Soil Science and Plant Nutrition

ISSN: 0038-0768 (Print) 1747-0765 (Online) Journal homepage: http://www.tandfonline.com/loi/tssp20

Phosphorus and nitrogen contents of azolla grown

in the Philippines

Iwao Watanabe & Corazon Ramirez

To cite this article: Iwao Watanabe & Corazon Ramirez (1990) Phosphorus and nitrogen
contents of azolla grown in the Philippines, Soil Science and Plant Nutrition, 36:2, 319-331, DOI:
10.1080/00380768.1990.10414998

To link to this article: http://dx.doi.org/10.1080/00380768.1990.10414998

Published online: 04 Jan 2012.

Submit your article to this journal

Article views: 323

View related articles

Citing articles: 4 View citing articles

Full Terms & Conditions of access and use can be found at

http://www.tandfonline.com/action/journalInformation?journalCode=tssp20

Download by: [Old Dominion University] Date: 01 March 2017, At: 08:00
Soil Sci. Plant Nutr., 36 (2), 319-331, 1990 319

Phosphorus and Nitrogen Contents of Azolla

Grown in the Philippines

I w a o W a t a n a b e and C o r a z o n R a m i r e z

Soil Microbiology Department, The International Rice Research Institute,

P.O. Box 933, Manila, Philippines

Received May 22, 1989

The distribution of azolla, its g r o w t h , and P-deficiency w e r e studied in the

Philippines by collecting 232 azolla samples f r o m ponds (33%) and rice fields
(67%: 40% with rice and 27% w i t h o u t rice) in 11 regions. The d o m i n a n t species,
coverage, color, healthiness, f e r t i l i z e r t r e a t m e n t , and use by f a r m e r s were
recorded. The N and P c o n c e n t r a t i o n s in azolla w e r e expressed on an a s h - f r e e
dry m a t t e r basis. The a v e r a g e N c o n c e n t r a t i o n was 4.5% and the median 4.5%.
The a v e r a g e P c o n c e n t r a t i o n was 0.385% and the median 0.332%. Region,
species, color, and visual j u d g m e n t of h e a l t h i n e s s w e r e c o r r e l a t e d with P
c o n c e n t r a t i o n . Red A z o U a p i n n a t a var. intbricata samples had a lower aver-
age P c o n t e n t ( n = 4 2 , P = 0 . 2 4 5 % ) t h a n g r e e n samples ( n = 4 1 , P = 0 . 4 6 % ) . A.
m i c r o p h y l l a was always g r e e n and had significantly h i g h e r N and P c o n c e n t r a -
tions t h a n A . p i n n a t a var. imbrieata. N i t r o g e n and P c o n t e n t s w e r e highly
c o r r e l a t e d (simple c o r r e l a t i o n coefficient=0.64, r a n k i n g c o r r e l a t i o n coeffi-
cient = 0.73). As t h e c o n t e n t of P increased, t h e c o n t e n t of N was likely to
a p p r o a c h a plateau. The N c o n c e n t r a t i o n at t h e p l a t e a u and t h e P c o n c e n t r a -
tion required to r e a c h this p l a t e a u were h i g h e r in the A . m i c r o p h y l l a - d o m i n a t -
ed samples t h a n in A . p i n n a t a var. i m b r i e a t a - d o m i n a t e d samples. W h e n t h e P
c o n c e n t r a t i o n c o r r e s p o n d i n g to 90% of the N % p l a t e a u was set as t h e critical
c o n c e n t r a t i o n f o r P deficiency, 53% of azolla plants was considered to be P
deficient. Soil samples were t a k e n f r o m 66 out of the t o t a l n u m b e r of azolla
s a m p l i n g sites, a n d t h e i r chemical p r o p e r t i e s w e r e analyzed. A v e r a g e available
soil P c o n t e n t of the soils, w h e r e the azolla samples w e r e t a k e n , was h i g h e r
t h a n t h e a v e r a g e f o r t h e Philippine soils. T h e r e was a distinct difference in the
c o n t e n t of available soil P (Olsen P) b e t w e e n t h e soils w h e r e A . p i n n a t a var.
i m b r i e a t a was d o m i n a n t (n--51) and those w h e r e A . raicrophylla was domi-
n a n t ( n = 8 ) . The a v e r a g e c o n t e n t of available soil P Qf t h e f o r m e r was 28 ppm
and the median 12.5 ppm, w h e r e a s the a v e r a g e of t h e l a t t e r was 54 p p m and the
median 47 ppm. No statistically significant differences w e r e observed in o t h e r
chemical p r o p e r t i e s . In soils w h e r e A . p i n n a t a v a t . i m b r i c a t a was d o m i n a n t ,
the simple c o r r e l a t i o n coefficient b e t w e e n p l a n t P c o n c e n t r a t i o n and soil
available P c o n t e n t was 0.31 and the r a n k i n g c o r r e l a t i o n coefficient 0.34. The
r a n k i n g c o r r e l a t i o n coefficient excluding samples f r o m ponds was 0.54 (n=42).
Key Words: A z o l l a , P and N contents, Philippines, ponds, rice fields, soil
available soil P.

