3 rd World Conference on Applied Sciences, Engineering & Technology 27-29 September 2014, Kathmandu, Nepal Study of Cyanobacterial Biodiversity in Rice Fields of Central Bihar P. KHARE, A. SINGH, C. PRABHA and S. KUMARI Patna University, Patna, Bihar, India E-mail: khare.pushpanjali2@gmail.com, singh.abhabt@gmail.com Abstract: The agroclimatic conditions of Bihar favours the growth of several rice cultivars along with luxuriant cyanobacterial population. This study was done to document the cyanobacterial diversity of central Bihar situated between 10 N and 85 32 E. Five soil samples per field per site were collected, pooled and subjected to isolation, characterization and identification of the isolates. Out of the total 19 isolates, 11 were nitrogen fixing forms (Aulosira fertilissima, Nostoc muscorum, Anabaena oryzae, Scytonema mirabile, Gloeotrichia ghosei, Cylindrospermum muscicola, Calothrix fusca, Tolypothrix tenuis, Rivularia sps., Hapalosiphon sps. and Stigonema sps.) and 08 were non-nitrogen fixing forms (Aphanocapsa sps., Aphanothece sps., Gloeocapsa sps. and Gloeothece sps., Oscillatoria sps., Plectonema sps., Phormidium sps. and Lyngbya sps.). Nitrogen fixer were multicellular, filamentals and heterosystous, while non-nitrogen fixers were unicellular, colonial and filamentous forms. High frequency of Aulosira fertilissima, Anabaena oryzae and Nostoc muscorum were isolated from almost all the investigated sites. At the time of harvesting the rice fields were specifically dominated by the Aulosira fertilissima and Cylindrospermum muscicola. The presence of different cyanobacterial forms is correlated with their periodicity supporting the growth and development of rice plants. Keywords: Cyanobacteria, Biodiversity, Central Bihar, Nitogen Fixers, Non-Nitogen Fixers, Periodicy, Rice Cultivation Introduction: India is a cradle of biodiversity due to its agroclimatic conditions, where in Bihar the Indo- Gangetic planes are very fertile and suitable for cultivation. Rice is one of the major crops that plays an important role in socio cultural life and economy of Bihar. The rice producing belts of central Bihar receives moderate rainfall with hot and humid climate serving as reservoir of cyanobacterial diversity. Cyanobacteria are able to photosynthesize, fix atmospheric nitrogen, highly adaptive and they also contribute to productivity in a variety of agricultural and ecological environments. Their role in sustaining fertility of flooded rice field soil is well established [1]. Their contribution in maintenance and enhancement of soil fertility is vital. They serve as potential biofertilizer, promoting paddy growth and yield [2]. Various workers have studied the cyanobacterial flora of rice fields of our country [3],[4],[5],[6],[7],[8]. The studies on cyanobacteria pool of central Bihar rice fields are largely unexplored, hitherto. This study is therefore, aimed to isolate, characterize, document and examine their distribution pattern, during rice cultivation period, along with screening of potential isolates serving as natural source of biofertilizers to be used in algalization to increase the yield. 2. Materials and Methods: The study covered seven districts (Muzaffarpur, Vaishali, Samastipur, Begusarai, Nalanda, Patna and Bhojpur) of Central Bihar (Figure 1). Sampling was done from 10 sites, ie Bakhtiyarpur, Biharsharif, Bihta, Begusarai, Jagdishpur, Fatuha, Nalanda, Muzaffarpur, Samastipur and Vaishali. The selected sites are situated between 10 N latitude and 85 E longitude. Meteorological data was collected from District Agriculture and Meteorological office. 2.1 Collection, identification, maintenance and preservation of samples: Soil samples were collected during the cultivation and pre-harvesting period, ie from July to December. The collection was done in every third week of the month from the selected paddy fields. Five samples per field were collected during cultivation period, pooled and subjected to isolation by established protocols. CHU-10 media was used as growth medium for routine sub culturing. The isolates were preserved in 4% formalin. Temporary slides from each soil samples were prepared by staining with methylene blue, mounted in glycerine and examined under high power of the compound microscope with Nikon E-200 photo micrographic attachment. The isolates were identified on the basis of their cell morphology, reffering the available keys [9],[10]. Screenings of heterocystous and non-heterocystous forms were carried out by dilution and pour-plate methods using nitrogen deficient (C-N) medium. 3. Results and Discussion: In the present study rich diversity of heterocystous cyanobacteria was recorded from rice fields of Central Bihar. Nineteen isolates comprising of heterocystous and non heterocystous were isolated. The morphological details of the isolates are as follows: WCSET 2014138 BASHA RESEARCH CENTRE. All rights reserved. http://basharesearch.com/wcset2014.htm
