SIST-TS CEN/TS 17722:2023
(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/specie determination [1], [2], [3] of mycorrhizal fungi in plant biostimulants products in accordance to the Regulation of EU fertilizing products.
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 Biostimulanzien für die pflanzliche Anwendung 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
SIST-TS CEN/TS 17722:2023
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
SIST-TS CEN/TS 17722:2023 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST-TS CEN/TS 17722:2023
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.
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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
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6.3.3 PCR amplification of ITS sequences . 37
6.3.4 Gel electrophoresis and PCR product visualization . 38
Bibliography . 39
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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.
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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.
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IMPORTANT — It is absolutely essential that tests conducted in accordance with this document be
carried out by suitably trained staff.
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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 endomycorrhiza) or extracellularly (as with ectomycorrhiza). This beneficial interaction brings several
advantages to the plants such as, for instance, enhancement of nutrients and water uptake.
[SOURCE: CEN/TS 17724:2022, 3.2.2.6]
3.2
endomycorrhiza
symbiotic association characterized by a filamentous fungal partner that colonizes the plants’ root tissues
intracellularly
EXAMPLE Four main groups of endomycorrhizal associations exist like arbuscular, ericoid, orchidoid and
sebacinoid mycorrhiza.
[SOURCE: CEN/TS 17724:2022, 3.2.2.6.1]
3.3
arbuscular mycorrhizal fungus
AMF
AM fungus
biotrophic microscopic fungus belonging to the Glomeromycota phylum (synonymous Glomeromycota)
that establishes obligate symbiotic associations with more than 70 % of plant species on Earth
Note 1 to entry: Arbuscular mycorrhizal fungi produce structures inside plant roots, such as vesicles and/or
endospores, but also specialized nutrient exchange structures called arbuscules.
Note 2 to entry: The hyphae do not penetrate the plant cell protoplast, but instead they invaginate the cortical cell
membrane, where they branch dichotomously to develop the arbuscule, which is meant to be the place where the
exchange of nutrients and water takes place between the plant and the fungus.
Note 3 to entry: Arbuscular mycorrhizal fungi extraradical mycelium forms an extensive network within the soil,
which increases plant nutrient availability and absorption.
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3.4
ericoid mycorrhizal fungus
filamentous fungus belonging to the Ascomycota phylum that establishes endomycorrhizal symbiotic
associations specifically with ericaceous plants (such as blueberry and cranberry)
Note 1 to entry: The intraradical growth phase is characterized by dense coil of hyphae in the outermost layer of
root cells. Ericoid mycorrhizal fungi also have saprotrophic capabilities which can enable the plant to access
nutrients not yet available.
3.5
orchidoid mycorrhizal fungus
filamentous fungus belonging to the Basidiomycota phylum that establishes endomycorrhizal symbiotic
associations specifically with orchids
Note 1 to entry: The hyphae of ochidoid mycorrhizal fungi penetrate the root cell and form dense coil of hyphae,
where the nutrient exchange takes place.
3.6
sebacinoid mycorrhizal fungus
endophytic filamentous fungus belonging to the Basidiomycota phylum, more specifically, the order
Sebacinales, which establishes mutualistic symbiotic relationship with a wide variety of plant hosts
EXAMPLE The model species Piriformospora spp.
Note 1 to entry: Sebacinoid mycorrhizal fungi colonize plant roots with intracellular mycelium, where the nutrient
exchanges take place.
3.7
serendipita mycorrhizal fungus
serendipitaceae (formerly Sebacinales Group B) belonging to a taxonomically, ecologically and
physiologically diverse group of fungi in the Basidiomycota (kingdom Fungi)
Note 1 to entry: While historically recognized as orchid mycorrhizae, recent based phylogenetic studies have
demonstrated both their pandemic distribution and the broad spectrum of mycorrhizal types they form.
Note 2 to entry: Serendipita mycorrhizal fungi are associated to all families of herbaceous angiosperms (flowering
plants) from temperate, subtropical and tropical regions.
Note 3 to entry: Serendipitaceae mycorrhizal fungi should be considered as a previously hidden but amenable and
effective microbial tool for enhancing plant productivity and stress tolerance.
