Plant biostimulants - Determination of Azospirillum spp.

This document provides the methodology for the enumeration and determination of Azospirillum spp. in plant biostimulant products in accordance to the Regulation of EU fertilising products [1].

Pflanzen-Biostimulanzien - Bestimmung von Azospirillum spp.

Dieses Dokument stellt die Methodik für die Zählung und Bestimmung von Azospirillum spp. in Pflanzen-Bio¬stimulanzien in Übereinstimmung mit der EU Düngeprodukteverordnung [1] bereit.

Biostimulants des végétaux - Détermination d'Azospirillum spp.

Le présent document fournit la méthodologie de dénombrement et de détermination d’Azospirillum spp. dans les produits biostimulants des végétaux conformément au Règlement sur les fertilisants [1].

Rastlinski biostimulanti - Določanje Azospirillum spp.

Ta dokument zagotavlja metodologijo za štetje in določanje bakterij Azospirillum sp. v rastlinskih biostimulantih v skladu z Uredbo EU o sredstvih za gnojenje [1].

General Information

Status
Published
Publication Date
22-Mar-2022
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Start Date
23-Mar-2022
Due Date
29-Aug-2022
Completion Date
23-Mar-2022

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SLOVENSKI STANDARD
SIST-TS CEN/TS 17713:2023
01-februar-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: CEN/TS 17713:2022
ICS:
65.080 Gnojila Fertilizers
SIST-TS CEN/TS 17713:2023 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
SIST-TS CEN/TS 17713:2023

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SIST-TS CEN/TS 17713:2023


CEN/TS 17713
TECHNICAL SPECIFICATION

SPÉCIFICATION TECHNIQUE

March 2022
TECHNISCHE SPEZIFIKATION
ICS 65.080
English Version

Plant biostimulants - Determination of Azospirillum spp.
Biostimulants des végétaux - Détermination Pflanzen-Biostimulanzien - Bestimmung von
d'Azospirillum spp. Azospirillum 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 NORMUN 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 17713:2022 E
worldwide for CEN national Members.

---------------------- Page: 3 ----------------------
SIST-TS CEN/TS 17713:2023
CEN/TS 17713:2022 (E)
Contents Page
European foreword . 3
Introduction . 4
1 Scope . 6
2 Normative references . 6
3 Terms and definitions . 6
4 Enumeration of Azospirillum spp. . 6
4.1 General . 6
4.2 Sampling . 6
4.3 Preparation of the sample . 6
4.4 Requirements (apparatus) . 7
4.5 Preparation of the culture media . 8
4.6 Preparation of serial dilution for MPN count . 8
4.7 Incubation of tubes . 8
4.8 Counting . 8
4.9 Method for estimating MPN count . 8
5 Species determination of Azospirillum spp. via genetic analysis . 9
5.1 General . 9
5.2 Preparation of the sample for the genomic DNA extraction . 9
Annex A (normative) Formula of the culture media . 11
Annex B (normative) Table of MPN values . 13
Bibliography . 15

2

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SIST-TS CEN/TS 17713:2023
CEN/TS 17713:2022 (E)
European foreword
This document (CEN/TS 17713:2022) 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.
3

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SIST-TS CEN/TS 17713:2023
CEN/TS 17713:2022 (E)
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 Azosprillum.
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
- 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
4

---------------------- Page: 6 ----------------------
SIST-TS CEN/TS 17713:2023
CEN/TS 17713:2022 (E)
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.

5

---------------------- Page: 7 ----------------------
SIST-TS CEN/TS 17713:2023
CEN/TS 17713:2022 (E)
1 Scope
This document provides the methodology for the enumeration and determination of
Azospirillum spp. in plant biostimulant products in accordance to the Regulation of EU fertilising
products [1].
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
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 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 details, however most
likely they constitute a coherent group.
Note 2 to entry: They are aerobic non-fermentative chemoorganotrophs, vibroid, 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: CEN/TS 17724:2021, term 3.2.2.1]
4 Enumeration of Azospirillum spp.
4.1 General
The goal of the method is the enumeration of Azosprillium spp. (CFU/g) in the given bio-
stimulant/formulation [2].
4.2 Sampling
Sampling is not part of the method specified in this document (see the specific European Standard
dealing with the product concerned). If there is no specific International or European Standard, it
is recommended that the parties concerned come to an agreement on this subject.
It is important that the laboratory receives a sample which is representative and has not been
damaged or changed during transport or storage.
4.3 Preparation of the sample
4.3.1 Sample preparation
A representative sample of the product to be analysed (see CEN/TS 17702-1) will be prepared
according to following procedure which takes into consideration the different formulations of
biostimulant based products:
6

---------------------- Page: 8 ----------------------
SIST-TS CEN/TS 17713:2023
CEN/TS 17713:2022 (E)
4.3.2 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 [4].
4.3.3 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 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 [4].
4.3.4 Solid - Wettable Powder (WP) formulations
Dispense 25 gr 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 [4].
4.3.5 Solid - Water dispersible granules (WDG) formulations
Dispense 25 g (or more for low concentrated products) of sample in 275 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 [4].
4.3.6 Solid – Pellets, granules, microgranules (slow release) formulations
Dispense 25 g (or more for low concentrated products) of sample 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 maxim
...

