SIST EN 17713:2025

Plant biostimulants - Determination of Azospirillum spp.

This document provides the methodology for the enumeration and determination of Azospirillum spp. in microbial plant biostimulants.
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 Azospirillum spp.

Dieses Dokument stellt die Methodik für die Zählung und Bestimmung von Azospirillum spp. in mikrobiellen Pflanzen-Biostimulanzien bereit.
Dieses Dokument ist auf 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 d'Azospirillum spp.

Le présent document fournit la méthodologie de dénombrement et de détermination d’Azospirillum spp. dans les biostimulants microbiens des végétaux.
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 Azospirillum spp.

Ta dokument določa metodo za štetje in določanje bakterij Azospirillum spp. v mikrobnih rastlinskih stimulantih.
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
• ICS: 65.080 - Fertilizers
• 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
• 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
• Mandate: M/564 AMD 1 - amending Implementing Decision C(2020) 612 as regards certain harmonised standards and standardisation deliverables, deadlines for their adoption and the duration of the validity of the standardisation request
• Mandate: M/XXX - COMMISSION IMPLEMENTING DECISION of XXX on a standardisation request to the European Committee for Standardization, to the European Committee for Electrotechnical Standardization and to the European Telecommunications Standards Institute as regards certain measuring instruments in support of Directive 2014/32/EU of the European Parliament and of the Council
• Ref Project: EN 17713:2024 - Plant biostimulants - Determination of Azospirillum spp.

Relations

• Replaces: SIST-TS CEN/TS 17713:2023 - Plant biostimulants - Determination of Azospirillum spp.
• Effective Date: 01-Feb-2025

Overview

EN 17713:2024 - "Plant biostimulants - Determination of Azospirillum spp." is a CEN standard that defines laboratory methods for the enumeration and species determination of Azospirillum spp. in microbial plant biostimulants. The standard supports implementation of Regulation (EU) 2019/1009 for EU fertilising products and applies to product blends where Plant Biostimulants is the dominant category by mass or dry mass.

Key topics and technical requirements

• Scope and applicability: Applies to microbial plant biostimulants and to blends when plant biostimulants are the highest-percentage component. Guidance on choosing the correct standard for mixed-category products is provided.
• Sample preparation: Procedures cover various formulations - water-based liquids, oil-based emulsions, wettable powders (WP), water dispersible granules (WDG), pellets/granules and substrates. Typical sample: 25 ml or 25 g into buffered diluent (e.g., 225 ml PBS); shaking and sieving steps are specified for uniform suspension.
• Enumeration method (MPN/CFU): A serial-dilution, plating-based Most Probable Number (MPN) approach is used to estimate colony-forming units (CFU) of Azospirillum per g or ml. At least five dilutions with five replicates each are recommended. Positive tubes are identified by a blue colour change and a white subsurface pellicle; values are read from the MPN table (Annex B).
• Culture media and diluents: Preparation of N‑free semisolid medium (Nfb) is given (Annex A). The 2024 edition adds Buffered Peptone Water as a diluent option and updates some buffer recipes (e.g., PBS/A5).
• Incubation & conditions: Example conditions include incubation of MPN tubes at (36 ± 1) °C for 3–4 days and colony isolation at (28 ± 1) °C for 48–72 h.
• Species determination (genetic analysis): Optional confirmation via amplified rDNA restriction analysis (ARDRA) of 16S rRNA genes is described, including isolation, DNA extraction and concentration steps.
• Equipment & quality: Lists required apparatus (pipettes, autoclave, incubator, pH meter) and references EN ISO 11133:2014 for media quality control. Annex C provides repeatability/reproducibility data; Annex ZA maps the standard to EU regulatory requirements.

Applications and users

• Quality control and batch testing by biostimulant manufacturers
• Independent testing laboratories and certification bodies
• Regulatory authorities verifying label claims under the EU Fertilising Products Regulation (FPR)
• Agritech R&D teams assessing microbial content and strain identity

Practical benefits include consistent Azospirillum spp. counts, robust product labelling, and regulatory compliance for microbial plant biostimulants.

Related standards

• EN 17702-1:2024 - Sampling and sample preparation for plant biostimulants (normative reference)
• EN ISO 11133:2014 - Media quality and performance guidance
• EN 17724:2024 - Taxonomic/terminology references for microbial plant biostimulants

Keywords: EN 17713:2024, Azospirillum spp., plant biostimulants, enumeration, MPN, CFU, ARDRA, 16S rRNA, EU fertilising products, FPR.

