CEN/TS 17722:2022

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 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.
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]
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
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

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.
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.
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).

Dehydrogenase-activated stain 3-(4,5-dimethylthiazol-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT).
Figure 2 — Sieves
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:
— 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).
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.
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;
— KOH solution (10 %);
KOH means potassium hydroxide.
— 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 roots;
— lactoglycerol (876 ml of lactic acid + 64 ml of glycerol + 60 ml of distilled water);
— Bunsen burner/spirit lamp.
4.3.3.2 Procedure
Wash root samples under running tap water thoroughly. Place the root material into a 50 ml
thermoresistant glass pill jar and cover the root material with 10 % of KOH (do not exceed 2/3 of the
glass pill jar volume). Incubate then for 30 min to 60 min at 80 °C in a dry oven.
Pour off the KOH solution and rinse the roots well in a glass 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 bleached.
2 2
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 samples 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 samples in glass Petri plate/multi well plate for de-staining. The de-staining solution used
is 50 % lactoglycerol or in alternative replace lactoglycerol 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 number Total
Number vesicles/ root number of vesicles in of vesicles in number of
root fragments in vesicles in ……….ml 25 g sample vesicles/g
fragment 1 ml 1 ml (dilution factor) 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 can be trapped in the edges of the sieves, otherwise the
observations can be faulty.
For a clearer explanation, see the following example:
— Take 25 g of the soil/sample suspended in 100 ml of sterile water.
— Obtain a homogenized suspension.
— Use the sieve as described in Figure 2.
— Collect the sieved contents and reconstitute the final volume of 100 ml.
— Homogenize again the suspension and take 1 ml of the suspension in a Petri dish (in triplicates: A, B,
and C).
— Observe under the stereomicroscope and calculate the number of spores in each replicate and
assume the number of spores observed as follows: 10 spores (A), 15 spores (B) and 14 spores (C).
— Calculate the average number of spores as follows: (number of spores in A+B+C)/3. In this case, the
average number of spores is 13 (this is the number of spores observed in 1 ml of the suspension). An
example is reported in Table 4.
— Calculate the number of spores in 100 ml of the suspension as follows: average number of spores/ml
× 100. In the present case, it is 13 × 100 = 1 300 spores (this is the number of spores present in 25 g
of the sample).
— Calculate the number of spores/g as follows: total number of spores present in 100 ml of the
sample/25. In this case, it is as follows: 1 300/25 = 52 spores.
Table 4 — Enumeration of number of intra-radical vesicles in the product
Serial Number of Frequency of Total Total number of Total number Total
number vesicles/ root number of vesicles in of vesicles in number of
root fragments in vesicles in 100 ml (dilution 25 g sample vesicles/g
fragment 1 ml 1 ml factor) in sample
1 1 10 10 4 500 4 500 180
2 2 4 8
3 3 2 6
4 4 2 8
5 5 1 5
6 6 0 0
7 7 0 0
8 8 1 8
9 9 0 0
10 10 0 0
Total 20 45
Thus, the following results are obtained:
— the number of spores/g of the sample is 52;
— total number of vesicles/g in the product equals 180;
— total number of UPM /g in the product equals total number of spores/g + total number of
vesicles/g equals 52 + 180;
— total number of UPM /g in the product equals 232.
4.3.4 Enumeration of the total number of UPM in the product using Method N°1 + Method N°2
Estimate the total number of UPM/g in the product as follows: total number of UPM/g in the
product equals total number of spores/g (Method 1) + total number of vesicles/g (Method 2).
Calculate the total number of UPM in the whole product as follows: total number of UPM/g in the product
× net weight (g) of the product.
4.3.5 Method N°3: Endomycorrhiza bioassay
4.3.5.1 Principle of the MPN (Most Probable Number of mycorrhizal propagule)
Determining the mycorrhizal potential of a soil or fungal inoculum by the MPN test (Most Probable
Number of mycorrhizal propagule) consists in quantifying the number of propagules (fungal propagation
unit such as spore, vesicle, sporocarp, mycorrhizal root fragments, living hypha or auxiliary cells). This is
a biological test, usually carried out in the presence of mycorrhizal host plants. The potential term is
related to the ability of a soil or fungal inoculum to generate mycorrhiza under predefined growth
condition and host plant, depending on the question asked [6].
