Plant and Soil 76, 115-124 (1984). Ms. BPSF 2.

4
© 1984 Martinus Nijhoff/Dr W. Junk Publishers, The Hague. Printed in the Netherlands.

Effect of liming on spore germination, germ tube growth

and root colonization by vesicular-arbuscular
mycorrhizal fungi*
J. O. SIQUEIRA**, D. H. HUBBELL and A. W. MAHMUD
Soil Science Department, University of Florida, Gainesville, FL, 32611, USA

Key words Al toxicity Fungistasis Liming Mycorrhizal fungi Spore germination VAM

Summary The effect of soil acidity on spore germination, germ tube growth and root coloniz-
ation of vesicular-arbuscular mycorrhizal (V AM) fungi was examined using a Florida Ultisol.
Soil samples were treated with 0, 4, 8 and 12meq Ca/MgC03/IOOg soil and each lime level
received 0, 240, and 720 ppm P as superphosphate. Corn (Zea mays 1.) was planted in the
soil treatments, inoculated with either Glomus mosseae or Gigagpora margarita spores and
grown for 31 days. Acid soil inhibits mycorrhizal formation by G. mosseae through its strong
fungistatic effect against the spores. The dolomitic lime increased mycorrhizal formation by
both fungal species. G. margarita is much less sensitive to acidic conditions than G. mosseae.
Al ions are a very important component of the fungistatic property against the V AM symbiosis.
V AM fungus adaptation may be important for plants growing on infertile acid soils if soil
inoculation with these fungi is to contribute significantly to low-input technology for tropical
agricultural systems.

Introduction
A possible alternative mechanism for maximizing fertilizer efficiency
is via inoculation with vesicular-arbuscular mycorrhizal (V AM) fungi.
They enhance nutrient uptake, and consequently plant growth, through
an extensive network of external mycelium which acts as an extension
of the root absorption system 17• This is particularly important in
infertile soils, commonly found in tropical regions, and appears to be
a promising technology for subsistence farmers 23 • If economical tech-
niques for inoculation or manipulation of the native population of
VAM fungi are to be developed, the adaptability of the fungi to edaphic
factors must be considered. Soil acidity is of immediate concern since
it is known to affect spore distribution, root colonization and mycor-
rhizal efficiency 4, 18, 19 • However, it is not dear if this results from direct
activity of the ionic concentration of H+ and/or from changes in the
chemical properties of the soil. Hubbe1P 2 suggests that liming may
influence the development of mycorrhizal associations either by de-
creasing the growth of fungal propagules in the rhizosphere or by
decreasing fungal colonization of root tissue .

.... Present address: Dept. of Soil Science, E.S.A.1., P.O. Box 37 37.200, Lavras-MG, Brazil.
115
116 SIQUEIRA, HUBBELL AND MAHMUD

Germination of VAM fungal spores on either agar media or soil is

affected by the pH8. However, soil acidity is not an independent factor;
pH alone may have little significance in understanding fungal spore
germination and root colonization by these fungi. We have recently
found significant interactions between pH and medium composition
for spore germination "in vitro"2S. This suggests that the effect of
soil acidity on VAM fungi may be the result of changes in solubility
of nutrients and toxic elements in the soil environment, rather than
pH "per se".
This study attempts to evaluate the effect of soil acidity on spore
germination, germ tube growth and colonization of corn root by two
VAM fungi.

