BIOHERBICIDAL POTENTIAL OF ROOT EXTRACTS OF
International Journal of Innovations in Biological and Chemical Sciences, Vol. 4: 1-10, 2013BIOHERBICIDAL POTENTIAL OF ROOT EXTRACTS OF TAGETUS MINUTAAGAINST PARTHENIUM HYSTEROPHORUS L.Riti Thapar Kapoor* and Ashwani Kumar SrivastavaPlant Physiology Laboratory, Amity Institute of Biotechnology, Amity University, Noida - 201 303,Uttar Pradesh, India.ABSTRACTThe present paper highlights the bioherbicidal property of the root extracts of Tagetus minuta againstthe obnoxious alien weed Parthenium hysterophorus L. Root extracts of Tagetus minuta significantlyinhibited seed germination, seedling length, vigour index, biomass and biochemical components ofParthenium weed. The phytotoxicity of the roots of Tagetus minuta against Parthenium shoots andseedlings was directly proportional to the concentration, immersion period and exposure period. It wasalso observed that phytotoxicity of the roots of Tagetus minuta collected at flowering stage was more incomparison to the roots of vegetative stage of Tagetus minuta and it may be due to the presence ofpotential allelochemicals in the roots of Tagetus minuta.Keywords : Allelochemicals, biopesticide, Parthenium hysterophorus and Tagetus minuta.5vegetation . A successful establishment ofParthenium in any ecosystem is attributed toseveral reasons such as high germination abilitythroughout the year, an enormous seed bank,rapid spread, plasticity in physiologicalbehaviour and extreme adaptability in a wide6,7range of habitats . The chemical analysis ofParthenium hysterophorus has indicated that allthe plant parts including trichomes and pollencontain several secondary metabolites such asalkaloids, parthenin, kaempferol, p - coumaricacid and caffeic acid being high in leaves8followed by inflorescence, fruit, root and stem .The sesquiterpene lactones namely partheninand coronopilin present in the trichomes ofleaves and stems of Parthenium are responsiblefor causing various allergies like contactdermatitis, hay fever, asthma and bronchitis in9human - beings . Parthenium hysterophorusinterferes with the growth of other species byreleasing allelochemicals like phenolic acids andsesquiterpene lactones which seize the growth8phenomenon of the co - existent species .INTRODUCTIONWeeds are undesirable and non - economicplants that compete with crops for naturalresources like water, nutrients and sunlight.Parthenium hysterophorus L., an obnoxiousweed has been reported as a main source ofnuisance and health hazard to mankind andanimals as well as threat to biodiversity and1dangertoenvironment .Partheniumhysterophorus L. popularly known as carrotweed, congress weed and feverfew, is native ofNorth - east Mexico, probably introduced inIndia along with wheat grains under the PL 480scheme and spread alarmingly like a wild blazeto almost all the states in India and establishedas a naturalized weed. Parthenium is commonlyseen lavishly growing in vacant sites, rockcrevices, city waste - dumped areas, roadsides,2railway tracks and construction sites . This plantbelongs to the division : Magnoliophyta, class :Magnoliopsida, order : Asterales and family :3Asteraceae . Parthenium hysterophorus L. hasbeen originated as a result of naturalhybridization between Parthenium confertum4and Parthenium bipinnatifidum . Partheniumcompletes its life - cycle within 3 - 4 months andit shows three to four generations in a yearwhich helps in quick spreading and generationof adverse impacts on the surroundingPlants are the natural treasure of thebiologically active chemicals (secondary metabolites) that affect the growth andpopulation - biology of individuals of otherspecies and they impose an environmentalstress on other plants growing in their vicinity.Tagetes minuta L. (Asteraceae) is an annualaromatic species native to South America,although it has become widespread throughout10the world .Thiophenes are sulphur containing*Corresponding author:Email: drriti bhu@yahoo.co.in1
