Micorriza Manual

22/11/2017 Procedures
Mycorrhiza Manual
prepared for the Workshop
Arbuscular mycorrhizal fungi in plant

production systems:
detection, taxonomy, conservation and

ecophysiology
held in Wuhan P.R. China at the
Laboratory of Agricultural Microbiology,

Huazhong Agricultural University, Wuhan, P.R. China
April 2001
ORGANISED BY
Dr. John C. Dodd, Dr. Justin P. Clapp - The International Institute of Biotechnology, Sittingbourne
Research Centre, Kent, UK
AND
Prof. Bin Zhao - Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan,
PRC
WITH THE COLLABORATION OF :
Dr. Mary Jo Farmer, Laboratoire de Phytoparasitologie, INRA-CMSE, Dijon, F
Dr. Eckhard George, Institute of Plant Nutrition, Hohenheim University & IGZ, Grossbeeren, G.
Dr. Silvio Gianinazzi, Laboratoire de Phytoparasitologie, INRA-CMSE, Dijon, F
Dr. Vivienne Gianinazzi-Pearson, Laboratoire de Phytoparasitologie, INRA-CMSE, Dijon, F
Prof. Xiaolin Li, China Agricultural University, Beijing, PRC
Ms. Elke Neumann, Institute of Plant Nutrition, Hohenheim University, G
Dr. Diederik van Tuinen, Laboratoire de Phytoparasitologie, INRA-CMSE, Dijon, F
Dr. Wing Kuen Chan, Hong Kong Polytechnic University, Kowloon, Hong Kong
UsefulWeb Links
BEG (Banque Européenne des Glomales) website
Laboratoire de Phytoparasitologie INRA/CNRS
Mark Brundrett's Working with Mycorrhizas in Forestry and Agriculture
INVAM
Mycorrhiza Information Exchange
https://www2.dijon.inra.fr/mychintec/Protocole/Workshop_Procedures.html#Annex1 1/14
David Sylvia
Reference database
National Centre for Biotechnology Information
Mychintec WEB page
International Science Fondation
UNESCO fellowships
COST Action 8.38 Managing arbuscular mycorrhizal fungi for improving soil quality
and plant health in agriculture
1.0 Manipulation and Staining of Spores and Roots

1.1. Spore and Root Extraction from Pot Cultures/Field Samples
Remove soil sample from the rhizosphere of the host plant growing in the pot with a 10-20mm
diameter core borer. If the sample is taken from the field larger quantities should be sieved
(100g-200g) and mixed into a 1L beaker of water before pouring through the sieves. Clay
based soils will block the finer sieve quickly and care must be taken to tap the base of that
sieve to encourage excess water to drain through. The same procedure used for pot culture
material should then be followed:
a. Wash the soil through 710µm and 45µm pore sieves with running water.
b. Remove root material trapped on the 710µm sieve to check for attached mycelium of
AMF with spores or for staining of roots (Trypan blue, Chlorazole Black E, Alkaline
Phosphatase, Acid Fuchsin etc.) if required.
c. Backwash the contents of the 45µm sieve into a small beaker. Try to keep the volume to
a minimum.
a. Swirl the beaker contents and quickly decant the contents into 50ml centrifuge tubes
up to a maximum half way up the tube.
b. Gently inject an equal amount of a 60% (w/v) commercial sugar (sucrose) solution into
the pellet at the bottom of each tube using a syringe with a plastic tube extension.
There should be a clear interface visible between the water (above) and sugar phase
(below).
c. Centrifuge the capped tubes at approx. 3000 rpm for 2 minutes in a bench centrifuge.
d. Remove the spores caught at the interface of the two layers with the syringe and tube
attachment. Start above the interface and work down into the sugar phase using a
circular motion as some species produce spores which can sink in the sugar solution while
others can float just above the interface.
e. Pour the contents of the syringe into a clean 45µm sieve, and wash thoroughly to remove
traces of sugar solution.
f. Backwash contents into a Petri dish and view under a stereomicroscope
1.2. Making a permanent slide mount for reference or BEG registration
a. After extracting spores from a fresh pot culture. Isolate a minimum of 10-20
spores.
b. On two clean microscope slides place one drop each of the mountant PVLG
(Polyvinyllactoglycerol) and Melzer's PVLG see annex 2.
Transfer half the spores to the first drop of mountant and the second half to the
second drop using fine tip forceps (e.g. VOMM forceps No. 999220: HWC 118-10
Hammacher Instruments, P. O. Box 120209, D-42677 Solingen, Germany)
c. Try and orientate the spores so that distinguishing features will be apparent
once the coverslip is added.
d. Carefully place a clean coverslip over each drop, making sure to lower the
coverslip at an angle to prevent air bubbles being trapped.
e. Gently apply a pressure to the coverslips of one of the slides to break open the
spores. Wait 30 seconds and then apply gentle pressure in a circular motion with a
soft (B) pencil to break spore walls open further (The pressure will depend on the
species of AMF). This should be done under a stereomicroscope.
f. If using PVLG, remember to allow the mountant to polymerise and top-up it up as

