the whole plant of p. amarus was collected from varanasi and dried in sun light . the dried , powdered whole plant ( 4 kg ) the methanol extract was dried on water bath and extracted with 7% aqueous citric acid . the chcl3 fraction was concentrated and chromatographed over sio2 gel column eluting with solvents of increasing polarity and each eluted fraction was monitored by thin layer chromatography for their homogeinity . fractions eluted from chcl3-methanol ( 1 : 2 ) were mixed together and crystallised from methanol which furnished an alkaloid ( 56 mg ) , as colourless granules , rf 0.45 ( chcl3-methanol , 4 : 1 ) , m.p . it exhibited ir absorption bands at 1840 and 1760 cm and uv absorption maxima at 256 nm . its molecular formula was determined as c13h15no2 from its molecular ion peak at m / z 217 [ m ] in the mass spectrum . the spectral data , i.e. 
 h - nmr , c - nmr and ms were identical with reported data of securinine . 1 ) ( manske , 1977 ) by direct comparison with an authentic sample ( mmp , co - tlc and superimposable ir ) . the fungi ( table 1 ) were isolated on pda ( peeled potato 250 g , dextrose 20 g , agar 15 g , distilled water 1 liter ) from their respective hosts collected from the experimental farm of banaras hindu university , varanasi , india . the cultures were purified by single spore isolation technique on pda slants and maintained by periodic transfer on the same medium for further experiments . the stock solution ( 2,000 ppm ) was prepared by dissolving 10 mg of the alkaloid securinine ( 1 ) initially with a few drops of methanol in a test tube . after the chemical was completely dissolved , approximately 5 ml of distilled water was added . the required concentrations ( 200 , 400 , 600 , 800 and 1,000 ppm ) of the chemical were prepared from the stock solution by diluting with distilled water . a drop ( 30~40 ml ) of the chemical solution fungal spores ( about 200~300 spores ) were mixed in a solution with the help of a sterile inoculation needle . e. pisi conidia were directly picked up from diseased plants and mixed in a solution . the slides were later placed in moist chamber made by placing two sterile moist filter papers on the lid and base of petri plates . the germination of the spores was observed after staining with cotton blue prepared in lactophenol under a binocular light microscope ( nikon , japan ) . the effect of securinine ( 1 ) on spore germination of some plant pathogenic fungi was studied ( table 1 ) . the sensitivity of different fungi to this chemical varied considerably . alternaria brassicicola , curvularia lunata , curvularia pallenscens and helminthosporium spiciferum were the most sensitive as complete inhibition of germination was observed in all the concentrations ( 200 , 400 , 600 , 800 and 1,000 ppm ) of the chemical . similar effect on curvularia maculans and colletotrichum musae was recorded at 600 , 800 and 1,000 ppm . however , only 1,000 ppm was effective against spore germination of alternaria alternata , alternaria brassicae , colletotrichum sp . and heterosporium sp . the chemical was mildly effective at all the concentrations studied against e. pisi and helminthosporium echinoclova . a number of alkaloids isolated from plants have already been reported in literature to possesses antifungal activity ( atta - ur - rahman et al . , 1997 ; bracher , 1994 ; goel et al . , we have reported the antifungal activity of the alkaloids , ent - norsecurinine ( goel et al . , 2002 ) , norsecurinine ( sahni et al . , 2005 ) and allosecurinine ( singh et al . , 2007 ) isolated from p. amarus . securinine ( 1 ) and allosecurinine ( 2 ) ( fig . 1 ) are isomers whereas norsecurinine and ent - norsecurinine have different structures compared to compounds 1 and 2 . securinine inhibited complete spore germination of fungi a. brassicicola , c. lunata , c. pallenscens and h. spiciferum , whereas the alkaloid allosecurinine inhibited complete spore germination of fungi c. lunata , collectotrichum sp . , c. musae and heterosporium sp . at very low concentrations . this is the first report of the antifungal activity of securinine , the fourth alkaloid isolated from p. amarus . the efficacy of the chemical is significantly high even at low concentrations which indicates a possibility of its use to control plant diseases under field conditions .
the alkaloid securinine was assessed against spore germination of some plant pathogenic and saprophytic fungi ( alternaria alternata , alternaria brassicae , alternaria brassicicola , curvularia lunata , curvularia maculans , curvularia pallenscens , colletotrichum musae , colletotrichum sp . 
 , erysiphe pisi , helminthosporium echinoclova , helminthosporium spiciferum , heterosporium sp . ) . 
 spore germinations of all the tested fungi were inhibited . 
 alternaria brassicicola , c. lunata , c. pallenscens and h. spiciferum were highly sensitive as complete inhibition of spore germination was observed at very low concentrations ( 200 ppm ) .