International Journal of Innovations in Biological and Chemical Sciences, Vol. 4: 1-10, 2013heterocyclic - compounds derived frompolyacetylene, are present in different plant11parts of Tagetus minuta . Higher concentrationof thiophene was observed in roots of Tagetus12.minuta in comparison to leaves and shootsThiophene acts as toxins against fungi, bacteria,insects and nematodes and toxicity of thiscompound is related with the generation ofreactive oxygen species that adversely affectbiological-membranes and other biochemicalcomponents of the target pest.between Parthenium hysterophorus andTagetus minuta in nature. The fresh and healthyplants of Tagetus minuta L. were collected atvegetativeandfloweringstagefromsurrounding areas of Amity University campus,Noida. Fresh roots of Tagetus minuta, bothvegetative and flowering stages were separatedcarefully from the intact plants. The roots ofTagetus minuta were washed gently with tapwater for 10 minutes and surface sterilized with70% ethyl alcohol for 30 seconds then immersedin 1% NaOCl solution containing two drops ofTween-20 for 15 minutes and finally rinsedthrice with sterilized distilled water and dryingwith clean absorbent paper. The sterilized rootswere cut into small pieces with sterilized knifeand air - dried under shade for 15 days. After airdrying, roots were homogenized to fine powderin a grinder and stored in airtight bottles forfurther bioassay tests and analysis ofbiochemical components.It has been found that the root extracts of T.minuta significantly inhibited seed germination,relative germination rate, vigour index, biomassand biochemical components of Partheniumhysterophorus. It was also observed thatphytotoxicity of the roots of Tagetus minutacollected at flowering stage was more incomparison to the roots collected at vegetativestage of Tagetus minuta. The rationale behindthe selection of Tagetus minuta plant as abiopesticide against Parthenium weed was dueto the reason that overuse of syntheticagrochemicals causes environmental hazards,an imbalance of soil microbes, nutrientdeficiency, change of physico - chemicalproperties of soil resulting in decrease of cropproductivity. The indiscriminate use ofhazardous pesticides have eroded the ecologicalsustainability and deleterious effect on humanhealth. Therefore, root extracts of Tagetusminuta may be use as natural herbicide for thebiologicalmanagementofPartheniumhysterophorus L. It is believed that crude extractof the plant are biologically more active thanisolated compounds due to synergistic13effects .Therefore, the present investigationwas carried out to study the bioherbicidalimpact of root extracts of Tagetus minuta onParthenium hysterophorus L. This study mayprovide baseline information for thedevelopment of future strategies for theproduction of herbicidal formulation for themanagement of obnoxious Parthenium weed.Preparation of aqueous extracts: Forpreparation of aqueous root extracts, 10 g of air- dried root powder of Tagetus minuta wasmacerated with 100 ml of sterilized distilledwater in a blender for 10 minutes thentransferred in conical flask and boiled on slowheat for 2 h. It was then filtered through doublelayered muslin cloth and centrifuged at 5000 gfor 10 minutes. The supernatant was filteredthrough Whatman No. 1 filter paper. Thesupernatant was collected and this procedurewas repeated twice. After 6 h, the supernatantwas collected at an interval of every 2 hours waspooled together and concentrated to make thefinal volume one - fourth of the original volume.0It was then autoclaved at 121 C temperature14and at 15 lbs pressure . The aqueous rootextracts of both vegetative and flowering stageswere preserved aseptically in amber coloured0bottle at 4 C until further use.Seed germination bioassay test: Seedgermination bioassay test was used to test theinhibitory effect of aqueous root extracts ofTagetus minuta on the seeds of Partheniumhysterophorus under laboratory conditions.Before seed germination test, empty andundevelopedseedsofPartheniumhysterophorus were discarded by floating in tapwater. Seeds of Parthenium hysterophorus werethoroughly washed with tap water to removedirt and dust for 5 minutes. To avoid possibleMATERIALS AND METHODSExperimental design: The experiment wasconducted during November 2010 - March 2012in Amity Institute of Biotechnology, AmityUniversity, Noida, India. The systematic surveyof the surroundings of Amity University Campus,Noida was made to study the interaction2
