Growth, nutrition and response to water stress of Pinus pinaster

Tree Physiology
10, 153-167
0 1992 Heron Publishing-Victoria,
Canada
Growth, nutrition and responseto water stress of Pinus pinaster
inoculated with ten dikaryotic strains of Pisolithus sp.
MOHAMMED S. LAMHAMEDI,’
J. ANDRE FORTIN
’ Centre
Laval,
2 Forestry
3 Author
de Recherche
en Biologie
Sainte Foy, Quebec GlK
Canada,
to whom
P. 0. Box 3800,
reprint
requests
PIERRE Y. BERNIER2s3 and
Forestiere,
Fact&
7P4, Canada
Sainte
should
Foy, Quebec
de Foresterie
G6V
lG7,
et de Gtomatique,
Universite
Canada
be addressed
4 Institut de Recherche
en Biologie
Vege’tale,
Montreal,
Quebec HlX 2B2, Canada
Universite’
de Montreal,
4101
est, rue Sherbrooke,
Received February 25, 1991
Summary
Reconstituted dikaryons of Pisolithus
sp. (Pers.) Coker & Couch from South Africa influenced growth
parameters (shoot length, shoot/root ratio and leaf area), nutrition and physiological indicators (transpiration rate, stomatal conductance and xylem water potential) of maritime pine (Pinus pinaster Ait.)
seedlings during drought and recovery from drought. Seedlings colonized with certain dikaryons were
more sensitive to water stress and showed less mycorrhiza formation under water stress than seedlings
colonized with other dikaryons. Control (uninoculated) seedlings were significantly smaller than those
inoculated with dikaryons. Transpiration rate, stomata1 conductance and xylem water potential varied
among mycorrhizaf treatments during the water stress and recovery periods. After rewatering, the
controls and seedlings inoculated with dikaryon 34 x 20 had a weaker recovery in transpiration rate,
stomatal conductance and xylem water potential than the other treatments and appeared to have
experienced damage due to the water stress. Concentrations of various elements differed in the shoots of
Pinuspinaster
colonized by the various dikaryons. It is suggested that breeding of ectomyconhizal fungi
could constitute a new tool for improving reforestation success in arid and semi-arid zones.
Introduction
The rapid increase in the desertification of arid and semi-arid lands is a problem of
international scope (Ben Salem 1985, Nelson 1990). Restoration of affected areas by
tree planting is an immediate priority. However, trees grown in these regions exhibit
both high mortality and poor growth (Felker 1986).
Seedlings planted on dry sites must possess characteristics that confer tolerance to
water stress if they are to survive. Nursery management practices, including inoculation with ectomycorrhizal fungi, can improve seedling vigor after outplanting
(Duryea and Landis 1984, Duryea 1985). Ectomycorrhizal fungi influence the water,
nutritional and physiological processes of their host (Dixon et al. 1980, Dixon et al.
1983, Reid et al. 1983, Mitchell et al. 1984) and can confer a greater tolerance to
water stress on the host plant (Hardie and Leyton 198 1, Pigott 1982, Brownlee et al.
1983, Boyd et al. 1986, Read and Boyd 1986). For example, survival and early
154
LAMHAMEDI,
BERNIER
AND
FORTIN
growth of pine and oak seedlings on a range of site types are significantly improved
following inoculation with ectomycorrhiza-forming
Pisolithus sp. (Pers.) Coker &
Couch (Marx et al. 1977, Marx 1980, Dixon et al. 1983, Marx et al. 1985). The ability
of ectomycorrhizal seedlings to exploit available nutrients and water in the soil
depends greatly on the soil characteristics and on the extension of the extramatrical
phase of the fungal mycelium, which is often in the form of mycelial strands (Skinner
and Bowen 1974a, 19746, Finlay and Read 1986).
Ectomycorrhizal fungi differ in their ability to improve host nutrient uptake and
tolerance to water stress. Parke et al. (1983) showed that Rhizopogon vinicolor
conferred greater water stress resistance on Douglas-fir (Pseudotsuga menziesii
France.) seedlings than Laccaria laccatu, Pisolithus sp., or native fungus. The
selection of superior genotypes of ectomycorrhizal fungi may further enhance the
performance of inoculated seedlings subjected to unfavorable post-planting conditions.
