SIST EN 17718:2025
(Main)Plant biostimulants - Determination of Rhizobium spp.
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 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.
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.
Ta dokument podaja metodologijo za štetje in določanje družine bakterij Rhizobiaceae (Rhizobium spp., Mesorhizobium spp., Ensifer spp., Bradyrhizobium spp.).
Ta dokument se uporablja za mešanice sredstev za gnojenje, ki vsebujejo najmanj dve od naslednjih kategorij sredstev za gnojenje EU: gnojila, sredstva za apnenje, izboljševalci tal, rastni substrati in rastlinski biostimulanti, pri čemer rastlinski biostimulanti predstavljajo največji masni ali prostorninski odstotek mešanice ali, v primeru tekoče oblike, odstotek suhe mase. Če rastlinski biostimulanti ne predstavljajo največjega odstotka v mešanici, se uporablja evropski standard za največji odstotek mešanice. Če vsebuje mešanica sredstev za gnojenje sestavine v enakih količinah ali če imajo posamezna sredstva za gnojenje EU, uporabljena za mešanico, enake formulacije1, standard za uporabo izbere uporabnik.
General Information
- Status
- Published
- Public Enquiry End Date
- 02-Jun-2023
- Publication Date
- 06-Jan-2025
- Technical Committee
- KAT - Soil quality
- Current Stage
- 6060 - National Implementation/Publication (Adopted Project)
- Start Date
- 20-Dec-2024
- Due Date
- 24-Feb-2025
- Completion Date
- 07-Jan-2025
Relations
- Effective Date
- 01-Feb-2025
Overview
EN 17718:2024 - "Plant biostimulants - Determination of Rhizobium spp." (CEN) specifies validated laboratory methods for the enumeration and identification of Rhizobiaceae in plant biostimulant products. The standard covers Rhizobium, Mesorhizobium, Ensifer and Bradyrhizobium and is intended to support conformity with the EU Fertilising Products Regulation (Regulation (EU) 2019/1009) for plant biostimulant products and applicable blends.
Key topics and requirements
- Scope and applicability
- Applies to plant biostimulants and blends where plant biostimulants represent the highest fraction by mass, volume or (for liquids) dry mass. If another category is dominant, the relevant standard for that category applies.
- Enumeration method
- Quantification of colony-forming units (CFU) per gram or millilitre using serial dilutions and spread-plate techniques.
- Typical sample preparation: 25 ml (liquid) or 25 g (solid) in 225 ml/g sterile diluent with magnetic stirring; times vary by formulation (10–40 min) and include specific guidance for emulsifiable concentrates, wettable powders, water-dispersible granules and pellets.
- Diluent: Phosphate Buffered Saline (PBS) is specified (Annex A); Buffered Peptone Water (BPW) has been added as a diluent option.
- Spread plating: inoculate 0.1 ml aliquots, use replicate plates, incubate at approx. 28 °C ± 2 °C for 2–8 days (species-dependent), and count plates with well-separated colonies (recommended 30–300 CFU for statistical robustness).
- Species determination
- Clause 5 provides genetic analysis procedures for species-level identification (Rhizobium, Mesorhizobium, Ensifer, Bradyrhizobium).
- Supportive material
- Annex A: culture media formulations and diluent recipes.
- Annex B: repeatability and reproducibility data.
- Annex ZA: relationship to EU Regulation 2019/1009.
Practical applications
- Verifying declared microbial content of plant biostimulant products and quality control testing during production.
- Routine microbiological testing for manufacturers, contract laboratories and conformity assessment bodies to demonstrate compliance with EU fertilising product rules.
- Research labs assessing survival, formulation effects and viability of Rhizobiaceae in different product matrices.
