SIST EN 17713:2025
(Main)Plant biostimulants - Determination of Azospirillum spp.
Plant biostimulants - Determination of Azospirillum spp.
This document provides the methodology for the enumeration and determination of Azospirillum
spp. in microbial plant biostimulants.
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 percentage in the blend by mass or volume, or in the case of liquid
form by dry mass. If Plant Biostimulants is not the highest percentage in the blend, the European
Standard for the highest percentage 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 formulations1, the user decides which standard to apply.
Pflanzen-Biostimulanzien - Bestimmung von Azospirillum spp.
Dieses Dokument stellt die Methodik für die Zählung und Bestimmung von Azospirillum spp. in Pflanzen-Biostimulanzien in Übereinstimmung mit der Verordnung (EU) 2019/1009 des Europäischen Parlaments und des Rates [1] bereit.
Biostimulants des végétaux - Détermination d'Azospirillum spp.
Le présent document fournit la méthodologie de dénombrement et de détermination d’Azospirillum spp. dans les produits biostimulants des végétaux conformément au Règlement (UE) 2019/1009 du Parlement Européen et du Conseil [1].
Rastlinski biostimulanti - Določanje Azospirillum spp.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
01-februar-2025
Nadomešča:
SIST-TS CEN/TS 17713:2023
Rastlinski biostimulanti - Določanje Azospirillum spp.
Plant biostimulants - Determination of Azospirillum spp.
Pflanzen-Biostimulanzien - Bestimmung von Azospirillum spp.
Biostimulants des végétaux - Détermination d'Azospirillum spp.
Ta slovenski standard je istoveten z: EN 17713:2024
ICS:
65.080 Gnojila Fertilizers
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
EN 17713
EUROPEAN STANDARD
NORME EUROPÉENNE
November 2024
EUROPÄISCHE NORM
ICS 65.080 Supersedes CEN/TS 17713:2022
English Version
Plant biostimulants - Determination of Azospirillum spp.
Biostimulants des végétaux - Détermination Pflanzen-Biostimulanzien - Bestimmung von
d'Azospirillum spp. Azospirillum 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 NORMALISATI O 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 17713:2024 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 Azospirillum spp. . 6
5 Species determination of Azospirillum spp. via genetic analysis . 8
Annex A (normative) Formula of the culture media . 11
Annex B (normative) Table of MPN values [4] . 14
Annex C (informative) Repeatability and reproducibility data . 16
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 . 20
Bibliography . 21
European foreword
This document (EN 17713:2024) 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 17713:2022.
CEN/TS 17713:2022:
— the European foreword has been updated;
— the Introduction has been updated;
— the Bibliography has been re-numbered;
— in Annex A, Buffered Peptone Water has been added as diluent for the enumeration;
— the recipe of Phosphate Buffered Saline (A5) has been modified;
— 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.
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.
IMPORTANT — It is absolutely essential that tests conducted in accordance with this document
be carried out by suitably trained staff.
1 Scope
This document provides the methodology for the enumeration and determination of Azospirillum
spp. in microbial plant biostimulants.
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 percentage in the blend by mass or volume, or in the case of liquid
form by dry mass. If Plant Biostimulants is not the highest percentage in the blend, the European
Standard for the highest percentage 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
3 Terms and definitions
For the purposes of this document, the following terms and definitions 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/
3.1
Azospirillum spp.
Gram-negative bacteria that belong to the alphaproteobacterial phylum
Note 1 to entry: Azospirillum is a Gram-negative, microaerophilic, non-fermentative and nitrogen-
fixing bacterial genus. Azospirillum spp. are Gram-negative, do not form spores, and have a slightly-twisted
oblong-rod shape. Azospirillum have at least one flagellum and sometimes multiple flagella. The genus has
about 20 species, the relationships between all the species have not been resolved in detail, however they
most likely constitute a coherent group.
Note 2 to entry: Azospirillum bacteria are aerobic non-fermentative chemoorganotrophs, vibroid, they
produce several hormones, mainly auxins (not described for all species yet), and most of them are
diazotrophic (fix atmospheric nitrogen gas into a more usable form).
[SOURCE: EN 17724:2024, 3.2.2.1 [2]]
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 Enumeration of Azospirillum spp.
4.1 General
This procedure is meant to determine the number of colony-forming units (CFU) of Azospirillum
spp., per gram or per millilitre. The method, in order to be fast, cheap and repeatable, is based on
serial dilutions and plating [3].
4.2 Sample preparation
4.2.1 General
A representative sample of the product to be analyzed as per 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.
4.2.2 Liquid (water-based) formulations
Dispense 25 ml of sample (or more for low concentrated products) in 225 ml of sterile phosphate
buffer saline (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 of maximum speed [3].
4.2.3 Liquid (oil–based) emulsifiable concentrate (EC) formulations
Dispense 25 ml of sample (or more for low concentrated products) in 225 ml of sterile phosphate
buffer saline (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 of maximum speed [3].
4.2.4 Solid wettable powder (WP) formulations
Dispense 25 g of sample (or more for low concentrated products) in 225 ml of sterile phosphate
buffer saline (PBS) maintained at room temperature in a flask and shake for 20 min or more until
the distribution is optimal, with a magnetic stirrer at half of maximum speed [3].
4.2.5 Solid water dispersible granules (WDG) formulations
Dispense 25 g (or more for low concentrated products) of sample in 275 ml of sterile phosphate
buffer saline (PBS) maintained at room temperature in a flask and shake for 40 min or more until
the distribution is optimal, with a magnetic stirrer at half of 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 [3].
4.2.6 Solid pellets, granules, microgranules (slow release) formulations
Dispense 25 g (or more for low concentrated products) of sample in 225 ml of sterile phosphate
buffer saline (PBS) maintained at room temperature in a sterile bag and disperse them using a
magnetic stirrer for 40 min at half of maximum speed. Then sieve the suspension in a 100 mesh
sieve (150 µm sieve corresponding to a 100 mesh sieve) and if material remains in the sieve,
repeat the process for a maximum of three times. Pay attention to the volume of buffer used to
make the exact final calculation [3].
4.2.7 Solid substrate
Dispense 25 g (or more for low concentrated products) of sample in 225 ml of sterile phosphate
buffer saline (PBS) maintained at room temperature in a flask and shake for 20 min or more until
the distribution is optimal, with a magnetic stirrer at half of maximum speed [3].
4.3 Requirements (apparatus)
— Graduated pipettes (1 ml and 10 ml);
— Dilution bottles or flasks;
— Petri dishes clear, uniform, flat-bottomed;
— Hot air oven - Capable of giving uniform and adequate temperatures, equipped with a
thermometer calibrated to read up to (250 ± 1) °C and with vents suitably located to ensure
prompt and uniform heating;
— Autoclave/steam sterilizer;
— Incubator;
— Hand tally or mechanical counting device;
— pH meter, having an accuracy of calibration of ±0,1 pH unit at 25 °C.
4.4 Serial dilution
The principle in counting bacteria by dilution is to serially dilute them to
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