There are substantial differences in the ability of monokaryotic and dikaryotic
strains of Pisolithus sp. to form mycorrhizae and mycelial strands, and in their effect
on the growth of maritime pine (Pinus pinaster Ait. ) (Lamhamedi et al. 1990,
Larnhamedi and Fortin 1991). Pisolithus sp. shows great potential for application to
reforestation programs on adverse sites (Marx and Cordell 1989). However, little is
known about the genetic variation underlying the ability of ectomycorrhizal fungi to
tolerate water stress. The objective of this study was to compare the influence of
different dikaryotic strains of Pisolithus sp. on the growth, nutrition, and ability of
Pinus pinaster seedling’s to tolerate water stress.
Materials
and methods
Monokaryotic cultures were obtained by germinating spores from a single
basidiomata of Pisolithus sp. from South Africa (Kope and Fortin 1990). The spores
were paired in all possible combinations on MNM agar medium. After incubation
for 3-4 weeks each plate was examined microscopically at the interface of the two
colonies for the presence of clamp connections. Number of nuclei was observed by
staining with 0.05% DAPI. Clamp connections and number of nuclei were taken as
evidence of the dikaryotic state. Dikaryotic cultures used in this research were the
same as those used in previous studies (Lamhamedi et al. 1990, Lamhamedi and
Fortin 1991). Characteristics of the cultures are presented in Table 1.
Plant inoculation and growth
The fungal inoculum of each dikaryotic strain included a vegetative mycelium grown
in a peat and vermiculite substrate (solid inoculum) and a mycelial suspension grown
in a mineral solution (liquid inoculum) prepared according to Gagnon et al. (1987,
1988).
Seeds of Pinus pinaster, obtained from an open-pollinated provenance in Morocco
RESPONSES OF PINE SEEDLINGS TO PISOLITHUS
STRAINS
15.5
Table 1. Characteristics of different reconstituted dikaryotic cultures of Pisolithus
sp.
Strain
Mycorrhizal
colonization’
(%I
Diameter of
mycelial
strands*
(Pm)
Mycelial
spread3
(cm* day-‘)
G14
(%)
3 x 28
37 x 34
34 x 25
2 x 36
8 x 28
11 x 15
34x20
Y x 22
27x34
17 x 20
64.5
32.5
67.9
61.6
89.9
32.5
41.2
66.0
59.1
79.9
40.3
23.7
49.7
63.9
123
33.2
-5
99.5
66.3
49.7
1.27
2.28
2.14
2.30
2.99
1.64
1.07
1.36
1.12
2.43
loo
52
64
100
0
0
92
66
’ Ectomycorrhizal colonization was studied using the growth pouch method (Lamhamedi et al. 1990).
’ Diameter of mycelial strands (n = 5) on MMN agar in the absence of the host plant after 5 weeks of
growth (Lamhamedi and Fortin 199 1).
’ Rates of mycelial spread (n = 5) of dikaryons determined axenically with their host plant on soil using
a large petri dish (150 x 15 mm).
’ GI is germination inhibition (%) of conidia of Truncatella
hartigii after 24 hours of incubation in
culture filtrates of dikaryons (Kope 1990).
5 Dikaryon did not produce mycelial strand.
(F. W. Schumacher Co, Inc., USA), were stratified in tap water at 4 “C for five days,
surface sterilized in 30% hydrogen peroxide for 15 min and rinsed with sterilized
distilled water. Seeds were germinated on a peat and vermiculite substrate (l/10 v/v)
saturated with mineral solution (Lamhamedi et al. 1990). Five weeks after germination, 1100 seedlings were transplanted to soil-filled plastic trays (80 x 15 x 15 cm),
ten seedlings per tray, and inoculated by placing 10 ml of each inoculum type in
direct contact with the roots of each seedling. Ten trays were used for each dikaryotic
strain. A single strain was used within each tray. Seedlings in 10 trays were left
uninoculated and served as controls of the inoculation treatment.
The soil used in the trays was collected from the C horizon of a podzolic soil, in
an area dominated by Abies bulsamea, north of Quebec City. Granulometric analysis
showed that it contained 83.8% sand, 15.8% loam and 0.4% clay. The soil was
fumigated with methyl bromide one month before planting (Gagnon et al. 1987).
