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eLibrary > BS EN 17718:2024 Plant biostimulants. Determination of Rhizobium spp. >

BS EN 17718:2024 Plant biostimulants. Determination of Rhizobium spp., 2024

BS EN 17718:2024 Plant biostimulants. Determination of Rhizobium spp., 2024

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European foreword
Introduction
1 Scope
2 Normative references
3 Terms and definitions
4 Enumeration of Rhizobium spp., Mesorhizobium spp., Ensifer spp., Bradyrhizobium spp. [Go to Page]
4.1 General
4.2 Sample preparation [Go to Page]
4.2.1 General
4.2.2 Liquid (water-based) formulations
4.2.3 Liquid (oil-based) emulsifiable concentrate (EC) formulations
4.2.4 Solid wettable powder (WP) formulations
4.2.5 Solid water dispersible granules (WDG) formulations
4.2.6 Solid pellets, granules, microgranules - slow release - formulations
4.2.7 Solid substrate
4.3 Serial dilution
4.4 Preparation of the culture media
Figure 1 — Scheme of serial dilutions [Go to Page]
4.5 Spread plate counting
4.6 Plate counts of Rhizobiaceae in sterile diluent
4.7 Growing media
Table 2 —Growing media for the growth of the different microorganisms [Go to Page]
4.8 Calculation
5 Species determination of Rhizobium spp., Mesorhizobium spp., Ensifer spp., Bradyrhizobium spp. via genetic analysis [Go to Page]
5.1 General
5.2 Preparation of the sample for genomic DNA extraction [Go to Page]
5.2.1 Isolation and preparation of the microorganism
5.2.2 Sample concentration
5.2.3 DNA extraction and storage
5.2.4 Partial PCR amplification of the 16S rRNA genes [Go to Page]
5.2.4.1 PCR amplification
5.2.4.2 Visualization of PCR products
5.2.4.3 Sequencing of partial 16S rRNA genes
5.2.4.4 Whole-genome sequencing
Annex A (normative) Formula of culture media7F
A.1 YT (Tryptone-Yeast Medium) for Rhizobium spp., Mesorhizobium spp. and Ensifer spp.
A.1.1 Ingredients of YT medium
Table A.1 — Ingredients of YT medium
A.1.2 Method to prepare YT media
A.2 Modified Yeast extract mannitol agar medium (YEMA)alternative medium for Rhizobium spp.
A.2.1 Ingredients of YEMA medium (and suggested suppliers)
Table A.2 — Ingredients of YEMA medium
A.2.2 Method to prepare YEMA medium
A.3 Reasoner's 2A Agar Medium (R2A) for Bradyrhizobium spp.
A.3.1 Ingredients of R2A medium (and suggested suppliers)
Table A.3 — Ingredients of R2A media
A.3.2 Method to prepare Reasoner's 2A Agar (R2A) medium
A.4 Congo Red stock solution for YEMA medium
A.4.1 Congo Red stock solution medium preparation
Table A.4 — Ingredients of Congo red stock solution medium
A.5 0,1 M Phosphate Buffered Saline (PBS)
A.6 Nutrient broth
A.7 Buffered Peptone Water
A.7.1 Composition
A.7.2 Preparation
Annex B (informative) Repeatability and reproducibility of the method
B.1 Materials used in the interlaboratory comparison study
Table B.1 — Materials tested in the interlaboratory study for the enumeration and determination of Rhizobium spp., Mesorhizobium spp., Ensifer spp., or Bradyrhizobium spp. in plant biostimulant
B.2 Interlaboratory comparison results
Table B.2 — Results of the interlaboratory comparison study of the enumeration of Rhizobium spp., Mesorhizobium spp., Ensifer spp., or Bradyrhizobium spp. in biostimulants
Table B.3 — Samples distributed for the interlaboratory comparison study of the determination of Rhizobium spp., Mesorhizobium spp., Ensifer spp., or Bradyrhizobium spp. in biostimulants
Annex ZA (informative) Relationship of this European Standard and the essential requirements of Regulation (EU) 2019/1009 making available on the market of EU fertilising products aimed to be covered
Table ZA.1 — Correspondence between this European Standard and Regulation (EU) 2019/1009
Bibliography [Go to Page]

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