Azolla has long been used in China and northern Vietnam as green manure for wetland
320 I. WATANABE and C. RAMIREZ

rice (Lumpkin and Plucknett 1982). Recently, successful use in the Philippines was reported
in the Koronadal area in South Cotabato Province (Kikuchi et al. 1984; Watanabe 1984).
Stimulated by the success in that area, the Philippine government launched a nationwide
program to promote azolla as N source for wetland rice (Quebral 1986; Mabbayad 1987).
Regional Azolla Propagation Centers were set up in many provinces and various strains of
azolla were distributed in malay villages. Preliminary studies using a simple bioassay
identified the soils suitable for azolla growth in various provinces (Callo et al. 1985; Ramirez
et al. 1986). Watanabe and Ramirez (1984) reported that the P supply limits the growth of
azolla in various soils and that the soils in the Koronadal area contain a large amount of
available P. It is likely that the high content of available P and short dry season favored the
growth of azolla in this area. Since the P content of azolla was proportional to the biomass
production in various soils (All and Watanabe 1986), the azolla P content may be the most
suitable index for assessing azolla growth conditions. The growth of azolla and its mineral
nutrient contents in farmers' fields may vary with habitat, soil, climates, and cultural
practices of farmers.
To study the distribution of Azolla species in the Philippines and their P nutrition, and
to identify suitable areas for azolla growth, azolla samples were collected from ponds,
canals, and flooded rice fields throughout the Philippines and their N and P contents were
measured. Various characteristics related to the azolla samples and sampling sites were
recorded to correlate these characteristics with the N and P contents. In some sites where
azolla samples were taken, soil samples were also collected to correlate azolla growth with
soil conditions.

MATERIALS AND METHODS

Samples were taken in 1985 and 1986. Azolla samples were collected from at least five
sites in a field plot (rice field, pond, or canal). The following characteristics of the sites were
recorded: region, village name, habitat (pond/canal, rice field with rice or rice field without
rice), growth media (floating on water or attached to the soil surface), azolla species and
their dominance (A. pinnata var. imbricata, A. microphylla, A. caroliniana/A, mexicana, or
A. pinnata vat. pinnata), color of azolla, coverage ( < [0, 10-50, 50-90, or >90%), presence
of other aquatic plants, treatments (fertilizer application), healthiness (unhealthy, healthy, or
very healthy), and usage by farmers (inoculum, fertilizer, feed, or fish feed). Color was
grouped into 1) green, 2) greenish red ol- reddish green, 3) purplish red or purplish brown,
and 4) red oi brown. Azolla health was rated 1 (unhealthy), when the plant size was less than
5 mm, plant was fragmented, and the roots were fragile. Red color was also used as a
criterion in open field samples. Plants larger than I cm, with thick ( > 1 mm) edge of frond,
and green color were rated 3 (very healthy). In some sites, azolla plants growing on water
and adhering to the soil surface were taken from the same field plot. The sampled azolla
plants were washed with water, blotted, and cleaned of the contaminating aquatic plants as
much as possible. It was impossible to remove microscopic algae. The samples were oven
dried at 80~ After grinding, a part of the plant materials was ashed at 450~ Total N
content was determined fi-om dried samples, and total P content was determined from ashed
samples. The concentrations were expressed on an ash-free dry eight basis.
In some sampling sites, soil samples of plowed layers (up to 10cm depth) or bottom
sediments were taken fiom at least 5 sites in a plot. After air-drying, the soil texture, pH
(water), electro-conductivity, cation exchange capacity, total N content, Olsen P content, P
 N and P of Azolla in the Philippines 321

a b s o r p t i o n capacity, contents o f a v a i l a b l e Zn and K, and active Fe2Oa were d e t e r m i n e d by

the A n a l y t i c a l Service L a b o r a t o r y at IRRI.

RESULTS

Distribution of samples
D i s t r i b u t i o n o f s a m p l e s in relation to the categories o f characteristics (attributes) is
shown in T a b l e 1. F r o m sites in 11 regions, i n c l u d i n g 25 provinces, 232 a z o l l a p l a n t s a m p l e s
were collected. F r o m 2 regions, o n l y one s a m p l e each was collected. S a m p l e s from p o n d s or
canals a c c o u n t e d for 33%, those from rice fields w i t h o u t s t a n d i n g rice crop, 27% and with rice
crop, 40% of the total n u m b e r o f samples.
A. p i n n a t a var. imbricata was detected in 181 s a m p l e s and was d o m i n a n t in 169 (72.8%)
o f them. O n l y this species was i n d i g e n o u s to the P h i l i p p i n e s , b u t the i n t r o d u c e d strains also
grew widely. A. microphylla, which was easily r e c o g n i z e d by its green c o l o r and m o r p h o l -
ogy, was detected in 34 samples, and d o m i n a n t in 28 (12%) o f them. Its frequency was high
in Regions I I (South C o t a b a t o : 55% o f the samples) and 5 (Bicol: 29% o f the samples).
Samples d o m i n a t e d by this species in these two regions a c c o u n t e d for 60% o f all the s a m p l e s
d o m i n a t e d by this species. Since the identification o f A. caroliniana and A. m e x i c a n a was
difficult, the records for both species were p o o l e d . This g r o u p was f o u n d in 36 samples, and
was d o m i n a n t in 27 (12%). A. p i n n a t a var. p i n n a t a was d o m i n a n t in o n l y 8 s a m p l e s (3.4%).
A z o l l a grown on the soil surface was taken from 23 sites. A z o l l a for i n o c u l u m p r o d u c t i o n

Table 1. Number of samples under categories of characteristics.

