MICROBIOLOGICAL IMAGE

https://doi.org/10.1016/j.ram.2018.04.002

Veil-like pellicle development by Azospirillum brasilense in semisolid NFb medium

Desarrollo de película típica en medio Nfb semisólido por Azospirillum brasilense

 

Luciana P. Di Salvo^a, Inés E. García de Salamone^b,*

^a CONICET - Cátedra de Microbiología Agrícola, Facultad de Agronomía, Universidad de Buenos Aires, Av. San Martín 4453, Ciudad Autónoma de Buenos Aires 1417, Argentina
^b Universidad de Buenos Aires, Facultad de Agronomía, Departamento de Biología Aplicada y Alimentos, Cátedra de Microbiología Agrícola, Av. San Martín 4453, Ciudad Autónoma de Buenos Aires 1417, Argentina

Received 26 February 2018; accepted 4 April 2018
Available online 14 July 2018*Corresponding author.

E-mail address: igarcia@agro.uba.ar (I.E. García de Salamone).

 

Azospirillum brasilense is a vastly studied plant growth-promoting rhizobacterium, which produces direct and indirect beneficial effects on several crops⁴. Historically, the culture of this bacterium has been performed using the semisolid NFb medium³, which is a nitrogen-free medium. Although other diazotrophs can grow in this culture medium², A. brasilense can be isolated and identified by a typical growth after 120h of incubation³. Other authors have exhaustively described the NFb medium composition and growth of many diazotrophs in semisolid media¹. Here, we show pictures of seven stages during the development of A. brasilense in the course of the incubation period.

A colony of A. brasilense was inoculated in a vial containing this culture medium. After 24h of incubation at 30°C, growth development can be observed by a halo formation around the colony as a 'ghostly-balloon' (Fig. 1A). The halo grows in size (Fig. 1B) and opens (Fig. 1C) after 48 h of incubation, approximately. By aerotaxis, cells move to the specific place in the medium (Fig. 1D) where the rate of oxygen diffusion and bacterial respiration allows the nitrogenase enzyme to fix N₂ without the irreversible inhibitory effect of oxygen². Then, bacteria continue growing and form a typical sub-superficial whitish 'veil-like' pellicle after five days of incubation (Fig. 1E). As growth occurs, the alkalization of the culture medium can be observed by a change in color from green (Fig. 1F) to blue (Fig. 1G) due to the bromothymol blue redox dye. This culturing technique can also be used for other purposes, such as the inoculation of serial dilutions (Fig. 1F and G) to perform the estimation of the most probable number of this plant beneficial bacterium in commercial inoculants or the count of diazotrophs from environmental samples.

Figure 1 Growth development by Azospirillum brasilense in vials with semisolid NFb medium during the incubation period (a, b, c, d and e). Growth starts with the formation of a halo (white arrows) around the colony. Typical growth as a 'veil-like' pellicle (black arrows) under the medium surface (dotted circles) is formed. After six (f) and seven (g) days of incubation, it can be observed the change of color indicating the alkalization of the culture medium. Vials showed in a to e were inoculated with one colony of A. brasilense. Vials showed in f and g were inoculated with 50 µl of the 10^-2 dilution of an inoculant sample.

Funding

This work was partially supported by FONCYT2008 PICT 1864

from theMINCyT and UBACyT project 20020090100255, Universidad de Buenos Aires (UBA) in Argentina.

Conflict of interest

The authors declare that they have no conflicts of interest.

References

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2. Di Salvo LP, Silva E, Teixeira KR, Esquivel-Cote R, Pereyra MA, García de Salamone IE. Physiological and biochemical characterization of Azospirillum brasilense strains commonly used as plant growth-promoting rhizobacteria. J Basic Microbiol. 2014;54:1310-21.         [ Links ]

3. Dobereiner J. Isolation and identification of aerobic nitrogenfixing bacteria from soil and plants. In: Alef K, Nannipieri P, editors. Methods in applied soil microbiology and biochemistry. London, UK: Academi Press; 1998. p. 134-41.         [ Links ]

4. García de Salamone IE. Use of soil microorganisms to improve plant growth and ecosystem sustainability. In: Caliskan M, editor. The molecular basis of plant genetic diversity. Rijeka, Croatia: INTECH; 2012. p. 233-58. http://www.intechopen.com/articles/show/title/use-of-soilmicroorganisms-to-improve-plant-growth-and-ecosystemsustainability        [ Links ]

0325-7541/© 2019 Asociación Argentina de Microbiología. Published by Elsevier Espana, S.L.U. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).