Seedlings were grown in a greenhouse with a maximum irradiance of 150 W cm-*
at 400-700 nm and with a natural day length of about 8 h. Day/night temperature
and relative humidity were 22/18 “C and 58%, respectively. One month after
inoculation, seedlings were fertilized weekly by direct injection of nutrient solution
into the irrigation system, applied at a dilution of 1.25/l 27 (nutrient solution/water,
v/v). For each injection, the nutrient solution contained 73 g 1-l of (30/10/10, N,P,K)
and 20 g 1-l of KzS04. Seedlings were watered four times each week with tap water
until drainage was observed.
After seven months, while all seedlings were still actively growing, a water-stress
156
LAMHAMEDI,
BERNIER
AND
FORTIN
treatment was applied to half of the seedlings by withholding water for 19 days (Days
0 to 19), followed by daily irrigation to field capacity (Days 20 to 26). The remainder
of the seedlings were watered normally.
Measurements
Seedlings were sampled on 10 different days during both the water stress and the
drought recovery periods (Days 1,4,8,15,19,21,22,23,24
and 26). Around sunrise,
on each of these days, five seedlings were selected randomly from each fungal strain
and watering regime treatment. Stomata1 conductance and transpiration were measured on the selected seedlings with a Li-1600 (Li-Cor, Lincoln, Nebraska, USA)
steady state porometer and a conifer foliage cuvette. The shoots were then severed
and shoot xylem water potential was measured with a pressure chamber (P.M.S.
Instrument, Corvallis, OR) by the procedures outlined in Ritchie and Hinckley
(1975) and Joly (1985).
Growth parameters (shoot length, shoot/root dry weight ratio and leaf area) were
measured and mycorrhiza classes were established following plant harvest. Each
seedling was rated according to the degree of mycorrhiza root colonization (Class 1
= O-10%, Class 2 = > lo-25%, Class 3 = > 25-50%, Class 4 = > 50-75% and
Class 5 = > 75-100%) as described by Kropp and Fortin (1988).
Leaf area of each seedling was estimated by volume displacement of fresh
seedlings (Burdett 1979). A linear regression equation (y = 15.94x; 12 = 0.96) was
used to describe the relationship between displaced water volume and the surface
area of needles and twigs. This equation had previously been established on a
subsample of 19 seedlings. Projected surface areas were measured with a Leitz Tas
Plus images analysis (Ernst Leitz Ltd., Midland, Ontario, Canada).
Soil water content of each tray was determined gravimetrically for each sampling
day. These values were converted to soil water potential from soil water curves
(Figure 1) obtained by the pressure plate method (Richards 1949).
The needles and twigs of all sampled seedlings were dried at 65 “C for 48 h. Total
N of the oven-dried shoots was determined by the standard micro-Kjeldahl procedure, using a Kjeltec auto 1030 analyser (Tecator AB, Hoganas, Sweden). Phosphorus was determined by the molybdenum blue technique (Chapman and Pratt 1961),
and K, Ca, and Mg were measured by atomic absorption spectrophotometry.
Experimental design and data analysis
The experiment was analyzed as a split-block design with the two watering regimes
(stressed and non-stressed) as the main plots and the 11 strains (10 dikaryotic strains
and one uninoculated or control) as the sub-plots. There were also 10 sampling dates
and five trays for each watering regime for a total of 1100 seedlings.
Minimum significance differences (MSD) between means were determined with
the Waller-Duncan test as shown in Montgomery (1984). Logarithmic (x* =
ln(x + 1)) and power transformation (Y* = Ya) were used to achieve homogeneity of
variances of potassium concentrations and growth parameters, respectively. The
analyses of variance were performed using SAS software (SAS Institute, Cary, NC,
RESPONSES OF PINE SEEDLINGS TO PISOWTHUS
STRAINS
157
-0.1
-0.5
Figure 1. Soil water potential in sandy soil during and after the drought cycle. Arrow indicates day of
re-watering.
USA, 1988). Significance differences are reported at P = 0.05.
