Azolla Fronds Research Articles

Effect of Water Sources on the Growth Performance and Chlorophyll Content of Azolla pinnata R.Br.


 
 
 Azolla pinnata R.Br. is an aquatic fern that grows on the surface of water bodies. The unique quality of A. pinnata is its symbiotic relationship with a prokaryotic cyanobacterium (Anabaena azollae), which confers a high rate of nitrogen fixation. It is important to find the growing conditions of Azolla in different water sources for its optimum biomass production for different usages such as biofertilizers, animal feeds, water purification and biofuel production. This preliminary study was conducted to investigate the effect of water sources on the growth performance (shoot biomass, root biomass, root length), and the chlorophyll content of Azolla. Presence of Anabaena in Azolla fronds was monitored by light microscope and number of filaments were counted per leaflet. The experiment utilized three types of water sources (tap water as control (T1), lake water (T2) and cattle manure mixed tap water (T3), in plastic buckets with 40 cm in diameter and 25 cm in height. Each treatment was triplicated in a completely randomized design and total biomass was collected weekly. Data were statistically analyzed using Minitab 17. The highest biomass of Azolla was obtained in T3 from the first week until 8th week and it was higher from T1 and T2. The highest chlorophyll content was obtained in T3 (6.4 SPAD value) showed a significant difference from T1 and T2 (p<0.05). The dry weight of roots and root length in T1 showed a significant difference from T2 and T3 (p<0.05). The highest root length was observed as 5 cm in T1 indicating that roots may be in search of nutrients and the lowest root length was observed as 0.8 cm in T3 with high nutrient concentrations in water. The highest number of Anabaena was observed in the leaflet of Azolla fronds in T3 which in turn increase N content in Azolla plants. It reveals that application of cattle manure in tap water significantly affects the plant biomass and the chlorophyll content of Azolla. Further studies are needed to conduct in large scale growing facilities with different nutrient concentrations of water sources to find sustainable and optimum growing conditions for Azolla and the nutrient concentrations of the plants.
 Keywords: Azolla, Chlorophyll, Growth performance, Water sources
 
 

English

This work proposes NADP-ME activity as bio-indices for herbicidal sensitivity in Azolla pinnata R.Br. under 2,4-D toxicity. Azolla fronds were illuminated under saturated photosynthetic flux and bicarbonate induction. There was significant variation in the NADP-ME activity under light and dark conditions. This increased more when varying concentrations of 2,4-D were used for the up regulation of enzyme activity especially under illumination. There was evident modulation through activators (citrate and succinate) and inhibitors (pyruvate and oxaloacetate) under light and dark conditions at 2,4-D concentration. This occurs at the same time in overriding the herbicidal stress linked to the adjustment of the cellular redox of regulatory sites using dithiothreitol. This shows that Azolla can quench 2,4-D, which suggests NADP-ME as a biomarker through its sensitivity and mode of activities under xenobiotic contaminated soil. Key words: NADP-malic enzyme, aquatic fern, herbicide, modulators, malate.

Changes in elemental content in fronds of Azolla filiculoides due to arsenic accumulation

This study analyzed the accumulation of arsenic and its influence on the content of P, Mn, Fe, Cu, Zn, S, Ca, Cl, and K in fronds of Azolla filiculoides via X-ray fluorescence (XRF). Azolla was exposed to increased concentrations of sodium arsenate (0, 5, 10, 20, and 30 μg ml − 1) in Yoshida's nutrient solution. After 96 h, fronds were collected and taken to constant weight to calculate the relative growth rate (RGR), the biomass duplication rate, the bioaccumulation factor (BAF), and the elemental concentration via XRF analysis. By increasing As concentrations the RGR and BAF were reduced. The greatest accumulation of As in fronds was achieved at 20 μg As ml − 1, which did not show significant differences with the highest concentration. Accumulation of As in fronds diminished concentrations of Mn, Fe, Zn, S, Ca, and K, while the concentration of P remained low but unchanging. The P content was significantly lower than the As content, thus the As:P ratio in fronds enhanced as the As concentration increased. Concentrations of Cl and Cu were unaffected due to As. This is one of the first reports about the influence of As-accumulation on the elemental content in Azolla fronds.

