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Soil Science and Plant Nutrition

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Differential phosphorus requirements of Azolla


species and strains in phosphorus-limited
continuous culture

Budunkila Prakash Rao Subudhi & Iwao Watanabe

To cite this article: Budunkila Prakash Rao Subudhi & Iwao Watanabe (1981) Differential
phosphorus requirements of Azolla species and strains in phosphorus-limited continuous culture,
Soil Science and Plant Nutrition, 27:2, 237-247, DOI: 10.1080/00380768.1981.10431275

To link to this article: http://dx.doi.org/10.1080/00380768.1981.10431275

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Soil Sci. Plant Nutr., 21 (2), 237-247, 1981

DIFFERENTIAL PHOSPHORUS REQUIREMENTS OF


AZOLLA SPECIES AND STRAINS IN PHOSPHORUS-
LIMITED CONTINUOUS CULTURE

Budunkila Prakash Rao SuBUDHI and Iwao WATANABE


The International Rice Research Institute,
Los Baflos, Laguna, Philippines

Received October 17, 1980

Phosphorus represents a major limiting factor in the field for the growth of the Azalia-
Anabaena symbiotic nitrogen-fixing system.
The yield of fresh matter increased up to S ppm P in batch culture, following a quadratic
type of relationship. However, the tissue phosphorus content increased with each incremental
increase in phosphorus concentration. The threshold concentration of the phosphorus in
Azolla appeared to be 0.2 to 0.3%. Below that, plant growth was proportional to the phos-
phorus content.
A continuous-flow device for determining the minimum phosphorus level was developed.
Azolla pinnata from Bangkok grew normally at 0.06 ppm P. At 0.03 ppm P, the biomass,
contents of phosphorus, nitrogen, and chlorophyll, and the acetylene-reduction activity were
decreased. Further phosphorus increases of more than 0.06 up to 0.12 ppm in the medium
had no impact on the growth of Azo//a.
Eleven Azolla strains, including A. pinnala, A. mexicana, A. caro/iniana, and A.fi/iculoides,
were compared at the limiting concentration of phosphorus (0.03 ppm) in continuous-flow
culture. Substantial differences among the species and strains were noted. For healthy
growth, different species required widely differing phosphorus concentrations in the water
medium. A. pinnata from Bangkok produced maximum yield and maximum frond area,
and exhibited the better nitrogen-fixing capacity in the phosphorus limiting condition than
did the other species compared. Other A. pinnata strains grew better in the phosphorus
limited condition than the New World Azo/la species.
Key Words: Azo/la, phosphorus nutrition, nitrogen fixation.

Recent detailed field studies in various countries have substantiated the potential
of Azo/la in rice cultivation (5-7, 13-17). Information on the nutrient requirements
of Azolla is essential in work aimed at improving the growth of Azolla in paddy fields.
However, the data on the mineral nutrition of Azolla are limited (1, 12, 15, 18, 19).
Field studies show that the addition of phosphorus is most effective in stimulating
the growth of Azol/a in the field (4, 7, 8, 17, 18). The threshold concentration of
phosphorus in the water and in the Azolla tissue must be studied in order to under-
stand the phosphorus nutrition of Azolla. Several authors (ll, 12, 19) have employed
batch culture to determine the phosphorus requirement.
237
238 B.P.R. SUBUDHI and I. WATANABE

The minimum phosphorus requirement for Azolla is more accurately determined


by continuous-flow culture than by batch culture. In the batch culture, the supply
of a nutrient depends on the concentration, volume, and frequency of renewal of the
water culture media. The amount of the nutrient is sufficient at the beginning but
becomes deficient later because of the increased requirement of the exponentially
growing biomass (3). The optimum concentration in water obtained by batch culture
is much higher than that found in the natural environment. In soil-water systems,
the phosphorus in the water is maintained at a low concentration, but is continuously
supplied from the solid phase. Continuous-flow culture can simulate the phosphorus
status of a soil-water system. Based on these considerations, we conducted contin-
uous-flow culture of Azolla to determine the minimum phosphorus requirement and
to evaluate the differences in phosphorus requirement among Azolla species or strains.
Part of the results reported here have been described previously (10).

MATERIALS AND METHODS

Experiment ]-Phosphorus requirement in batch culture.


