Isolation and characterization of bacterial isolates recovered from surface sterilized alfalfa (Medicago sativa) seeds

LÓPEZ, J. L.; GIUSTI, M. A.; MARTINI, C; SALAS, M, E.; LOZANO, M. J.; DEL PAPA, M.F.; PISTORIO, M.; LAGARES, A.

Mar del Plata

Congreso; VIII Congreso Argentio de Microbiología General; 2012

SAMIGE

The use of inoculant microorganisms in agriculture is a growing practice directed to increase culture yields, and to preserve soil fertility. In addition to the use of rhizobia with legumes, the application of other biofertilizers is currently extended to different microorganisms which, from the rhizosphere or from within the plants (endophytes), improve plant growth and plant health. Such microorganisms, that inhabit the underground, live both in the bulk soil and associated with the plant roots that they colonize. In addition to the plant-associated soil microorganisms, there is a growing piece of evidence showing that plant seeds also contain quite diverse bacteria, though their role in plant growth/development has not yet been properly characterized. In the frame of our studies on the microbiota associated with alfalfa, we investigated the bacterial diversity present within seeds of this plant species. Handelsman and Brill (1985) had previously reported the presence Erwinia herbicola within alfalfa seeds. In the work that we present here, surface sterilized seeds (Medicago sativa cv Monarca, INTA) were prepared using either sulfuric acid (98% P/P, 10 min) or NaClO (9 g Cl2/l, 10 min). Those seeds were then washed with abundant water, PBS (pH = 7), and incubated 4 hs at room temperature in the same buffer to facilitate water entrance to the seeds and bacterial release. The resulting supernatant was finally plated on different solid media (LB, TSA, YEM) where several colonies with different morphologies could be observed. PCR amplification and sequencing of a partial rDNA fragment from representative clones demonstrated the presence of the following bacterial genera: Arthrobacter, Bacillus, Enhydrobacter, Janibacter, Kokuria, Microbacterium, Moraxella, Paenibacillus, Pantoea, Rhizobium, Sphingomonas, and Streptomyces. Interestingly, we could isolate bacteria belonging to 8 of these genera from either roots or shoots of alfalfa plants grown in vermiculite with mineral medium. The results strongly suggest that bacteria from the seeds could have been the source of the plant colonizing microorganisms. Most bacteria recovered from the aerial part of the plant could be also isolated from roots and/or seeds. Bacteria belonging to the genera Corynebacterium, Exiguobacterium, Flavobacterium, and Pseudomonas could only been isolated from roots. Based on these findings we are now investigating if any the seed isolates either produce plant growth promoting enzymes/compounds or display specific tropism for the colonization of alfalfa (root/shoots) in single inoculation experiments.