necessary before sealing with clear nail varnish or white/silver car paint.
g. Label the slide at one end with the species name and reference code, date, your
name, and the mountant used.
1.3. Histochemical Staining of Total AMF Mycelium in Roots
The presence of arbuscular mycorrhizal fungi in roots is not visible without appropriate
staining. Different non-vital strains are available (eg trypan blue, chlorazole black, fuschin) to
detect intraradical mycelium and they enable an estimation of the abundance of arbuscular
mycorrhizal fungi within a root system (Trouvelot et al, 1986). However, they stain both dead
and living fungal structures.
A fuller understanding of AM functioning requires consideration of the metabolic states of

both internal and external hyphae, and the relationship between these, because the
physiological interactions will necessitate the presence of an active symbiotic fungus.

Activity of succinate dehydrogenase (SDH), a mitochondrial enzyme, is considered as an
indicator of viability of mycorrhiza but does not appear to reflect mycorrhizal efficiency for
plant growth enhancement (Vierheilig & Ocampo, 1989). Alkaline phosphatase (ALP) activity,
located within the phosphate-accumulating vacuoles of AM hyphae (Gianinazzi et al., 1979)
has been proposed as a physiological marker for analysing the efficiency of mycorrhiza
(Tisserant et al., 1993). Measurements of these two enzyme activities make it easy to
directly compare the total production of fungal tissue with the proportion that is living or
functional, and to compare simultaneously the production of mycelium within roots and in soil
in order to determine whether (i) biomass produced in the two compartiments is
interdependent and, (ii) the proportion of metabolically active hyphae differs with time.
1.3.1 Root Preparation
a. Wash the roots free of soil.
b. Cut roots into 1cm long segments.
1.3.2 Trypan blue staining of total mycelium
1. Clear roots in 2% (w/v) KOH (10% can be used for very pigmented tree roots) for
15 min at 120°C in a pressure cooker (1h at 90°C in a water bath or oven) (Do not
use samples that are more than 2g.
2. Rinse roots with water three times on a fine sieve or using a mesh and forceps.
3. Cover roots with 2% (v/v) HCl for at least 30 mins and preferably longer.
4. Throw away the HCl and cover roots with 0.05% (w/v) trypan blue in lactoglycerol
(1:1:1 lactic acid, glycerol and water 5:1:1 may be used if tree roots are to be
stained) for 15min at 120°C in a pressure cooker or 15min to 1h at 90°C in water
bath or oven.
5. Place roots into Petri dish with 50% (v/v) glycerol for destaining and viewing under
stereomicroscope.
Figure 3
with the permission of Mark Brundett
1.4 Histochemical staining of active AMF mycelium in roots
1.4.1 Succinate dehydrogenase (SDH)- and alkaline phosphatase (ALP) staining of

intraradical hyphae
1. Wash roots from soil using ice cold water and keep in ice
2. Cut roots into 1cm lengths and mix the roots sample uniform
3. Take two 0.2-0.5g root samples
4. Clear roots in the following solution 2h at room temperature :
20ml 0.05 M Tris/citric acid pH 9.2
50mg/ml sorbitol
15 units/ml cellulase (from A. niger)
15 units/ml pectinase (from A. niger).
5. Rinse roots with water on a fine sieve.
6. Put the roots sample into two bottles marked with SDH and ALP separately, and
add 20ml solution A and B separately
7. Incubate roots pieces overnight at room temperature
8. Pour out mixture solution, wash with distilled water
9. Put the roots marked SDH and ALP in sodium hypochlorite solution(containing 3%
and 1% active chlorine separately) 5min, then wash with distilled water
10. Transfer the roots into a Peri dish
11. Observe purple-black or dark-brown particles in roots under microscope
12. Estimate root length containing stained hyphae (see section 1.5 and figure 3)
Solution A for SDH staining
Chemical Concentration Volume(ml)
Tris/HCl (pH 7.4) 0.2 mol.l-1 5
MgCl2 5 mmol.l-1 2
NBT 4 mg.ml-1 5
H2O 6
Na-succinate 2.5 mol.l-1 2
*NBT------ Nitro-blue Tetrazonium, prepared daily.
Solution B for ALP staining
Chemical Concentration Volume(ml)
Tris/citric acid (pH 0.05 mol.l-1 18ml