Results
Mycorrhizal colonization
All Pisofithus sp. cultures tested formed ectomycorrhizae under both watering
regimes. By Day 26, mycorrhiza classes ranged from 3.9 to 4.4 depending on the
dikaryotic culture and water regime (Table 2). Seedlings inoculated with dikaryon
11 x 15 showed the highest mycorrhiza formation when non-stressed, but colonization was reduced under water stress. Only dikaryotic culture 8 x 28 showed high
colonization regardless of the watering regime. Cultures 37 x 34, 17 x 20 and 34 x
20 were more sensitive to water stress and showed less colonization under water
stress than the other dikaryotic cultures.
Seedlings growth
Growth parameters were influenced by mycorrhizal treatment and watering regime.
At the end of the experiment, on Day 26, statistically significant differences
(P < 0.05) among mycorrhiza strains were observed in both watering regimes for
shoot length, root collar diameter, root dry weight and leaf area of seedlings
(Table 2). Non-stressed seedlings generally had greater growth than water-stressed
seedlings. The control seedlings were significantly smaller, both in height and in dry
matter, than most of the inoculated seedlings in the water-stress treatment. The tallest
seedlings in the water-stressed and non-stressed treatments were those inoculated
with dikaryotic cultures 2 x 36 and 34 x 20, respectively. Leaf areas were greater in
the inoculated seedlings than in the controls. Seedlings inoculated with dikaryotic
culture 34 x 20 exhibited the highest leaf areas in both watering regimes; these leaf
158
LAMHAMEDI,
BERNIER AND FORTIN
Table 2. Growth parameters and mycorrhizal colonization of water-stressed and non-stressed Pinus
pinaster seedlings inoculated with different dikaryotic strains of Pisolithus sp. on Day 26 at the end of
the recovery period. Different letters indicate a significant difference (P = 0.05) according to the
Wailer-Duncan K ratio t-test.
Treatment
Non-stressed
3x28
37x34
34x25
2x36
8x28
11x15
34x20
9x22
21x 34
17x20
Control
Water-stressed
3x28
37x34
34x25
2x36
8x28
11x15
34x20
9x22
27x34
17x20
Control
Shoot
length
Total DW
(mg)
Root DW
(mg)
8.9 c
9.0 abc
8.2 d
9.2 ab
8.9 bc
8.1~
9.3 a
8.3 d
8.9 bc
9.0 abc
6.4 e
110a
101 b
91.6 cde
50.3
48.8
46.8
38.5
38.5
43.4
44.4
42.5
46.8
47.8
44.4
cd
bc
cd
cd
cd
91.5 ab
96.6 a
79.4 d
82.3 cd
79.6d
72.2 e
43.5 cd
45.1 abc
41.9 cd
37.6 e
41.7 cd
41.62 ab
48.9 a
40.3 de
41.8 cd
43.9 bed
31.3 e
7.8 cd
7.7 cde
7.4 e
8.8 a
8.3 b
7.8 cd
7.7 cde
7.7 c
7.6 de
7.7 cde
6.0 f
97.8 cd
90.8 de
83.0 f
89.1 ef
101 b
87.9 ef
103 ab
98.4bc
85.3
85.7
83.5
80.6
80.0
a
a
abc
d
d
c
bc
c
abc
ab
bc
Shoot/root
ratio
Leaf area
(cd
Mycorrhiza’
class
1.17 bed
de
1.08 cde
1.35 a
1.14cde
1.05 de
1.25 ab
1.02e
1.20 abc
1.10 cde
1.05 de
27.73
28.8
28.7
24.2
25.68
33.95
24.0
29.6
25.7
23.6e
31.9 ab
cd
4.1 bed
3.9 d
4.2 abc
4.3 ab
4.2 abc
4.4 a
4.2 abc
4.0 cd
1.05
b
b
l.OOb
1.05 a
0.98 b
1.01 ab
1.01 ab
0.99 b
0.98 b
0.97 b
0.98 b
0.99
0.97
bc
c
e
de
a
e
bc
de
bed
b
21.7 bc
25.6 a
21.33
23.0
21.7 bc
25.0 a
26.4 a
20.8 cd
22.3 bc
19.9 d
l7.8e
4.1 bed
4.3 ab
1
4.2 ab
3.5 d
3.8 c
4.2 a
4.2 a
3.8 c
3.4 d
3.9bc
4.0 abc
3.8 c
1
1 Mean mycorrhiza classes were determined by visual assessment of short root colonization in percentage classes (1 = O-l O%, 2 = > 1O-25%, 3 = 225550%, 4 = >50-75%, and 5 = >75-100%) as described
by Kropp and Fortin (1988).
areas were 48 and 44% greater than those of the controls in the non-stressed and
water-stress treatments, respectively. Regardless of the watering regime, strain
2 x 36 had the highest shoot/root ratio, primarily because of a low root dry weight.
