Soil Biol. Bioch~m.Vol. 27. No. 9. pp. 1153-l 159.

1995
Copyright c 1995 Elsevier Science Ltd
Pergamon 0038-0717(95)00047-x Printed in Great Britain. All rights reserved
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GROWTH AND EXTRACELLULAR PHOSPHATASE

ACTIVITY OF ARBUSCULAR MYCORRHIZAL HYPHAE
AS INFLUENCED BY SOIL ORGANIC MATTER

E. J. JONER* and I. JAKOBSEN

Plant Biology Section, Environmental Science and Technology Department, Risra National Laboratory,
DK-4000 Roskilde, Denmark

(Accepted 16 February 1995)

Summary-Two experiments were set up to investigate the influence of soil organic matter on growth of
arbuscular mycorrhizal (AM) hyphae and concurrent changes in soil inorganic P, organic P and phosphatase
activity. A sandy loam soil was kept for I4 months under two regimes (outdoor where surplus precipitation
leached through the soil, or indoor at constant moisture) with or without 9% (w/w) chopped wheat straw
plus mineral N. Then the soils were partially sterilized and placed in two-compartment pots where
mycorrhizal or non-mycorrhizal cucumber plants were grown in one root compartment (RC), and soils
differing in organic matter were placed in six parallel hyphal compartments (HC) separated from the RC
with a 37 pm mesh. In the first experiment, using Glomus caledonium, hyphal length densities were measured
in the HC after 3 I days. Added straw increased hyphal length densities by 34 and 62% for soil kept outdoors
and indoors. respectively. In the second experiment, using G. invermaium and only soil kept outdoors, three
treatments were included: soil with no added straw with or without a new addition of 0.5% (w/w) of ground
clover leave!, and soil with 9% straw plus mineral N. After 41 days hyphal length density was twice as high
in soil with added straw compared to the two other treatments. Mycorrhizal colonization resulted in lower
activity of acid phosphatase in the HC for two out of three treatments. Alkaline phosphatase activity was
only decreased by mycorrhiza in soil without organic matter additions. In soil with added clover alkaline
phosphatase activity increased due to the presence of mycorrhizal hyphae. We suggest that mycorrhizas may
influence the exudation of acid phosphatase by roots. Hyphae of G. imermaium did apparently not excrete
extracellular phosphatases, but their presence may have influenced alkaline phosphatase excreted by other
microorganisms, probably through competition for nutrients. Phosphatase activity was not correlated with
the concentration of labile organic P in soil extracts

INTRODUCTION organic P (PO) are conflicting (Tarafdar and

External hyphae of arbuscular mycorrhizal (AM) Marschner, 1994; Joner et al., 1995). The quality of
fungi are responsible for a large part of the P uptake soil organic matter may be one determinant to the
reported differences, as phosphatase activity is
in mycotrophic plants as they bridge the P depletion
zone around roolts and acquire P for their host influenced by the availability of P, to hydrolytic
plants from unexploited soil volumes (Sanders and cleavage (Stewart and Tiessen, 1987). Thus, extracellu-
Tinker, 1971). Accordingly, amounts of external lar phosphatase activity of roots may be stimulated in
hyphae have been proposed as a better determinant the presence of easily hydrolyzed substrates (Tarafdar
of mycorrhizal efficiency than the extent of root and Claassen, 1988), but repressed by non-hydrolyz-
colonization (Gra:ham et al., 1982), although large able forms of P, (Azcon et al., 1982). The host plant
differences in P transport rates may exist between (Azcon et al., 1982) and fungal species (Dodd et al.,
different fungi (Jakobsen et al., 1992). Organic 1987; Rao and Tarafdar. 1993) may also influence
matter has been reported to enhance proliferation of phosphatase activities.
AM fungal hyphae in soil (St. John et al., 1983). We report two experiments where the effect of plant
Organic matter is also a major pool of P in soil, material added to soil on proliferation and function of
but information on the possible role of AM fungal AM fungal hyphae was measured. In one of these
hyphae in utiliza.tion of this P pool is scarce. experiments plant materials of different quality were
Recent reports on the role of AM in production added to soil. Fresh clover represented an easily
of extracellular phosphatases and mobilization of degradable organic input, while wheat straw mixed
and kept with soil for 14 months represented a stable
organic matter enrichment. The experiments were
*Author for correspondence, permanent address: Depart-
ment ofBiotechnological Sciences. Microbiology Section,
carried out in a compartment system where growth
Agricultural Umversity of Norway, P.O. Box 5040. and influence of AM fungal hyphae could be studied
N-1432 Aas, Norway. in the absence of roots.

