SIST EN 17718:2025
(Main)Plant biostimulants - Determination of Rhizobium spp.
Plant biostimulants - Determination of Rhizobium spp.
This European Standard establishes the methodology for the determination of Rhizobium spp. in plant biostimulant products in accordance to the future Regulation of EU fertilising products.
Pflanzen-Biostimulanzien - Bestimmung von Rhizobium spp.
Dieses Dokument wurde entwickelt, um die Methodik für die Zählung und Bestimmung von Rhizobium sp., Mesorhizobium sp., Ensifer sp. oder Bradyrhizobium sp. in Pflanzen-Biostimulanzienprodukten in Übereinstimmung mit der Verordnung (EU) 2019/1009 des Europäischen Parlaments und des Rates [1] bereitzustellen.
Biostimulants des végétaux - Détermination de Rhizobium spp.
Le présent document fournit la méthodologie de dénombrement et de détermination de Rhizobium sp., Mésorhizobium sp., Ensifer sp. ou Bradyrhizobium sp. dans les produits biostimulants des végétaux conformément au Règlement (UE) 2019/1009 du Parlement européen et du Conseil [1].
Rastlinski biostimulanti - Določanje Rhizobium spp.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
01-februar-2025
Nadomešča:
SIST-TS CEN/TS 17718:2023
Rastlinski biostimulanti - Določanje Rhizobium spp.
Plant biostimulants - Determination of Rhizobium spp.
Pflanzen-Biostimulanzien - Bestimmung von Rhizobium spp.
Biostimulants des végétaux - Détermination de Rhizobium spp.
Ta slovenski standard je istoveten z: EN 17718:2024
ICS:
65.080 Gnojila Fertilizers
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
EN 17718
EUROPEAN STANDARD
NORME EUROPÉENNE
November 2024
EUROPÄISCHE NORM
ICS 65.080 Supersedes CEN/TS 17718:2022
English Version
Plant biostimulants - Determination of Rhizobium spp.
Biostimulants des végétaux - Détermination de Pflanzen-Biostimulanzien - Bestimmung von
Rhizobium spp. Rhizobium 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
E UR OPÄISCHES 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 17718:2024 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 Rhizobium spp., Mesorhizobium spp., Ensifer spp.,
Bradyrhizobium spp. . 6
5 Species determination of Rhizobium spp., Mesorhizobium spp., Ensifer spp.,
Bradyrhizobium spp. via genetic analysis .10
Annex A (normative) Formula of culture media .13
Annex B (informative) Repeatability and reproducibility of the method .17
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 17718:2024) 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 17718:2022.
— 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;
— in 4.5 a general description of the colonies of the different genera has been included;
— in Clause A.5, the recipe of Phosphate Buffered Saline 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.
In accordance with Regulation (EU) 2019/1009 of the European Parliament and of the Council [1], only
Rhizobium spp. is required.
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 Rhizobiaceae
(Rhizobium spp., Mesorhizobium spp., Ensifer spp., Bradyrhizobium spp.).
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
EN 17724:2024, Plant biostimulants — Terminology
EN ISO 11133:2014 , Microbiology of food, animal feed and water — Preparation, production, storage and
performance testing of culture media (ISO 11133:2014)
3 Terms and definitions
For the purposes of this document, the terms and definitions given in EN 17724:2024 and the following
apply.
ISO and IEC maintain terminology 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
Rhizobium
beneficial bacteria belonging to the group named Rhizobiaceae, where the most relevant genera are
Rhizobium, Mesorhizobium, Ensifer and Bradyrhizobium [2] [3] [4] [5] [6]
Note 1 to entry: Genera belonging to this group are Rhizobium spp., Mesorhizobium spp., Ensifer spp.,
Bradyrhizobium spp.
Note 2 to entry: Legumes (Leguminosae or Fabaceae) are considered the second most cultivated crop, covering 14 %
of the total cultivated land worldwide and providing an important source of food for human beings via direct
consumption or indirect consumption via animal feed. Leguminosae can ensure high quality protein-rich food and
feed due to a special symbiosis they have with specific microorganisms present in the soil that can fix in the
rhizosphere, atmospheric nitrogen. Those microorganisms can account for 65 % of the total fixed nitrogen. Those
microorganisms were originally called Rhizobium. The word ‘rhizobium’ is actually derived from two Greek words
‘rhizo’ meaning root and ‘bium’ meaning home. Since the late nineteenth century, all legume root-nodule bacteria
An example of such a blend is a product with 2 claimed functions consisting of a non-microbial plant biostimulant
and an organic fertiliser composed of 1 kg/kg of plant biostimulant from seaweed.
As impacted by EN ISO 11133:2014/A1:2018 and EN ISO 11133:2014/A2:2020.
were placed in the genus ‘Rhizobium’. Gradually it was realized that they were rather diverse. A few slow-growing
rhizobia were split off into a new genus ‘Bradyrhizobium’. In the 1984 edition of Bergey’s Manual of Systematic
Bacteriology [9], all rhizobia were placed in the family Rhizobiaceae which included Bradyrhizobium and Rhizobium.
Since then, the number of bacterial genera representing rhizobia has increased rapidly; Rhizobia are plant root
nodule inhabiting, associative symbiotic, nitrogen fixing bacteria. Today the classification of the different Rhizobia
species is based on the sequence of the 16S rDNA sequence comparison and physiological and biochemical
properties. Considering that taxonomy and phylogeny of bacteria are in continuous evolution and considering that
any current classification scheme is subject to future revision and considering moreover that most of the species in
the phylum Pseudomonadota, class Alphaproteobacteria, family Rhizobiaceae are in the genera Rhizobium,
Mesorhizobium, Ensifer and Bradyrhizobium, for the purpose of this document we will consider the above-
mentioned genera as referring to the Rhizobiaceae group.
Note 3 to entry: Other nodule-forming bacteria belong to the genus Frankia and interact with
non-leguminous species, including woody species of the Betulaceae and Casuarinaceae families. Such bacteria
should be included in the general wording of “Rhizobium” according to the terms of this document.
[SOURCE: EN 17724:2024, 3.2.2.14]
4 Enumeration of Rhizobium spp., Mesorhizobium spp., Ensifer spp.,
Bradyrhizobium spp.
4.1 General
This procedure is meant to determine the number of colony-forming units (CFU) of the above-mentioned
bacteria, per gram or per millilitre. The method, in order to be fast, cheap and 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 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
25 ml of sample (or more for low concentrated products) 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 maximum speed [7].
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 maximum speed [7].
4.2.4 Solid wettable powd
...
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