Plant and Soil 121, 187-196 (1990).

~) Kluwer Academic" Publishers. Printed in the Netherlands'. PLSO 8243

Vesicular-arbuscular mycorrhizae in a wastewater-irrigated oldfield
ecosystem in Michigan*
G.R. SAFIR, J.O. SIQUEIRA ~ and T.M. BURTON
Department of Botany and Plant Pathology and the Department of Zoology, Michigan State University,
East Lansing, MI 48824, USA. IOn sabbatical leave.from ESAL, Lavras-MG, Brazil
Received 16 May 1989. Revised August 1989
Key words: Glomus, irrigation, mycorrhizal fungi, soil microorganisms, wastewater disposal
Abstract
The incidence of vesicular-arbuscular mycorrhizae (VAM) in wastewater irrigated and non-irrigated
oldfield soils in Michigan was studied. Soil and root samples were taken monthly from field plots on the
second and third years of consecutive irrigation with municipal wastewater at rates of 0, 5 and 10 cm wk J.
The oldfield ecosystem contained a high VAM fungal spore population density, but low species diversity.
The most common VAM fungal species were Glomus mosseae and G. fasciculatum. Both spore density and
root colonization were higher in irrigated than in non-irrigated plots. Irrigation effects were largest early in
the growing season. In addition to increasing VAM incidence, wastewater irrigation shifted VAM fungal
species composition. Irrigation favored G. mosseae over G. fasciculatum. Bioassays using either Sorghum
vulgate or Daucus carota, an oldfield native species, indicated that the VAM systems were still functioning
after the third year of consecutive wastewater irrigation. The data from experiments using nutrient solutions
at wastewater concentrations suggest that the effects of wastewater irrigation on VAM are due to the effects
of both water and nutrients. Since VAM are a very important component of the plant's water and nutrient
uptake system and equally important in structuring plant communities under limiting growth conditions, it
is suggested that the stimulatory effect of wastewater irrigation on VAM in an oldfield ecosystem enhances
the ecosystem's ability to function as a living filter for wastewater clean up.
Introduction
Soil and its vegetation act as a wastewater filter
and can provide a desirable alternative for disposal
of secondary treated wastewater (Bower and
Chaney, 1974). Application ofwastewater to either
natural or agricultural ecosystems often increases
plant biomass production (see overview by Brock-
way et al. and other individual papers in D'Itri,
1982). However, this increased biomass production
has to be balanced against possible contamination
of aquatic and groundwater systems, especially
with nitrate and chloride (Burton, 1978; Burton
and King, 1981), possible problems with increased
* Michigan Agricultural Experimental Station Journal Article
No 13137.
187
spread of human pathogens (Kowal et al., 1981;
Kristensen and Bonde, 1977; Shuval, 1977), and
possible deleterious effects on the plant com-
munities within natural ecosystems including
changes in species composition (Burton and Hook,
1982) and injury and vegetation decline if
wastewater is applied in excess (Burton, 1982).
Other adverse effects of wastewater application on
vegetation may result from over fertilization (Baier
and Fryer, 1973; Neary et al., 1975), increased
disease incidence (Epstein and Safir, 1982) and
depression of biological nitrogen fixation in
legumes (Tesar et al., 1982). Despite these potential
problems, reuse of wastewater is a widespread
phenomenon worldwide (Heaton, 1981) and offers
substantial advantages over discharge into aquatic
ecosystems, especially in low precipitation areas of
188 Safir et al.
the world. An area of concern to us that has been
largely overlooked is the potential impact of
wastewater nutrients, toxins, pathogens and anti-
microbial agents, such as chlorine, on the desirable
microbiological processes in the soil ecosystems. Of
particular interest is the impact on vesicular-
arbuscular mycorrhizae (VAM).