Region Shown in Table 3
Species A. mexicana and A. caroliniana (27), A. microphylla (28), A. pinnata vat.
bnbricata (169), and A. pinnata var. pinnata (8)
Color Green (68), reddish green or greenish red (62), purplish (48), red or brown
(54)
Coverage Less than 10% (54), 10-50% (45), 50-90% (75), more than 90% (58)
Habitats Pond or canal (76), rice field with rice (93), rice field without rice (63)
Mixed with other plants Mixed (72), not mixed (160)
Healthiness Unhealthy (56), intermediate (104), healthy (48), not rated (24)
Use by farmers Inoculum (58), feeds (8), feed and fertilizer (6), fertilizer (160)
Treatments Treatment (34), no treatment (68), no record (130)
Grown on soil or water On soil (23), on water (209)

Fig. 1. Frequency distribution of N % (left top), P % (right top), and ash % (bottom) oF azolla collected
in Philippines.
322 I. W A T A N A B E and C. R A M I R E Z

F i g . 2. Frequency distribution of ash-free N and P % ( n = 2 3 2 ) .

T a b l e 2. Correlation coefficients of N and P contents with attributes.

Attribute ~ N % P %
N % I 0,64
P % 0.64 1
Region (I 1 } 0.29 0.27
Species (4) 0,30 0.125
C o l o r (4) 0,39 0,37
Coverage (4) 0.11 -0.01
Habitat (3) 0.06 0.06
Multiple regression r-" 0.327 0.242
" Numbers in tile parentheses are tile numbers of categories.

a c c o u n t e d for 25% o f the samples. A z o l l a was used for a n i m a l or fish feed in o n l y 6% o f the
sites. A t o t h e r sites, a z o l l a was used as green m a n u r e i n t e n t i o n a l l y o r not used by farmers.
O n l y 16% o f the sites received fertilizer o i - i n s e c t i c i d e treatments to s t i m u l a t e the g r o w t h o f
azolla. At o t h e r sites, no a d d i t i o n was made.

Distribution of N and P %
On the average a z o l l a c o n t a i n e d 3.32% N, 0.284% P, and 25.4% ash on a dry weight basis.
The m e d i a n s were 3.29% N, 0.242% P, and 24.2% ash. T h e f i e q u e n c y d i s t r i b u t i o n s o f N, P,
and asia percentages on a dry weight basis are shown in Fig. 1. F r e q u e n c y d i s t r i b u t i o n o f N
c o n c e n t r a t i o n s was n o r m a l , but that o f P c o n c e n t r a t i o n s was a s y m m e t r i c a l d u e to the p l a t e a u
of N c o n c e n t r a t i o n s a b o v e a certain level o f P c o n c e n t r a t i o n s . Ash c o n t e n t ranged from 8.8
to 62% and its d i s t r i b u t i o n was not symmetrical. Since a z o l l a g r o w n in water c u l t u r e
c o n t a i n s less than 15% ash ( o b s e r v a t i o n s by authors), the high ash c o n t e n t suggests that some
c o n t a m i n a t i o n with soil occurred. Then, the N and P c o n c e n t r a t i o n s were expressed on an
asia free dry weight basis.
Average o f N c o n c e n t r a t i o n was 4.47 and m e d i a n was 4.54%. M a x i m u m and m i n i m u m
were 6.57 and 1.95%. A v e r a g e P c o n c e n t r a t i o n was 0.385 and m e d i a n 0.332%. M a x i m u m and
m i n i m u m were 1.74 and 0.063%. T h e frequency d i s t r i b u t i o n s o f N and P c o n c e n t r a t i o n s on
an ash fi-ee basis are s h o w n in Fig. 2.
T h e d i s t r i b u t i o n o f the N c o n c e n t r a t i o n s was n o r m a l and that o f the P c o n c e n t r a t i o n s
was a s y m m e t r i c a l , as in the case o f fi-equency d i s t r i b u t i o n s h o w n in Fig. I.
 N and P of Azolla in the Philippines 323

Table 3. Average N and P contents ofazolla in II regions in the Philippines.

Number N % P%
Regions Provi noes"
of samples Aver. Aver. Median
I Abra. and Mountain Province 12 4.2 0.33 0.32
Ifugao, and Nueva Viscaya 14 4.2 0.49 0.47
3 Tarlac 1 6.5 0.88 0.88
4 Laguna. Occidental Mindoro, Palawan. and Quezon 35 4.2 0.33 0.29
5 Albay, Camarines Norte, Camarines Sur, and Sorsogon 41 4.5 0.39 0.34
6 Alkan, Antique, Capiz, lloilo, and Negros Occidental 30 4.4 0.40 0.26
7 Bohol, Cebu, and Negros Occidental h 12 5.1 0.49 0.39
8 Leyte and South Leyte 50 4.3 0.36 0.34
9 Zamboanga del Norte, and Zamboanga del Sur 27 4.5 0.33 0.29
II South Cotabato 9 5. I 0.52 0.57
I2 Sultan Kudarat I 6.0 0.77 0.77
" Only the provinces where samples were taken are shown, t, High ash content.

Table 4. Ash free N and P contents of Azolla species.

