Plant biostimulants - Determination of Azotobacter spp.

This document was developed to provide the methodology for the enumeration and determination of Azotobacter sp. in plant biostimulant products in accordance with the Regulation (EU) 2019/1009 of the European Parliament and of the Council [1].

Pflanzen-Biostimulanzien - Bestimmung von Azotobacter spp.

Dieses Dokument wurde entwickelt, um die Methodik für die Zählung und Bestimmung von Azotobacter sp. in Pflanzen-Biostimulanzienprodukten in Übereinstimmung mit der Verordnung (EU) 2019/1009 des Europäischen Parlaments und des Rates [1] bereitzustellen.

Biostimulants des végétaux - Détermination d'Azotobacter spp.

Le présent document a été élaboré pour fournir la méthodologie de dénombrement et de détermination d’Azotobacter sp. dans les produits biostimulants des végétaux conformément au Règlement (UE) 2019/1009 du Parlement européen et du Conseil [1].

Rastlinski biostimulanti - Določanje Azotobacter spp.

General Information

Status
Published
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Due Date
23-Mar-2022
Completion Date
23-Mar-2022

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SLOVENSKI STANDARD
kSIST-TS FprCEN/TS 17709:2021
01-november-2021
[Not translated]
Plant biostimulants - Determination of Azotobacter spp.
Biostimulanzien für die pflanzliche Anwendung - Bestimmung von Azotobacter spp.
Biostimulants des végétaux - Détermination d'Azotobacter spp.
Ta slovenski standard je istoveten z: FprCEN/TS 17709
ICS:
65.080 Gnojila Fertilizers
kSIST-TS FprCEN/TS 17709:2021 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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kSIST-TS FprCEN/TS 17709:2021
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kSIST-TS FprCEN/TS 17709:2021
FINAL DRAFT
TECHNICAL SPECIFICATION
FprCEN/TS 17709
SPÉCIFICATION TECHNIQUE
TECHNISCHE SPEZIFIKATION
September 2021
ICS 65.080
English Version
Plant biostimulants - Determination of Azotobacter spp.
Biostimulanzien für die pflanzliche Anwendung -
Bestimmung von Azotobacter spp.

This draft Technical Specification is submitted to CEN members for Vote. It has been drawn up by the Technical Committee

CEN/TC 455.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,

Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,

Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and

United Kingdom.

Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are

aware and to provide supporting documentation.

Warning : This document is not a Technical Specification. It is distributed for review and comments. It is subject to change

without notice and shall not be referred to as a Technical Specification.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. FprCEN/TS 17709:2021 E

worldwide for CEN national Members.
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kSIST-TS FprCEN/TS 17709:2021
FprCEN/TS 17709:2021 (E)
Contents Page

European foreword ............................................................................................................................... 3

Introduction ............................................................................................................................................ 4

1 Scope ............................................................................................................................................ 6

2 Normative references ............................................................................................................ 6

3 Terms and definitions ........................................................................................................... 6

4 Enumeration of Azotobacter spp ........................................................................................ 6

4.1 General ........................................................................................................................................ 6

4.2 Sample preparation ................................................................................................................ 6

4.2.1 General ........................................................................................................................................ 6

4.2.2 Liquid (based water) formulations ................................................................................... 6

4.2.3 Liquid - based oil, emulsifiable concentrate (EC) formulations ............................. 6

4.2.4 Solid - Wettable Powder (WP) formulations ................................................................. 7

4.2.5 Solid - Water dispersible granules (WDG) formulations .......................................... 7

4.2.6 Solid – Pellets, granules, microgranules - slow release - formulations ............... 7

4.2.7 Solid - substrate ....................................................................................................................... 7

4.3 Serial dilution ........................................................................................................................... 7

4.4 Plate counts of Azotobacter sp in sterile diluent .......................................................... 8

4.5 Spread-plate counting with ASHBY SUCROSE AGAR [6] ............................................ 8

4.6 Calculation ................................................................................................................................. 9

5 Species Determination of Azotobacter sp.. via genetic analysis ............................. 9

5.1 Preparation of the sample for the genomic DNA extraction .................................... 9

5.1.1 Isolation and preparation of the microorganism ........................................................ 9

5.1.2 Sample concentration ............................................................................................................ 9

5.1.3 DNA extraction and storage ................................................................................................. 9

5.1.4 Partial PCR Amplification of the 16S rRNA Genes ..................................................... 10

Annex A (informative) Formula of colture media ..................................................................... 11

A.1 Ashby’s sucrose Agar medium .......................................................................................... 11

A.1.1 Ingredients .............................................................................................................................. 11

A.1.2 Method to prepare YT media ............................................................................................ 11

A.2 Procedure for preparing nutrient broth medium ..................................................... 11

A.3 Procedure for preparing 0,1 M Phosphate Buffer Saline (PBS)............................ 12

Bibliography .......................................................................................................................................... 13

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kSIST-TS FprCEN/TS 17709:2021
FprCEN/TS 17709:2021 (E)
European foreword
This document (FprCEN/TS 17709:2021) has been prepared by Technical Committee
CEN/TC 455 “Plant biostimulants”, the secretariat of which is held by AFNOR.
This document is currently submitted to the Vote on TS.