VAM are widely recognized as components of all
terrestrial ecosystems (Safir, 1987), and known to
be essential to above- (Allen and Allen, 1984) and
below-ground processes in oldfield ecosystems
(Crowell and Boerner, 1988). Since VAM for-
mation and function are sensitive to soil moisture
(Nelson, 1987) and fertility (Hayman, 1982), the
effect of wastewater irrigation on naturally occur-
ing VAM is of interest. In this paper, we report the
effects of three consecutive years of wastewater
irrigation on the incidence of VAM fungi and
VAM infectivity in an oldfield ecosystem in
Michigan.
Material and methods
Field studies
This study was conducted at the water quality
management facility at Michigan State University,
on an abandoned farm field site that had not been
cultivated for approximately 10 years. Site veg-
etation was domi nat ed by a mixture of quackgrass
(Agropyron repens) and goldenrod (Solidago
graminifolia and Solidago canadensis) with a
diverse mixture of other oldfield species. This field
was divided into 24 x 27 m plots that received 0, 5,
and 10 cm wk- 1 of chlorinated municipal
Table 1. Mean annual concentration (mg L -~) of wastewater
applied to the oldfield site (from Hook and Burton, 1978)
Constituent Second year Third year
Nitrate-nitrogen 10.5 9.1
Ammonium-nitrogen 1.4 1.2
Total nitrogen 13.7 13.6
Total phosphorus 2.7 2.7
Potassium 10.0 10.5
Calcium 69.9 57.9
Magnesium 24.4 25.6
Sodium 86.5 97.1
Chloride I 12.0 120.0
pH = 7.5; alkalinity - equivalent CaCO 3 = 150.
Table 2. Average soil analyses/~g g dry soil ~, for the top 30 cm
of soil of the oldfield site after two years of wastewater irrigation
(after Hook and Burton, 1978)
Constituent 0 cm wk -t 5 cm wk -~ 10 cm wk -~
Phosphorus (Bray) 18.1 11.0 17.5
Potassium (NH4OAc) 135.8 75.1 73.6
Calcium (NH4OAc) 806.8 932.4 944.8
Magnesium (NH4OAc) 89.3 130.6 160.4
Sodium (NH4OAc) 27.9 87.0 99.7
Chloride (K2 SO4) 9.6 32.2 32.7
NO 3-nitrogen 1.3 1.9 2.3
(Kjeldahl)
wastewater irrigation by spray. The wastewater
originated from the East Lansing, Michigan sewage
treatment facility from the first of four lakes used to
store this wastewater prior to irrigation (see Table'
1 for chemical analysis of this wastewater). Spray
irrigation continued from April to Oct ober each
year.
Soil chemistry for these plots determined after
the first and second years of irrigation, indicated
that wastewater irrigation increased calcium, mag-
nesium, sodium, and chloride in these soils (Table
2). Mass balance budgets for the site also indicated
that substantial amount s of N and P were being
retained by the vegetation and soils (Burton and
Hook, 1982; Hook and Burton, 1978).
Precipitation averaged 77cm year -~ with an
average snowfall of 124cm year -~. Temperat ure
averaged 8.2C with a mean monthly low of
- 5. 5 C in January and a mean monthly high of
27.6C in July. The average growing season in this
part of Michigan extends from May 7 to October 8
(154 days).
To evaluate VAM incidence, 20 soil and root
samples were removed mont hl y from May to
October, for a two year period from each of six
randomly assigned 24 x 27 m plots that received
0, 5 or 10 cm wk- a of wastewater. Wastewater ir-
rigation had been applied in the year before sam-
pling and was applied at the same rate during the
two years of sampling. They are referred to as the
second and third years of consecutive wastewater
irrigation. Plots were not harvested during the sam-
pling period. Samples were taken to a depth of
25cm and 5cm sections were separated and
analyzed for VAM spore numbers. Fifteen gram
samples from each 5 cm section were thoroughly
mixed in 100 mL of distilled water and wet-sieved
through 40 and 325-mesh screens (Gerdemann and
VA mycorrhizae in a waste-water irrigated oldfield 189
Nicolson, 1963) and the spores separated by
sucrose (density = 1.18gcm 3) centrifugation.