3.8
ectomycorrhiza
hyphal sheath, or mantle, covering the root tip and an extracellular Hartig net of hyphae surrounding the
plant cells within the root cortex
Note 1 to entry: Beneficial symbiotic associations established by filamentous fungi belong mainly to the
Ascomycota and Basidiomycota phylum with around 5 % to 10 % of coniferous and deciduous trees.
Note 2 to entry: In some cases, the hyphae can also penetrate the plant cells, in which case the mycorrhiza is called
an ectendomycorrhiza. Outside the root, ectomycorrhizal extraradical mycelium forms an extensive network within
the soil, which increases plant nutrient availability and absorption. Since these fungi have septate hyphae, hyphal
fragments along with spores are considered long-term effective propagation structures.
[SOURCE: CEN/TS 17724:2022, 3.2.2.6.2]
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3.9
spore
very small and very tough cell able of germination under favourable conditions, caused by the fungi which
ensure its dissemination
Note 1 to entry: There are sexual, asexual or vegetative spores [1].
3.10
propagule
component of the fungus able to initiate a symbiosis with root
3.11
in vivo
production performed in open area (greenhouse, tunnel, open field)
3.12
in vitro
production performed in monoxenic conditions
3.13
Unit Potential Mycorrhizal
UPM
unit of counting for mycorrhiza
where
U is unit, spore or propagule of any type able to initiate mycorrhiza formation in a host plant’s root;
P is potential, since the development of the symbiosis depend on different factors (soil, plant,
agriculture practises, competition with other soil borne microorganisms, etc.);
M is mycorrhizal, since the inoculum is able to synthesize new mycorrhizae in association with plant
roots depending on factors previously cited.
EXAMPLE UPM per gram (% spores, % propagules) (in vivo, in vitro).
4 Methods for the quantification of mycorrhiza
4.1 General
According to the type of mycorrhiza analysed (see Figure 1), the method to be used is listed in the Table 2
to obtain the quantification in UPM.
Figure 1 — Different types of mycorrhizas and propagules
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Table 2 — Methods to use for enumeration of UPM with plant cultures and without plant cultures
Origin of SPORES Other Endo Ectomycorrhiza Ericoid Orchidoid Sebacinoid Serendipita
product propagules,
Extractable mycorrhiza
roots extractable
in vitro 1 Yes NO Method N°1 Method N°4 Method N°4
in vitro 2 Yes Yes Method N°1 to count the spores
and Method N°2 to count
propagules
in vivo 1 NO NO Method N°3
in vivo 2 Yes NO Method N°1 Method N°4 Method N°3
in vivo 3 Yes Yes Method N°1 to count the spores
and Method N°2 to count
propagules
in vivo 4 NO Yes Method N°2 Method N°3 Method N°3 Method N°3
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4.2 How to prepare the initial sample
4.2.1 General
A base concentration of a product is a product of 500 UPM/g. All the preparation should be made
according to this.
H High concentration means higher than 100 000 UPM/g;
M Medium means between 1 000 UPM/g and 100 000 UPM/g;
L Low means below 1 000 UPM/g.
For samples with different concentrations, different amounts should be taken in a proportionate amount
of tap water in order to maintain the proportion 1 : 10 as follows:
— for H, take 2,5 g in 22,5 millilitre of tap water;
— for M, take 25 g in 225 ml of tap water;
— for L, take 250 g in 2,250 ml of tap water.
A representative sample of the product shall be prepared according to the following procedure which
takes into consideration the different formulations of biostimulants based products.
4.2.2 Liquid – water-based formulations
Dispense the quantity of sample depending on the concentration of the product as described in 4.2.1 of
tap water 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.
4.2.3 Liquid – oil-based (emulsifiable concentrate EC) formulations
Dispense the quantity of sample depending on the concentration of the product as described in 4.2.1 of
tap water 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.
4.2.4 Solid – wettable powder (WP) formulations
Dispense the quantity of sample depending on the concentration of the product as described in 4.2.1 of
tap water 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.
4.2.5 Solid – water dispersible granules (WDG) formulations
Dispense the quantity of sample depending on the concentration of the product as described in 4.2.1 of
tap water 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
®1
other apparatus such as a Stomacher after having sieved (100 mesh sieve) the particles and resuspend
them in the same suspension.
1 ®
Stomacher 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.
