Quantification of natural populations of Gluconacetobacter diazotrophicus and Herbaspirillum spp. In sugar cane (Saccharum spp.) Using differente polyclonal antibodies

Abstract

The species Gluconacetobacter diazotrophicus, Herbaspirillum seropedicae and H. rubrisubalbicans are endophytic N 2 -fixing [diazotrophic] bacteria which colonise not only roots, but also the aerial tissue of sugar cane. However, the technique most commonly used to quantify the populations of these microbes in plants is by culturing serial dilutions of macerates of plant tissues in N free semi-solid media which are only semi-selective for the species/genera [the Most Probable Number (MPN) Technique] and each culture must be further subjected to several tests to identify the isolates at the species level. The use of species-specific polyclonal antibodies with the indirect ELISA (enzyme-linked immunosorbent assay) can be an alternative which is rapid and specific to quantify these populations of bacteria. This study was performed to investigate the viability of adapting the indirect ELISA technique to quantify individually the populations of these three species of diazotroph within the root and shoot tissues of sugarcane. The results showed that species-specific polyclonal antibodies could be obtained by purifying sera in protein-A columns which removed non-specific immuno-globulins. It was possible to quantify the three bacterial species in the Brazilian sugarcane variety SP 70-1143 in numbers above 10 5 cells per g fresh weight in roots, rhizomes and leaves. The numbers of the different bacterial species evaluated using the ELISA technique were found to be higher than when the same populations were evaluated using the MPN technique, reaching 1400 times greater for G. diazotrophicus and 225 times greater for Herbaspirillum spp. These results constitute the first quantification of Herbaspirillum using immunological techniques.

Research Progress and Perspectives of Nitrogen Fixing Bacterium,Gluconacetobacter diazotrophicus, in Monocot Plants

“…Growth of G. diazotrophicus under laboratory conditions is primarily achieved through plating on LGIP medium due to the fact that it contains high sugar levels which are very similar to those found within sugarcane (quantities per litre: K 2 HPO 4 , 0.2 g; KH 2 PO 4 , 0.6 g; MgSO 4 ⋅7H 2 O, 0.2 g; CaCl 2 ⋅2H 2 O, 0.02 g; Na 2 MoO 4 ⋅2H 2 O, 0.002 g; FeCl 3 ⋅6H 2 O, 0.01 g; bromothymol blue in 0.2 M KOH, 0.025 g; sucrose, 100 g; yeast extract, 0.025 g; agar, 15 g; 1% acetic acid, pH 5.5) [18]. Other media capable of sustaining G. diazotrophicus growth include but are not limited to DYGS, C2, ATGUS, modified potato, SYP, AcD, GYC, and EYC media [22][23][24][25][26][27][28][29]. The biochemical characteristics of G. diazotrophicus are listed in Table 2.…”

Development of a real-time PCR assay for the detection and quantification of Gluconacetobacter diazotrophicus in sugarcane grown under field conditions

“…Quantification of G. diazotrophicus is usually performed using the culture medium LGI-P where bacterial growth is confirmed by the presence of characteristic pellicles formed on the surface of a vial containing semisolid LGI-P medium and the number of bacteria is estimated using the McCrady table . However, as discussed by Silva-Froufe et al (2009) this method presents several limitations, such as the presence of other microorganisms that can inhibit G. diazotrophicus growth, which can lead to an underestimation of the actual population. Furthermore, the method assume that all bacteria present in plant are isolated and not form aggregates, so the maceration extracts contain all of the bacteria present in the plant tissue in a homogeneous suspension of individual bacterial cells.…”

“…Furthermore, the method assume that all bacteria present in plant are isolated and not form aggregates, so the maceration extracts contain all of the bacteria present in the plant tissue in a homogeneous suspension of individual bacterial cells. These premises could be not real, but are important for dilutions based method (Silva-Froufe et al, 2009). Moreover, bacterial cells may be present in a viable but non-culturable form that may also lead to an underestimation of bacterial numbers, as was observed for the bacteria of the genus Herbaspirillum (Olivares et al, 1996).…”

“…Many techniques have been applied with success to monitor the presence (Oliveira et al, 2009;Silva-Froufe et al, 2009) or even quantify (Gomes et al, 2005;Munoz-Rojas and Caballero-Mellado, 2003) the inoculated bacterial strains within plants.…”

The survival of plant growth promoting microorganisms in peat inoculant as measured by selective plate counting and enzyme-linked immunoassay

“…Although immunological methods do not generally distinguish between viable and non-viable cells, attempts have been made to incorporate additional steps such as vital staining (Olsen and Rice 1996) or fermentation (Mijajlovic et al unpublished) to determine the extent of cell viability. Immunological techniques have been used to evaluate the quality of rhizobial inoculants (Nambiar and Anjaiah 1985;Lochner et al 1988) and localize and quantify PGPR in soil-plant systems (Levanony et al 1987;Silva-Froufe et al 2009), but references to their use for evaluating the quality of nonrhizobial inoculants are limited (Bashan and Gonzalez 1999). Given their greater diversity, associative (rather than symbiotic) nature, and poorer nutritional and serological characterization as inoculants to date, we believe a focus on non-rhizobial compared to rhizobial inoculants currently deserves attention.…”