The effects of four phosphorus rates (0, 2.5, 5, and 10 ppm) on three Azo/la pinnata
R. Br. strains from Bangkok (Thailand), Sri Nagor (Bangladesh) and Cuttack (India)
were studied.
The Azolla inoculum was starved by keeping it in demineralized water for 5-6
days before inoculating it into the test medium. One-half gram of fresh, starved
Azo/la was inoculated into each 318-cm2 tray containing 1 liter of medium. Phos-
phorus-free mineral medium was prepared using 40% Hoagland's nitrogen-free solu-
tion with the following composition (in g liter- 1): CaCI 2 2H20, 22.1; KCl, 14.9;
MgS0 4 7H20, 19.7; MnS04 7H20, 0.05; ZnS0,7H 20, 0.05; CuS0,5H20, 0.02;
H 3B03 , 0.5; Mo0 3 , 0.01; CoC1 2 , 0.04; and Fe-EDTA, 5. The pH was adjusted to
5.5. Three trays were used for each treatment. The medium was changed every 3
days. The experiment was carried out in a greenhouse. After 12 days, the plants
were washed and blotted to dry, and their fresh weight, doubling time, and phosphorus
content were determined.
Experiment 2-Minimum level of phosphorus requirement determined by continu-
ous-flow culture.
All culture vessels and glassware used in the experiment were washed with I : 1
HCl and then rinsed 3 to 4 times with demineralized water. Three reservoirs, each
containing a 50-liter culture solution of one fifth Hoagland's nitrogen-free medium
with 2.5 ppm Fe as Fe-EDTA and 0.25, 0.5, and 1 ppm phosphorus were connected to
318-cm2 culture trays. The solution flowed through dextrose intravenous solution
administration tubes, covered with aluminum foil to prevent algal growth. The outlet
hole was situated so that about 1 liter of culture solution was maintained in each tray.
The outgoing solution was received into a sink tank. The culture solution was peri-
odically returned to the reservoirs. The flow rate of the culture medium was adjusted
Phosphorus Nutrition of Azo/la 239

with a regulator. Preliminary studies were made with 1, 0.5, and 0.25 ppm P at a
flow rate of 1 liter he 1 using Azalia pinnata Kumaltar. Detailed studies with more
dilute phosphorus solutions (0, 0.03, and 0.06 ppm P) were conducted with A. pinnata
Bangkok.
Because of the dilute solution, the flow rate was kept sufficiently high to ensure
that the concentration remained constant as it passed over the roots. At 0.06 ppm P,
the flow rate was 800 mi hr- 1 (48 pg P hr- 1); and at 0.03 ppm P, it was 1,000 ml hr-1
(30 pg p bel). In the nonphosphorus control, the flow rate was 1,000 ml hr- 1. Each
unit was drained and refilled with fresh solution 3 times during the experiment.
Azalia pinnata Bangkok was grown in a phosphorus-free medium for 3 days before
the experiment in a greenhouse, and 1.5 g fresh weight was inoculated into each tray.
The phosphorus content of the inoculum was 0.16% on a dry matter basis. After 11
days, the plants were washed and blotted to dry, and their fresh weight and area were
determined. The acetylene reduction and chlorophyll content were determined. Ni-
trogen and phosphorus were analyzed after drying.
Experiment 3-Growth characteristics of different Azalia species and strains at 0.03
ppm P by continuous-flow culture.
The strains used in this study (Table 1), selected from an Azalia collection from
different countries, are maintained by the IRRI Soil Microbiology Department. The
experiment was conducted in a greenhouse to select Azalia strains which grew best at
a low phosphorus level (0.03 ppm). The experimental set-up was similar to that of
experiment 2 with 3 replicated plates. The strains of Azalia were evaluated with A.
pinnata from Bangkok as reference. The flow rate was maintained at 1,000 ml hr-1
(30 pg P hr- 1).

Table 1. Azo/la species and strains used in the experiments.