Materials and methods

Samples of a virgin Dothan fine sandy loam (thermic Plinthic Paleudult) soil were collec-
ted at 0-20 cm depth from an area under a 20-year-old stand of slash pine in Escambia County,
Florida. The samples received the equivalent of 0, 4, 8 and 12 meq Ca/MgC0 3 /IOO g of soil
(as dolomitic limestone containing 54% CaC0 3 and 43% MgC0 3 ). After incubation for 3
months with the water content maintained at 22% by weight, 0, 240, and 720 ppm of P as
superphosphate (46% PP s) was applied simultaneously with 200 ppm of K as KCl and 20 ppm
of trace elements (frit) as FTE 503 (18%Fe, 7.5%Mn, 7.0%Zn, 3%B and 0.2%Mo). The
samples were again incubated for 3 months, then air dried and stored in plastic bags until
used.
For the greenhouse experiments, 250 g of soil was packed into styrofoam cups. Fifty
spores per cup of either Glomus mosseae or Gigaspora margarita were applied 3 cm below the
soil surface, and 25% w/w water was added. This was a factorial experiment composed of
4 lime treatments X 3 P levels X 3 V AM treatments X 3 replications. Three imbibed seeds of corn
(Zea mays L. var. Dekalb) were planted per pot and thinned to one plant five days after emer-
gence. After growing for 31 days under greenhouse conditions, plants were harvested and
individual root samples were cleared and stained 20 for root colonization assessment using the
grid intersect method.
At the same time, soil samples with no applied P and different limestone treatments were
brought to the laboratory for spore germination studies. Twenty spores of either G. mosseae
or G. margarita were placed between two Gelman membrane filters (.45 J,tm) and incubated
between two 50 g layers of soil in 100 XIS mm Petri dishes. Each limestone treatment was
triplicated. Washed river sand was used as control. Soil moisture was adjusted to 25% w/w,
the plates were wrapped with aluminium foil and incubated at 28° C for 20 days. After in-
cubation, filters with spores were removed from the plates and flooded with either trypan
blue or acid fuchsin (both 0.01%) staining solution for 5 min. Following staining, spores were
observed under a dissecting microscope at X 12 or 25 and spore germination and germ tube
growth rate were recorded according to Siqueira et al. ".
Water extracts from the different soil samples were obtained using 100 g/lOO ml of de-
ionized water, shaken for 48 hours and prepared according to Ko and Hora 13 • These were
incorporated in agar plates for studies of their effects on spore germination and growth features.
Experimental details are given by Siqueira 24 and Siqueira et al. 2s but only data for germin-
ation are included here (Fig. 2).
EFFECT OF LIMING ON V AM 117

50 ~-----------------,-------------------, 50

Glomus mosseae Gigospora margarita

40 40

-
c 240 ppm
.2
o
N
·2
o
8
~

Fig. 1. Effect of liming (0.4,8 and 12 meq CaMgCO,/100 g soil) and P fertilization (0, 240 and
720 ppm) on V AM formation on corn roots growing in a Ultisol infested with V AM fungi.
Bars represent standard deviation for 3 replications.

OOr---------------77--~~:~=_~~----~~------_, ~
501 L EXTRACT AGAR
GigoS{JOro mat'gorilo

60

40

(F

:~~~m~~~
o 4 8 12 ~O 0 4 8 12 2H0

Fig. 2. Effect of water extracts from soils incubated with different levels of lime (0, 4, 8 and
12 meq CaMgCO,/lOO g soil) on spore germination of two VAM fungal species "in vitro".

Results and discussion

Root colonization
Addition of dolomitic limestone to the acid soil increased root
colonization by the introduced VAM fungi except for G. margarita,
at the highest level of applied P (Fig. I). Colonization by the indigenous
mycorrhizal fungi reached a maximum of 7% and was not affected by
either lime or P applications (data not shown). The mycorrhizal in-
oculum potential, as determined by the most probable numbers method
118 SIQUEIRA, HUBBELL AND MAHMUD

Table 1. Water pH and 0.5N H 2S0 4 + 0.025N HCl soluble elements (ppm) for the different
soil samples with no P application, and their relationship with spore germination, germ tube
growth, and root colonization by G. mosseae
Lime level pH Al Ca Mg P K Mn Fe Zn Cu
(meq/IOO g) (HP)

Sand 7.0 4 13 4 10 I 0 19 0.92 0.08

0 5.5 772 26 12 3 29 0.42 0.72 1.08 0.16
4 5.9 766 382 222 3 16 0.92 0.92 0.68 0.16
8 6.1 746 702 378 3 14 1.14 1.14 0.56 0.12
12 6.4 652 1120 550 3 11 1.44 1.44 0.64 0.12