P. KHARE, A. SINGH, C. PRABHA, S. KUMARI Nostoc muscorum :Nostocales / Nostocaceae Colony: Bluish green, gelatinous. Trichomes: tightly packed and densely entangled in common mucilage, cells oblong, irregular, globose, filamentous, heterocystous. Heterocyst: sub-spherical and intercalary, [Figure 2a]. Anabaena oryzae : Nostocales / Nostocaceae - Colony: Soft, green, gelatinous, membranous. Trichomes: Short, straight, densely aggregated, cells barrel shaped and parallel (2.5-3µm), filamentous, heterocystous. Heterocyst: Broad, terminal and intercalary, [Figure 2b]. Aulosira fertilissima: Nostocales / Nostocaceae- Colony: Expanded, membranous, dark bluish green. Trichomes: Straight, parallel, unbranched, densely intricate, cells elongated (7-11 µm) and broad (7-8 µm), filamentous, heterocystous. Heterocyst: Oblong or elliptical, intercalary, [Figure 2c]. Rivularia sps. : Nostocales / Rivulariaceae- Colony: Expanded, vescicular, lobed, hyaline, gelatinous, bluish green. Trichomes: long, basal cells broad, filaments closely adpressed, heterocystous. Heterocyst: Basal and intercalary, [Figure 2d]. Gloeotrichia ghosei : Nostocales / Rivulariaceae- Colony: Free floating, spherical, thallus 4-5 cm in diameter, filaments thin, brown, stratified. Trichomes: Constricted, broader at the base, heterocystous. Heterocyst: Spherical, single, terminal, [Figure 2e]. Cylindrospermum muscicola: Nostocales / Nostocaceae Colony: Expanded, mucilaginous, blackish green. Trichomes: Broad (3-4.7 µm) and constricted, filamentous, heterocystous. Heterocyst: Oblong with epispore, present at both ends of trichomes, [Figure 2f]. Calothrix fusca : Nostocales / Rivulariaceae- Colony: Yellowish brown. Trichomes: single, filamentous, tapering, irregularly curved, bulbous at the base (11-14 µm), filamentous, heterocystou. Heterocyst: Single, hemispherical, basal, [Figure 2g]. Scytonema mirabile: Nostocales / Scytonemataceae- Colony: Expanded, spongy, blackish green. Trichomes: Broad (6-12 µm), filamentous, cells cylindrical, heterocystous. Heterocyst: Cylindrical, convex, intercalary, [Figure 2h]. Hapalosiphon sps. : Stigonematales / Mastigocladaceae- Colony: Floccose, bluish green or brown, highly interwoven filaments, richly branched, heterocystous. Heterocyst: Cylindrical, intercalary, rare. Stigonema sps. : Stigonemales / Stigonemataceae Colony: Thallus thin, crustaceous, brown to black. Trichomes: Arranged in row 2-4, prostrate filamentous, richly branched, hormogones on lateral branches, heterocystous. Heterocyst: Broad, intercalary or lateral. Tolypothrix tenuis : Nostocales / Nostocaceae- Colony: cushion like, filamentous, yellowish brown, thin sheath. Trichomes: Long (2 cm), repeatedly branched, heterocystous. Heterocyst: Rounded cylindrical, solitary or in a row. Oscillatoria sps. : Nostocales / Oscillatoriaceae- Colony: Free swimming thallus, spongy, shining, dull blue green or olive green. Trichomes: Single, straight, filamentous, non-heterocystous, [Figure 2i]. Lyngbya sps. : Nostocales / Oscillatoriaceae- Colony: Massive, thick, blue to purple-red. Trichomes: Single, free, filamentous, spirally coiled, covered with thick and firm sheath, nonheterocystous, [Figure 2j]. Plectonema sps. : Oscillatoriales / Oscillatoriaceae.