E P I P H Y T I C NITROGEN F I X A T I O N ON WEEDS I N A
R I C E F I E L D ECOSYSTEM
S.A. Ku1asooriya'gJP.A. Roger', W.L. Barraquio3 and
I . Watanabe3
University o f Peradeniya, S r i Lanka.
' t ~ = ~ = ~ m
'
'~ Ox f f~i c es de~
France,
l ~a Recherche
S c i e n t i f i q u e e t Technique Outre-Mer,
a
F i g . 1. Diagram
onents i n a r i c e
Bacteria
11 rhizosphere
2) s o i l
31 epiphytic on
r i ce
4) epiphytic on
weeds
I
^
_
e r n a t i o n a l Rice Research I n s t i t u t e , Los Banos,
ABSTRACT
- -__.._-I
1O)Azolla
.
.
.
eval&ting t h e weed biomass i n planted and fallow f i e l d s ;
measuring s p e c i f i c dark and light-&pendent acetylene
reducing a c t i v i t y (ARA);
enmerating and i d e n t i f y i n g epiphytic N2-fzking microorganisms.
Submerged weeds y d u e e d a mean biomsses o f 1 t ha-l a t r i c e
t i l l e r i n g and 3 t ha- a t r i c e harvest stage; wider fallow they reached
7.5 t ha-1. Corresponding biomasses of non-submerged tJeeds were 1.7 t
ha-1 under r i c e and 7.7 t ha-1 wzder fallow a t r i c e harvest stage.
Dominant Nz-fixicing Cyanobacteria were G l o e o t r i c h i a s p . , Nostoc s p p .
and C a l o t h r i x s p p . Epiphytism by G l o e o t r i c h i a was predominantly on Chara
whereas that by other Cyanobacteria d i d not e x h i b i t any host s e l e c t i v i t y .
Submerged weeds harboured both aerobic and micro-aerophilic N z - f i ~ h g
bacteria. Growth on glucose medium showed the presence of acid-gasproducing organisms (probably Enterobacteriaceae), while growth on malate revealed Azospirillum-like organisms. Light ARA on the submerged
weeds (29-35 nmoZeC2Hq ( g f r e s h weight)-1 h-1) was about t e n times higher
thwi t h a t on the non-submerged ones (2.8-4.4 nmokC2Hq (g f m s h weight)-l
h-1). Dark a c t i v i t y uas about the same f o a~ l l the weed types studied
(0.9-2.5 nmohC2H4 ( g f r e s h we+ght)-l 12-1),
Relating s p e c i f i c ARA to
weed biomass measurements it was f o m d t h a t the non-submerged weeds
e x h i b i t a very low a c i i v i t y (0.4-2.3 g N ha-1 d-1) while the a c t i v i t y
on submerged weeds (5-34 g N haq1 d-1) makes an appreciable N2 input
i n t o t7iis ecosystem.
of NZ-fixing compf i e l d ecosystem.
Cyanobacteria
51 S o i l water i n t e r face
6) f r e e f l o a t i n g
7) water a i r interface
8 ) epiphytic on r i c e
91 epiphytic on weeds
MATERIALS AND METHODS
ExperimentuZ
An experiment was conducted i n 1.5 m2 p l o t s with f o u r treatments i n t r i p l i c a t e , d i s t r i b uted on a randomized block design. Each p l o t was p l a n t e d s e p a r a t e l y with e i t h e r submerged
weeds (67zara s p . o r Najas s p . ) o r non-submerged weeds (Monochoria vaginazis o r Cyperus i r i a ) .
The p l o t s with submerged weeds were sampled by h a r v e s t i n g t h e t o t a l p l a n t m a t e r i a l i n a p l o t ,
nixing them t o g e t h e r and removing random t r i p l i c a t e 10-g samples f o r subsequent a n a l y s i s . In
the case o f non-submerged weeds, t h e r o o t system and t h e a e r i a l p a r t s above t h e flood water
levé1 were f i r s t c u t o f f , and t h e remaining m a t e r i a l was mixed t o g e t h e r before sampling. The
samples were s t u d i e d i n r e g a r d t o t h e i r s p e c i f i c a c e t y l e n e reducing a c t i v i t y (ARA) and t h e i r
epiphytic a l g a l and b a c t e r i a l f l o r a .