Species Strain designation Site of collection Year of collection

A. pinnata R. Brown Bangkok Bangkok, Thailand 1977


Kumaltar Kumaltar, Nepal 1978
Vietnam greena Hanoi, Vietnam 1978
Malaysia Bumbong Lima, Malaysia 1977
Cuttack Cuttack, India 1978
Pu Tiang Zhu Fujieng, China 1979
Vietnam wild Tai Yin, Vietnam 1979
Sri Nagor Sri Nagor, Dacca, Bangladesh 1978
Bogor Bogor, Indonesia 1977
A. mexicana Prest. California Glaylodge, California, U.S.A. 1978
A. filicu/oides Lamarck Hawaii Hawaii, U.S.A. 1977
A. caro/iniana Wild Ohio Ohio, U.S.A. 1978

a Evergreen variant.
240 B.P.R. SUBUDHI and I. WATANABE

The growth and phosphorus absorption ability of the test Azolla species in con-
tinuous-flow culture at the limiting level of 0.03 ppm P were also compared.
Analytical procedures.
Chlorophyll: Chlorophyll was extracted by grinding I g of blotted Azolla with
45 ml acetone and centrifuging at 1,000 rpm for 30 min. The chlorophyll was meas-
ured at 652 nm in a Hitachi model 200-20 double-beam spectrophotometer (20).
Plant area: The fresh, blotted Azolla was placed on graph paper, its outline was
traced and the area calculated.
Morphology of Anabaena azollae: Plants were dissected and the individual leaves
were teased with needles and examined under a microscope in order to determine the
morphology of Anabaena azollae (9).
Nitrogenase activity: Nitrogenase activity was determined by acetylene reduction
assay. Harvested plants of fresh, blotted Azolla (0.3 g) were placed in 25 ml Corning
flasks fitted with rubber septa and containing 5 ml of distilled water. Ten percent of
the gas in the flasks was replaced by acetylene and placed in a constant environment
of 30 klux light at 26C for 30 min.
Analysis of total nitrogen and phosphorus: The nitrogen was analyzed by the
micro-Kjeldahl method and the phosphorus by the stannous chloride method after
dry ashing (20).

RESULTS AND DISCUSSION

Experiment 1-E.ffect of varying phosphorus concentration on growth


Table 2 shows the fresh matter production and phosphorus content in the dry
matter of three Azolla pinnata strains after 12 days of growth in doses of phosphorus
varying from 0 to I0 ppm.
Phosphorus deficiency limits Azolla growth. Without phosphorus, the growth of
all varieties was severely affected. With phosphorus, the fresh matter production
increased from the lower to the higher doses up to 5 ppm P, following a quadratic
type of relationship (Table 2). At 2.5 ppm P, the collection from Cuttack exhibited
deficiency symptoms-browning, fragility, and poor growth. However, other strains
showed no deficiency symptoms and remained green, suggesting that the Cuttack
strain may require more phosphorus or is more susceptible to a low-phosphorus con-
dition.
The phosphorus content in Azolla increased linearly with increased external phos-
phorus concentration (0 to 10 ppm) in all strains (Fig. I). Similar observations have
been made by SuBUDHI and SINGH (1 2). Increasing the phosphorus to more than 5
ppm in medium did not increase the growth significantly, indicating a luxury consump-
tion of phosphorus. From Table 2 and Fig. 1, it appears likely that the fern growth
was not increased by phosphorus application when the phosphorus content exceeded
0.3%. The A. pinnata collected from Bangkok revealed the highest phosphorus con-
tent among the three strains at the four given levels. A I2-day phosphorus starvation
Phosphorus Nutrition of Azolla 241

Table 2. Effect of varying doses of phosphorus in batch culture on the fresh matter.

Variety P level Fresh mattera (g) Doubling time (days)

A. pinnata Bangkok 0 4.10.45 4.0


2.5 20.00. 75 2.43
5 22.01.15 2.39
10 22.50.32 2.35
A. pinnata Cuttack 0 2.60. 70 5.46
2.5 9.60.47 3. 10
5 19. 70. 93 2.45
10 20.00.84 2.44
A. pinnata Sri Nagor 0 3. 50. 06 4.61
2.5 15.1 0. 53 2.64
5 17. 30. 50 2.54
10 18.60.11 2.49

a Average and standard error.

0.9.------------,

CJ)
:::>
_gc. 0.3
CJ)
0
.c
a..

