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Water-retentive polymers increase nodulation
of actinorhizal plants inoculated with Frankia
Article in Plant and Soil · August 1999
DOI: 10.1023/A:1004634804354
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Plant and Soil 214: 105–115, 1999.
© 1999 Kluwer Academic Publishers. Printed in the Netherlands.
105
Water-retentive polymers increase nodulation of actinorhizal plants
inoculated with Frankia
Steven J. Kohls1, Dwight D. Baker2 , Douglas A. Kremer3 and Jeffrey O. Dawson1,∗
1 Department of Natural Resources and Environmental Sciences, University
of Illinois, W-503 Turner Hall, 1102 S.
Goodwin, Urbana, Illinois 61801, USA; 2 New Chemical Entities, Inc., 11804 North Creek Parkway South, Bothell,
Washington 98011-8805, USA and 3 MiTech Research and Development Inc, 3522 Labore Rd., St. Paul, Minnesota
55101, USA
Key words: Actinorhiza, Alnus, Casuarina, Frankia, Nodulation, Water-retentive polymers
Abstract
Actinorhizal plants form a nodular, nitrogen-fixing root symbiosis with the actinomycete Frankia and are economically and ecologically important due to their ability to improve the nitrogen fertility of disturbed and infertile
substrates. In this study, water-retentive polymer inoculum carriers were applied as a root dip. This treatment
significantly increased nodulation and in some cases early growth of Alnus glutinosa (L.) Gaertn. and Casuarina
equisetifolia var. equisetifolia Forst. & Forst. in a controlled environment and also of A. glutinosa under field
conditions. Nodule number and nodule dry weight per plant were at least two to three times greater after 56 to 140
days for plants inoculated with Frankia carried in a water-retentive polymer base compared with plants inoculated
with Frankia in water. Nodules on the roots of the plants that were inoculated with Frankia in a polymer slurry were
distributed throughout the entire root system, rather than concentrated near the root collar. When amended with
water-retentive polymers, actinorhizal plants inoculated with 5- to 10-fold lower titers of Frankia exhibited early
growth and nodule numbers equal to or greater than those plants inoculated with standard titers without polymers.
The water-retentive, superabsorbent polymers clearly increased the nodulation of two actinorhizal plant species.
Introduction
Actinorhizal plants are unique in their ability to establish root nodules with the nitrogen-fixing actinomycete Frankia, a widespread soil actinomycete that is
symbiotic with at least 8 families, 25 genera and over
200 species of primarily woody angiospermous plants
(Baker, 1988; Baker and Schwintzer, 1990; Moiroud
and Gianinazzi-Pearson, 1984; Torrey, 1988). A number of actinorhizal plants are noted for their ability
to fix ecologically significant quantities of nitrogen
(Bormann and Gordon, 1989).
The infectious population of Frankia, or of an effective Frankia microsymbiont, is often low or absent
in certain soils (Dawson et al., 1989; Houwers and
Akkermans, 1981; Kohls et al., 1994; Smolander and
Sundman, 1987; Visser et al., 1990). Thus, inoculation with a desired microsymbiont may be necessary
∗ FAX No: +1 217 244 3219. E-mail: jdawson2@uiuc.edu
to insure or to optimize the nitrogen fixing capabilities
of these species. Inoculation methods have included
crushed nodule suspensions, substrate injection with
the microsymbiont, large scale spraying of cultured
Frankia, seed inoculation, rhizospheric soil inoculation, dry inoculum, alginate beads, peat carriers or,
most frequently, aqueous suspensions of Frankia at
0.01–0.05 µL packed cell volume (PCV) per plant
(Akkermans and Howers, 1979; Berry and Torrey,
1985; Burleigh and Dawson, 1994; Diem et al., 1988;
Martin et al., 1991; Perinet et al., 1985; Stowers and
Smith, 1985; Thomas, 1986; Wheeler et al., 1991).
Carriers for Rhizobium and mycorrhizal organisms
are known to improve the degree of infection and to
reduce competition from indigenous strains of the microorganism for inoculated woody host plants (Torrey,
1988). However less is known about methods to improve nodulation and growth or to reduce microbial
competition in the inoculation of actinorhizal plants
(Benoit and Berry, 1990). The autecology of Frankia
106
and many of the factors influencing infection by
the microsymbiont remain poorly understood (Baker,
1988). In contrast to Rhizobium and their leguminous
host species, commercial inoculation methods using
Frankia and its host plants have yet to be fully developed or tested, though some research has been done
on methods for developing economic techniques for
large-scale inoculation of actinorhizal plants (Martin
et al., 1991). Rapid growth and the increased soil nitrogen fertility characteristic of many actinorhizal plants
are often dependent on the establishment of a symbiotic partnership with the microsymbiont (Wheeler and
Miller, 1990). Standard inoculation procedures have
often been ineffective in the field for reclamation and
amenity plantings of actinorhizal species (Reddell et
al., 1991), and no commercial Frankia inoculants are
readily available (Benoit and Berry, 1990).
