T.P. McGonigle, M.H. Miller Applied Soil Ecology 14 2000 147–155 149
effects of disturbance on colonization that have been found.
Tillage of soil can itself affect the inoculum den- sity in soil during the growth of the subsequent crop.
The densities in soil of numbers of AM spores, and of lengths of structurally and metabolically stained hy-
phae, was lower in the top 5 cm of plots in Quebec, Canada, following conventional tillage as compared to
no-till Kabir et al., 1998. No difference in coloniza- tion of maize roots in the field by AM fungi was found
Kabir et al., 1998. However, a difference in inoculum density following tillage may have led to a difference
in colonization earlier in the growth season. Soil and roots were sampled during grain filling Kabir et al.,
1998, whereas differences for colonization of maize by AM fungi in response to tillage are typically seen
only up to the six-leaf stage McGonigle and Miller, 1993, 1996b.
Our aim was to evaluate the role that inoculum den- sity plays in determining the impact of disturbance
on colonization of roots by AM fungi in agricultural soils. We used maize and the soil of the Elora Re-
search Station in Ontario, Canada, which we have studied extensively. The hypothesis was as follows:
colonization of roots in D soil will be reduced, rela- tive to that for plants sown in U soil, but only when
the inoculum potential of the soil is low. On this basis, high inoculum should produce well-colonized roots
in both U and D soils. The approach was to select a batch of soil for which a high inoculum density
was expected, and to lower it by manipulation. Soil was collected mid-season from the rooting zone in
a maize field. Plant growth and the colonization of roots by AM fungi after two cycles of growth in the
laboratory were determined, with or without distur- bance between cycles. As an exploratory exercise, soil
biomass-C was estimated for available samples using the fumigation–extraction technique, in order to see if
it would reflect the behavior of the mycorrhizal fungi, and by doing so aid our understanding of mycorrhizal
relationships.
2. Materials and methods
2.1. Preparation of soil Soil was collected in early July 1995, from directly
below plants in an actively growing maize field at the Elora Research Station 43
◦
31
′
N, 80
◦
14
′
W, Ont., Canada. This soil is a Conestogo silt loam 300 g kg
− 1
sand, 500 g kg
− 1
silt, 200 g kg
− 1
clay, and it is clas- sified as an Aquic Hapludalf Ketcheson, 1980. The
soil has a pH of 7.5 in a saturated soil–water paste. Soil was taken from the upper 15 cm of the profile
and air-dried to approximately 0.18 g H
2
O g
− 1
dry soil. The soil was passed through a 2 cm sieve, using
scissors to cut the roots as necessary, and mixing them back into the soil. This soil was then confirmed
to have abundant colonized root on the basis of the following values mean±S.D.: root-length density
was 1.8±0.2 cm g
− 1
; the proportions of root length colonized with arbuscules and hyphae McGonigle
et al., 1990b were 43.1±8.6 and 67.1±14.5, re- spectively. This root length density and extent of
colonization are close to the seasonal maximums for maize in this region McGonigle and Miller,
1993.
The soil was mixed and divided into two parts, one of which was then pasteurized in an electrical
soil-heating unit. Pasteurization involved heating the soil slowly to 80
◦
C, maintaining at this tempera- ture for 2 h, and allowing it to cool. Pasteurized and
non-pasteurized soils were analyzed using quadru- plicate sub-samples for extractable concentrations
of macronutrients. The only differences p0.05 between the nutrients of the two soils were for ex-
tractable NH
4
–N and NO
3
–N. The pasteurized soil had NH
4
–N and NO
3
–N concentrations extracted with 2.0 M KCl of 33 and 24 mg kg
− 1
; correspond- ing values for the non-pasteurized soil were 4 and
18 mg kg
− 1
. Overall values means±S.D.; n=8 for other nutrients were as follows: P extracted in 0.5 M
NaHCO
3
at pH 8.5 was 5.4±0.7 mg kg
− 1
; using neu- tral 1.0 M ammonium acetate for the extraction, the
soil had 75±13 mg K kg
− 1
, 450±16 mg Mg kg
− 1
, and 2.8±0.2 g Ca kg
− 1
. The pasteurized and non-pasteurized fractions were
then blended to give four inoculum-density treatments, which contained 10, 35, 65 or 90 of pasteurized
soil by volume. The soil blends were fertilized with 50 mg N kg
− 1
as KNO
3
powder, and 50 mg P kg
− 1
as calcium monophosphate CaH
2
PO
4 2
· H
2
O powder. The soil blends were packed to a bulk density of 1.1 g
dry soil cm
− 3
in pots so that each pot had 4730 g dry soil. The diameter of a pot at the soil surface was
20.5 cm, and the soil depth was 15 cm. A 300 g layer
150 T.P. McGonigle, M.H. Miller Applied Soil Ecology 14 2000 147–155
of clean sand was added to the surface of all soils to restrict evaporation.