Correlation coefficients
Germination 0.97* - 0.83* 0.07 0.05 0.18 0.68* 0.22 -0.79 - 0.27 - 0.93*
GT growth 0.72 - 0.48* 0.31 0.30 0.08 - 0.78* 0.09 - 0.49 - 0.56 - 0.81 *
% Coloniz-
ation 0.63 - 0.13 0.09 0.60 0.67 - 0.82 0.67 - 0.12 - 0.95* - 0.48
Soil organic matter = 3.7%; Al saturation = 68%.
* p;;; 0.05.
No significant relationship was found between these parameters for G. margarita.

on fresh soil samples, was 47 propagules per 100 g soil, and the native
spore population was dominated by Gigaspora calospora, Acaulospora
spp., and Entrophospora Spp.lS. The low infectivity exhibited by the
indigenous mycorrhizal fungi may be the result of decreased viability
caused by the long term storage and incubation (9 months) of the soil
without the host plant. As already reported 4,27 G. margarita was less
affected by liming and showed greater colonization rates than G.
mosseae. These results suggest that G. margarita is more adapted
to low-P acid soils than G. mosseae, which does not establish well in
acid soils, sometimes even after liming. Soil pH and chemical elements
extracted by 0.5 N HCI + 0.025 N H 2S0 4 (double acid) did not explain
the variability in root colonization except for a significant correlation
(P = 0.05) found for AI, Zn and root colonization by G. mosseae
(Table I). Yawney et al. 27 also reported no significant correlations
between soil pH adjustment and percent root colonization on sweet-
gum plants inoculated with G. margarita. Mcilveen and Cole 16 showed
that addition of a small amount of ZnS04 enhanced soybean root
colonization by G. mosseae, while rates higher than 45 mg Zn/kg
soil resulted in decreased colonization. The low root colonization
by G. mosseae in soil with no limestone could be due to Zn and Al
toxicity caused by their higher solubility under acid conditions
(Table I). The relationship of these elements to spore germination
will be discussed in a later section of this paper. Different mechanisms
may operate at different H+ ion concentrations; thus, for the unlimed
EFFECT OF LIMING ON VAM 119

soil root colonization may be affected by high Al and Zn concen-

trations, while at high lime rates the high basic cations and low available
Mn could be the operative factors (see Table 1). Elmes et al. 5, using
a nutrient film technique, found that Ca at 75 mg/l gave better corn
root colonization than at 15 or 150 mg/l: they indicated tht the highest
level was approaching toxicity. Soils that received high rates of lime had
Ca levels high enough to reduce spore germination24 .
VAM fungi have been reported to occur in plants growing in bitu-
minous mine spoil at pH 2.7 3 and in alkaline soils at pH 9.22. Sparling
and Tinker26 found no effect of pH on root colonization in grassland
sites of pH 4.9, 5.9 and 6.2. These observations support the concept
that pH alone is not the controlling factor for root colonization in
acid soils. However, roots from these sites may be colonized by strains
of different fungal species tolerant of heavy metals 6 • Indeed, Glomus
species and isolates differed in their ability to form mycorrhizae at
low pH!4. The lack of correlation between soil chemical properties
and root colonization, also found by Yawney et al. 27, suggests that
the effect of soil acidity on the VAM symbiosis is very complex and
it is more likely to operate on the fungus than on the host plant. Soil
acidity may reduce not only mycorrhizal formation, but also the pro-
duction of VAM fungal spores and their viability in natural soils 2!.
Due to Al and Mn toxicities and low P in soil solution most crop
species growing in acid soils have coarse, stunted root systems con-
fined to the top soil layer. Because of their beneficial effect on P
uptake and plant-water relationships, VAM associations would play
a very important role in crop production under acid soil conditions
if both host and fungus species could be selected for adaptation to such
an environment 24 . Sanchez and Salinas23 have speculated that the
ability to form VAM associations may be important for crop species
and varieties adaptable to low-input systems. Depression of plant
growth by over-liming highly weathered tropical soils has been at-
tributed to reduced P uptake and induced trace element deficiency22.
However, our results and those of Yawney et al. 27 show that appli-
cations of high levels of limestone can depress root colonization by
G. mosseae and G. margarita. This factor may be important since VAM
fungi normally increase P and trace element uptake by the host plant!7.