Spores were grouped by appearance and counted
under a dissecting microscope. These groups were
later classified to the species level as described in
Gerdemann and Trappe (1974) and Trappe (1977).
In the third year of consecutive application, root
samples were taken from 20 plants, separated by
species, in three 2 2m random sections from
each plot. Quackgrass (Agropyron repens) the
predominant species, was sampled on a monthly
basis, while other species were sampled only in
September. Roots were separated from soil,
washed, cleared, and stained according to Phillips
and Hayman (1970). For colonization assessment
stained segments were mounted on microscope
slides, scanned for VAM fungal structures, and
rated as 3, 2 and 1 according to the intensity of
fungal structures in the root as follows. Rating 3
when fungal structures were present in at least
130 mm/200 mm of root; 2 if they were present in
60-129mm/200mm and 1 if present in 1-59mm/
200 mm of root. The data were subjected to a one
way ANOVA and the means for each sampling
time separated by the Student-Newman-Keuls'
multiple range test at the 0.05 level.
Greenhouse experiments
To evaluate the effects of wastewater application
on VAM infectivity and effectiveness of oldfield
ecosystems, VAM formation potential and effects
on plant growth were assessed using sorghum
(Sorghum vulgare) (routinely used for infection
studies) and Queen Anne's lace (Daucus carota), a
mycorrhizal oldfield species, as test plants. Both of
these species are easily produced from seeds.
Quackgrass was not used for these studies because
of the difficulty in obtaining uniform plant material
for propagation. The first experiment was conduc-
ted for 10 weeks in waxed cups containing 800 g of
soil collected from each irrigation treatment (0, 5
and 10cmwk-I), by the end of the third consecu-
tive year of irrigation. In addition soil from the
highest irrigation level (10cmwk -t) and a sandy
loam soil (with pH = 7.7 and 8 ppm of P) were
autoclaved and included as treatments. Each treat-
ment had 8 replications. In the second experiment,
sorghum remained untreated or was inoculated
with VAM fungi and planted in either 10 cm wk -I
irrigated or greenhouse autoclaved soil. Highly in-
fective VAM fungus inoculum (a mixture of
Glomus mosseae and Glomus fasciculatum) originat-
ing from sudangrass greenhouse pot cultures, was
applied at a rate of 200 g of soil inoculum per 800 g
of either field or greenhouse soil. Each treatment
had 6 replications and the experiment was conduc-
ted for 10 weeks. A third set of experiments was
conducted using the same treatments as the second
experiment, except that the oldfield native plant
Queen Anne's lace (D. carota) was used as a test
plant. At the end of each experiment plants were
harvested and shoot dry weights were determined.
Roots were separated from the soil for assessment
of VAM colonization as previously described.
In additional greenhouse experiments, nutrient
solutions were adjusted to simulate wastewater (ac-
cording to Table 1) using the following salts:
(NH4)2NO3, KNO3, NaH2PO4, NaC1, CaC12
2H20, Ca(NO3)' 4H20, MgSO4" 7H20, CaSO4,
CaCO3, MnSO4.4H2 and FeSOa' 7H20. Pre-
germinated seeds of D. carota were planted in
waxed cups containing 800 g of oldfield soil, to
which the following treatments were applied: 1)
water-stressed control (100mL of distilled water);
2) water irrigated (190mL of distilled water); 3)
simulated wastewater (100 mL of nutrient solution)
and; 4) water and nutrient (190mL of nutrient
solution). These treatments were applied in the pres-
ence and absence of VAM inoculum (G. mosseae
and G. fasciculatum) as previously described.