Water-retentive polymers have been available to
tree growers for some time. They are generally used
as a dip to reduce moisture stress and to increase
nutrient availability in newly transplanted seedlings
(Adams and Lockaby, 1987). Water-retentive polymers include either starch-based agents or polyacrylamide combinations. These materials have the ability
to adsorb moisture up to 400 times their dry weight,
allowing them to capture water normally lost to gravitational forces or to evaporation in the rhizosphere
of the plant (Magnusen, 1986). Without intervention,
substantial losses of plants are often incurred in transplanting tree seedlings on harsh sites resulting from
root desiccation and subsequent lack of root establishment in outplants (Adams and Lockaby, 1987).
Superabsorbent, water-retentive polymers have been
proposed widely as a tool for mine-spoil reclamation and tree and shrub transplantation, to minimize
moisture stress in young transplants. A number of
experiments employing superabsorbent polymer root
treatments have resulted in increased tree seedling survival rates in comparison with control plants when
treated trees were transplanted under harsh environmental conditions (Pritchard, 1984). Positive results
led us to explore the use of superabsorbent polymer root treatments together with Frankia inocula in
increasing nodulation of actinorhizal plants.
We chose two species of actinorhizal plants for our
experiments, Alnus glutinosa (L.) Gaertn. and Casuarina equisetifolia var. equisetifolia Forst. & Forst.
A. glutinosa and other Alnus spp. have been widely
used as nurse trees, for biomass production and for reclamation purposes, most notably on coal mine spoils
(Dawson, 1986; Dawson, 1990; Dawson et al., 1983;
Fessenden, 1979; Heilman and Ekuan, 1982; Silvester,
1977). A. glutinosa tolerates soil pH extremes which
can pose a problem in revegetating mine spoils (Vogel,
1981).
Casuarina spp. are used in arid or semiarid landscapes for the reclamation of disturbed land, as amenity plantings, in agroforestry and to stabilize desert,
riparian, and coastal soils. A number of the casuarinas have been widely planted throughout tropical and
warm temperate regions in Egypt, India, southeastern China, Australia, Mexico, the mild, subtropical
fringes of the United States, and throughout the Pacific. Casuarinas outside their native range usually
must be inoculated when first planted in order to obtain nodulation (Diem and Dommergues, 1990). Some
of the casuarinas are also found to lack nodules even
in their native habitats such as Australia (Dawson et
al., 1989; Lawrie, 1982). Species of Casuarina have
been noted for their salt and drought tolerance and
have been used extensively on dry and sodic soils for
fuelwood and agroforestry purposes. Moreover, the
caloric value of the wood (5000 kcal/kg) is higher than
for most other tree species (Reddell et al., 1991; U.S.
National Research Council, 1984).
In an effort to evaluate the potential of superabsorbant polymers as a carrier for symbiotic microorganisms, we incorporated Frankia into superabsorbants which were used as an inoculant for Alnus and Casuarina plants. We hypothesized that the
polymer-Frankia delivery system would allow for increased infection, nodule development and growth of
actinorhizal plants compared to standard inoculation
methods.
Materials and methods
Experiment I
In this experiment polymer-amended Frankia inoculum was compared with Frankia inoculated in a
standard aqueous suspension for resulting nodulation
of A. glutinosa under controlled conditions. Seeds
of A. glutinosa (F. W. Schumacher, Inc., Sandwich,
Mass.) were soaked in warm sterile water for 12 h,
surface sterilized in 30% (v/v H2 O2 for five minutes,
washed in sterile water, and germinated in sterile sand.
Seedlings 3–4 weeks old were subsequently treated
as described below, transferred to sterile perlite in
15×20 cm white plastic pots and watered immediately
with sterile deionized water.
107
Cells of Frankia strain ArI4 (DDB01310210) isolated by D. Baker from Alnus rubra root nodules were
used in this experiment. Frankia strain ArI4 is in
the same cross-infectivity group as most other isolates from Alnus root nodules and studies including
that of Baker (1987) have shown this strain to be
a viable inoculant for A. glutinosa. Strain ArI4 was
grown in the dark in an incubator at 27 ◦ C and at pH
6.5 in ‘Frankia Broth’ (Baker and O’Keefe, 1984).