N% P%
Species Number
Aver. STDEV Aver. STDEV Median
A. phTnata vat. imbrieata 169 4.5 0.96 0.38 0.22 0.33
A. caroliniana and ,4. mexicana 27 4.4 0.67 0.41 0.31 0.34
,4. mierophylla 28 4.9 0.92 0.49 0.27 0.42
,4. phTnata v'~r. pinnata 8 2.8 0.70 0.18 0.06 0.18

S i g n i f i c a n c e of a t t r i b u t e s
To determine whether the attributes to describe sample site and azolla characteristics
were correiated with the N and P contents of azolla, Hayashi's m u l t i v a r i a t e q u a n t i f i c a t i o n
analysis-I (regression of q u a n t i t a t i v e data on q u a l i t a t i v e data) was applied (Hayashi 1952;
T a n a k a et al. 1984). A t t r i b u t e s that do not have missing r e c o r d s - - r e g i o n s , species, color,
coverage, and h a b i t a t s - - w e r e used (Table 2). Both the N and P c o n c e n t r a t i o n s were
correlated with the region, species, and color. Partial c o r r e l a t i o n coefficients of the N
c o n c e n t r a t i o n s were 0.30 with the region, 0.36 with the species, and 0.40 with the color.
Those of the P c o n c e n t r a t i o n s were 0.30 with the region, 0.18 with the species, and 0.39 with
the color.
Region. Averages of N and P c o n c e n t r a t i o n s a n d m e d i a n s of the P c o n c e n t r a t i o n s are
s h o w n in T a b l e 3. Ash-free P percentages were high in the samples from Region 2 (Ifugao
and Nueva Viscaya) and South C o t a b a t o ( K o r o n a d a l Valley area). T h e N c o n c e n t r a t i o n s
were also high in the samples from S o u t h C o t a b a t o , but not in Region 2. Since the ash
contents in Region 6 were high (average, 42%), the i n t e r p r e t a t i o n of the data from this region
is not conclusive. The same holds in Region 7 (average ash content, 35.8%). T h e averages
tended to be high in the regions where the n u m b e r of samples was small like in Regions 2,
7, and 11.
Species. Statistics on N and P c o n c e n t r a t i o n s of species are s h o w n in T a b l e 4. A.
pinnata var. pinnata had lower N and P c o n c e n t r a t i o n s t h a n A. pinnata vat. imbricata, and
A. microphylla had higher N and P c o n c e n t r a t i o n s .
Color. C o l o r of azolla was classified into 4 categories. Averages of N c o n c e n t r a t i o n s
of green samples were 4.9 ( n = 6 8 ) ; greenish red or reddish green, 4.7 ( n = 6 2 ) ; purplish red
324 I. WATANABE and C. R A M I R E Z

Table 5. Correlation coefficients of ash free N and P contents in A. pinnata var. imbricata
with attributes (number of samples= 169).
Attributesh N % P%
N% I
P% 0.63 I
Region (10) 0.24 0.21
Color (4) 0.46 0.43
Coverage (4) 0.16 0.07
Habitat (3) 0.06 0.05
Multiple regression r~ 0.300 0.292
" Number of attributes in parentheses. Color: green (41 samples), reddish green or greenish red (42), purplish
(44), red or brown (42). Coverage: < I0% (46), I0-50% (32), <:50% (61), <90% (30). Habitats: rice field
without rice (43), rice field with rice (74). and pond or canal (52).

% Nitrogen % Nzlrogen

e, A microphylla

m a m~n --

F ~ ~ ~ o
: ~O~ o A. pinnata vat. irnbricata

?
T

A caroliniana
A pinnata vat pinnata

3
K M
N N

K
2 N

I I , I I I O
G.0 02 04 0.6 08 10 12 14 16 18 O0 02 04 06 08 10 12 14 16 18

% Phosphorus % Phosphorus

Fig. 3. N and Pconcentrations ofazoI1a in the Philippines.

or purplish b r o w n , 4.15 ( n = 4 8 ) ; and red or brown, 3.9 (17=54) ( c o m m o n average s t a n d a r d

error is 0.14). Green samples of azolla had a significantly higher N c o n c e n t r a t i o n than the
red ones. Averages of P c o n c e n t r a t i o n were 0.45, 0.44, 0.31, and 0.26, respectively. The
average P c o n c e n t r a t i o n of green azolla plants was also significantly higher than that of the
red ones ( c o m m o n s t a n d a r d error is 0.04).
Coverage and habitats. No significant difference in the N and P c o n c e n t r a t i o n s was
detected either for the coverage, or habitat.
Other attributes. Healthiness was recorded in most samples. Samples d o m i n a t e d by A.
microphylla were recorded as intermediate or- healthy (26 out of 28). In 148 samples of A.
pinnata var. hnbricata, healthiness was significantly correlated with N ( r = 0 . 4 8 ) and P
 N and P of Azolla in the Philippines 325

T a b l e 6. Regression equations o r N concentrations on P concentrations.