Soil incubation
The effect of soil acidity on spore germination, germ tube (GT)
growth and theoretical mycorrhizal colonization index (MCI = % ger-
mination x GT growth rating) are presented in Table 2: additions
of limestone enhanced all three indices. The germination of G. mosseae
120 SIQUEIRA, HUBBELL AND MAHMUD

Table 2. Spore germination percent (%G) , mean germ tube growth rating spore (GT) and
theoretical mycorrhizal colonization index (Mel) by VAM fungi incubated for 20 days on
acid soil treated with different limestone levels
Limestone Glomus mosseae Gigaspora margarita
level %G GT Mel %G GT Mel
meq/100g
0 43c 0.9d 39d SOb 3.8b 190b
4 60b 1.8c 108c SOb 4.3ab 215b
8 68b 3.8a 259a 63a 4.7a 296a
12 67b 2.Sb 168b 38c 4.4ab 168b
sand 88a 3.4a 299a 39c 3.0c 117c
Mel = %G XGT growth rating.
Means followed by the same letter are not statistically different by Duncan's test at 0.05 level.

was increased above that of unlimed soil by as much as 58%, but

still remained below the control (sand), while the maximum increase
for G. margarita was 26%, in both cases with 8 meq Ca/MgC03/1 00 g
of soil. Germination rates and GT growth for G. margarita were higher
than the control treatment (sand) except for the highest level of liming.
In general, G. mosseae spores that germinated in unlimed soil had
stunted GT; liming increased their GT growth by as much as 320%,
while in G. margarita maximum increase was about 24%. These data
suggest that acid soil is fungistatic for VAM fungal spores and that
this fungistasis is far more active on G. mosseae than on G. margarita,
which apparently has adapted to the acid environment. Correlation
analysis showed significant (P ~ .05) relationships of G. mosseae,
spore germination with pH and K( +), Al and Cu(-); while the GT
growth correlated negatively with K and Cu (Table I). For G. mar-
garita spores no significant relationship was found between either
spore germination or GT growth and soil chemical properties. This
suggests the germination process and GT growth in G. mosseae is
more sensitive to chemical factors under acidic conditions. In fact,
G. mosseae does not form GT on agar medium with pH adjusted
to 4.5 with 1M HCl (Siqueira, unpublished); shows very low ger-
mination rates at pH 5.0 8 . and is not usually found in soils with pH
below 5.0 1• The toxicity of Cu ions to VAM fungal spores has been
demonstrated by Hepper9. Amendment with Cu at 0.5 mg/l reduced
the germination of Glomus caledonicum to 16% of the controllO.
The results in Table 3 indicate that the inhibitory effect of un-
limed soil on G. mosseae spore germination was not removed by the
presence of the host plant, by addition of root exudates, or by auto-
claving (Table 3).
Volatile compounds produced by soil microorganisms can be either
fungistatic or stimulatory for spore germination and GT growth.
EFFECT OF LIMING ON V AM 121

Table 3. Effect of different treatments and experimental conditions on germination and GT

growth by G. mosseae after 12 days of incubation. Means for 3 replications ± standard de-
viation
Treatments % Germination GT Growth rating
Sand control 90 ± 6.4 4.7 ± .60
Unlimed soil 31 ± 5.1 .5 ± .31
4 meq/100 g CaC0 3 soil 68 ± 9.2 1.5± .40
Unlimed; autoclaved soil 17 ± 3.5 .5 ± .63
4 meq/l00 g CaC0 3 ; autoclaved soil 44 ± 1.3 1.1± .48
Unlimed; transferreda 69 ± 12.7 4.6 ± .65
Unlimed; membraneb 82 ± 9.2 4.2 ± .36
4 meq/100 g CaC0 3 ; membraneb 77± 3.5 4.1 ± .19
Unlimed + PlantC 19 ± 1.5 .9 ± .14
Unlimed + root exudates d 18 ± 1.4 .5 ± .40
Sand + unlimed soil extracts e 67 ± 2.1 3.6 ± .21
Volatile; from unlimed soilf 69 ± 8.5 4.2 ± .35
Volatile; 4 meq/l00 g CaC0 3 soilf 71± 7.7 4.8 ± .21
Volatile; 8 meq/lOO g CaC0 3 soilf 68 ± 18.1 3.9 ± 1.01
Volatile; 12 meq/l00 g CaC0 3 soilf 76 ± 13.2 4.0 ± 1.41
Volatile; Hp (control)f 61 ± 3.8 3.8 ± .91
a Spores were incubated on unlimed soil for 5 days, then transferred to washed sand plates.
b 50 g of unlimed soil and limed (4 meq Ca/MgC0 3 /lOO g soil) was packed into a dialysis
tube (14K cut off) and spores placed outside on MF filter in sand.
C Spores were placed to germinate in the rhizosphere of Phaseolus vulgaris growing in fungi-
static soil (no lime).
d Root exudates from Phaseolus vulgaris were used to bring soil moisture to an optimum
for spore germination on plates with unlimed soil.
e Unsterile soil extracts from unlimed soil samples were used to moisten sand plates where
spores had been plated to germinate.
f Effect of volatile compounds from soil samples incubated with different levels of limestone.