Simulation treatments had 8 replications and were
applied 5 times per week to approximate the 5 cm
irrigation rates. After 12 weeks growth, plants were
harvested and dry weight and root colonization
assessed as previously described. Every experiment
was repeated once. All the data were subjected to
statistical analysis and significant effects separated
by the Student Newman-Keuls multiple range test
at the .05 probability level.
Results
Incidence of VAM
The oldfield site from Michigan had spore
population densities as high as 25 spores/g soil
(Table 3, 4). The predominant fungal species
190 Safir et al.
Table 3. Effect of wastewater irrigation on soil spore density for the three predominant VAM fungal species in a oldfield ecosystem
in Michigan. Data (number of spores/15 g soil) for the second consecutive year of irrigation in the 0- - 10c m top soil layer
Sampling G. fasciculatum G. mosseae G. constrictum
time
0 5 cm wk -~ 10 cm wk ~ 0 5 cm wk -~ 10 cm wk -t 0 5 cm wk -~ 10 cm wk -~
May 109 be 331 a 392 a 41 cd 154 b 111 b 29 d 84 c 57 cd
June 134 b 203 ab 282 a 49 cd 188 ab 170 ab 47 c 22 c 38 c
July 167 b 161 b 417 a 53 cd 127 b 411 a 43 b 21b 54 b
August 132 c 169 b 163 b 60 cd 170 b 282 a 40 cd 47 cd 50 cd
September 98 cde 160 bcd 207 b 105 ode 270 b 353 a 56 de 32 de 58 de
October 107 c 151 b 164 b 66 cd 223 a 281 a 57 cd 57 cd 55 cd
Means in the same line (month) followed by the same letters are not statistically different by the Student-Newman-Keuls' multiple range
test at .05 level.
Table 4. Effect of wastewater irrigation on soil spore density for the three predominant VAM fungal species in an oldfield ecosystem
in Michigan. Data (number of spores/15 g soil) for the third year of consecutive irrigation in the 0- - 10 cm top soil layer
Sampling G. fasciculatum G. mosseae G. constrictum
time
0 5 cm wk t 10 cm wk -t 0 5 cm wk -~ 10 cm wk -~ 0 5 cm wk -~ i0 cm wk t
May 31 b 33 b 18 c 22 c 81 a 28 b 2 d 5 d 10 d
June 13c 44a 23b 7c 75a 29b 4c 4c 7c
Jyly 8 c 19 b 15 b 8 c 35 a 23 a 6 c 2 c 4 c
August 17 c 34 a 20 bc 14 c 38 a 17 c 6 c 18 c 7 d
September 19 c 30 b 20 d 22 cd 60 a 28 bc 6 f 16 de 11 e
October 11 c 28 a 20 a 10 c 87 a 23 a 2 d 12 b 6 c
Means in the same line (month) followed by the same letters are not statistically different by the Student-Newman-Keuls' multiple range
test at .05 level.
recovered were identified as Glomus mosseae,
Glomus fasciculatum and Glomus constrictum. A
fourth species Sclerocystis rubiformis was found in
low numbers and for this reason was not con-
sidered further. Spore numbers were higher in the
second (Table 3) than in the third year of consecu-
tive wastewater irrigation (Table 4), even in non-
irrigated plots.
In the second year of consecutive irrigation
(Table 3), the numbers of G. mosseae and G.
fasciculatum spores were higher in irrigated than in
non-irrigated plots. G. constrictum was only sig-
nificantly affected by irrigation in May of the
second year. In non-irrigated plots, G. fasciculatum
had higher spore numbers in June and July, while
in irrigated plots, at both 5 and 10 cm wk-t , spore
numbers were higher earlier in the growing season
and decreased toward the end of the season. G.
mosseae did not change throughout the season in
non-irrigated plots, but increased late in the season
in irrigated plots. G. constrictum had a lower spore
density than the other VAM fungus species and
was less affected by either sampling time or irri-
gation treatment.