Beforehand, the MPN test requires to clarify the question of the term “potential”, especially for a fungal
inoculum. The MPN test can be strongly influenced by different parameters (choice of host plant, plant
density, choice of substrate, choice of fertilization, light intensity, growing condition, volume of growing
pots and timing of implementation or seasonality). Any condition that inhibits the germination of
propagules and intraradical mycorrhiza (high phosphate content, type of fertilizer, type of substrate used,
use of plants with little or medium mycorrhiza, reduced light intensity, low plant density, etc.) can more
or less strongly influence the value of the number of propagules calculated through the MPN test
(depending on a factor of 1 to 1 000), potentially de-loading from the actual number of propagules
contained in the sample. The term “potential” is therefore synonymous with “the ability to generate
mycorrhiza, related to the conditions of the implementation of the test” but also the “ability of propagules
(which takes into account the number, viability and dormant state) to generate mycorrhiza.”
When it comes to determine the closest number of propagules actually contained in an inoculum (which
can be called the “maximum mycorrhizal potential”), it would be necessary to set up conditions that
promote the germination of propagules and intraradical colonization. For this, the use of plants highly
susceptible to mycorrhization such as Plantago lanceolata, Trifolium pratensis or Medicago truncatula
developing on a chemically and biologically inert substrate (most often sterilized sand) is recommended,
as well as the controlled application of a low available phosphate fertilizer (Hoagland-based solution).
Plant density is also an important factor: the denser the root system is, the greater are the chances of
contact with a fungal propagule. On the other hand, especially for a fungal inoculum, if one wishes to
determine the mycorrhizal potential in the frame of a specific application (which can be called mycorrhiza
potential relative to a designed target), the test should be implemented by approaching the target
conditions, notably in terms of host plant, substrate and fertilization.
The principle of the MPN test is to establish successive dilutions of the sample (see Figure 3) with a
sterilized substrate (either the same target soil or sand depending on the question asked, sterilized twice
at 120 °C for 6 h), using a fast-growing plant species (most often an herbaceous or legume, highly
susceptible to mycorrhizal colonization). The dilution factor, the number of successive dilutions and the
number of repetitions depend on the time available, the type of sample and the degree of accuracy
desired. The dilution factor influences the accuracy of the MPN test value; the number of repetitions
influences the confident interval as well as standard error (particularly for comparison purposes); the
number of successive dilution “caps” the maximum value that can be calculated.
After 6 weeks, a scoring is performed by observation under binocular microscope of intact root systems
after washing and staining with China Ink. Each notation is denoted by “+” or “-”, depending on whether
or not there is a visible mycorrhizal colonization [7]. The number of propagules per g or per kg of soil is
then deducted from this reading by the use of tables which give a range of statistical estimation [8] or,
more quickly, through free and available programs on the internet [16],[17].
Two protocols are described in the Figure 1, which differ depending if the inoculum is crude or based on
mycorrhizal root fragments powder. In the latter case, the first dilution is specifically set due to the
inability of plants to grow in the pure powder material and due to the high cost of the powder inoculum.
The inoculum (especially the powder based with mycorrhizal root fragments) can saturate the MPN test
set with 6 successive dilutions. In this case, the test is adapted with 7 to 10 successive dilutions,
depending on the data obtained from the microwave assisted staining method.
MPN test can be more or less difficult to implement depending on the inoculum formulation, in particular
those with big particle size or liquid/gel formulation (issues could happen during a proper
homogenization when preparing the dilution). In this case, as the formulation is an important integrative
part of a mycorrhizal inoculum, it is not advised to extract mycorrhizal propagules to then perform the
MPN test.