9.2)
a -naphthyl acid 1 mg.ml-1 20mg

phosphate
Fast Blue RR salt 1 mg.ml-1 20mg
MgCl2 0.5 mg.ml-1 1 ml
MnCl2.4H2O 0.8 mg.ml-1 1 ml
1.5. Estimation of AMF colonisation
Estimation of mycorrhizal colonization according to Trouvelot et al
a. Mount 15 root fragments on one slide; prepare two slides (30 root fragments total).
b. Observe these fragments under the microscope and rate according to the range of classes
indicated in figure 4 and Annex 1. These classes give a rapid estimation of the level of
mycorrhizal colonisation of each root fragment and the abundance of arbuscules.
c. Put the values into the computer program 'Mycocalc' to calculate the parameters: %F,
%M, %m, %a and %A, according to Trouvelot et al.. 1986. (see Figure 4 from Trouvelot et al
1986)
Frequency of mycorrhiza in the root system

F% = ( nb of fragments myco/total nb)*100
Intensity of the mycorrhizal colonisation in the root system
M% = (95n5+70n4+30n3+5n2+n1)/(nb total)
where n5 = number of fragments rated 5; n4 = number of fragments 4 etc.
Intensity of the mycorrhizal colonisation in the root fragments
m% = M*(nb total)/(nb myco)
Arbuscule abundance in mycorrhizal parts of root fragments
a% = (100mA3+50mA2+10mA1)/100
where mA3, mA2, mA1 are the % of m, rated A3, A2, A1, respectively, with mA3=
((95n5A3+70n4A3+30n3A3+5n2A3+n1A3)/nb myco)*100/m and the same for A2
and A1.
Arbuscule abundance in the root system
A% = a*(M/100)
Figure 4
1.6. Histochemical Staining of Total and Active Soil Mycelium

1.6.1. Extraction and measurement of AM fungal hyphae in soil
1. Take soil cores (1x6cm) randomly from pots.

2. Mix the soil sample well and then put a 2 gram sample in a 500ml beaker.
3. Suspend the soil in 250ml distilled water.
4. Filter the soil suspension through a 300m m mesh sieve.
5. The washings are collected and blended 30 seconds at high speed in a blender.
6. Transfer the suspension to a flask, shake by hand and then stand on the bench for one
minute.
7. Pipette 10ml (5mlx2) aliquots onto a millipore filter(1.0m_m pore size) and filter under
vacuum using the filter holder.
8. Place the filter on a microscope slide and let dry
9. Stain the hyphae on the filter in lactic glycerol-trypan blue (0.05% (v/v)) for 5 minutes.
10. Observe the stained filter under a coverslip at 200X magnification.
11. Examine 30 random fields and estimate hyphal length by using a grid line interception
method as used for evaluating mycorrhizal root lenthes (see Figure 5 from Brundett bet
al 1996).
Figure5
with the permission of Mark Brundett
1.6.2. Estimation of succinate dehydrogenase (SDH)- and alkaline phosphatase (ALP)-

active hyphae in soil
1. Take soil cores and put in a beaker on ice.

