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 fertilizing products categories: Fertilizers, Liming Materials, Soil Improvers, Growing Media, Plant Biostimulants and where the following category Plant Biostimulants is the highest percentage in the blend by mass or volume, or in the case of liquid form by dry mass. If Plant Biostimulants is not the highest percentage in the blend, the European Standard for the highest percentage of the blend applies. In case an end 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 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 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.

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oSIST prEN 17713:2023
Rastlinski biostimulanti - Ugotavljanje prisotnosti in števila 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: prEN 17713
65.080 Gnojila Fertilizers
oSIST prEN 17713:2023 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

oSIST prEN 17713:2023
oSIST prEN 17713:2023
prEN 17713
April 2023
ICS 65.080 Will supersede 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 draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee
CEN/TC 455.
If this draft becomes a European Standard, CEN members are bound to comply with the CEN/CENELEC Internal Regulations
which stipulate the conditions for giving this European Standard the status of a national standard without any alteration.

This draft European Standard was established by CEN in three official versions (English, French, German). A version in any other
language made by translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC
Management Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and
United Kingdom.
Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are
aware and to provide supporting documentation.

Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without
notice and shall not be referred to as a European Standard.



CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2023 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 17713:2023 E
worldwide for CEN national Members.

oSIST prEN 17713:2023
prEN 17713:2023 (E)
Contents Page
1 Scope . 7
2 Normative references . 7
3 Terms and definitions . 7
4 Enumeration of Azospirillum spp. . 7
4.1 General . 7
4.2 Sample preparation . 8
4.2.1 General . 8
4.2.2 Liquid -based water- 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, microgranuls (slow release) formulations . 8
4.2.7 Solid - substrate . 8
4.3 Requirements (apparatus) . 8
4.4 Serial dilution . 9
4.5 Preparation of the culture media . 9
4.6 Preparation of serial dilution for MPN count . 9
4.7 Incubation of tubes . 9
4.8 Counting . 9
4.9 Method for estimating MPN count . 10
5 Species Determination of Azospirillum spp. 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 . 11
5.2.4 Partial PCR Amplification of the 16S rRNA Genes . 11
Annex A (normative) Formula of the culture media . 13
A.1 0,1 M Phosphate Buffer Saline (PBS) . 13
A.2 Composition of Nfb semisolid medium . 13
A.2.1 Ingredients . 13
A.2.2 Method to prepare Trace element solution . 13
A.2.3 Method to prepare Vitamin solution. 14
A.2.4 Sterilization and preparation of MPN tubes . 14
A.3 Nutrient agar . 14
A.4 Nutrient broth . 14
Annex B (normative) Table of MPN values [9] . 15
Annex C (informative) Repeatability and reproductibility data . 17
C.1 General . 17
C.2 Microbial analysis-Materials used in the interlaboratory comparison study . 17
C.3 Microbial analysis-Interlaboratory comparison results . 18
C.4 Genetic analysis-Materials used in the interlaboratory comparison study . 19
oSIST prEN 17713:2023
prEN 17713:2023 (E)
C.5 Genetic analysis- Interlaboratory comparison results . 20
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 . 21
Bibliography . 22

oSIST prEN 17713:2023
prEN 17713:2023 (E)
European foreword
This document (prEN 17713:2023) 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 17713:2022.
In comparison with the previous edition, the following technical modifications have been made:
This document has been prepared under a Standardization Request given to CEN by the European
Commission and the European Free Trade Association, and supports essential requirements of EU
Directive(s) / Regulation(s).
For relationship with EU Directive(s) / Regulation(s), see informative Annex ZA, which is an
integral part of this document.

oSIST prEN 17713:2023
prEN 17713:2023 (E)
This document was prepared by the experts of CEN/TC 455 ‘Plant Biostimulants’. The European
Committee for Standardization (CEN) was requested by the European Commission (EC) to draft
European standards or European standardization deliverables to support the implementation of
Regulation (EU) 2019/1009 of 5 June 2019 laying down rules on the making available on the
market of EU fertilizing 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”. The Working
Group 5 “Labelling and denominations”, was created to develop a work program as part of this
Standardization Request. The 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 plant, 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 genera Azospirillum.
The Table 1 below summarizes many of the agro-ecological principles and the role played by
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

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.
oSIST prEN 17713:2023
prEN 17713:2023 (E)
IMPORTANT — It is absolutely essential that tests conducted in accordance with this document
be carried out by suitably trained staff.

oSIST prEN 17713:2023
prEN 17713:2023 (E)
1 Scope
This document provides the methodology for the enumeration and determination of Azospirillum
spp. 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.
prEN 17702-1:— , 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
— IEC Electropedia: available at
— ISO Online browsing platform: available at
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 are gram-negative, do not form spores, and have a slightly-twisted
oblong-rod shape. Azospirillum have at least one flagellum and sometimes mult

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