Chroococcalean blue green algae from the paddy fields of Satara District, Maharashtra, India

Blue green algae are the photosynthetic prokaryotes representing a wide distribution in habitat, i.e., temperate, tropical, and polar region. Paddy fields are the best studied aquatic ecosystems on earth which fulfill all the necessary demands required for blue green algal growth. Blue green algal role in enhancement of paddy yield has been studied worldwide. Sustainable utilization of an organism for community use depends on how successfully the ecology of that organism is understood. Twenty-eight chroococcalean blue green algal taxa were recorded from the study area. They were taxonomically investigated and found to belong to two families and 11 genera. The first family Chroococcaceae was the largest family with 10 genera and 26 species while the second family Entophysalidaceae had only one genus and two species. The genus Gloeocapsa from the family Chroococcaceae exhibited largest species diversity (21.42%), as well as taxa Chlorogloea fritschii of family Entophysalidaceae showed species abundance from the study area. All heterocystous blue green algal forms are capable of fixation of atmospheric N2. Many of the non-heterocystous or unicellular blue green algae also have the capacity of N2 fixation. The taxonomical documentation of chroococcalean blue green algae provide information about such indigenous unicellular blue green algae which will help in the development of niche specific inoculants as biofertilizers for rice fields of the study region.


INTRODUCTION
Blue green algae are important components of soil microflora in paddy fields. They play an important role in maintaining and improving soil fertility, as they have the ability to fix atmospheric nitrogen and transform it to nitrate/nitrite (Anand 1990). The rice fields provide ideal environment for luxuriant growth of blue-green algae. They are found in paddy field soil throughout the year at various growth stages of the rice crop (Nayak et al. 2001). There is huge structural diversity found in class Cyanophyta (blue green algae) which is the main reason for attracting algologists to develop a keen interest in their taxonomic study.

MATERIALS AND METHODS
Two-hundred-and-eighty-eight paddy fields were selected from Patan and Karad tehsils of Satara, Maharashtra. Patan is 65km away to the south-west of Satara and is located at 17.370N & 73.900E. Most of Patan Tehsil is hilly with deep valleys while some parts are plains and receives heavy rainfall. The common soil is red lateritic soil, in the plains it is black cottony soil while at elevations it is the basaltic and lateritic type. This tehsil is famous for the cultivation of local varieties of paddy, viz.: Dombya, Dodkya, Kolambya, Bhados, Panwel, Indrayani, Champakali, Ghansal, Jiresal, Teliansh, Kaveri, Krishnakusal, Basmati, and Ambemohar. Karad is 52km to the south-east of Satara and is located at 17.289N & 74.181E. Karad city situated at southern part of Satara District near Agashiva, at the confluence of Koyna and Krishna rivers called 'Preeti sangam'. The tehsil receives moderate rainfall and the common soil type is black cottony soil. It is famous for the cultivation of local varieties of rice, viz.: Indrayani, Rethare Basmati, Pusa Basmati, Hansa, Khadkil Kolhapuri, Kolhapuri R-24, and Kaveri.
Frequent and timely collection of soil and algal samples were undertaken during the rainy season (2012)(2013)(2014)(2015)(2016)(2017). Soil samples were collected from paddy fields of the study area ( Fig. 1). Soil from rice fields were collected randomly from both the tehsils as per Somawanshi et al. (1999). The collected soil samples were brought into the laboratory using polythene bags, dried at room

J TT
temperature in diffused sunlight, and crushed with the help of a mortar and pestle. About 10g of sieved soil was inoculated in culture bottles containing 100ml culture media like BG -11±, Foggs and Chu 10. We found good results in BG 11 medium, so for further culturing and sub culturing we prefer BG 11 ± medium. These cultures were incubated at 22±2˚c with 16/8 light dark cycle under 5 Klux intensity of light, after incubation algal growth appeared in the enriched cultures in laboratory. Cyanobacterial growth from enriched cultures were examined microscopically and identified with the help of standard literature (Dasikachary 1959;Anagnostidis & Komarek 1985;Anand 1990;Santra 1993). Photographs were taken by using photomicrography unit of Olympus CH20i (Photoplates I, II, III).
The species diversity % was calculated by using the following formula.