In the third year of consecutive irrigation (Table
4), 5 cm wk- ~ plots had more of G. mosseae and G.
fasciculatum spores than the 10cmwk -~ plots at
most sampling times. G. constrictum spore numbers
were significantly different in August and Septem-
ber when 5 cmwk -~ plots had more spores than
either non-irrigated or 10 cm wk-~ treatments.
Despite the monthly variation, irrigated plots
had higher numbers of G. mosseae and G.
fasciculatum population densities than non-
irrigated ones for both G. mosseae and G.
fasciculatum during both sampling years but to a
lesser extent in the third year (Fig. 1). G. constric-
turn, however responded very little to irrigation.
Spore numbers were also affected by soil depth.
Spores were concentrated in the upper 15 cm of soil
and their distribution in the soil profile was dif-
ferentially affected by irrigation and sampling time
(Figure 2). In May, irrigated plots had an average
of 4.6 times more spores than non-irrigated ones in
VA mycorrhizae in a waste-water irrigated oldfield 191
400
0 G, fasci cul atum
"6 aoo
0' )
t O
}
iI~ 2OO
.1o
g
0 5 10
G. mos s eae
0 5 10
I--] Second year
[ ] Third year
G. consrri ctum
o 5 l O
I r r i gat i on Tr eat ment s, cm/ wk
Fig. 1. Overall effects of wastewater irrigation levels on the spore
densities of G. fasciculatum, G. mosseae and G. constrictum in an
oldfield soil duri ng the second and third years of consecutive
irrigation.
5.0
o
t~ 4. 0
I f :
"E
~ 3.0
Z
ca
~ 2.0
September
,~/ , h , L ' 115 ' 0 i , 0
5 10 2 25
Soi l Dept h, cm
Fig. 2. Effect of wastewater irrigation on VAM spore density at
increasing soil dept h at the begi nni ng and at the end of the
growing season duri ng the second year of consecutive
wastewater application. Each data point represents the ratio of
mean spore density for irrigated (5 and 10cmwk -t plots) over
non-irrigated plots.
the 0-5 cm soil layer. This ratio dropped to less
than 2.0 at higher depth (20-25 cm). By September
this trend reversed, i.e. irrigation favored fungal
spore production at deeper layers in the profile.
In addition to effects on total spore number,
irrigation altered VAM fungal community struc-
ture in the oldfield soil (Fig. 3). The calculated ratio
between numbers of G. mosseae/G, fasciculatum
spores indicated very little effect of sampling time
O
=.
t 2
81
c
May
a, irrigated - 0 - Non-irrigated
qr i ..Q- -- I-- O- -- O-- --8
Second Year Third Year
I i I I J L I I I I I
Jul Sept May Jul Sept
Sampl i ng Ti me
Fig. 3. Rat i o of mean spore numbers for G. mosseae (MOS)
versus G.fasciculatum (FAS) in irrigated and non-irrigated plots
t hroughout the growing season in the second and third year of
consecutive wastewater irrigation.
in non-irrigated plots, but a considerable effect in
irrigated plots. In the second year of wastewater
application, the G. mosseae/G, fasciculatum spore
number ratio increased from 0.36 early in the
season (May) to 2.0-2.5, late in the season (Septem-
ber and October). In the third year, however, this
ratio was not as high as was found in the second
year, but was much less affected by the sampling
time.
Since plant species composition differs in ir-
rigated and non-irrigated plots, individual species
can not be compared, except for quackgrass, which
3.0
ca
t-
e,-
i-
._o
C
2
O
tJ
2.5
2. 0
1. 5
1. 0
o. of
A I r r i gat ed ,~
- - - 0- - - Non- i r r i gat ed / /
/ /
/ / /
I I
May June July
Sampl i ng Ti me
Au;ust I
September
Fig. 4. VAM root colonization rating of quackgrass (Agropyron
repens) after the third year of consecutive wastewater irrigation
and from non-irrigated plots in an oldfield ecosystem duri ng the
growing season.