Actively growing strains that had been subcultured
biweekly were washed twice via centrifugation for
5 min at 2000×g in sterile distilled water, homogenized manually using a glass tissue grinder, then
centrifuged for 10 min at 2000×g in graduated centrifuge tubes to determine packed cell volume (PCV).
Then 100:1 v:v dilutions were aseptically prepared by
mixing 100 parts sterile water or polymer slurry with
one mL Frankia PCV according to Berry and Torrey
(1985). The polymer slurry consisted of 0.35 grams of
cross-linked potassium polyacrylate/polyacrylamide
copolymer (MiTech Research and Development, Inc.,
St. Paul, Minnesota) per 100 mL of sterile water.
The three treatments consisted of (1) control plants
the roots of which were dipped for 5 s in sterile water, (2) seedlings dipped in a sterile water suspension
of Frankia, and (3) seedlings dipped in the polymer
– Frankia slurry. Neither brief suspension in distilled
water prior to inoculation into a soil substrate nor immersion in the water-retentive polymers used in this
study have exhibited a reduction in infectious capacity
suggestive of osmotic stress. The macromolecules of
water retentive polymers form gels that do not possess the negative water potentials of low molecular
weight solutes, allowing plants and microorganisms to
avail themselves of the adsorbed water without suffering osmotic stress under our experimental conditions.
There were 3 pots per treatment with 3 seedlings in
each pot resulting in 9 seedlings per treatment. Within
each treatment group, the remainder of the water or
inoculum not absorbed onto the roots after dipping
was applied in equal aliquots to the base of each plant,
so that each plant received 1 mL of treatment solution by dipping or substrate application. The plants
were grown in a growth chamber and were irrigated 5
times weekly with 250 mL of 1/4 strength Hoagland’s
nitrogen-free solution (Hoagland and Arnon, 1950) at
pH 5.5 supplemented with 0.05 mM KNO3 , a beneficial low level of nitrogen that prevents chlorosis prior
to establishment of the symbiois but does not affect
nodulation (Baker, 1987). Twice weekly plants were
flushed with 400 mL of deionized water. Treated trans-
plants were grown for a total of 8 weeks. The growth
chamber was illuminated by 1000-Watt High Pressure
Sodium and Metal Halide Lamps in GE Duraglow fixtures in an alternating array. Photosynthetic photon
flux density at plant level was determined to be 700–
800 µmoles m−2 s−1 with a light:dark cycle of 14:10
h and a day:night temperature cycle of 27:22 ◦ C.
Treatments were completely randomized by pot in the
growth room. At 8 weeks, plants were harvested and
shoot length, root length, and nodule number per plant
were determined. Dry weights of shoots, roots and
nodules were determined after drying for 24 h at 70
◦ C.
Experiment II
A second experiment was performed with A. glutinosa to ascertain the effectiveness of two different
polymer formulations and the use of reduced Frankia
titers in the polymer slurries. The same methods for
plant propagation, inoculation, and controlling environmental conditions used in the first experiment
were also employed in the second experiment. Immediately upon transfer from the sand germination
medium to perlite, plants were subjected to seven different treatments. The treatments, with three plants per
pot, consisted of corn-starch based copolymer slurry
(MiTech Research and Development, Inc., St. Paul,
MN) with the 100:1 dilution PCV in aqueous solution
and another treatment consisting of one fifth of this
Frankia concentration (0.01 mL PCV/plant) with a
five-fold (5×) reduction in titer (0.002 mL PCV/plant)
plus the corn-starch based copolymer slurry. The remaining treatments consisted of the same concentrations of the polyacrylate/polyacrylamide copolymer
with standard and reduced concentrations of Frankia
inoculum and control plants without polymers containing no Frankia, standard inoculum density or a
5× reduced Frankia titer. Seed of A. glutinosa was
germinated as described previously before transferring
to the 15×20 cm pots. Plants were randomized on a
greenhouse bench and grown using the same regime
as described in Experiment I. Plants were harvested
10 weeks after inoculation and evaluated for growth
and nodulation parameters as in Experiment I.
Experiment III
A field trial was performed with outplanted A. glutinosa to complement laboratory studies. Seeds were
germinated as described previously and 3–4 week old
108
seedlings were transferred to 15×20 cm pots containing autoclaved perlite. Plants were grown for 24
weeks under the same conditions as in the previous
experiments. The roots were then washed with sterile
water to remove the substrate after which plants were
transferred from the laboratory to the field with roots
immersed in sterile distilled water.