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4.2.6 Solid – pellets, granules, microgranules (slow release) formulations
Dispense the quantity of sample depending on the concentration of the product as described in 4.2.1 of
tap water 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.
4.2.7 Solid – substrate
Dispense the quantity of sample depending on the concentration of the product as described in 4.2.1 of
tap water 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.
The time required for some analyses is too long and the cost too high and therefore fast and economical
methods are proposed.
According to the diversity of the types of mycorrhizae several methods are proposed for the
quantification, depending on the origin of products and the extraction of spores and propagules.
4.3 Enumeration methods
4.3.1 General
The methods described in 4.3.2, are those listed in Table 2.
2
4.3.2 Method N° 1: Spore isolation and counting MTT
4.3.2.1 Procedure for enumeration of spores
Use the following procedure for enumeration of spores:
— Decant the suspension through a series of sieves arranged in descending order of opening size:
250 μm, 150 μm and 25 μm. The coarse particles are collected on a coarse sieve, while spores are
captured on one or more finer sieves.
— Vigorous washing with water is necessary to free spores from aggregates of clay or organic materials.
— Collect the sieved contents in jars. Transfer a known volume (for example, 1 ml/10 ml of the sieved
contents) onto the gridded Petri dishes/plates and observe under a stereomicroscope.
— Count the number of spores in plate/dish. Accordingly, calculate the total number of spores in the
100 ml of the suspension.
— Express the number of spores in spores/(g of the sample).
2
Dehydrogenase-activated stain 3-(4,5-dimethylthiazol-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT).
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Figure 2 — Sieves
4.3.2.2 Viability of spores
4.3.2.2.1 General
3
The spore sample shall be absolutely free of any other particles since MTT like INT can react with some
other components.
4.3.2.2.2 Materials and equipment
The materials and equipment for spores isolated from the inoculum are the following:
— MTT as a first option, INT only if MTT is not available;
— sterile distilled water;
— aluminium foil;
® 4
— Falcon tube (15 ml);
— micropipette (1 ml);
®4
— Eppendorf tubes ;
— forceps;
— Petri dish;
— microscope with external light source;
— incubator (28 °C).
3
INT means iodonitrotetrazolium.
4 ® ®
Falcon tube and Eppendorf tube are examples of suitable products available commercially. This information
is given for the convenience of users of this document and does not constitute an endorsement by CEN of these
products.
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4.3.2.2.3 Procedure
Use the following procedure for viability of spores:
— Prepare stock solution of MTT having 0,1 % concentration in sterile distilled water and cover with
aluminium foil.
— Take the original suspension and pick about from 50 spores to 100 spores with the pipette.
— Keep this suspension overnight at 28 °C in the incubator, before using.
®
— In an Eppendorf tube , add 500 μl of spore suspension.
— Add 500 μl of MTT from 0,1 % stock to make 1 ml final volume.
®
— Incubate the Eppendorf tubes in dark at 28 °C from 42 h to 48 h.
— Observe after 24 h and from 42 h to 48 h.
— Viable spores in absence of an external source appear dark pink in colour and red/purple when
observed with an external source.
— Percentage viability can be calculated as follows:
Spore viability (%) means (number of viable spores/total number of spores) × 100.
4.3.3 Method N° 2: Procedure for clearing and staining root samples and enumeration of vesicles
in the stained root samples
4.3.3.1 Materials and equipment
The materials and equipment for the enumeration of vesicles are:
— mycorrhizal based product/sample;
— 25 µm to 50 µm sieve for small sized vesicles;
— 150 µm sieve for medium-sized vesicles;
— 250 µm sieve for very large vesicles;
— glass jars for collecting the sieving;
— stereo zoom (stereomicroscope) and simple compound microscope;
— Petri dishes (90 mm or 30 mm) for observing the sieving under a stereomicroscope;
— micropipettes for spore picking;
— centrifuge;
5
— KOH solution (10 %);
5
KOH means potassium hydroxide.
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— glass beaker (250 ml);
— scissors and needles;
— water bath;
— coarse sieves (250 µm, 150 µm and 25 µm) to prevent root loss during washing/changing solutions;
— plastic vials with tight-sealing lids for storage of stained samples in 50 % glycerol;
— alkaline H O (3 ml of 25 % ammonia solution + 30 ml of 10 % H O + 67 ml of distilled water);
2 2 2 2
— 1 % HCl;
— 50 % glycerol-water (volume fraction) solution for de-staining and storage of stained
...