2. Mix the soil sample well and take two 2 gram subsamples.
3. Put the sub-samples in two bottles marked with SDH and ALP separately, and cover the
soil with 20ml ice-cold water immediately
4. Add 20ml incubation solution A in the bottle marked with SDH, and add 20ml solution B
in the bottle marked with ALP.
5. Incubate the soil suspension at room temperature for 3 hours.
6. Filter the suspension through a 300m m mesh sieve with 210ml distilled water.
7. The collected washings are blended at high speed for 25 minutes.
8. Transfer the suspension to a flask and leave to stand on the bench for one minute.
9. Pipette 10ml (5mlx2) aliquots on a millipore filter, filter under vacuum as above.
10. Transfer the filter onto a microscope slide.
11. Counterstain AMF hyphae on the filter with 0.1% basic fuschin for 5 minutes.
12. Cover the filter with a coverslip and observe under the microscope at 200X
magnification.
13. Estimate stained hyphal length using the gridline intersect method.
Solution A for SDH staining

Chemical Concentration Volume
Tris/HCl(pH7.4) 0.2M 5ml
MgCl2 5mM 2ml
*NBT 4mg/ml 5ml
H2O 2.5M 6ml
Na-succinate 2.5M 2ml
*NBT:Nitro-blue Tetrazonium,prepared daily.
Solution B for ALP staining

Chemical Concentration Volume
Tris/citric acid (pH 0.05M 18ml

9.2)
a-naphthyl acid 1mg/ml 20mg

phosphate
Fast Blue RR salt 1mg/ml 20mg

10%MgCl2 0.5% 1ml
10%MnCl2 0.5% 1ml
1.7. References
Boddington,C.L.; Bassett,E.E.; Jakobsen,I.; Dodd,J.C. 1999 Comparison of techniques for the extraction and quantification of extra-radical
mycelium of arbuscular mycorrhizal fungi in soils. Soil Biol.Biochem. 31: (3) 479-482.
Brundrett, M., N. Bougher, B. Dell, T. Grove and N. Malajczuk 1996 Working with mycorrhizas in forestry and agriculture. ACIAR Monograph
32.374. ISBN 1 86320 181 5.
Gianinazzi S, Gianinazzi-Pearson V & Dexheimer J (1979) Enzymatic studies on the metabolism of vesicular-arbuscular mycorrhiza. 3.
Ultrastructural location of acid and alkaline phosphatase activity in onion roots infected by Glomus mosseae (Nicol. & Gerd.). New Phytol 82,
127-132.
Tisserant B, Gianinazzi-Pearson V, Gianinazzi S & Gollotte A (1993) In planta histochemical staining of fungal alkaline phosphatase activity
for analysis of efficient arbuscular mycorrhizal infections. Mycol. Res. 97, 245-250.
Trouvelot A, Kough JL & Gianinazzi-Pearson V (1986) Mesure du taux de mycorhization VA d’un système radiculaire. Recherche de méthodes
d’estimation ayant une signification fonctionnelle. In : Physiological and Genetical Aspects of Mycorrhizae, V. Gianinazzi-Pearson and S.
Gianinazzi (eds.). INRA Press, Paris, pp. 217-221.
Vierheilig H. & Ocampo JA (1989) Relationship between SDH-activity and VA mycorrhizal infection. Agriculture, Ecosystems and
Environment 29, 439-442.
see these website for further informations:
BEG (Banque Européenne des Glomales) website
Mark Brundrett's Working with Mycorrhizas in Forestry and Agriculture
2.0 - DNA Techniques: PCR of ribosomal DNA from spores