Standards Content (Sample)

SLOVENSKI STANDARD
01-februar-2025
Nadomešča:
SIST-TS CEN/TS 17713:2023
Rastlinski biostimulanti - Določanje Azospirillum spp.
Plant biostimulants - Determination of Azospirillum spp.
Pflanzen-Biostimulanzien - Bestimmung von Azospirillum spp.
Biostimulants des végétaux - Détermination d'Azospirillum spp.
Ta slovenski standard je istoveten z: EN 17713:2024
ICS:
65.080 Gnojila Fertilizers
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN 17713
EUROPEAN STANDARD
NORME EUROPÉENNE
November 2024
EUROPÄISCHE NORM
ICS 65.080 Supersedes CEN/TS 17713:2022
English Version
Plant biostimulants - Determination of Azospirillum spp.
Biostimulants des végétaux - Détermination Pflanzen-Biostimulanzien - Bestimmung von
d'Azospirillum spp. Azospirillum 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 NORMALISATI O N

EUROPÄISCHES 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 17713:2024 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 Azospirillum spp. . 6
5 Species determination of Azospirillum spp. via genetic analysis . 8
Annex A (normative) Formula of the culture media . 11
Annex B (normative) Table of MPN values [4] . 14
Annex C (informative) Repeatability and reproducibility data . 16
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 17713:2024) 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 17713:2022.
CEN/TS 17713:2022:
— 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;
— the recipe of Phosphate Buffered Saline (A5) 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.
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 Azospirillum
spp. in microbial plant biostimulants.
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
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological 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
Azospirillum spp.
Gram-negative bacteria that belong to the alphaproteobacterial phylum
Note 1 to entry: Azospirillum is a Gram-negative, microaerophilic, non-fermentative and nitrogen-
fixing bacterial genus. Azospirillum spp. are Gram-negative, do not form spores, and have a slightly-twisted
oblong-rod shape. Azospirillum have at least one flagellum and sometimes multiple flagella. The genus has
about 20 species, the relationships between all the species have not been resolved in detail, however they
most likely constitute a coherent group.
Note 2 to entry: Azospirillum bacteria are aerobic non-fermentative chemoorganotrophs, vibroid, they
produce several hormones, mainly auxins (not described for all species yet), and most of them are
diazotrophic (fix atmospheric nitrogen gas into a more usable form).
[SOURCE: EN 17724:2024, 3.2.2.1 [2]]