1153
1154 E. J. Joner and I. Jakobsen

and Gerdemann (isolate RIS 42) and one pot was left
uninoculated. In one inoculated pot the HC were filled
with soil kept outdoors (3 HC with no addition and 3
HC with 9% straw) and in the other two pots the HC
were filled with corresponding soil kept indoors.
Mycorrhizal inoculum was placed only in the RC
and consisted of 200 g per pot of the described soil
from a pot culture of G. caledonium propagated on
subterranean clover (Trifolium subterraneum L.). The
non-mycorrhizal pot received 200 g soil from a pot
culture of non-mycorrhizal clover. Soil in the RC
received 10 mg P kg - ’as KH?PO., and all soil received
Fig. I. Diagram showing pot divided by 37 pm mesh into one 10 ml kg- ’of a freshly prepared soil filtrate (10 g soil
root compartment (RC) and six hyphal compartments (HC). in 200 ml water, blended 2 min, filtered through
Dimensions are given in mm.
Whatman no. 40 filter) as microbial inoculum without
AM fungal propagules. Hyphal length densities in
the HC were measured 31 days after sowing. In
MATERIALS AND METHODS this experiment measurements of triplicate HC for
each treatment cannot be regarded as independent
Pots and soil replicates as they all belonged to the same pot.
Pots consisted of square PVC boxes divided In the second experiment, three pots were
vertically into one root compartment (RC) and six inoculated with 200 g soil from pot cultures of G.
parallel hyphal compartments (HC) (reconstructed invermaium Hall (isolate WUM 10, obtained form
from Wyss et al., 1991; Fig. 1). RC and HC were L.K. Abbott, Univ. of Western Australia) propagated
separated by a 37 pm nylon mesh. The outer wall of on subterranean clover and three pots with soil from
each HC could be removed to section soil at harvest. uncolonized clover. Two HC of each pot were filled
The RC contained 1800 g of a sieved (< lmm) with either: (1) soil kept outdoors without straw;
partially sterilized (10 kGy, 10 MeV electron beam) 1: 1 (2) the same soil as (l), but with newly added
mixture (w/w) of a moderately P-deficient sandy loam (0.5% w/w) ground (<OS mm) lo-week-old clover
(Jakobsen and Nielsen, 1983) and quartz sand. The shoots (43.85% C, 2.27% N, 0.15% P); or (3) soil kept
HC contained 225 g or 200 g of another sandy loam outdoors with 9% straw and mineral N. All soil
soil (Jensen, 1994) that had been kept for 14 months received soil filtrate as in the first experiment, but no
mixed with 0 or 9% (w/w), respectively, of chopped P was added. Plants were grown for 41 days before
wheat straw and NH,NO3 (9.8 g N kg-’ straw). The measuring acid and alkaline phosphatase, extractable
soil was either kept indoors at 60-80% of water P, pH and hyphal length densities in two soil sections
holding capacity (WHC) and average temperature of each HC.
of 15’C, or outdoors exposed to precipitation (ca.
800 mm y-‘) and leaching of N. After incubation, the Plants and growth conditions
soil was partially sterilized as described above. Some Pots were watered to 60% of WHC of each
data for this soil after organic matter addition and compartment and incubated for 8 days for mycor-
incubation are given in Table 1. Nutrients except N rhizal propagules to germinate. Four germinating
and P were added to soil in RC at optimum rates for seeds of cucumber (Cucumis sativus L., cv. Aminex)
plant growth (Joner and Jakobsen, 1994). Soil in HC were sown in each pot and thinned to two per pot after
received no nutrients. seedling emergence. The plants were placed in a
growth chamber with a l&8 h light-dark cycle
(500 pmol rn-~’ s - I, Osram HQI-T 250 W/D) at
E.uperinwntal design
20-15X and watered daily by weight. N (NH,NO,)
In the first experiment, two pots were inoculated was added weekly to RC to a total amount of 200 mg
with Glomus caledonium (Nicol. and Gerd.) Trappe N per pot.