International Journal of Innovations in Biological and Chemical Sciences, Vol. 4: 1-10, 2013inhibition caused by toxins from fungi orbacteria, seeds were surface sterilized with 10 :1 distilled water/ bleach (commercial NaOCl)solution for 5 minutes and then washed 6 - 7times with distilled water. Parthenium seedswere soaked in different concentrations ofaqueous root extracts of T. minuta for 4 hours.Two pieces of filter paper were placed insterilized petri - dishes (20 cm diameter) andParthenium seeds which were soaked inaqueous root extracts transferred intopetridishes. The petridishes were covered andplaced in sterilized polythene bags to preventfurther loss of volatiles and kept in a SeedGerminator for 10 days under 70% relative0humidity at 25 2 C with a 12 h photoperiod15following a guidelines of ISTA to test the seedgermination under different concentrations ofaqueous root extracts in three replicates withcompletely randomized block design.bioassay test. Parthenium shoots were cut andwashed in tap water and dipped in 1% NaOClsolution for 3 minutes. The tips of the shootswere immediately washed in sterilized distilledwater to remove any residual trace of thechemical. An inclined cut was made at the tip ofthe shoots and the shoots were placed in testtubes containing 10 ml of 50 and 100%concentrations of aqueous root extracts ofvegetative and flowering stages of Tagetusminuta. In control, Parthenium hysterophorusshoots were dipped in 10 ml of distilled water.The tubes were sealed with cotton buds andaluminium foil to make it airtight. The effect ofroot extracts of Tagetus minuta on Partheniumshoots was observed at regular interval of 24, 48and 72 hours at room temperature. In shoot cutbioassay, phytotoxic damage was recorded onthe basis of a rating scale of 0 - 5; where0 No effectGermination percentage: Total number ofseeds germinated / Total number of seeds takenfor germination x 1001 Slight chlorosis / wilting of leaves2 Marked chlorosis and slight necrosis3 Acutechlorosisandnecrosis/drooping of entire twigsDetermination of growth parameters: Differentgrowth characteristics such as radicle andplumule length, relative germination rate, vigourindex and biomass were determined in controland treatment by the following methods :marked4 Falling of petals and leaves/high necrosisand chlorosis5 Acute chlorosis and very high necrosisleading to death of the whole shoot.Seedling length: After 10 days of seed sowing,radicle and plumule length of the seedlings were15measured as per standard methods of ISTA .Seeds were considered to be germinated withthe emergence of both plumule and radicle. Theradicle and plumule length were measured witha measuring scale and values were expressed incentimeters.Seedling bioassay: Parthenium hysterophorusseedlings were raised in plastic pots (depth 7 cmand diameter 7 cm) containing 150 gms ofsterilized soil, sand and peat (1 : 1 : 1) andplaced in growth chamber at 26 1 C.Parthenium seedlings were sprayed with 5 ml of50 and 100% concentrations of aqueous rootextracts of vegetative and flowering stages ofTagetus minuta and irrigation was carried outfor 3 days with root extracts in treatment andwith distilled water in control ptoms on the Parthenium seedlings weremade at regular interval of 24, 48 and 72 hoursat rating scale of 0 - 5; whereVigour index: Vigour index of the seedlings wasestimated according to the formula: Vigourindex Total seedling length (mm) x16germination percentage .Biomass estimation: Fresh weight of theseedlings of control and treatment wasmeasured after 10 days of sowing. After that,0the seedlings were oven dried at 65 C for 72hours and dry weight was also estimated. Themoisture content of the tissue was calculated bythe formula : (FW - DW/FW x 100).0 No effect1 Slight chlorosis / wilting of leavesShoot cut bioassay: Parthenium hysterophorusshoots (30 days old) with one inflorescence and15 cm in length were taken for the shoot cut2 Marked chlorosis and slight necrosis3
