New Phytol.

(1990), 115,

A new method which gives an objective

measure of colonization of roots by
vesicular-arbuscular mycorrhizal fungi
MILLERS, D. G. EVANS^
G. L. FAIRCHILD^ AND J. A. SWAN^

BY T . P. M C G O N I G L E S M . H.

^ Department of Land Resource Science, University of Guelph, Guelph, Ontario,

NIG 2W1, Canada
^Department of Statistics, Oregon State University, CorvalUs, OR 97331-4606, USA
^ International Institute of Tropical Agriculture, PMB 5320, Oyo Road, Ihadan, Nigeria
* Organization Studies and Applied Psychology Division, Aston University, Birmingham,
B4 7ET, UK
{Received 19 October 1989; accepted 1 March 1990)
SUMMARY

Previously described methods to quantify the proportion of root length colonized by vesicular-arbuscular (VA)
mycorrhizal fungi are reviewed. It is argued that these methods give observer-dependent measures of colonization
which cannot be used to compare, quantitatively, roots examined by different researchers. A modified method is
described here to estimate VA mycorrhizal colonization on an objective scale of measurement, involving inspection
of intersections between the microscope eyepiece crosshair and roots at magnification x 200; it is referred to as
the magnified intersections method. Whether the vertical eyepiece crosshair crosses one or more arbuscules is
noted at each intersection. The estimate of colonization is the proportion of root length containing arbuscules,
called the arbuscular colonization (AC). The magnified intersections method also determines the proportion of
root length containing vesicles, the vesicular colonization (VC), and the proportion of root length containing
hyphae, the hyphal colonization (HC). However, VC and HC should be interpreted with caution because vesicles
and hyphae, unlike arbuscules, can be produced in roots by non-mycorrhizal fungi.
Key words: Magnified intersections, percent infection, fractional infection, method, arbuscules.

INTRODUCTION

Procedures to determine visually the proportion of

root length colonized by VA mycorrhizal fungi can
be divided into three groups: (i) subjective estimation, (ii) calculation of the percentage of root
segments or microscope fields of view that contain any
colonization, and (iii) grid-line intersect methods.
Techniques using chemical estimation of chitin
(Hepper, 1977; Bethlenfalvay, Pacovsky & Brown,
1981) are not considered here.
Subjective visual estimation has been used frequently (Mosse, 1973; Sanders et aL, 1977; Abbott &
Robson, 1978; Buwalda & Goh, 1982; Hopkins,
1987), and has been modified to estimate the
intensity of arbuscular proliferation in colonized
regions (Trouvelot, Kough & Gianinazzi-Pearson,
1986). By repeatedly scoring root systems,
Giovanetti & Mosse (1980) calculated a standard

error of between 2 and 5 % colonization for subjective visual estimation. However, no investigation
has yet been undertaken to assess the accuracy of this
method. In addition, this method has not yet been
considered in terms of the relationship between
perceived magnitude (^) and stimulus magnitude
{(j)), known as Stevens' power law (Teghtsoonian &
Teghtsoonian, 1971):
^ = hij/,

(1)

where /J is a constant which depends on the choice of

units, and ^ is dependent on the type of stimulus
characteristic that is perceived (Stevens, 1975). Part
of the work presented here was designed to investigate the accuracy of subjective estimation and to
consider it in terms of Stevens' power law.
Several studies have calculated colonization as the
number of root segments with any colonization,
divided by the total number of segments examined

