Evaluation of Aqueous Extract of Seaweed as an Elicitor of Plant Defence Mechanism

Abstract

Algal plans viz: Hypnea musciformis, Botryocladia leptopoda, Acanthophora delili and Dictyota haupia of Red algae, Sagasum tererumium, lyengaria stellata and Padina tetrastromatica of brown algae and Codium elongatum, Caulerpa texiflora and Ulva lactulus of green algae were collected from coastal area of Karachi. Water (85-95%) moisture (6-12%) and ash contents (6-3.8%) were found. Air dried materials were sequentially extracted with water, dilute alkali and acid. High Molecular Weight Crude Elicitor Preparations ‘HMWCEP’ were obtained by ethanol precipitation and lyophilisation. Yields were high in NaOH fractions, as (13.32%) in brown, (14-49%) in red and (10-69% in green algae. Chemical composition of various HMWCEP revealed that acidic and aqueous preparations of H.musciformis and A.delili (red algae) are rich, 25-69% and 5.5-6.1% in sugar and protein content respectively. Sugar contents were high 18-40% in P.tetrastromatic as compare to other two plants of brown algae. Protein was not found in acidic extracts of brown algae. U.lactulus are quiet rich in protein (1-2%), sugar (18-16%) and uronic acid (1-8%) in comparison of C.elongatum and C.texiflora. Acid hydrolysis as function of time (0.5, 1.5, 3 and 5 hours) and paper chromatography was carried out to identify the constituent monosaccharides and for optimization of hydrolysing condition. Galactose was found as major sugar component of red algae, significant amount of fructose was found in brown and green algal plants along with glucose, mannose, arabinose, xylose and in some cases rhamnose, in varying proportion. Monosaccharide composition of various algal polysaccharide was determined by acid hydrolysis, alditol acetates preparation and analysed by gas liquid chromatography. Elicitor activity of seaweed polysaccharides was determined and established for first time. Both qualitative and quantitative differences were observed in terms of induced browning and phytoalexin production by cotyledons of Cicer arietinum, on treatment with the preparations obtained from the seaweeds. A variable dose of response was observed for various elicitor preparations. The level and timing of induced browning and induced metabolites, were estimated as a function of time, results are presented and discussed. A simple hplc separation method was developed to analyse the major and minor components of phytoalexin mixture, the individual components were identified by LC- Electrospray Mass Spectrometry. Two components previously reported as product of C.arietinum were identified viz: Formononetin and Biochanin. In addition, glycoconjugates were identified but not fully characterised. Large scale elicitations and fractional extraction provided, yellow precipitate, collected when total alcoholic extract was kept in the fridge and a total alcoholic ext.-1, Pet ether ext.-2, chloroform-ethyl acetate ext.-3 and residual aqueous ext.-4. These Fractions wee identified for their antimicrobial activity against some pathogenic and nonpathogenic fungi, results indicated that Extract-3, 1 and 2 exhibited minimum inhibition zone. Fractions were separated by hplc. In repeated experiments HMW preparations (polysaccharides) of Hypnea musciformis (red algae) were found as the most active elicitors. Dialysis and anion-exchange chromatographic techniques were used to provide homogenous fraction. The estimated molecular weight obtained by gel permission chromatography indicated Mr of c/h extracts of NaOH as <20,000 Dalton, and cold acidic and aqueous fractions extract were as Mr>70,000 Dalton, values are lower than those of commercial products. 1H-NMR spectra recorded, were not a great help in characterization of these polysaccharides. 13C-NMR spectrum analysis of crude HMW polysaccharides revealed, overabundance signals (12-24peaks), of variable intensities in the anomeric region, and on the basis of data available in literature, the identification of these polysaccharides was carrageenan of λ, as dominating repeating units, along with other minor precursors and reflected the heterogeneity in construction of these polysaccharides. IR data supported these findings. Twelve signals corresponding to the disaccharide repeating units were visible in the 13C-NMR spectra of partially purified carrageenan of H.musciformis. Two anomeric signals 98.7 ppm and 104.56ppm are prominent and show a close proximity to G4S-DA2S or due to G4S-DA2S, 6S, building unit of λ carrageenan.