CEN/TS 17722:2022
(Main)Plant biostimulants - Determination of mycorrhizal fungi
Plant biostimulants - Determination of mycorrhizal fungi
This document was developed to provide a horizontal method for enumeration and genera/species determination [1], [2], [3] of mycorrhizal fungi in plant biostimulants products in accordance with the EU Fertilising Products Regulation.
Pflanzen-Biostimulanzien - Bestimmung von Mykorrhizapilzen
Dieses Dokument wurde entwickelt, um ein horizontales Verfahren für die Zählung und die Bestimmung der Gattung/Spezies [1] [2] [3] von Mykorrhizapilzen in Produkten der Pflanzen Biostimulanzien in Übereinstimmung mit der EU Düngemittelverordnung bereitzustellen.
Biostimulants des végétaux - Détermination des champignons mycorhiziens
Le présent document a été élaboré pour fournir une méthode horizontale pour le dénombrement et la détermination du genre/de l’espèce [1], [2], [3] des champignons mycorhiziens dans les produits biostimulants des végétaux conformément au Règlement UE sur les fertilisants.
Rastlinski biostimulanti - Določanje mikoriznih gliv
Ta dokument zagotavlja vodoravno metodo za štetje in določanje rodov/vrst [1], [2], [3] mikoriznih gliv v rastlinskih biostimulantih v skladu z Uredbo EU o sredstvih za gnojenje.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
01-februar-2023
Rastlinski biostimulanti - Določanje mikoriznih gliv
Plant biostimulants - Determination of Mycorrhizal fungi
Pflanzen-Biostimulanzien - Bestimmung von Mykorrhizapilzen
Biostimulants des végétaux - Détermination des champignons mycorhiziens
Ta slovenski standard je istoveten z: CEN/TS 17722:2022
ICS:
65.080 Gnojila Fertilizers
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
CEN/TS 17722
TECHNICAL SPECIFICATION
SPÉCIFICATION TECHNIQUE
March 2022
TECHNISCHE SPEZIFIKATION
ICS 65.080
English Version
Plant biostimulants - Determination of mycorrhizal fungi
Biostimulants des végétaux - Détermination des Biostimulanzien für die pflanzliche Anwendung -
champignons mycorhiziens Bestimmung von Mykorrhizapilzen
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 NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
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 17722:2022 E
worldwide for CEN national Members.
Contents Page
European foreword . 4
Introduction . 5
1 Scope . 7
2 Normative references . 7
3 Terms and definitions . 7
4 Methods for the quantification of mycorrhiza . 9
4.1 General . 9
4.2 How to prepare the initial sample .11
4.2.1 General .11
4.2.2 Liquid – water-based formulations .11
4.2.3 Liquid – oil-based (emulsifiable concentrate EC) formulations .11
4.2.4 Solid – wettable powder (WP) formulations .11
4.2.5 Solid – water dispersible granules (WDG) formulations .11
4.2.6 Solid – pellets, granules, microgranules (slow release) formulations.12
4.2.7 Solid – substrate .12
4.3 Enumeration methods .12
4.3.1 General .12
4.3.2 Method N° 1: Spore isolation and counting MTT .12
4.3.3 Method N° 2: Procedure for clearing and staining root samples and enumeration of
vesicles in the stained root samples .14
4.3.4 Enumeration of the total number of UPM in the product using Method N°1 + Method
N°2 .17
4.3.5 Method N°3: Endomycorrhiza bioassay .17
4.3.6 Method N°4: Ectomycorrhiza and ericoid count .25
5 Molecular characterization and identification of mycorrhiza isolates .29
5.1 General .29
5.2 Materials and equipment .29
5.3 Method for the molecular characterization and identification of mycorrhiza isolates
....................................................................................................................................................................29
5.3.1 Spores cleaning .29
5.3.2 DNA extraction .30
5.3.3 Preparation for PCR .31
5.3.4 Preparation for gel-electrophoresis .33
5.3.5 Direct sequencing (outsourced sequencing lab) .34
6 Method of molecular characterization and identification for ectomycorrhiza and
ericoid .34
6.1 General .34
6.2 Materials .35
6.2.1 Fungal material .35
6.2.2 Molecular biology kits/chemicals .35
6.2.3 Equipment .36
6.3 Detailed description of method .36
6.3.1 Material preparation .36
6.3.2 DNA extraction and quality check .37
6.3.3 PCR amplification of ITS sequences . 37
6.3.4 Gel electrophoresis and PCR product visualization . 38
Bibliography . 39
European foreword
This document (CEN/TS 17722:2022) 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 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 (“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”. Working Group 5 “Labelling and
denominations” was created to develop a work program as part of this standardization request.
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.
The biostimulants used in agriculture can be applied in multiple ways: on soil, on plants, 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 mycorrhiza.
Table 1 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. Mycorrhiza
— symbiotic exchanges i.e. Rhizobiaceae/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.
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 a horizontal method for enumeration and genera/species
determination [1], [2], [3] of mycorrhizal fungi in plant biostimulants products in accordance with the EU
Fertilising Products Regulation.
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.
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
3.1
mycorrhiza
symbiotic relationship between a filamentous fungus and a plant
Note 1 to entry: In a mycorrhizal association, the fungus colonizes the plants’ root tissues either intracellularly (as
with endom
...
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