496

T. P. McGonigle and others

fore be expected to give a relative measure of

colonization.
20%
To determine unequivocally whether arbuscules
are present in all cases requires examination at x 200
magnification. Ambler & Young (1977) described a
grid-line intersect method involving the compound
30%
microscope, but this still has the difficulty that some
intersections must be classified as colonized or not
when hyphae but no arbuscules are seen. Since
arbuscules are the only unique feature of VA
60%
mycorrhizas, decisions as to whether hyphae seen
alone are mycorrhizal may vary from person to
Figure 1. A schematic drawing of three 10 cm lengths of
root. Each one has the same proportion of root length person. This method is therefore vulnerable to bias
colonized, which expressed as a percentage is 20 %. The and probably generates a relative measure of
stippled regions represent colonized portions of the roots, colonization.
and dashed lines indicate where the root lengths are to be
We describe below a technique which enables
cut into ten 1 cm root segments. When each of the
colonization to be scored objectively because the
segments is rated as a plus or a minus for colonized or not
only decision necessary at each intersection is
colonized respectively, the estimates of colonization will be
20, 30 or 60%, as shown for the three arrangements.
whether or not arbuscules are present. The method
requires that the roots are observed at a magnification
sufficient to easily discern the presence of arbuscules,
(Daft & Nicolson, 1972; Khan, 1974; Read, and that the intersection line is thin relative to the
Koucheki & Hodgson, 1976; Biermann & width of the arbuscules. We refer to this technique as
Linderman, 1981; Malibari, Al-Fassi & Ramadan, the magnified intersections method.
1988). This is the same in principle as calculating the
number of microscope fields of view with any
MATERIALS AND METHODS
colonization divided by the total number offieldsof
Investigation into the subjective method
view examined, a method also used (Baylis, 1967;
Sutton, 1973; Newman, Heap & Lawley, 1981; The accuracy of subjective estimation was assessed
Plenchette, Furlan & Fortin, 1982; Dodd & Jeffries, using test diagrams so that the true value for the
1986). These methods almost always overestimate proportion being judged by eye was known. Seventy
percentage colonization, the degree of overestimation students were asked to participate as subjects. The
depending on the lengths of the segments, and on the diagrams (a) and (b) in Figure 2 were chosen to
lengths of the regions of colonization (Fig. 1).
represent schematically in two dimensions, roots
The grid-line intersect method (Giovannetti & under a microscope. All subjects were allowed 20 s to
Mosse, 1980) or various modifications of it, is the visually examine each of (a) and (b) one at a time and
technique most frequently used to estimate in that order. Subjects were instructed to estimate,
colonization. We found that over two-thirds of a during the 20 s allocated to each diagram, the
sample of 80 papers, published after 1980 and proportion of the total length of the straight line,
reporting colonization data, used this technique. enclosed within the circle, that is thick.
The procedure is to note the presence or absence of
colonization at each intersection of root and gridCollecting and processing roots
line, after dispersing the roots above a grid of squares
drawn on a petri dish, and observing under a Roots were collected from a field of Zea mays L. cv.
dissecting microscope at x 40 magnification. In Pioneer 3949 at the Elora Research Station
many cases colonization by VA mycorrhizal fungi (43 31' N, 80 14' W) at 15 days after 50 % silking in
can readily be determined. However, even at x 80 the summer of 1988. Sampling was effected by
magnification it is not possible to ascertain if the taking 5 cm diameter soil cores directly over the cut
roots are mycorrhizal at all intersections. This is stems of randomly selected plants. To obtain roots
because cortical cells or parts of the stele can become with a reasonably broad range of colonization,
stained (Dodd & Jeffries, 1986), the roots may be samples were taken from areas within the maize field
crowded with hyphae, and because arbuscules can be differing in phosphorus fertilizer history and from
diflicult to detect when they are small. Structures depths up to 30 cm. Roots were washed from the
formed by other fungi may also be confused with soil, and stored in formyl acetic alcohol. For
arbuscules
at low magnification.
Different subsampling the roots were cut into 1 cm fragments
researchers are unlikely to be consistent in the way and dispersed in water. T h e water was stirred
they record these difficult intersections, and may vigorously and a subsample collected in a beaker
arrive at different answers. The grid-line intersect immersed in the water for a few seconds. Root
method using the dissecting microscope can there- samples were cleared for 20 min at 121 C in 10%

Objective measurement of colonization by VA mycorrhizal fungi

497

Root

Direction
of
travel
across

slide

Horizontal
crosshair
Perimeter
of field
of view
Vertical
crosshair
ib)

Position to which
vertical crosshair
is moved

Figure 3. A diagram to show how a magnified intersection

perpendicular to the long axis of the root can be made
when the root is aligned with its long axis at an angle to the
vertical crosshair. The stage is moved until the centre of
the crosshairs is contiguous with the first edge of the root
reached. To make the perpendicular intersection, the
vertical crosshair is then rotated as shown.