Azolla domestication towards a biobased economy?

Due to its phenomenal growth requiring neither nitrogen fertilizer nor arable land and its biomass composition, the mosquito fern Azolla is a candidate crop to yield food, fuels and chemicals sustainably. To advance Azolla domestication, we research its dissemination, storage and transcriptome. Methods for dissemination, cross-fertilization and cryopreservation of the symbiosis Azolla filiculoides-Nostoc azollae are tested based on the fern spores. To study molecular processes in Azolla including spore induction, a database of 37 649 unigenes from RNAseq of microsporocarps, megasporocarps and sporophytes was assembled, then validated. Spores obtained year-round germinated in vitro within 26 d. In vitro fertilization rates reached 25%. Cryopreservation permitted storage for at least 7 months. The unigene database entirely covered central metabolism and to a large degree covered cellular processes and regulatory networks. Analysis of genes engaged in transition to sexual reproduction revealed a FLOWERING LOCUS T-like protein in ferns with special features induced in sporulating Azolla fronds. Although domestication of a fern-cyanobacteria symbiosis may seem a daunting task, we conclude that the time is ripe and that results generated will serve to more widely access biochemicals in fern biomass for a biobased economy.

POTENTIAL OF THE AQUATIC FERN AZOLLA FILICULOIDES IN BIODEGRADATION OF AN AZO DYE: MODELING OF EXPERIMENTAL RESULTS BY ARTIFICIAL NEURAL NETWORKS

The potential of an aquatic fern, Azolla filiculoides, in phytoremediation of a mono azo dye solution, C.I. Acid Blue 92 (AB92), was studied. The effects of operational parameters such as reaction time, initial dye concentration, fern fresh weight, pH, temperature and reusability of the fern on biodegradation efficiency were investigated. The intermediate compounds produced by biodegradation process were analyzed using GC–MS analysis. An artificial neural network (ANN) model was developed to predict the biodegradation efficiency. The findings indicated that ANN provides reasonable predictive performance (R2 = 0.961). The effects of AB92 solutions (10 and 20 mg L−1) on growth, chlorophylls and carotenoids content, activity of antioxidant enzymes such as superoxide dismutase, peroxidase and catalase and formation of malondialdehyde were analyzed. AB92 generally showed inhibitory effects on the growth. Moreover, photosynthetic pigments in the fronds significantly decreased in the treatments. An increase was detected for lipid peroxidation and antioxidant enzymes activity, suggesting that AB92 caused reactive oxygen species production in Azolla fronds, which were scavenged by induced activities of antioxidant enzymes.

Differential responses of hydrogen peroxide, lipid peroxidation and antioxidant enzymes in Azolla microphylla exposed to paraquat and nitric oxide

The present investigation was carried out to decipher the interplay between paraquat (PQ) and exogenously applied nitric oxide (NO) in Azolla microphylla. The addition of PQ (8 μM) increased the activities of superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPX), ascorbate peroxidase (APX) by 1.7, 2.7, 3.9 and 1.9 folds respectively than that control in the fronds of Azolla. The amount of H2O2 was also enhanced by 2.7 times in the PQ treated plants than that of control. The supplementation of sodium nitroprusside (SNP) from 8–100 μM along with PQ, suppressed the activities of antioxidative enzymes and the amount of H2O2 compared to PQ alone. The drop in the activity of antioxidative enzymes — SOD, GPX, CAT and APX was highest (39.9%, 48.4%, 41.6% and 41.3% respectively) on the supplementation of 100 μM SNP with PQ treated fronds compared to PQ alone. The addition of NO scavengers along with NO donor in PQ treated fronds neutralized the effect of exogenously supplied NO. This indicates that NO can effectively protect Azolla against PQ toxicity by quenching reactive oxygen species. However, 200 μM of SNP reversed the protective effect of lower concentration of NO donor against herbicide toxicity. Our study clearly suggests that (i) SNP released NO can work both as cytoprotective and cytotoxic in concentration dependent manner and (ii) involvement of NO in protecting Azolla against PQ toxicity.