SLOVENSKI STANDARD
kSIST-TS FprCEN/TS 17722:2021
01-november-2021
[Not translated]
Plant biostimulants - Determination of Mycorrhizal fungi
Biostimulanzien für die pflanzliche Anwendung - Bestimmung von Mykorrhizapilzen
Ta slovenski standard je istoveten z: FprCEN/TS 17722
ICS:
65.080 Gnojila Fertilizers
kSIST-TS FprCEN/TS 17722:2021 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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kSIST-TS FprCEN/TS 17722:2021
FINAL DRAFT
TECHNICAL SPECIFICATION
FprCEN/TS 17722
SPÉCIFICATION TECHNIQUE
TECHNISCHE SPEZIFIKATION
September 2021
ICS 65.080
English Version
Plant biostimulants - Determination of Mycorrhizal fungi
Biostimulanzien für die pflanzliche Anwendung -
Bestimmung von Mykorrhizapilzen
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 17722:2021 E
worldwide for CEN national Members.
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FprCEN/TS 17722:2021 (E)
Contents
European foreword . 4
Introduction . 5
1 Scope . 6
2 Normative references . 6
3 Terms and definitions . 6
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 . 11
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 specimens and
enumeration of vesicles in the stained root specimens . 14
4.3.4 Enumeration of the total number of U.P.M 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 . 24
5 Molecular characterization and identification of mycorrhiza isolates . 27
5.1 General . 27
5.2 Materials and equipment . 27
5.3 Method for the molecular characterization and identification of mycorrhiza
isolates . 27
5.3.1 Spores cleaning . 27
5.3.2 DNA extraction . 28
5.3.3 Preparation for PCR . 29
5.3.4 Preparation for gel-electrophoresis . 31
5.3.5 Direct sequencing (outsourced sequencing lab) . 32
6 Method of Molecular characterization and identification for Ectomycorhiza
and ericoid . 32
6.1 General . 32
6.2 Materials . 32
6.2.1 Fungal material . 32
6.2.2 Molecular biology kits/chemicals . 33
6.2.3 Equipments . 33
6.3 Detailed description of method . 34
6.3.1 Material preparation . 34
6.3.2 DNA extraction and quality check . 34
6.3.3 PCR amplification of ITS sequences . 35
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6.3.4 Gel electrophoresis and PCR product visualization . 35
Bibliography . 36
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European foreword
This document (FprCEN/TS 17722:2021) 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 Standardization Request given to CEN by the
European Commission and the European Free Trade Association.
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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 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.
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 Fertilizing
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. 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.
IMPORTANT — It is absolutely essential that tests conducted in accordance with this document
be carried out by suitably trained staff.
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1 Scope
This document was developed to provide a horizontal method for enumeration and genera/specie
determination [1], [2], [3] of mycorrhizal fungi in plant biostimulants products in accordance to
the Regulation of EU fertilizing products.
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 endomycorrhiza) or extracellularly (as with ectomycorrhiza). This beneficial
interaction brings several advantages to the plants such as, for instance, enhancement of nutrients and
water uptake.
[SOURCE: FprCEN/TS 17724, 3.2.2.7]
3.2
endomycorrhiza
symbiotic association characterized by a filamentous fungal partner that colonizes the plants’ root
tissues intracellularly
EXAMPLE Four main groups of endomycorrhizal associations exist like arbuscular, ericoid, orchidoid
and sebacinoid mycorrhiza.
[SOURCE: FprCEN/TS 17724, 3.2.2.7]
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3.3
arbuscular mycorrhizal fungi
AMF
AM fungi
biotrophic microscopic fungi belonging to the Glomeromycota phylum (synonymous
Glomeromycota) that establish obligate symbiotic associations with more than 70% of plant
species on Earth
Note 1 to entry: Arbuscular mycorrhizal fungi produces structures inside plant roots, such as vesicles
and/or endospores, but also specialized nutrient exchange structures called arbuscules.
Note 2 to entry: The hyphae do not penetrate the plant cell protoplast, but instead, it invaginates the
cortical cell membrane where it branches dichotomously to develop the arbuscule which is mean to be the
place where the exchange of nutrients and water takes place between the plant and the fungus.