Drought tolerance and recovery from water stress
The effects of the dikaryotic cultures of Pisolithus sp. on physiological parameters
during and after recovery from water stress are shown in Figure 2. The decrease in
soil water potential between Days 1 and 19 in the water-stress treatments was
accompanied by pronounced decreases in transpiration, stomata1 conductance and
xylem water potential (Figures 2a-c). By comparison, transpiration, stomatal conductance and xylem water potential of non-stressed treatments were very stable
between Days 1 and 19 (Figures 3a-c).
Figure 2a shows large variation in transpiration rates among seedlings inoculated
RESPONSES OF PINE SEEDLINGS TO PISOLITHUS STRAINS
159
03
10
0
0
I
f
20
Days
30
Ic
*
.4 +=zc!Z-T
0
3x28
37x34
34x25
2x36
8x28
11x15
34x20
9x22
27x34
17x20
Y
10
20
Days
30
Figure 2. Transpiration rate (a), stomata1conductance (b) and predawn xylem water potential (c) of Pinus
pinaster seedlings inoculated with different dikaryons of Pisolithus sp. and of control seedlings(T) when
subjected to drought. Arrow indicates day of re-watering. Symbols as in Figure 2c.
160
LAMHAMEDI.
0
10
*---
0
20
Days
BERNDER AND FORTIN
30
a
----_
10
20
Days
30
Figure 3. Transpiration rate (a), stomata1 conductance (b) and xylem water potential (c)for non-stressed
treatments. Symbols as in Figure 2c.
RESPONSES
OF PME
SEEDLINGS
TO PISOLITHUS
STRAINS
161
with different strains, even on Day 1 of the water stress treatment. Strain 34 x 20 had
the highest transpiration throughout the drought period. The transpiration rate of
seedlings inoculated with 34 x 25 dropped sharply 4 days after water was withheld.
By Day 19, seedlings in all treatments had nearly stopped transpiring. However, in
the non-stressed treatments, seedlings inoculated with dikaryon 34 x 20 had a higher
transpiration rate than seedlings inoculated with the other strains (Figure 3a). The
lowest transpiration rates were measured in non-stressed seedlings inoculated with
dikaryon 17 x 20.
By the end of the drought recovery period, three statistically different groups were
observed (MSD = 4.21 g cm-* s-l). Seedlings inoculated with dikaryons 2 x 36,
27 x 34,9 x 22, 11 x 15 and those inoculated with 8 x 28,3 x 28,34 x 25, 17 x 20
and 37 x 34 exhibited high and medium rates of transpiration, respectively, but
seedlings inoculated with 34 x 20, along with the controls, had the lowest transpiration rates and showed signs of severe damage due to water stress.
Stomatal conductance also varied among mycorrhizal treatments during both the
water stress and recovery periods (Figure 2b). During the water-stress period,
inoculated seedlings showed a lower stomata1 conductance than control seedlings,
with the exceptions of 17 x 20 and 2 x 36 on Day 15, and 9 x 22 and 17 x 20 on
Day 1. Stomata of all water-stressed seedlings were apparently closed by Day 19. By
the end of the drought recovery period, controls and seedlings inoculated with
dikaryotic culture 34 x 20 had the lowest stomata1 conductance. Seedlings inoculated with dikaryons 8 x 28,34 x 25,37 x 34,3 x 28,9 x 22,27 x 34 and 17 x 20
had higher stomata1 conductances at the end of the recovery period than on Day 1 of
the drought cycle. In the non-stressed treatment, significant differences between
strains (P < 0.05) were initially observed in stomata1 conductance, but these differences had disappeared by the end of the experiment (Figure 3b).