was missing, all intersections between roots and the

vertical eyepiece crosshair were considered. The
position on the root surface at which the centre of the
eyepiece crosshairs entered through the side of the
root was taken as the point of intersection. Rotation
of the vertical crosshair ensured each intersection
Figure 2. The two diagrams (a) and {b) shown to the 70 was at right angles to the long axis of the root (Fig.
subjects in the investigation into subjective visual es- 3). Where the centre of the crosshairs entered a root
timation. See text for further details.
through an end rather than a side, the point of exit
from the root through its side was taken as the point
KOH, and stained in Chlorazol Black E (Brundrett, of intersection. Roots too wide to fit into the field of
Piche & Peterson, 1984). Roots of a selection of 12- view at x 200 magnification were examined in two or
week-old leek {Allium porrum L.) seedlings were also more width portions.
examined. Leek seeds were germinated for 1 week on
To examine each intersection, the plane of focus
moist paper towels. For the 5th-7th week inclusive was moved completely through the root and a note
they were transplanted into mature pot cultures of made of whether the vertical crosshair actually cut
Glomus versiforme Karsten (Berch). At other times any arbuscules, vesicles and hyphae. Intersections
they were grown in inoculum free calcined clay and were counted in the following categories; ' negative'
given water and nutrients as described previously (no fungal material in root), 'arbuscules', 'vesicles',
(Brundrett, Piche & Peterson, 1985).
and ' hyphae only'. If the vertical crosshair cut one or
more arbuscules or vesicles, the appropriate category
was incremented by one, and similarly for
Estimating colonization using the magnified
intersections where hyphae only were crossed. When
intersections method
both vesicles and arbuscules were scored at an
Roots were mounted in glycerin on microscope intersection, the total number of intersections was
slides and covered with 40 x 22 mm coverslips. only increased by one. The arbuscular colonization
Between 2 and 4 slides were used for each subsample, (AC) and vesicular colonization (VC) were calculated
but all slides for a subsample were treated as a single by dividing the count for the 'arbuscules' and
unit, and not as subsubsamples. Roots were aligned ' vesicles' categories respectively by the total number
parallel to the long axis of the slides and observed at of intersections examined. Hyphal colonization (HC)
magnification x 200 as follows. The field of view of was calculated as the proportion of non-negative
the microscope was moved using the stage graticule intersections.
to make four, six or eight complete passes across each
All of the data collected using the magnified
slide perpendicular to its long axis. The number and intersections method was for subsamples examined in
distance between passes is not critical, but should be a random order with the identity of the roots
constant for a subsample. Except where the cortex unknown to the observer.
33

A N P 115

498

T. P. McGonigle and others

Table 1. A summary of the variables in the investigation into subjective visual estimation using test diagrams {a)
and {b)
Variable
Score for {a)
Score for {b)
Score for (6)
score for {a)
Score for {b)
-r-score for (a)

True
value

Subject
mean

Standard
deviation

Coefficient of
variation (%)

0-45
0-63
0-18

0-49
0-66
0-17

0-11
0-11
0-09

22
17
53

1-40

1-37

0-23

17

Assessment of the variability of data from the

magnified intersections method

To determine the variability associated with the

score for a given subsample, one subsample was
scored ten times using different starting points for
the first pass of the field of view in each case. This
ensured difTerent intersections were examined for
each of the ten estimates. To assess the variability
between subsamples, 10 subsamples were scored for
each of two root samples. To determine variability
between observers within subsamples, one
subsample was scored by eight observers. These
varied in experience of examining mycorrhizas from
5 years to none at all. For this comparison each
observer used the same stage graticule locations for
the passes across the slides. To assess the number of
intersections required for a root subsample, one root
sample (analysed by three observers using different
root subsamples) was recorded so that a running
mean of AC could be calculated for each increment
of 10 intersections.
Comparison of the magnified intersections method to
the dissecting microscope grid-line intersect method

Prior to mounting on microscope slides, 30 root

subsamples (one for each of 30 samples) were scored
for colonization under the dissecting microscope
using the grid-line intersect method (Giovannetti &
Mosse, 1980). Roots were dispersed in glycerin in a
9 cm Petri dish marked into 1 cm grid-squares. At
least 100 intersections were examined for each
subsample. Each intersection was counted in the
following categories; colonized, not colonized and
unclear. Two values for proportion of root length
colonized were calculated. For the lower estimate,
the unclear scores were treated as not colonized; for
the upper estimate, they were treated as colonized.

0-8

07
0-6

o
CO

0-5
0-4

0-3
0-2

04

0-6
Score for (b)

08

10

Figure 4. A scatterplot of the scores for diagrams {a) and

(6) in Figure 2 in the investigation into subjective visual
estimation. Each of the 70 points on the graph corresponds
to the estimates made by one subject. The dashed
horizontal and vertical lines indicate the true values for (a)
and (b).

variation, almost all subjects (64 out of 70) were able

to tell that the proportion in {b) was larger than that
in {a). The scores for {a) and {b) were positively
correlated (correlation coefficient = 0-648, P <
0-001) with one another (Fig. 4), indicating that most
of the subjects either underestimated or overestimated both proportions. The difference in scores
[score for {b) score for (a)] and the ratio of scores
[score for (6)/score for ()] were normally distributed, with mean values close to the true values,
but once again there was much variation among
subjects (Table 1). Averaged across all subjects, fi
[equation (1)] was found to have a mean + SD of
1-02 + 0-06 for diagram {a) and 1-01+0-04 for
diagram {b), where ^ = the subjects score and (j) =
the true value.