Note 3 to entry: Arbuscular mycorrhizal fungi extraradical mycelium forms an extensive network within
the soil which increase plant nutrient availability and absorption.
3.4
ericoid mycorrhizal fungi
filamentous fungi belonging to the Ascomycota phylum that establish endomycorrhizal symbiotic
associations specifically with Ericaceous plants (such as blueberry and cranberry)
Note 1 to entry: The intraradical growth phase is characterized by dense coil of hyphae in the outermost
layer of root cells. Ericoid mycorrhizal fungi also have saprotrophic capabilities which can enable plant to
access nutrients not yet available.
3.5
orchidoid mycorrhizal fungi
filamentous fungi belonging to the Basidiomycota phylum that establish endomycorrhizal
symbiotic associations specifically with Orchids
Note 1 to entry: The hyphae of ochidoid mycorrhizal fungi penetrates the root cell and forms dense coil of
hyphae where the nutrient exchange take place.
3.6
sebacinoid mycorrhizal fungi
endophytic filamentous fungi belonging to the Basidiomycota phylum, more specifically the order
Sebacinales, which establishes mutualistic symbiotic relationship with a wide variety of plant host
EXAMPLE The model species Piriformospora spp.
Note 1 to entry: Sebacinoid mycorrhizal fungi colonizes plant roots with intracellular mycelium where the
nutrient exchanges take place.
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3.7
serendipita mycorrhizal fungi
serendipitaceae (formerly Sebacinales Group B) belong to a taxonomically, ecologically and
physiologically diverse group of fungi in the Basidiomycota (kingdom Fungi)
Note 1 to entry: While historically recognized as orchid mycorrhizae, recent based phylogenetic studies
have demonstrated both their pandemic distribution and the broad spectrum of mycorrhizal types they
form.
Note 2 to entry: Serendipita mycorrhizal fungi is associated to all families of herbaceous angiosperms
(flowering plants) from temperate, subtropical and tropical regions.
Note 3 to entry: Serendipitaceae mycorrhizal fungi should be considered as a previously hidden, but
amenable and effective microbial tool for enhancing plant productivity and stress tolerance.
3.8
ectomycorrhiza
hyphal sheath, or mantle, covering the root tip and an extracellular Hartig net of hyphae
surrounding the plant cells within the root cortex
Note 1 to entry: Beneficial symbiotic associations established by filamentous fungi belonging mainly to the
Ascomycota and Basidiomycota phylum with around 5 - 10 % of coniferous and deciduous trees.
Note 2 to entry: In some cases the hyphae may also penetrate the plant cells, in which case the mycorrhiza
is called an ectendomycorrhiza. Outside the root, ectomycorrhizal extraradical mycelium forms an
extensive network within the soil which increase plant nutrient availability and absorption. Since these
fungi have septate hyphae, hyphal fragments along with spores are considered long-term effective
propagation structures.
[SOURCE: FprCEN/TS 17724, 3.2.2.7]
3.9
spores
very small and very tough cells able of germination under favourable conditions, caused by the
fungi which ensure their dissemination
Note 1 to entry: There are sexual, asexual or vegetative spores [1].
3.10
propagules
component of the fungus able to initiate a symbiosis with root
3.11
in vivo
production performed in open area (greenhouse, tunnel, open field)
3.12
in vitro
production performed in monoxenic conditions
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3.13
Unit Potential Mycorrhizal
UPM
unity of counting for mycorrhiza
where
U is unit, spore or propagule of any type able to initiate mycorrhiza formation in a host
plant’s root;
P is potential, since the development of the symbiosis depend on different factors (soil, plant,
agriculture practises, competition with other soil borne microorganisms, etc);
M is mycorrhizal, since the inoculum is able to synthesize new mycorrhizae in association
with plant roots depending on factors previously cited.
EXAMPLE U.P.M per gram (% spores, % propagules) (in vivo, in vitro).
4 Methods for the quantification of Mycorrhiza
4.1 General
According to the type of mycorrhiza analysed (see Figure 1), the method to be used is listed in the
Table 2 to obtain the quantification in U.P.M.