Who should use this standard
- Quality control teams in biostimulant and fertiliser manufacturing
- Accredited testing laboratories and microbiologists
- Regulatory compliance officers and notified bodies assessing conformity with EU FPR
- R&D groups studying microbial inoculants and formulation stability
Related standards
- EN 17702-1:2024 - Sampling and sample preparation for plant biostimulants
- EN 17724:2024 - Terminology for plant biostimulants
- EN ISO 11133:2014 - Preparation and performance testing of culture media
Keywords: EN 17718:2024, plant biostimulants, Rhizobium spp., Rhizobiaceae, enumeration, CFU, serial dilution, spread plate, culture media, PBS, CEN, EU fertilising products.
Frequently Asked Questions
SIST EN 17718:2025 is a standard published by the Slovenian Institute for Standardization (SIST). Its full title is "Plant biostimulants - Determination of Rhizobium spp.". This standard covers: 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.
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.
SIST EN 17718:2025 is classified under the following ICS (International Classification for Standards) categories: 65.080 - Fertilizers. The ICS classification helps identify the subject area and facilitates finding related standards.
SIST EN 17718:2025 has the following relationships with other standards: It is inter standard links to SIST-TS CEN/TS 17718:2023. Understanding these relationships helps ensure you are using the most current and applicable version of the standard.
SIST EN 17718:2025 is associated with the following European legislation: EU Directives/Regulations: 2019/1009; Standardization Mandates: M/564, M/564 AMD 1, M/XXX. When a standard is cited in the Official Journal of the European Union, products manufactured in conformity with it benefit from a presumption of conformity with the essential requirements of the corresponding EU directive or regulation.
You can purchase SIST EN 17718:2025 directly from iTeh Standards. The document is available in PDF format and is delivered instantly after payment. Add the standard to your cart and complete the secure checkout process. iTeh Standards is an authorized distributor of SIST standards.
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 powder (WP) formulations
25 g 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 20 min or more until the
distribution is optimal, with a magnetic stirrer at half of maximum speed [7].
4.2.5 Solid water dispersible granules (WDG) formulations
25 g of sample (or more for low concentrated products) shall be dispensed in 225 g of sterile diluent
(Annex A) maintained at room temperature in a flask and shaken 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 (150 µm sieve
corresponding to a 100 mesh sieve) the particles and resuspended them in the same suspension [7].
4.2.6 Solid pellets, granules, microgranules - slow release - formulations
25 g of sample (or more for low concentrated products) shall be dispensed in 225 g of sterile diluent (see
Annex A) maintained at room temperature in a sterile bag and dispersed using a magnetic stirrer for
40 min at half of maximum speed and then sieved in a 150 µm sieve corresponding to a 100 mesh sieve
and, if there is still sample material in the sieve, repeat the process for a maximum of three times. Pay
attention to all the buffer used to make the exact final calculation [7].
4.2.7 Solid substrate
25 g of sample (or more for low concentrated products) shall be dispensed in 225 g of sterile diluent (see
Annex A) maintained at room temperature in a flask and shaken for 20 min or more until the distribution
is optimal, with a magnetic stirrer at half of maximum speed [7].
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, colonies that grow on plates from single cells, or cells estimated through the plant-infection
technique (with the principle that a single cell can multiply to initiate an infection). Serial dilutions may
be applied to all kinds 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 of the culture media is described in Annex A. The preparation and performance testing
of culture media are fundamental steps to ensure the integrity of microbiological examination. When
ready-to-use media are used, the manufacturers of these available media should have a quality program
that ensures the quality of the media they supply, according to EN ISO 11133:2014 . 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 shall be evaluated according
to EN ISO 11133:2014 .
Key
A Suspension of the sample
B Serial dilutions
C Petri plates
Figure 1 — Scheme of serial dilutions
A sample of the product is shaken in a bulk diluent (PBS or BPW)) which represents the first level of
dilution. This is then serially diluted with a sample at each level of dilution directly plated [10].
4.5 Spread plate counting
The choice of medium is made according to the genus researched by referring to Table 2.
−5 −6 −7
1) Inoculate 0,1 ml of the serial dilutions desired (e.g. 10 , 10 and 10 ) on the surface of the culture
medium in Petri dishes (Figure 1).