All fungal treatments had similar xylem pressure potentials at the beginning of the
drought cycle (Figure 2~). As the soil dried, xylem water potential declined gradually
for both control and inoculated seedlings. At the end of the drying period, on Day 19,
a wide range of xylem water potentials was observed, with the control seedlings
showing intermediate values. After re-watering, all treated seedlings returned to
approximately their original values by Day 25, with the exception of the control and
the 34 x 20-inoculated seedlings. The non-stressed seedlings showed xylem water
potentials above -0.075 MPa throughout the experiment (Figure 3~).
The after-effects of water stress on transpiration, stomata1 conductance and xylem
water potential were more pronounced in the controls and in seedlings inoculated
with dikaryotic strain 34 x 20.
Shoot nutrient analysis
Elemental concentrations in shoots of Pinus pinaster varied according to dikaryons
and watering regime (Table 3). In the non-stressed treatments, nutrient concentrations were higher in seedlings inoculated with dikaryon 34 x 20 than in any other
seedlings. In the water-stress treatments, only control seedlings had concentrations
of N, K and Ca equal to or greater than those in the 34 x 20-inoculated seedlings.
162
LAMHAMEDI.
BERNIER AND FORTIN
Table 3. Concentrations of elements in water-stressed and non-stressed Pinus pinaster seedlings either
uninoculated (control) or inoculated with different dikaryotic cultures of Pisolithus
sp. at Day 26 at the
end of the recovery period. Different letters for each treatment indicate a significant difference (P = 0.05)
according to the Waller-Duncan K ratio t-test.
Treatment
N
(%I
P
(%I
K
(%I
Ca
C%)
Mg
(%)
5.26 b
4.71 bc
4.94 bc
4.87 bc
4.34 bc
5.08 bc
6.87 a
4.19 c
4.83 bc
4.60 bc
4.24 c
1.58 bc
1.53 bc
1.62 b
1.59 bc
1.43 cd
1.50 cd
1158 bc
1.37 d
1.48 bed
1.59 bc
2.15 a
0.86
0.84
0.93
0.84
0.76
0.90
0.95
0.86
0.87
0.76
0.74
ab
ab
a
ab
b
a
a
ab
ab
b
b
4.2 a
3.90 a
4.48 a
3.84 a
4.28 a
4.49 a
4.71 a
4.24 a
4.lOa
4.09 a
4.00 a
1.51 b
1.38 b
1.41 b
1.44 b
1.41 b
1.38 b
1.53 b
1.49 b
1.44 b
1.44 b
2.13 a
0.82
0.93
0.78
0.77
0.82
0.93
0.86
0.84
0.76
0.73
abc
Non-stressed
3 x 28
37x34
34x25
2 x 36
8 x 28
11x15
34 x 20
9 x 22
21 x 34
11x20
Control
0.78
0.73
0.71
0.76
0.77
0.80
0.92
0.72
0.70
0.73
0.71
ab
ab
b
ab
ab
ab
a
b
b
ab
b
0.95
0.91
0.92
0.87
0.89
0.97
0.99
0.84
0.85
0.86
0.74
ab
ab
ab
ab
ab
ab
a
ab
ab
ab
b
0.75
0.86
0.86
0.78
0.78
0.77
0.76
0.77
0.67
0.76
0.64
a
a
a
a
a
a
a
a
a
a
a
0.93 abc
0.89 bc
0.94 abc
0.93 abc
0.98 abc
1.04 ab
1.11 a
0.96 abc
0.88 bc
0.90 bc
0.79 c
Water-stressed
3 x 28
37 x 34
34 x 25
2 x 36
8 x 28
11x15
34 x 20
9 x 22
21 x 34
17 x 20
Control
-
a
bc
bc
abc
a
ab
abc
bc
c
Discussion
Inoculation of seedlings with Pisolithus sp. reduced the detrimental effects of
drought on leaf area, shoot length and shoot/root dry weight ratio. Similar effects of
Pisolithus sp. on Douglas-fir and white oak seedlings have been reported by Parke
et al. (1983) and Dixon et al. (1983). In addition, in this experiment, we have shown
that differences in growth, nutrition, and drought tolerance of Pinus pinaster seedlings result when different dikaryotic cultures of Pisolithus sp. are used (Tables 2,3;
Figures 2a-c, 3a-c). This agrees with recent results showing the high intraspecific
variability of monokaryotic and dikaryotic cultures of Laccariu bicolor in their
ability to form mycorrhizae and to promote the growth of the host plant (Kropp et al.