N %=a{l-exp(-bP%)} Correlation coefficients
Samples Number Regression
a b Simple Ranking correlation
coefficients
All 232 5.32_+0.08 6.29+0.41 0.64 0.73 0.89
A. pirlnata var. imbricata 169 5.22_+0.11 6.92_+0.49 0.63 0.68 0.89
A. microphylla 28 5.75_+0.21 5.16_+0.63 0.63 0.81 0.83
A. mexieaJla 27 5.11_+0.18 6.71 _+0.76 0.77 0.81 0.76
A. phTnata var. phT#.lata 8 Not applicable 0.74 0.83

T a b l e 7. Threshold values for P defici/'ncy estimated by 2 methods and percentage of P deficient

azolla in each species.
Methods For defining critical level
90% of N plateau in regression Cate and Nelson method
Species
P % Percentage of p % Percentage of
P deficiency P deficiency
A. phTnata var. imbricata 0.33 50 0.40 62
A. microph)'lla 0.45 53 0.49 57
A. carofiniana and A. mexicaf~a 0.34 52 0.48 74
A. phlnata var. pinnata 0.30 100 0.40 100
Total 53 64

T a b l e 8. Chemical properties of soils where azolla samples were taken and

N and P concentrations of azolla.
Azolla Soil
EC Total-N CEC Av P Av Zn P abs Act Fe
N % P % Ash %
pH (mS/cxr~) (%) (meq/100g)(ppm) ( p p m ) ( g P / k g ) (%)
All data
Average 4.58 0.40 26. 6.1 0.55 0.19 27. 24. 1.3 5.15 1.5
STDEV 0.94 0.25 I3. 0.8 0.51 0.07 13. 25. 1.35 2.4 0.8
Median 4.55 0.32 23. 5.8 0.39 0.18 25. 14 0.8 5.5 1.4
Number 66 66 66 66 65 66 66 66 63 63 66
A. p#l#lata var. bllbricata
Average 4.49 0,37 27 6.0 0.51 0.19 27. 28 1.3 5.2 1.5
STDEV 0.97 0.23 12 0,8 0.41 0.07 12. 20 1.4 0.23 0.82
Mediala 4.48 0.32 25 5.8 0.35 0.18 26 13 0.80 5.4 1.4
Number 51 51 51 51 50 51 51 51 49 48 51
A. microphylla
Average 5.43 0.65 28 6.4 0.91 0.21 22 54 0.8 4.4 1.0
STDEV 0.4 0.34 17 0.7 0.9 0.07 12. 20 1.4 0.23 0.48
Median 5.43 0.57 23 6.4 0,28 0.18 12.7 47 0.9 3.0 0.9
Number 8 8 8 8 8 8 8 8 7 8 8
EC. electroconductivity; CEC, cation exchange capacity; Av P, awtilable P (Olsen); Av Zn, available Zn; P
abs. P absorption capacity: Act Fe, active Fe.,O:~.

concentrations (r=0.37). In t h e s a m p l e s r a t e d as u n h e a l t h y , t h e N c o n t e n t s w e r e 4 . 0 5
(n--46) and the P content, 0.32 In t h e s a m p l e s r a t e d as v e r y h e a l t h y , t h e N c o n t e n t
was 5 04 90% ( n - - 2 9 ) and the P content, 0.46
326 1. W A T A N A B E and C. R A M I R E Z

T a b l e 9. Correlation coefficients between P concentration of A. pinnata var. imbricata

and chemical properties of soils.
P % pH EC a T-N CEC Av P Av Zn ~' P abg ~ Act Fe
N % 0.77 0.35 0,18 0.16 0,07 0.32 -0.32 0.17 -0.04
P % I 0.34 0.11 -0.03 0.20 0.31 --0.33 0.15 - 0 . I0
pH I 0.18 0.01 0,11 0.20 --0.49 0.32 --0.15
EC a I 0.25 0.25 0.22 -0.002 0.06 -0.21
T-N I 0.16 0.35 -0.16 0.37 --0.06
CEC I 0.16 -0.51 0.87 0.41
Av P I -0.09 0.17 0.003
Av Zn ~ I --0.56 --0.10
P abs" 1 0.46
u Because of missing data, 46 data were used. In other variables, 51 data were used.

T a b l e 10. Simple and ranking correlation coefficients of P contents of A. pinnata var. imbricata
with available P in soils under different categories of azolla samples.
Averages Correlation coefficients
Item and categories
Number P% Av. P Simple Ranking
Without treatment 43 0.35 18 0,38 0.54
Habitats
Without rice 17 0,34 15 0.67 0.55
With rice 25 0.35 14,5 0.46 0,61
Ponds 9 0,44 35 0,06 0.13
Rice fields 42 0,37 14 0.49 0.53
Without treatment 37 0.34 14 0.46 0.55

1.0-

0.8

0.6

0.4 9 :.: 9
0.2-
9 .!. " - ' z Fig. 4. Available P in soils and P concen-
0.0 i J i i 1 i trations in A. phmata vat. hnbricata grown
10 20 30 40 5D 60 in rice fields without treatments ( n = 3 7 , r =
Available P 0.46),

S i n c e all t h e d. microphylla s a m p l e s w e r e g r e e n a n d t h i s s p e c i e s h a d h i g h e r N a n d P
concentrations than others, the color factor may be confounded with species factor. Also
since the N and P contents differed among the species, the relationships of various attributes
to N a n d P c o n c e n t r a t i o n s of the most widely distributed A. pinnata var. irnbricata are
d i s c u s s e d in d e t a i l .