Phosphorus (ppm)
Fig. 1. Effect of phosphorus in batch culture on the phosphorus content in the dry matter.

led to 10-12 times lower phosphorus contents than those in plants treated with 5 ppm P.
The visible symptoms of phosphorus deficiency were reduced growth, decreased
frond size, fragile fronds, browning of roots, and marked browning of dorsal and
ventral lobes. Phosphorus-deficient plants also showed reduction in the number of
heterocysts of the symbiont Anabaena azol!ae, a fainter color, and decreased chlorophyll
content and nitrogenase activity.
242 B.P.R. SUBUDHI and I. WATANABE

Experiment 2-Minimum level of phosphorus in water for growth of Azolla


Preliminary continuous-flow culture experiments (Table 3) with varied phosphorus
concentrations of 1, 0.5, and 0.25 ppm revealed no phosphorus deficiency symptoms
in Azolla. However, the fresh matter production gradually decreased with increased
dose of phosphorus. The phosphorus content in the Azolla body also increased with
the increased dose. The minimum phosphorus level in the water was determined
by further decreasing the phosphorus level, using A. pinnata from Bangkok as the test
plant.
The growth at 0 and 0.03 ppm P was lower by 63 and 22%, respectively, than
that at 0.06 ppm P (Table 4). The Azol/a fronds were smaller and browner and root
tip detachment was more apparent in the non-P treatment than in the 0.03 pp P treat-
ment. Fronds grown at a 0.06-ppm P concentration were larger and greener, and
exhibited less root tip detachment than those at 0.03 ppm. The browning of the
ventral lobes was the most severe in the non-P treatment, less in the 0.03 ppm P treat-
ment, and least in the 0.06 ppm P treatment. The growth rate and nitrogen content
of the fern body suggested that Azolla grows normally at 0.06 ppm P. The fresh
matter yield did not increase (Table 5) with further increase of the phosphorus con-
centration up to 0.09, or 0.12 ppm in the external medium.
The heterocysts of Anabaena azollae were paler, thinner, and less numerous in
phosphorus-deficient plants grown at 0 and 0.03 ppm P than in healthy plants.
Table 4 shows the change in chlorophyll, nitrogen and phosphorus and the nitro-
genase activity of phosphorus-deficient Azolla grown in 0 and 0.03 ppm P. The phos-

Table 3. Effect of phosphorus on the growth of Azol/a pinnata from Nepal in


continuous-flow culture (initial inoculum 0.5 g, cultured for 11 days).

P level (ppm) Fresh weight (g) Doubling time (days) P % in dry matter

0.25 15.96 2.20 0.13


0.50 14. 76 2.25 0.22
1.00 12.30 2.38 0.34

Table 4. Influence of low phosphorus levels on the growth, composition, and nitrogen
fixation of Azol/a pinnata from Bangkok in continuous-flow culture. a

nmol of
P level Fresh Doubling Frond mgCh g-1 N%in P%in C1 H,g-1
(ppm) weight time size Color of ventral of fresh dry dry fresh
(g) (days) (cm2) lobe of each leaf Azol/a matter matter Azol/a
hrl

0 11.5 3.75 0.38 Dark brown 0.34 2. 78 0.028 200


0.03 24.0 2.75 0.47 Brown 0.58 3.30 0.060 290
0.06 30.8 2.52 1.00 Greenish white 0. 74 4.53 0.105 370

a The initial inoculation had 0.16% of Pin the dry matter.


Phosphorus Nutrition of Azol/a 243

Table 5. Effect of phosphorus on the growth of Azol/a pinnata from Bangkok


in continuous-flow culture.

P level Fresh weight Doubling time Frond size Color of ventral


(ppm) (g) (days) (cm2) lobe of each leaf

0.06 29.30 2.72 0.92 Green


0.09 29.39 2.72 0.90 Green
0.12 28.16 2. 75 0.95 Green

phorus uptake of Azolla in the 0.03 and 0.06 ppm P treatments amounted to 7.6 and
11.8% of the totalsupply to the trays. It appears likely, therefore, that the total
supply of phosphorus was adequate but its concentration was limiting. A small
amount of phosphorus was taken up, probably through contamination by Azolla grown
in the 0 ppm treatment.
Our results indicate that the growth-limiting phosphorus concentration for Azolla
pinnata is below 0.06 ppm or 2 pM. Recent studies in the field by WATANABE et al.
(17) revealed that the growth of Azolla was improved with split application of phos-
phorus fertilizer when the phosphorus content in the floodwater was maintained at
about 0.3-1.0 mg P 20sliter- 1 (0.13-0.43 ppm P).
When Azolla was deficient in phosphorus without phosphorus fertilization, the
floodwater contained between 0.05 and 0.3 mg P 20 5 litec 1 (0.022-0.13 ppm P). The
threshold value of the phosphorus concentration in the water indicated by this experi-
ment is likely to be close to the value predicted by analysis of the paddy field flood-
water. RAINS and TALLEY (6) found that 0.8 mg P 20 5 liter- 1 (0.34 ppm P) in the
floodwater was required for maximal growth of Azolla mexicana and Azollafiliculoides.

