Directory UMM :Data Elmu:jurnal:S:Scientia Horticulturae:Vol84.Issue1-2.Apr2000:
Scientia Horticulturae 84 (2000) 151±162
Growth and ¯owering in Petunia hybrida, Callistephus
chinensis and Impatiens balsamina inoculated with
mixed AM inocula or chemical fertilizers
in a soil of low P fertility
Anupama Gaura, Atimanav Gaurb, Alok Adholeyac,*
a
Forestry and Agricultural Biotechnology Institute (FABI), Faculty of Biological and Agricultural
Sciences, University of Pretoria, Pretoria 0002, South Africa
b
Mycology Lab, Department of Botany, University of Pretoria, Pretoria 0002, South Africa
c
Microbial Biotechnology, Tata Energy Research Institute, India Habitat Centre,
Lodhi Road, New Delhi-110 003, India
Accepted 13 August 1999
Abstract
Three seasonal ornamental plants, namely Petunia hybrida, Callistephus chinensis and Impatiens
balsamina, were tested for their response to inoculation with mixed indigenous AM culture when
grown on a marginal wasteland amended with organic matter.
Subsequently, the treatments consisting of AM inoculations were compared with those consisting
of recommended chemical fertilizers in terms of growth response and cost economics. In all the
three plant species, mycorrhizal inoculation led to marked improvement in both reproductive
(number of ¯owers) and vegetative (dry matter) phase of the plants. P. hybrida showed a threefold
increase over uninoculated plants in the reproductive growth as compared to twofold in C. chinensis
and I. balsamina. Application of the recommended dose of chemical fertilizers produced a
comparable response.
The inoculated plants produced greater dry matter, grew taller, ¯owered at least 15 days earlier
and produced more ¯owers when compared to uninoculated plants. In addition there was a
signi®cant increase in P and K uptake in shoots of all the three ornamentals. AM inoculation could
be at least 30% cost economic as compared to the chemical fertilizers. Therefore, mycorrhizal
inoculation is recommended at the nursery level for nutrient-de®cient soil conditions because it is a
cost-effective substitute for chemical fertilizers, either partly or fully, which makes the approach
*
Corresponding author. Tel.: 91-11-460-1550; fax: 91-11-462-1770.
E-mail address: [email protected] (A. Adholeya).
0304-4238/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 2 3 8 ( 9 9 ) 0 0 1 0 5 - 3
152
A. Gaur et al. / Scientia Horticulturae 84 (2000) 151±162
particularly suitable to marginal farmers with their low-input farming system. # 2000 Elsevier
Science B.V. All rights reserved.
Keywords: Petunia hybrida; Impatiens balsamina; Callistephus chinensis; Indigenous AM
consortium; Organic amendment; Chemical fertilizers; Cost economics
1. Introduction
AM fungal inocula hold tremendous potential in increasing crop production as
an integral component of sustainable crop production systems (Bagyaraj, 1992).
They have been shown to confer a variety of bene®ts on their hosts including
growth and yield enhancement (Furlan, 1993). AM fungi are found in a wide
variety of habitats (Brundrett, 1991).
Wasteland are large stretches of land de®cient in nutrients and bene®cial
microorganisms and account for approximately 20% of India's total geographical
area (Sharma et al., 1996). Mycorrhizal fungi are likely to be bene®cial in
bringing these habitats under cultivation. The current study was designed to
assess, in economic and physical terms, the bene®t of cultivating economically
important ornamental and oil seed plants on such wastelands using bene®cial
microorganisms and to see how it compares with the cultivation based on
applying chemical fertilizers in terms of costs.
Ornamental plants are often grown from seedlings and cuttings grown in
disinfected soils or on inert substrates, mainly to lower the risk of contamination
and to ensure controlled conditions to obtain homogenous material. Such
activities and other horticultural practices that tend to eliminate AM fungi create
the conditions best suited to using mycorrhizal biotechnology (Johnson et al.,
1980). In addition, production of ornamental species in nutrient-de®cient arid
soils is more dif®cult because these plants generally have a high fertilizer
requirement. Adopting management practices to increase plant production with
low fertilizer input will minimize adverse effects on the environment and keep
production costs low, making it suitable for marginal farmers with low incomes.
AM fungi are known to increase plant growth in arid and semi-arid regions
(Hirrel and Gerdemann, 1980). Although many studies of host plant responses to
mycorrhizal infection have been performed using a single AM fungal isolate, the
present study used a mixed inoculum of AM fungi so as to simulate the natural
conditions more closely, where an assemblage of many species is more common
(Brundrett, 1991).
The current experiment was planned with the following objectives: (1) to study
the ef®cacy of an indigenous, mixed AM inoculum in infecting and promoting the
growth of selected ornamental plants; (2) to study the effect of mycorrhizal
A. Gaur et al. / Scientia Horticulturae 84 (2000) 151±162
153
inoculation on ¯owering, and nutrient uptake and compare; (3) the cost
effectiveness of using mycorrhizal inoculation and chemical fertilizers.