SLOVENSKI STANDARD
kSIST-TS FprCEN/TS 17713:2021
01-november-2021
[Not translated]
Plant biostimulants - Determination of Azospirillum spp.
Biostimulanzien für die pflanzliche Anwendung - Bestimmung von Azospirillum spp.
Biostimulants des végétaux - Détermination d'Azospirillum spp.
Ta slovenski standard je istoveten z: FprCEN/TS 17713
ICS:
65.080 Gnojila Fertilizers
kSIST-TS FprCEN/TS 17713:2021 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
kSIST-TS FprCEN/TS 17713:2021

---------------------- Page: 2 ----------------------
kSIST-TS FprCEN/TS 17713:2021


FINAL DRAFT
TECHNICAL SPECIFICATION
FprCEN/TS 17713
SPÉCIFICATION TECHNIQUE

TECHNISCHE SPEZIFIKATION

September 2021
ICS 65.080
English Version

Plant biostimulants - Determination of Azospirillum spp.
 Biostimulanzien für die pflanzliche Anwendung -
Bestimmung von Azospirillum spp.


This draft Technical Specification is submitted to CEN members for Vote. It has been drawn up by the Technical Committee
CEN/TC 455.

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.

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 Technical Specification. It is distributed for review and comments. It is subject to change
without notice and shall not be referred to as a Technical Specification.


EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. FprCEN/TS 17713:2021 E
worldwide for CEN national Members.

---------------------- Page: 3 ----------------------
kSIST-TS FprCEN/TS 17713:2021
FprCEN/TS 17713:2021 (E)
Contents Page
European foreword . 3
Introduction . 4
1 Scope . 6
2 Normative references . 6
3 Terms and definitions . 6
4 Enumeration of Azospirillum spp . 6
4.1 Sampling . 6
4.2 Preparation of the sample . 7
4.2.1 Sample preparation . 7
4.2.2 Liquid -based water- formulations . 7
4.2.3 Liquid – oil based (emulsifiable concentrate -EC) formulations . 7
4.2.4 Solid - Wettable Powder (WP) formulations . 7
4.2.5 Solid - Water dispersible granules (WDG) formulations . 7
4.2.6 Solid – Pellets, granules, microgranuls (slow release) formulations . 7
4.2.7 Solid - substrate . 7
4.3 Requirements (apparatus) . 7
4.4 Preparation of the culture media . 8
4.5 Preparation of serial dilution for MPN count . 8
4.6 Incubation of tubes . 8
4.7 Counting . 8
4.8 Method for estimating MPN count . 8
5 Species Determination of Azospirillum sp. via genetic analysis . 9
5.1 General . 9
5.2 Preparation of the sample for the genomic DNA extraction . 9
5.2.1 Isolation and preparation of the microorganism . 9
5.2.2 Sample concentration . 9
5.2.3 DNA extraction and storage . 9
5.2.4 Partial PCR Amplification of the 16S rRNA Genes . 10
Annex A (normative) Formula of the culture media. 11
A.1 0,1 M Phosphate Buffer Saline (PBS) . 11
A.2 Composition of Nfb semisolid medium . 11
A.2.1 Ingredients . 11
A.2.2 Method to prepare Trace element solution . 11
A.2.3 Method to prepare Vitamin solution . 12
A.2.4 Sterilization and preparation of MPN tubes . 12
A.3 Nutrient Agar . 12
A.4 Nutrient Broth . 12
Annex B (normative) Table of MPN values. 13
Bibliography . 15

2

---------------------- Page: 4 ----------------------
kSIST-TS FprCEN/TS 17713:2021
FprCEN/TS 17713:2021 (E)
European foreword
This document (FprCEN/TS 17713:2021) 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 Vote on TS.
This document has been prepared under a mandate M/564 given to CEN by the European
Commission and the European Free Trade Association.

3

---------------------- Page: 5 ----------------------
kSIST-TS FprCEN/TS 17713:2021
FprCEN/TS 17713:2021 (E)
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 request,
presented as SR 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 Fertilizing
Products Regulation.
This document is applicable to all biostimulants in agriculture based on live microorganisms
belonging to the genera Azosprillum.
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. mycorrhize
- symbiotic exchanges i.e. rhizobiaciae/fava
- 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.
4

---------------------- Page: 6 ----------------------
kSIST-TS FprCEN/TS 17713:2021
FprCEN/TS 17713:2021 (E)
IMPORTANT — It is absolutely essential that tests conducted in accordance with this document
be carried out by suitably trained staff.

5

---------------------- Page: 7 ----------------------
kSIST-TS FprCEN/TS 17713:2021
FprCEN/TS 17713:2021 (E)
1 Scope
This document provides the methodology for the enumeration and determination of Azospirillum
sp. in plant biostimulant products in accordance to the Regulation of EU fertilising products [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.
There is no normative reference in this document.
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:
• IEC Electropedia: available at https://www.electropedia.org/
• ISO Online browsing platform: available at https://www.iso.org/obp
3.2
azospirillum sp
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 multiple flagella. The genus has
about 20 species, the relationships between all the species have not been resolved in details, however most
likely they constitute a coherent group.
Note 2 to entry: They are aerobic non fermentative chemoorganotrophs, vibroid, 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: FprCEN/TS 17724:2021, term 3.2.2.1]
4 Enumeration of Azospirillum spp
The goal of the method is to Enumeration of Azosprillium spp. (CFU/g) in the given bio-
stimulant/formulation [2].
4.1 Sampling
Sampling is not part of the method specified in this document (see the specific European Standard
dealing with the product concerned). If there is no specific International or European Standard, it
is recommended that the parties concerned come to an agreement on this subject.
It is important that the laboratory receives a sample which is representative and has not been
damaged or changed during transport or storage.
6

---------------------- Page: 8 ----------------------
kSIST-TS FprCEN/TS 17713:2021
FprCEN/TS 17713:2021 (E)
4.2 Preparation of the sample
4.2.1 Sample preparation
A representative sample of the product to be analysed (see FprCEN/TS 17702-1) will 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 [4].
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 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 [4].
4.2.4 Solid - Wettable Powder (WP) formulations
Dispense 25 gr 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 [4].
4.2.5 Solid - Water dispersible granules (WDG) formulati
...

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