CEN/TS 17718:2022

Plant biostimulants - Determination of Rhizobium spp.

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 the Regulation (EU) 2019/1009 of the European Parliament and of the Council [1].

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.

Ta dokument zagotavlja metodologijo za štetje in določanje bakterij Rhizobium sp., Mesorhizobium sp., Ensifer sp. ali Bradyrhizobium sp. v rastlinskih biostimulantih v skladu z Uredbo (EU) 2019/1009 Evropskega parlamenta in Sveta [1].

General Information

• Status: Withdrawn
• Publication Date: 22-Mar-2022
• Withdrawal Date: 13-Apr-2025
• ICS: 65.080 - Fertilizers
• Technical Committee: CEN/TC 455 - Plant Biostimulants and Agricultural Micro-Organisms
• Drafting Committee: CEN/TC 455/WG 3 - Pathogenic and non-pathogenic microorganisms
• Current Stage: 9960 - Withdrawal effective - Withdrawal
• Start Date: 27-Nov-2024
• Completion Date: 14-Apr-2025
• Directive: 2019/1009 - Regulation (EU) 2019/1009 of the European Parliament and of the Council of 5 June 2019 laying down rules on the making available on the market of EU fertilising products and amending Regulations (EC) No 1069/2009 and (EC) No 1107/2009 and repealing Regulation (EC) No 2003/2003
• Mandate: M/564 - Fertilising products in support of Regulation (EU) 2019/1009
• Ref Project: SIST-TS CEN/TS 17718:2023 - Plant biostimulants - Determination of Rhizobium spp

Relations

• Replaced By: EN 17718:2024 - Plant biostimulants - Determination of Rhizobium spp.
• Effective Date: 15-Feb-2023

Standards Content (Sample)

SLOVENSKI STANDARD
01-februar-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: CEN/TS 17718:2022
ICS:
65.080 Gnojila Fertilizers
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

CEN/TS 17718
TECHNICAL SPECIFICATION
SPÉCIFICATION TECHNIQUE
March 2022
TECHNISCHE SPEZIFIKATION
ICS 65.080
English Version
Plant biostimulants - Determination of Rhizobium spp.
Biostimulants des végétaux - Détermination de Biostimulanzien für die pflanzliche Anwendung -
Rhizobium spp. Bestimmung von Rhizobium spp.
This Technical Specification (CEN/TS) was approved by CEN on 3 January 2022 for provisional application.

The period of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested to
submit their comments, particularly on the question whether the CEN/TS can be converted into a European Standard.

CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS
available promptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in
parallel to the CEN/TS) until the final decision about the possible conversion of the CEN/TS into an EN is reached.

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, Turkey and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATIO N

E U R OP ÄI SCHES KOMITEE FÜR NORMUN G

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2022 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TS 17718:2022 E
worldwide for CEN national Members.

Contents Page
European foreword . 3
Introduction . 4
1 Scope . 6
2 Normative references . 6
3 Terms and definitions . 6
4 Enumeration of Rhizobium sp., Mesorhizobium sp., Ensifer sp., Bradyrhizobium sp. . 7
4.1 General . 7
4.2 Sample preparation . 7
4.2.1 General . 7
4.2.2 Liquid – water-based formulations . 7
4.2.3 Liquid – oil-based, emulsifiable concentrate (EC) formulations . 7
4.2.4 Solid – wettable powder (WP) formulations . 7
4.2.5 Solid – water dispersible granules (WDG) formulations . 7
4.2.6 Solid – pellets, granules, microgranules - slow release - formulations . 7
4.2.7 Solid – substrate . 8
4.3 Serial dilution . 8
4.4 Plate counts of rhizobia in sterile diluent . 8
4.5 Spread-plate counting . 9
......... 9
4.6 Growth Media .
4.7 Calculation . 9
5 Species determination of Rhizobium sp., Mesorhizobium sp., Ensifer sp.,
Bradyrhizobium 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 .10
5.2.4 Partial PCR Amplification of the 16S rRNA Genes .11
Annex A (informative) Formula of culture media .12
Bibliography .16

European foreword
This document (CEN/TS 17718:2022) has been prepared by Technical Committee CEN/TC 455 “Plant
Biostimulants”, the secretariat of which is held by AFNOR.
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 has been prepared under a Standardization Request given to CEN by the European
Commission and the European Free Trade Association.
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 announce this Technical Specification: 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, Turkey and the United
Kingdom.
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 the European Parliament and of the Council 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 M/564, 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 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.
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 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 biostimulants.
Table 1 — Agro-ecological principles and the role played by biostimulants
Increase biodiversity
By improving soil microorganism quality/quantity
Reinforce biological regulation and interactions
By reinforcing plant-microorganism interactions
— symbiotic exchanges i.e. Mycorrhizae
— symbiotic exchanges i.e. Rhizobiaceae/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
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 Rhizobium sp.,
Mesorhizobium sp., Ensifer sp., or Bradyrhizobium sp. in plant biostimulant products 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.
CEN/TS 17702-1, Plant biostimulants - Sampling and sample preparation - Part 1: Sampling
CEN/TS 17724, Plant biostimulants - Terminology
3 Terms and definitions
For the purposes of this document, the terms and definitions given in CEN/TS 17724 and the following
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
3.1
rhizobium
beneficial bacteria belonging to the group named rhizobia, where the most relevant genera are
Rhizobium, Mesorhizobium, Ensifer and Bradyrhizobium
Note 1 to entry: Rhizobium belonging to this group are Rhizobium sp., Mesorhizobium sp., Ensifer sp.,
Bradyrhizobium sp.
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 a 65 % of the total fixed nitrogen. Those
microorganisms have originally been 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 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, 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 the sequence of the 16S rDNA sequence comparison and physiological and biochemical
properties. Considering that taxonomy and phylogeny of bacteria is in continuous evolution and considering that
any current classification scheme is subject to future revision and considering moreover that most of the rhizobial
species in the alpha-proteobacteria class of phylum proteobacteria in Rhizobiaceae family are in the Rhizobium,
Mesorhizobium, Ensifer, or Bradyrhizobium genera, for the purpose of this document we will consider the above-
mentioned genera as referring to the Rhizobium sp. 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 families Betulaceae and Casuarinaceae. Such bacteria
should be included in the general wording of “Rhizobium” according to the terms of this document.
[SOURCE: CEN/TS 17724:2022, 3.2.2.7]
4 Enumeration of Rhizobium sp., Mesorhizobium sp., Ensifer sp.,
Bradyrhizobium sp.
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 CEN/TS 17702-1 will
be prepared according to following procedure, which takes into consideration the different formulations
of biostimulants based products.
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
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 [9].
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 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 [9].
4.2.4 Solid – wettable powder (WP) formulations
Dispense 25 g of sample (or more for low concentrated products) in 225 ml of sterile 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 speed [9].
4.2.5 Solid – water dispersible granules (WDG) formulations
Dispense 25 g of sample (or more for low concentrated products) in 275 g of sterile PBS maintained at
room temperature in a flask and shake for 40 min or more until the distributio
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