Since p-hydroxybenzoic acid is unlikely to be present at such a high concen- .... zolium chloride in darkness at 30 + 1 °C for 4 hr, formazan was extracted, and.
Journal oat` Chemical Ecology, VoL 20, No. 12, 1994
INHIBITION OF S A L V I N I A (Salvinia molesta MITCHELL) BY P A R T H E N I U M (Parthenium hysterophorus L.). I. E F F E C T OF L E A F R E S I D U E A N D A L L E L O C H E M I C A L S l
D.K. PANDEY National Research Centre fi~r Weed Science (L C.A.R.) Adhartal, Jabalpur (M.P.)-482 004, India (Received February 23, 1994; accepted July 25, 1994)
Abstraet--Parthenium (Parthenium hysterophorus L.) leaf residue (LP, leaf powder) inhibited salvinia (Salvinia molesta Mitchell) biomass and the number of healthy fronds at 0,25% (w/v) and killed the treated plants at and above 0.75% (w/v) in about 5 15 days, depending on the quantity of the residue. At the lethal dose, the LP caused an abrupt desiccation of above-water plant parts, probably due mainly to root dysfunction, This was concurrent with the loss of dehydrogenase activity in, and an increase in solute leakage from, the roots and loss of chlorophyll a, b, and total chlorophyll contents in the fronds, resulting in death of the treated plants. The LP appears inhibitory to salvinia through affecting macromolecules--proteins, lipids, and nucleic acids. The inhibitory activity of LP at the lethal dose suspended in water was completely lost when allowed to stand for 30 days under outdoor conditions and promoted growth of the salvinia plants placed in it. The standard alleiocbemicals, including those present in parthenium LP, except parthenin and p-hydroxybenzoic acid. did not inhibit growth up to 100 ppm. However, parthenin and p-hydroxybenzoic acid killed salvinia plants at 100 and 50 ppm, respectively. Since p-hydroxybenzoic acid is unlikely to be present at such a high concentration, parthenin appears to be one of the main allelochemicals responsible for the inhibitory effect of parthenium leaf residue on salvinia. Key Words--Sah'inia molesta, growth inhibition, Parthenium hysterophorus, leaf residue, membrane integrity, dehydrogenase activity, chlorophyll, water absorption, allelochemicals, phenolics, parthenin. A portion of this work was presented at the International Symposium on Weed Management for Sustainable Agriculture held at C.C.S. Haryana Agricultural University, Hissar, India, November 18-20, I993. 3111 00984)331/9411200-31 I I $07.0010 ~c~ 1994 Plenum Publishing C~rporatlon
3112
PANDEY INTRODUCTION
Parthenium (Parthenium hysterophorus L.) is an aggressive tropical weed of the Asteraceae, endemic to the Americas, and has spread to Africa, Australia, and Asia (Towers et al., t977). The weed commonly infests pastures, wastelands, and agricultural fields. The weed has spread throughout India, posing serious threats to the environment and may affect natural diversity and cause extinction to natural flora. The plants and their residues show a range of biological activities including affecting various surrounding plants, some economically important, through allelopathy (Kanchan, 1975; Kanchan and Jayachandra, 1979a,b; Sharma et al., 1976, Mersie and Singh, 1987, 1988; Pandey et al., 1993a,b). The allelopathy is caused by the predominant allelochemicals, mainly sesquiterpene lactones and phenolics (Towers et al., 1977; Picman, 1986; Kanchan and Jayachandra, 1979a,b) released from aerial and underground parts of the plants and their residue into the soil environment. Interestingly, the parthenium plant residue showed inhibitory effects on an aquatic weed, water hyacinth, at a much lower level than that required to inhibit growth of wheat seedlings in aquaculture bioassay. Such inhibitory activity indicated the potential for possible biological control of water hyacinth by the strongly allelopathic terrestrial weed (Pandey et al., 1993a,b). Parthenium plant residue either inhibited growth of water hyacinth at lower doses or killed the plants at higher doses, resulting in clearing the water surface (Pandey et