International Journal of Innovations in Biological and Chemical Sciences, Vol. 4: 1-10, 20133 Acutechlorosisandmarkednecrosis/drooping of entire seedling.D. Folin - Ciocalteu reagent (Diluted Folin Ciocalteu reagent with equal volume ofdistilled water just before use).4 Falling of petals and leaves/high necrosisand chlorosisE.5 Acute chlorosis and very high necrosisleading to death of the whole seedling.Ten (10) mg fresh Parthenium leaves of theshoot cut bioassay of 50 and 100%concentrations of vegetative and floweringstages of aqueous root extracts werehomogenized with 1ml of 1N NaOH and kept for5 minutes at 100 C into the boiling water bath.Added 5 ml of alkaline copper reagent to it andallowed the mixture to stand at roomtemperature for 10 minutes. Added 0.5 ml ofFolin - Ciocalteu reagent immediately and mixedthe contents properly in the test tube. Theabsorbance of the solution was measured at 650nm after 30 minutes. The amount of protein wascalculated with reference to standard curve oflysozyme.Quantitative estimation of chlorophyll: Theamount of chlorophyll was determined by the17following method . 10 mg of fresh Partheniumleaves of the shoot cut bioassay treated with 50and 100% concentrations of aqueous rootextracts of vegetative and flowering stages ofTagetus minuta were grounded with neutralsand and 10 ml of 80% acetone and centrifugedat 3000 rpm for 10 minutes. The volume ofsupernatant was recorded. Optical density wasmeasured at 645 nm and 663 nm. For thedetermination of chlorophyll a, chlorophyll band total chlorophyll following formulae wereemployed :Statistical analysis: The treatment in all theexperiments were laid out in a completerandomized block design with a three replicatesand Duncans Multiple Range Test was employedto test the effect of treatment over the19control .Total chlorophyll (mg/g) 20.2 OD645 8.02 OD663 V1000 WChlorophyll a (mg/g) RESULTS AND DISCUSSION12.7 OD663 2.69 OD645 V1000 WChlorophyll b (mg/g)The aqueous root extracts of Tagetus minutaboth at vegetative and flowering stagessignificantly affected the growth andphysiological parameters of Partheniumhysterophorus L. 22.9 OD645 4.68 OD663 V1000 WWhere, V Volume of the supernatant in ml,W Fresh weight of the Parthenium leaves ingm and OD Optical density.Seed germination bioassay: The treatment ofParthenium seeds with aqueous root exracts ofvegetative and flowering stages of Tagetusminuta of 100% concentration exhibited markedvariation on seed germination over control.Root extracts of flowering stage were moreinhibitory than root extracts of vegetative stageof Tagetus minuta. Reduction in seedgermination was more with roots extracts offlowering stage in comparison to vegetativestage. 34.41% reduction in seed germinationwas observed with 50% concentration of rootextracts of Tagetus minuta at vegetative stage,which increased to 70.97 % in 100%concentration. The 100% concentration of rootextracts of Tagetus minuta at flowering stageexhibited maximum 84.95% reduction and 50%concentration exhibited 59.14% reduction inQuantitativeestimationofprotein:Quantitative estimation of protein was done by18the following method . Stock solution of thefollowing reagents was prepared :Reagents:A. Alkaline sodium carbonate solution (0.2%Na2CO3 in 0.1 N NaOH).B.Copper sulphate - sodium potassiumtartarate solution (0.5% CuSO4. 5H2O1% sodium potassium tartarate).C.1 N NaOH.Alkaline copper reagent: Mixed 50 ml ofreagent A and 1 ml of reagent B.4
