Isolation of the algal symbiontAnabaena azollae and the role of vitamins in growth,heterocyst development and nitrogen-fixing activity

Nitrate-free medium supplemented with growth regulators such as 2,4-D, NAA and IBA was used to isolateAnabaena azollae. TwoA. azollae isolates grew adequately in the N-free Allen and Arnons medium. Incorporation of biotin and pantothenic acid stimulated the growth and heterocyst frequency of one of the isolates. This isolate also exhibited higher nitrogenase activity and excreted a higher amount of ammonia.     

Phylogenetic analysis of symbiotic and free-living cyanobacterial cultures using DNA amplification fingerprinting

DNA amplification fingerprinting (DAF) of 17 cyanobacterial isolates belonging to symbiotic and free-living forms of 6 different genera was done. The dendrogram analysis of 17 cyanobacterial cultures revealed three major clusters. All Westiellopsis cultures formed the first major cluster and their nucleotide relatedness ranged between 71-93%. In the second major cluster, the symbiotic cyanobacterium Anabaena sp dominated and all Anabaena cultures showed 60-75% similarity with each other. Nostoc muscorum is related to Anabaena variabilis by 85% and formed the third major cluster. The dendrogram analysis of cyanobacterial isolates clearly revealed that free-living cyanobacterial cultures were closely related with each other and were diverse from the symbiotic forms.     

Pigment analysis and ammonia excretion in herbicide tolerant cyanobacteria

Isolation of cyanobacteria was attempted from herbicide applied rice soils. The predominant genera were Westiellopsis followed by Anabaena, Nostoc and Oscillatoria. The herbicide tolerance was further tested by growing the cyanobacterial cultures in BG-11 medium supplemented with varying concentrations of the commonly used rice herbicide, viz butachlor under in vitro condition. The chlorophyll-a, phycobiliproteins and ammonia excretion were assessed at periodic intervals. Westiellopsis showed the maximum tolerance followed by Anabaena, Nostoc and Oscillatoria.     

Variation revealed by RAPD‐PCR profiles of microsymbiont Anabaena azollae strains from four N2 fixing Azolla cultures

Nitrogen fixing Anabaena azollae strains isolated from four different Azolla cultures were characterized based on their total protein profile and RAPD profile to study the existing variation among them. As expected, the isolates showed almost similar protein banding patterns, but exhibited differences in 40–70 KDa protein subunits. Polymerase chain reaction of the DNA of the isolates, using four different primers, amplified specific sequences of DNA and showed clear polymorphism among the isolates. The RAPD profile generated the fingerprinting pattern characteristic of each strain based on the sequence of the primers used. Common band sharing observed between the strains A. azollae‐RS‐KK‐SK‐AM and A. azollae‐RS‐KK‐SK‐RP probably represents maternal inheritance of DNA to the progeny. The polymorphic bands were generated specifically for the isolates A. azollae‐RS‐KK‐SK‐RP and A. azollae‐RS‐KK‐SK‐AM with primers numbered 2 and 4, respectively, which could be developed as possible markers for these isolates.     

Influence of nitrogen and phosphorus fertilizers on ammonia-assimilating enzymes of Azolla

Summary A field experiment was conducted and studied the effect of nitrogen and phosphorus on ammonia assimilating enzymes of Azolla. Nitrogen and phosphorus at 30 and 60 kg/ha respectively were tested andAzolla pinnata was inoculated at 200 g/m². The Azolla samples were drawn on 24th hr, 7th day and 14th day and the ammonia assimilating enzymes glutamine synthetase (GS), glutamate synthase (GOGAT) and glutamine dehydrogenase (GDH) were estimated. Nitrogen and phosphorus have markedly suppressed the GDH activity but fertilizer nitrogen has no significant influence in inhibiting the enzyme activity of GOGAT and GS. In general phosphorus application also has stimulated the GS activity significantly during the first sampling period of 24th hour.     