Table I. Total C, total N and 2 M KCI-extractable inorganic N in soil placed in hyphal compartments after indoor or outdoor
storage with 0 or 9% (w/w) wheat straw + inorganic N
Extractable N
StOrdge regime Straw additmn (%) Total C (%) Total N (%) NH,-N (mg N kg- ‘) NO?-N (mg N kg ‘)
Indoors 9 2.51 0.23 19 227
0 I .30 0.14 I 58
Outdoors 9 2.88 0.21 54 3
0 1.33 0.14 I2 27
0 + chwert I.54 0.15 ND* ND
tClover added after storage.
*ND = not determined
 Soil organic matter
Sectioning 13f’soil
Soil in the HC of the first experiment was not
sectioned, whereas in the second experiment the soil of
the HC was cut into three sections: O-10, 10-20 and
20-30 mm from the mesh interface. Only soil from
the two sections closest to the roots were analyzed.
Immediately after sectioning, soil was packed in
plastic bags and st’ored at - 18°C until samples were
taken for phosphatase measurements.
Phosphatase measurements
In the second experiment, phosphatase activity was
measured by a procedure modified after Tabatabai
and Bremner (1969) using 1 g moist soil and 12 ml
buffer-substrate per sample. Acid phosphatase was
measured at pH 5.2 using a 0.1 M acetate-acetic acid
buffer containing 1 mg p-nitrophenyl phosphate
(PNPP) ml- I. Buffered substrate with soil was kept for
1 h at 37’C in a shaking water bath before adding
10 ml 0.5 M NaOH and 2 ml 0.5 M CaCl?. Alkaline
phosphatase was measured similarly at pH 8.5
using 0.1 M NaHCO? as buffer. Blanks were prepared
by adding NaOH to buffer and soil from either HC
prior to substrate addition. Soil suspensions were
centrifuged in the sample tubes at 2750 x g for 10 min
beforep-nitrophenol in the supernatant was measured
spectrophotometrically at 405 nm. Enzyme activity
was expressed as (enzyme units (EU; 1 pmol PNPP
hydrolysed g - ’soil h- ‘) on a soil dry wt basis after
subtracting blanks.
C, N and P measurements
Soil P was extracted from 2 g moist soil with 40 ml
0.5 M NaHCO, at pH 8.5 (Olsen etai., 1954). Inorganic
P was analyzed using ammonium molybdate-ascorbic
acid (Murphy and Riley, 1962). Total Pin the extracts
was measured similarly after oxidizing an aliquot in
0.5 M K&O8 in an autoclave at 120°C for 45 min
(Ebina et al., 1983’).Organic P was calculated as the
difference between total and inorganic P. Total C and
N in soil and plants were measured on a Carlo Erba
C and N analyzer. Soil inorganic N was measured in
2 M KCl-extracts (Keeney and Nelson, 1982) using a
Technicon Auto-Analyzer II.
Measurements of roots and hyphae
Subsamples of roots were used to determine total
and colonized root length (Newman, 1966) after
staining with trypan blue (Kormanik and McGraw,
1982). Length of mycorrhizal hyphae was measured
as the difference between mycorrhizal and non-
mycorrhizal samples of each soil using a membrane
filter-grid intersect method modified after Abbott
et a/. (1984). Modifications included the use of 2 ml
aliquots and 20 mm dia filter area. Duplicate filters
were made from each of two samples per HC, and
hyphae were counted in 35 or 25 fields of vision at
200 x magnifical.ion in the first and the second
experiment, respectively.
and AM fungal hyphae 1155
Treatment effects were tested by two or three-
way analysis of variance and differences between
means compared by the F-test or by LSD-values
(P = 0.05).
RESULTS
The C content was increased in soil which had been
incubated with straw for 14 months at two incubation
regimes (Table 1). This was accompanied with an
increase in WHC from 25 to 3 1%) a reduction in bulk
density from 1.48 to 1.19g cm-’ and a pH(H20)
decline from 7.3 to 6.6. Leaching of surplus
precipitation had led to loss of N in the soil incubated
outdoor with 9% straw (Table 1).
In the first experiment, inoculation with G.
caledonium resulted in an average colonization of 85%
of the root length. Uninoculated plants remained
uncolonized. Shoot and root dry wt were 40 and 30%
higher, respectively, in mycorrhizal than in non-myc-
orrhizal plants (data not shown). AM hyphal length
densities were higher in soil amended with wheat
straw for both incubation treatments (Fig. 2). The
hyphal length densities measured in HC of the
non-mycorrhizal treatment were 6.0 and 4.9 m cm - 3
in soil with no addition and 9% wheat straw,
respectively. These values were subtracted to give the
data in Fig. 2.
In the second experiment, inoculation with G.
inoermaium resulted in an average colonization of 66%
of the root length. Uninoculated plants remained
uncolonized. Dry wt of mycorrhizal and non-
mycorrhizal plants were not significantly different
(data not shown). Also in this experiment the hyphal
length densities were highest in soil where straw had
been added (Fig. 2). There was no difference in hyphal
length density between soil with or without 0.5% of
freshly added clover. The subtracted hyphal length
__
1 T
Indoors Outdoors Outdoors
G. catedonium G. invermaium
Fig. 2. Hyphal length density of G. caldonium (first
experiment: O-30 mm from roots) and G. irwerrwiwt (second
experiment: I&20mm from roots) after subtraction of
hyphal lengths in corresponding non-mycorrhizal compart-
ments. Soil additions: no addition W, 0.5% clover leaves ? ,?
9% wheat straw ‘J. Storage regime prior to soil sterilization
(indoors/outdoors) is indicated. Bars show LSD,,,,S.
 1156 E. J. Joner and 1. Jakobsen

