The Australian Centre for International Agricultural Research (AClAR) was established in June

1982 by an Act of the Australian Parliament. Its mandate is to help identify agricultural
problems in developing countries and to commission collaborative research between Australian
and developing country researchers in fields where Australia has a special research
competence.
Where trade names are used this does not constitute endorsement of nor discrimination
against any product by the Centre.

ACIAR MONOGRAPH SERIES

This peer-reviewed series contains the results of original reseach supported by AClAR, or
material deemed relevant to ACIAR's research and development objectives. The series is
distributed internationally, with an emphasis on developing countries.

© Australian Centre for International Agricultural Research

GPO Box 1571, Canberra, ACT 2601

Brundrett M., Bougher N., Dell B., Grove T. and Malajczuk N. 1996. Working with Mycorrhizas
in Forestry and Agriculture. AClAR Monograph 32. 374 + x p.

ISBN 186320 181 5

Editorial management: Peter Lynch

Illustrations: Mark Brundrett
Design and art production: BPD Graphic Associates, Canberra, Australia
Printed by: Pirie Printers, Canberra, Australia
Working Ylith
Mycorrhizas
in Forestry and
Agriculture

Mark Brundrett,
Neale Bougher,
Bernie Dell,
Tim Grove and
Nick Malajczuk
 Chapter 4 E.xamining
MYCORRHIZAS FOR FORE.STRY AND AGRICULTURE. Mycorrhizal Associations

Chapter 4

EXAMINING MYCORRHIZAL
ASSOCIATIONS
This section contains information on methods commonly used to
sample roots and to examine their mycorrhizal associations. A general
introduction to root sampling, processing and microscopy methods used
to examine mycorrhizal associations is provided. Further information is
available in standard botanical microtechnique and histology references
such as Jensen (1962) and O'Brien & McCully (1981). The book Practical
Methods in Mycorrhiza Research (Brundrett et al. 1994), contains
additional information about microscopy techniques used to study
mycorrhizas.
The study of plant roots has received much less attention than it
deserves, due to the technical difficulties of studying events within the
soil (Lyr & Hoffmann 1967, Harper et al. 1991). Data concerning root
systems and mycorrhizal associations can be of great value in forestry
and agriculture for the reasons outlined in Chapter I. There is also a
need for more information about the role of mycorrhizal associations in
various plant communities (St John & Coleman 1983, Brundrett 1991).
This information can be provided by sampling roots in natural
ecosystems, forest plantations, agricultural situations and disturbed
habitats by growing seedlings of plants in intact cores of soil from their
natural habitats, or by inoculating them with particular mycorrhizal fungi.
These alternative procedures were compared during a survey of
mycorrhizal associations of jarrah forest plants (Brundrett & Abbott
1991). Experiments with seedlings were found to be more time-
consuming (due to the time required for plant growth and mycorrhiza
formation), but resulted in root samples of superior quality to those
collected from the field (where it is difficult to obtain unmixed samples
of fine, young roots). Root parameters that can be used to evaluate the
performance of mycorrhizal and non-mycorrhizal root systems are
outlined in Table 4. 1.

Table 4.1. Root system information that could be used in mycorrhizal

studies.
fresh or dry root mass (g/plant)
root length (m/plant) and density (m rootlL soil)
specific root length (m rootlg root)
branching orders and branching frequency of laterals
root hair length (mm) and frequency (root hairs/mm root)
growth rates (mm/day)
phenology (seasonal changes in activity)
life span (weeks. years) and turnover rates (m/week)
mycorrhizal roots:
- total length (m/plant. m/L soil)
- proportion of root length (%)
- biomass (% of root biomass. mg/kg soil)
nutrient contents (Chapter 6)

