The characterization and diversity of bacterial endophytes of grapevine

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.

Impact of metagenomic DNA extraction procedures on the identifiable endophytic bacterial diversity in Sorghum bicolor (L. Moench)

“…Seven plant mDNA extraction protocols were tested; two classical protocols (SDS-or CTAB-based) and five commercial kits (MoBio PowerPlant Pro® DNA Isolation Kit, Qiagen DNeasy R Plant Mini Kit, Fermentas GeneJet Plant Genomic DNA Purification Kit, MoBio PowerSoil™ DNA Purification Kit and MoBio UltraClean® Soil DNA Isolation Kit). These protocols have previously been used to study endophytic bacterial communities (Drabkova et al, 2002;Green et al, 1999;Krechel et al, 2002;West et al, 2010). All kit-based DNA extrac-tion protocols were performed according to the manufacturer's instruc-tions, with the exception that starting plant material quantities were always 0.1 g or 0.3 g, and the final elution was performed in 50 μL buffer for normalization purposes.…”

Fungal Endophytic Communities in Grapevines (Vitis vinifera L.) Respond to Crop Management

“…However, the relation between communities of endophytic fungi and host plants is still poorly studied, and as of yet, is far from being fully understood. In grapevines, recent studies have shed some light upon the bacterial endophytic communities (13,14,20,47,72), while investigations on fungal communities have been rare and often limited to culture-dependent methods (20,30,43,71). Furthermore, research has mainly focused on subsoil plant-microbe associations (36,65).…”

Screening of rhizosphere bacteria from grapevine for their suppressive effect on Xiphinema index Thorne & Allen on in vitro grape plants

“…Investigations have been undertaken to isolate from grapevines the endophytic bacteria present in internal tissues, such as xylem, roots, trunk, cordon and crown, and the epiphytic bacteria in canes and leaves, but no association with either pests or diseases affecting the plants or their incidence on growth have been performed (Bell et al 1995;West et al 2010).…”