Effects of attributes on the N and P contents o f A. pinnata v a r . i m b r i c a t a

C o l o r a f f e c t e d m o s t l y t h e N a n d P c o n t e n t s o f a z o l l a ( T a b l e 5). G r e e n a z o l l a h a d highel-
N concentration (5.0 and P concentration (0.46+__0.18%) t h a n red a z o l l a (3.9_+0.93%
 N and P oF Azolla in the Philippines 327

N and 0.25__+0.13% P). In the Bicol and South Cotabato Regions, the averages of all the
samples were high, but the averages of A. pinnate vat-. imbricate were not high (0.33___0.21
in Bicol and 0.33_+0.20 in South Cotabato Regions) due to high N and P concentrations of
A. microphylla samples.
Azolla from Region 2 (Mountain Province 0.50_+0.26) and Region 6 (mostly Panay
Island, 0.45-t-0.34) had higher P concentrations than others. The N and P concentrations
were related to the visual rating of healthiness. In the samples rated as very healthy, the N
content was 5.04--+0.9% and the P content, 0.46___0.23% ( n = 2 9 ) ; in the samples rated as
intermediate, the N content was 4.48+0.85% and the P content, 0.39--+0.18% ( n = 7 3 ) ; in the
plants with low rating, the N content was 4.05-t-0.95% and the P content, 0.32--+0.29% ( n =
46).

Relationship between N and P contents

The N concentration in azolla was highly correlated with the P concentration. The
simple correlation coefficient was 0.64 (n=232), and the ranking correlation coefficient 0.73
(Fig. 3). As the P concentration increased, the N concentration approached a plateau, which
explains why ranking correlation coefficient was higher than simple correlation coefficient.
An exponential regression Y = a ( l - e x p ( - b X ) ) was, therefore, used. The regression and
correlation coefficients are shown in Table 6. For the samples dominated by A. pinnata vat.
pinnata, the application of this equation was not possible, because the N concentrations of
all the samples were below a plateau value. The plateaus in the N concentrations and P
concentrations required to reach the plateaus were higher in A. microphylla and A.
mexicana/A, caroliniana than in A. pinnate vat. imbricate.

D i s t r i b u t i o n of a z o l l a p l a n t s deficient in P or N
The P concentration at 90% of the N concentration plateau can be considered as the
critical level of P deficiency in each species, which enables to classify the azolla samples into
P-deficient or P-sufficient ones.
Critical level of P was 0.33% for A. pinnata vat. imbricata, 0.45% for A. microphylla and
0.34% for A. caroliniana or A. mexicana. Although the number of samples of A. pinnate vat.
pinnate was too small to determine the critical level, the same value as that used for A.
pinnate var. hnbricata may be applied.
Another way of determining the critical level is by using the Cate and Nelson method
(Care and Nelson 1971). The critical levels of P deficiency in azolla estimated by this method
were 0.40% P for A. pinnate vat-. bnbricata, 0.49% for A. microphylla, and 0.48% for A.
mexicana or A. caro#niana. For A. pinnate var. pinnate, the application was not possible,
because no plateau in the N content was observed. It is, however, assumed that the critical
level may not be lower than 0.4% P. The critical levels of P content for P deficiency estimated
by the two methods and the percentage of P deficient azolla in each species (Table 7) showed
that at least 50% of the azolla samples were deficient in P.

R e l a t i o n s h i p w i t h soil c h a r a c t e r i s t i c s
Soil samples were taken from 66 sites where azolla samples were collected. Among
them, 9 were taken From the sediments of ponds or canals. The average N and P concentra-
tions in the azolla plant from the sites where the soil samples were taken were 0.395% P and
4.58% N. Medians were 0.318% P and 0.455% N. These values are similar to those of all the
azolla samples, and this statistics suggests that the soil samples were representative. The
328 I. WATANABE and C. RAMIREZ

averages of N and P concentrations of azolla, and soil chemical properties are shown in
Table 8. The average of available P (Olsen) was 24.3 ppm and the median was 14 ppm.
Correlation coefficients of available P concentrations with attributes were 0.13 for the
region, 0.53 for the species, 0.05 for the color, 0.36 for the coverage and 0.15 for the habitats.
Partial correlation coefficients were all significant, being the highest with species. A. pinnata
var. imbricata was dominant in 77% of the samples. The average value of available P
concentration of 8 soils dominated by A. microphylla was 54 ppm and the median was 47
ppm, while those of soils dominated by A. pinnata var. imbricata were 18 and 13 ppm. This
difference suggests that A. microphylla grew in the soils with a higher concentration of
available P than A. pinnata var. hnbricata. Other soil characteristics were not significantly
different between the soils with A. microphylla and A. pinnata var. hT~bricata. Average and
median values in the soils where A. caroliniana or A. rnexicana dominated ( n = 6 ) were also
higher (41 and 33 ppm, respectively) than those in which A. pinnata var. imbricata domi-
nated.
Due to the difference in P requirements among Azolla species, details on the relation-
ship of N and P concentrations with the soil chemical properties were examined in the soil
samples dominated by A. pinnata var. imbricata. The correlation matrix (Table 9) shows
that the P concentrations in azolla was correlated with the available P content of soil ( r =
0.31) and soil pH (r=0.34). The correlation coefficients were not high, though statistically
significant. The ranking coefficients were 0.34 with available P, and 0.46 with pH.
Some soil samples were taken from the bottom of ponds, and in the pond samples the
correlation was not significant (Table 10). Thus, the removal of data from ponds increased
the value of the correlation coefficient to 0.49. The samples included those that received P
fertilizers to stimulate azolla growth. The removal of data including fertilizer treatments
increased the correlation coefficient ( n = 4 3 , simple correlation coefficient=0.38, and ranking
correlation coefficient=0.54). In the data excluding samples from ponds or with fertilizer
treatments, the correlation coefficient was high ( n = 37, simple correlation coefficient=0.46,
and ranking correlation coefficient=0.55). The relationship between the P concentration of
azolla and available soil P contents in flooded rice fields without treatments is shown in Fig.
4. Grouping of these data by categories of various attributes did not increase the correlation
coefficients substantially.