Hora and Baker l l reported that increasing the pH of acidic soils re-
sulted in the release of volatile compounds inhibitory to two test
fungi and stimulatory to three others. Germination of Acaulospora
laevis spores was induced by soil volatile compounds while germination
of G. margarita spores was inhibited by volatile compounds from soil
actinomycetes (Siqueira and Bleakley, unpublished). The effect of
volatile compounds released from soil samples treated with different
limestone rates on either spore germination of GT growth of G. mos-
seae chlamydospores (Table 3), cannot account for the differences
found in our experiments.

Nature of the inhibitory factor

In the case of G. mosseae the results obtained for spore germination
and GT growth in the soil (Tables 2 and 3) and soil extract-agar (Fig. 2)
suggest the presence of a water-insoluble fungistatic factor, very similar
122 SIQUEIRA, HUBBELL AND MAHMUD

to that reported for Neurospora terrasperma by Ko and Hora 13. Evi-

dently, Al toxicity is a very important component of the fungistasis
complex present in acid mineral soils. Indeed, in other experiments
using the same soil, G. mosseae, spore germination was negatively
correlated with exchangeable Al more than with either com root
colonization or shoot dry weight 1S • Soil acidity appears to affect
VAM fungal propagules, mostly on the fungus in the rhizosphere
before root penetration takes place rather than on the host itself.
These results and others those of Hepper9 and Hepper and Smith10
indicate that metals such as AI, Zn, Cu, and Mn and H+ ions present
in soil solution may be toxic under acid conditions and may reduce
plant growth by reducing spore germination, GT growth, and hence
root colonization by the VAM fungi. In acid tolerant host-fungus
combinations, metal toxicity may result in poor growth of external
mycelium, nutrient uptake by fungal hyphae, and hence non-efficient
VAM symbiosis4,7.

Conclusions

Liming acid soils to reduce H, Al and other metal toxicities reduces

the fungistatic effect against some VAM fungi, favours mycorrhizal
formation, and plant growth.
Species and isolates of VAM fungi differ in their tolerance to soil
acidity. Because VAM symbiQsis increases plant nutrient uptake and
growth, acid-soil tolerant species of VAM fungi may play an important
role in plant resistance to low P in soil solution and high Al saturation
found in most tropical soils. Overliming and heavy fertilizer appli-
cations of highly weathered soils of the tropics may reduce mycorrhizal
formation and depress plant growth.
Soil acidity appears to be an edaphic factor with great influence
on the distribution of VAM fungal species and may be a serious con-
straint for introduction and longevity of VAM fungi as efficient plant
growth promoters and biocontrol agents such as G. mosseae, into
tropical agricultural production systems.
Inoculations under greenhouse and laboratory conditions, and
monitoring spore germination and saprophytic growth by these fungi
in the soil, may be a useful way to select species and isolates for further
inoculation studies.

Acknowledgement The authors thank Dr. Geraldo A. A. Guedes for providing the soil samples,
and MEC/CAPES-Brasilia for financial support.
EFFECT OF LIMING ON V AM 123

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