density measured in non-mycorrhizal treatments

constituted 3.5, 2.8 and 4.9 m crne2 for soil with no
addition, clover leaves and wheat straw, respectively.
For both experiments the water content of HC was
measured at harvest and showed small deviations
(14% of WHC) from the initially added water content
(60% of WHC), in spite oflow water content in the RC
(2&32% of WHC), which received no water at the day
of harvest.
Phosphatase activity at both pH values was
increased as a result of organic matter addition
(Table 2). The highest activities of acid phosphatase
was found when dried clover leaves were added, while
addition and incubation of wheat straw resulted
in the highest activities of alkaline phosphatase.
Acid phosphatase activity was higher in the soil
layer closer to roots than in the 10-20 mm layer for
soil with no addition of organic matter and for soil
with 9% wheat straw. The same effect was seen for
alkaline phosphatase for soil without organic matter
addition. In contrast, alkaline phosphatase activity
was lower in the soil layer closer to the roots than in
the lo-20 mm layer when soil was amended with
clover leaves. Mycorrhiza led to a decrease in acid
phosphatase activity in soil with no additions and in
soil with added clover leaves. Alkaline phosphatase
activity decreased similarly in the presence of
mycorrhiza only when no organic matter was added.
However, mycorrhiza led to an increase in alkaline
phosphatase activity when clover leaves were added.
No effect of mycorrhiza was seen in soil amended with
wheat straw.
Depletion of NaHCO,-extractable inorganic P
(P,) was evident in the soil layer closer to the roots
(Table 3). Increased depletion in the presence of
mycorrhiza was seen only in soil with 9% wheat straw.
No depletion of NaHCOi-extractable P, was detected
irrespective of organic matter addition or the presence
of mycorrhiza.
The pH was significantly lower in soil incubated
with 9% wheat straw compared to the two other
treatments (P < 0.001) but there were no differences
in pH between soil O-10 and IO-20 mm from the roots
or between soil with and without mycorrhizal hyphae
(Table 2).

