IN SPECIES

Indian J. Plant PhYlliol., Vol. XXIX No. J pp. ,99-103 (Marcb 1986)

SHORT COMMUNICATION

GROWTH AND NITROGENASE ACTiVITY IN t4,ZOLLA SPECIES

D.P. SINGH AND P.K. SINGH

Laboratory of Blue-green Algae, Central Rice Research Institute, Cuttack-7S3 006

Growth and nitrogenase activity Were determined in thirteen collections of Azolla; eleven from A. pinnata and one each from

A. mexicana and A. filiculoidell. under identical condition in mineral medium. Many isolates of A. pinnata. 8Jld A. filicrlloidell grew better than A. mexicana and among different collections of A. pinnata, isolates of Thailand. BangIadesh and Vietnam produced more fresh and dry

matter while the minimum growth was recorded in Nepal (I> isolate. Though A. mexicana showed poor growth, however, nitrogenase activity was maximum in this isolate whereas A. filicuioidell showed a low leve) of nitrogenase activity. AmoDg A. p;nnata isolates. Bangla­ desh (Tangail isolate exhibited highest nitrogenase activity and the

lowest Was recorded in Indonesia (Bogor) isolate.

INTRODUCTION

Water fern Azolla is known to fix atmospheric nitrogen by its symbiotic association with Anabaena azollae, a blue-green alga found in the cavities of dorsal lobe of the fronds (Becking, 1979; Singh, 1979; Peters et al.. 1982;

Watanabe, 1982). Although growth and nitrogenase activity of individual isolates (Peters and Mayne, 1974; Becking, 1976; Talley et al., 1971; Watanable

, et aI., 1977) have been reported little is known on the comparative performance of different isolates. The present stUdy was aimed to compare growth and nitrogenase activity amongest thirteen isolates of Azalia grown under identical i environmental and nutritional conditions.

I

Eleven isolates of A. pinnata and one each of A. mexicana and A. jiliCfl­ ( loides collected from different countries were used in this study. Three

hundred mg fresh Azolla was inoculated in 600 ml corning glass beakers con­ taining 300 ml of modified Hoagland's N-free medium supplemented with trace elements. The composition of the medium used was as follows: P (KH.zPO ..) 10 ppm. K (KCn 15 ppm. Ca (CaCls.2H20) 20 ppm, Mg (MgSo...7HaO) 30 ppm~ Fe (Fe-EDTA) 2 ppm, Mo (NasMo04.2H.zO) 0.01 ppm, Co (CoCl1·2H10) 0.01

100 D.P. SINGH ,AND P.K. SINGH

under identical conditions in a net house. Each Azolla isolate was grown in

3 replications and the experiment was repeated twice. Results are reported in Table I and are the means of two experiments. .Azolla plants were harvested

Table I: Growth and nitrogenase activity in .AzoDa species

Fresh Dry Total Ethylene

weight weight chlorophyll produced

"bol/a sps. (g) (mg) (mg/g fresh (nmoles/mg

Azolla) chl./min)

A. pinnaill.

Vietnam 2.68 96 0.744 14.9

Nepal (I) 1.86 58 0.613 20.4

Nepal (II) 2.17 '4' 0.686 11.0

Nepal (III) 2.60 69 0.710 13.0

India (Cuttack) 2.29 56 MIl 12.0

Thailand (Bangkok) 2.81 79 0.637 16.1

Africa 1.90 57 0.714 12.4

Bangladesh (Serigor) 2.77 73 0.629 17.0

Bangladesh (Tangai) 2.60 33 0.671 29.5

Indonesia (Bogor) 2.10 74 0.642 9.8

Philippines (Bicol) 2.04 69 0.743 11.9

A. mixico1fll 1.37 56 0.625 29.9

A. /iliculoMes 2.4] 83 0.620 16.9

C.D.5% 0.18 10 0.044 6.9

after 10 days of inoculation. blotted with blotting paper to remove surface water and the fresh weight was recorded. Dry weight was recorded after drying the samples in an oven. Total chlorophyll was measured following the method described by Yoshida et 01. (1972). Nitrogenase activity was estimated

by acetylene reduction assay method using a gas chromatograph (Schollharn and Burris. 1967). To estimate nitrogenase activity. fresh .Azolla (100 mg) was

placed in 20 ml capacity coming glass tubes. fitted with air tight rubber stoppers, containing 5 ml of N-free medium. Purified acetylene (0.5 mt) was injected after removing an equal volume of air. The reaction mixture was incubated for 2 hours in a culture room maintained at 25±loC and 2.5 Klux. Sample of 0.5 ml of gas mixture was injected in a gas chromatograph (AIMIL.