Page 173

Equipment
• Clear plastic dishes with
inscribed grid lines to
measure colonisation
• Fine screen (100 j..Im) with
nylon mesh for transferring
roots from solutions
• Fine forceps and probes for
manipulating roots
• Microscope slides. long cover
slips and PVLG mountant
(Section 3.3)
• Plastic vials with tight-sealing
lids for storage of samples
• Dissecting microscope with a
transmitted light illumination
- a clear plastic panel over
the microscope base is
recommended to provide a
stable platform and
protection from spilled
liquids
• Compound microscope with
an eyepiece cross-hair
Page 184
MYCORRHIZAS FOR FORE.STRY AND AGRICULTURE.
4.3. MEASURING ROOT COLONISATION
BY MYCORRHIZAL FUNGI
Mycorrhizal studies often require procedures for estimating the
proportion of roots in a sample that contains mycorrhizal
structures. after clearing and staining them (Section 4.2). Root
length can be measured simultaneously with mycorrhizal
colonisation by a gridline intersection procedure (Giovannetti &
Mosse 1980). or separately by making slides and viewing them
with a compound microscope (McGonigle et al. 1990).
The length of mycorrhizal roots present in a sample should be
presented along with data on the proportion (%) of root length
occupied by these fung i. because mycorrhizal root length is more
directly correlated with association costs/benefits and inoculum
production by the fungus. Root-length data can be used to
calculate root production (growth) rates. root densities (within a
volume of soil) and specific root lengths (root length:weight
ratiOS) which provide valuable information about the capacity of
roots to obtain water or nutrients from soils and their ability to
form mycorrhizal associations (Table 4.1).
Analysis of colonisation data
Data on mycorrhizal colonisation of roots and the
distribution of fungal propagules such as spores is often highly
variable. with a non-normal frequency distribution of data
points (St John & Hunt 1983. Friese & Koske 1991). Thus
statistical analysis of such data may require data to be
transformed. or non-parametric statistics to be used
(Chapter 7). The aggregated distribution pattern of
propagules of soil fungi must be considered during field
sampling. experimental design and data analysis (St John &
Hunt 1983. Campbell & Noe 1985. Dutilleul 1993).
A. Roots and VAM fungi
I. The most frequently used root measuring procedure is a
modification of the grid line intersect method (Newman
1966. Tennant 1975. Giovannetti & Mosse 1980). in which
roots are randomly dispersed in a 9-cm diameter Petri plate
with grid lines (Fig. 4.3A). The observer scans along these
grid lines with a dissecting microscope to quantify
intersections between grid lines and roots - which are
deSignated as either colonised or non-mycorrhizal (Fig. 4.3A).
2. The proportion of root length that is mycorrhizal and total
root length can then be calculated from a conversion facto r
derived from the total length of grid lines and the area of the
dish (Newman 1966. Tennant 1975). If a 14111 cm (approx.
1/2 inch) grid is used the number of intersects will provide
values of mycorrhizal and non-mycorrhizal root length in cm.
3. Giovannetti & Mosse (1980) recommend that at a minimum
100 intersections should be used to assess a sample. and
found that accuracy was improved if samples were re-
randomised and counted several times. It is also possible to
 Chapter 4 Examining
MYCORRHIZAS FOR FORESTRY AND AGRICULTURE Mycorrhizal Associations

MICROSCOPIC EXAMINATION OF ROOTS

A. Mounting roots on slides

Fine forceps

I. Arrange root segments

,
lengthwise on slide
with fine forceps

-ILA
GlaSSSI
Cover
slip Mo"nuot

2. Add small drops of PVLG

mountant at one end, then
:: - '..,
c slowly lower cover slip at
that end first

. ... 3. Allow mountant to flow around

roots before gently tapping
= .
.--=::P.= = coverslip to flatten roots
and remove air bubbles

B. Assessing mycorrhizas mounted on slides

Random ly selected microscope field of view and cross-hair positions

Possible I. hyp hae and 2. hyphae 3. hyphae and 4. root only

intersects: vesicle only arbu scules

Figure 4.4. Microscopic examination of roots.

Page 185
 MYCORRHIZAS FOR FORESTRY AND AGRICULTURE

use this method with larger circular or rectangular dishes .