2.1. Introduction to the Polymerase Chain Reaction
The Polymerase Chain Reaction (PCR) is an in vitro technique enabling chemical amplification
of DNA. With the improvement brought by the use of the heat stable Taq DNA polymerase
of Thermus aquaticus and automation it is possible to obtain quick amplification even of single
copy genes, starting from minute amounts of material. The impact of this technique in
molecular biology is comparable to that which followed the discovery of restriction enzymes.
It has been adapted for a wide variety of applications, and in particular PCR has opened the
possibility to analyse organisms at the nucleic acid level even when only small amounts of
nucleic acid can be obtained, as in the case of arbuscular mycorrhizal (AM) fungi.
Furthermore, although the efficiency of PCR amplification is dependent on the purity of the
target DNA, Taq DNA polymerase is less sensitive to template purity than other molecular
biology techniques so that partially purified nucleic acid can be used. This feature is a great
advantage for plant/soil microbiology research, as investigations can be made directly on
partially purified biological material, like fungal spores or infected plant roots.
Ribosomal genes are multicopy genes tandemly organised in the genome. Each ribosomal genes
encodes for three subunits (18S[SSU], 5.8S and 28S[LSU]) separated from each other by a
Inter Non Transcribed region (ITS). The genes themselves are separated from each other by
an Inter Genic Spacer (IGS) (see figure).
The various characteristics of rRNA and rDNA have made them a choice target for
phylogenetic and taxonomic studies, and comparative studies of the nucleotide sequences in
ribosomal genes has provided data for the analysis of phylogenetic relationships over a wide
taxonomic range of organisms. The nucleotidic polymorphism is not evenly distributed
throughout the ribosomal genes and the three regions evolve at different rates. ITS and IGS
are variable regions which mutate more frequently than the three conserved coding subunit
regions (18S, 5.8S, 25S). This generally makes the former more informative for analyses of
closely related genomes, whereas the coding regions of the small and the large ribosomal
subunit are considered to be more useful for understanding more distant relationships at the
species/order level.
The internal transcribed spacer region like the intergenic spacer region, evolved much faster
and sequence differences between different populations of one species, or in a single spore in
the case of the Glomales, can be detected. The 5' end of the large ribosomal subunit
harbours two informative polymorphic domains (D1 and D2). The polymorphism observed in
these domains between and in a taxa, allows also to identify specific nucleotidic sequences
which can be used to design primers with different level of specificity or discrimination (van
Tuinen et al 1998a).
2.2. Practical aspects of PCR
The Polymerase Chain Reaction is an in vitro technique which allows the amplification of a
specific region of DNA located between two known sequences. After each cycle of
denaturation, annealing and extension the amount of DNA is double. Potentially, after 20
cycles of PCR, there will be a 220- fold amplification (or 1.106). This illustrates the sensitivity
of this method, and the potential artifactual amplification of DNA, as any traces of DNA can
be amplified.
SCHEMATIC REPRESENTATION OF THE POLYMERASE CHAIN REACTION
Before the discovery of thermostable polymerase, DNA polymerases such as the Klenow
fragment of E. coli DNA polymerase I or T4 DNA polymerase were used. Due to their heat
lability, fresh aliquots of enzymes had to be added after each denaturation cycle. The first
heat stable DNA polymerase (Taq polymerase) was purified from Thermus aquaticus . Today
several heat stable polymerase are available, they are of natural or recombinant origin and
vary in their biochemical properties such as extension rate, thermal stability, 5'?3' or 3'?5'
exonuclease activity. The specificity and activity of the same enzymes is also very dependent
on the producer. Some enzymes such as Tth, have a reverse transcriptase activity, they
cannot therefore be used for the synthesis of cDNA.
Beside the enzyme the other factors that can affect the PCR reaction are:
Primers
MgCl2 concentration
Primer concentration
Primer sequence
Reaction stringency
Length of the amplification product
Number of PCR cycles
other unknown factors
For each PCR reaction the optimal conditions can vary depending mainly on the primer-DNA
combination.
The dNTP's are generally used at a concentration of 100µM, although at lower concentrations
(10-100 µM) Taq polymerase has a higher fidelity.
The most common buffer used with the Taq polymerase is:
10 mM Tris/HCl pH 8,3
50 mM KCl
1.5 mM MgCl2
0.1% (w/v) gelatin
The MgCl2 concentration affects the specificity of the PCR reaction. A too low concentration
affects the final yield whereas a too high concentration reduces the specificity of the
reaction. Other components often present in DNA extraction buffer can affect the enzyme
activity. SDS at a concentration > 0.01% inhibits the polymerase. The inhibition of SDS
(0.01%) can be reversed by some non-ionic detergents (0.5 % (v/v) Tween 20, NP 40).
The primer working concentration is generally of 0.5 - 1 µM. If the primer concentration is
too high primer dimerisation can occur.The primer composition is very important. In most PCR
applications, the primers are designed to be exactly complementary to the template DNA.
The general rules for the primer design are: a length of about 20 - 30 nucleotides. Shorter
primers can be used with success and primers longer than 30 do not increase the specificity
of the binding
the GC content should be about 50%
the 3' ends should not be complementary, as primer dimerisation will occur
the 3' of the primer should be as homologous as possible
the 5' can be modified to add a restriction site or a GC clamp, in this case, both primers
should be equivalent in their melting temperatures
The number of the cycles can be increased to increase the amount of product recovered, but
this will also increase non-specific amplification.
Beside all these factors, some primer combinations will work very well, and others not. As so
many factors affect the PCR reaction it is very important to have a positive and negative
control in an PCR reaction.
2.3. Contamination
As the PCR reaction is so sensitive, precautions have to be taken to avoid undesirable

amplifications., such as using DNA free water and negative controls with every set of
amplifications.
Thermostable DNA polymerases and their sources
DNA polymerase Natural/recombinant Source
Taq Natural Thermus aquaticus
Amplitaq® Recombinant T. aquaticus
Amplitaq® (Stoffel Recombinant T. aquaticus

fragment)
Hot TubTM Natural Thermus flavis
PyrostaseTM Natural T. flavis
VentTM Recombinant Thermoccucus litoralis
DeepVentTM Recombinant Pyrococcus GB-D
Tth Recombinant Thermus thermophilus