2) Spread the 0,1 ml aliquot over the culture medium with a sterilized L-shaped glass spreader (or
equivalent, e.g. a Drigalski loop).
3) There should be at least three separate replicate plates for each dilution.
4) After inoculation and absorption of the inoculum into the agar, the plates are placed in an incubator
at approximately 28 °C ± 2 °C for a period of two to eight days (according to the growth rate of the
species; see Table 2).
5) Count the number of colonies on the plates where colonies are well separated. If colony numbers are
low, the variation between plates and errors may be large. If colony numbers are too high,
overcrowding may result in an underestimation of numbers. Many texts recommend counting
between 30 and 300 CFU per plate to give statistical robustness.
4.6 Plate counts of Rhizobiaceae in sterile diluent
The counting of microorganisms on plates, following dilution, is also called direct counting. Count only
plates where there are between 30 and 300 colonies. The morphology of the colonies is convex, circular
and regular. The colour of the colonies varies with the genus and the media but in general is from whitish
to pinkish.
4.7 Growing media
For the different Rhizobiaceae species use different growing media according to Table 2: YT media for
Rhizobium spp. (alternatively, use YEMA media), YT media for Mesorhizobium spp., YT media for Ensifer
spp. and R2A media for Bradyrhizobium spp.
The Table 2 describes the different growing media to be used for the different microorganisms.
Table 2 —Growing media for the growth of the different microorganisms
Genus Media Bibliographic Conditions Time of Temperature
Reference for incubation
the Growing
Media
Rhizobium spp. YT [11] Aerobic 7 to 8 days 28 °C ± 2 °C
(Tryptone-Yeast
Media)
YEMA [8] [7] Aerobic 7 to 8 days 28 °C ± 2 °C
(Modified Yeast
extract mannitol
agar media)
(alternative
media)
Mesorhizobium spp. YT (Tryptone- [11] Aerobic 7 to 8 days 28 °C ± 2 °C
Yeast Media)
Ensifer spp. YT (Tryptone- [12] Aerobic 7 to 8 days 28 °C ± 2 °C
Yeast Media)
Bradyrhizobium spp. R2A (Reasoner's [13] Aerobic 7 to 8 days 28 °C ± 2 °C
2A Agar Media)
4.8 Calculation
Divide the average number of CFU on the three Petri dishes by the inverse of the dilution that gave the
reading in the range 30 to 300 CFU and then multiply with 10 (to correct for the 0,1 ml or 0,1 g used).
...
La norme SIST EN 17718:2025 offre une méthodologie précise pour l’énumération et la détermination des Rhizobiaceae, incluant les genres Rhizobium spp., Mesorhizobium spp., Ensifer spp., et Bradyrhizobium spp. Cette norme est particulièrement pertinente pour les mélanges de produits fertilisants, un domaine crucial dans l'agriculture moderne, où l’utilisation de biostimulants végétaux prend de plus en plus d'importance. L’un des points forts de cette norme est son approche systématique qui garantit une évaluation et un contrôle de qualité rigoureux des biostimulants d'origine microbienne. En fournissant des lignes directrices claires sur la manière de déterminer la présence et l’abondance de ces micro-organismes, la norme promeut des pratiques agricoles durables et efficaces. De plus, la norme se distingue par sa flexibilité, permettant aux utilisateurs de prendre en compte la composition spécifique des mélanges. En identifiant que dans les cas où les biostimulants végétaux constituent le pourcentage le plus élevé, cette norme s'applique de manière prioritaire, elle aide à assurer que les produits respectent les exigences réglementaires tout en priorisant l’optimisation des résultats agronomiques. En cas d'égalité entre les composants, la possibilité pour l'utilisateur de choisir la norme appropriée ajoute un niveau de personnalisation qui peut être apprécié par les producteurs. En somme, la norme SIST EN 17718:2025 joue un rôle essentiel dans le secteur des biostimulants végétaux, en fournissant des méthodes robustes pour l'analyse microbienne, tout en respectant les diversités des applications et des formulations de produits fertilisants. Sa pertinence devient donc manifeste dans le contexte actuel de recherche de solutions agronomiques durables et économiquement viables.