The planting site was an old pasture dominated by
grasses, including Agropyron repens and some remnant native Andropogon spp., in a xeric oak-savanna
vegetation zone directly adjacent to the Anoka Sand
Plain in Minnesota (45◦ , 35′ N, 93◦ , 10′ W). The
climate of the area is characterized by short, warm
summers and long, cold winters. Annual precipitation
in the area averages 660 mm, with a mean annual
temperature of 22 ◦ C (Baker et al., 1967). The soil
at this site is a well-drained sandy loam classified as
a Mollisol in the Zimmerman series. The soil has a
pH of 5.5–6.0 and less than 0.5% total N in the surficial meter of the soil. Plant growth on these soils is
considered to be nitrogen limited (Grigal et al., 1974).
The alder seedlings were planted in a completely
randomized design with two replicates of each of
four treatments. Each of the eight randomized plots
was 5×5m with even spacing of 4–5 plants per treatment plot and a one meter buffer zone between each
plot. The four treatments consisted of a control group,
plants that were treated with only a cross-linked
potassiumpolyacrylate/polyacrylamide copolymer, a
group receiving 0.01 mL PCV of Frankia per plant,
and a treatment combining this same inoculum titer
with the cross-linked potassium polyacrylamide coploymer. The treatments were applied by dipping
individual root systems in sterile water (the control
group), polymer only, Frankia in sterile water, or the
polymer-Frankia slurry as previously described. The
seedlings were planted in mid-June in soil tilled to approximately 40 cm in depth, heeled-in, and each was
watered with 100 mL of deionized water. The plants
were grown for 4 months with no fertilizer, watering,
or weed control. The small trees were carefully excavated so that the entire root systems were removed
and all soil was gently washed from the root systems.
Growth and nodulation parameters were determined as
described previously.
Experiment IV
This experiment was designed to evaluate the potential of a water-retentive polymer inoculum carrier to
enhance nodulation, nodule development and growth
of C. equisetifolia under controlled conditions. Two
different polymer formulations with both a standard
and a 10-fold (10×) reduction in Frankia inoculum
(0.001 mL PCV/plant) were used.
Seeds were obtained from a plantation of C. equisetifolia located in Hawaii. Seedlings (3–4 weeks after
germination on sterile sand) were transplanted to premoistened sterile perlite, with nine plants per treatment and 3 plants per pot. Casuarina seedlings were
inoculated (root dipped) with either 1 mL each of
a 100:1 PCV dilution or a further 10× dilution of
washed and homogenized cells as described in Experiment I, but using Frankia strain Cc13 (HFP020203)
(Zhang et al., 1984) grown in defined propionate medium (Baker and O’Keefe, 1984) at 30 ◦ C. Cells
of Frankia strain CcI3 used in this experiment readily and effectively nodulate C. equisetifolia, which
has the broadest microsymbiont affinity of all Casuarina and Allocasuarina spp., and studies including
that of Baker (1987) have shown this strain to be
a viable inoculant for C. equisetifolia. There were
9 seedlings per treatment and treatments consisted
of untreated controls; polymer only (using both the
polyacrylate/polyacrylamide copolymer or the cornstarch based copolymer individually); and one treatment each of Frankia only at the standard titer (0.01
mL PCV plant) and a 10× dilution (0.001 cc PCV)
of treatments with each polymer and the reduced inoculum density. Plant culture was the same as in
previous experiments except that day temperature for
plant growth was 30 ◦ C instead of 27 ◦ C. The same
plant nutrient solution was used as in the preceeding inoculation experiments, however, at 8 weeks the
application of the nutrient solution was reduced to
once weekly for the remainder of the 12-week growth
period. Casuarina spp. are slower to nodulate than
many other actinorhizal species (Kohls and Baker,
1989), and this longer, reduced nitrogen regime was
employed to promote nodulation of the plants. The
plants were harvested at 12 weeks, and the nodulation and growth patterns were evaluated as previously
described.
Statistical analyses
Data were analyzed using ANOVA and a protected
Fisher’s Least Square Differences method to detect
statistically significant (p ≤ 0.05) differences among
individual treatment means. Unless noted in the tables,
ANOVA indicated significant overall treatment effects
(P ≤ 0.05).
109
Results
Nodules appeared 10–12 days after inoculation for Alnus and 21–28 days after inoculation for Casuarina.
The time required for nodule initiation in the alder
field trial was not determined to avoid damaging the
plants. In all experiments, nodules of the polymerFrankia treated plants were distributed throughout the
entire root system. In these treatments, nodules on the
lower and distal regions of the tap and lateral roots
were smaller than those on the upper portions of the
root system. In contrast, the majority of nodules on
plants inoculated with Frankia alone were confined to
the upper sections of the tap and lateral roots. Few,
if any, nodules were present on the distal portions of
these root systems. This pattern was the same for both
Alnus and Casuarina.
