The characterization and diversity of bacterial endophytes of grapevine

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Abstract
The diversity of culturable and nonculturable bacterial endophytes of grapevine ( Vitis vinifera L.) was examined using a combination of cultivation and molecular methods. Entire grapevines were sampled to characterize bacterial diversity from different locations throughout the vine. Gas chromatography of fatty acid methyl esters (FAMEs) was used to identify culturable isolates prior to subsequent further microbiological characterization, whilst denaturing gradient gel electrophoresis (DGGE) was used to profile the ribosomal DNA of the bacterial endophyte community extracted from grapevines. Gas chromatography of FAMEs identified 75% of culturable bacterial endophytes to genus level (similarity index >0.3). Many isolates were identified as Bacillus spp., Pseudomonas spp., and Curtobacterium spp. Additionally, actinomycetes are reported for the first time as endophytes of grapevines, with a number of isolates identified as Streptomyces spp. DGGE was successfully used to identify major bands present in samples and indicated a degree of homogeneity of bacterial endophyte community profiles within the grapevines sampled. The major bacterial bands were sequenced and used in identification. Comparison with bacterial markers produced from cultured bacterial endophytes suggested that bacteria in the DGGE profiles were not the species most commonly cultured. Additional research demonstrated similarities between epiphytic and endophytic populations and examined potential entry vectors. Endophyte entry was demonstrated in both field-grown and potted grapevines (‘Chardonnay’) using a rifampicin-resistant Bacillus cereus mutant. The possibility of grapevine epiphytes becoming endophytes, if the opportunity arises, was supported by comparison of gas chromatography of FAMEs from epiphytic and endophytic populations. This research adds grapevine bacterial endophyte communities to those that have been characterized by a multifaceted approach.