2. Materials and methods
2.1. Preparation of AM inocula
A mixed indigenous culture (containing native populations of Glomus,
Gigaspora and Scutellospora spp.) collected from the experimental site was
used as the AM inoculum and multiplied for 1 year in clay pots (5 kg capacity)
®lled with soil similar to the poting mixture, with Sorghum bicolor as the host
plant. At maturity, the tops of the sorghum plants were removed and substrate was
allowed to dry for a week at 25 58C. The roots were ®nely chopped and the
dried root/soil mixture was thoroughly mixed to obtain a homogeneous inoculum.
Spores were isolated by wet sieving and decanting (Gerdemann and Nicolson,
1963) and counted on a ®lter paper (Gaur and Adholeya, 1994). The percentage
of root colonization by mycorrhizae was assessed as described by Biermann and
Lindermann (1981) after staining the roots with acid fuchsin (Phillips and
Hayman, 1970). The total number of infectious propagules (IPs) per gram of
inoculum was also counted (Sharma et al., 1996) and the value was found to be 15
IPs/g. The colonization percentage was 62 and the spore density was 20 spores/g
inoculum.
2.2. Preparation of seedlings
Seeds of Petunia hybrida cv. blue bird, Callistephus chinensis cv. dwarf
chrysanthemum and Impatiens balsamina were surface sterilized with 10% H2O2
for 5 min. Subsequently, the seeds were washed repeatedly with sterile water and
kept for germination on moist sand in sterile petriplates at 308C in dark for 48 h.
On germination, the seedlings were given half-strength Hoagland solution
(Hoagland and Arnon, 1950) for even 15 days.
2.3. Preparation of growth substrate
The experimental site is located at Gwal Pahari in Haryana state, India
(778120 E and latitude 288350 N) 255 m above the mean sea level and receives a
mean annual rainfall of 500 mm.
Two experiments were conducted. In Experiment 1, the poting mixture was
prepared by mixing two parts of soil (sandy loam Hyperthermic Typic Haplustalf,
pH 7.5, NO3N 124 mg/kg, available P 0.53 mg/kg, available K 124 mg/
kg) from the experimental site and one part of compost (made from leaves of
154
A. Gaur et al. / Scientia Horticulturae 84 (2000) 151±162
Table 1
Soil characteristics of the substrate used in both the experiments
Substrate
pH
Phosphorus
(ppm)
Nitrogen
(ppm)
Potassium
(%)
With chemical fertilizers
With organic manure only
7.3
7.4
30
2.4
128
72
4
2.6
Albizzia and Poplars, pH 7.2, available P 12 ppm, organic C 11.40% and
N 0.75%). The compost amended substrate showed a pH of 7.3, available
P 2.5 ppm (Olsen et al., 1954), and organic C 3.46% (Datta et al., 1962). The
substrate was autoclaved before inoculation (1218C for 1 h at 15 psi). Clay pots,
®lled with 5 kg of substrate, were used in the experiment.
In Experiment 2, the same potting mixture was used and one set of pots was
given chemical fertilizers in the form of SSP (single super phosphate),
micronutrients (commercially available), ammonium nitrate and potassium
chloride at the recommended levels (Table 1).
2.4. Experiment layout and mycorrhizal inoculation
Experiment 1 followed a completely randomized design consisting of a 3 2
structure three types of ornamental plants and two treatments (inoculated and
uninoculated). Each treatment was replicated six times. The substrate was
inoculated by thoroughly mixing the crude inoculum with soil in each pot (at
2000 IP per pot). The inoculum consisted of spores, hyphae and infected root bits.
Five kg of soil containing the inoculum mixture was transferred to earthen pots
(17 cm diameter). Uninoculated plants served as controls.
Experiment 2 also followed a completely randomized design consisting of a
3 3 structure (having an additional treatment, inoculated, uninoculated and
uninoculated with chemical fertilizer) each treatment was replicated six times.
Six 15-day-old germinated seedlings of each host were selected for uniformity
and transplanted to the pots. All the plants were grown under 70% daylight in a
greenhouse and watered to maintain the soil at 60% of its water holding capacity.
The pots were rotated regularly to avoid any positional effect. All the parameters
were analysed at harvest except the ¯ower count, which was carried out as
described below.
2.5. Observation, harvest and analysis
Flowers were counted (non-destructively) at 10 days intervals for all the six
replicates in each treatment for 120 days (Experiment 1 and 2). Shoots were
severed just above the crown, weighed while fresh, rinsed in distilled water, dried
A. Gaur et al. / Scientia Horticulturae 84 (2000) 151±162
155
at 708C for 48 h and weighed again, which were then ground ®ne enough to pass
through a 0.5 mm screen and digested in H2SO4. The P and K content in the
digest was determined chemically (Jackson, 1973) and using ¯ame photometry
(Chapman and Pratt, 1978).