at., 1993a,b). However, growth of wheat seedlings continued even at much higher levels of parthenium residue than the lethal dose for water hyacinth. Salvinia (Salvinia motesta Mitchell) is a rapidly growing South American free-floating fern (Fomo and Harley, t979) that has become a serious weed in Africa, Southeast Asia, and Australia (Harley and Mitchell, 1981). This weed is now a major problem in irrigated lowland rice (Or3,za sativa L.) in some parts of the world (Pablico et al., 1989). In India, it is a major problem in the state of Kerala, where it chokes canals, rivers, ponds, and even paddy fields, affecting the life of more than 5 million people (Joy, 1978). The present investigation was undertaken to study the inhibitory effects of a strongly allelopathic terrestrial weed, parthenium leaf residue, and the possible implications of such inhibition in the management of salvinia through biological control. Attempts have been made to study some selected physiological processes associated with and the allelochemicals involved in inhibition. METHODS AND MATERIALS
Salvinia Plants. Salvinia (Satvinia molesta Mitchell) plants were obtained from the collection of Dr. K.P~ Jayanth, Indian Institute of Horticultural Research, Bangalore, India. The plants were grown and maintained in an aqueous
I N H I B I T I O N OF S A L V I N I A BY P A R T H E N I U M 1
3113
medium that consisted of a 5% (w/v) mixture of farmyard manure powder and field soil (3 : 1, v/v) in water in 20-liter plastic tubs under outdoor conditions. The growing medium was changed at regular intervals, and the plants from this culture were used for the experiments. Collection and Preparation of Parthenium Leaf Residue. Parthenium leaf residue (LP, leaf powder) was prepared according to the procedure described earlier (Pandey et al., 1993a). Anal~,sis of Medium. LP was suspended in water at 0.25-1.25% (w/v) and allowed to stand under outdoor conditions for 24 hr. Samples were drawn and analyzed for pH, electrical conductivity (EC), water potential (if',,), and OD at 215 and 340 nm, corresponding to parthenin, a sesquiterpene lactone in the parthenium plant by the procedure described earlier (Pandey et al., 1993a). Total phenolics were measured colorimetrically using the Folin-Denis reagent method (Swain and Hillis, 1959). Inhibitory activity of the samples was tested by using the wheat (Triticurn aestivum L. var. Sujata) coleoptile growth bioassay in the seeds germinated for 48 hr at 30°C in the dark as described earlier (Pandey et al., 1993a). Inhibitory. Effect of Parthenium Leaf Residue on Salvinia. Leaf residue (LP) was dispersed in 20 liters of tap water in plastic tubs to make suspensions of 0.25, 0.50, 0.75, 1.00, and 1.25% (w/v, the convention used throughout). Plants grown in tap water served as controls. Preweighed salvinia plants with uniform fronds were loaded in each of the tubs and allowed to grow. Tap water was used because such a large quantity was required, its quantity in the tubs being kept constant by regularly replenishing the water lost due to evapotranspiration. Biomass and healthy frond numbers (HFN) were monitored. A frond that did not show desiccation and drying from the margins or appear dull green and flaccid was considered to be a healthy frond. Effect of pH, Salt, and Water Stress on Salvinia Plants. Preweighed salvinia plants were placed in 1 liter of tap water (EC 0.750 mS/cm, pH about neutrality) with pH adjusted at 1-14 by adding HCI or NaOH solution and were allowed to grow. The HFN and biomass were monitored. For salinity stress studies, salvinia plants were allowed to grow in tap water or in NaC1 concentrations ranging from 2.40 to 22.4 mS/cm, and HFN and biomass were monitored. The effect of water stress on salvinia plants was studied by allowing the plants to grow in water or in polyethylene glycol (PEG) -6000 solutions with different water potentials (q,,) (Michael and Kaufmann, 1973) ranging from about - 0 . 0 1 3 to - 0 . 2 5 0 MPa, and HFN and biomass were monitored.