BIOACCUMULATION OF HEAVY METALS BY THE WATER FERN AZOLLA PINNATA

Azolla pinnata aquatic fronds were tested for their ability to bioaccumulate heavy metals from Yoshida medium supplemented with different heavy metals, i.e. Pb +2, Cd+2 and Cu+2 at different concentrations 0.0, 0.5, 1.0, 1.5 and 2.0 ppm, at different growth periods of 5, 10, 15, 20, 25 and 30 days. Results revealed that azolla can grow healthy with the accumulation of these metals. The highest fresh and dry weights of azolla were observed after 30 days at 2.0 ppm of Cu+2. These values were significantly higher than those for Pb+2 and Cd+2 at 1.0 and 0.5 ppm , respectively. Also, azolla fronds exerted higher nitrogenase activity when grown in culture medium without heavy metals, or supplemented with 1.5, 1.5 and 0.5 ppm of Pb +2, Cd+2 and Cu+2 , respectively. The highest records of nitrogen uptake were obtained, in the presence of Cu +2 (2.0 ppm) treatment, while phosphorus uptake increased with increasing the concentrations of Pb+2 and Cu+2 from 0.5 ppm to 2.0 ppm. On the other hand, phosphorus uptake decreased with increasing the concentrations of Cd+2 from 0.5 to 2.0 ppm. Potassium uptake in response to different Cd+2 concentrations was lower than the other treatments including the control, from 5 to 30 days of growth periods. The greatest contents of Pb +2, Cd+2 and Cu+2 were observed in azolla fronds grown in presence of 2.0 ppm of any of these elements and collected after 30 days. Pb+2 uptakes were higher at the concentration of 2.0 ppm compared with other concentrations of Cd+2 and Cu+2 . Thus, Azolla pinnata showed tolerance to the tested heavy metals at concentration up to 1mg/l that supposed to pollute the medium. Therefore, azolla can serve as a phytoremediation agent in the heavy metal polluted habitats.

Metabolic responses of Azolla pinnata to cadmium stress: photosynthesis, antioxidative system and phytoremediation

This present study deals with the growth, photosynthesis, oxidative stress and phytoremediation character of Azolla pinnata L. exposed to different levels (0.05, 0.1, 0.5, 1.0, 1.5 and 2.0 mg·L−1) of cadmium (Cd). Significant accumulation of Cd in Azolla fronds was noticed after 24 and 96 h of exposure and the accumulation rate was dose and time dependent. Growth of A. pinnata increased significantly after both exposure times with and without metal. At lower Cd doses (0.05 and 0.1 mg·L−1), growth and photosynthesis of A. pinnata showed a marginal increase over the respective control, however, at higher Cd doses (0.5, 1.0, 1.5 and 2.0 mg·L−1), a decreasing trend was noticed. At lower doses, Azolla fronds could counterbalance the negative effect of enhanced levels of superoxide radicals (SOR) and hydrogen peroxide (H2O2) through the greater activity of antioxidative enzymes. The decaresing trends in catalase and peroxidase activity at higher Cd doses suggest that Azolla fronds were not able to mitigate the negative effects of H2O2, hence an increase in malondialdehyde content was noticed. The study concludes that up to 0.1 ,mg·L−1 Cd, A. pinnata can flourish and be used as biofertiliser and for phytoremedial purposes in Cd-contaminated fields; beyond this concentration poor growth may restrict its application.