Experiment I
Nodulation
The number of nodules per plant, nodule dry weight
per plant and nodule weight as a percentage of whole
plant dry weight were all significantly greater, with
a 2-fold increase in the polymer-Frankia treatments
compared to the Frankia treatments without polymer
(Table 1). In this controlled study with young seedlings, the total number and percentage of nodule mass
relative to whole plant mass were quite high in the
early stages of nodule development. The nodule mass
proportion decreases with plant development.
Plant growth
The control plants were not nodulated and were
chlorotic as is common for nitrogen deficient plants.
Their growth was significantly less than that of the
two other treatments. No statistically significant differences were found between the plants treated with
polymer-Frankia and those treated with Frankia alone
with respect to the biomass of the plant components.
However, there was a significant difference in the
root/shoot ratios (Table 1).
Experiment II
Nodulation
Nodule number per plant was highest in the polymerFrankia standard concentration treatment and lowest
in the 5× dilution with Frankia alone (Table 2).
Among the various inoculum titers employed, approximately 2-fold increases were observed for nodule
number and nodule weight per plant for the polymerFrankia inoculated outplants compared with plants
inoculated with Frankia alone. Irrespective of the inoculum titer, the percentage of nodule weight per plant
was equal to or significantly greater for plants inoculated with the polymer-Frankia treatments than for
plants inoculated with Frankia alone (Table 2). As in
Experiment I, the total number and percentage of nodule mass relative to whole plant mass were quite high
in the early stages of nodule development.
Plant growth
Initial alder growth increased dramatically as a result of the combined polymer-Frankia treatments
(Table 3). There was a 2–3-fold increase in plant shoot
weight compared with the controls for the polymerFrankia treated plants. The use of Frankia alone increased shoot, root, and whole plant weights when
compared to the controls. However, the shoot/root
ratio was not significantly altered (Table 3). The
use of standard and reduced titers of the polymerFrankia slurries resulted in significantly greater shoot
and whole plant dry weights than the use of Frankia
alone. In addition, lower root:shoot ratios were observed in the polymer-Frankia treated plants compared
with the other treatments (Table 3). Moreover, the
mean shoot length of plants inoculated with both polymer and Frankia exceeded that of plants inoculated
with Frankia alone, which in turn exceeded the mean
shoot length of control plants (Table 2). As in Experiment I, with heavily inoculated young seedlings, the
total number and percentage of nodule mass relative
to whole plant mass were quite high in the early stages
of nodule development. The nodule mass proportion
decreases with plant development.
Experiment III
Nodulation
The presence of a few nodules on upper portions of the
root systems of the controls and of the alder treated
with polymer alone indicate that an indigenous population able to nodulate Alnus glutinosa was present
at the Minnesota planting locale. The nodule number
and degree of nodule development apparently induced
by the indigenous microsymbiont were significantly
lower when compared to nodulation of the inoculated
plants (Table 4). After one full growing season, plants
treated with the polymer-Frankia inoculum had significantly greater mean nodule numbers and nodule
weight than did the other treatments. Furthermore,
110
Table 1. Experiment I: Mean shoot length, mass, mass ratios and nodulation values of Alnus glutinosa seedlings with Frankia inoculum or
Frankia plus polymer inoculation
Treatment1
n
Shoot length2 (cm)
Nod. #/Plant
Nod. Weight (g)
Nod. wt./Plant wt. (%)
Controls
Frankia alone
Frankia+Polymer
9
9
9
10.4a
12.2b
13.1b
0
203.9a
489.7b
0
0.07a
0.13b
N/A
13.1a
23.6b
Treatment1
n
Shoot2 (g)
Root3 (g)
Whole Plant (g)
Root/Shoot
Controls
Frankia alone
Frankia+Polymer
9
9
9
0.20a
0.30b
0.33b
0.22a
0.24a
0.21a
0.42a
0.54b
0.54b
1.13a
0.87b
0.67c
1 Definitions of treatments: Controls, no inoculum or polymer; Frankia alone, standard inoculum titer without polymer; Frankia+Polymer,
standard inoculum titer with cross-linked potassium polyacrylamide/polyacrylate copolymer.
