Growth and Nitrogen Fixation in Azolla pinnata . under Field Conditions

The growth and nitrogen fixation of two indigenous and two exotic s t ra i~s of Azollapinnata under rice field condttions, were examined at Ambalantota, in the dry zone of Southern Sri Lanka where the terrain is undulating to flat, tlie rice soils are of the low humic gley type and the 75 % expectancy value of annual rainfall is less than 500 rnm. A seven to eight fold increase in biomass was recorded in 15 days, giving a doubling time of 4.8 days. In situ nitrogenase activity (measured by the acetylene reduction technique) gave values equivalent to 3.1 to 4.6 kgN/ha/day. These resnlis lend strong support to the potential use of A.yinnata as a biofertilizer for rice in Sri Lanka.


I. Introduction
Azolla is a free floating aquatic fern which contains a nitrogen fixing blue-green alga (Anabaena azollae) as an endosymbiont.It grows well under flooded conditions and is used in celtain Asian countries as an organic f~rtilizer for r i ~e .~> ~ There are reports which indicate that Azolla performs better under low light and low temperature c~nditions,l>~ but cartain strains of Azollapinnata the species native to Asia, have been found to grow well under conditions of relatively high light and tempera.tu ~e , ~, ~ and such strains may have a greater agronomic potential in tropicalhabitats.
This paper reports on certain field investigations conducted on four strains of Azolla pinnata R. Brown, at the Rice Research Station at Anlbalantota, in the d ~y zone of Southern Sri Lanka, where the terrain is undulating to flat, the rice soils are of the lob humic gley type and the 75% expectancy value of annual rainfall is less than 500 mm.

Experimental
The four strains of Azolla piranata examined, have been originally collected from Peradeniya, Debokkawa (a village 20 knl away fiom Ambalantota), India and Bangkok (the latter two strains were obtained from the Azolla collection at the International Rice Research Institute, Los Banos, Philippines).These strains were initially grown under laboratory conditions in the Department of Botany, University of Pe~adeniya, and then adapted to outdoor conditions at Peradeniya growing them in galvanized-iron trays in soil-water culture.
A 2 cm layer of rice soil was added to each tray; 240 cm long, 60 cnl wide and lOcm high and tap water was slowly added to fill up to 5 cm.These trays wcre jnoculated with fresh.Azolla and left outdoors, with periodic additions of con.centrated superphosphate fertilizer.A bent glass tubing attached to one end of the tray was positioned in such a manner to provide drainage of water above the 6 cm level (Figure l), in order to prevent Azolla spilling over during rainy wea-ther.Material thus grown at Pera.den.iya was t~.a.nsported fresh to Ambalantota.and gradually adapted to field conditions there.They were initially grown i n small galvanized-iron trays in soil-water culture, under a partial shade and the11 transferred to 8m X 3m plots jn the rice fields, and allowed to grow a.nd adapt themselves for three nzonths.Relatively heavy inocula, (1 to 2 kg fresh.Azollaper m 3 , were used at this stage, to ensure its propsr estab1ishmen.t in the fields.The original 8n1 x 3m field plots were subdivided to sina.ller plots, sing bamboo poles t o keep the Azolh fronds together, because sapid fraginentation and separation of the frosnds have been reported t o retard the growth of Azol1a.l.jAs the Azolla carpet increased, the bamboo poles were moved gradually to increase tlze surface area of the plots (Figures 2 a, b,  & c).The bamboo poles were placed in relation to the wind direction, the blowing of which would assist the Azolla cover to spiead once a pole is lemoved.Initially, a large number of Azolla fronds turned red and many died, but certain fronds remained green and produced luxuriant growth, to cover the plots completely within.three weeks.
These nursery cultures were maintained with psriodic additions of triple superphosphate (TSP) powder to provide phosplzorus and Carbofuran granules (3 to prevent any insect attack.Material from these nurseries were used for the experimental evaluation of the ~r o w t h and nitrogen fixation of Azollu in monoculture, under field conditions.

