Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/30/2008
Publication Date: 12/1/2008
Citation: Duan, Y., Sun, X., Zhou, L., Gabriel, D., Benyon, L.S., Gottwald, T.R. 2008. Bacterial brown leaf spot of citrus, a new disease caused by Burkholderia andropogonis. Plant Disease. 93:607-614. Interpretive Summary: This is the first report of bacterial brown leaf spot (BBLS) of citrus, a new bacterial disease found in Florida. Typical symptoms of the disease are flat, circular and brownish lesions with water-soaked margins surrounded by a chlorotic halo, and some of them displayed canker-like symptoms. The phenotypic similarities with citrus canker led to samples being collected by field inspectors and tested as canker suspects. PCR and ELISA screening using X .citri pv. citri-specific primers and antibody (data not shown) revealed that the causal agent was not Xanthomonas and, therefore, the causal agent was not related to bacteria normally associated with citrus canker disease. All results confirmed the causal agent of the disease belongs to Burkholderia andropogonis. Both Biolog metabolic profiles and the FAME similarity indices identified Burkholderia andropogonis as the causal agent. A score of 0.8 is considered an exact FAME match and the 6 isolates tested had cellular fatty acid composition indices that ranged from 0.692 to 0.934. 16S rDNA sequence analysis of two representative isolates of BBLS from Florida citrus (6269 and 6369) revealed greater than 99% identities to previously reported nucleotide sequences of the 16S rDNA gene from B. andropogonis (DQ123835). Our B. andropogonis-specific PCR primers amplified the gyrB and rpoD genes in all thirteen isolates and sequence analysis of these isolates found them to be over 99% homologous to the previously reported sequences of these two genes of B. andropogonis, respectively (AB190572; AB190669). Sequence analysis of 16S rDNA, gyrB and rpoD did not differentiate the BBLS isolates, even those that elicited different host responses. However, when using the DG04 invert repeat primer, we obtained different PCR products from these isolates. Primer DG04 was designed for genetic typing of X. citri pv. citri based on the terminal inverted repeat sequences that flanks the pathogenicity gene pthA. Four of the eleven isolates examined shared a similar pattern of PCR products and these same isolates were found to be more pathogenic to citrus. When inoculated at high concentrations they caused more obvious canker-like symptoms than the other seven isolates. Sequence analysis of DNA fragments from isolates with different PCR patterns revealed the presence of a phage-integrase-like protein, and an ABC transporter-like protein from isolate 6269, and a putative iron-sulfur-binding reductase-like protein from isolate 6177. The different genetic elements present within these PCR products revealed genetic diversity among these isolates. Future sequencing and characterization of B. andropogonis citrus BBLS strains may reveal their effector gene(s) response for these pathogenic variations.
Technical Abstract: A new bacterial disease of citrus was recently identified in Florida and named as bacterial brown leaf spot (BBLS) of citrus. BBLS-infected citrus displayed flat, circular and brownish lesions with water-soaked margins surrounded by a chlorotic halo on leaves. Based on Biolog carbon source metabolic “fingerprinting”, fatty acid analysis, and sequence analysis of partial 16S rDNA, gyrB and rpoD genes, the causal agent of the disease was identified as Burkholderia andropogonis. The pathogenicity of the bacterial isolates was tested by various inoculation methods on three species of citrus as well as on carnation, corn and sorghum. All isolates infected carnation, corn and sorghum with varying degrees of pathogenicity. Significant pathogenic variations were also observed in citrus, ranging from a hypersensitive response to the appearance of canker-like lesions, when leaves were infiltrated with high (108 CFU/ml) concentrations of the bacteria. When the inoculum concentration was low (106 CFU/ml) necrotic spots or small lesions were elicited. In citrus, B. andropogonis grew relatively slowly, indicating that this wide host range bacterium can be a weak pathogen of citrus, but with strain variation. Specific primers targeting gyrB and rpoD genes of B. andropogonis were also developed.