1987, Kropp and Fortin 1988, Kropp and Langlois 1990). They also extend earlier
in vitro observations on the intraspecific variability of Pisolithus sp. (Lamhamedi et
al. 1990).
It has been suggested that mycorrhizal associations can increase transpiration flux
and the ability to tolerate water stress in their host trees by increasing the absorbing
surface area of the roots and by decreasing resistance to water flow from soil to roots
RESPONSES OF PINE SEEDLINGS TO PISOLITHUS
STRAINS
163
(Reid 1979, Nelsen 1987). Seedlings inoculated with certain dikaryons exhibited
higher transpiration rates than the control seedlings with no large change in xylem
water potential before peak drying on Day 19 (Figures 2a and 2~). When water stress
became severe, only uninoculated seedlings (control) and seedlings inoculated with
34 x 20 wilted. Seedlings inoculated with strain 34 x 20 had the largest leaf area at
the onset of the drying treatment (Table 2). Strain 34 x 20 had a relatively low
capacity for the formation of mycelial strands and for mycelial spread (Table 1). All
other dikaryotic strains are capable of extensive mycelial spread and formation of
mycelial strands under controlled conditions (Lamhamedi and Fortin 1991).
The poor performance of seedlings inoculated with 34 x 20 suggests that genotypes of Pisolithus sp. differ in their ability to confer drought tolerance to Pinus
pinuster seedlings. It apppears that mycelial strands associated with the host root
system can transport physiologically significant quantities of water through the
soil-plant continum (Duddridge et al. 1980, Brownlee at al. 1983, Dixon et al. 1983,
Read 1984, Boyd et al. 1986, Read and Boyd 1986). Differences in conductance and
transpiration among the different dikaryons could therefore have resulted from
differences in mycelial spread or strand formation (Table l), or from differences in
the number of mycorrhizal entry points per unit of mycorrhizal root length (Read and
Boyd 1986, Wong et al. 1989).
In addition to a possible physical effect on water supply to the plants, dikaryons of
Pisolithus sp. could have affected stomatal functions through phytohormone concentrations. Xylem concentrations of abscisic acid and proline have been linked to
stomatal regulation in mycorrhizal and nonmycorrhizal plants (Levy and Krikun
1980, Johnson 1987, Coleman et al. 1990, Zhang and Davies 1990). Full stoma&l
function lagged several days behind the recovery of xylem water potential depending
on the dikaryotic culture (Figures 2b and 2c), possibly because of the presence of
residual abscisic acid in the vicinity of the guard cells (Johnson 1987). Others factors
such as stomata1 anatomy, temperature and the epidennal cells can also affect
stoma&l aperture (Spence 1987).
Seedlings inoculated with certain dikaryons (3 x 28, 34 x 20, 17 x 20, 9 x 22,
11 x 15,37 x 34,34 x 25) exhibited lower xylem water potentials than the controls,
especially when water availability was extremely low (cf. Sands and Theodorou
1978, Dixon et al. 1980 and Parke et al. 1983). Xylem water potentials lower than
those of the control seedlings could reflect a greater osmotic adjustment (Nguyen and
Lamant 1989). Recently, it has been shown that, in Douglas-fir seedlings,
Rhizopogon sp. and Hebeloma sp. caused decreased leaf osmotic potential compared
to controls, whereas Laccariu did not (Dosskey et al. 1990). Vesicular-arbuscular
mycorrhizae also enable plants to maintain leaf turgor and conductance to lower leaf
and soil water potentials than can nonmycorrhizal plants (AugC et al. 1986).