RESULTS

Subjective visual estimation

Magnified intersections method

The mean scores for diagrams {a) and {b) were close Two examples of the data and of the calculation of
to their true values, although there was considerable AC, VC, and HC are given in Table 2. For the 30
variation across the subjects (Table 1). Despite this maize field samples AC ranged from 010 to 0-70, but

Objective measurement of colonization by VA mycorrhiza

499

Table 2. Examples of data obtained using the magnified intersections method. The subsamples shown are for
separate maize root samples
Number of intersections

Root
subsample
1

Microscope
slide
A
B
C
D
Sum
A
B
C
D

Sum

Negative"
59
54
46
43

Arbuscules*

Vesicles"

Hyphae
only"*

Total'

73

0
0
0

6
6

67
56

4
20
6

52
248
62

44
51
43
200

202
33
25

5
25
23
13

21
23
102

16
14
66

1
0
1
1

13

30

Calculation of arbuscular colonization (AC), vesicular colonization (VC), and hyphal colonization (HC) proceeds as
follows. T h e respective values for subsamples 1 and 2 for AC are 25/248 = 0 1 0 and 66/200 = 0-33; for VC are
1/248 = 0-004 and 2/200 = 0-010; and for H C are ( 2 4 8 - 2 0 2 ) / 2 4 8 = 0-19 and ( 2 0 0 - 1 0 2 ) / 2 0 0 = 0-49.
"Negative: crosshair did not cut through any hyphae nor arbuscules nor vesicles.
"Arbuscules: crosshair cut through at least one arbuscule.
''Vesicles: crosshair cut through at least one vesicle.
''Hyphae only: crosshair cut through at least one hypha, but no arbuscules nor vesicles.
''Total: total number of intersections examined. This must be recorded separately and not calculated as a sum of
categories a, b, c and d, because when examining a given intersection a score of 1 can sometimes be added to both
categories b and c

Table 3. Arbuscular colonization {AC), vesicular

colonization {VC) and hyphal colonization {HC) of
12-week old leek seedlings

upper estimates from the dissecting microscope for

23 out of 30 samples (Fig. 6).
DISCUSSION

Pot

AC

VC

HC

10
11
12

0-31 (0-06)
0-27 (0-05)
0-38 (0-03)

0-31 (0-07)
0-23 (0-08)
0-34 (0-05)

0-66 (0-04)
0-66 (0-10)
0-71 (0-07)

Data are backtransformed means (sample size = 4) with

standard errors in parentheses. Plants were transplanted
into mature Glomus versiforme-leek pot cultures (numbered
10, 11 and 12) for the 5th-7th week inclusive.

there were few vesicles in the roots, with VC having

a mean + SD of 0-011+0-008. In the leek seedlings
VC was considerably higher (Table 3).
The variability within a subsample was low, with
a coefficient of variation for AC of 8 % and for HC of
10% (Table 4). Variation in AC between observers
was also small (coefficient of variation 6 %), although
that seen between subsamples was somewhat higher
(Table 4). The three observers who each examined a
separate subsample of one root sample obtained very
similar final estimates of AC (Fig. 5). Inspection of
the running means for AC, and how they change
with the number of intersections examined (Fig. 5),
suggests that 150 (or at the very least 100)
intersections should be assessed for each subsample.
AC values determined by the magnified
intersections method fell between the lower and

Any individual subjective estimate of a proportion is

likely to be inaccurate, but a set of estimates gave a
mean close to the actual value (Table 1). Most
observers either overestimated or underestimated
both proportions (Fig. 4). By being consistent in this
way an observer will correctly detect relative levels
of colonization across experimental treatments, but
these data will be observer-dependent and so should
not be directly compared across experiments conducted by different researchers. Despite the closeness of fi to unity when averaged across all subjects,
which would suggest that \}r = (j) when identical units
are used for both parameters, it seems that fi is
subject-dependent and can be expected to be consistently > 1 or < 1 for those who tend toward
overestimation or underestimation, respectively.
Similar findings have been obtained for the visual
estimation of line length (Verrillo, 1983; DaSilva,
Ruiz & Marques, 1987).
We argued from a theoretical standpoint that
other previously described methods to quantify
colonization also yield relative measures. Scoring the
percentage of root segments or fields of view with any
colonization should lead to overestimation, with the
extent of overestimation varying from study to study.
The grid-line intersect method (Giovanetti & Mosse,
1980) and an analogous method using the compound
33-2