DISCUSSION
The use of one soil with or without amendments
with two types of organic matter in combination
with a compartmented growth system allowed com-
parisons of hyphal length and phosphatase activity as
influenced by soil organic matter quality.
Previous reports that hyphae of AM fungi
proliferate in and around aggregates of organic matter
(Nicolson, 1959; Koske ef al., 1975; St. John er al.,
1983) and in soil amended with phytate (Tarafdar and
Marschner, 1994) were based on qualitative obser-
vations. However, Abbott er ul. (1984) concluded that
it is difficult to distinguish hyphae of AM fungi from
 Soil organic matter and AM fungal hyphae
Table 3. Inorganic and organic P extractable with 0.5 MNaHCO, from soil in hyphal compartments unamended or amended with 0.5% clover
or 9% wheat straw at different distances from roots of non-mvcorrhizal (control) or mvcorrhizal (G. inwrmaiunt) cucumber (second experiment)
Inorganic
Mycorrhizal Distance from No addition Clover leaves
treatment roots (mm) (ma P kg-‘)
Control &IO 9.0 9.3
IO-20 9.8 9.7
G. iruwwtniunt &IO 9.0 8.8
I &20 9.8 9.6
Statistical effectst
Mycorrhiza NS NS
Distance from roots * *
Interactions M x D NS NS
tNS = not significant. *P < 0.05.
those of other fungi. A non-mycorrhizal control, as
used in our work, is therefore essential in order to
correct any treatment effect on hyphal length for
effects on saprophytic fungi. Increased hyphal growth
in the soil with increased organic C content was
consistent for the two AM fungi. This effect seemed
to be exerted directly on the external hyphae as
mycorrhizal colonization of plants grown in separate
pots with the same soils did not vary (results not
shown). The mechanism behind stimulated hyphal
growth is not obvious. A chemotactic response to
nutrients, CO? or other microbially-mediated factors
are possible explanations. The lack of response in
hyphal length density due to added clover leaves
indicates that nutrients did not account for the
observed effect. This is supported by the findings of
Abbott et al. (1984) and Li et al. (1991) that increased
P concentrations in soil do not increase hyphal length
densities, and of the findings of Johansen et al.
(1992) that N added as NH: had no influence on
hyphal lengths. In the first experiment, the absence
of an effect of soil N concentrations on hyphal
lengths can also be deduced by comparing hyphal
lengths for soil Ikept outdoors (low N) or indoors
(high N).
Enhanced microbial activity and changes in the
partial pressures of O2 and CO? are likely to result
from mineralization of organic matter. Although
being slowly degradable, wheat straw added in large
amounts could well have resulted in higher concen-
trations of CO? and lower concentrations of O2 in
soil than those present after the addition of clover
leaves. Elevated CO1 concentrations have been
reported to enhance hyphal growth of AM fungi
(Btcard and Piche, 1989), but a simultaneous lack of
O1 could affect AM fungi negatively (Saif, 1983). AM
have the potential to alter bacterial species compo-
sition in rhizosphere soil (Meyer and Linderman,
1986) and some bacteria can positively stimulate
certain aspects of the symbiosis (Azcon-Aguilar and
Barea, 1985). However, it is unknown whether
microbial effects can be exerted directly on AM fungal
hyphae.
The beneficial role of organic matter may also be
related to an improvement of physical properties like
increased soil porosity and reduced mechanical
1157
P Organic P
Wheat straw No addition Clover leaves Wheat straw
(mg P kg-‘)
12.6 10.4 9.6 II.3
14.1 9.6 IO.1 12.9