However. with rectangular dishes care must be taken to
ensure that the number of vertical and horizontal lines are
fully representative of the area of the dish. It is best to test
new dishes with a sample of precisely known length. as shown
in Table 4.2.
4. A much simpler procedure. where an observer simply
provides a visual estimate of the degree of mycorrhizal
colonisation (within 5 or 10%) can also be reliable
(Giovannetti & Mosse 1980). While this method is subjective
and prone to operator bias. it still can provide sufficient
information when precise values are not required (for pot
culture quality control. or when looking at samples from
the field).
5. When mycot-rhizal colonisation is being assessed using a
dissecting microscope. it is always a good idea to make slides
irom randomly selected subsamples of roots for observation
with a compound microscope (Fig. 4.4A). This will allow fungi
that do not stain well (such as many Acaulospora and some
Glomus species) to be seen. and the contribution of saprobic
or parasitic fungi to root colonisation to be determined. The
contribution of different types of mycorrhizal fungi to root
colonisation can also be estimated by counting different VAM
fungi (recognised by root morphology) separately
(Section 3. 1).
6. Assessing mycorrhizal root segments can be done using a
compound microscope with an eye-piece cross-hair which is
moved to randomly selected positions (McGonigle et al.
1990). This allows the length of arbuscules. vesicles and
inte rnal hyphae within roots to be separately determined
(F ig. 4.4B).

Table 4.2. Grid line intersection method example using an

8.5 cm-d iameter round Petri dish with a 1/2 in ch (14/ II cm) grid
an d a I-m test sample of thread cut into fragments and randomly
redistributed 10 t imes.
Redistribution 2 3 4 5 6 7 8 9 10
Intersects (cm) 102 107 9 1 98 92 114 108 99 104 94
Average 100.9 cm ± 2.5 (standard error)

Page 186
 Chapter 4 Examining
MYCORRHIZAS FOR FORESTRY AND AGRICULTURE Mycorrhizal Associations

B. Quantifying ectomycorrhizal associations

I. A variety of methods has been used to quantify ECM roots
(Grand & Harvey 1982). Unstained ECM roots can usually be
distinguished from non-mycorrhizal roots by differences in
their colour, thickness, texture and branching patterns.
However, a clearing and staining (Section 4.2) or sectioning
procedure (Section 4.5) is necessary to visualise the Hartig
net to confirm that an ECM association is present (see 1.6 B).
A post-clearing bleaching step to remove excess tannins often
helps reveal the Hartig net in ECM roots (see 4.2C).
2. ECM roots are usually quantified by sampling seedlings, or
washing roots from soil cores, taking care to exclude
contaminating roots of non-target species (Section 4.2C).
Assuming that roots are young and healthy. each mycorrhizal
root tip will contain an active Hartig net zone (where active
exchange processes are thought to occur). These tips can
be counted to quantify the intensity of the association, and
their numbers should be expressed relative to root length
and soil volume.
3. The root length of a sample can be measured with the
gridline intersect method. while either simultaneously
measuring the length of mycorrhizal roots or separately
counting the total number of mycorrhizal tips (Fig. 4.5).
4. For some mycorrhizas, the intensity of branching within a
mycorrhizal cluster varies considerably (Fig. 4.5), and can be
quantified with a branching density index that was developed
for pine roots (Marx 1969). However, branching intensity will
be taken into account by the gridline intersection method if
ECM root tips are counted.
5. Ectomycorrhizal association of eucalypts may be more difficult
to recognise than those typical of conifer roots, in cases
where there are minimal changes to the host root (see 1.6 B).
This occurs when mycorrhizal colonisation results in minimal
root swelling, limited root branching, a shallow Hartig net,
and a thin 'superficial' mantle. These associations can be
recognised with practice, but require more careful
examination of roots.

Page 1S7
 MYCORRHIZAS FOR FORE.STRY AND AGRICULTURE.