Effects of paraquat on lipid peroxidation and antioxidant enzymes in aquatic fern Azolla microphylla

Paraquat is most extensively used methyl viologen herbicide to control weeds in the rice-Azolla ecosystem. The effects of different paraquat (PQ) dosages on growth, lipid peroxidation, and activity of antioxidant enzymes of Azolla microphylla Kaul. were investigated. The results indicated that Azolla fronds survived only at the concentrations of 2–6 μM PQ. Frond fragmentation and browning occurred after 24 h at 8 μM PQ. At 24 h, the amount of proteins decreased by 48.7 % in Azolla fronds exposed to 10 μM PQ than that in control fronds. The supplementation of 10 μM PQ increased the activities of superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPX), and ascorbate peroxidase (APX) by 2,4-, 1,8-, 3,0-, and 2,2-fold, respectively, as compared with control. The content of PQ and activities of SOD, CAT, GPX, and APX were found to be positively correlated. Our study showed that PQ (2–6 μM) caused ROS overproduction in Azolla fronds, which were scavenged by induced activities of antioxidant enzymes.

Salinity stress induced physiological and biochemical changes in Azolla pinnata

On brief exposure of Azolla fronds to salinity stress, a significant decrease in photosynthetic pigment like chlorophyll and carotenoid with a decrease in ascorbate and glutathione content was observed. Lipid peroxidation increases with doses of NaCl stress resulting a greater membrane damage supported by increase in superoxide radical. However, increase in activities of superoxide dismutase, catalase, guaiacol peroxidase and glutathione reductase showed the development of biochemical defence mechanism against free radicals generated during exposure to short-term salinity stress. K+ ion was found to be decreased with increasing NaCl concentration, with a decrease in relative water content. An increase in fresh mass was observed, with a significant increase in dry mass suggested a development of salt tolerance in Azolla exposed to short-term salinity stress.

Feeding deterrence of Azolla in relation to deoxyanthocyanin and fatty acid composition

Higher levels of deoxyanthocyanins in Azolla fronds correlated with feeding deterrence against adult Lymnea swinhoei snails and Polypedates leucomystax tadpoles. After 7 days in co-culture, the growth yield of Azolla filiculoides was almost twice that of Azolla pinnata, which contained approximately 20-fold higher levels of deoxyanthocyanins. Inclusion of snails upon initiating a plant co-culture resulted in an A. pinnata growth yield that was 1.5-fold higher than that of A. filiculoides. The two plant species did not differ significantly in fatty acid composition. However, snails incubated for 1 week solely with A. pinnata showed a characteristic starvation-like fatty acid profile, with a two-fold lower proportion of polyunsaturated fatty acids (PUFAs) compared to snails that had access to A. filiculoides, either as a sole food source or in co-culture with A. pinnata. A 3-month cultivation of A. pinnata with tadpoles resulted in fronds having a 260% higher concentration of deoxyanthocyanins and a 10-fold lower proportion of PUFAs in comparison to plants unexposed to tadpoles, indicating that prolonged feeding reduces the desirability of the surviving plants as a food source.

Genetic diversity and phylogeny analysis of Anabaena azollae based on RFLPs detected in Azolla-Anabaena azollae DNA complexes using nif gene probes

The cyanobacterium Anabaena has both symbiotic and free-living forms. The genetic diversity of Anabaena strains symbiotically associated with the aquatic fern Azolla and the evolutionary relationships among these symbionts were evaluated by means of RFLP (restriction fragment length polymorphism) experiments. Three DNA fragments corresponding to nif genes were cloned from the free-living cyanobacterium Anabaena PCC 7120 and used as probes. A mixture of Azolla, Anabaena and bacterial DNA was extracted from Azolla fronds and digested with two restriction enzymes. Single-copy RFLP signals were detected with two of the probes in all Azolla Anabaena examined. Multiple-copy RFLP signals were obtained from the third probe which corresponded to a part of the nif N gene. A total of 46 probe/enzyme combinations were scored as present or absent and used to calculate pairwise Nei's genetic distances among symbiotic Anaebaena strains. Phylogenetic trees summarizing phenetic and cladistic relationships among strains were generated according to three different evolutionary scenarios: parsimony, UPGMA and neighbour joining. All trees revealed identical phylogenetic relationships. Principal component analysis was also used to evaluate genetic similarities and revealed three groups: group one contains the cyanobacteria associated with plants from the Azolla section, group two contains those associated with plants from the pinnata species and group three contains those associated with plants from the nilotica species. The same groups had already been identified earlier in a random amplified polymorphic DNA (RAPD) analysis of Azolla-Anbaena DNA complexes, suggesting that the present Azolla taxonomy should be revised. We now suggest a taxonomy of Anabaena azollae that is parallel to such a revised Azolla taxonomy. An Azolla chloroplast DNA sequence derived from Oryza sativa was also used as an RFLP probe on Azolla DNA to confirm the presence of plant DNA in the total genomic DNA extracted from ferns with or without the symbiont. Our results also suggest that total DNA extracted from the Azolla-Anabaena complexes includes both plant and symbiont DNA and can be used equally well for RFLP analysis of host plant or symbiotic cyanobacteria.