The harvested roots were washed clean or cut into 1 cm segments, and
homogenized thoroughly. Samples of root segments were analysed for
mycorrhizal colonization. Colonization percentage was determined on 100 root
segments from each sample. Roots were stained using the method of Phillips and
Hayman (1970). Root pieces were mounted between the glass slides and
examined under a microscope at X40 for AM hyphae, arbuscules, vesicles and
spores. The extent of colonization was assessed by using the method of Biermann
and Lindermann (1981) and expressed as the percent of root segment colonized
for each root piece (Experiment 1 and 2). Spores of AM fungi were extracted
(Gerdemann and Nicolson, 1963) from 50 ml samples of the homogenized
substrate from three replicates for each treatment (Experiment 1). The spores
retained on different sieves were collected in a beaker and recovered by sucrose
density centrifugation. Only visually intact spores were counted under a
stereoscopic microscope (Gaur and Adholeya, 1994). The average number of
spores in 50 ml of the substrate soil was used to estimate the spores per gram soil
(gÿ1 soil) in each of the treatments. Sporocarps were gently crushed to count the
number of spores in each sporocarp.
2.6. Statistical analysis
The data were analysed using one-way ANOVA using the least signi®cant
difference (Duncan's multiple range test at 5% signi®cant level). The data were
also analysed for observing the standard deviation within the treatments using
Costat software (Cohort, Berkeley, CA, USA).
3. Results
3.1. Flowering
The inoculated plants ¯owered signi®cantly earlier than uninoculated controls
in both the experiments though the difference was not signi®cant in Experiment 2.
The inoculated plants of C. chinesis ¯owered 27 DAT (days after transplanting),
whereas the uninoculated plants took 22 days longer. Those of I. balamina
¯owered 37 DAT, which was 16 days earlier than uninoculated plants and those of
P. hybrida ¯owered 29 DAT, 12 days before the uninoculated plants (Experiment
1, Table 2). The inoculated host plants not only ¯owered earlier but also produced
signi®cantly more ¯owers, 190%, 106% and 75% more in P. hybrida, I.
balsamina and C. chinensis, respectively (Fig. 1).
156
A. Gaur et al. / Scientia Horticulturae 84 (2000) 151±162
Table 2
In¯uence of inoculation with an indigenous arbuscular mycorrhizal (AM) consortium on growth
and nutrient uptake of the three ornamentals (Experiment 1)a
Hosts
MCP
(%)
AM propagules
Spores
IPd
ÿ1
(g soil) (gÿ1 soil)
Flower
initiation
(DATe)
Nutrient uptake
Shoot P
(mg gÿ1)
Shoot
K (%)
Shoot
height
(cm)
Dry
matter
(g/plant)
P. hybrida
AMb
67.3
NMc
±
8.6a
±
6.5a
±
29
41
4.5a
3.17b
6.21a
5.32b
70.57a
64.53b
0.84a
0.64b
I. balsamina
AM
59.8
NM
±
6.9b
±
6.0b
±
37
53
2.84a
2.54b
5.0a
4.02b
53.06a
33.12b
0.72a
0.62b
C. chinensis
AM
51.6
NM
±
6.7b
±
5.3c
±
27
49
2.65a
2.07b
5.33a
3.72b
16.01a
11.50b
0.34a
0.27b
a
Means followed by the same letters are not signi®cantly different (p < 0.05).
AM Ð mycorrhizal.
c
NM Ð non-mycorrhizal.
d
IP Ð infectious propagule.
e
DAT Ð days after transplanting.
b
In Experiment 2, the number of ¯owers in AM inoculated P. hybrida was
signi®cantly higher, though the plants that had received chemical fertilizers also
produced more ¯owers, as compared to control plants (Fig. 2).
3.2. Plant dry matter
In both the experiments, dry matter was signi®cantly higher in the inoculated
plants than in their uninoculated counterparts (Tables 2 and 3).
3.3. Nutrient uptake
In Experiment 1, the P and K content of the three hosts tested was signi®cantly
higher in the inoculated plants (Table 2). In Experiment 2, both the AM fungal
inoculation and addition of chemical fertilizer increased P and K uptake over
the uninoculated controls, though the increase was greater in the later plants
(Table 3).
3.4. Mycorrhizal parameter
In both the experiments, AM consortia produced the maximum colonization in
P. hybrida followed by I. balsamina and C. chinensis (Tables 2 and 3). In terms of
A. Gaur et al. / Scientia Horticulturae 84 (2000) 151±162
157
Fig. 1. Effect of mycorrhizal inoculation on the number of ¯owers produced by: (a) P. hybrida; (b)
I. balsamina; (c) C. Chinensis in Experiment 1. Error bars denote standard errors of mean.
158
A. Gaur et al. / Scientia Horticulturae 84 (2000) 151±162
Fig. 2. Effect of mycorrhizal inoculation on the number of ¯owers produced by: (a) P. hybrida; (b)