9.0 10.2 9.5 10.9
10.0 8.9 9.3 10.7
* NS NS NS
NS NS NS NS
NS NS NS NS
resistance to hyphal growth trough the soil. Giovanetti
and Avio (1985) found that additions of different
materials which increased the pore volume in soil had
a beneficial effect on mycorrhizal growth response,
colonization and spore numbers.
Acid and alkaline phosphatase activities responded
differently to the quality of the added organic matter,
though both amendments caused a dramatic increase
compared to unamended soil. Thus, the increased
phosphatase activity at acid pH, being the character-
istic optimum of several saprophytic fungi (Casida,
1959) and plant roots in the absence of microorgan-
isms (Tarafdar and Claassen, 1988; Tadano et al.,
1993) was highest when the readily degradable clover
leaves were added. With added straw higher acid
phosphatase activity was found closest to the roots.
Increased phosphatase activity in the rhizosphere is
commonly found (Helal and Sauerbeck, 1984;
Tarafdar and Jungk, 1987) though differences are
usually larger. The small differences in this experiment
were attributed to the coarse sectioning of rhizosphere
soil resulting in a dilution of an assumed high activity
extending l-2 mm from the roots (Tarafdar and
Jungk, 1987; Joner et al., 1995). Studies on the
influence of AM on extracellular acid phosphatase
have shown variable responses caused by AM both in
rhizosphere (Azcon et al., 1982; Dodd et al., 1987; Rao
and Tarafdar, 1993) and hyphosphere soil (Tarafdar
and Marschner, 1994; Joner et al., 1995). Such
variation could be related to the use of different
host-endophyte combinations. In our study acid
phosphatase activity was not altered by the presence
of AM hyphae, but the quantity exuded by the
mycorrhizal roots was reduced. Seemingly, AM fungi
regulate root phosphatase exudation as suggested by
Dodd et al. (1987).
Few studies of extracellular phosphatases of AM
plants have considered their activity in the alkaline
range. Alkaline phosphatase has been ascribed to soil
bacteria, as it is absent from the rhizosphere of plants
grown axenically (Tarafdar and Claassen, 1988). We
found that alkaline phosphatase activity in general
was highest in soil containing increased amounts of
organic C (straw), and lowest in soil without organic
matter additions. In the latter soil alkaline phospha-
tase activity was lowest when AM fungal hyphae were
1158 E. J. Joner and I. Jakobsen

present. In contrast, hyphae increased alkaline extracellular phosphatases are not produced by
phosphatase activity when clover was added, and here extraradical hyphae of the AM symbiosis. The
soil beyond the rhizosphere had the highest activity. influence of AM hyphae on phosphatase activity in
Increased alkaline phosphatase activity in the presence root-free soil seem to be indirect through interactions
of AM hyphae was also found by Tarafdar and with other microorganisms.
Marschner (1994) when Na-phytate was used as a P,
source, but in their experiment phosphatase activity Acknowledgements-We thank E. S. Jensenand M. Brink for
was highest in the rhizosphere. AM hyphae deplete soil help with soil C and N analyses, and U. Weinreich for skilful
of labile P, (Li et al., 1991; Joner et al., 1995) and we technical assistance. E. J. Joner received a visiting scientist
propose that the increased alkaline phosphatase grant from The Norwegian Research Council for the
duration of this investigation.
activity in clover-amended soil with AM hyphae could
have been caused by a starvation-induced increase
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