Systematic enumeration of Chroococcalean blue green algae
Order: Chroococcales Wettstein Forms of this order are unicellular or colonial, not differentiated into base and apex; as well as trichome organization totally absent. Endospores or exospores not present. Thallus irregularly spherical, bluish green in color. Cells 6.5-7.5 µm diameter, somewhat oval, sheath mucilaginous.

Mundhe, Vijaynagar.
Patan and Karad tehsils of Satara districts are famous for paddy cultivation. An extensive study was made in search of diversity, distribution and occurrence of chroococcalean cyanobacteria. Order chroococcales contains two families-Chroococcaceae and Entophysalidaceae. Eleven genera and 28 eight species were recorded by screening 288 paddy field localities of study area. From family chroococcaceae 10 genera and 26 species were recorded. Genus Gloeocapsa with six species; followed by genera Chroococcus with five species, Gloeothece and Aphanothece with three species were dominant. While from family Entophysalidaceae only one genus Chlorogloea with two species, i.e., Chlorogloea fritschii and C. microcystoides were reported. But these two forms were frequently recorded from the study area. Genus Gloeocapsa showed species diversity i.e., six species of single genera recorded in study area while genus Chlorogloea with two species reported to be dominant taxa i.e., reported in most of the paddy fields repeatedly.
Some interesting observations were made while isolating and culturing of unicellular forms from paddy field soils of Patan and Karad tehsils. Generally at the beginning filamentous forms appear in the culture bottles while unicellular forms appeared in old cultures. The members from family Chroococcaceae showed coccoid appearance, they form smooth gelatinous loose colonies while members of family Entophysalidaceae are also coccoid but show colonial growth habit. The cells grow to give dense parenchymatous mass. The growth habitat in culture condition of these taxa become helpful to differentiate them visually. Wyatt & Silcey (1969) also studied nitrogen fixation of chroococcalean blue green alga Gloeocapsa species while Zhou & Chen (1991) recorded their efficiency for nitrogen fixation. Our study area also showed J TT  (Figure 2). Nitrogen fixation by Gloeothece species was noted by Maryan et al. (1986). The least diverse species did not show their adaptability for changing pH condition of the cultures and they vanish very soon. But Gloeocapsa, Chroococcus, Gloeothece, and Aphanothece proved their adaptability to changing pH. Chlorogloea fritschii showed high dominance (reported from 11 localities of Patan Tehsil and 26 localities of Karad Tehsil) followed by Microsytis, Chroococcus & Gloeothece. This data would provide the knowledge about such indigenous chroococcalean species which showed species diversity and occur frequently in paddy soil cultures. This will help in development of niche specific inoculants as biofertilizers for rice fields of the study region. The abundant growth of chroococcophyceae members in aquatic environment especially planktonic state than in terrestrial environment was recorded by Naz et al. (2003). They surveyed fresh water cyanophyta from certain areas of northern region of Pakistan and Azad Kashmir. Naz et al. (2004) reported 46 planktonic, edaphic, epipsammic, epioikotic, epilithic & epiphytic blue green algae belonging to class Chroococcophyceae (cyanophyta) from various fresh water habitats of Pakistan. They reported these chroococcalean forms from various habitalts; but we recorded 28 chroococcalean taxa from paddy field J TT   Singh (1973). Majority taxa found in paddy in fresh form as well as in soil cultures was of filamentous heterocystous and filamentous non-heterocystous type. Non-heterocystous chroococcalean cyanobacteria, however, also fixes atmospheric nitroge (Wyatt & Silvery 1969). Huang & Chow (1988) showed comparative account of nitrogen fixing unicellular cyanobacteria from rice fields. Capacity of nitrogen fixation by chroococcalean blue green algae Aphanothece pallida was recorded by Van et al. (1988) by isolating it from paddy fields. Unicellular forms were not recorded from paddy fields in the study region (Not as field collected specimens). They showed their occurrence in paddy soil cultures only and especially when cultures become 3-4 weeks old. The reason behind less number of chroococcalean taxa is, majority of the chroococcalean forms occur in soil cultures; not in field conditions and especially when soil cultures becomes 3-4 weeks old. At first filamentous heterosystous and filamentous non-heterosystous forms occur in cultures and when cultures became old (3-4 weeks) and when the nitrogen content of the medium slow down, chroococcalean forms grow upward direction in the culture bottles. Out J TT of 28 chroococcalean blue green algal forms, 18 forms show common occurrence, nine restricted to paddy field soils of Patan Tehsil and only one taxa restricted to paddy field soils of Karad Tehsil. Detailed distribution of chroococcalean blue green algae in study area is given in tabular form (Table 2).