Absorption of Water and Change in Fresh Weight Over a Brief Period. Preweighed plants were placed in 1 liter of aqueous medium containing a lethal dose of LP and allowed to grow. Plants similarly kept in tap water served as the control. Water was similarly kept for measuring evaporative loss from the free open surface. The plants were removed, weighed, and the volume of aqueous
3114
PANDEY
medium was measured at each observation. Then the volume of the open medium or water was replenished to 1 liter and the plants were replaced. The quantity of water absorbed by the plants was calculated by measuring the evaporative loss. Solute Leakage from Roots. The roots (about 1 g) from the control and LPtreated plants were sampled, washed with distilled water, blotter-dried, weighed, and steeped in 100 ml distilled water at 30°C for 4 hr. Then the roots were removed, the steep water was filtered through Whatman No, 1 filter paper, and OD at 264 nm (corresponding to UV-absorbing materials, e.g., amino acids, nucleotides, polypeptides, etc.) was measured. An OD of 0.01 was considered as one unit of UV-absorbing substances and was expressed on a per gram root (fresh weight) basis. This was taken as an index of solute leakage and, thus, of cellular membrane integrity. Dehydrogenase Activit3, in Roots. For dehydrogenase activity determination, about 0.5 g roots were soaked in 5 ml of 1% (w/v) 2,3,5-triphenyl tetrazolium chloride in darkness at 30 + 1°C for 4 hr, formazan was extracted, and OD at 520 nm was read following the method described earlier (Pandey et al., 1993a). An enzyme unit was defined as 0.01 OD at 520 nm and was expressed on a per gram root (fresh weight) basis. Determination of Chlorophyll a, b, and Total Chlorophyll. Freshly sampled frond disks (about 5 cm z) were weighed and ground with a small quantity of acid-washed silica sand in 25 ml of 80% (v/v) acetone, using a mortar and pestle as described earlier (Pandey et al., 1993a). Optical density of the final extract was measured at 645, 652, and 663 nm and chlorophyll a, b, and total chlorophyll were calculated considering equal area equal weight by the method of Arnon (1949). Isolation and Purification ofParthenin. Parthenin was isolated and purified by the procedure described by Picman et al. (1980) with modifications (P.V. Subbarao, personal communication, 1992). Dried plant material was refluxed with methanol in a Soxhlet apparatus for 12 hr. The crude methanolic fraction was evaporated to dryness and extracted with petroleum ether. The residue was extracted with chloroform. The chloroform fraction was evaporated to dryness, dissolved in hot ethanol and an equal volume of 4% lead acetate, and allowed to stand for 20 rain. This was filtered through diatomaceous earth. The supernatant was concentrated to half the volume and extracted with chloroform. The chloroform layer, comprising the terpene fraction, was dehydrated with anhydrous sodium sulfate, evaporated to dryness, dissolved in benzene-acetone (2 : I, v/v), and subjected to chromatography over a silica gel column equilibrated with benzene. The column was washed with benzene and eluted successively with portions of a solvent containing increasing amounts of acetone in benzene (10100%, v/v). The fractions were evaporated to dryness and analyzed by TLC on silica gel-G plates using benzene-acetone (4: 1, v/v) as a solvent system. The
I N H I B I T I O N O F S A L V I N I A BY P A R T H E N I U M 1
3115
compounds were located on TLC plates by exposure to iodine vapors or spraying with 5% aqueous KMnO 4. The fractions yielded one major and a few minor compounds, which were detected on TLC plates. The fractions containing the major compound parthenin, as identified by cochromatography with parthenin, were pooled and subjected to preparatory TLC using a benzene-acetone (4: 1) solvent system. The areas corresponding to the major compound on individual TLC plates were scraped and eluted with chloroform. Pooled chloroform eluate was evaporated to dryness. The residue was dissolved in a small amount of ethyl acetate, the compound