Madras Agric. J. 91 (1-3) : 142-144 January-March 2004 Research Notes

Response of rice (ASD 16) seedlings to inoculation of acid tolerant cyanobacterial mutants G. GOPALASWAMY AND S. MALATHY Algal Biotechnology Laboratory, Tamil Nadu Agricultural University, Coimbatore-641 003, Tamil Nadu The agronomic potential of cyanobacterial N2 fixation in rice fields was first recognized in India by De (1939). The rice field ecosystem provides favourable environment for light, water, high temperature and nutrient availability. However cyanobacteria in rice fields are subjected to various abiotic stresses viz. acidity, herbicide, salinity etc. which reduced the population and nitrogen fixation. To overcome the stresses, much emphasis was given for the development of location specific cultures adapted to the stress environment (Gopalaswamy, 2000). Though several promising stress tolerant cyanobacterial cultures were isolated, their performance could still be improved by genetic manipulation to get their full potential. Induced mutagenesis is one such genetic improvement technique. Recently several acid tolerant cyano-bacterial cultures were isolated from acidic rice soils of Tamil Nadu and characterized (Gopalaswamy et al. 2002). Several acid tolerant cyanobacterial mutants were developed using EMS. In this study, an attempt was made to find out the response of rice seedlings to inoculation of these cyanobacterial mutants. A pot cultures experiment was conducted at the Algal Biotechnology Laboratory, Dept. of Biotechnology, Centre for Plant Molecular Biology, TNAU, Coimbatore during 2000 in plastic tubs (15cm dia and 6 cm height) with three replications in a completely randomized design. The tub were filled with 2kg of unsterilized acid soil and sown with pregerminated seeds of rice variety ASD 16 at 15 seeds tub-1. The acid tolerant cyanobacterial mutants viz. AnabaenaAT-MGK-5A6-E200, Nostoc-AT-MGK-4C4-E250, Oscillatoria-AT-MGK-5B8-E200, Westiellopsis-ATMGK-4A2-E250 and Westiellopsis-AT-MGK-4A7-

E250 were used for the study. For comparison, the normal soil culture Anabaena NS-5G2 and acid tolerant wild culture Anabaena-AT-TGK5A6 were used. All cyanobacterial cultures were inoculated individually at 1g fresh weight tub-1 on 7th day after sowing. An uninoculated control was maintained for comparison. The water level was maintained at 2 cm throughout the study period. Plant height, ammonia excretion (Solarzano, 1969), and the total chlorophyll (Winterman and Demotes, 1965) of the seedlings were taken at 20 and 40 days after sowing, while the total nitrogen content (Humphries, 1956) of the seedlings was assessed on the 40th day after sowing. Inoculation of acid tolerant cyanobacterial mutants increased the plant height, chlorophyll content, total nitrogen content of ASD 16 rice seedlings and flood water ammonia significantly over uninoculated control as well as Anabaena NS-5G2. Among the cyanobacterial cultures Westiellopsis increased all the above parameters (Table 1 and 2). Between the Westiellopsis cultures, Westiellopsis-AT-MGK-4A2-E250 registered significant increase in all the above parameters besides excreting higher levels of ammonia in the flood water. The cultures grew well in the tubs and covered the soil surface in 30 days of inoculation. This clearly showed the adaptive nature of the mutants to acidic conditions. The higher chlorophyll and nitrogen content in rice seedlings might possibly be attributed to the vigorous growth of cyanobacteria, which facilitated the uptake of nutrients, by rice seedlings. Similar observation in improving the growth, chlorophyll and total nitrogen content of rice due to inoculation of acid tolerant cyanobacterial cultures was observed by Tamilselvan (1998).

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Response of rice (ASD 16) seedlings to inoculation of acid tolerant cyanobacterial mutants

Table 1. Effect of inoculation of acid tolerant cyanobacterial mutants on seedling height, total chlorophyll and total N of ASD 16 rice seedlings Treatments

Uninoculated Anabaena-NS-5G2 Anabaena-AT-TGK-5A6 Anabaena-AT-MGK-5A6-E200 Nostoc-AT-MGK-4C4-E250 Oscillatoria-AT-MGK-5B8-E200 Westiellopsis-AT-MGK-4A2-E250 Westiellopsis-AT-MGK-4A7-E250 Days Treatment Days x Treatment

Seedling height (cm)

Total chlorophyll (mg g-1 fresh wt.)