Growth and nitrogen fixation
This experiment was conducted in l m s 5n1 plots wit11 independent irrigation and drainage facilities.These plots had bunds 20 cm wide and 15 cm high, and the Aood water level in the111 was maintained between 5 to 10 cm.There were four replicates for each dzolln strain tested and these were arranged in a randomized-complete-block design.
One week after preparation, the plots were inoculated at the rate of 180 g (fresh Azolla) per in2 (i.e.900 g per plot or 1.8 t/ha), using material from the field nu~series.
Each inoculum was nlixed wit11 6 g/kg (fresh Azolla) of TSP powder and 1 g/kg (fresh Azolla ) of Carbofuran.TSP powder a t the rate of 3 g/m2 was broadcast over the Azolla cover, every three days and Carbofuran was applied at the rate of 0.5 g / ~, ~ at the initial sign of any pest attack.Every 3 days, the fresh weight of Azolla was measured separately in each plot.The few weeds that showed up were hand removed.Within 15 days.most of the plots were completely covered by Azolla and further biomass measurements were stopped at this stage.Nitrogen fixing activity of these 15-day old material was measured by the acetylene reductionassay.

Measurement of Acetylene Reducing Activity (ARA)
ARA measurements on field grown Azolla are frequently condl~cted in glass bottles, conical flasks and other transparent containers.Although suclz measurements zre sometimes carried out in the field, it is not possible to incubate the samples in a floating position together with the natural Azolla cover.In the present study, a novel method of incubation was adopted, wh~reby it was possible to keep the samples floating in situ with the rest of the Azolla mat.In this case, transp~rent, plastic baby feeding bottles of approxima-tely 300 ml were used as sample containers.Short pieces of flexible PVC tubing stoppered at one end wit11 subaseal caps, were firmly fixed to the lids of the bottles, so as to permit the injection and withdrawal of gases.Each feeding bottle was filled upto the 100 ml mark with Azolla and water from the plot and this was allowed to float in a horizontal position with the rest of the Azolla in the field (Figure 3)." --o m -O . c . r   ---------r --------.-----*--*----o-----._---------*  Using a 50 ml graduated syringe.a volume of air equivalent to 10% of the gas phase in the bottle was first withdrawn and an equal volume of acetylene was then injected.In. this manner, the test Azolla samples were incubated under 10 % acetyene in air.Eight bottles were ~a n d o ~n l y thrown into each plot, and the values given 4 'in Table 1 are the mean of elght such leading~.A set of 'blank samples' containing Azollu-free paddy water was also Included, to monitor 2ny background nitrogenase activity.Incubation with acetylene was carried out for 20 minutes, under a light intensity of 90 klx a.nd a temperature whicli Zuctuated between 34°C to 37OC.At the end of the incubation period, 2 ml gas samples were withdrawn and stored in vacutainers for subsequent gas chromatographic analyses.The samples were analysed at 80°C, in a Perkin-Elmer Sigma-4 gas chromatograph, fitted with a 2m, 80/100 mesh column of Poropak T. Detection of ethylene and acetylene was done on a Hydrogen-Flame-Ionization detector, w~th n~trogen at a flow rate of 30 ml per minute serving as the carrier gas.

Results
The growth patterns of the four Azolla strains are shown in Figure 4.It can be seen from this f i y ~r e that all th.e four Azolla strains have followed a similar pattern of growth.; after an initial, short lag phase, they have m~rltiplied q i t e rapidIy until the 15th day, whpn F-jrther nleasgrements were stopped, because limitation of space would have retarded the rate of growth in the plots that were completely covered by this time.It is clcar from this curve that the biomass of Azolla, has increased 7 to 8 fold within 15 days.Although.tlaerp, is not mi-~ch difference in the final biomasses produced, by the differen.tstlains, the strain from Debokkawa appears to have pzrformed marginally above all the others.Nitrogenase activity (ARA) of the 4 Azolla strains of 15 day, are in Table 1.There are no significant differences in ARA among the four strains.

Figure 1 .
Figure 1.Outdoor, soil-water culture of Azolla pinnata in a galvanized-iron tray.(a) Cross sectional view of the draining system.(b) View of tray showing drain tube attachment.

( 6 )
2nd stage : Bamboo poles moved to double the area of each subplot.

Figure 3 .
Figure 3. Use of a 'baby feeding ' bottle for the field incubation of AzoIIa with acetylene.

TABLE I .
Biomass and Acetylene Reducing Activity (ARA) of 15 day old monocultures of Azolla pinnata strains grown in 5m2 field plots at Am'balantota, in the lowcountry dry zone of Sri Lanka.Growth conditions are the same as in Figure4.Mean value of eight samples incubated with 20 % acetylene in air from 1330 to 1430 CST under 90 klx 34 to 370C. (b)