A s i t was observed t h a t more algae were p r e s e n t on t h e o l d e r p a r t s o f submerged weeds,
o l d and young p a r t s were s e p a r a t e d and a l i q u o t samples were analyzed f o r ARA and N2-fixing
microflora. To e x t r a p o l a t e t o t h e f i e l d from t h e s p e c i f i c ARA measurements (expressed i n terms
of a c t i v i t y p e r gram of h o s t ) , an assessment o f t h e wccd biomass and i t s v:iri:ibj l i t y i n tho
f i o l d wcis tloiio rit two s t n g o s o r t h o growtliqoT r l c o und i n r n l l o w p l o t s .
I NTRODUCTI ON
Biological n i t r o g e n f i x a t i o n c o n t r i b u t e s s i g n i f i c a n t l y t o t h e f e r t i l i t y o f r i c e s o i l s .
Microorganisms o p e r a t i v e i n t h i s process and t h e i r s p a t i a l d i s t r i b u t i o n within a r i c e fte'ld
ecosystem a r e i l l u s t r a t e d i n Fig. 1. S t u d i e s have been conducted i n most of t h e components
depicted i n t h i s Figure and have been r e c e n t l y reviewed by Dommergues & Rinaudo (1979) on
t h e rhizosphere; by Matsuguchi (1979) on h e t e r o t r o p h i c b a c t e r i a ; by Roger E Reynaud (1979)
and Venkataraman (1979) on t h e blue-green algae (BGA), and by Watanabe (1978) and Decking
(1979) on Azolla. There a r e a few r e p o r t s on n i t r o g e n - f i x i n g b a c t e r i a a s s o c i a t e d with r i c e
stems (Watanabe e t a l . , 1979; Watanabe fr Barraquio, 1979) and n i t r o g e n f i x a t i o n by bluegreen algae e p i p h y t i c on f r e s h water macrophytes (Finke & Seeley, 1978), but we a r e unaware of
any s t u d i e s on n i t r o g e n f i x a t i o n by e p i p h y t i c BGA i n r i c e f i e l d s .
The e p i p h y t i c m i c r o f l o r a appears t o occupy an e c o l o g i c a l niche with c e r t a i n d i s t i n c t i v e
f e a t u r e s . Being a t t a c h e d i n a somewhat permanent submerged p o s i t i o n , BGA a r e p r o t e c t e d from
d e s s i c a t i o n and i n h i b i t o r y e f f e c t s of high s o l a r r a d i a t i o n (Reynaud E Roger, 1979). This e p i
p h y t i c h a b i t i s advantageous t o t h e h e t e r o t r o p h i c b a c t e r i a , which may o b t a i n nourishment from
t h e i r h o s t s , b u t no n u t r i t i v e a s s o c i a t i o n between t h e algae and t h e h o s t p l a n t s has y e t been
found.
The submerged weed p o p u l a t i o n i n a r i c e f i e l d c>* develop i 5 t o a considerable biomass
( S a i t o & Watanabe, 1978) and t h e n i t r o g e n f i x e d by t h e i r e p i p h y t i c microflora could then
make a s i g n i f i c a n t c o n t r i b u t i o n t o t h e t o t a l n i t r o g e n i n p u t . S t u d i e s were t h e r e f o r e undertaken t o i n v e s t i g a t e t h e e p i p h y t i c N p f i x a t i o n on weeds i n a r i c e f i e l d .
-- -
Acetylene reducing a c t i v i t y
ARA measurements were conducted i n 250-ml Erlenmeyer f l a s k s , under an atmosphere o f 10%
acetylene i n a i r . Incubation w a s done e i t h e r under 800 l x provided by f l u o r e s c e n t l i g h t s ,
or i n t h e dark by wrapping t h e f l a s k with aluminium f o i l . P l a n t m a t e r i a l d e s t i n e d f o r t h e
dark i n c u b a t i o n was covered i n s i t u with a black c l o t h t h e day b e f o r e h a r v e s t i n g , i n o r d e r t o
eliminate any r e s i d u a l a l g a l a c t i v i t y . Gas samples were removed a f t e r 0.5, 1, 2 , 4, and 6
h o u r s o f incubation and analysed by gas chromatography.
Algal counts
Algae were enumerated by p l a t i n g on B G I I medium (Allen & S t a n i e r , 1968) with and without
combined n i t r o g e n t o e s t i m a t e r e s p e c t i v e l y t h e t o t a l and t h e N2-fixing a l g a l populations.
A f t e r i n c u b a t i n g f o r t h r e e weeks a t 30 C under continuous f l u o r e s c e n t l i g h t (800 l x ) t h e
p l a t e s were observed under a s t e r e o s c o p i c microscope, a l g a l colonies were i d e n t i f i e d an¿
s e p a r a t e l y counted.