was twice crystallized by dropwise addition of cyclohexane in cold, and the purified toxin was obtained and used in the experiments. Effect of Standard Allelochemicals. Allelochemicals tested included all the major constituents reported in parthenium plant residue, viz., p-hydroxybenzoic acid, anisic acid, cinnamic acid, salicylic acid, coumaric acid, fumaric acid, tannic acid, gallic acid, chlorogenic acid, vanillic acid, caffeic acid, ferulic acid, and parthenin, which was isolated and purified in the laboratory. For preparing required concentrations of 10, 25, 50, and 100 ppm, the allelochemicals were, wherever necessary, dissolved in a small quantity of acetone or ethanol or directly prepared in distilled water. The solutions were then made up to one half the total volume with distilled water and the remaining half with the nutrient medium described by Jain et al. (1989). The small quantity of solvent used for dissolving the allelochemicals did not affect growth of the salvinia plants. The plants grown in half-strength nutrient medium served as controls. The HFN and biomass were monitored. In all experiments the plants were grown under outdoor conditions. All experiments and determinations were repeated at least three times. The data were statistically analyzed for indices of significance (LSD) using a completely randomized block design. Treatments causing death of salvinia plants (i.e., 100% reduction in value) in five days were not included in the statistical analysis.
RESULTS
Analyses of the medium gave the following results. The LP at 0.25-1.25 % did not change the pH of the medium much to either side of neutral (range 7.0 _ 0.6). Electrical conductivity (EC) ranged from 0.7 to 2.7 mS/cm. Water potential (q'w) ranged from - 2 3 5 × 10 -4 to - 9 4 0 × 10 -4 MPa. Total phenolic acids were 18-108 ppm. Optical density at 215 and 340 nm (corresponding to absorption maxima of parthenin), probably indicating the presence of parthenin, and inhibitory activity as shown by reduction in wheat coleoptile length (after 48 hr) bioassay showed a steady increase with the increase of LP in the medium.
3116
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Both the number of healthy fronds (HFN) and biomass increased in the control (Figure la,b), At 0.25 and 0.50%, the LP inhibited both HFN and biomass. The treatment with LP was relatively more inhibitory to HFN than to biomass. While at lower levels (0.25 % and 0.50%) the LP dramatically reduced HFN, a slight increment in biomass continued at 0.25% and was reduced marginally at 0.50%. However, at and above 0.75%, the treatment drastically affected the plants. The results showed very high LSD values due to much fluctuation in the response of plants to LP and, in part, probably due to elimination of treatments causing death (i.e., 100% reduction) from statistical analyses. The LP killed treated plants in about five days and resulted in the subsequent sinking of dead plants. Appearance, disappearance, persistence, and magnitude of the symptoms depended on the light, concentration of the LP, and treatment duration. The inhibitory activity of LP at 0.25% ceased completely in aqueous medium in about 30 days under outdoor conditions as freshly placed plants in the medium showed a rapid increase both in HFN and biomass (data not presented). In the controls, although initial growth was rapid, it subsequently became static, probably due to unavailability of nutrients. Salvinia plants were able to grow in a wide range of pHs, from 3 to 10. The growth was inhibited at pH 11. At extreme pHs viz., 1, 2, and 12, the plants were killed, showing bleaching and desiccation in about one to five days following death. Salinity levels below 12 mS/cm did not inhibit growth of salvinia. In the range above 12 and below 22.4 mS/cm, depending on the EC, growth of the treated plants was marginally affected. At and above 22.4 mS/
~4o I
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FIG. 1. Effect of parthenium leaf residue (LP, leaf powder) on (a) number of healthy fronds (HFN) and (b) biomass of salvinia.