This study has shown that preselected dikaryons of Pisolithus sp. modified ecophysiological responses during and after an episode of low water availability and
improved the host plant’s response to these conditions. Others have reported that
differences in leaf water potential, transpiration rate, leaf resistance and the ability to
recover rapidly from water stress were due to differences in hydraulic conductivities
164
LAMHAMEDI,
BERNIER
AND
FORTIN
and plant nutrition (Nelsen and Safir 1982, Nelsen 1987). Soil water potential at the
soil-root interface and hydraulic conductance, in addition to physiological parameters, would have been helpful in evaluating and understanding the ecophysiological
responses of the host plant. From the data presented here, it is apparent that certain
dikaryons of Pisolithus sp. such as 3 x 28, 9 x 22, 27 x 34, 17 x 20, 37 x 34 and
8 x 28 can improve the tolerance of plants to drought conditions and aid their
recovery after watering. The results recorded here and in previous studies (Kope and
Fortin 1989,1990, Kope 1990, Lamhamedi et al. 1990, Lamhamedi andFortin 1991)
demonstrate that the variability within Pisofithus sp. could be used for breeding
improved mycorrhizal fungi with characteristics such as mycorrhiza formation,
production of mycelial strands, production of antibiotic substances and tolerance to
water stress. The encouraging results obtained on dry and harsh sites by using
Pisolithus sp. as an inoculum (Marx et al. 1977, Dixon et al. 1983, Marx et al. 1985,
Marx and Cordelll989) could be further improved by breeding the symbiont with a
view toward reforestation programs of arid and semi-arid zones.
Mycorrhizal infection also influences total plant dry mass. Lodgepole pine seedlings inoculated with Pisolithus sp. have shown significantly greater photosynthesis
and growth rates than nonmycorrhizal plants (Ekwebelam and Reid 1983). In
contrast, other research has shown interspecific variability in the photosynthesis of
Douglas-fir seedlings inoculated with different ectomycorrhizal fungi (Dosskey et
al. 1990). Although different dikaryons influence stomatal conductance (Figure 2b),
it is not clear whether this response is of a magnitude that will affect photosynthesis
and growth. No clear relation could be found between dry mass accumulation and
stomatal conductance of seedlings inoculated with the different dikaryons. Read et
al. (1983) suggested that ectomycorrhizal fungi influenced photosynthesis and
growth through source-sink effects. Differences in growth rates could be due to the
effect of different dikaryons on the partitioning of carbohydrates among organs. The
low total dry mass of seedlings inoculated with the vigorous dikaryon 8 x 28 (Tables
1 and 2) and other dikaryons could have been due to the high energy cost of this strain
(Fogel 1980). Bowen (1984) postulated that ectomycorrhizal plants compensate for
high energy costs by having greater photosynthesis per day.
The higher nutrient contents of seedlings inoculated with dikaryon 34 x 20 may be
attributed to the efficiency with which this strain exploits the soil surrounding the
roots. Dikaryon 34 x 20 produces tine and densely packed hyphae, whereas the other
dikaryotic cultures exploit the soil by forming relatively long but widely spaced
mycelial strands (Lamhamedi and Fortin 1991). Similar differences among different
isolates for nutrient uptake were reported by Mitchell et al. (1984). The increase in
explored soil volume by dikaryons having long mycelial strands could have significant effects on the uptake of poorly mobile nutrients. Under such conditions,
mycelial strand extension could compensate for decreased root growth during periods of drought. The lower calcium content of inoculated seedlings compared to
controls for both watering regimes (Table 3) may be the result of accumulation of
this nutrient in the hyphae in the form of polyphosphate granules (Stark 1972, Strullu
et al. 1983). Harley and Smith (1983) reported that ectomycorrhizal development
RESPONSES OF PINE SEEDLINGS TO PISOLJTHUS
STRAINS
165
increased the internal phosphate concentration of seedlings but reduced the excessive
intake of calcium.
Seedlings inoculated with certain dikaryons maintained physiological functions at
reduced tissue water potentials. Thus, the high mortality of non-inoculated Pinus
pinaster seedlings planted on most sites in Morocco (Ruehle et al. 1981) could be
improved by planting seedlings inoculated with preselected dikaryons.
Acknowledgments
We thank Dr. J. M. Girard and S. Boisclair for advice on experimental design and for statistical analyses.
We also thank Dr. A. Gonzalez and M. Gauthier (Forestry Canada, Quebec) for soil and plant analyses,
Dr. D. Perry and Pierre Davignon (Forestry Canada, Quebec) for technical guidance with the Leitz Tas
Plus image analysis and technical assistance, respectively. We are grateful to Dr. B. Kropp, D. Boyle and
M. Coyea for editorial comments. Support for this project was provided by NSERC through a grant to
Dr. J. A. Fortin. Additional support was provided by the Canadian International Development Agency.
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