2 Values within a column are not significantly different (P ≤ 0.05) if they share a lower case letter.
3 No significant differences for treatments according to ANOVA (P ≤ 0.05).
Table 2. Experiment II: Mean shoot length and nodulation values for Alnus glutinosa seedlings differing in Frankia inoculum density
and copolymer treatment
Treatment1
n
Shoot length2 (cm)
Nod. #/Plant
Nod. Weight (g)
Nod. wt./Plant wt. (%)
Controls
FR
F
FRP1
FRP2
FP1
FP2
9
9
9
9
9
9
9
6.36a
8.02b
8.61b
9.50c
10.80c
10.20c
10.50c
0
43.0a
93.0b
110.0c
129.0c
272.0d
266.0d
0
0.017a
0.022b
0.033c
0.041d
0.058e
0.060e
N/A
9.9a
13.9b
17.9c
13.2b
18.4c
21.2c
1 Definitions of treatments: Controls, no inoculum or polymer; FR, 5× reduction in inoculum titer without polymer; F, standard inoculum
titer without polymer; FRP1, 5× reduction in inoculum titer with cross-linked potassium polyacrylamide/polyacrylate copolymer; FRP2,
5× reduction in inoculum titer with starch-based polymer; FP1, standard inoculum titer with cross-linked potassium polyacrylamide/polyacrylate copolymer; FP2, standard inoculum titer with starch-based polymer.
2 Values within a column are not significantly different (P ≤0.05) if they share a lower case letter.
Table 3. Experiment II: Mean mass or mass ratios of Alnus glutinosa seedlings differing in Frankia inoculum density and copolymer
treatment
Treatment1
n
Shoot2 (g)
Root (g)
Whole Plant (g)
Root/Shoot
Controls
FR
F
FRP1
FRP2
FP1
FP2
9
9
9
9
9
9
9
0.077a
0.111a
0.096a
0.134b
0.207c
0.179c
0.230c
0.058a
0.084b
0.062a
0.083b
0.108c
0.095b
0.066a
0.136a
0.195b
0.158c
0.217d
0.315e
0.274e
0.296e
0.74a
0.75a
0.68a
0.60a
0.53b
0.52b
0.37b
1 Definitions of treatments: Controls, no inoculum or polymer; FR, 5× reduction in inoculum titer without polymer; F, standard
inoculum titer without polymer; FRP1, 5× reduction in inoculum titer with cross-linked potassium polyacrylamide/polyacrylate
copolymer; FRP2, 5× reduction in inoculum titer with starch-based polymer; FP1, standard inoculum titer with cross-linked potassium
polyacrylamide/polyacrylate copolymer; FP2, standard inoculum titer with starch-based polymer.
2 Values within a column are not significantly different (P ≤0.05) if they share a lower case letter.
111
Table 4. Experiment III: Mean shoot length and nodulation values of Alnus glutinosa seedlings grown in the field after Frankia inoculation and
copolymer root treatments
Treatment1
n
Shoot length2 (cm)
Nod. #/Plant
Nod. Weight (g)
Nod. wt./Plant wt. (%)
Controls
Polymer alone
Frankia alone
Frankia+Polymer
8
7
9
9
18.6a
21.3a
20.5a
24.9b
7.0a
8.8a
46.0b
131.0a
0.038a
0.046a
0.070b
0.160c
0.96a
0.95a
1.20a
3.82b
1 Definitions of treatments: Controls, no inoculum or polymer; Polymer alone, cross-linked potassium polyacrylamide/polyacrylate copolymer
without inoculum; Frankia alone, standard inoculum titer without polymer; Frankia+Polymer, standard inoculum titer with cross-linked
potassium polyacrylamide/polyacrylate copolymer.
2 Values within a column are not significantly different (P ≤0.05) if they share a lower case letter.
nodule weight as a percentage of whole plant weight
was increased more than 3-fold over other treatments
in the polymer-Frankia treatments (Table 4).
was a significant difference in shoot length between
the inoculated and control plants (Table 6).
Plant growth
Following one growing season in the field, alder shoot
dry weight and shoot length for plants treated with
the polymer Frankia slurry were significantly greater
than those of all other treatments (Tables 4 and 5).
A decrease in the root dry weights and corresponding significant decrease in the root:shoot ratios for
the polymer-Frankia treatment group accounts for the
lack of a significant difference in whole plant dry
weight. There was a significant increase in shoot
length for inoculated compared with control plants
(Table 4).
Discussion
Experiment IV
Nodulation
For each Frankia inoculum concentration, the polymer treatment resulted in a 2 to 3 fold increase in
nodule number and weight for C. equisetifolia plants
compared with treatments of Frankia inoculum alone
(Table 6). The increase in the number of nodules for
the polymer treated plants (Table 6) indicated that the
polymer treatment definitely enhanced nodulation of
C. equisetifolia just as polymer root treatments had
enhanced nodulation of A. glutinosa.