2.2. Plant growth All pots were taken for two successive cycles
of maize growth. Plants were kept in a growth chamber with a 17 h day at 28
◦
C, a 7 h night at 23
◦
C, and with daytime irradiation of approximately 400 m mol cm
− 2
s
− 1
. Pots were watered by mass every 1 or 2 days to keep them close to but not exceeding
0.24 g H
2
O g
− 1
dry soil. The first growth cycle was as follows. After soaking
for 48 h in aerated water, six maize seeds were planted per pot at a depth of 2 cm on 21 July 1995. Thinning
was to three seedlings per pot at the two-leaf stage. The leaf-stage counting system used here excluded the
spike leaf within the whorl. The shoots were cut at the soil surface and removed from all pots on 10 August
1995, when they were at the seven-leaf stage. The soil was then broken up by hand and passed through a
5 mm sieve to give the D treatment, or left intact to give the U treatment. In the D treatment, scissors were
used to cut roots so they could pass through the 5 mm sieve, and the roots were mixed back into the soil.
Disturbance was in three 5 cm depth fractions, which were handled separately and returned in order to each
pot. For the second growth cycle, seeds were sown on 16 August 1995, and maize was grown in all pots as for
the first growth cycle. While thinning at the two-leaf stage, root samples were taken by loosening gently
the surface soil below a seedling and withdrawing the roots that were connected to the plants being removed.
Root samples were taken from all pots at the three-, four-, and five-leaf stages using a 33 mm diameter soil
corer, which was inserted midway between adjacent plants to the full pot depth. Holes made by coring
at the three- and four-leaf stages were plugged using wooden dowels. Shoots were harvested at the five-leaf
stage on 31 August 1995.
2.3. Laboratory measurements All procedures refer to samples taken in the second
growth cycle. Roots collected at the two-leaf stage were rinsed in tap water and stored in formyl-acetic
alcohol FAA, which consists of an 18:1:1 mixture of 95 ethanol, 28 wv formaldehyde, and glacial
acetic acid. All soil cores were handled separately on their day of harvest as follows. The soil in each core
was passed through an 8 mm sieve, cutting the roots as before. Each sample was well mixed, the fresh weight
taken, and a 50 g fresh weight sub-sample was put to one side. All roots in the remainder of the sample
were collected by rinsing against a 0.5 mm screen. The length of root was determined Tennant, 1975
immediately, and the roots were then stored in FAA. The soil in the 50 g sub-samples was passed through
a 2 mm sieve, brushing soil from the surfaces of roots and discarding those roots. The sieved soils were then
stored in plastic bags for up to 3 days at 5
◦
C in the dark.
Shoots were rinsed in deionized water and then dried in a forced-air oven at 65
◦
C. Dried shoots were weighed and then ground to a fine powder. Shoot-P
and shoot-N concentrations were determined follow- ing the method of Thomas et al. 1967.
Stored roots were rinsed free of FAA and sub-sampled as necessary for assessment of coloniza-
tion by AM fungi. A sub-sample was taken in each case by stirring roots in 3 l of water in a pitcher,
and collecting the sub-sample in a 500 ml beaker plunged quickly in and out of the water. Roots were
cleared by autoclaving in 10 KOH for 15 min. at 121
◦
C, and stained as described by Brundrett et al. 1984. The percentage of root length with arbus-
cules, which is the arbuscular colonization AC, was determined as described by McGonigle et al.
1990b.
Stored 50 g soil samples at the three-, four-, and five-leaf stages were used for determination of
moisture content and of soil biomass-C using the fumigation–extraction method Vance et al., 1987
using an efficiency factor of 0.25 Voroney et al., 1993 in the calculation.
2.4. Statistical design and analysis Based on the soil batch used for handling dur-
ing preparation, and based on position in the growth-chamber, the pots were divided into four
blocked replicates in a randomized complete block design. Each replicate had eight pots, one for each
combination of the four soil-blend treatments and the two soil-disturbance treatments. Data were evaluated
using separate analyses of variance for each time,
T.P. McGonigle, M.H. Miller Applied Soil Ecology 14 2000 147–155 151
with separation of means using Tukey’s test Steel and Torrie, 1980.
3. Results