An example of such a blend is a product with 2 claimed functions consisting of a non-microbial plant
biostimulant and an organic fertilizer composed of 1kg/kg of plant biostimulant from seaweed.
4 Enumeration of Azospirillum spp.
4.1 General
This procedure is meant to determine the number of colony-forming units (CFU) of Azospirillum
spp., per gram or per millilitre. The method, in order to be fast, cheap and repeatable, is based on
serial dilutions and plating [3].
4.2 Sample preparation
4.2.1 General
A representative sample of the product to be analyzed 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
Dispense 25 ml of sample (or more for low concentrated products) in 225 ml of sterile phosphate
buffer saline (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 [3].
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 phosphate
buffer saline (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 [3].
4.2.4 Solid wettable powder (WP) formulations
Dispense 25 g of sample (or more for low concentrated products) in 225 ml of sterile phosphate
buffer saline (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 [3].
4.2.5 Solid water dispersible granules (WDG) formulations
Dispense 25 g (or more for low concentrated products) of sample in 275 ml of sterile phosphate
buffer saline (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 another apparatus such as a laboratory paddle blender after
having sieved (150 µm sieve corresponding to a 100 mesh sieve) the particles, and resuspend
them in the same suspension [3].
4.2.6 Solid pellets, granules, microgranules (slow release) formulations
Dispense 25 g (or more for low concentrated products) of sample in 225 ml of sterile phosphate
buffer saline (PBS) maintained at room temperature in a sterile bag and disperse them using a
magnetic stirrer for 40 min at half of maximum speed. Then sieve the suspension in a 100 mesh
sieve (150 µm sieve corresponding to a 100 mesh sieve) and if material remains in the sieve,
repeat the process for a maximum of three times. Pay attention to the volume of buffer used to
make the exact final calculation [3].
4.2.7 Solid substrate
Dispense 25 g (or more for low concentrated products) of sample in 225 ml of sterile phosphate
buffer saline (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 [3].
4.3 Requirements (apparatus)
— Graduated pipettes (1 ml and 10 ml);
— Dilution bottles or flasks;
— Petri dishes clear, uniform, flat-bottomed;
— Hot air oven - Capable of giving uniform and adequate temperatures, equipped with a
thermometer calibrated to read up to (250 ± 1) °C and with vents suitably located to ensure
prompt and uniform heating;
— Autoclave/steam sterilizer;
— Incubator;
— Hand tally or mechanical counting device;
— pH meter, having an accuracy of calibration of ±0,1 pH unit at 25 °C.
4.4 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.
For an example of suitable diluents see A.1.
4.5 Preparation of the culture media
The preparation and the composition of N-free semisolid medium (Nfb) is described in Annex A.
The preparation and quality of culture media is a fundamental step 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 [5]. Under these conditions, the user/laboratory does not need to run
additional testing on such media but shall ensure storage conditions according to the ones
recommended by the manufacturers.
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 may be evaluated according to EN ISO 11133:2014 [5].
4.6 Preparation of serial dilution for MPN count
Prepare the sample as described in 4.2. Make serial dilutions in order to cover a range including
the concentration expected/given by the manufacturer. Inoculate at least 5 dilutions. Five
replicates of each dilution shall be carried out, making it a total of at least 25 tubes per sample.
Pipette out 1 ml aliquots of the dilutions and deliver them to test tubes containing 5 ml of Nfb
media. The operator or laboratory shall check with the applicant (manufacturer, distributor) of
the expected concentration of Azospirillum spp.
4.7 Incubation of tubes
Label the tubes and incubate at (36 ± 1) °C for 3 days to 4 days in vertical position in a test tubes
stand. Do not disturb the medium during the entire period of incubation.
4.8 Counting
Count the tubes which have turned blue and which have developed the typical white sub-surface
pellicle.
Count the tubes as positive or negative for the presence or not presence of sub-surface pellicle
respectively, and consider the number of tubes for the calculation (4.9).
4.9 Method for estimating MPN count
To calculate the most probable number of organisms in the original sample, select as P the
number of positive tubes in the least concentrated dilution in which all tubes are positive, or in
which the greatest number of tubes is positive and let P and P represent the numbers of positive
2 3
tubes in the next two higher dilutions.
Then find the row of numbers in Table B.1 in Annex B in which P and P correspond to the values
1 2
observed experimentally. Follow that row of numbers across the table to the column headed by
the observed value of P.
The figure at the point of intersection is the most probable number of organisms in the quantity
of original sample represented in the inoculum added in the second dilution. Multiply this figure
by the appropriate dilution factor to obtain the MPN value.
Value from MPN table X Dilution level
Azospirillum count per gof carrier=
Dry mass of product g
( )
5 Species determination of Azospirillum spp. via genetic analysis
5.1 General
If required, this method shall be used for the determination of Azospirillum spp. using amplified
rDNA restriction analysis (ARDRA) of the 16S rRNA genes.
5.2 Preparation of the sample for the genomic DNA extraction
5.2.1 Isolation and preparation of the microorganism
— From a positive tube, streak a non-selective nutrient agar in order to obtain typical colonies
of Azospirillum spp.
— Put 1 g of the product in 100 ml of sterile normal saline.
— Keep the suspension in agitation for 5 min under sterile conditions.
— Under sterile conditions, strike a loop of the suspension in a Petri dish prepared with nutrient
agar, in order to isolate a few single colonies.
— Incubate the plate for 48 h to 72 h at (28 ± 1) °C.
— Check for a single typical colony (convex, with margins, colour).
— Under sterile conditions, take a loop of the typical isolated colony in a flask with 100 ml of
nutrient broth and keep in agitation at 130 rpm at (28 ± 1) °C for 48 h.
5.2.2 Sample concentration ®
— Take a 0,5 ml aliquot of the broth and put it in 1,0 ml of sterile saline in a 1,5 ml Eppendorf
and centrifuge at 13 000 rpm for 10 min.

2 ®
Eppendorf is an example of a suitable product available commercially. This information is given for the
convenience of users of this document and does not constitute an endorsement by CEN of this product.
— The supernatant should be poured out carefully, keeping the cell pellets at the bottom of the
® 2
tube .
Eppendorf
— The cell pellets should be washed five times with normal saline to remove the slimy
exopolysaccharide (EPS).
5.2.3 DNA extraction and storage
— Molecular biology kits/chemicals. The bacteria DNA can be extracted with microbial DNA
extraction kits following the manufacturer’s instructions. For example: innuPREP Bacteria
DNA Kit (Analytik Jena) .
— The PCR reactions can be performed with any standard polymerase following the
manufacturer’s instructions. For example: MTP™ Taq DNA Polymerase (Merck).
— The PCR product clean-up can be done by any standard kit according to the manufacturer’s
instructions.
— It shall then be stored at −20 °C.
5.2.4 Partial PCR amplification of the 16S rRNA genes
5.2.4.1 PCR amplification
— The PCR amplification should be carried out in 30 μl reaction with 23,55 μl sterile PCR water,
®4
3,0 μl buffer (Biolabs ), 0,6 μl dNTP (10 mM), 0,
...