I. balsamina; (c) C. chinensis in Experiment 2. Error bars denote standard errors of mean.
159
A. Gaur et al. / Scientia Horticulturae 84 (2000) 151±162
Table 3
In¯uence of inoculation with an indigenous arbuscular mycorrhizal (AM) consortium on the growth
and nutrient uptake of three ornamentals (Experiment 2)a
Hosts
P. hybrida
AM
CFb
NM
I. balsamina
AM
CFa
NM
C. chinensis
AM
CF
NM
a
b
MCP (%)
Nutrient uptake
Shoot
height (cm)
Dry matter
(g/plant)
Shoot P
(mg gÿ1)
Shoot
K (%)
66.8
±
±
4.36b
5.52a
3.21c
6.18b
6.52a
5.27c
70.43b
75.62a
65.3c
0.86b
0.91a
0.63c
59
±
±
2.89b
3.15a
2.58c
5.08b
5.21a
4.11c
54.96b
58.32a
35.16c
0.72a
0.72a
0.62b
52.2
±
±
2.69b
3.1a
2c
5.37b
5.52a
3.68c
16.8b
17.32a
11.8c
0.34a
0.33b
0.28c
Means followed by the same letters are not signi®cantly different (p < 0.05).
CF Ð chemical fertilizer.
AM multiplication (number of spores and infectious propagules), the trend was
the same, with maximum multiplication in P. hybrida followed by I. balsamina
and C. chinensis (Table 2).
3.5. Cost analysis
Application of chemical fertilizers proved to be 30% more expensive than
inoculation. The total cost of growing a chemically fertilized plant was $4.15,
compared to $2.93 in the case of a mycorrhizal plant, a saving of 30%.
4. Discussion
The result of the present study demonstrated that indigenous mycorrhizal
endophytes established well in the targeted plants and the occurrence of these
endophytes in plant roots and soil varied with host plant. The data also indicate
the potential role of AM fungi in the growth and mineral nutrition of the host
plants tested.
In both the experiments, the soil used as a substrate was low in P (except in the
treatment with chemical fertilizers). However, mycorrhization by the indigenous
AM consortia enhanced vegetative growth, nutrient uptake (P and K) and ¯ower
production in all the three ornamental spp. tested. Several workers, namely
160
A. Gaur et al. / Scientia Horticulturae 84 (2000) 151±162
Boerner (1990), Davies (1987), Gianinazzi et al. (1989), Gianinazzi et al. (1990),
and Jeffries and Dodds (1991) have reported similar results in a number of
ornamental plants. In Experiment 2, though the application of chemical fertilizers
led to increased uptake of nutrients (non-signi®cant), the plants produced fewer
¯owers. This can perhaps be explained by the common observation that luxurious
consumption does not help in better ¯owering.The extent of colonization was
substantially higher in all the three host species with P. hybrida showing the
highest value. It is likely that P. hybrida relies more on AM fungi for the uptake
of phosphorus than I. balsamina and C. chinensis. Also, it was observed that these
horticultural plants have a fairly well-developed ®ne root system, offering a larger
surface area for the AM to colonize. By contrast, our observation of high MCP
values in all three hosts in both the experiments supports the hypothesis of Baylis
(1970) that plant with poorly developed ®ne root system may be mycotrophs in
P de®cit soils.
Mycorrhizal inoculation improves P (Sharma et al., 1996) and K (Johnson
et al., 1980) uptake because roots are supplemented with the AM fungal hyphae
in tapping soil resource (Abbot and Robson, 1982). This is shown in the present
study by the increased P and K recovery in the inoculated plants as compared to
the uninoculated controls. The inoculated plants were able to obtain greater
quantities of soil phosphorus and produce more plant dry matter. In addition,
increased K concentration in plants has been shown to increase the number of
¯owers (Dufault et al., 1990) and plant yield (Albregts et al., 1991).
Mycorrhizal inoculation had a pronounced in¯uence on the time required for
¯ower initiation, as studied in Experiment 1. Also, the infection led to prolonged
¯owering and produced signi®cantly greater number of ¯owers in the inoculated
plants than in their uninoculated counterparts. Our ®ndings are consistent with
those in many other species that the number of ¯owers produced by a plant is
proportional to plant size and nutrient content (Lee and Bazzaz, 1982).
Thus, the two experiments showed that these ornamental species performed
equally well with chemical fertilizers and mycorrhizal inoculation. Application of
fertilizers accounts for 30% of the cash expenses in nurseries (Thakur and
Panwar, 1997). This will continue to be high. With inoculation, the expenses on
phosphorus fertilizers could be reduced to 70%. Current levels of N, K and
micronutrients could also be reduced. Thus, there is a wide potential in exploiting
mycorrhizal fungi for improved establishment, survival, nutrient uptake and
growth of plants especially in nutrient de®cient soils.
Acknowledgements
The present study was supported by the Department of Biotechnology,
Government of India. Thanks are due to the Director of Tata Energy Research
A. Gaur et al. / Scientia Horticulturae 84 (2000) 151±162
161
Institute for providing support. Copy editing by Yateen Joshi is thankfully
acknowledged.
