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

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Due Date
23-Mar-2022
Completion Date
23-Mar-2022

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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
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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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kSIST-TS FprCEN/TS 17722:2021
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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kSIST-TS FprCEN/TS 17722:2021
FprCEN/TS 17722:2021 (E)
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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kSIST-TS FprCEN/TS 17722:2021
FprCEN/TS 17722:2021 (E)
Introduction

This document was prepared by the experts of CEN/TC 455 ‘Plant Biostimulants’. The European

Committee for Standardization (CEN) was requested by the European Commission (EC) to draft

European standards or European standardization deliverables to support the implementation of

Regulation (EU) 2019/1009 of 5 June 2019 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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FprCEN/TS 17722:2021 (E)
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.
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

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:

• 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;
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;
• 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.
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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