Key
A volume adapted with crude inoculum
B volume adapted with powder based with mycorrhizal root fragments
Figure 3 — Example of a schematic presentation of the MPN test
In Figure 3 the inoculum to analyse is diluted in 6 successive dilutions (by 10-fold), with five replicates
per dilution. The evaluation of the MPN test depends on the ability to observe the entire root system in
order to search for mycorrhizal propagules. When the root system is colonized (containing typical fungal
structures such as vesicles, arbuscule, spores and eventually auxiliary cells around roots), the notation is
easily noted as positive (see Figure 4 a)). Then, when no fungal mycorrhizal structure is observed, the
rating is negative (Figure 4 c)). However, propagules can consist of spores, which can germinate but
whose development of hyphae can surround and/or contact the root without penetration into the cortex.
When the presence of an extra radical mycorrhizal propagule is observed, the notation should be
considered positive since it is present, provided that such structures are well recognized as mycorrhizal
(which means observing the presence of spore and/or root fragment containing vesicles/spores
connected to the hypha that contacts or surrounds the root). A distinction can be made between the
ability to efficiently generate mycorrhiza in the root (relative potential, see Figure 4 a)) and the total
number of propagules (maximum potential) that take into account the propagules colonizing the cortical
tissues (Figure 4 a)) with those detected around the root system (Figure 4 b)).

a) b) c)
Key
a) Positive notation where intraradical mycorrhizal colonization is clearly visible.
b) Positive notation by the presence of a hypha surrounding the root and connected with a mycorrhizal spore
(within the limit of being able to recognize such structures), but without colonization within the root
system.
c) Negative notation where no mycorrhiza fungal structure is observed.
Figure 4 — Definition of MPN rating from 3 case studies
4.3.5.2 Protocols and examples
4.3.5.2.1 General
There are 4 steps to obtain the MPN.
4.3.5.2.2 Step 1
Considering only mycorrhizal inoculum, two sample types are provided below: one using the so-called
“crude” inoculum, i.e. a substrate-based inoculum, containing mycorrhizal propagules in the form of root
fragments, spores and/or hypha; and one which consists of a pure powder made of mycorrhizal root
fragments, containing mycorrhizal propagules in the form of intraradical spores and vesicle. It is usually
expected that the number of propagules is higher within pure powder based mycorrhizal inoculum than
with crude inoculum. In the example below, 50 ml pots (multiarray pots) are used, hence each dilution is
prepared from 300 ml mix (to be adjusted according to the volume of the pot). If the mass density of the
sample and the diluent differ greatly (such as a vermiculite-based inoculum and a sand-based diluent), it
is preferable to mix by volume and not by mass.
Step 1 a): crude inoculum
The first dilution (so-called pure) needs 5 pots of 50 ml, i.e. 250 ml in total. As the preparation of the next
dilution requires a fraction of the present dilution, it requires a supplement (50 ml, thus a total of 300 ml).
Fill each of the 50 ml pots with the pure product of the mycorrhizal inoculum. It shall therefore remain
50 ml after filling the first dilution series.
Step 1 b): powder-based inoculum
Mix 1 g of the powder inoculum in 300 ml sterilized sand. Then, fill each of the 50 ml pots with the pure
product of the mycorrhizal inoculum. It shall therefore remain 50 ml after filling the first dilution series.
4.3.5.2.3 Step 2: preparation of the dilution
From the remaining 50 ml obtained from both methods in Step 1, prepare the second dilution (1 : 10 in
this example): dilute 30 ml of the inoculum with 270 ml sterilized sand (total is 300 ml). Then, fill in the
50 ml pot with this mix. If all goes well, there are 50 ml left. From the remaining 50 ml, prepare the third
dilution as for the second and so on, until desired final dilution is reached. The final MPN test should
contains 8 dilutions series, by 10-fold dilutions with 5 repetitions per dilution, to cover most of inoculum
product richness.
4.3.5.2.4 Step 3: plant seeding
Seeds from strong mycorrhizal plant sp
...