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Growth and ¯owering in Petunia hybrida, Callistephus
chinensis and Impatiens balsamina inoculated with
mixed AM inocula or chemical fertilizers
in a soil of low P fertility
Anupama Gaura, Atimanav Gaurb, Alok Adholeyac,*
a
Forestry and Agricultural Biotechnology Institute (FABI), Faculty of Biological and Agricultural
Sciences, University of Pretoria, Pretoria 0002, South Africa
b
Mycology Lab, Department of Botany, University of Pretoria, Pretoria 0002, South Africa
c
Microbial Biotechnology, Tata Energy Research Institute, India Habitat Centre,
Lodhi Road, New Delhi-110 003, India
Accepted 13 August 1999
Abstract
Three seasonal ornamental plants, namely Petunia hybrida, Callistephus chinensis and Impatiens
balsamina, were tested for their response to inoculation with mixed indigenous AM culture when
grown on a marginal wasteland amended with organic matter.
Subsequently, the treatments consisting of AM inoculations were compared with those consisting
of recommended chemical fertilizers in terms of growth response and cost economics. In all the
three plant species, mycorrhizal inoculation led to marked improvement in both reproductive
(number of ¯owers) and vegetative (dry matter) phase of the plants. P. hybrida showed a threefold
increase over uninoculated plants in the reproductive growth as compared to twofold in C. chinensis
and I. balsamina. Application of the recommended dose of chemical fertilizers produced a
comparable response.
The inoculated plants produced greater dry matter, grew taller, ¯owered at least 15 days earlier
and produced more ¯owers when compared to uninoculated plants. In addition there was a
signi®cant increase in P and K uptake in shoots of all the three ornamentals. AM inoculation could
be at least 30% cost economic as compared to the chemical fertilizers. Therefore, mycorrhizal
inoculation is recommended at the nursery level for nutrient-de®cient soil conditions because it is a
cost-effective substitute for chemical fertilizers, either partly or fully, which makes the approach
*
Corresponding author. Tel.: 91-11-460-1550; fax: 91-11-462-1770.
E-mail address: [email protected] (A. Adholeya).
0304-4238/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 2 3 8 ( 9 9 ) 0 0 1 0 5 - 3
152
A. Gaur et al. / Scientia Horticulturae 84 (2000) 151±162
particularly suitable to marginal farmers with their low-input farming system. # 2000 Elsevier
Science B.V. All rights reserved.
Keywords: Petunia hybrida; Impatiens balsamina; Callistephus chinensis; Indigenous AM
consortium; Organic amendment; Chemical fertilizers; Cost economics
1. Introduction
AM fungal inocula hold tremendous potential in increasing crop production as
an integral component of sustainable crop production systems (Bagyaraj, 1992).
They have been shown to confer a variety of bene®ts on their hosts including
growth and yield enhancement (Furlan, 1993). AM fungi are found in a wide
variety of habitats (Brundrett, 1991).
Wasteland are large stretches of land de®cient in nutrients and bene®cial
microorganisms and account for approximately 20% of India's total geographical
area (Sharma et al., 1996). Mycorrhizal fungi are likely to be bene®cial in
bringing these habitats under cultivation. The current study was designed to
assess, in economic and physical terms, the bene®t of cultivating economically
important ornamental and oil seed plants on such wastelands using bene®cial
microorganisms and to see how it compares with the cultivation based on
applying chemical fertilizers in terms of costs.
Ornamental plants are often grown from seedlings and cuttings grown in
disinfected soils or on inert substrates, mainly to lower the risk of contamination
and to ensure controlled conditions to obtain homogenous material. Such
activities and other horticultural practices that tend to eliminate AM fungi create
the conditions best suited to using mycorrhizal biotechnology (Johnson et al.,
1980). In addition, production of ornamental species in nutrient-de®cient arid
soils is more dif®cult because these plants generally have a high fertilizer
requirement. Adopting management practices to increase plant production with
low fertilizer input will minimize adverse effects on the environment and keep
production costs low, making it suitable for marginal farmers with low incomes.
AM fungi are known to increase plant growth in arid and semi-arid regions
(Hirrel and Gerdemann, 1980). Although many studies of host plant responses to
mycorrhizal infection have been performed using a single AM fungal isolate, the
present study used a mixed inoculum of AM fungi so as to simulate the natural
conditions more closely, where an assemblage of many species is more common
(Brundrett, 1991).
The current experiment was planned with the following objectives: (1) to study
the ef®cacy of an indigenous, mixed AM inoculum in infecting and promoting the
growth of selected ornamental plants; (2) to study the effect of mycorrhizal
A. Gaur et al. / Scientia Horticulturae 84 (2000) 151±162
153
inoculation on ¯owering, and nutrient uptake and compare; (3) the cost
effectiveness of using mycorrhizal inoculation and chemical fertilizers.
