EN 17709:2024
(Main)Plant biostimulants - Determination of Azotobacter spp.
Plant biostimulants - Determination of Azotobacter spp.
This document was developed to provide the methodology for the enumeration and determination of Azotobacter spp. [2] [3] in microbial plant biostimulants in accordance with the Regulation (EU) 2019/1009 of the European Parliament and of the Council [1].
This document is applicable to the blends of fertilizing products where a blend is a mix of at least two of the following component EU fertilising products categories: Fertilizers, Liming Materials, Soil Improvers, Growing Media, Plant Biostimulants and where the following category Plant Biostimulants is the highest % in the blend by mass or volume, or in the case of liquid form by dry mass. If Plant Biostimulants is not the highest % in the blend, the European Standard for the highest % of the blend applies. In case a blend of fertilizing products is composed of components in equal quantity or in case the component EU fertilising products used for the blend have identical formulations , the user decides which standard to apply.
Pflanzen-Biostimulanzien - Bestimmung von Azotobacter spp.
Dieses Dokument wurde entwickelt, um die Methodik für die Zählung und Bestimmung von Azotobacter spp. [2] [3] in mikrobiellen Pflanzen-Biostimulanzien in Übereinstimmung mit der Verordnung (EU) 2019/1009 des Europäischen Parlaments und des Rates [1] bereitzustellen.
Dieses Dokument ist für die Mischungen von Düngeprodukten anwendbar, in denen eine Mischung aus mindestens zwei der folgenden Kategorien von Komponenten-EU Düngeprodukten besteht: Düngemittel, Kalkdünger, Bodenverbesserungsmittel, Kultursubstrate, Pflanzen-Biostimulanzien und wenn die folgende Kategorie Pflanzen-Biostimulanzien prozentual den höchsten Massen- oder Volumenanteil in der Mischung bzw., bei flüssiger Form, den höchsten Trockenmasseanteil aufweist. Wenn Pflanzen-Biostimulanzien nicht den höchsten prozentualen Anteil in der Mischung ausmachen, ist die für den höchsten prozentualen Anteil geltende Europäische Norm anwendbar. Falls eine Düngemittelmischung aus Komponenten gleicher Menge besteht oder falls die für die Mischung verwendeten Komponenten-EU Düngeprodukte identische Formulierungen haben, entscheidet der Anwender, welche Norm angewendet wird.
Biostimulants des végétaux - Détermination d'Azotobacter spp.
Le présent document a été élaboré pour fournir la méthodologie de dénombrement et de détermination d’Azotobacter spp. [2] [3] dans les biostimulants microbiens des végétaux conformément au Règlement (UE) 2019/1009 du Parlement européen et du Conseil [1].
Le présent document est également applicable aux mélanges de fertilisants constitués d’au moins deux composants appartenant aux catégories de fertilisants UE suivantes : engrais, amendements minéraux basiques, amendements du sol, supports de culture, biostimulants des végétaux et, dans lesquels la catégorie Biostimulants des végétaux représente le % le plus élevé du mélange en masse ou en volume, ou dans le cas d’un liquide, en masse sèche. Si les biostimulants des végétaux ne représentent pas le % le plus élevé du mélange, la Norme européenne relative au composant représentant le % le plus élevé du mélange s’applique. Dans le cas où un mélange de fertilisants contient des composants en quantité égale ou dans le cas où les composants des fertilisants UE utilisés pour le mélange ont des formulations identiques, l’utilisateur décide quelle norme appliquer.
Rastlinski biostimulanti - Določanje Azotobacter spp.
Ta dokument podaja metodologijo za štetje in določanje bakterij
Azotobacter spp. [2] [3] v mikrobnih rastlinskih biostimulantih v skladu z Uredbo (EU) 2019/1009 Evropskega parlamenta in Sveta [1].
Ta dokument se uporablja za mešanice sredstev za gnojenje, ki vsebujejo najmanj dve od naslednjih kategorij sredstev za gnojenje EU: gnojila, sredstva za apnenje, izboljševalci tal, rastni substrati in rastlinski biostimulanti, pri čemer rastlinski biostimulanti predstavljajo največji masni ali prostorninski odstotek mešanice ali, v primeru tekoče oblike, odstotek suhe mase. Če rastlinski biostimulanti ne predstavljajo največjega odstotka v mešanici, se uporablja evropski standard za največji odstotek mešanice. Če vsebuje mešanica sredstev za gnojenje sestavine v enakih količinah ali če imajo posamezna sredstva za gnojenje EU, uporabljena za mešanico, enake formulacije1, standard za uporabo izbere uporabnik.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
01-februar-2025
Nadomešča:
SIST-TS CEN/TS 17709:2023
Rastlinski biostimulanti - Določanje Azotobacter spp.
Plant biostimulants - Determination of Azotobacter spp.
Pflanzen-Biostimulanzien - Bestimmung von Azotobacter spp.
Biostimulants des végétaux - Détermination d'Azotobacter spp.