T. P. McGonigle and others

500

Table 4. Means and standard deviations fmr arbuscular colonization {AC) and hyphal colonization {HC) in the
study to assess the variability within a subsample, between subsamples, and between observers. A separate mai
root sample was used for each of the four cases {n = sample size)
AC

HC

Variation

Mean

SD

Within a subsample
Between subsamples

10
10
10
8

0-51
0-13
0-28
0-34

0-039
0-027
0-050
0-020

Between observers

Mean
0-66
0-22
0-39
0-52

SD
0-065
0-055
0-057
0-081

Figure 6. A comparison of grid-line intersect scores,

obtained using the dissecting microscope, with arbuscular
colonization (AC) values from the magnified intersections
method. The scores for the dissecting microscope have
been presented as two lines, one for the minimum (--)
and one for the maximum (
) estimates (see text). The
data have been ranked into ascending order of values of AC
for the 30 maize field samples,
, compound microscope.

been shown here to vary little from observer to

observer. We recommend that with this method 100
to 150 intersections be taken for each root subsample.
For routine work one subsample per root sample
should be adequate. To increase precision it would
be advisable to increase the number of subsamples
per root sample, and not the number of intersections
per subsample or the number of observers per
subsample (Table 4).
Arbuscules are the principle sites of transfer of
phosphorus from endophyte to plant (Hayman,
1983), and so knowledge of the proportion of root
length containing arbuscules is likely to be of use in
physiological studies. However, it is the mycorrhizal
hyphae which extend beyond the root and ramify
into the surrounding soil that are important for
nutrient uptake (Harley & Smith, 1983). The degree
of colonization of roots by mycorrhizal hyphae might
be expected to give the best indication of the extent
of development of the external mycelium and so, in
turn, for the capacity of the association to benefit the
plant. The grid-line intersect method does not
estimate the 'true' proportion of the root length
colonized (Giovannetti & Mosse, 1980; Kormanik
and McGraw, 1982). The magnified intersections
method similarly cannot estimate the ' t r u e ' percentage colonization, for which it would be necessary
to distinguish between mycorrhizal and other hyphae
at all intersections. The magnified intersections
method perhaps offers the best quantitative description of colonization that can be made, given the
inherent difficulties associated with the interpretation of fungal material seen in roots. Since it is
difficult to estimate the proportion of root length
containing mycorrhizal fungi (arbuscules, vesicles
and hyphae), we believe it preferable to adopt a more
reliable estimate, namely the proportion of root
length containing arbuscules, or AC.

microscope (Ambler & Young, 1978) are likely to

generate observer-dependent data because of the
type of interpretations required during the analysis.
The data obtained for the grid-line intersect method
(Fig. 6) support this contention.
The magnified intersections method provides an
objective measure of colonization, AC, which has

The main drawback to the magnified intersections

method is that it takes up to one hour per subsample
in total to mount the roots on microscope slides and
score the colonization at x 200 magnification. Using
a multi-channel electronic counter helps to speed the
analysis because the eyes do not need to look away
from the microscope to record the status of each
intersection. The magnified intersections method

< 060
o
c 040

Runnii

1 020

Observer A
Observer B
~ Observer C

0
20

40

60
80 100 120 140 160
Number of intersections

180

Figure 5. Estimates of arbuscular colonization (AC) made

using the magnified intersections method by three
observers (A, B and C). Each observer worked with a
separate subsample of the same maize root system. The
data are presented as the running mean values for AC
calculated after each additional 10 intersections were
made.

c
o
(0

E
_o
o
u

04-

._

02

Rank

Objective measurement of colonization by VA mycorrhizal fungi

should be of use when objective assessment of the
extent of formation of arbuscules, vesicles and
hyphae are required. An important advantage of our
method is the elimination of the need for a subjective
interpretation of whether hyphae seen in the absence
of arbuscules are mycorrhizal. The method also
permits direct comparisons of colonization data from
one researcher to another.
ACKNOWLEDGEMENTS
We are grateful to Ranee Pararajasingham for valuable
technical assistance. Thanks to Ken Alexander, Dean
Barry, Peter Braunberger, Cheng-Yong Zhang and Shen
Lu who acted as observers, to the 70 students of the
University of Guelph who acted as subjects, and to Sharon
Lackie who provided the leek roots. T.P.M. is grateful to
Dr S. E. Smith for her comments about this project.

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