Molecular Tools to Study Azospirillum sp. and Other Related Plant Growth Promoting Rhizobacteria

Molecular methods have been used in the study of 'Azospirillum' and other related PGPRs to carry out gene functional analysis, create gene knockouts, generate genetically engineered strains, and carry out gene expression studies. Genetic transformation has routinely been carried out using conjugation, while chromosomal modifi cations have been performed using unstable, suicide plasmids, or more stable, broad host-range vectors. Gene expression studies are often carried out using promoter-bound reporter genes; however, quantitative methods such as reverse transcribed polymerase chain reaction can now be used to directly study gene expression. In this chapter we describe the common types of vectors used in 'Azospirillum', as well as methods for transformation and mutagenesis. We also describe the use of promoter-bound reporter genes and the applications of quantitative RT-PCR for 'Azospirillum' gene expression studies. Methods for the isolation of DNA and RNA from 'Azospirillum' for use in molecular and gene expression studies are also described

Azospirillum Cell Aggregation, Attachment, and Plant Interaction

'Azospirillum' cellular and morphological transformation in culture as well as cyst formation, aggregation, and flocculation in response to nutritional limitations and increasing oxygen levels are discussed and typical protocols for flocculation and aggregation are presented. An overview of the mechanisms of attachment to plant roots and other surfaces is followed by protocols for labeling 'Azospirillum' cells with reporter genes and using such genetically labelled cells in qualitative and quantitative assays of 'Azospirillum'-plant associations. The potential of 'Azospirillum' in plant pathogen and disease suppression is discussed