Ta slovenski standard je istoveten z: EN 17709:2024
ICS:
65.080 Gnojila Fertilizers
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
EN 17709
EUROPEAN STANDARD
NORME EUROPÉENNE
November 2024
EUROPÄISCHE NORM
ICS 65.080 Supersedes CEN/TS 17709:2022
English Version
Plant biostimulants - Determination of Azotobacter spp.
Biostimulants des végétaux - Détermination Pflanzen-Biostimulanzien - Bestimmung von
d'Azotobacter spp. Azotobacter spp.
This European Standard was approved by CEN on 26 August 2024.
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. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists 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.
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
© 2024 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 17709:2024 E
worldwide for CEN national Members.
Contents Page
European foreword . 3
Introduction . 4
1 Scope . 5
2 Normative references . 5
3 Terms and definitions . 5
4 Enumeration of Azotobacter spp. . 5
4.1 General . 5
4.2 Sample preparation . 5
4.2.1 General . 5
4.2.2 Liquid – water-based formulations . 6
4.2.3 Liquid – oil-based, emulsifiable concentrate (EC) formulations . 6
4.2.4 Solid – wettable powder (WP) formulations . 6
4.2.5 Solid – water-dispersible granules (WDG) formulations . 6
4.2.6 Solid – pellets, granules, microgranules (slow release) formulations. 6
4.2.7 Solid – substrate . 6
4.3 Plate counts of Azotobacter spp. in sterile diluent with serial dilutions . 6
4.4 Preparation of the culture media . 7
4.5 Spread plate counting with Ashby’s Sucrose Agar [5] (for the different media, see
Annex A) . 7
4.6 Plate counts of Azotobacter spp. in sterile diluent . 8
4.7 Calculation . 8
5 Species determination of Azotobacter spp. via genetic analysis . 8
5.1 General . 8
5.2 Preparation of the sample for the genomic DNA extraction . 8
5.2.1 Isolation and preparation of the microorganism . 8
5.2.2 Sample concentration . 9
5.2.3 DNA extraction and storage . 9
5.2.4 Partial PCR amplification of the 16S rRNA genes . 9
Annex A (normative) Composition and preparation of culture media and reagents .11
A.1 Ashby’s Sucrose Agar medium .11
A.2 Procedure for preparing nutrient broth medium .12
A.3 Procedure for preparing 0,1 M Phosphate Saline (PS) .12
A.4 Procedure for preparing 0,1 M Buffered peptone water .12
Annex B (informative) Repeatability and reproducibility of the method .13
B.1 Materials used in the interlaboratory comparison study .13
B.2 Interlaboratory comparison results .14
Annex ZA (informative) Relationship of this European Standard and the essential
requirements of Regulation (EU) 2019/1009 available on the market of EU fertilising
products aimed to be covered .16
Bibliography .17
European foreword
This document (EN 17709:2024) has been prepared by Technical Committee CEN/TC 455 “Plant
biostimulants”, the secretariat of which is held by AFNOR.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by May 2025, and conflicting national standards shall be
withdrawn at the latest by May 2025.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document supersedes CEN/TS 17709:2022.
— the European foreword has been updated;
— the Introduction has been updated;
— in Annex A, buffered peptone water has been added as diluent for the enumeration;
— in 4.5, a general description of the colonies of the different genera has been included;
— the recipe of phosphate buffered saline has been modified;
— the Bibliography has been updated;
— Annex ZA has been added.
This document has been prepared under a standardization request addressed to CEN by the European
Commission. The Standing Committee of the EFTA States subsequently approves these requests for its
Member States.
For the relationship with EU Legislation, see informative Annex ZA, which is an integral part of this
document.
Any feedback and questions on this document should be directed to the users’ national standards body.
A complete listing of these bodies can be found on the CEN website.
According to the CEN-CENELEC Internal Regulations, the national standards organisations of the
following countries are bound to implement this European Standard: 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 the United
Kingdom.
Introduction
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 [1] laying down rules on the making available on the market
of EU fertilising products (“FPR” or “Fertilising Products Regulation”).
This standardization request, presented as SR M/564 and relevant amendments, also contributes to the
Communication on “Innovating for Sustainable Growth: A Bio economy for Europe”. 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 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.
1 Scope
This document was developed to provide the methodology for the enumeration and determination of
Azotobacter spp. [2] [3] in microbial plant biostimulants in accordance with the Regulation (EU)
2019/1009 of the European Parliament and of the Council [1].
This document is applicable to the blends of fertilizing products where a blend is a mix of at least two of
the following component EU fertilising products categories: Fertilizers, Liming Materials, Soil Improvers,
Growing Media, Plant Biostimulants and where the following category Plant Biostimulants is the highest
% in the blend by mass or volume, or in the case of liquid form by dry mass. If Plant Biostimulants is not
the highest % in the blend, the European Standard for the highest % of the blend applies. In case a blend
of fertilizing products is composed of components in equal quantity or in case the component EU
fertilising products used for the blend have identical formulations , the user decides which standard to
apply.
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.