Plant growth
Shoot weights and whole plant weights were significantly greater for the polymer-Frankia treatments than
for treatments using Frankia alone irrespective of inoculum concentration (Table 7). Root:shoot ratios for
standard and reduced titers of polymer-Frankia treatments were found to be significantly lower than for
the standard Frankia titer alone (Table 7). Also, there
The results of all experiments indicate that greater
nodulation and, in some cases, early growth of actinorhizal plants are obtained by inoculating with water
retentive, superabsorbent polymer slurries used as a
carrier for cultured Frankia. The number of nodules
per plant is the best measure of microsymbiont infection (Streeter, 1988). On this basis, our data support
the premise that polymer-Frankia formulations promote increased infection. Our data are consistent with
the concept that overall nodule weight corresponds
to plant productivity in nitrogen limited environments
(Hielman and Ekuan, 1982). Lower root:shoot ratios
for plants inoculated with the polymer-Frankia slurries indicates a shift in dry weight allocation towards
shoot growth (Arnone et al., 1994). Hydrated polymer
amended with reduced Frankia inoculum concentrations induced equal or greater nodulation and growth
of the host plant than did polymer-free standard titer
inoculation methods for Alnus and Casuarina. We observed the polymer slurry to migrate with and adhere
to the developing roots. Apparently as the polymerFrankia slurry migrated with the developing root system, it permitted the nodulation of roots away from the
root collar. This nodulation pattern is unusual and did
not occur in the polymer-free plants. Moisture deficits can adversely affect Frankia growth (Shipton and
Burgraff, 1982), so perhaps rhizosphere moisture conditions are ameliorated by the water retaining capacity
of the polymer.
Alnus treated with the Frankia polymer slurry in
the first experiment did not exhibit the relative growth
increases for shoots observed in the other experiments.
112
Table 5. Experiment III: Mean mass or mass ratios of Alnus glutinosa seedlings grown in the field after Frankia inoculation and copolymer
root treatment
Treatment1
n
Shoot2 (g)
Root (g)
Whole Plant3 (g)
Root/Shoot
Controls
Polymer alone
Frankia alone
Frankia+Polymer
8
7
9
9
1.31a
2.02b
2.35b
2.90c
2.83a
3.22a
2.90a
1.74b
4.14a
5.24a
5.25a
4.64a
2.41a
1.96a
1.37a
0.60b
1 Definitions of treatments: Controls, no inoculum or polymer; Polymer alone, cross-linked potassium polyacrylamide/polyacrylate copolymer
without inoculum; Frankia alone, standard inoculum titer without polymer; Frankia+Polymer, standard inoculum titer with cross-linked
potassium polyacrylamide/polyacrylate copolymer.
2 Values within a column are not significantly different (P ≤ 0.05) if they share a lower case letter.
3 No significant difference for treatments according to ANOVA (p ≤ 0.05).
Table 6. Experiment IV: Mean shoot length and nodulation value for Casurarina equisetifolia seedlings differing in Frankia inoculum
density and copolymer treatment
Treatment1
n
Shoot length2 (cm)
Nod. #/Plant
Nod. Weight (g)
Nod. wt./Plant wt. (%)
Controls
P1
P2
FR
FR1
FR2
F
F1
F2
8
8
9
9
8
8
9
9
9
12.2a
14.5b
14.7b
15.8b
17.4c
17.2c
17.6c
22.2d
22.6d
0
0
0
3a
8b
6b
4a
24c
28c
0
0
0
0.009a
0.022b
0.020b
0.019b
0.051c
0.048d
N/A
N/A
N/A
1.47a
3.27b
3.28b
1.88c
3.96d
4.31d
1 Definitions of treatments: Controls, no inoculum or polymer; P1, cross-linked potassium polyacrylamide/polyacrylate copolymer
without inoculum; P2, starch-based polymer without inoculum; FR, 10× reduction in inoculum titer without polymer; FR1, 10×
reduction in inoculum titer with cross-linked potassium polyacrylamide/polyacrylate copolymer; FR2, 10× reduction in inoculum
titer with starch-based polymer; F, standard inoculum titer without polymer; F1, standard inoculum titer with cross-linked potassium
polyacrylamide/polyacrylate copolymer; F2, standard inoculum titer with starch-based polymer.