A. Using the gridline intersection method to count ECM roots

I. Measure root length of stained (A) or unstained (B) roots with a dissecting microscope
using the gridline intersect method

2. Count the length of mycorrhizal roots or the number of mycorrhizal root tips

Results: 65 = Root length (cm)

24 = Mycorrhizal root length (cm)
36 = Mycorrh izal root tips

B. Classifying ECM roots by branching patterns and appearance

Pine mycorrhizas Eucalypt mycorrhizas

Dichotomously
branched \/ Unbranched
............
<::-=

Unbranched

Sparsely Pinnate branching

branched

Tuberoid
mycorrhizas

Dense clusters
of branches

Figure 4.5. A. Using the gridline intersection method to count ECM roots, B. Classifying ECM roots by branching patterns
and appearance.

Page /88
 Chapter 4 Examining
MYCORRHIZAS FOR FORESTRY AND AGRICULTURE Mycorrhizal Associations

c. Identifying ectomycorrhizal fungi

Characteristic features of fresh samples of ECM root tips can be
used to identify particular ECM fungi (Figs 4.6, 4.7). Some of these
characteristics are outlined in Table 4.3, and include features
which are also used to identify fungal fruiting bodies (Chapter 2).
Many features in Table 4.3 can be observed with a dissecting
microscope, but more information can be obtained by sectioning
roots, using procedures described in the following sections.
Atlases illustrating ECM root types which are associated with
particular fungi in some habitats have been produced (Agerer
1986, Ingleby et al. 1990, Haug & Pritsch 1992). Before
mycorrhizal fungi can be recognised on roots from the field, they
must be characterised by observations of roots which are
colonised by known ECM fungi as described below.
I. Roots can be obtained from synthesis experiments by using
inoculum of a particular fungus (Chapter 5) and excluding
other fungi by pasteurising soil (Chapter 6). Examples of
mycorrhizal roots from syn-thesis experiments are shown in
Figures 4.6 and 4.7.
2. Alternatively, roots can be collected from under a particular
fungal sporocarp in the field as described by Agerer (1986,
1991). First, an undisturbed block of soil (up to 10 cm 3) is
excavated from under a sporocarp and transported back to
the laboratory. Next, a dissecting microscope is used to trace
hyphal connections between that sporocarp and mycorrhizal
roots presumed to be formed by that fungus . A voucher
collection of fruit bodies of fungi should also be made and
their identity confirmed by microscopic investigations
(Chapter 2).
3. A uniform collection of ECM roots can be characterised using
anatomical details (Table 4.3) which can be photographically
recorded (Ingleby et al. 1990, Agerer 1991, Haug & Pritsch
1992). External features are observed with a dissecting
microscope, while internal features are documented with a
compound microscope by progressively squashing roots or by
making thin longitudinal sections through the inner and outer
mantle and the Hartig net layers (Ingleby et al. 1990, Agerer
1991 ).

Page 189
Page 190
 Chapter 4 Examining
MYCORRHIZAS FOR FORESTRY AND AGRICULTURE Mycorrhizal Associations

Figure 4.6. Examples of ECM root systems of eucalypt species.

A. Roots of Eucalyptus marginata growing in forest soil amended with burnt
litter in a root chamber. Abundant extramatrical mycelium of a
Hysterangium species can be seen (arrows).
B-F. In situ examples of synthesised ECM associations of eucalypt seedlings
grown in the glasshouse (B,D,E), or nursery (C,F).
B. Pisolithus sp. and E. diversicolor in white sand.
C. Laccaria laccata and E. globulus in a potting mix.
D. Setchelliogaster sp. and E. globulus in white sand.
E. Scleroderma sp. and E. globulus in yellow sand.
F. Laccaria fraterna and E. regnans in a potting mix.
G, H. Tuberculate ECM of Eucalyptus pilularis collected in Queensland.
G. Young (Y) and old (0) tuberculate ECM attached to woody roots.
H. C/oseup of tuberculate mycorrhizas showing numerous ECM roots
(arrows) encased with an outer rind (R).