Effect of Benlate and Rhizoctonia interactions on the growth, chlorophyll contents and nitrogen fixation in three species of Azolla

Azolla is an aquatic fern that fixes atmospheric nitrogen in symbiotic association with the cyanobacterium Anabaena azollae. Azolla fronds are commonly affected by a soil-borne pathogen, namely Rhizoctonia solani. Benlate, a systemic fungicide, was used for treating three species of Azolla with a view to assessing its effect on growth, chlorophyll synthesis and nitrogen-fixing potential besides checking the Rhizoctonia solani infection. Benlate at 2.5 p.p.m. induced the growth of Azolla microphylla – BR–GI, Azolla caroliniana – WT–V and Azolla sp. – ST–SI. The growth rate induction in Azolla sp. – ST–SI due to Benlate treatment was significant. The culture Azolla sp. – ST–SI showed maximum nitrogenase activity due to Benlate treatment, followed by A. microphylla – BR–GI. A. caroliniana – WT–V and Azolla sp. – ST–SI, when treated with Benlate, have shown higher chlorophyll. Benlate treatment reduced the nitrogen content in A. microphylla – BR-GI and Azolla sp. – ST–SI, while carbon content was not affected much. Azolla is ’n watervaring wat atmosferiese stikstof bind in simbiotiese assosiasie met die sianobakterie Anabaena azollae. Azolla blare word algemeen aangetas deur die grondpatogeen Rhizoctonia solani. Benlate, ’n sistemiese swamdoder, is gebruik om drie Azolla spesies te behandel, nie net om die Rhizoctonia solani infeksie te stuit nie, maar ook om die uitwerking daarvan op die groei, chlorofilsintese en stikstofbindingspotensiaal te bepaal. Benlate, teen 2.5 d.p.m., het die groei van Azolla microphylla – BR–GI, Azolla caroliniana – WT–V en Azolla sp. – ST–SI geïnduseer. Die groeitempo-induksie by Azolla sp. – ST–SI as gevolg van Benlate-behandeling was betekenisvol. Die kultuur Azolla sp. – ST–SI het maksimum nitrogenase-aktiwiteit as gevolg van Benlate-behandeling getoon, gevolg deur A. microphylla – BR–GI. Wanneer met Benlate behandel, het A. caroliniana – WT–V en Azolla sp. – ST–SI hoër chlorofil getoon. Benlate-behandeling het die stikstofinhoud van A. microphylla – BR–GI en Azolla sp. – ST–SI verminder, terwyl die koolstofinhoud nie veel beïnvloed is nie.

Disappearance of symbiotic algae in theAzolla-Anabaenaassociation subjected to transitory-exposure to ammonium

Abstract When Azolla japonica was grown on media containing ammonium at high concentrations, the ammonium levels within the Azolla tissue increased rapidly and reached the same levels as those in the media. When these Azolla fronds were transferred to a N-free medium, yellow spots were observed. In the crushed yellow spots, hardly any filaments of symbiotic algae could be detected under a microscope. Furthermore, the yellow spots cut from whole plants were able to regenerate to normal fronds only on a medium containing combined nitrogen at low concentrations. These regenerated fronds were free from Anabaena, did not exhibit acetylene reduction activity and failed to survive on a N-free medium.

