CEN/TS 17709:2022

SLOVENSKI STANDARD
01-februar-2023
Rastlinski biostimulanti - Določanje Azotobacter spp.
Plant biostimulants - Determination of Azotobacter spp.
Pflanzen-Biostimulanzien - Bestimmung von Azotobacter spp.
Biostimulants des végétaux - Détermination d'Azotobacter spp.
Ta slovenski standard je istoveten z: CEN/TS 17709:2022
ICS:
65.080 Gnojila Fertilizers
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

CEN/TS 17709
TECHNICAL SPECIFICATION
SPÉCIFICATION TECHNIQUE
March 2022
TECHNISCHE SPEZIFIKATION
ICS 65.080
English Version
Plant biostimulants - Determination of Azotobacter spp.
Biostimulants des végétaux - Détermination Pflanzen-Biostimulanzien - Bestimmung von
d'Azotobacter spp. Azotobacter spp.
This Technical Specification (CEN/TS) was approved by CEN on 3 January 2022 for provisional application.

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

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

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATIO N

EUROPÄISCHES KOMITEE FÜR NO RMU N G

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2022 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TS 17709:2022 E
worldwide for CEN national Members.

Contents Page
European foreword . 3
Introduction . 4
1 Scope . 6
2 Normative references . 6
3 Terms and definitions . 6
4 Enumeration of Azotobacter spp . 6
4.1 General . 6
4.2 Sample preparation . 6
4.3 Serial dilution . 7
4.4 Plate counts of Azotobacter sp in sterile diluent . 8
4.5 Spread-plate counting with ASHBY SUCROSE AGAR [6] . 8
4.6 Calculation . 8
5 Species determination of Azotobacter sp. via genetic analysis . 8
5.1 General . 8
5.2 Preparation of the sample for the genomic DNA extraction . 8
Annex A (informative) Formula of culture media . 11
Bibliography . 13

European foreword
This document (CEN/TS 17709:2022) has been prepared by Technical Committee CEN/TC 455
“Plant biostimulants”, the secretariat of which is held by AFNOR.
Attention is drawn to the possibility that some of the elements of this document may be the subject
of patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document has been prepared under a Standardization Request given to CEN by the European
Commission and the European Free Trade Association.
Any feedback and questions on this document should be directed to the users’ national standards
body. A complete listing of these bodies can be found on the CEN website.
According to the CEN/CENELEC Internal Regulations, the national standards organisations of the
following countries are bound to announce this Technical Specification: Austria, Belgium,
Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece,
Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain,
Sweden, Switzerland, Turkey and the United Kingdom.
Introduction
This document was prepared by the experts of CEN/TC 455 “Plant Biostimulants”. The European
Committee for Standardization (CEN) was requested by the European Commission (EC) to draft
European standards or European standardization deliverables to support the implementation of
Regulation (EU) 2019/1009 of 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 M/564, 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 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 biostimulants has increased significantly in
Europe as a valuable tool to use in agriculture. Standardization was identified as having an
important role in order to promote the use of biostimulants. The work of CEN/TC 455 seeks to
improve the reliability of the supply chain, thereby improving the confidence of farmers, industry,
and consumers in biostimulants, and will promote and support commercialisation of the
European biostimulant industry.
Biostimulants used in agriculture can be applied in multiple ways: on soil, on 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 biostimulants in agriculture based on live microorganisms
belonging to the genera Azotobacter.
The Table 1 below summarizes many of the agro-ecological principles and the role played by
biostimulants.
Table 1 — Agro-ecological principles and the role played by biostimulants
Increase biodiversity
By improving soil microorganism quality/quantity
Reinforce biological regulation and interactions
By reinforcing plant-microorganism interactions
- symbiotic exchanges i.e. Mycorrhizae
- symbiotic exchanges i.e. Rhizobiaceae/Faba
- secretions mimicking plant hormones (i.e. Trichoderma)
By regulating plant physiological processes
- for ex growth, metabolism, plant development…
Improve biogeochemical cycles
- improve absorption of nutritional elements
- improve bioavailability of nutritional elements in the soil
- stimulate degradation of organic matter

WARNING — Persons using this document should be familiar with normal laboratory practice.
This document does not purport to address all of the safety problems, if any, associated with its
use. It is the responsibility of the user to establish appropriate safety and health practices and to
ensure compliance with any national regulatory conditions.
IMPORTANT — It is absolutely essential that tests conducted in accordance with this document
be carried out by suitably trained staff.

