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Title: Identification, phylogenetic analysis and biological characterization of Serratia marcescens strains causing cucurbit yellow vine disease

Author
item Rascoe, J.e.
item Berg, M.
item Melcher, U.
item Mitchell, F.l.
item Bruton, Benny
item Pair, Sammy
item Fletcher, J.

Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/1/2003
Publication Date: 10/1/2003
Citation: Rascoe, J., Berg, M., Melcher, U., Mitchell, F., Bruton, B.D., Pair, S.D., Fletcher, J. 2003. Identification, phylogenetic analysis and biological characterization of Serratia marcescens strains causing cucurbit yellow vine disease. Phytopathology. 93:1233-1239.

Interpretive Summary: Cucurbit yellow vine disease (CYVD) is caused by a phloem-inhabiting bacterium that was recently isolated onto agar media. Isolation of the bacterium allows a more precise classification of the pathogen. To more clearly describe and identify the CYVD organism, two cucurbit isolates and selected reference isolates were characterized phenotypically using fatty acid profiling (FAME) and substrate utilization tests (BIOLOG and Vitek). To more precisely place the CYVD bacterium phylogenetically, we determined the sequences from two regions of the DNA from two CYVD isolates and eight other bacteria preliminarily characterized as S. marcescens. Phylogenetic trees were generated using DNA sequences of cucurbit yellow vine disease-associated bacterial isolates and eight additional bacterial strains preliminarily characterized as S. marcescens. The phylogenetic placement of the two CYVD strains, W01-A and Z01-A, as S. marcescens by DNA sequencing data is in striking contrast to characterization based on automated BIOLOG and Vitek assays and fatty acid analysis. The latter two tests indicate that the metabolic capabilities and fatty acid profiles of these cucurbit bacteria are much different from those of the type strain of S. marcescens as well as from the reference strains. The disparity in isolate identification results yielded by the different analyses illustrates the difficulty in assigning taxonomic identities from a single test, especially one based on characters other than DNA sequence.

Technical Abstract: A serious vine decline of cucurbits known as yellow vine disease (CYVD) is caused by rod- shaped bacteria that colonize the phloem elements. Sequence analysis of a CYVD-specific PCR amplified 16S rDNA product showed the microbe to be a gamma-proteobacterium related to the genus Serratia. To identify and characterize the bacteria, one isolate each from watermelon and zucchini and several non-cucurbit derived reference strains were subjected to sequence analysis and biological function assays. Taxonomic and phylogenetic placement was investigated by analysis of the groE and 16S rDNA regions, which were amplified by PCR and directly sequenced. For comparison, eight other bacterial strains identified by others as Serratia spp. also were sequenced. These sequences clearly identified the CYVD isolates as Serratia marcescens. However, evaluation of metabolic and biochemical features revealed that cucurbit-derived strains of S. marcescens differ substantially from strains of the same species isolated from other environmental niches. Cucurbit strains formed a distinct cluster, separate from other strains, when their fatty acid methyl ester profiles were analyzed. In substrate utilization assays (BIOLOG, Vitek and API 20E) the CYVD isolates lacked a number of metabolic functions characteristic for S. marcescens, failing to catabolize 25-30 compounds that were utilized by S. marcescens reference strains. These biological differences may reflect gene loss or repression that occurred as the bacterium adapted to life as an intracellular parasite and plant pathogen