Immobilized cyanobacteria as a biofertilizer for rice crops; Intl. Conference on Applied Algology, Knysna, South Africa, April 1996.

N₂-fixing cyanobacteria (Anabaena azollae, symbiont strains) were immobilized in polyurethane foam and ammonia production by the cyanobacteria was investigated in the laboratory and rice field. The cyanobacterial symbiont, A. azollae - MPK-SK-AM-24 showed the highest growth rate and biomass production amongst the 5 isolates examined while A. azollae-AS-DS showed the highest nitrogenase activity followed by A. variabilis - SA₀ (wild type, non-symbiotic). Treatment of the foam-immobilized cyanobacteria with the systemic fungicide Bavistin stimulated nitrogenase activity while inhibiting glutamine synthetase (GS) activity. Free-living A. azollae-MPK-SK-AF-38, A. azollae - MPK-SK-AM-24 and A. azollae-MPK-SK-AM-27 excreted the highest amounts of ammonia into the growth medium; under foam - immobilized conditions the ammonia production increased further. Treatment of the foam - immobilized cyanobacteria with the fungicides Bavistin and Vitavax resulted in ammonia production at significantly higher rates. Rice seedlings (var. ADT 36) grown in the laboratory in conjunction with foam - immobilized A. azollae showed increased growth. A field experiment with paddy rice and foam - immobilized A. azollae strains indicated that the cyanobacteria excreted significant amounts of ammonia into the flood water in the rice fields resulting in increased chlorophyll content of the plants and increased the rice grain and straw yields. A combination of fertilizer nitrogen and inoculation with foam - immobilized cyanobacteria also significantly increased the rice grain and straw yield. Additionally, both A. azollae and A. variabilis were immobilized in sugarcane waste (bagasse), added to rice paddy and resulted in increased rice grain yield. This revised version was published online in September 2006 with corrections to the Cover Date.     

Intercellular colonization and growth promoting effects of Methylobacterium sp. with plant-growth regulators on rice (Oryza sativa L. Cv CO-43)

The Methylobacterium sp. strain NPFM-SB3, isolated from Sesbania rostrata stem nodules possessed nitrogenase activity and nodA genes. Pure culture of NPFM-SB3 strain produced indole-3-acetic acid, cytokinins and on inoculation to rice plants resulted in numerous lateral roots. Inoculation of synthetic auxins 2,4-dichlorophenoxy acetic acid, naphthalene acetic acid or flavonoids naringenin and dihydroxy-4-methoxyisoflavone individually or to bacterial inoculated rice seedlings improved the plant growth and lateral root formation under hydroponic condition. The formation of nodule-like structure and nitrogenase activity which is purely auxin dependent was observed in 2,4-dichlorophenoxy acetic acid treatments to Methylobacterium sp. NPFM-SB3 inoculated rice plants. The rhizobia entered through fissures formed due to lateral root emergence and spread intercellularly in the nodular structures concluded that the effect of 2,4-dichlorophenoxy acetic acid treatment for rice seedlings grown under gnotobiotic conditions is to create a niche in which these bacteria can grow.     

Cryopreservation of embryonic axes of trifoliate orange (Poncirus trifoliata [L.] RAF.)

Seeds of trifoliate orange (Poncirus trifoliata (L.) Raf.) are sensitive to desiccation, and could not withstand reduction in moisture level below 20%, whereas the excised embryonic axes could be easily desiccated to moisture levels as low as 14% without much loss in viability. Axes could be successfully cryopreserved in liquid nitrogen (–196°C) for eight months. The viable embryonic axes exhibited good growth on modified Murashige and Skoog medium supplemented wiith 1-Naphthalene acetic acid (NAA) and 6-Benzylaminopurine (BAP). Growth of cryopreserved axes was promoted in the presence of charcoal in the medium allowing for plant recovery.Abbreviations NAA Napthaleneacetic acid - BAP 6-Benzylaminopurine - MS Murashige and Skoog (1962) - LN Liquid nitrogen