2 Values within a column are not significantly different (P <0.05) if they share a lower case letter.
Table 7. Experiment IV: Mean mass or mass ratios of Casurarina equisetifiolia seedlings differing in Frankia inoculum density and
copolymer treatment
Treatment1
n
Shoot2 (g)
Root (g)
Whole Plant (g)
Root/Shoot
Controls
P1
P2
FR
FR1
FR2
F
F1
F2
8
8
9
9
8
8
9
9
9
0.184a
0.287b
0.241b
0.286b
0.368c
0.391c
0.433c
0.783d
0.666d
0.306a
0.369a
0.321a
0.395a
0.332a
0.269b
0.502c
0.576c
0.469c
0.490a
0.657b
0.563b
0.682b
0.700b
0.661b
0.935c
1.359d
1.135d
1.68a
1.23b
1.34c
1.57a
0.93d
0.73e
1.15b
0.73e
0.75e
1 Definitions of treatments: Controls, no inoculum or polymer; P1, cross-linked potassium polyacrylamide/polyacrylate copolymer
without inoculum; P2, starch-based polymer without inoculum; FR, reduction in inoculum titer without polymer; FR1, 10× reduction in inoculum titer with cross-linked potassium polyacrylamide/polyacrylate copolymer; FR2, reduction in inoculum titer with
starch-based polymer; F, standard inoculum titer without polymer; F1, standard inoculum titer with cross-linked potassium polyacrylamide/polyacrylate copolymer; F2, standard inoculum titer with starch-based polymer.
2 Values within a column are not significantly different (P <0.05) if they share a lower case letter.
113
It is likely that relative increases in shoot growth
would have been observed if the plants had been allowed to grow for longer periods as in subsequent
experiments. Abundant nodulation in the treated plants
in the first experiment may have caused an initial shift,
not noted in the longer experiments, in the allocation
of photosynthates away from the shoots to the numerous developing nodules, subsequently inhibiting an
early phase of shoot growth. The high proportional
mass of nodules in Experiments I and II would also
be expected to decrease with subsequent plant growth.
The results of Experiment III indicated that significant increases in nodulation can be obtained with
the polymer-Frankia treatments under field conditions. The difference in mean shoot weights between
treatments was statistically significant, though overall
biomass did not differ significantly among treatments
owing to a shift in growth allocation of polymerFrankia treated plants from roots to shoots. The results of inoculation studies at this site indicate that
the polymer-Frankia treated plants had greatly increased nodulation numbers over those induced by the
indigenous Frankia population alone.
The relative contributions of the polymer-Frankia
slurry to both nodulation and growth in Casuarina indicate that the polymers are superior to standard inoculation techniques in a controlled environment. A field
trial using these same treatments will be necessary to
assess the practical benefits of this inoculum formulation for Casuarina seedlings. We obtained increased
nodulation with polymer carriers as was previously
obtained in studies using Frankia entrapped in alginate
beads and supplied to Casuarina seedlings (Sougoufara et al., 1989). The lower cost of the superabsorbant
formulation, high moisture retention capability benefitting both Frankia and seedling host, and adherence
of the polymer to the root system are advantages not
afforded by the alginate bead technique. However alginate beads are easier to store and deliver to remote
field sites.
Though the use of superabsorbant polymers has
been proposed as a means for increasing transplant
survival (Callaghan et al., 1988), we did not observe significant differences in survival rates between
plants with and without polymer treatment. Plants in
the laboratory experiments were grown under favorable conditions and early survival of transplants was
not problematic. Plants treated with polymer alone in
Experiments III and IV showed significantly greater
shoot growth and correspondingly lower root/shoot ratios. This suggests that the polymer itself may serve to
facilitate the uptake of water and associated nutrients.
Furthermore, we did not observe any significant differences between the effectiveness of the varied starch
based and polyacrylate polymer formulations used.
In summary, we have demonstrated that (1) infection, nodule development, and in some cases, early
actinorhizal plant growth can be enhanced by the use
of superabsorbent polymers amended with Frankia,
(2) for Alnus and Casuarina, superabsorbent polymers amended with reduced titers of Frankia inocula
are at least as effective as the higher tiers used with
standard inoculation techniques, and (3) alder seedlings grown in the field exhibited increased infection,
nodule development, and a higher shoot to root mass
ratio when inoculated with the combination of Frankia
and polymer.
The development of these Frankia-polymer formulations could further the use of actinorhizal species
for the reclamation of disturbed landscapes, amenity
plantings, and at locales where indigenous populations of Frankia are absent by increasing nodulation
and thereby enhancing the survival and growth of
actinorhizal plants.
Acknowledgements
Appreciation is expressed to the late Prof D. B.
Lawrence of the University of Minnesota, St. Paul,
MN for critical evaluation of the manuscript. We are
indebted to the family of the late J. Arthur Holm for
the use of their farmstead for the field experiment.
Finally, we wish to thank Carolyn K. Curti, Douglas
Harbrect, and Vicki L. Raffle, for editorial assistance.
The delivery system is part of U.S. Patent #4,975,105.
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