Bacterial counts
Aerobic h e t e r o t r o p h i c N2-fixing b a c t e r i a were enumerated by t h e most probable number
(MPN) technique a s described by Watanabe e t a l . (1979). I n o c u l a t i o n was done i n t o semi-solid
glucose-yeast e x t r a c t medium, which u s u a l l y gives h i g h e r counts than malate medium (Watanabe
e t a l . , 1979) and i n t o m a l a t e - y e a s t - e x t r a c t medium t o d e t e c t t h e presence o f Azospirillum
(Day E DÖbereiner, 1976). A f t e r i n c u b a t i n g f o r two days a t 30 C, the tubes were exposed t o
I
,
57
I
10% a c e t y l e n e i n a i r f o r 24 hours and t h e ethylene formed w a s measured. The tubes with ethyl e n e values twice t h a t of t h e uninoculated c o n t r o l s were considered a s p o s i t i v e ,
T o t a l a e r o b i c h e t e r o t r o p h i c b a c t e r i a were enumerated b y spreading on t r y p t i c - s o y ( O . 1%)
a g a r (1.5%) p l a t e s (Watanabe E Barraquio, 1979). A f t e r one week o f incubation a t 30 C, t h e
colonies were counted on t h e p l a t e s containing 30 t o 300 c o l o n i e s .
'5
a c t i v i t i e s on young p a r t s , s p e c i a l l y on Chara, d e s p i t e t h e f a c t t h a t t h e populations o f N2f i x i n g b a c t e r i a on t h e o l d e r p a r t s were h i g h e r (Table 1).
I
Fig, 2. Distribution o f submerged ueeds
biomass ( t ha-1 fresh weight) among
A: 20 p l o t s a t end of t i l l e r i n g ; the
plots had been hanheeded four ueeks
before the measurement,
B: 15 p l o t s a t harvesting stage; no
weeding was perfomled,
C: 9 fallow p l o t s a t h a r u e s t h g stage of
rice.
3
6
9
O
3
6
Bmmass of Submerged weed (1hä'l
O
9
1
2
RESULTS
Biomass o f weeds
The d i s t r i b u t i o n o f t h e biomass o f submerged weeds (Clara and Najas) i s shown i n Fig. 2
a t t i l l e r i n g s t a g e , h a r v e s t i n g s t a g e and i n a fallow p l o t a t h a r v e s t i n g s t a g e o f r i c e . This
Figure shows t h a t t h e submerged weed population under a r i c e crop a t t h e end o f t i l l e r i n g
had a mean biomass of about 1 t h a - I w i t h i n a range o f 0.4 t o 3 t h a - l and t h a t i t had
i n c r e a s e d a t maturity t o a mean o f 3 t ha-l w i t h i n a range o f 0 . 2 t o 4 . 5 t h a - l . The h i g h e s t
v a l u e s , which ranged from 2.7 t o 12 t ha-1 with a mean of 7.5 t ha-1, were recorded i n t h e
fallow p l o t s . Twenty f i e l d measurements o f non-submerged weed biomass under r i c e cropping
I n fallow p l o t s , completely
gave a mean value o f 1 . 7 t h a - l and a maximum o f 4 . 1 t h a - l .
covered e i t h e r with M. vaginalis o r C. i r i a , t h e values obtained were 7.7 t h a - l and 2 . 8 t
h a - l r e s p e c t i v e l y , o f which about 10% was found t o remain submerged. These f i g u r e s give an
i d e a o f t h e weed biomass t h a t i s a v a i l a b l e f o r c o l o n i z a t i o n by e p i p h y t i c microorganisms i n a
rice field.
Plate 1
al
G l o e o t r i c h i a colonies epiphytic
on Chara.
b)
-
58
[ ) W l l C P l / , OF1 O l t / / J ( l P / , l J : (
Enmration of epiphytic microorganisms on submerged weeds
.
N2-fixing blue-green
algae
B a c t e r i a on:
Chara
Najas
Old
Young
Old
Young
p a (Number
rts
pg a- ir t sf r e s h weight
p a r t s of h ops at )r t s
7 ~ 3 . 1 0 ~ 19.104
13.104
4.10~
glucosea
70.104
16*104
6.104
5.104
malateb
413.10~
25*104
25.104
11*104
32.10'
99-10'
t r y p t i c soy agar'
Chara
140.106
39
210.106
I)
absent on young parts: (2)
r
Table 1.
