The application of fluorescent single-strand conformation polymorphism (F-SSCP) to assess the diversity of Burkholderia species in sugarcane rhizosphere.
Résumé
The genus Burkholderia is the dominant component of the sugarcane rhizosphere microbial community. The Burkholderia population is complex and assessments performed on these populations do not always reveal specific members. Moreover, current cultivation based methods are limited because they do not assess the non-culturable fraction of rhizosphere microbiota. The aim of this work was to develop root DNA-based PCR fluorescent singlestrand conformation polymorphism (F-SSCP), a molecular method for studying the diversity of microbial communities, for analysis of Burkholderia diversity in root samples. Speciesspecific primers for Burkholderia were developed, based on a small region of the 16S rRNA gene sequence. F-SSCP analyses of the PCR products obtained showed that there were sufficient differences in migration to distinguish all 39 Burkholderia species tested. For microbial rhizosphere community studies, six-month-old variety N29 from the existing Burkholderia field trial at Zinkwazi was used. Roots from plots inoculated with Burkholderia isolates N8.2 and LM1-376.8 and from a corresponding control plot were sampled. Total genomic DNA extracted from roots was subjected to PCR and assessed using F-SSCP. FSSCP profiles on amplicons generated from root DNA were aligned with Burkholderia reference species profiles. The root DNA exclusively revealed profiles affiliated with profiles of Burkholderia reference species, and demonstrated that the F-SSCP method befits the study of diversity in Burkholderia spp. in natural samples. The dominant Burkholderia spp. in the root samples were B. caledonica, B. saccharri, B. cepacia, B. graminis and B. ambifaria. No differences in profiles between control and Burkholderia inoculated plots were distinguished.