Bacterial diversity analysis of Huanglongbing pathogen-infected citrus, using PhyloChip arrays and 16S rRNA gene clone library sequencing

Abstract

The bacterial diversity associated with citrus leaf midribs was characterized for citrus groves that contained the Huanglongbing (HLB) pathogen, which has yet to be cultivated in vitro. We employed a combination of high-density phylogenetic 16S rRNA gene microarrays and 16S rRNA gene clone library sequencing to determine the microbial community composition for symptomatic and asymptomatic citrus midribs. Our results revealed that citrus leaf midribs can support a diversity of microbes. PhyloChip analysis indicated that 47 orders of bacteria in 15 phyla were present in the citrus leaf midribs, while 20 orders in 8 phyla were observed with the cloning and sequencing method. PhyloChip arrays indicated that nine taxa were significantly more abundant in symptomatic midribs than in asymptomatic midribs. "Candidatus Liberibacter asiaticus" was detected at a very low level in asymptomatic plants but was over 200 times more abundant in symptomatic plants. The PhyloChip analysis results were further verified by sequencing 16S rRNA gene clone libraries, which indicated the dominance of "Candidatus Liberibacter asiaticus" in symptomatic leaves. These data implicate "Candidatus Liberibacter asiaticus" as the pathogen responsible for HLB disease.

FIG. 1.

Agarose gel electrophoresis of PCR products amplified using primers specific for “Ca. Liberibacter asiaticus.” Specific primers A2 and J5 target the 16S rRNA gene of “Ca. Liberibacter asiaticus,” resulting in a 703-bp amplicon (24). Total DNA extracted from midribs of symptomatic and asymptomatic leaves of sweet orange trees were used as templates for PCR amplification. Lanes 1 to 6, symptomatic leaf midribs; lanes 7 to 12, asymptomatic leaf midribs; lane M, DNA molecular weight markers. (Upper panel) Grove 1; (lower panel) grove 2.

FIG. 2.

Mean hybridization scores (hybe score) for 9 of 117 taxa detected in the leaf midrib microbial community. These nine taxa were significantly different (P < 0.05) for the symptomatic and asymptomatic leaves in each grove. The error bars indicate standard errors.

FIG. 3.

Prevalence of “Ca. Liberibacter asiaticus” in clone libraries for asymptomatic and symptomatic trees in each of the two groves sampled.