Plant biostimulants - Determination of Rhizobium spp.

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 fertilizing 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.

Pflanzen-Biostimulanzien - Bestimmung von Rhizobium spp.

Dieses Dokument stellt die Methodik für die Zählung und Bestimmung von Rhizobiaceae (Rhizobium spp., Mesorhizobium spp., Ensifer spp., Bradyrhizobium spp.) bereit.
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 de Rhizobium spp.

Le présent document fournit la méthodologie de dénombrement et de détermination des Rhizobiaceae (Rhizobium spp., Mésorhizobium spp., Ensifer spp., Bradyrhizobium spp.).
Le présent document est 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 pourcentage 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 pourcentage le plus élevé du mélange, la Norme européenne relative au composant représentant le pourcentage 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 Rhizobium spp.

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Standards Content (Sample)

oSIST prEN 17718:2023
Rastlinski biostimulanti - Ugotavljanje prisotnosti in števila 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: prEN 17718
65.080 Gnojila Fertilizers
oSIST prEN 17718:2023 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

oSIST prEN 17718:2023
oSIST prEN 17718:2023
prEN 17718
April 2023
ICS 65.080 Will supersede 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 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.



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 17718:2023 E
worldwide for CEN national Members.

oSIST prEN 17718:2023
prEN 17718:2023 (E)
Contents Page
European foreword . 4
Introduction . 5
1 Scope . 7
2 Normative references . 7
3 Terms and definitions . 7
4 Enumeration of Rhizobium spp., Mesorhizobium spp., Ensifer spp.,
Bradyrhizobium spp. . 8
4.1 General . 8
4.2 Sample preparation . 8
4.2.1 General . 8
4.2.2 Liquid - Water based formulations . 8
4.2.3 Liquid - Oil based, 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 . 9
4.3 Serial dilution . 9
4.4 Preparation of the culture media . 9
4.5 Plate counts of Rhizobia in sterile diluent .10
4.6 Spread-plate counting .10
4.7 Growth Media .11
4.8 Calculation .11
5 Species determination of Rhizobium spp., Mesorhizobium spp., Ensifer spp.,
Bradyrhizobium spp. via genetic analysis .11
5.1 General .11
5.2 Preparation of the sample for the genomic DNA extraction .12
5.2.1 Isolation and preparation of the microorganism .12
5.2.2 Sample concentration .12
5.2.3 DNA extraction and storage .12
5.2.4 Partial PCR Amplification of the 16S rRNA Genes .12
Annex A (informative) Formula of culture media .14
A.1 YT (Tryptone-Yeast Media) for Rhizobium spp., Mesorhizobium spp. and Ensifer spp.
A.2 AYEM (Modified Yeast extract mannitol agar media) alternative media for Rhizobium
spp. .14
A.3 R2A (Reasoner's 2A Agar Media) for Bradyrhizobium spp. .15
A.4 Congo Red stock solution for AYEM Media .16
A.5 0,1 M Phosphate Buffer Saline (PBS).16
A.6 Nutrient broth .17
Annex B (informative) Repeatability and reproducibility of the method .18
B.1 Materials used in the interlaboratory comparison study .18
B.2 Interlaboratory comparison results .18
oSIST prEN 17718:2023
prEN 17718:2023 (E)
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
oSIST prEN 17718:2023
prEN 17718:2023 (E)
European foreword
This document (prEN 17718:2023) 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 17718:2022.
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.
oSIST prEN 17718:2023
prEN 17718:2023 (E)
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 standardization request,
presented as SR M/564 and M/564 Amd1, also contributes to the Communication on “Innovating for
Sustainable Growth: A Bio economy for Europe”. Working Group 5 “Labelling and denominations”, was
created to develop a work program as part of this standardization request.
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 biostimulant
Plant biostimulants used in agriculture can be applied in multiple ways: on soil, on plants, 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 group Rhizobia.
Table 1 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
— e.g. 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
oSIST prEN 17718:2023
prEN 17718:2023 (E)
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.
oSIST prEN 17718:2023
prEN 17718:2023 (E)
1 Scope
This document provides the methodology for the enumeration and determination of Rhizobium sp.,
Mesorhizobium sp., Ensifer sp., or Bradyrhizobium sp. in plant biostimulant products in accordance with
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.
EN 17702-1:— , Plant biostimulants — Sampling and sample preparation — Part 1: Sampling
EN 17724:— , Plant biostimulants — Terminology
3 Terms and definitions
For the purposes of this document, the terms and definitions given in EN 17724:— and the following
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
• ISO Online browsing platform: available at
• IEC Electropedia: available at
beneficial bacteria belonging to the group named Rhizobia, where the most relevant genera are
Rhizobium, Mesorhizobium, Ensifer and Bradyrhizobium [2] [3] [4] [6] [7]
Note 1 to entry: Rhizobium belonging to this group are Rhizobium spp., Mesorhizobium spp., Ensifer spp.,
Bradyrhizobium spp.
Note to entry 2: Legumes (Leguminosae or Fabaceae) are considered the second most cultivated crop, covering
14 % of the total cultivated land worldwide an

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