2. Materials and methods
2.1. Preparation of AM inocula
A mixed indigenous culture (containing native populations of Glomus,
Gigaspora and Scutellospora spp.) collected from the experimental site was
used as the AM inoculum and multiplied for 1 year in clay pots (5 kg capacity)
®lled with soil similar to the poting mixture, with Sorghum bicolor as the host
plant. At maturity, the tops of the sorghum plants were removed and substrate was
allowed to dry for a week at 25 58C. The roots were ®nely chopped and the
dried root/soil mixture was thoroughly mixed to obtain a homogeneous inoculum.
Spores were isolated by wet sieving and decanting (Gerdemann and Nicolson,
1963) and counted on a ®lter paper (Gaur and Adholeya, 1994). The percentage
of root colonization by mycorrhizae was assessed as described by Biermann and
Lindermann (1981) after staining the roots with acid fuchsin (Phillips and
Hayman, 1970). The total number of infectious propagules (IPs) per gram of
inoculum was also counted (Sharma et al., 1996) and the value was found to be 15
IPs/g. The colonization percentage was 62 and the spore density was 20 spores/g
inoculum.
2.2. Preparation of seedlings
Seeds of Petunia hybrida cv. blue bird, Callistephus chinensis cv. dwarf
chrysanthemum and Impatiens balsamina were surface sterilized with 10% H2O2
for 5 min. Subsequently, the seeds were washed repeatedly with sterile water and
kept for germination on moist sand in sterile petriplates at 308C in dark for 48 h.
On germination, the seedlings were given half-strength Hoagland solution
(Hoagland and Arnon, 1950) for even 15 days.
2.3. Preparation of growth substrate
The experimental site is located at Gwal Pahari in Haryana state, India
(778120 E and latitude 288350 N) 255 m above the mean sea level and receives a
mean annual rainfall of 500 mm.
Two experiments were conducted. In Experiment 1, the poting mixture was
prepared by mixing two parts of soil (sandy loam Hyperthermic Typic Haplustalf,
pH 7.5, NO3N 124 mg/kg, available P 0.53 mg/kg, available K 124 mg/
kg) from the experimental site and one part of compost (made from leaves of
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Table 1
Soil characteristics of the substrate used in both the experiments
Substrate
pH
Phosphorus
(ppm)
Nitrogen
(ppm)
Potassium
(%)
With chemical fertilizers
With organic manure only
7.3
7.4
30
2.4
128
72
4
2.6
Albizzia and Poplars, pH 7.2, available P 12 ppm, organic C 11.40% and
N 0.75%). The compost amended substrate showed a pH of 7.3, available
P 2.5 ppm (Olsen et al., 1954), and organic C 3.46% (Datta et al., 1962). The
substrate was autoclaved before inoculation (1218C for 1 h at 15 psi). Clay pots,
®lled with 5 kg of substrate, were used in the experiment.
In Experiment 2, the same potting mixture was used and one set of pots was
given chemical fertilizers in the form of SSP (single super phosphate),
micronutrients (commercially available), ammonium nitrate and potassium
chloride at the recommended levels (Table 1).
2.4. Experiment layout and mycorrhizal inoculation
Experiment 1 followed a completely randomized design consisting of a 3 2
structure three types of ornamental plants and two treatments (inoculated and
uninoculated). Each treatment was replicated six times. The substrate was
inoculated by thoroughly mixing the crude inoculum with soil in each pot (at
2000 IP per pot). The inoculum consisted of spores, hyphae and infected root bits.
Five kg of soil containing the inoculum mixture was transferred to earthen pots
(17 cm diameter). Uninoculated plants served as controls.
Experiment 2 also followed a completely randomized design consisting of a
3 3 structure (having an additional treatment, inoculated, uninoculated and
uninoculated with chemical fertilizer) each treatment was replicated six times.
Six 15-day-old germinated seedlings of each host were selected for uniformity
and transplanted to the pots. All the plants were grown under 70% daylight in a
greenhouse and watered to maintain the soil at 60% of its water holding capacity.
The pots were rotated regularly to avoid any positional effect. All the parameters
were analysed at harvest except the ¯ower count, which was carried out as
described below.
2.5. Observation, harvest and analysis
Flowers were counted (non-destructively) at 10 days intervals for all the six
replicates in each treatment for 120 days (Experiment 1 and 2). Shoots were
severed just above the crown, weighed while fresh, rinsed in distilled water, dried
A. Gaur et al. / Scientia Horticulturae 84 (2000) 151±162
155
at 708C for 48 h and weighed again, which were then ground ®ne enough to pass
through a 0.5 mm screen and digested in H2SO4. The P and K content in the
digest was determined chemically (Jackson, 1973) and using ¯ame photometry
(Chapman and Pratt, 1978).
