Abstract
Azospirillum has been one of the most studied genera of plant growth promoting rhizobacteria (PGPR) worldwide over the past 50 years. The use of these microorganisms in agriculture practices has been adopted due to their ability to associate in rhizospheric, epiphytic, or endophytic ways with roots and promote whole plant growth or crop productivity. The biological treatment of seeds (inoculation) in more than a hundred species of economic or ecological interest has become a common practice in many countries. In Argentina, the Az39 strain of Azospirillum brasilense, belonging to the Culture Collection of the Instituto de Microbiología y Zoología Agrícola (IMYZA) of INTA Castelar, was selected in the 1980s after an intensive program to isolate and identify microorganisms for agriculture, according to their agronomic behavior. Since then, its ability to cover the premise for which it was selected has determined that Az39 is largely adopted by inoculant companies in Argentina with the aim of producing biological products for the treatment of several crops. In this chapter, those methods developed and standardized by the network Red de Control de Calidad de Inoculantes (REDCAI) of the Asociación Argentina de Microbiología (AAM) have been adapted as a guide for the quantification of Azospirillum spp. and the detection of contaminating microorganisms in biological products, as two of the most basic and important quality control parameters of inoculants.
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Methodological Appendix
Methodological Appendix
1.1 Agar-RC Medium (Agar–Congo Red)
Component | Amount |
|---|---|
K2HPO4 | 0.5 g |
MgSO4·7H2O | 0.2 g |
NaCl | 0.1 g |
Yeast extract | 0.5 g |
FeCl3·6H2O | 0.015 g |
dl-malic acid | 5.0 g |
KOH | 4.8 g |
Congo red solutiona | 15.0 mL |
Agar | 20.0 g |
H2O | 1,000 mL |
1.2 NFb Semisolid Medium to Evaluate the Microaerophilic Diazotrophic Activity
Component | Amount |
|---|---|
d-malic acid | 5.0 g |
K2PO4H | 0.5 g |
MgSO4·7H2O | 0.2 g |
NaCl | 0.1 g |
Ca2Cl·2H2O | 0.02 g |
Bromothymol blue solutionb | 2.0 mL |
Micronutrient solutionc | 2.0 mL |
FeCl3·6H2O | 0.015 g |
KOH | 4.5 g |
Agar | 1.8 g |
Distilled water | 1,000 mL |
1.3 Tween 80 Stock Solution at 2.5 % (w/v)
Component | Amount |
|---|---|
Tween 80 | 5 g |
Distilled water | 200 mL |
1.4 Gram Staining Technique Modified by Hucker
1.4.1 Crystal Violet Reaction
Crystal violet mother solution (A) | |
Crystal violet | 5 g |
Alcohol 95° | 25 mL |
Ammonium oxalate mother solution (B) | |
Ammonium oxalate | 2 g |
Distilled water | 200 mL |
Prepare solutions A and B separately and use a working solution prepared with 4 mL of solution A, 36 mL of distilled water, and 160 mL of solution B.
1.4.2 Lugol’s Iodine Solution
Iodine | 1 g |
Potassium iodide | 2 g |
Distilled water | 300 mL |
1.4.3 Safranin Reagent
Safranin | 2.5 g |
Alcohol 95° | 100 mL |
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Cassán, F.D. et al. (2015). Protocol for the Quality Control of Azospirillum spp. Inoculants. In: Cassán, F., Okon, Y., Creus, C. (eds) Handbook for Azospirillum. Springer, Cham. https://doi.org/10.1007/978-3-319-06542-7_27
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DOI: https://doi.org/10.1007/978-3-319-06542-7_27
Publisher Name: Springer, Cham
Print ISBN: 978-3-319-06541-0
Online ISBN: 978-3-319-06542-7
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