Pfu Natural Pyrococcus furiosus
Pfu Cloned Pyrococcus furiosu
Exo-PFU Recombinat Pyrococcus furiosu
UITmaTM Recombinant Thermotoga maritima
Properties of DNA polymerases commonly used in PCR
Taq/ Stoffel VentTM Deep- Pfu Tth UITmaTM

Vent
Amplitaq® fragment TM
Thermostability- 40 80 400 1380 >120 20 >50

half-life at 95°C
5’ - 3’ Yes No
exonuclease
activity

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22/11/2017 Procedures

prepared for the Workshop

Arbuscular mycorrhizal fungi in plant

detection, taxonomy, conservation and

Laboratory of Agricultural Microbiology,

BEG (Banque Européenne des Glomales) website

Laboratoire de Phytoparasitologie INRA/CNRS

Mark Brundrett's Working with Mycorrhizas in Forestry and Agriculture

Mycorrhiza Information Exchange

National Centre for Biotechnology Information

Mychintec WEB page

International Science Fondation

and plant health in agriculture

1.0 Manipulation and Staining of Spores and Roots

1.2. Making a permanent slide mount for reference or BEG registration

f. If using PVLG, remember to allow the mountant to polymerise and top-up it up as

1.3. Histochemical Staining of Total AMF Mycelium in Roots

A fuller understanding of AM functioning requires consideration of the metabolic states of

physiological interactions will necessitate the presence of an active symbiotic fungus.

1.3.1 Root Preparation

a. Wash the roots free of soil.

b. Cut roots into 1cm long segments.

1.3.2 Trypan blue staining of total mycelium

with the permission of Mark Brundett

1.4 Histochemical staining of active AMF mycelium in roots

1.4.1 Succinate dehydrogenase (SDH)- and alkaline phosphatase (ALP) staining of

Solution A for SDH staining

Chemical Concentration Volume(ml)

Tris/HCl (pH 7.4) 0.2 mol.l-1 5

Na-succinate 2.5 mol.l-1 2

*NBT------ Nitro-blue Tetrazonium, prepared daily.

Solution B for ALP staining

Chemical Concentration Volume(ml)

Tris/citric acid (pH 0.05 mol.l-1 18ml

a -naphthyl acid 1 mg.ml-1 20mg

Fast Blue RR salt 1 mg.ml-1 20mg

MgCl2 0.5 mg.ml-1 1 ml

MnCl2.4H2O 0.8 mg.ml-1 1 ml

1.5. Estimation of AMF colonisation

Estimation of mycorrhizal colonization according to Trouvelot et al

Frequency of mycorrhiza in the root system

1.6. Histochemical Staining of Total and Active Soil Mycelium

1. Take soil cores (1x6cm) randomly from pots.

with the permission of Mark Brundett

1.6.2. Estimation of succinate dehydrogenase (SDH)- and alkaline phosphatase (ALP)-

1. Take soil cores and put in a beaker on ice.

Solution A for SDH staining

Tris/HCl(pH7.4) 0.2M 5ml

MgCl2 5mM 2ml

*NBT 4mg/ml 5ml

H2O 2.5M 6ml

Na-succinate 2.5M 2ml

*NBT:Nitro-blue Tetrazonium,prepared daily.

Solution B for ALP staining

Tris/citric acid (pH 0.05M 18ml

a-naphthyl acid 1mg/ml 20mg

Fast Blue RR salt 1mg/ml 20mg

10%MnCl2 0.5% 1ml

see these website for further informations:

BEG (Banque Européenne des Glomales) website

Mark Brundrett's Working with Mycorrhizas in Forestry and Agriculture

2.0 - DNA Techniques: PCR of ribosomal DNA from spores

2.2. Practical aspects of PCR

SCHEMATIC REPRESENTATION OF THE POLYMERASE CHAIN REACTION