The harvested roots were washed clean or cut into 1 cm segments, and
homogenized thoroughly. Samples of root segments were analysed for
mycorrhizal colonization. Colonization percentage was determined on 100 root
segments from each sample. Roots were stained using the method of Phillips and
Hayman (1970). Root pieces were mounted between the glass slides and
examined under a microscope at X40 for AM hyphae, arbuscules, vesicles and
spores. The extent of colonization was assessed by using the method of Biermann
and Lindermann (1981) and expressed as the percent of root segment colonized
for each root piece (Experiment 1 and 2). Spores of AM fungi were extracted
(Gerdemann and Nicolson, 1963) from 50 ml samples of the homogenized
substrate from three replicates for each treatment (Experiment 1). The spores
retained on different sieves were collected in a beaker and recovered by sucrose
density centrifugation. Only visually intact spores were counted under a
stereoscopic microscope (Gaur and Adholeya, 1994). The average number of
spores in 50 ml of the substrate soil was used to estimate the spores per gram soil
(gÿ1 soil) in each of the treatments. Sporocarps were gently crushed to count the
number of spores in each sporocarp.
2.6. Statistical analysis
The data were analysed using one-way ANOVA using the least signi®cant
difference (Duncan's multiple range test at 5% signi®cant level). The data were
also analysed for observing the standard deviation within the treatments using
Costat software (Cohort, Berkeley, CA, USA).
3. Results
3.1. Flowering
The inoculated plants ¯owered signi®cantly earlier than uninoculated controls
in both the experiments though the difference was not signi®cant in Experiment 2.
The inoculated plants of C. chinesis ¯owered 27 DAT (days after transplanting),
whereas the uninoculated plants took 22 days longer. Those of I. balamina
¯owered 37 DAT, which was 16 days earlier than uninoculated plants and those of
P. hybrida ¯owered 29 DAT, 12 days before the uninoculated plants (Experiment
1, Table 2). The inoculated host plants not only ¯owered earlier but also produced
signi®cantly more ¯owers, 190%, 106% and 75% more in P. hybrida, I.
balsamina and C. chinensis, respectively (Fig. 1).
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A. Gaur et al. / Scientia Horticulturae 84 (2000) 151±162
Table 2
In¯uence of inoculation with an indigenous arbuscular mycorrhizal (AM) consortium on growth
and nutrient uptake of the three ornamentals (Experiment 1)a
Hosts
MCP
(%)
AM propagules
Spores
IPd
ÿ1
(g soil) (gÿ1 soil)
Flower
initiation
(DATe)
Nutrient uptake
Shoot P
(mg gÿ1)
Shoot
K (%)
Shoot
height
(cm)
Dry
matter
(g/plant)
P. hybrida
AMb
67.3
NMc
±
8.6a
±
6.5a
±
29
41
4.5a
3.17b
6.21a
5.32b
70.57a
64.53b
0.84a
0.64b
I. balsamina
AM
59.8
NM
±
6.9b
±
6.0b
±
37
53
2.84a
2.54b
5.0a
4.02b
53.06a
33.12b
0.72a
0.62b
C. chinensis
AM
51.6
NM
±
6.7b
±
5.3c
±
27
49
2.65a
2.07b
5.33a
3.72b
16.01a
11.50b
0.34a
0.27b
a
Means followed by the same letters are not signi®cantly different (p < 0.05).
AM Ð mycorrhizal.
c
NM Ð non-mycorrhizal.
d
IP Ð infectious propagule.
e
DAT Ð days after transplanting.
b
In Experiment 2, the number of ¯owers in AM inoculated P. hybrida was
signi®cantly higher, though the plants that had received chemical fertilizers also
produced more ¯owers, as compared to control plants (Fig. 2).
3.2. Plant dry matter
In both the experiments, dry matter was signi®cantly higher in the inoculated
plants than in their uninoculated counterparts (Tables 2 and 3).
3.3. Nutrient uptake
In Experiment 1, the P and K content of the three hosts tested was signi®cantly
higher in the inoculated plants (Table 2). In Experiment 2, both the AM fungal
inoculation and addition of chemical fertilizer increased P and K uptake over
the uninoculated controls, though the increase was greater in the later plants
(Table 3).
3.4. Mycorrhizal parameter
In both the experiments, AM consortia produced the maximum colonization in
P. hybrida followed by I. balsamina and C. chinensis (Tables 2 and 3). In terms of
A. Gaur et al. / Scientia Horticulturae 84 (2000) 151±162
157
Fig. 1. Effect of mycorrhizal inoculation on the number of ¯owers produced by: (a) P. hybrida; (b)
I. balsamina; (c) C. Chinensis in Experiment 1. Error bars denote standard errors of mean.
158
A. Gaur et al. / Scientia Horticulturae 84 (2000) 151±162
Fig. 2. Effect of mycorrhizal inoculation on the number of ¯owers produced by: (a) P. hybrida; (b)