Taxonomic as well as ecological study of chroococcalean blue green algae was done from paddy fields of many regions of the world. Roger (1985) made a report on mucilaginous bloom of unicellular blue green algae and its application as a biofertilizer. Majority forms recorded at field and cultures are filamentous heterosystous and filamentous non-heterosystous type. Ahmed & Kalita (2002) recorded abundance of unicellular chroococcalean forms in paddy fields. They isolated 53 chroococcalean forms from paddy fields of Nagaon. Our observations differ from them, we did not find abundance of chroococcalean forms in the field, only paddy field soil cultures showed their presence and especially when cultures become old. Cyanobacterial distribution pattern from paddy field soils of Konkan region, Maharashtra has been studied by Sardeshpande & Goyal (1981). Roger & Reynaud (1979) reported luxuriant growth of blue green algae from rice fields of Japan. Mukhopadhyay & Chatterjee (1980) published a checklist of paddy field blue green algae from West Bengal. Nitrogen fixing potential in rice fields of Sri Lanka studied by Kulasooriya & De Silva (1978). Cyanobacterial taxa from Tripura was studied by Singh et al. (1997). Aerobic growth and nitrogenase activity of marine unicellular blue green alga Synechococcus was reported by Duerr & Mitsui (1980). Dhanya & Ray (2015) studied cyanobacterial diversity and ecology from Kuttanadu paddy wetlands of Kerala. Prasad & Prasad (2003) showed increase in rice yield up to 5-24 % by applying cyano-biofertilizers in paddy fields of Nepal. A large variety of cyanobacterial species fix nitrogen and their importance to improve soil fertility for sustainable agriculture in submerged and irrigated rice cultivation is well recognized by Saikia & Bordoloi (1994). The great majority of cyanobacteria that fixed atmospheric nitrogen were probably heterocystous (Rodrigo & Eberto 2007), however non-heterocystous unicellular cyanobacteria also fixed atmospheric nitrogen (Wyatt & Silvery 1969). Aerobic nitrogen fixation without heterocyst was studied by Carpenter & Price (1976) in Marine Oscillatoria (Trichodesmium species). In our study area we found high diversity of Gloeocapsa species and dominance of Chlorogloea species which could serve as the best nitrogen fertilizer for paddy. Our observations differed with those proposed by Chudhary (2009) that members of Chroococcaceae are dominant in paddy fields. We found least abundance of chroococcaceae members in field condition as well as in culture condition. Majority taxa recorded was filamentous type. Algae stabilize the surface layer of soil, prevent soil erosion, improve infiltration of water, produce organic matter in the soil by death and decay of algae & hence increase soil fertility (Dawes 1998). Thus the role of unicellular blue green algae in nitrogen economy of paddy fields is recorded by many studies all over the world. Culture study of these unicellular taxa showed that the rate of survival and N 2 fixing capacity of chroococcaceae members, viz., Gloeocapsa, Oscillatoria (Trichodesmium species & Synechococcus is more (Wyatt & Silvery 1969;Carpenter & Price 1976;Duerr & Mitsui 1980). Therefore taxonomic documentation of unicellular blue green algae will provide the knowledge about such sturdy and durable indigenous species of chroococcalean blue green algae which will help in development of niche specific inoculants as biofertilizers for rice fields in the study region

CONCLUSION
The present study showed diversity and dominance of chroococcalean blue green algae. Overall the data obtained by thorough screening of paddy field soils indicates the dominance of heterocystous filamentous taxa followed by non-heterocystous taxa; besides these unicellular taxa also showed diversity and abundance of taxa from paddy soil cultures of study region. Study reports also showed beneficial role of many unicellular blue green algae in nitrogen economy of paddy soil. Our study area showed genus Gloeocapsa with high species diversity (21.42%) followed by Chroococcus, Aphanothece & Gloeothece from family Chroococcaceae & species dominance with Chlorogloea followed by Microsystis, Chroococcus & Gloeothece from family Entophysalidaceae. This survey on chroococcalean blue green algae will help in developing niche specific inoculum of indigenous species of the study area. These local strains should be cultured on a large scale for their mass production which would serve the best and low cost biofertilizer especially for paddy fields.