EN 17702-1:2024, Plant biostimulants — Sampling and sample preparation — Part 1: Sampling
EN 17724:2024, Plant biostimulants —Terminology
3 Terms and definitions
For the purposes of this document, the terms and definitions given in EN 17724:2024 apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https://www.iso.org/obp
— IEC Electropedia: available at https://www.electropedia.org/
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, meeting the requirements of EN 17702-1:2024
shall be prepared according to the following procedure, which takes into consideration the different
formulations of plant biostimulants.
An example of such a blend is a product with 2 claimed functions consisting of a non-microbial plant biostimulant
and an organic fertilizer composed of 1kg/kg of plant biostimulant from seaweed.
4.2.2 Liquid – water-based formulations
25 ml of sample (or more for low concentrated products) shall be dispensed in 225 ml of sterile diluent
(see Annex A), maintained at room temperature, in a flask, and shaken for 10 min or more until the
distribution is optimal, with a magnetic stirrer at half of the maximum speed [5].
4.2.3 Liquid – oil-based, emulsifiable concentrate (EC) formulations
25 ml of sample (or more for low concentrated products) shall be dispensed in 225 ml of sterile diluent
(see Annex A), maintained at room temperature, in a flask, and shaken for 10 min or more until the
distribution is optimal, with a magnetic stirrer at half of the maximum speed [5].
4.2.4 Solid – wettable powder (WP) formulations
25 g of sample (or more for low concentrated products) shall be dispensed in 225 ml of sterile Phosphate
Buffer Solution (PBS), maintained at room temperature, in a flask, and shaken for 20 min or more until
the distribution is optimal, with a magnetic stirrer at half of the maximum speed [5].
4.2.5 Solid – water-dispersible granules (WDG) formulations
25 g of sample (or more for low concentrated products) shall be dispensed in 225 ml of sterile diluent
(see Annex A), maintained at room temperature, in a flask, and shaken for 40 min or more until the
distribution is optimal, with a magnetic stirrer at half of the maximum speed. If required, help the
dispersion of the formulations with another apparatus such as a laboratory paddle blender after having
sieved (150 µm sieve corresponding to a 100 mesh sieve) the particles and resuspend them in the same
suspension [5].
4.2.6 Solid – pellets, granules, microgranules (slow release) formulations
25 g of sample (or more for low concentrated products) shall be dispensed in 225 ml of sterile diluent
(see Annex A), maintained at room temperature, in a sterile bag, and dispersed using a magnetic stirrer
for 40 min at half of the maximum speed and then sieved in a 150 µm sieve corresponding to a 100 mesh
sieve. If material remains in the sieve, repeat the process for a maximum of three times. Put attention to
all the buffer used to make the exact final calculation [5].
4.2.7 Solid – substrate
25 g of sample (or more for low concentrated products) shall be dispensed in 225 ml of sterile diluent
(see Annex A), maintained at room temperature, in a flask, and shaken for 20 min or more until the
distribution is optimal, with a magnetic stirrer at half of the maximum speed [5].
4.3 Plate counts of Azotobacter spp. in sterile diluent with serial dilutions
The principle of counting bacteria by dilution is to serially dilute them to reduce the bacterial density to
the level where individual cells can be differentiated. This may be, for example, live cells under the
microscope, colonies that grow on plates from single cells, or cells estimated in the plant-infection
technique (considering that a single cell can multiply to initiate an infection). Serial dilution can be
applied to all kinds of formulations. A 10-fold serial dilution is most often used (Figure 1) but if the
number of Azotobacter spp. cells is expected to be low, then a lower number of dilutions can be adopted.
A sample of the product is shaken in a bulk diluent (see Annex A) which represents the first level of
dilution. This is then serially diluted with a sample at each level of dilution directly plated.
4.4 Preparation of the culture media
The preparation and the composition of N-free agar medium (Ashby’s Sucrose Agar) is described in
Annex A. The preparation and performance of culture media is a fundamental step to ensure the integrity
of microbiological examination. When ready-to-use media are used, the manufacturers of these available
media should have a quality program that ensures the quality of the media they supply, according to
EN ISO 11133:2014 . Under these conditions, the user/laboratory does not need to run additional testing
on such media but shall ensure storage conditions according to the ones recommended by the
manufacturers. For diluents and media prepared by the user/laboratory directly from commercially
available dehydrated formulations and/or from basic individual components, the performance of these
diluents/media should be evaluated according to EN ISO 11133:2014.
Key
A product suspended in sterile diluent
−8
B prepare dilution series at 10
C 3 replicates of Petri dishes with media
Figure 1 — Scheme of a serial dilution series
4.5 Spread plate counting with Ashby’s Sucrose Agar [5] (for the different media, see
Annex A)
The steps of the procedure are the following:
−5 −6 −7
1) inoculate 0,1 ml of the serial dilutions desired (e.g. 10 , 10 and 10 ) on the surface of the culture
medium in Petri dishes (Figure 1);
2) spread the 0,1 ml aliquot over the cult
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