- Colony: Expanded, filamentous, entangled, lamellated, bluish green in colour, covered with thick sheath, non-heterocystous, [Figure 2k]. Phormidium sps. : Nostocales / Oscillatoriaceae- Colony: Mucilaginous, lamellated, brownish bluegreen. Trichomes: Entangled, flexous, nearly parallel, diffluent sheath, non-heterocystous, [Figure 2l]. Gloeocapsa sps. : Chroococcales / Chroococcaceae- Colony: cells spherical, 2-4 together, colourless sheath, thick, lamellated, blue-green, nannocytes present, unicellular, non-filamentous, nonheterocystous, [Figure 2m]. Gloeothece sps. : Chroococcales / Chroococcaceae- Colony: Thallus mucilaginous, group of unicellular cells (4-8, 16-32), cells ellipsoidal to cylindrical, blue-green to brown cells, sheath brown, nannocytes present, non-heterocystous, [Figure 2n]. Aphanocapsa sps. : Chroococcales / Chroococcaceae- Colony: Mucilaginous, unicellular cells, loosely arranged in a group, gelatinous mass, blue-green, yellowish, nannocytes present, nonheterocystous, [Figure 2o]. Aphanothece sps. : Chroococcales / Chroococcaceae- Colony: Extended mass, shapeless thallus, homogenous mucilage, lamellated individual envelopes, densely arranged, yellowish or colourless, nannocytes present, non-heterocystous, [Figure 2p]. Eleven filamentous heterocystous forms were identified as Nostoc muscorum, Anabaena oryzae, Aulosira fertilissima, Rivularia sps., Gloeotrichia ghosei, Cylindrospermum muscicola, Scytonema mirabile, Calothrix fusca, Hapalosiphon sps. Stigonema sps. and Tolypothrix tenuis, and eight nonheterocystous forms as species of Oscillatoria, Lyngbya, Plectonema, Phormidium, Gloeocapsa, Gloeothece, Aphanocapsa, and Aphanothece (Figure 2 a-p). The isolates belonged to different orders: Nostocales, Chroococcales and Stigonematales and the families Nostocaceae, Rivulariaceae, Oscillatoriaceae, Chroococcaceae and Scytonemataceae. The non nitrogen fixers, ie the species of Aphanocapsa, Aphanothece., Gloeocapsa, Gloeothece are unicellular, colonial and Oscillatoria, Plectonema, Phormidium and Lyngbya are filamentous non-heterocystous forms. The percentage presence recorded by species of Nostoc, Anabaena
Study of Cyanobacterial Biodiversity in Rice Fields of Central Bihar and Aulosira were 94.5, 96.0 and 93.4 respectively during rice cultivation period (Figure 3). Periodicity of cyanobacteria during rice cultivation months gave variable results in all the selected sites. Some of the species were: completely present (P), invariably present (IP) and absent (A) during the cultivation months, ie July-December. Species of Lyngbya was recorded throughout the cultivation period, Nostoc, Oscillatoria, Gloeocapsa and Hapalosiphon in August and from October to December, Aphanocapsa and Aphanothece from August to December, Anabaena and Aulosira from September to December, whereas, Gloeothece and Rivularia October to December. Calothrix and Stigonema, were completely absent from the soil samples of Bihta and Bakhtiyarpur, respectively throughout the cultivation period, but on other hand, Stigonema with other species, eg Cylindrospermum, Plectonema, Scytonema, Tolypothrix were invariably isolated from all the sampling sites. Maximum cyanobacterial genera were present during tillering to harvesting stages, ie from August to December (Table 1). Cyanobacteria are the characteristic features of rice fields, which provide a potential source of nitrogen fixation without any cost for sustainable agriculture [11]. Their taxonomic classification is based upon morphological features and their diversity on biochemical and physiological properties, which make them capable of thriving under different habitats. Cyanobacterial abundance, diversity, distribution and presence of several heterocystous forms might be used as indicator of low nitrogen content in rice fields [2],[7]. Several workers have reported both richness and evenness of cyanobacterial diversity from different rice fields of India [12]. 58 taxa belonging to 20 genera out of which 19 forms were heterocystous in rice fields of Orissa [13]. Presence of high numbers of heterocystous forms suggest the presence of some limiting factors in heterocyst development. The present study revealed rich cyanobacterial diversity and dominance of heterocystous forms, which may be due to environmental conditions with respect to their requirement for light, temperature, water and nutrients as pointed by previous workers. The occurrence of less number of forms during early cultivation stage (Table 1) may be due to inhibitory effect of high light intensity, whereas in the later period of cultivation it is due to depletion of nutrients and low light intensity [2]. Maximum numbers of species (14-15) were recorded during mid-cultivation cycle (September-October), which is in conformity with the earlier findings [2]. Species of Aulosira and Cylidrospermum dominate the soil surface by their dark, matty appearance at the time of harvesting. Predominance of heterocystous forms in this study is attributed to dry