Die Norm SIST EN 17718:2025 befasst sich mit der Bestimmung von Rhizobium spp. und bietet eine umfassende Methodik zur Enumeration und Bestimmung der Rhizobiaceae, einschließlich Rhizobium spp., Mesorhizobium spp., Ensifer spp. und Bradyrhizobium spp. Der Geltungsbereich dieser Norm ist besonders relevant für die Herstellung und Anwendung von Mischungen von Düngemitteln, insbesondere wenn die Kategorie der Pflanzenbiostimulanzien den höchsten Anteil an der Mischung ausmacht. Ein wesentlicher Vorteil dieser Norm liegt in ihrer Klarheit und Präzision, da sie eine standardisierte Methode bereitstellt, die für diverse Düngemittelprodukte anwendbar ist. Dies fördert nicht nur die Konsistenz in den Testergebnissen, sondern verbessert auch die Vergleichbarkeit zwischen verschiedenen Produkten, die Pflanzenbiostimulanzien enthalten. Die Möglichkeit, diese Methodik für Mischungen anzuwenden, die aus mindestens zwei der angegebenen Produktkategorien bestehen, hebt ihre Vielseitigkeit hervor und zeigt, dass sie den Bedürfnissen der modernen Landwirtschaft gerecht wird. Darüber hinaus verspricht die Norm eine verbesserte Sicherheit und Effizienz bei der Verwendung von Pflanzenbiostimulanzien, indem sie klare Richtlinien für die Prüfung und Qualifizierung bietet. Die Betonung auf der höchsten prozentualen Menge der Pflanzenbiostimulanzien in der Mischung unterstreicht die Relevanz der Norm, da sie sicherstellt, dass die Wirksamkeit und die Eigenschaften der Produkte entsprechend bewertet werden. Dies ist besonders wichtig in Zeiten, in denen Nachhaltigkeit und ökologische Landwirtschaft an Bedeutung gewinnen. Insgesamt zeigt die SIST EN 17718:2025 eine solide Grundlage für die Qualitätskontrolle und Bewertung von Pflanzenbiostimulanzien auf und unterstützt die Hersteller dabei, den aktuellen Anforderungen des Marktes gerecht zu werden.
SIST EN 17718:2025 표준은 식물 생물자극제에서 Rhizobium spp.를 측정하고 결정하는 방법론을 제공합니다. 이 문서는 Rhizobiaceae에 속하는 종들인 Rhizobium spp., Mesorhizobium spp., Ensifer spp., Bradyrhizobium spp.의 수를 세고 정량하는 데 필요한 절차를 체계적으로 정리하고 있습니다. 이 표준은 최소 두 가지 이상의 EU 비옥도 제품 범주가 혼합된 비료 제품 혼합물에 적용됩니다. 여기서 비료 제품은 비료, 석회 자재, 토양 개선제, 성장 매체 및 식물 생물자극제와 같은 카테고리를 포함합니다. 또한 해당 혼합물에서 식물 생물자극제가 가장 높은 비율을 차지하는 경우에만 이 표준이 적용됩니다. 이는 비료 제품 혼합물의 품질 관리를 돕고, 강력한 성장과 생장 촉진을 위한 균형 잡힌 영양소 공급을 보장하는 데 중요한 요소입니다. SIST EN 17718:2025 표준의 강점 중 하나는 정량화된 방법론을 제공하여 실험실 검토 및 제품 개발에 적용할 수 있다는 점입니다. 이는 연구자와 개발자가 식물 생물자극제를 최적화할 수 있는 기초를 마련하며, 관련 산업에서의 활용성을 극대화합니다. 또한, 유럽 연합의 다른 비료 제품 범주와의 조화를 이루어 이 표준의 실제적인 응용 가능성을 높이고 있습니다. 이 표준은 특히 식물 생물자극제의 비료 제품으로서의 중요성을 강조하며, 환경 친화적인 농업 기술을 추진하는 데 있어 필수적인 요소입니다. 다양한 비료 혼합물 내에서 식물 생물자극제를 효과적으로 관리하고 분석할 수 있는 가이드라인을 제공함으로써, 지속 가능한 농업 발전에 기여할 수 있습니다. 이러한 요소들은 SIST EN 17718:2025 표준이 현대 농업에서 중요한 기준으로 자리잡게 하는 데 기여하고 있습니다.
