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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
Download by: [Old Dominion University] Date: 01 March 2017, At: 07:56
Soil Sci. Plant Nutr., 21 (2), 237-247, 1981
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
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).
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).
Table 2. Effect of varying doses of phosphorus in batch culture on the fresh matter.
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
P level (ppm) Fresh weight (g) Doubling time (days) P % in dry matter
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
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.
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.
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
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