This document has been prepared under a mandate given to CEN by the European Commission

and the European Free Trade Association.
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kSIST-TS FprCEN/TS 17709:2021
FprCEN/TS 17709:2021 (E)
Introduction

This document was prepared by the experts of CEN/TC 455 ‘Plant Biostimulants’. The European

Committee for Standardization (CEN) was requested by the European Commission (EC) to draft

European standards or European standardization deliverables to support the implementation of

Regulation (EU) 2019/1009 of 5 June 2019 laying down rules on the making available on the

market of EU fertilising products (“FPR” or “Fertilising Products Regulation”). This request,

presented as SR M/564, also contributes to the Communication on “Innovating for Sustainable

Growth: A Bio economy for Europe”. The Working Group 5 “Labelling and denominations”, was

created to develop a work program as part of this Request. The technical committee CEN/TC 455

‘Plant Biostimulants’ was established to carry out the work program that will prepare a series of

standards. The interest in biostimulants has increased significantly in Europe as a valuable tool to

use in agriculture. Standardization was identified as having an important role in order to promote

the use of biostimulants. The work of CEN/TC 455 seeks to improve the reliability of the supply

chain, thereby improving the confidence of farmers, industry, and consumers in biostimulants,

and will promote and support commercialisation of the European biostimulant industry.

Biostimulants used in agriculture can be applied in multiple ways: on soil, on plant, as seed

treatment, etc. A microbial plant biostimulant consists of a microorganism or a consortium of

microorganisms, as referred to in Component Material Category 7 of Annex II of the EU Fertilizing

Products Regulation.

This document is applicable to all biostimulants in agriculture based on live microorganisms

belonging to the genera Azotobacter.

The Table 1 below summarizes many of the agro-ecological principles and the role played by

biostimulants.
Table 1 — Agro-ecological principles and the role played by biostimulants
Increase biodiversity
By improving soil microorganism quality/quantity
Reinforce biological regulation and interactions
By reinforcing plant-microorganism interactions
- symbiotic exchanges i.e. mycorrhize
- symbiotic exchanges i.e. rhizobiaciae/fava
- secretions mimicking plant hormones (i.e. trichoderma)
By regulating plant physiological processes
- for ex growth, metabolism, plant development…
Improve biogeochemical cycles
- improve absorption of nutritional elements
- improve bioavailability of nutritional elements in the soil
- stimulate degradation of organic matter

WARNING — Persons using this document should be familiar with normal laboratory practice.

This document does not purport to address all of the safety problems, if any, associated with its

use. It is the responsibility of the user to establish appropriate safety and health practices and to

ensure compliance with any national regulatory conditions.
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kSIST-TS FprCEN/TS 17709:2021
FprCEN/TS 17709:2021 (E)

IMPORTANT — It is absolutely essential that tests conducted in accordance with this document

be carried out by suitably trained staff.
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kSIST-TS FprCEN/TS 17709:2021
FprCEN/TS 17709:2021 (E)
1 Scope
This document was developed to provide the methodology for the enumeration and

determination of Azotobacter sp. in plant biostimulant products in accordance with the Regulation

(EU) 2019/1009 of the European Parliament and of the Council [1].
2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements of this document. For dated references, only the edition cited applies.

For undated references, the latest edition of the referenced document (including any

amendments) applies.

FprCEN/TS 17702-1, Plant biostimulants — Sampling and sample preparation — Part 1: Sampling

FprCEN/TS 17724, Plant biostimulants — Terminology
3 Terms and definitions

For the purposes of this document, the terms and definitions given in FprCEN/TS 17724 apply.

ISO and IEC maintain terminological databases for use in standardization at the following

addresses:
• IEC Electropedia: available at https://www.electropedia.org/
• ISO Online browsing platform: available at https://www.iso.org/obp
4 Enumeration of Azotobacter spp
4.1 General

This procedure is meant to determine the number of colony-forming units (CFU) of the above

mentioned bacteria«Pathogen», per gram, per millilitre, per square centimetre, or per sampling

device. The method, in order to be fast, cheap, repeatable, is based on serial dilutions and plating.

4.2 Sample preparation
4.2.1 General

A representative sample of the product to be analysed according to FprCEN/TS 17702-1 shall be

prepared according to following procedure which takes into consideration the different

formulations of biostimulants based products.
4.2.2 Liquid (based water) formulations

Dispense 25 ml of' sample (or more for low concentrated products) in 225 ml of sterile Phosphate

Buffer Solution (PBS) maintained at room temperature, in a flask and shake for 10 min or more

until the distribution is optimal, with a magnetic stirrer at half speed [6].
4.2.3 Liquid - based oil, emulsifiable concentrate (EC) formulations

Dispense 25 ml of' sample (or more for low concentrated products) in 225 ml of sterile Phosphate

Buffer Solution (PBS) maintained at room temperature, in a flask and shake for 10 min or more

until the distribution is optimal, with a magnetic stirrer at half speed [6].
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