I. balsamina; (c) C. chinensis in Experiment 2. Error bars denote standard errors of mean.
159
A. Gaur et al. / Scientia Horticulturae 84 (2000) 151±162
Table 3
In¯uence of inoculation with an indigenous arbuscular mycorrhizal (AM) consortium on the growth
and nutrient uptake of three ornamentals (Experiment 2)a
Hosts
P. hybrida
AM
CFb
NM
I. balsamina
AM
CFa
NM
C. chinensis
AM
CF
NM
a
b
MCP (%)
Nutrient uptake
Shoot
height (cm)
Dry matter
(g/plant)
Shoot P
(mg gÿ1)
Shoot
K (%)
66.8
±
±
4.36b
5.52a
3.21c
6.18b
6.52a
5.27c
70.43b
75.62a
65.3c
0.86b
0.91a
0.63c
59
±
±
2.89b
3.15a
2.58c
5.08b
5.21a
4.11c
54.96b
58.32a
35.16c
0.72a
0.72a
0.62b
52.2
±
±
2.69b
3.1a
2c
5.37b
5.52a
3.68c
16.8b
17.32a
11.8c
0.34a
0.33b
0.28c
Means followed by the same letters are not signi®cantly different (p < 0.05).
CF Ð chemical fertilizer.
AM multiplication (number of spores and infectious propagules), the trend was
the same, with maximum multiplication in P. hybrida followed by I. balsamina
and C. chinensis (Table 2).
3.5. Cost analysis
Application of chemical fertilizers proved to be 30% more expensive than
inoculation. The total cost of growing a chemically fertilized plant was $4.15,
compared to $2.93 in the case of a mycorrhizal plant, a saving of 30%.
4. Discussion
The result of the present study demonstrated that indigenous mycorrhizal
endophytes established well in the targeted plants and the occurrence of these
endophytes in plant roots and soil varied with host plant. The data also indicate
the potential role of AM fungi in the growth and mineral nutrition of the host
plants tested.
In both the experiments, the soil used as a substrate was low in P (except in the
treatment with chemical fertilizers). However, mycorrhization by the indigenous
AM consortia enhanced vegetative growth, nutrient uptake (P and K) and ¯ower
production in all the three ornamental spp. tested. Several workers, namely
160
A. Gaur et al. / Scientia Horticulturae 84 (2000) 151±162
Boerner (1990), Davies (1987), Gianinazzi et al. (1989), Gianinazzi et al. (1990),
and Jeffries and Dodds (1991) have reported similar results in a number of
ornamental plants. In Experiment 2, though the application of chemical fertilizers
led to increased uptake of nutrients (non-signi®cant), the plants produced fewer
¯owers. This can perhaps be explained by the common observation that luxurious
consumption does not help in better ¯owering.The extent of colonization was
substantially higher in all the three host species with P. hybrida showing the
highest value. It is likely that P. hybrida relies more on AM fungi for the uptake
of phosphorus than I. balsamina and C. chinensis. Also, it was observed that these
horticultural plants have a fairly well-developed ®ne root system, offering a larger
surface area for the AM to colonize. By contrast, our observation of high MCP
values in all three hosts in both the experiments supports the hypothesis of Baylis
(1970) that plant with poorly developed ®ne root system may be mycotrophs in
P de®cit soils.
Mycorrhizal inoculation improves P (Sharma et al., 1996) and K (Johnson
et al., 1980) uptake because roots are supplemented with the AM fungal hyphae
in tapping soil resource (Abbot and Robson, 1982). This is shown in the present
study by the increased P and K recovery in the inoculated plants as compared to
the uninoculated controls. The inoculated plants were able to obtain greater
quantities of soil phosphorus and produce more plant dry matter. In addition,
increased K concentration in plants has been shown to increase the number of
¯owers (Dufault et al., 1990) and plant yield (Albregts et al., 1991).
Mycorrhizal inoculation had a pronounced in¯uence on the time required for
¯ower initiation, as studied in Experiment 1. Also, the infection led to prolonged
¯owering and produced signi®cantly greater number of ¯owers in the inoculated
plants than in their uninoculated counterparts. Our ®ndings are consistent with
those in many other species that the number of ¯owers produced by a plant is
proportional to plant size and nutrient content (Lee and Bazzaz, 1982).
Thus, the two experiments showed that these ornamental species performed
equally well with chemical fertilizers and mycorrhizal inoculation. Application of
fertilizers accounts for 30% of the cash expenses in nurseries (Thakur and
Panwar, 1997). This will continue to be high. With inoculation, the expenses on
phosphorus fertilizers could be reduced to 70%. Current levels of N, K and
micronutrients could also be reduced. Thus, there is a wide potential in exploiting
mycorrhizal fungi for improved establishment, survival, nutrient uptake and
growth of plants especially in nutrient de®cient soils.
Acknowledgements
The present study was supported by the Department of Biotechnology,
Government of India. Thanks are due to the Director of Tata Energy Research
A. Gaur et al. / Scientia Horticulturae 84 (2000) 151±162
161
Institute for providing support. Copy editing by Yateen Joshi is thankfully
acknowledged.
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