SERIRS~SSOO) Nitrogenase activity was expressed as n moles C1l:I4/mg Ch1./min

r

GROWm AND NITROGENASE ACTIVITY IN AZOLLA SPECIES 101

From the data it was observed that .4.. pinnata (all isolates) and .4.. filiculoides

produced more fresh weight than .4.. mexicana whereas .4.. pinnata isolates from Thailand (Bangkok), Bangladesh (Seigor and Tangai). Vietnam and Nepal lIT were significantly superior to .4.. filiculoides. .4.. filiculoides and vietnam. Nepal II, Nepal III, Thailand (Bangkok), Bangladesh (Serigor and Tangai), Indonesia (Bogor), PhiJippines (Bicol) isolates of .4.. pinnata produced more . dry matter than .4.. mexicana while only Vietnam isolate of .4.. pinnata was superior to .4.. filiculoides • .4.. pinnata isolates from Vietnam. Nepal II. Nepal

nl.

and.4.. mexicana did not differ. Among.4.. pinnata isolates, Thailand (Bangkok). Bangladesh (Serigor) and Vietnam recorded higher fresh weight while isolate of Bangladesh (Tangai) and Nepal (III) did not differ with that of Vietnam in fresh weight. Maximum dry matter was produced in Vietnam isolate followed by Thailand (Bangkok). Bangladesh (Tangai), Nepal (II) and Indonesia (Bogor) however they did not differ in dry matter production among them­ selves. Significantly higher total chlorophyll was obtained in the isolates of Vietnam, Philippines (BicoI), Africa, India (Cuttack) and Nepal (In) followed by Bangladesh (Tangai). Indonesia (Bogor) and Nepal (II) isolates. Nepal (I) 'produced minimum fresh and dry matter with lower chlorophyll content among.4.. pinnata collections. Further the fresh weight of Philippines (Bicot) isolate did not differ with that of Nepal (I). Though growth of the isolate from Africa was poor its chlorophyll content was fairly high. Singh (1982) reported better growth of.4.. pinnata in rice fields. Further amongpinnata isolates, Vietnam green isolate grew well during summer months.

Maximum nitrogenase activity of 29.9 and 29.S n moles CsRl/mg chl./min. respecti vely (Table I) was recorded in .4.. mexicana and Bangladesh (Tangai) isolate of A. pinnata while .4.. filiculoides showed moderate enzyme activity. Highest nitrogenase activity among.4.. pinnata isolates was obtained in Tangai isolate of Bangladesh and the minimum was recorded in Bogor isolate of Indonesia. The isolates from Nepal (I) and Bangladesh (Serigor) showed more enzyme activity than Indonesian isolate. The isolates from Vietnam, Thailand (Bangkok). Nepal (I) and Bangladesh (Scrigor) showed similar enzyme activities.

Peters and Mayne (1974) reported the nitrogenase activity of 25-30 n moles CIIH4/IDI chl./min. in A. caro/iniana wild grown under controlled

conditions. Talley et al. (1977) obtained the acetylene reduction rates of 800 and 773 n moles CIiRI/g dry weight/min. in.4.. me:Kicana and.4.. filiculoides

102

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D.P. SfNGH AND P.K. SINGH

study are lower than that of Talley et al. (1977), however. A.. mexicana showed comparatively better enzyme activity. In A.. pinnata isolate from Philip~

pines, the nitrogenase activity of 340 n moles CtH4/g fresh weight/h was reported by Watnable et aI. (1977) wbereas with present study it was 528 n moles CsH4/g fresb weight/h. Becking (1979) summarized the work on growth and nitrogenase activity of different Azolla species but a comparison possibly can not .be made because of the differences in the growth conditions of Azolla

and acetylene reduction assays method. In the present study the growth and nitrogenase activity of A.zolla isolates of different species were compared under identical growth and assay conditions. Azolla grown in the net house under natural environment follows similarity under field conditions as well.

ACKNOWLEDGEMENT

We gratefully acknowledge Dr. H.K. Pande, Director, C.R.R.I. for providing necessary facilities.

REFERENCES

Decking, J.H. (J976). Contribution of plant algal association. In "Proceedings of the 1st International Symposium on Nitrogen Fixation", (eds .. W.E. Newton and C.J. Nyman). Washington State University Press Pullman, Washington, Vol. II, pp. 581-591.

Becking, J.H. (1979). Environmental requirements of A.zoJla for use in tropical rice production. In "Nitrogen and Rice", IRRl, Laguna. Philippines, pp. 345-374.

Peters, G.A. and Mayne, B.C. (1974). The Azolla-Anabaena azolJae relationship. II Locali­ zation of nitrogenase activity as assayed by acetylene reduction. Plant Ph),siol.,

53: 820-824.

Peters, G.A., Calvert, H.E .• Kaplans, D., Ito, O. and Toia, Jr. R.E. (1982). The Azolla­

Anabaena symbiosis: morphology, physiology and use. Israel J. /Jot•• 31 : 30'-323. SchoUham, R. and BUrris, R.H. (1967). Acetylene as a competitiVe inhibitor of Ni-fixation,

Proc. Natl. A.cad. Sci, USA 58 : 213-216.

Singh, P.K. (1979). Use of A.zolla in rice production in India. In "Nitrogen and Rice", IRRI. Laguna philippines, pp. 407-4J8.

Singh, P.K:. (J980). Introduction of green Azolla biofertilizer in India. CUrr, Sci., 49: ISS-IS6.

Singh. P.K. (1982). AzolJa as an organic nitrogen fertilizer for medium and lowland rice. In "Review of soil Research in India". IARI, New Delhi Vol. II, pp. 236-242.

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OROwm AND NITROGENASE .ACTIVITY IN AZOILA SPBCIBl

Watanabe, I. (1982). Azol/a-Anobaena symbiosis-its physiolosy and use in tropicaiagricuUure.

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Watanabe. L •• Bspinas, C.R., Borja, N.S. aDd Alimango, B.V. (1977). Utilization of tho

Azolia-Anabaena complex 88 a nitrogen fertilizer for rice. IRRI Paper Series II: 15 p.