The SIST EN 17718:2025 standard provides a comprehensive methodology for the enumeration and determination of Rhizobiaceae, specifically targeting Rhizobium spp., Mesorhizobium spp., Ensifer spp., and Bradyrhizobium spp. This ensures a focused approach to analyzing essential components of plant biostimulants, thereby facilitating better understanding and utilization in agricultural applications. One of the primary strengths of this standard is its clear scope, which outlines its applicability to blends of fertilizing products. By defining a blend as a mix of at least two categories of EU fertilising products-including fertilizers, liming materials, soil improvers, growing media, and plant biostimulants-it sets a robust framework for application across various product types. The specific emphasis on plant biostimulants, when they constitute the highest percentage by mass or volume, underscores their significance in modern agricultural practices. Furthermore, the standard is adaptable to various scenarios. In cases where the plant biostimulants category is not predominant, it establishes a logical hierarchy for applying the appropriate European Standard for the component with the highest percentage. This flexibility is critical for users as it accounts for different formulation complexities and balances in product composition. The relevance of SIST EN 17718:2025 cannot be overstated in the context of current agricultural practices. As the demand for sustainable and effective agricultural solutions grows, the identification and quantification of beneficial microbial communities, such as Rhizobium spp., become increasingly important. The standard's focus on plant biostimulants and their contributions to soil health and crop productivity aligns well with contemporary initiatives aimed at improving agricultural sustainability. In conclusion, the SIST EN 17718:2025 standard not only streamlines the analysis of Rhizobiaceae within plant biostimulants but also enhances understanding of how these microorganisms can positively impact agricultural productivity. Its detailed scope and adaptable nature make it a vital resource for professionals in the field seeking to optimize fertilizer blends and improve soil health.
SIST EN 17718:2025は、植物バイオスティミュラントに関連する標準であり、特にRhizobium属の決定と数え上げに関する方法論を提供しています。この標準は、肥料製品の混合において、植物バイオスティミュラントが最も高い割合で含まれている場合に適用されます。これにより、多様な農業環境における最適な栄養源の管理と改善が可能となります。 この標準の強みは、Rhizobiaceae（Rhizobium spp.、Mesorhizobium spp.、Ensifer spp.、Bradyrhizobium spp.）の正確な測定方法を提供する点にあります。これは、農業や園芸において重要な窒素固定を行う微生物の管理に直結しており、作物の生産性を高めるために不可欠です。特に、農業の持続可能性を推進する上で、植物バイオスティミュラントとしての役割がますます重要視されている現代において、この標準の適用はますます関連性が増しています。 また、肥料のブレンドに関する明確な指針を提供しているため、農業者は適切な製品を選択する際に混乱することが少なくなり、品質基準に基づいた製品の開発と使用が促進されます。このように、SIST EN 17718:2025は、地元および国際レベルでの農業生産性向上に向けた重要な枠組みを提供しています。加えて、ユーザーがブレンドの成分に基づいてどの標準を適用するかを選択できる柔軟性も、この標準の重要な特徴です。 総じて、SIST EN 17718:2025は、植物バイオスティミュラント市場における信頼性の高い指標を提供し、農業分野における新たなスタンダードとしての位置づけを確立しています。
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