Title Endophytic Bacterial Diversity in Grapevine (Vitis vinifera L.) Leaves Described by 16S rRNA Gene Sequence Analysis and Length Heterogeneity-PCR
Author Daniela Bulgari1, Paola Casati1, Lorenzo Brusetti2, Fabio Quaglino1, Milena Brasca3, Daniele Daffonchio4, and Piero Attilio Bianco1*
Address 1Dipartimento di Produzione Vegetale (Di.Pro.Ve.) – sez. Patologia Vegetale, Università degli Studi, via Celoria 2, 20133 Milan, Italy, 2Facoltà di Scienze e Tecnologie, Libera Università di Bolzano, Sernesistrasse 1, I-39100, Bozen, Italy, 3CNR - Istituto di Scienze delle Produzioni Alimentari, via Celoria 2, 20133 Milan, Italy, 4Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche (DISTAM), Università degli Studi, via Celoria 2, 20133 Milan, Italy
Bibliography Journal of Microbiology, 47(4),393-401, 2009,
Key Words Vitis vinifera L., plant endophytes, 16S rRNA gene, length heterogeneity-PCR, bacterial enrichment
Abstract Diversity of bacterial endophytes associated with grapevine leaf tissues was analyzed by cultivation and cultivation-independent methods. In order to identify bacterial endophytes directly from metagenome, a protocol for bacteria enrichment and DNA extraction was optimized. Sequence analysis of 16S rRNA gene libraries underscored five diverse Operational Taxonomic Units (OTUs), showing best sequence matches with gamma-Proteobacteria, family Enterobacteriaceae, with a dominance of the genus Pantoea. Bacteria isolation through cultivation revealed the presence of six OTUs, showing best sequence matches with Actinobacteria, genus Curtobacterium, and with Firmicutes genera Bacillus and Enterococcus. Length Heterogeneity-PCR (LH-PCR) electrophoretic peaks from single bacterial clones were used to setup a database representing the bacterial endophytes identified in association with grapevine tissues. Analysis of healthy and phytoplasma- infected grapevine plants showed that LH-PCR could be a useful complementary tool for examining the diversity of bacterial endophytes especially for diversity survey on a large number of samples.