OM
fx 2 0 ) .
h ' p i p l y Liu miu~ioor~gwrioiriuarid Llrcis d i o LriibuLiun
Two types o f a l g a l epiphytism, (1) v i s i b l e t o t h e naked eye and (2) observable only
under t h e microscope, were n o t i c e d , s p e c i a l l y i n t h e case of t h e subme ged weeds. I n t h e
f i r s t type, globose g e l a t i n o u s colonies of Gloeotrichia (2-10 mm i n &meter)
were a t t a c h e d
t o t h e Clara filaments ( P l a t e l a ) . The d i s t r i b u t i o n o f t h e colonies on t h e h o s t was freque n t l y unequal, t h e o l d e r p a r t s being more h e a v i l y colonized ( P l a t e l a , l b ) . The second
e p i p h y t i c h a b i t , which could be seen only under t h e microscope, was predominantly due t o
Nostoc, Calothrix and Anabaena sp., whose filaments grew f i r m l y attached t o t h e h o s t s u r f a c e .
Even i n t h i s case, c o l o n i z a t i o n by t h e epiphytes became p r o g r e s s i v e l y h i g h e r from apex t o base
o f t h e h o s t and t h i s was q u i t e apparent among t h e young, i n t e r m e d i a t e and o l d l e a v e s o f Najas.
These observations were confirmed by a l g a l enumerations done s e p a r a t e l y on o l d and young p a r t s
o f Chara and Najas.
Table 1 shows t h a t t h e t o t a l a l g a l population on t h e o l d p a r t s w a s four
times t h a t on t h e young p a r t s .
Submerged weeds harboured both a e r o b i c and micro-aerophilic N2-fixing b a c t e r i a . Growth
on glucose medium showed t h e presence o f acid-gas-producing organisms (probably Enterobacteri a c e a e ) , while growth on malate revealed AzospiriZZwn-like organisms.
Based on t h e MPN method t h e number o f NZrfixing b a c t e r i a was i n t h e o r d e r o f lo5 c e l l
(g f r e s h w e i g h t ) - l o f h o s t . There was very l i t t l e d i f f e f e n c e i n t h e c e l l numbers on t h e
d i f f e r e n t weeds and between o l d and young p a r t s (Table 1 except, on o l d p a r t s o f f i a r a ,
where t h e N2-fixing b a c t e r i a l population was approximate y t h r e e times t h a t on t h e young
parts.
ARA measurements i n t h e l i g h t , c a r r i e d o u t s e p a r a t e l y (Table 2), showed t h a t t h e a c t i v i t y
on young p a r t s o f Chma was much h i g h e r than on o l d p a r t s , while on Najas both o l d .and young
p a r t s had t h e same a c t i v i t y . ARA measurements i n t h e dark (Table 2) also showed h i g h e r
G l o e o t r i c h i a epiphytism
l
TaLle 2 .
ARAa by epiphytes
s u i t a b l e f o r t h e attachment o f Gloeotrichia s p . This blue-green a l a forms f l o a t i n g , flobose,
colonies t h a t could develop considerable biomasses o f s e v e r a l t habf (Watanabe e t aZ., 19781,
b u t a r e f r e q u e n t l y washed o u t o f t h e f i e l d by heavy r a i n s o r bleached by high l i g h t i n t e n s i t i e s . Epiphytic Gloeotrichia a r e p r o t e c t e d from t h e s e adverse conditions and provide an
inoculum from which r e g e n e r a t i o n o f t h e bloom i s p o s s i b l e . I t i s t h e r e f o r e c l e a r t h a t i n
t h e n i t r o g e n cycle o f a r i c e f i e l d , t h e submerged weeds p l a y a p o s i t i v e r o l e i n t h e N 2 f i x a t i o n process.
on o l d and young p a r t s o f submerged w e d s , i n
the l i g h t and i n the dark
Chara
Na jas
Light
Dark
Light
Dark
(nmok C2H2 h-1 g-1 f r e s h weight)
Old p a r t s
Young p a r t s
1.51
5 .O5
23.6
49.6
27.2
26.2
1.97
1.57
a Difference between 60 a t 30 minCltes measurements.
-
Ac'ety lene-reducing a c t i v i t y
Cumulative production o f e t h y l e n e by epiphytes on Cham
Time course of incubation.
and Najas incubated i n t h e l i g h t e x h i b i t e d a n o n - l i n e a r i n c r e a s e a f t e r one hour o f incubati o n , as r e p o r t e d by David & Fay (1977). Therefore, s p e c i f i c a c t i v i t i e s were c a l c u l a t e d using
t h e d i f f e r e n c e between 60 and 30 minutes measurements.
