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Applied and Environmental Microbiology, March 2009, p. 1566-1574, Vol. 75, No. 6
0099-2240/09/$08.00+0     doi:10.1128/AEM.02404-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Bacterial Diversity Analysis of Huanglongbing Pathogen-Infected Citrus, Using PhyloChip Arrays and 16S rRNA Gene Clone Library Sequencing{triangledown} ,{dagger}

Uma Shankar Sagaram,1,{ddagger} Kristen M. DeAngelis,2,{ddagger} Pankaj Trivedi,1 Gary L. Andersen,2 Shi-En Lu,3 and Nian Wang1*

Citrus Research and Education Center, Department of Microbiology and Cell Science, University of Florida, IFAS, 700 Experiment Station Road, Lake Alfred, Florida 33850,1 Lawrence Berkeley National Laboratory, Earth Sciences Division, One Cyclotron Road MS-70A3317, Berkeley, California 94720,2 Department of Entomology and Plant Pathology, Mississippi State University, Mississippi State, Mississippi 397623

Received 20 October 2008/ Accepted 6 January 2009

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.

* Corresponding author. Mailing address: Citrus Research and Education Center, Department of Microbiology and Cell Science, University of Florida/IFAS, 700 Experiment Station Road, Lake Alfred, FL 33850. Phone: (863) 956-1151. Fax: (863) 956-4631. E-mail: nianwang{at}crec.ifas.ufl.edu

{triangledown} Published ahead of print on 16 January 2009.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

{ddagger} U.S.S. and K.M.D. contributed equally to this work.

Applied and Environmental Microbiology, March 2009, p. 1566-1574, Vol. 75, No. 6
0099-2240/09/$08.00+0     doi:10.1128/AEM.02404-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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