192 Safir et al.
was present in both situations. The overall species
root colonization means for non-irrigated and ir-
rigated plots were 1.6 + 0.4 and 2.5 +__6, respec-
tively. The monthly colonization rating, for quack-
grass from non-irrigated and irrigated plots are
given in Figure 4. Irrigated plots were more heavily
colonized from May to July, but no differences
were evident late in the growing season. Although
non-irrigated plots reached the same colonization
levels as irrigated ones late in the season, higher
VAM formation earlier in the season may be of
great advantage for plant nutrient uptake and
growth.
Effects on VAM formation and function
The greenhouse assays using sorghum
demonstrated that three years of consecutive
wastewater irrigation had no significant effect on
either VAM formation or plant growth (Table 5).
However, when 10cmwk -~ irrigated soil was
autoclaved to eliminate VAM propagules, plant
growth was significantly reduced. Re-infestation of
the same soil with mixed VAM inoculum, restored
its infectivity and greatly increased sorghum and D.
carota growth. This suggests that wastewater ir-
rigation for three consecutive years at rates as high
as 10cmwk -~ had no adverse effects on VAM
formation and function.
Simulation experiments using D. carota showed
significant plant growth responses (Figure 5). Root
colonization was at the same level as those of D.
carota inoculated plants in Experiment 3 (Table 5)
and was not affected by any of the treatments. Plant
dry weight was lower when nutrients alone (NUT)
were applied in the absence of VAM, but was im-
proved by joint application of nutrients and water
(DW + NUT). VAM fungus inoculation im-
proved plant growth in comparison to non-mycorr-
hizal plants in every treatment. Its effects were
more pronounced when either distilled water
(DW), nutrients (NUT) or both (DW + NUT)
were applied. Plant growth in these treatments was
equally high and significantly greater than every
non-inoculated treatment. Simulated wastewater
(DW + NUT) improved plant growth in com-
parison to the control (CON) plants whether they
Tabl e 5. Infectivity and pl ant growt h pr omot i on of wastewater-irrigated and non-irrigated oldfield soils from Mi chi gan under
greenhouse conditions
Soil origin Treat ment Pl ant dry wt Root
colonization
Experi ment t - sorghum -
Non-irrigated field soil
5 cm wk -~ irrigated field soil
10 cm wk -~ irrigated field soil
10 cm wk- t irrigated field soil
Greenhouse VAM conductive soil
Experi ment 2- sorghum -
10 cm wk -t irrigated field soil
10 cm wk -1 irrigated field soil
Greenhouse soil
Greenhouse soil
Experi ment 3 - Daucus carot a -
10 cm wk -I irrigated field soil
10 cm wk- i irrigated field soil
10 cm wk -1 irrigated field soil
Greenhouse
None 7.0 a b 1.6 a b
None 6.6 a 1.2 a
None 6.5 a 1.7 a
Aut ocl aved 4.0 b 0.0 b
Aut ocl aved 3.2 b 0.0 b
Aut ocl aved 5.1 b 0.0 b
VAM inoculated a 10.5 a 1.8 a
Aut ocl aved 5.7 b 0.0 b
VAM inoculated a 11.9 a 2.0 a
None 0.87 b 1.5 a
Aut ocl aved 0.45 c 0.0 c
Aut ocl aved and 1.33 a 1.0 b
VAM inoculated a
Aut ocl aved and 1.05 b 1.0 b
VAM inoculated"
a Inoculated with highly infective soil i nocul um f r om pot culture cont ai ni ng a mi xt ure of Gl omus mosseae and Gl omusf asci cul at um.
b Means followed by t he same letters are not statistically different within experi ment s by the St udent -Keul s' multiple range test at .05
level.
5
I j Non-mycorrhizal
~ Mycorrhizal
4
a
iiii~iii~iiii~il
b ~ ~ !
1
c
d }
i ............
i
0 ...............