DISCUSSION

Due to the efforts of the Department Agriculture and the University of Philippines to
introduce azolla technology to rice culture in the Philippines, various species of azolla have
been grown over wide areas in the Philippines. Only A. pinnata var. imbricata was indige-
nous to the Philippines (Tan et al. 1986). Some strains of this species were also introduced
from abroad. A. pinnata vat. imbricata still dominates in fields and ponds in the Philip-
pines. Identification of A. microphylla from Paraguay is easy based on its green color and
plant shape. This species was distributed in limited areas in Bicol and South Cotabato,
where available soil P content was high. In the Koronadal area in South Kotabato, the
farmers adopted azolla technology as a substitute for N fertilizer (Kikuchi et al. 1984). A.
microphylla was introduced to the Koronadal area in 1984, and by 1986, it almost replaced
A. pinnata var. irnbricata, which had been introduced earlier. Median of the concentration
of available soil P in the sites where this species was grown was 40 ppm. This fact suggests
that A. microphylla requires a higher P content in soil than A. pblnata var. imbricata. Water
 N and P of Azolla in the Philippines 329

culture experiments carried out in the laboratory have recently shown that A. pinnata var.
imbricata (Acc. No. 5) grew better and fixed a larger amount of N under P-deficient
conditions than A. mierophylla (D.P. Kushari and 1. Watanabe, IRRI, unpublished data).
Azolla grown in the field turns red under stress conditions like P-deficiency, high solar
radiation, or a combination of these (Hoist and Yopp 1979; Zimmerman 1985). A survey of
azolla samples showed that the N and P contents of the green A. pinnata var. bnbricata
plants were highe," than those of the red ones. It was often observed that azolla turned green
under tile shade. Kannaiyan et al. (1983) and Rother and Whitton (1988), who compared a
few azolla plants grown in the same site stated that the color of azolla was not related to its
N-fixing ability or to P deficiency. The number of the samples they used was, however, too
small to enable an examination of the relationship between the azolla color and the
chemical composition. The average value of the content of available P in soils where A.
pinnata vat. imbricota was red was 7.7 pprn ( n = 10); which is significantly lower than that
in soils where the azolla plant with other colors grew. There were more red azolla plants
than green ones in P deficient soils. When a large number of samples were compared, the N
and P contents tended to be lower in the red azolla plants than in the green ones.
The N and P contents differed among the regions, indicating that azolla technology is
more suitable in some regions than in others such as Bicol (except Sol-sogon area), Mountain
Provinces and South Cotabato. Some parts of Partly Island (Region 6) may be also suitable.
Detailed analysis of regional differences will be reported in another paper.
Critical concentrations of P for P deficiency may vary with the method of determina-
tion. Two methods were used in this report: P concentration corresponding to 90% of the
plateau of N concentration and Care and Nelson method. The former gave a slightly lower
value (0.33%) than the latter (0.49%). By adopting the lower value, it can be considered that
at least 50% of the azolla samples were deficient in P.
To compare the current data expressed on an ash-free dry weight basis with others, N
and P concentrations should be converted to values on a dry weight basis. Since the average
ash content was about 25%, the P concentration on a dry weight basis would be three-fourths
of that on an ash-free dry weight basis. All and Watanabe (1986) set the value of 0.15% P
on a dry weight basis as the critical level based on experiments, in which the starved azolla
plants were inoculated to soils with various levels of available P. In the field, azolla might
have grown either from starved or P-rich conditions. The experiments of inoculation of
P-enriched azolla in a P-deficient soil showed a similar value for the critical P concentration
(0.1-0.2% on a dry weight basis), below which the N concentration decreased (1. Watanabe,
unpublished data). It seems that the critical P level estimated in field samples was higher
than that determined in more refined greenhouse experiments. In the field, other factors like
insect and fungal damage, and high temperature may have also limited the growth of azolla.
Under these conditions, a larger amount of P may be required for N2-fixation than under
controlled and optimum conditions.
The average value of available P in the soils at the sampling sites was 25 ppm P and the
median value was 14ppm. These values appear to be higher than the average values of
Philippine soils. The average value of 59 soils, where algal samples were taken, was 11 ppm
and the median value was 8.5 ppm (Roger et at. 1986). It is likely that the soils where azolla
grew had a higher content of available P than the other soils, where azolla could not grow.
To confirm this assumption, samples should be taken from adjacent fields with and without
azolla.
Correlation coefficients of azolla P concentration and soil available P content were
330 I. WATANABE and C. RAMIREZ