Figure 1 — Different type of mycorrhizas and propagules
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Table 2 — Methods to use for enumeration of U.P.M. with plant cultures and without plant cultures
Origin of SPORES Other Endo Ectomycorrhiza Ericoïd Orchidoid Sebacinoid Serendipita
product propagules,
Extractable mycorrhiza
roots
extractable
IN Yes NO Method N°1 Method N°4 Method N°4
VITRO 1
IN Yes Yes Method N°1 to count the
VITRO 2 spores and Method N°2 to
count propagules
IN NO NO Method N°3
VIVO 1
IN Yes NO Method N°1 Method N°4 Method N°3
VIVO 2
IN Yes Yes Method N°1 to count the
VIVO 3 spores and Method N°2 to
count propagules
IN NO Yes Method N°2 Method Method N°3 Method N°3
VIVO 4 N°3
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4.2 How to prepare the initial sample
4.2.1 General
A base concentration of a product is a product of 500 U.P.M./g. All the preparation should be made
according to this.
H High concentration = higher than 100,000 U.P.M./g;
M Medium = between 1000 and 100,000, U.P.M./g;
L Low = below 1000 U.P.M./g.
For samples with different concentration different amount should be taken in a proportionate
amount of tap water in order to maintain the proportion 1:10 as follow:
• For H take 2,5 g in 22,5 millitre of tap water;
• For M take 25 g in 225 ml of tap water;
• For L take 250 g in 2,250 ml of tap water.
A representative sample of the product shall be prepared according to the following procedure
which takes into consideration the different formulations of biostimulants based products.
4.2.2 Liquid — water based- formulations
Dispense the quantity of sample depending on the concentration of the product as described in
4.2.1 of tap water 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.
4.2.3 Liquid — oil based (emulsifiable concentrate EC) formulations
Dispense the quantity of sample depending on the concentration of the product as described in
4.2.1 of tap water 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.
4.2.4 Solid — Wettable Powder (WP) formulations
Dispense the quantity of sample depending on the concentration of the product as described in
4.2.1 of tap water 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.
4.2.5 Solid — Water dispersible granules (WDG) formulations
Dispense the quantity of sample depending on the concentration of the product as described in
4.2.1 of tap water 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 [5].
4.2.6 Solid — Pellets, granules, microgranules (slow release) formulations
Dispense the quantity of sample depending on the concentration of the product as described in
4.2.1 of tap water 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 [5].
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4.2.7 Solid — substrate
Dispense the quantity of sample depending on the concentration of the product as described in
4.2.1 of tap water 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.
The time required for some analyses is too long and the cost too high and therefore fast and
economical methods are proposed.
According to the diversity of the types of mycorrhizae several methods are proposed for the
quantification, depending on the origin of products and the extraction of spores and propagules.
4.3 Enumeration methods
4.3.1 General
The methods described in 4.3.2, are those listed in Table 2.
1
4.3.2 Method N° 1: Spore isolation and counting MTT
4.3.2.1 Procedure for enumeration of spores
Use the following procedure for enumeration of spores:
• Decant the suspension through a series of sieves arranged in descending order of opening
size: 250 μm, 150 μm and 25 μm. The coarse particles are collected on a coarse sieve, while
spores are captured on one or more finer sieves;
• Vigorous washing with water is necessary to free spores from aggregates of clay or organic
materials;
• Collect the sieved contents in jars. Transfer a known volume (for example 1 ml/10 ml of the
sieved contents onto the gridded petri dishes/plate and observe under stereomicroscope);
• Count the number of spores in plate/dish. Accordingly, calculate the total number of spores
in the 100 ml of the suspension;
• Express the number of spores in spores/ (g of the sample).
Figure 2 — Sieves
1
Dehydrogenase-activated stain 3-(4,5-dimethylthiazol-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT).
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4.3.2.2 Viability of spores
4.3.2.2.1 General
The spore sample shall be absolutely free of any other particles since MTT like INT can react with
some other components.
4.3.2.2.2 Materials and equipment
The materials and equipment for spores isolated from the inoculum are the following:
2
• MTT as a first option, INT only if MTT is not available;
• Sterile distilled water;
• Aluminium foil;
• Falcon tube (15 ml);
• Micropipette (1 ml);
• Eppendorf tubes;
• Forceps;
• Petri dish;
• Microscope with external light source;
• Incubator (28 °C).