1 Scope
This document was developed to provide the methodology for the enumeration and
determination of Azotobacter sp. in plant biostimulant products in accordance with the Regulation
(EU) 2019/1009 of the European Parliament and of the Council [1].
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies.
For undated references, the latest edition of the referenced document (including any
amendments) applies.
CEN/TS 17702-1, Plant biostimulants — Sampling and sample preparation — Part 1: Sampling
CEN/TS 17724, Plant biostimulants — Terminology
3 Terms and definitions
For the purposes of this document, the terms and definitions given in CEN/TS 17724 apply.
4 Enumeration of Azotobacter spp.
4.1 General
This procedure is meant to determine the number of colony-forming units (CFU) of the above
mentioned bacteria, per gram, per millilitre, per square centimetre, or per sampling device. The
method, in order to be fast, cheap, 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 according to CEN/TS 17702-1 shall be
prepared according to following procedure which takes into consideration the different
formulations of biostimulants based products.
4.2.2 Liquid (based water) formulations
Dispense 25 ml of sample (or more for low concentrated products) in 225 ml of sterile Phosphate
Buffer Solution (PBS) maintained at room temperature, in a flask and shake for 10 min or more
until the distribution is optimal, with a magnetic stirrer at half speed [6].
4.2.3 Liquid - based oil, emulsifiable concentrate (EC) formulations
Dispense 25 ml of sample (or more for low concentrated products) in 225 ml of sterile Phosphate
Buffer Solution (PBS) maintained at room temperature, in a flask and shake for 10 min or more
until the distribution is optimal, with a magnetic stirrer at half speed [6].
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 Solution (PBS) maintained at room temperature, in a flask and shake for 20 min or more
until the distribution is optimal, with a magnetic stirrer at half speed [6].
4.2.5 Solid - Water dispersible granules (WDG) formulations
Dispense 25 g of sample (or more for low concentrated products) in 225 g of sterile Phosphate
Buffer Solution (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 speed. If required help the
dispersion of the formulations with other apparatus such as a stomacher after having sieved (100
mesh sieve) the particles and resuspend them in the same suspension [6].
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 Phosphate
Buffer Solution (PBS) maintained at room temperature, in a sterile bag and disperse them using a
magnetic stirrer for 40 min at half speed and then sieve in a 100 mesh sieve and if material remain
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 [6].
4.2.7 Solid - substrate
Dispense 25 g of sample (or more for low concentrated products) in 225 g of sterile Phosphate
Buffer Solution (PBS) maintained at room temperature, in a flask and shake for 20 min or more
until the distribution is optimal, with a magnetic stirrer at half speed [6].
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 can be applied to all kind of formulations. A 10-fold serial dilution is most often
used (Figure 1) but if the number of Azotobacter sp. cells is expected to be low then a lower
number of dilutions can be adopted.
A sample of the product is shaken in a bulk diluent (PBS) which represents the first level of
dilution. This is then serially diluted with a sample at each level of dilution directly plated.

Key
A product suspended in sterile diluent
−8
B
prepare dilution series at 10
C 3 replicates of Petri dishes with media
Figure 1 — Scheme of a serial dilution series
4.4 Plate counts of Azotobacter sp in sterile diluent
The counting of microorganisms on plates, following dilution, is also called direct counting. Count
only plates where there are between 30 to 300 colonies.
4.5 Spread-plate co
...