significant, but not high. A m o n g the samples from rice fields w i t h o u t treatment (fertilizer
a p p l i c a t i o n of azolla), the r a n k i n g correlation coefficient was 0.55 ( T a b l e 10). F r o m Fig. 4,
it appears that the critical level of the c o n t e n t of a v a i l a b l e soil P falls between 20 and 30
ppm. These values were consistent with the early findings, using a smaller n u m b e r of soil
samples ( W a t a n a b e and Ramirez 1984). In the field, the r e l a t i o n s h i p may be masked by m a n y
factors such as the d u r a t i o n of stay in the field, the inflow of azolla from adjacent fields and
irrigation canals, the d i l u t i o n of floodwater by rain and irrigation water and the floating of
azolla plants attached to soils, and so on.
The azolla surveys in the P h i l i p p i n e s clearly showed that P deficiency in azolla is
widespread. Nitrogen contents of azolla, which may affect the a v a i l a b i l i t y of azolla N to
rice, varied markedly. T h e effect o f azolla on rice yield as reported by researchers may be
overestimated, because the N c o n c e n t r a t i o n of azolla in c o n t r o l l e d experiments may be
higher than that f o u n d in the field. This survey also revealed that in some regions in the
Philippines, the use of azolla is p r o m i s i n g due to a high c o n t e n t of a v a i l a b l e P in soil. T h e
data o b t a i n e d c o n t r i b u t e s to the d i s s e m i n a t i o n of azolla technology.

Acknowledgments. Surveys were made in cooperation with Regional Offices in the Departments of
Agriculture, the Philippines and the National Azolla Action Program in the University of Philippines, Los
Bafios. We would like to thank for Dr. O. Mochida, an entomologist at IRRI and currently at the National
Agriculture Research Center, Tsukuba, Japan, who accompanied us for the simultaneous surveys on the
incidence of insect damage to azolla. The projects were supported by a grant from United Nations
Development Programme.

REFERENCES

All, S. and Watanabe, I. I986: Response of Azolla to P, K and Zn in different wetland soils in relation to
chemistry of floodwater. Soil Sci Plant Nutr., 32, 239-253
Cello, D.P., Dilag, R.T., Necesario, R.S., Wood, D.M., Tagalog, F.C., Gapasin, D.P., Ramirez, C.M., and
Watanabe, I. 1985: A 14 day Azolla adaptability test on farmers field. Philipp. J. Crop Sci., 10, 129-I34
Cote, R.B. and Nelson, L.A. 1971: A simple statistical procedure for partitioning soil test correlation data
into two classes. Soil ScL Soc. Am. Proc., 35, 658-660
Hayashi, C. 1952: On the prediction of phenomena from qualitative data on the quantification ofqualitative
data from the mathematico-statistical point of view. Ann. Inst. Stat. Math., 3, 69-98
Hoist, R.W. and Yopp, J.H. 1979: Studies of the Azolla-Anabaena symbiosis using Azolla me_ricana. I.
Growth in nature and laboratory. Am. FeJw J., 69, 17-25
Kannaiyan, S., Viswanathan, G., and Rajeswari, N. 1983: Influence of green and brown color variation on
NPK contents of Azolla pinnata L. ScL Cult., 49, 25t-252
Kikuchi, M., Watanabe, I., and Haws, L.D. 1984: Economic evaluation of Azolla use in rice production. In
Organic Matter and Rice, Ed. IRRI, p. 569-592, The International Rice Research Institute, Los Bafios,
Laguna, Philippines
kumpkin, T.A. and Plucknett, D.A. 1982: Azolla as a Green Manure: Use and Management in Crop
Production, p. 1-230, Westview Press Boulder, Colorado
Mabbayad, B.B. 1987: The Azolla program in the Philippines. 117 Azolla Utilization, Ed. IRRI, p. 101-108,
The International Rice Research Institute, Los Bafios, Philippines
Quebral, F.C. 1986: The national Azolla action program (NAAP). Philipp. Agric., 69, 449-451
Ramirez, C., Dilag, R.T., Jr., Haws, L.D., Cello, D.P., Jr., and Watanabe, I. 1986: Additional notes on the
14-day test Azolla adaptability on soils from farmer's field. PhlTipp. ,4gric., 69, 665-573
Roger, P.A., Tirol, A., Ardales, S., and Watanabe, I. 1986: Chemical composition of cultures and natural
samples of N2-fixing blue-green algae from rice fields. Biol. Fertil. Soils, 2, 131-146
Rother, J.A. and Whitton, B.A. 1988: Mineral composition of Azolla pinnata in relation to composition of
floodwaters in Bangladesh. Arch. Hydrobiol., 113, 371-380
Tan, B.C., Payawal, P., Watanabe, l., Lacdan, N., and Ramirez, C. 1986: Modern taxonomy of Azolla: A
 N and P of Azolla in the Philippines 331

review. Philipp. Agric., 69, 491-512

Tanaka, Y., Tarumidzu, T., and Wakimoto. K. 1984: Handbook for Statistical Analysis by Personal
Computers. II. Multivariate Analysis, p, 1-403, Kyouritsu Shuppan Co., Tokyo (in Japanese)
Watanabe, I. 1984: Use of symbiotic and free-living blue-green algae in rice culture. Outlook Agric., 13, 166-
172
Watanabe, I. and Ramirez, C. 1984: Relationship between soil phosphorus availability and Azolla growth.
Soil Sei. Plant Nutr., 30, 595-598
Zimmerman, W.J. 1985: Biomass and pigment production in three isolates of Azolla, II. Response to light
and temperature stress, Ann. Bot., 56, 701-709