4.3.2.2.3 Procedure
Use the following procedure for viability of spores:
• Prepare stock solution of MTT having 0,1 % concentration in sterile distilled water and cover
with aluminium foil;
• Take the original suspension and pick about from 50 spores to 100 spores with the pipette;
• Keep this suspension overnight at 28 °C in the incubator, before using;
• In an eppendorf tube, add 500 μl of spore suspension;
• Add 500 μl of MTT from 0,1 % stock to make 1 ml final volume;
• Incubate the eppendorf tubes in dark at 28 °C from 42 h to-48 h;
• Observe after 24 h and from 42 h to 48 h;
• Viable spores in absence of external source appear dark pink in colour and red/purple when
observed with external source;
2
INT = iodonitrotetrazolium
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• Percentage viability can be calculated as follows:
Spore viability (%) = (Number of viable spores/total number of spores) x 100.
4.3.3 Method N° 2: Procedure for clearing and staining root specimens and enumeration
of vesicles in the stained root specimens
4.3.3.1 Materials and equipment
The materials and equipment for the enumeration of vesicles are:
• Mycorrhizal based product/sample;
• 25 µm to 50 µm sieve for small sized vesicles;
• 150 µm sieve for medium-sized vesicles;
• 250 µm sieve for very large vesicles;
• Glass jars for collecting the sieving;
• Stereo zoom (stereomicroscope) and simple compound microscope;
• Petri dishes (90 mm or 30 mm) for observing the sieving under stereomicroscope;
• Micropipettes for spore picking;
• Centrifuge;
3
• KOH solution (10 %);
• Glass beaker (250 ml);
• Scissors and needles;
• Water bath;
• Coarse sieves (250 µm, 150 µm and 25 µm) to prevent root loss during washing/changing
solutions;
• Plastic vials with tight-sealing lids for storage of stained samples in 50 % glycerol;
• Alkaline H O (3 ml of 25 % ammonia solution + 30 ml of 10 % H O + 67 ml of distilled
2 2 2 2
water);
• 1 % HCl;
• 50 % glycerol-water (v/v) solution for de-staining and storage of stained roots;
• Lactoglycerol (876 ml of Lactic acid + 64 ml of Glycerine + 60 ml of distilled water);
• Bunsen burner/spirit lamp.
3
KOH = potassium idroxide.
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4.3.3.2 Procedure
Wash root specimens under running tap water thoroughly. Place the root material into a 50 ml
thermoresistant glass pill and cover the root material with 10 % of KOH (do not exceed 2/3 of the
glass pill volume). Incubate then for 30 min to 60 min at 80 °C in dry oven.
Pour off the KOH solution and rinse the roots well in a beaker using at least three complete
changes of tap water or until no brown colour appears in the rinse water.
If required cover the roots with alkaline H O at room temperature for 10 min or until roots are
2 2
bleached.
Rinse the roots thoroughly using at least three complete changes of tap water to remove the H O
2 2.
Cover the roots with 1 % HCl and soak for 3 min to 4 min and then pour off the solution. Do not
rinse after this because the specimens shall be acidified for proper staining.
Incubate the roots with staining solution (5 % black ink + 8 % acetate in osmosed water) and keep
them overnight for staining.
Place the root specimens in glass petri plate/multi well plate for de-staining. The de-staining
solution used is 50 % lacto glycerol or in alternative replace lacto glycerol with tap water for
minimum 2 h up to 24 h.
Calculate the total number of root fragments/g containing intra-radical vesicles using Table 3.
Table 3 — Enumeration of number of intra-radical vesicles in the product
Serial Number of Frequency of Total Total number of Total Total
Number vesicles/ root number of vesicles in number of number of
root fragments in vesicles in ……….ml vesicles in vesicles/g
fragment 1 ml 1 ml (dilution factor) 25 g sample in sample
1 1
2 2
3 3
4 4
5 5
6 6
7 7
8 8
9 9
10 10
Every effort shall be made to:
• ensure that sieved contents do not get washed away along with the water.
• carefully isolate/recover the spores that may be trapped in the edges of the sieves, otherwise
the observations may be faulty.
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For a clearer explanation see the following example:
• take 25 g of the soil/sample suspended in 100 ml o
...
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