International Journal of Innovations in Biological and Chemical Sciences, Vol. 4: 1-10, 2013Parthenium seed germination as compared tocontrol. Relative germination rate was alsoreduced with 100% concentration in comparisonto 50% concentration of aqueous root extractsof Tagetus minuta.Table 1. Effect of root extracts of Tagetus minuta on the seed germination of Partheniumhysterophorus L.ConcentrationGermination percentage (%)of root extractAqueous root extractRelativeAqueous root extractRelativeof Tagetus minuta at germination of Tagetus minuta at germinationvegetative stagerateflowering stagerateControl93 0.8693 0.6550%61 0.57 (34.41)0.6638 0.21 (59.14)100%27 0.32 (70.97)0.2914 0.22 (84.95)Values are mean of three replicates semFigures in parentheses indicate percent inhibition over control.0.410.15Table 2. Effect of root extracts of Tagetus minuta on the seedling length of Partheniumhysterophorus L.ConcentrationSeedling length (cms)of rootAqueous root extract of TagetusAqueous root extract of Tagetusextractminuta at vegetative stageminuta at flowering stageRadiclePlumuleRadiclePlumuleControl2.8 0.093.9 0.092.9 0.054.3 0.0950%2.4 0.063.5 0.062.1 0.032.5 0.06100%1.5 0.012.7 0.070.9 0.021.2 0.04Where R radicle length and P plumule lengthValues are mean of three replicates semFigures in parentheses indicate percent inhibition over control.Table 3. Effect of root extracts of Tagetus minuta on the vigour index of Parthenium hysterophorusL.ConcentrationVigour indexof rootAqueous root extract of Tagetus Aqueous root extract of Tagetusextractminuta at vegetative stageminuta at flowering stageControl6231669650%3599 (42.24)1748 (73.89)100%1134 (81.80)294 (95.61)Values are mean of three replicates semFigures in parentheses indicate percent inhibition over control.Table 4. Effect of root extracts of Tagetus minuta on the biomass of Parthenium hysterophorus L.Concentration ofrootextractBiomass (gm)Aqueous root extract of Tagetus minuta at Aqueous root extract of Tagetus minuta atvegetative stageflowering stageFreshDry weightMoistureFreshDry weightMoistureweight (gm)(gm)content (%) weight (gm)(gm)content (%)Control1.214 0.09 0.9410 0.0522.491.350 0.021.064 0.0121.1950%0.9821 0.08 0.5613 0.0442.850.8952 0.05 0.4816 0.0246.20100%0.7639 0.06 0.5482 0.0228.240.6548 0.05 0.4319 0.0334.04Values are mean of three replicates semFigures in parentheses indicate percent inhibition over control.5
International Journal of Innovations in Biological and Chemical Sciences, Vol. 4: 1-10, 2013Table 5. Effect of root extracts of Tagetus minuta on the shoots of Parthenium hysterophorus L. inshoot cut bioassay.ExposureShoot cut bioassayTime (h)Aqueous root extract of Tagetus minuta atAqueous root extract of Tagetusvegetative stageminuta at flowering stageC50%100%C50%100%24 h0.00 0.000.66 0.542.33 0.270.00 0.00 3.33 0.27 3.90 0.0548 h0.00 0.001.00 0.473.00 0.470.00 0.00 3.66 0.72 4.50 0.2472 h0.00 0.001.50 0.244.00 0.470.00 0.00 3.90 0.05 5.00 0.09Values are mean of three replicates semPhytotoxicity rating scale : 0 no effect, 1 slight chlorosis/wilting of leaves, 2 marked chlorosis andslight necrosis, 3 acute chlorosis and marked necrosis/drooping of entire shoot, 4 falling of petalsand leaves/high necrosis and chlorosis, 5 acute chlorosis and very high necrosis leading to the deathof Parthenium shoots.Table 6. Effect of root extracts of Tagetus minuta on the seedlings of Parthenium hysterophorus L. inseedling bioassay.ExposureSeedling bioassaytime (h)Aqueous root extract of TagetusAqueous root extract of Tagetus minutaminuta at vegetative stageat flowering stage24 h48 h72 hC0.00 0.000.00 0.000.00 0.0050%0.00 0.000.66 0.541.00 0.47100%0.66 0.541.50 0.243.66 0.72C0.00 0.000.00 0.000.00 0.0050%2.50 0.282.80 0.093.33 0.27100%3.33 0.273.66 0.724.00 0.47Values are mean of three replicates semPhytotoxicity rating scale : 0 no effect, 1 slight chlorosis/wilting of leaves, 2
Riti Thapar Kapoor* and Ashwani Kumar Srivastava . It was then autoclaved at 1210C temperature and at 15 lbs pressure14. The aqueous root extracts of both vegetative and flowering stages . humidity at 25 20C with a 12 h phot
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