prEN 17718
(Main)Plant biostimulants - Determination of Rhizobium spp.
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 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 - Ugotavljanje prisotnosti in števila Rhizobium spp.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
oSIST prEN 17718:2023
01-maj-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
ICS:
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.
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oSIST prEN 17718:2023
DRAFT
EUROPEAN STANDARD
prEN 17718
NORME EUROPÉENNE
EUROPÄISCHE NORM
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.
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
© 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.
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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.
.14
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
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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
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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.
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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 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
industry.
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
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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.
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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.
1
EN 17702-1:— , Plant biostimulants — Sampling and sample preparation — Part 1: Sampling
2
EN 17724:— , Plant biostimulants — Terminology
3 Terms and definitions
2
For the purposes of this document, the terms and definitions given in EN 17724:— 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 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 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 spp. group.
1
Under preparation
2
Under preparation
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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.
2
[SOURCE: EN 17724:— , 3.2.2.7]
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
1
A representative sample of the product to be analysed as per the requirements of EN 17702-1:— shall
be prepared according to following procedure, which takes into consideration the different formulations
of plant 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 of maximum 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 of maximum 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 of maximum 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 distribution is optimal, with a
magnetic stirrer at half of maximum speed. If required, help the dispersion of the formulations with other
apparatus such as a laboratory paddle blender after having sieved (100 mesh sieve) the particles and
resuspend them in the same suspension [9].
4.2.6 Solid - Pellets, granules, microgranules - slow release - formulations
Dispense 25 g of sample (or more for low concentrated products) in 225 g of sterile PBS maintained at
room temperature, in a sterile bag and disperse them using a magnetic stirrer for 40 min at half of
maximum speed and then sieve in a 100 mesh sieve and, 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 [9].
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4.2.7 Solid - substrate
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 20 min or more until the distribution is optimal, with a
magnetic stirrer at half of maximum speed [9].
4.3 Serial dilution
The principle in 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, as live cells under the
microscope, as colonies that grow on plates from single cells, or estimated in the plant-infection technique
(with the principle that a single cell can multiply to initiate an infection). Serial dilution may be applied
to all kind of formulations. A 10-fold serial dilution is most often used (Figure 1), but if the number of
rhizobia is expected to be low then a lower number of dilutions may be adopted [8].
The diluent is the PBS (see Annex A, A.5).
4.4 Preparation of the culture media
The preparation and the culture media 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 this available media should have a quality program that
3
ensure the quality of the media they supply, according to EN ISO 11133:2014 [15]. Under these
conditions, the user/laboratory does not need to run additional testing on such media but shall ensure
the storage condition according to the ones recommended by the manufactures. 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
3
according to EN ISO 11133:2014 [15].
3
As impacted by EN ISO 11133:2014/A1:2018 and EN ISO 11133:2014/A2:2020
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Key
A Suspension of the sample
B Serial dilutions
C Petri plates
Figure 1 — Scheme
...
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