period caused by low temperature, light and water deficiency during winter [13],[14]. The present investigation throws light on the cyanobacterial diversity in rice fields of Central Bihar. Due to important property of nitrogen fixation, documented heterocystous cyanobacterial forms could be used as potential biofertilizers. The future prospect lies in the algalization of heterocystous cyanobacterial forms, ie species of Nostoc, Anabaena, and Aulosira, which being indigenous, can be used from pot level to field level by means of mass culture, to enhance productivity because they can established themselves permanently in the field, if the soil is inoculated consecutively for 3-4 cropping seasons [5]. Acknowledgements: The authors are immensely grateful to the Head, Department of Botany, Patna University for providing necessary laboratory facilities and also first author (Dr. Pushpanjali Khare) gratefully acknowledge Principal, Magadh Mahila College, Patna University for supporting throughout the research work. References: [1] Muruga B.N., Wagacha J.M., Kabaru J.M., Amugune N. and Duboise S.M, Effects of physiochemical conditions on growth rates of cyanobacteria species isolated from Lake Magadi a soda lake in Kenya. Web Pub Journal of Scientific Research, 2(5), 41, 2014. [2] Selvi K.T. and Sivakumar K, Distribution of cyanobacteria in rice fields of Cuddalore District, Tamilnadu. International Journal of Life Science and Pharma Research, 2(4), 30, 2012. [3] Nayak S., Prasanna R., Dominic T.K. and Singh P.K, Floristic abundance and relative distribution of different cyanobacterial genera in rice field soil at different crop growth stages. Phykos, 40, 14, 2001. [4] Kaushik B.D. and Prasanna R, Improved cyanobacterial biofertilizer production and N- saving in rice cultivation. In Sustainable Aquaculture, 145, 2002. [5] Mishra U. and Pabbi S, Cyanobacteria: a potential biofertilizers for rice. Resonance, 6, 2004. [6] Chaudhary A.T.M.A. and Kennedy I.R, Nitrogen fertilizer losses from rice soils and control of environmental pollution problems. Communications in Soil Science and Plant analysis, 36, 1625, 2005. [7] Nayak S. and Prasanna R, Soil ph and its role in cyanobacterial abundance and diversity in rice field soils. Applied Ecological and Environmental Research, 5,103, 2007. [8] Digambar R.B., Srinivas D., Padmaja O. and Rani K, Blue-green algae of rice fields of South Telangana region, Andhra Pradesh. Indian Hydrobiology, 11(1), 79, 2008.
P. KHARE, A. SINGH, C. PRABHA, S. KUMARI [9] Presscott G.W, Algae of the Western great lakes area. Pubb.Otto Koellz Science publishers, Koenigstein, 1951. [10] Desikachary T.V, Cyanophyta. ICAR, New Delhi, 1959. [11] Chaudhary K.K., Occurrence of nitrogen fixing cyanobacteria during different stages of paddy cultivation. Bangladesh Journal of Plant Taxon, 18(1), 73, 2011. [12] Prasanna R., Jaiswal P., Nayak S., Sood A. and Kaushik B.I, Cyanobacterial diversity in the rhizosphere of rice and its ecological significance. Indian Journal of Microbiology, 49, 89, 2009. [13] Dey H.S., Tayung K. and Bastia A.K, Occurrence of nitrogen-fixing cyanobacteria in local rice fields of Orissa. India Ecoprint, 17, 77, 2010. [14] Prasanna R. and Nayak S, Influence of diverse rice soil ecologies on Cyanobacterial diversity and abundance. Wetlands and Ecological management, 15, 127, 2007. Table 1. The population diversity of cyanobacteria during rice cultivation period of Central Bihar No. of organisms Months Present * Invariably Present ** Occasionally Present *** (P) (IP) (OP) July 3 11-5 August 12 3 1 3 September 4 15 - - October 14 5 - - November 12 7 - - December 13 6 - - * 10+: P ; **6+: IP ; ***4+: OP ; ****8- : A Absent **** (A) MAP OF BIHAR MUZAFFARPUR VAISHALI SAMASTIPUR BHOJPUR PATNA BEGUSARAI NALANDA Figure 1 : Map showing selected districts of Central Bihar
Study of Cyanobacterial Biodiversity in Rice Fields of Central Bihar
P. KHARE, A. SINGH, C. PRABHA, S. KUMARI 94.5% 96% 93.4% 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Figure 3 : Percentage occurence of organisms during rice cultivation period 1 : Anabaena; 2 : Aphanocapsa; 3 : Aphnothece; 4 : Aulosira ; 5 : Calothrix ; 6 : Gloeothece; 7 : Cylindrospermum ; 8 : Gloeotrichia; 9 : Gloeocapsa; 10 : Hapalosiphon ; 11 : Lyngbya; 12 : Nostoc; 13 : Oscillotoria ; 14 : Phormidium ; 15 : Plectonema; 16 : Rivularia ; 17 : Scytonema; 18 : Tolypothrix ; 19 : Stigonema