EN 17709:2024

SLOVENSKI STANDARD
oSIST prEN 17709:2023
01-maj-2023
Rastlinski biostimulanti - Ugotavljanje prisotnosti in števila Azotobacter spp.
Plant biostimulants - Determination of Azobacter spp.
Pflanzen-Biostimulanzien - Bestimmung von Azotobacter spp.
Biostimulants des végétaux - Détermination d'Azotobacter spp.
Ta slovenski standard je istoveten z: prEN 17709
ICS:
65.080 Gnojila Fertilizers
oSIST prEN 17709:2023 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

oSIST prEN 17709:2023
oSIST prEN 17709:2023
DRAFT
EUROPEAN STANDARD
prEN 17709
NORME EUROPÉENNE
EUROPÄISCHE NORM
April 2023
ICS 65.080 Will supersede CEN/TS 17709:2022
English Version
Plant biostimulants - Determination of Azobacter spp.
Biostimulants des végétaux - Détermination Pflanzen-Biostimulanzien - Bestimmung von
d'Azotobacter spp. Azotobacter spp.
This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee
CEN/TC 455.
If this draft becomes a European Standard, CEN members are bound to comply with the CEN/CENELEC Internal Regulations
which stipulate the conditions for giving this European Standard the status of a national standard without any alteration.

This draft European Standard was established by CEN in three official versions (English, French, German). A version in any other
language made by translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC
Management Centre has the same status as the official versions.

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, Türkiye 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 European Standard. It is distributed for review and comments. It is subject to change without
notice and shall not be referred to as a European Standard.

EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATIO N

EUROPÄISCHES KOMITEE FÜR NORMUN G

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2023 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 17709:2023 E
worldwide for CEN national Members.

oSIST prEN 17709:2023
prEN 17709:2023 (E)
Contents Page
European foreword . 4
Introduction . 5
1 Scope . 7
2 Normative references . 7
3 Terms and definitions . 7
4 Enumeration of Azotobacter spp. . 7
4.1 General . 7
4.2 Sample preparation . 7
4.2.1 General . 7
4.2.2 Liquid (based water) formulations . 7
4.2.3 Liquid - based oil, emulsifiable concentrate (EC) formulations . 8
4.2.4 Solid - Wettable Powder (WP) formulations . 8
4.2.5 Solid - Water dispersible granules (WDG) formulations . 8
4.2.6 Solid – Pellets, granules, microgranules - slow release - formulations . 8
4.2.7 Solid - substrate . 8
4.3 Plate counts of Azotobacter sp. in sterile diluent with serial dilutions . 8
4.4 Preparation of the culture media . 8
4.5 Plate counts of Azotobacter sp. in sterile diluent . 9
4.6 Spread-plate counting with ASHBY SUCROSE AGAR [5] (for the different media, see
Annex A) . 9
4.7 Calculation . 10
5 Species determination of Azotobacter sp. via genetic analysis . 10
5.1 General . 10
5.2 Preparation of the sample for the genomic DNA extraction . 10
5.2.1 Isolation and preparation of the microorganism . 10
5.2.2 Sample concentration . 10
5.2.3 DNA extraction and storage . 11
5.2.4 Partial PCR amplification of the 16S rRNA Genes . 11
Annex A (informative) Formula of culture media . 13
A.1 Ashby’s sucrose Agar medium . 13
A.1.1 Ingredients . 13
A.2 Method to prepare Ashby’s sucrose Agar medium . 13
A.3 Procedure for preparing nutrient broth medium . 14
A.4 Procedure for preparing 0,1 M Phosphate Buffer Saline (PBS). 14
Annex B (informative) Repeatability and reproducibility of the method. 15
B.1 Materials used in the interlaboratory comparison study . 15
B.2 Interlaboratory comparison results . 15
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 . 18
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prEN 17709:2023 (E)
Bibliography . 19

oSIST prEN 17709:2023
prEN 17709:2023 (E)
European foreword
This document (prEN 17709:2023) 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 CEN Enquiry.
This document will supersede CEN/TS 17709:2022.
This document has been prepared under a Standardization Request given to CEN by the European
Commission and the European Free Trade Association, and supports essential requirements of EU
Directive(s) / Regulation(s).
For relationship with EU Directive(s) / Regulation(s), see informative Annex ZA, which is an
integral part of this document.

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prEN 17709:2023 (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 fertilizing products (“FPR” or “Fertilising Products Regulation”).
This standardization request, presented as SR M/564and M/564 Amd1, 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 Plant 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 Plant 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 Plant Biostimulants, and will promote and
support commercialisation of the European Plant 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 Fertilising
Products Regulation.
This document is applicable to all Plant 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 Plant
Biostimulants.
Table 1 — Agro-ecological principles and the role played by Plant 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
oSIST prEN 17709:2023
prEN 17709:2023 (E)
use. It is the responsibility of the user to establish appropriate safety and health practices and to
ensure compliance with any national regulatory conditions.
IMPORTANT — It is absolutely essential that tests conducted in accordance with this document
be carried out by suitably trained staff.

oSIST prEN 17709:2023
prEN 17709:2023 (E)
1 Scope
This document was developed to provide the methodology for the enumeration and
determination of Azotobacter sp. [2] [3] in microbial plant biostimulants 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.
prEN 17702-1:— , Plant biostimulants — Sampling and sample preparation — Part 1: Sampling
prEN 17724:— , Plant biostimulants —Terminology
3 Terms and definitions
For the purposes of this document, the terms and definitions given in prEN 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. 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 prEN 17702-1:— shall be
prepared according to following procedure which takes into consideration the different
formulations of plant 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 [5].

Under preparation.
Under preparation.
Under preparation.
Under preparation.
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prEN 17709:2023 (E)
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 Solut
...