CON DW
O)
J
i
E'
l
"E 2
a
i , ! . . . .
i~;~ ~i~i;:
!,iiiiiiiiiii~i~i~'i~i ,~'
NUT
i : : /
~i~ i: ~ I
DW + NUT
Fig. 5. Growth of mycorrhizal and non-mycorrhizal Daucus
carota in a simulated wastewater experiment. CON = control
water stressed, DW = distilled water only, NUT = nutrients
only and DW + NUT = distilled water + nutrients. Means
with similar letters do not differ by the Student-Newman-Keuls'
test at the .05 level.
were mycorrhizal or not. However, if they were
mycorrhizal, biomass product i on was much
greater.
Discussion
As reported for other non-agricultural ecosys-
tems (Read et al., 1976; Sparling and Tinker, 1978)
VAM fungi occur quite abundantly in oldfields in
Michigan. The two predomi nant GIomus species,
G. mosseae and G. f asci cul at um, found in our
systems are also commonl y found in Michigan cul-
tivated soils (Wacker, 1988) and in the grassland/
shrubland of Nort h America and other parts of the
world (Miller, 1987). Also, in asparagus fields in
Michigan, the relative density of G. mosseae
decreases with field age, while G. f asci eul at um is
more frequent in older fields (Wacker, 1988). These
community shifts can result from the action of
selective factors such as moisture (Anderson et al.,
1983; Dickman et al., 1983) soil chemical charac-
teristics (Schenk and Siqueira, 1987) and al-
VA mycorrhi zae in a wast e-wat er irrigated oldfield 193
lelopathic compounds (Wacker, 1988) that
accumulate over time. Since this study was
concluded, G. f asci eul at um as previously described
(Gerdemann and Trappe, 1974), has been shown to
include several additional distinct taxa (Walker and
Koske, 1987). This suggests that additional
taxonomic studies may increase the number of
species reported herein.
The beneficial effects of VAM on plant-water
relationships is well known (Safir et al., 1971) and
has been the subject of many recent publications as
reviewed by Nelson (1987). Nevertheless, the influ-
ence of soil moisture on VAM development and
function has not attracted the same attention. The
results presented here show that root colonization
and spore numbers in the soil increased in response
to wastewater irrigation. Wastewater application
also resulted in increased plant biomass in these
sites (Burton and Hook, 1982). Water shortage in
the soil can reduce and delay VAM fungal spore
germination (Tommerup, 1984) root growth (Reid
and Bowen, 1979), and subsequent VAM for-
mation (Paula and Siqueira, 1987; Reid and
Bowen, 1979). Because colonization progresses to a
maximum t hroughout the season even under limit-
ing conditions (Hayman, 1970; Sparling and
Tinker, 1978), no effect of irrigation was found in
our study late in the growing season as was found
early in the season.
Soil moisture is known to affect VAM fungal
sporulation under controlled environments (Paula
and Siqueira, 1987) and to be a domi nant factor in
spore abundance in natural ecosystems (Miller,
1987). Soil moisture, however, may have a greater
effect on root growth than on the sporulation
process (Paula and Siqueira, 1987). Therefore, it is
possible that the higher spore numbers in our
wastewater irrigated plots are simply a result of an
increased amount of root growth (Burton and
Hook, 1982) and VAM fungal root colonization.
Wastewater application also affected spore
distribution in the soil profile. Similar effects have
been reported for plain water irrigation of Citrus
rootstock (Levi et al., 1983). However, in our study,
this effect varied during the growing season in that
it increased spore density at lower depths late in the
season. Increasing soil infectivity of lower soil
layers may be of ecological significance.
