Azospirillum isolation from barley seeds

DÍAZ HERRERA S.; ZAWOZNIK M.S.; VÁZQUEZ S.; BENAVIDES M.P.; GROPPA M.D.

Taller; TALLER INTERACCIONES MICROORGANISMOS-PLANTAS; 2012

Endophytic bacteria are plant-associated bacteria that live inside plant tissues without causing any harm. Among them, members of Azospirillum genus promote plant growth and help plants to thrive under adverse climate conditions. The present investigation was carried out to isolate diazotrophic bacteria from the seeds of three malting barley cultivars recommended at Buenos Aires productive area. A total of 8 endophytic bacterial isolates were finally retrieved from barley seeds using RC and Nfb media. All of them were screened for Gram reaction and motility, IAA production by Salkowski technique and nitrogen fixation ability, estimated as acetylene reduction activity (ARA). Genotypic identification was performed by 16S rRNA gene sequencing and genetic relationships between isolates were further investigated by random amplified polymorphism analysis (RAPD) using three different primers. An adaptation of the double agar layer plate assay allowed to screen potential antagonistic effects of the isolates obtained against Azospirillum brasilense strain Az39 (Az39), a well-known collection strain included in some biofertilizers used in Argentina. Preliminary characterization by morphological studies revealed that two of the 8 isolated endophytes could be Azospirillum. The molecular characterization by PCR amplification of 16S confirmed that these two isolates had a 99% similarity with the reference strain of Azospirillum doebereinerae. RAPD analysis showed slightly different band pattern using three different primers. In vitro IAA production levels were comparable to that of Az39; however, ARA was negligible. No ouvert antagonistic effects were detected, but the Azospirillum strain retrieved from Quilmes Carisma cultivar delayed Az39 growth for 72 h, as compared to the other strains tested.