Experiment 3-Growth characteristics of Azolla strains at 0.03 ppm phosphorus in con-


tinuous-flow culture
Fresh matter yield. Table 6 shows the fresh matter yields of 11 Azolla strains
after 11 days growth in flow culture with 0.03 ppm P. A wide diversity in response
to phosphorus was observed among the strains and species. All the strains showed
slight or severe symptoms of phosphorus deficiency. A. pinnata from Bangkok yielded
the highest amount of fresh matter. The remaining species and strains yielded from
77% (A. pinnata Kumaltar) to 13% (A. caroliniana Ohio) of that of the Bangkok strain.
The doubling time varied from 2.78 days in A. pinnata from Bangkok to 9.01 days
in A. caroliniana (Table 6).
Browning of dorsal and vental lobes. Dorsal lobe browning was inversely related
to growth. The browning was more intense in A. mexicana, A. caroliniana and A.
filiculoides, and much less in A. pinnata Bangkok and A. pinnata Kumaltar. At the
phosphorus level of 0.03 ppm, all strains except A. mexicana exhibited browning of
the central lobes, which spread to the peripheral lobes in all species. In A. pinnata
from Cuttack, however, the browning started in the central lobes, then the plant de-
244 B.P.R. SUBUDHI and I. WATANABE

Table 6. Fresh weight, doubling time, frond area, and root characteristics of different
Azolla species and strains at 0.03 ppm P in continuous-flow culture.

Doubling Frond Root character


Species Fresh matter time size Old roots New roots
(days) (cm 2) fallen observed

A. pinnata Bangkok 23.63.1* 2.78 0.47 a a


Kuma! tar 20.3** 2.92 0.39 a a
Vietnam green 15.0 3.31 0.41 a a
Malaysia 14.5 3.36 0.43 a*** a
Cuttack 11.9 3.69 0.07 a c
Pu TiangZhu 10. 7 3.88 0.40 a a
Vietnam wild 10. 7 3.88 0.35 a*** a
Sri Nagor 9. 7 4.09 0.28 a a
Bogor 8.5 4.38 0.23 a*** a
A. mexicana California, U.S.A. 7.8 4.56 0.37 a*** b
A. filiculoides Hawaii 5.3 6.01 0.36 b c
A. caroliniana Ohio 3.5 9. 01 0.26 b c

a, Phenomena found; b, Few new roots observed; c, Phenomena not found.


*Average of 5 trials each with 3 replicates and the standard deviation. **Average of 3 replicates.
***Some old roots did not fall.

tached into small pieces and turned brown. In A. mexicana, it began in the peripheral
lobes and advanced to the central lobes.
Browning of the ventral lobes was observed in all species except A. pinnata
Vietnam green, but some of its central dorsal lobes exhibited browning.
Roots and root tips. Differences in root morphology were observed during the
experiment. Two to 3 days after inoculation into the medium, the roots turned brown.
Then the root cap and, subsequently, the root itself became detached. There was
simultaneous development of young roots with a large capsulated root cap without
any root hairs. After 1 or 2 days, these young capsulated roots turned into active
roots with many root hairs. However, deviations were noted in certain Azolla plants
(Table 6). In A. pinnata from Malaysia, Bogor, and Sri Nagor, some of the old roots
did not fall although new ones were formed. No new root formation was seen in
A. caro/iniana or A. fi/iculoides, although curling tips were observed in the old roots
in contrast to the other strains that maintained straight roots. In A. pinnata from
Cuttack, all the old roots were detached and no new roots were formed.
Frond area. Those strains which grew better at the limiting phosphorus con-
centration generally had larger fronds (Table 6). With phosphorus deficiency, the
central dorsal lobes turned brown, dried up, and fell apart, thereby reducing the frond
size. The A. pinnata from Cuttack had the smallest fronds, while the A. pinnata from
Bangkok had the largest {Table 6). Similar observations with the Cuttack strain were
Phosphorus Nutrition of Azolla 245

made in batch-culture studies.