-
Results p r e s e n t e d i n Tab'le 3
show t h a t t h e a c t i v i t y p e r u n i t f r e s h weight i n t h e l i g h t on t h e submerged weeds i s much
h i g h e r than t h a t on t h e non-submerged ones. Compared t o t h e l i g h t a c t i v i t i e s , t h e dark acti v i t i e s a r e v e r y low and a r e o f t h e same o r d e r among t h e d i f f e r e n t weeds. The l i g h t a c t i v i t i e s measured under l a b o r a t o r y c o n d i t i o n s were m u l t i p l i e d by 1.8 t o e x t r a p o l a t e them t o t h e
f i e l d , as i t was found t h a t t h e ARA measured i n t h e l a b o r a t o r y was, on an average, 55% o f t h e
outdoor a c t i v i t y . Relating t h e s p e c i f i c ARA t o t h e biomasses o f t h e weeds, i t was found t h a t
e p i p h y t i c N 2 f i x a t i o n on t h e submerged weeds could c o n t r i b u t e 11 t o 24 g N ha-' d - l under r i c e
and 41 t o 63 g N ha-1 d-l under fallow, whereas t h e a c t i v i t y on t h e non-submerged weeds contr i b u t e s only n e g l i g i b l e q u a n t i t i e s o f n i t r o g e n t o t h i s ecosystem.
S p e c i f i c a c t i v i t i e s and extrapolation t o the f i e l d scale.
Table 3.
~
Habitat
Weed type
Light
Submerged
Chara
Naja
Non-submerged
Monochoria
vaginalis
Cyperus i r i a
a Assumes C2H2 : N 2 = 3:1,
35
Biomass o f submerged
host material ,
( t ha-1)
ARA values e x t r a p o l -
Under r i c e
crop
Fallow
Under r i c e
2.0
7.5
f
i t i o n s (nmole C2H4 h-!
(9-1 f r e s h weight)
Dark
7
(131
Fallow
22
( 34)
29
1.8
4.4
-
-
a t e d t o f i e l d condi t i o n (g N ha-1 d - l ) a
crop
0.9
-
-
S p e c i f i c ARA on veeds and extrapolation o f NFA t o f i e l d l e v e l using mean and madmwn
( i n parentheses) values o f weed b i o m e s recorded
ARA under lab. cond-
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Watanabe, I . 1978. Azolla and i t s use i n lowland r i c e c u l t u r e . - Tsuchi t o B i s e i b u t s u 20:
- 1-10.
Watanabe, I . & Barraquio, W.L. 1979. Low l e v e l s o f fixed n i t r o g e n required f o r i s o l a t i o n o f
free l i v i n g N2-fixing organisms from r i c e r o o t s . - Nature 277:565-566.
Watanabe, I . , Lee, K . K . E Alimagno, B.V. 1978. Seasonal change o f NZ-fixing r a t e i n r i c e
f i c l d ass:iycd by i n o i t u ucctylcno reduction tcchniyuc. I . lixpcrinicnts i II Iong-tcriii
S o i l S c i . Plant Nutr. 24:1-13.
f e r t i l i t y plots.
Watanabe, I . , Barraquio, W.L., De Guzman, M.R. & Cabrera, D.A. 1979, Nitrogen f i x i n g (acetylene reduction) a c t i v i t y and populations o f aerobic n i t r o g e n - f i x i n g b a c t e r i a a s s o c i a t e d
with wetland r i c e . - Appl. Environ. Microbiol. 37:813-819.
-
1.3
1.7
(4.1)
. .
2.5
7.7
(n ,d.)
0.4
(1.0)
(n.d.1
2.8
(n.d.)
1.0
(2.3)'
1.6
(n.d.1
2
'
n o t determined.
DISCUSSION AND CONCLUSION
Results i n d i c a t e t h a t both N2-fixing algae and b a c t e r i a were p r e s e n t on weeds b u t most
o f t h e a c t i v i t y w a s due t o blue-green algae. Although h i g h e r d e n s i t i e s o f t h e s e organisms
were observed on o l d p a r t s o f submerged weeds, ARA measurements showed t h a t the a c t i v i t y on
This perhaps i n d i c a t e s
young p a r t s was e i t h e r h i g h e r (Chura) o r o f t h e same o r d e r (Najas).
a h i g h e r c o n c e n t r a t i o n o f q u i e s c e n t o r l e s s a c t i v e populations on t h e o l d p a r t s .
Among t h e d i f f e r e n t weeds s t u d i e d , only submerged ones e x h i b i t e d a s i g n i f i c a n t a c t i v i t y ,
approximating t o an i n p u t o f 2 kg N ha-' crop-' under r i c e and 4 kg N ha-l under fallow
(C2112:N2 = 3 ) .
Another important r o l e o f t h e submerged weeds, mainly Chara, i s t o o f f e r a substratum
I'
60
,
'
e
-
61