significance for the ecology and pathogenesis of this bacterium