Table 4.3. Diagnostic features of ectomycorrhizal roots that may be

associated with particular mycorrhizal fungi.
Mycorrhizal development
• root branching patterns and density
• root thickening
• reduction in root elongation
External hyphae
• presence. abundance. distribution
• arrangement - single. strands. rhizomorphs
• structure - colour. wall. thickness. clamp connections. crystals
• sclerotia
Mantle
• thickness
• colour. changes with bruising. or age
• hyphal organisation in surface and in deeper layers
- loose or compact hyphae or pseudo-parenchyma. prosenchyma
• hyphae - thickness. wall structure. clamp connections. septae
• cystidia. crystals. exudates
• reactions to chemicals (Melzer's reagent. KOH. ete.)
• staining reactions
• odour
• fluorescence
Hartig net
• presence. thickness
• hyphal organisation
• hyphal structure

Page 191
Page 192
 Chapter 4 Examining
MYCORRHIZAS FOR FORESTRY AND AGRICULTURE Mycorrhizal Associations

Figure 4.7. Ectomycorrhizal roots washed from soil, illustrating characteristic

features of different inoculated fu ngi.
A-C. Pinus radiata roots with Sui llus brevipes (A), Amanita mascaria (B)
and Boletus edulis (C). Dichotomously branched root tips are shown
(arrows).
D. Eucalyptus maculata and Astraeus pterid is association with numerous
mycelial strands (star).
E. Tylopilus sp. and E. urophylla association with black roots (arrows) and
abundant soil mycelium (star).
F. Redd ello myces sp. and E. grandis association with shiny brown roots
(arrows).
G. Pisolithus tin ctori us and E. urophylla with yellow-orange roots (arrows)
and mycelial strands (asterisk).
H. Ama nita sp. and E. urophylla with yellow roots (arrows) covered with a
dense covering of short hyphae (star).

4.4. BIOASSAY MEASUREMENTS OF

MYCORRHIZAL INOCULUM IN SOILS
Propagules of vesicular-arbuscular mycorrh izal (VAM) fungi are
thought to incl ude spores, dead root fragme nts and other
co lonised o rganic material, as well as networks of hyphae in soil
(Brun drett 1991). Hyphal networks are consi dered to be
especially important in natural ecosystems, where they can
survive drought conditions, but are more sensitive than other
propagu les to soi l distu r bance Uasper et al. 1989, 1991).
Pro pagules of ectomycorrhizal (ECM) fungi include networks of
mycelial strands, o ld mycorrhizal roots, sclerotia and spores
(Ski nn er & Bowen 1974, Ba et al. 1991).
Ino culum potential is defined as t he energy fo r growth of an
organ ism at the surface of its host, and is a conseque nce of th e
numbers of active propagu les of that organism and their
nutritio nal status (Garrett 1956). In direct est imat ions of t he
in ocu lum potential of mycorrhizal fungi have been obtained by
meas uring mycorrhizal fo rm ation afte r serial dilutions of soil, or
by co unting propagules suc h as spores, but results of these
procedu res do not always correspon d with myco rrhiza formatio n
in the same soi l (Abbott & Robson 1991, Bru ndrett 1991 ). The
total myco rrhizal inoc ulum pote nt ial of so il s can be det e rmined
by growi ng bait plants in intact cores of soil to meas ure the rate
of mycorrhiza formation (Brundrett 1991, Abbott & Robson
1991 ). Th is metho d is supe rior to 'most probable number' assay
proced ures (which involve serial dilutions of so il), because it will
measu re infectivity due to intact hyphal netwo rks, as well as that
from distu rbance-tolerant propagules such as spores.
Intact soi l core bioassays have bee n used to quantify seasonal
and spatial variations in VAM inocu lu m potential and the relative
cont ribution of different VAM fungi t o these levels (by com pari ng
mycorrhizal morphology with in roots), in a wid e range of

Page 193