Effect of some physiological factors on the nitrogenase and hydrogenase of the Azolla—Anabaena Azollae association

Nitrogenase (acetylene reducing) activity, nitrogenase-mediated hydrogen evolution and hydrogenase-catalysed hydrogen uptake activity was studied in intact Azolla fronds. Nitrogenase activity was significantly influenced by various physiological parameters. When incubated under light, the rate of H 2 evolved by the fronds under anaerobic conditions was significantly higher than that under aerobic condition. H 2 uptake was significantly inhibited by both acetylene and carbon monoxide incubations.

Mucilage acts to adhere cyanobacteria and cultured plant cells to biological and inert surfaces

The surfaces of cells of several species of cyanobacteria have been studied using low-temperature scanning electron microscopy (SEM), and have been shown to be covered in a layer of hydrated mucilage. This mucilage is observed in specimens ofAnabaena azollae adhering to plant cells in their natural symbiotic niche (the cavity of the fronds ofAzolla species) and in samples of the various species of cyanobacteria immobilised on polyurethane and polyvinyl support matrices. The mucilage appears to maintain the close contact observed between the cyanobacteria and these surfaces. Comparable films observed surrounding plant cells immobilised on similar polymeric surfaces are considered to be performing a similar function.

无藻满江红(Azolla)和满江红鱼腥藻(Anabaena azollae)的分离与培养

Shoot tips of Aeolia were excised and inoculated into sterilized Nickell's liquid medium with nitrate. Algae and bacteria-free fronds of Azolla have been obtained and subcultured successfully. Anabaena azollae pipetted from the leaf cavity of axenie Azolla by micromanipulator to a nitrogen-free medium and grow normally. They have been proved to be free from bacteria by scanning electromicroscopie examination and cultivation in a bacterium culture medium.

The Azolla, Anabaena azollae Relationship

Cultures of Azolla caroliniana Willd. free of the symbiotic blue-green alga, Anabaena azollae, were obtained by treatment of Azolla fronds with a regimen of antibiotics. These symbiontfree plants can be maintained only on medium containing a combined nitrogen source.Morphological aspects of the symbiotic association show the confinement of the Anabaena azollae within the leaf cavity of the Azolla. Procedures were established for the isolation of pure preparations of Anabaena azollae and Azolla chloroplasts. It has not yet been possible to grow the isolated alga in independent culture.Photochemical activities of the isolated alga and fern chloroplasts were measured by spectrophotometric assays for photosystems I and II as well as by P700-content (photosystem I) and delayed light emission (photosystem II). In the algal fraction, both photosystems were repressed when compared to freeliving Anabaena cylindrica, but the relative ratio of photosystem I to photosystem II may be appreciably greater in Anabaena azollae. Azolla chloroplasts were generally comparable to spinach chloroplasts.A comparison of the chlorophyll a and b content of Azolla fronds with and without the symbiotic alga resulted in an estimate that in the symbiotic association, the Anabaena azollae accounts for from 7.5 to 15% of the total chlorophyll.

A Cobalt Requirement for Symbiotic Growth of Azolla filiculoides in the Absence of Combined Nitrogen

Cobalt was shown to be essential for the symbiotic growth of Azolla filiculoides and Anabacna azollac in the absence of fixed nitrogen. Addition of 0.01 mug/liter cobalt resulted in large increases in yield, chlorophyll content and nitrogen fixation as compared to control cultures without cobalt. Cobalt was not required for the growth of Azolla when nitrate nitrogen was supplied. The number of Anabaena azollae cells in the fronds of Azolla appeared to be decreased by ommission of cobalt from the culture medium containing nitrate nitrogen. It is concluded that cobalt is essential for the symbiotic growth of Azolla in the absence of combined nitrogen and it is suggested that the cobalt requirement is associated with the growth of Anabaena azollae.