Microscopic observations. At the end of each experiment, the brown lobes were
ruptured under a microscope and the morphology of the alga Anabaena azollae (9)
was investigated. In all species they were light colored, the number of heterocysts
had decreased, and the cell contents had shrunk. SUBUDHI and SINGH (12) reported
similar results for phosphorus-deficient plants. The phosphorus-deficient vegetative
algal cells in A. filiculoides were 10 times (40--50 nm) as long as normal vegetative cells
(4-5 nm). The number of heterocysts is dependent on the C : N ratio in the plant
(2). At a 0.03-ppm phosphorus concentration, the probable alteration of the C : N
ratio in the brown lobes led to the disappearance of heterocysts.
Phosphorus uptake, nitrogen content, and nitrogen fixation. The phosphorus uptake
varied from 0 to 6.8% from the supplied solution (Table 7). The A. pinnata from
Bangkok showed the highest fresh matter yield with a generation time of 2.8 days
and a phosphorus uptake of 6.8% from the solution. A. filiculoides and A. caroliniana
had 0 and 1.1% uptakes, respectively. The highest yields were associated with the
highest phosphorus uptakes.
Strains with more than 3% body N had higher yields and nitrogen fixation; those
with below 3% body N showed severe phosphorus deficiency and less nitrogen-fixation
capacity. The lowest nitrogen fixation was 0.05 pmol C2H 4 g- 1 fresh weight hr- 1 in
the Azolla pinnata from Cuttack, the strain with the smallest frond size. The nitrogen
fixation in Azolla fi/iculoides was 0.095 pmol C 2H 4 g- 1 fresh weight hr- 1 A low rate
was attributable to the deformed cells of symbiotic alga.
Considerable differences exist among species of the genus Azolla as regards growth
and nitrogen fixation. The species used in this experiment could be separated into
two major categories with respect to their efficiency in using phosphorus at a limiting
level: A. pinnata species, on the one hand, grew well, whereas the New World species
like A. mexicana, A. caroliniana, and A. filiculoides could not grow satisfactorily. A.

Table 7. Phosphorus uptake, nitrogen content, and nitrogenase activity of different


species and strains at 0.03 ppm P in continuous-flow culture.

%Pin % P uptake %Nin pmol C 8H, g- 1


Species dry matter from dry matter fresh Azo/la hr 1
solutions.

A. pinnata Bangkok 0.064 6.8 3.66 0.58


(reference)
Kumaltar 0.05 5.0 4.08 0.62
Malaysia 0.065 2.4 3.41 0.32
Cuttack 0.053 2.2 2.46 0.05
Pu TiangZhu 0.073 2.5 3.42 0.30
A. filiculoides Hawaii 0.085 0 2.83 0.095
A. caroliniana Ohio 0.09 1.1 2.42 0.23

a. Increase in P content of Azolla during experiment x 100/total supply of P in flow culture.


246 B.P.R. SUBUDHI and I. WATANABE

mexicana, A.filiculoides and A. caroliniana require a lower temperature than A. pinnata


(Watanabe and Berja, in preparation). The poor growth of these species in the phos-
phorus limited condition might be due partly to the high greenhouse temperatures,
which exceeded 30 or 35C in the daytime. We grew A. mexicana and A. caroliniana
in a temperature-controlled greenhouse (20C dayfl5C night) at 0.03 ppm phosphorus
concentration. The plants showed severe phosphorus deficiency under these condi-
tions. However, in batch culture with the complete culture solution placed at the
same temperature, the plants grew normally. It seems unlikely therefore that the
poor growth of the New World species was due simply to the high temperature.
Obviously, in determining the optimum phosphorus concentrations for various species
of Azolla, the phosphorus concentration in the flow culture medium should be varied.
A. caroliniana and A. filiculoides exhibited little or no phosphorus uptake during
the experiment, but their phosphorus content was slightly higher than in other strains
(Table 7). These species may require a high level of tissue phosphorus to maintain
normal metabolic activity.
The ability of different species to grow in minimum phosphorus may depend on
their root development. A. pinnata from Cuttack, A. filiculoides, and A. caroliniana
did not develop new roots and did not grow well. However, the strain from Bangkok
showed better root development.
Acknowledgments. The authors gratefully acknowledge the following persons for their help in
procuring the various species and strains from their respective countries: T.A. Lumpkin, University
of Hawaii, U.S.A.; Dao The Tuan, Central Agricultural Research Institute, Vietnam; P.K. Singh,
Central Rice Research Institute, Cuttack, Indica; Liu Chung-chu, Soil and Fertilizer Institute, Fujieng,
China; D.W. Rains, University of California, U.S.A.; and H.D. Catling, Bangladesh Rice Research
Institute, Bangladesh.

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