In addition to increasing VAM spore numbers,
wastewater irrigation resulted in a change in spore
194 Safir et al.
numbers for the predominate species. Irrigation
favored G. mosseae over G. f asci cul at um. After
September of the second year of consecutive irri-
gation the relative density of G. mosseae was twice
as high as that of G. f asci cul at um. This trend con-
tinued during the third year, however, the numbers
of spores were reduced. Population shifts between
these two fungi have been reported to occur in
other systems (Visser et al., 1984; Wacker, 1988),
and is probably due to changes in the edaphic
environment. Hayman and Mosse (1979) reported
that lime and phosphate amendments induced a
greater reduction in the population of indigenous
fungi than of introduced G. mosseae and G.
f asci cul at um in grassland soils in Wales. VAM
fungi do not exhibit habitat specificity, but may
have a narrow range of tolerance to environmental
conditions. G. mosseae is well known for its prefer-
ence for neutral to alkaline soils (Siqueira et al.,
1984), while G. f asci cul at um seems to tolerate a
broader pH range. In fact, either lime or Ca 2
application has been shown to increase root
colonization by G. mosseae (Hepper and O'shea,
1984; Siqueira et al., 1984). Wastewater has a high
pH and base content and its application to the
oldfield ecosystem increases Ca 2 and Mg 2 levels
in the soil (Table 2), thus favoring G. mosseae over
G. f asci cul at um.
Greenhouse experiments indicated that both ir-
rigated and non-irrigated field soils were very infec-
tive and had an effective VAM native population,
because plant growth dropped significantly when
VAM was eliminated by autoclaving soil. Growth
promotion effects were restored by the reinfestation
of autoclaved soil with a mixed inoculum of G.
mosseae and G. f asci cul at um. This suggests that
after three years of consecutive wastewater irri-
gation no anti-VAM fungal factor had built up in
the soil and that the native VAM fungi were func-
tioning. It also suggests that application of
chlorinated wastewater by spray irrigation does not
lead to reduction in VAM populations, due to
chlorination effects. Although D. carota is not the
predominant species in our oldfield irrigated plots,
the fact that it showed a high degree of mycorrhizal
dependency indicates the importance of VAM in
this system. The experiment with simulated
wastewater suggests that the wastewater irrigation
effects on VAM formation as reported here, and
biomass production (Burton and Hook, 1982) are
due to both nutrients and the water supply. The
3.5-fold increase in D. carota growth due to
inoculation with G. rnosseae and G. f asci cul at um
when water and nutrients were applied (Fig. 5), is
evidence of the importance of VAM in oldfield
irrigated ecosystems.
Urban and industrial disposal on land has been
of major concern among environmentalists because
of their potential to harm the ecosystem. For in-
stance, the percent of VAM colonization in barley
was reduced by 6-fold due to land sludge appli-
cation (Boyle and Paul, 1988). Because formation
and function of VAM are greatly affected by soil
nutrient status, especially P and N availability
(Hayman, 1982), and because considerable
amounts of these nutrients were applied in the
wastewater, between 150-270 kg ha- ~ yr- 1 of N and
30-70kgha -l yr -l of P (Burton and Hook, 1982),
it was expected that wastewater irrigation would
reduce VAM in the oldfield ecosystem. Instead, it
favored root colonization and increased VAM
spore population densities in the top soil layer and
also deeper in the soil profile. Such effects were also
found in a 50 year old beech-maple stand in north-
western Michigan with effluent irrigation up to
7.6 cm wk-~ (Otto, 1980). Below-ground activity, is
of importance to any system acting potentially as a
living filter for wastewater disposal. Since VAM are
crucial components of such a system in terms of its
nutrient and water uptake capacity, and equally
important in structuring plant communities under
limiting conditions for adequate growth (Allen and
Allen, 1984), the stimulatory action of wastewater
on VAM in oldfield ecosystems may enhance the
efficiency of these sites as living filters for
wastewater clean up. This suggests a need for ad-
ditional long term studies.
Acknowledgments
Here we would like to thank Ms Barbara Car-
penter for technical assistance on this project, and
CNPq-Brazil for the scholarship to J O S.
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