Molecular tools for monitoring Bacillus subtilis in the rhizosphere of crop plants

Molecular tools were developed for unambiguous detection and quantification of the biological control agent B. subtilis DCB101 strain in the environment, in order to evaluate its effects on plant growth and on soil and rhizosphere microbial community. The Random Amplified Polymorphic DNA (RAPD) technique was used to find a sequence characterized amplified region (SCAR) molecular marker enabling to detect the microorganism at strain level. SCAR markers have been commonly used for developing monitoring methods of microbial inoculants because they are natural sequences present in the genome allowing a simple specific detection by PCR procedure. The developed molecular marker was then used, coupled with classical microbiological methods and cytological analysis, to evaluate (a) the colonization ability and the persistence of the strain at rhizosphere level, (b) its effects on plant growth and on root tip development in tomato plants, both in individual inoculation as well as in dual combination with the plant growth promoting rhizobacteria Azospirillum brasilense. Furthermore, the effects of B. subtilis in multi-microbial combination with arbuscolar mycorrhizal fungi was also evaluated in sorghum plants. The DGGE analysis was performed in order to assess the impact of the various microbial inoculants on the structural composition and dynamics of microbial communities both in non-rhizosphere and in rhizosphere soil. The results show that when B. subtilis is co-inoculated with A. brasilense a not yet known mutual interaction between the two rhizobacteria takes place leading to the neutralization of the plant growth promotion effects due to the single inocula. These results along with the cytological observations of inoculated root tips lead to hypothesize that each rhizobacteria might operate in a distinct manner to promote plant growth and might influence, directly or indirectly, phytohormone content of plant root. As regards DGGE analysis of soil microbial community structure, B. subtilis DCB101 showed no impact on soil and rhizosphere microbial community of tomato and sorghum plants as well as on AMF establishment. Overall considered, the molecular monitoring tools developed in this work represented a useful approach, when coupled with classical microbiological methods, for evaluating the behaviour of B. subtilis DCB101 in rhizosphere and in soil. Studies are ongoing to develop a real-time PCR with fluorescent probes targeted in the SCAR sequence. This method will allow further field studies, including colonization and survival of the strain in the rhizosphere of plant roots, and its effects on plant health.