20th * day

40th day

20th day

40th day

12.60 13.46 15.48 16.74 17.54 13.51 18.48 19.45 SED 0.07 0.10 0.19

32.62 34.49 35.40 37.66 39.39 35.66 43.70 45.57 CD 0.14 0.20 0.39

0.79 0.86 0.95 1.05 1.15 0.86 1.25 1.27 SED 0.01 0.01 0.02

1.26 1.45 1.65 1.75 1.85 1.54 2.06 2.16 CD 0.02 0.01 0.04

Total N 40th day 0.49 0.53 0.56 0.66 0.67 0.57 0.69 0.73 CD 0.01 -

* Days after sowing Table 2. Effect of inoculation of acid tolerant cyanobacterial mutants on ammonia excretion in flood water of soils planted with ASD 16 rice seedlings Treatments

Uninoculated Anabaena-NS-5G2 Anabaena-AT-TGK-5A6 Anabaena-AT-MGK-5A6-E200 Nostoc-AT-MGK-4C4-E250 Oscillatoria-AT-MGK-5B8-E200 Westiellopsis-AT-MGK-4A2-E250 Westiellopsis-AT-MGK-4A7-E250 Days Treatment Days x Treatment

Ammonia excretion (n moles ml-1) 20th day*

40th day

20.56 53.15 68.61 73.36 77.64 65.57 84.49 87.68 SED 0.10 0.17 0.29

15.48 21.49 31.62 43.44 45.57 32.69 49.68 50.54 CD 0.21 0.33 0.58

* Days after sowing

The improved performance of the inoculated seedlings might be due to the production of the growth promoting substances and the continuous photo production of ammonia leading to enhance nutrient availability. The authors are thankful to ICAR, New Delhi for financial assistance under National

Agricultural Technology Project-Team of Excellence on Biofertlizers for rice based cropping systems. References De, P.K. (1939). The role of blue green algae in nitrogen fixation in rice fields. Proc. Roy. Soc. London, 127: 121-139.

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Gopalaswamy, G. (2000). Blue green algae for improving the fertility of soils. CAS training on soil microbial interaction. Tamil Nadu Agric. Univ., Coimbatore, 77-86 pp. Gopalaswamy, G., Tamilselvan, B. and Kannaiyan, S. (2002). Biomass production and biochemical composition of acid tolerant cyanobacteria. Indian J. Microbiol. 42: 121-124. Humphries, E.C. (1956). Mineral composition and ash analysis. In: Modern Method of Plant Analysis (Eds.) K.Peach and M.V.Tracey, Springer Verlag, Berlin, 468-502 pp. Solarzano, L. (1969). Determination of ammonia in natural waters by the phenol hypochlorite method. Limnol. Oceanogr. 14: 799-801.

Tamilselvan, B. (1998). Selection of fast growing and higher nitrogen fixing acid tolerant cyanobacteral cultures and their utility as biofertilizers for rice. M.Sc.(Ag.) Thesis, Tamil Nadu Agrl. Univ. Coimbatore, Tamil Nadu, India. Winterman, J.F.G.M. and Demotes, A. (1965). Spectrophotometric characteristics of chlorophyll a and b and their phenophytins in ethanol. Biochem. Biophy.Acta. 190: 448-453.

(Received: March 2003; Revised: December 2003)

Madras Agric. J. 91 (1-3) : 144-147 January-March 2004 Research Notes

Influence of organic manures, biofertilizers and plant density on growth, yield and alkaloid content of bhumyamalaki (Phyllanthus amarus Schum. and Thonn.) N. CHEZHIYAN AND S. SARASWATHY Hortl. College and Research Institute, Tamil Nadu Agrl. University, Coimbatore-641 003, Tamil Nadu Phyllanthus amarus belongs to the family Euphorbiaceae known popularly in the Indian Systems of Medicine as Bhumyamalaki. The plant has been reported to exhibit a marked antihepatitis B virus antigen activity, antibacterial, anticrustacean, antifungal and antiviral activity. The lignans particularly phyllanthin and hypophyllanthin are present in the plant and the whole plant is used in herbal drug preparation. There is ample scope for large-scale cultivation of P. amarus as a pure crop. As the whole plant is used as such in drug preparation, there is a need for producing the crop chemical free, which necessitates the organic farming technology. The present study was carried out to assess the role of organic manures, biofertilizers and plant density and their interaction effects on the growth and yield of P. amarus. The experiment was carried out in the Department of Spices and Plantation Crops,

Horticultural College and Research Institute, Tamil Nadu Agricultural University, Coimbatore during 2000-2001 to study the effects of organic manures, biofertilizers and plant density on growth herbage and alkaloid (lignans) yield of P. amarus. Based on the preliminary study results, the main experiment was carried out. The treatment consists of manures (M1-Farmyard manure-1.0 kg m-2; M2-Poultry manure-0.5 kg m-2), biofertilizers (B1-with Azospirillum-2 kg ha-1; B3-with Phosphobacteria-2 kg ha-1; B4-Both Azospirillum & Phosphobacteria-each @ 2 kg ha-1) and plant density (S1-15 x 10 cm, S230 x 10 cm, S3- 45 x 10 cm). The manures were applied at the time of land preparation and the biofertilizers were applied at the time of transplanting. Thirty five days old vigorous healthy uniform size seedlings were transplanted in the main field. After three months of planting, when plants were at drying stage followed completion of vegetative growth, the crop was