INHIBITION OF SALVINIA BY P A R T H E N I U M 1
31 17
cm, the plants were inhibited drastically, resulting in death. Results of the effect of xI, on salvinia plants showed that growth of the treated plants was unaffected from - 0 . 1 5 to - 0 . 2 5 MPa except that at the latter some leaflets of the treated plants initially showed desiccation along the frond margins; subsequently, the dryness disappeared. LP treatment at the lethal dose reduced water use by the plants. Over a 24-hr treatment period, the water use per gram fresh weight was 2.24 _+ 0.16 ml in control plants and 1.85 -I- 0.29 ml in LP-treated plants. Biomass increased considerably in the control plants (by about 10.18 +_ 0.62%) and decreased (by about 19.9 + 8.48%) in the treated plants. Treatment with LP at the lethal dose, 0.75%, for 12 hr caused massive leakage of solutes from the roots (Figure 2a). The leakage was further enhanced
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3118
PANDEY
by treatment for 36 hr, showing massive loss o f cellular membrane integrity due to the treatment. LP at the lethal dose drastically reduced dehydrogenase activity in the roots (Figure 2b). The treatment for 12 and 48 hr caused about 50% and near total loss of dehydrogenase activity, respectively, in the roots. Chlorophyll a, b, and total chlorophyll were considerably reduced in the fronds of the plants treated with LP at the lethal dose (Figure 2c). At 50 ppm, none of the alleiochemicals tested, except p-hydroxybenzoic acid, was lethal for salvinia plants (Figure 3). p-Hydroxybenzoic acid caused bleaching, browning, and desiccation, tbllowed by death of the treated plants in about 5-15 days. Parthenin proved to be a potential inhibitor at 50 ppm, as it almost contained the HFN and biomass increment over the original value. At 100 ppm (Figure 3), parthenin was also lethal. Although, except for gallic and ferulic acids, all other allelochemicals were slightly inhibitory to biomass, their effect on HFN was relatively less. Total phenolic acids in the medium at lethal dose of LP were about 64 ppm. Except for p-hydroxybenzoic acid at 50 ppm and parthenin at 100 ppm, none of the other allelochemicals was lethal at concentrations up to 100 ppm.
DISCUSSION The xI,. and EC values of the medium at even the highest concentration of the LP (i.e., 1.25%) were far lower than those that can cause water and salinity stresses to the salvinia plants. Similarly, the LP in aqueous medium did not change the pH much to either side of neutrality, whereas the salvinia plants were able to grow in a wide range of pH from 3 to 10. Thus, LP at the lethal dose did not result in water, salinity, or pH stress. The inhibitory activity of LP could be attributed solely to the allelochemicals present. An increase in inhibitory activity of the medium with an increase in the level of LP was evident from the results of the bioassay for inhibitory activity and was probably due to a rise in phenolic acids and sesquiterpene lactones, as these are the dominant classes of allelochemicals in the residue of the parthenium leaf (Kanchan and Jayachandra, 1980; Picman, 1986). The phytotoxic allelochemicals leached out of the LP into the aqueous medium inhibited growth of salvinia plants at lower doses and killed them at higher doses. An immediate effect of the treatment was a dull green appearance of the fronds, drastic loss of turgidity, and resultant flacid texture, as could be easily felt by touching. This was followed and accompanied by desiccation and browning of the frond margins followed by a change of color of the entire frond to pale brown or black. The symptoms started appearing after 6-8 hr of exposure to sunlight. The appearance of these symptoms was much delayed in the dark;
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FIG. 3. Effect of different allelochemicals (parthenin and organic and phenolic acids) on number of healthy fronds (HFN) and biomass of salvinia. Except for parthenin, all allelochemicals are acids. A 100% reduction in biomass indicates lethal dose.
Ferullc ParthenLn L S D at 5%
Caffe±c
Anisic Cinnamlc Salicylic Coumarlc Fumaric Tannlc Ga|lic Chlorogen,c Vani]|ic
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Allelochemical
3120
PANDEY
nevertheless they appeared, and the plants were affected as under outdoor conditions (unpublished work). It was interesting that untreated plants used more water than treated ones and that the fronds of the plants immersed in the medium containing a lethal dose of LP in l-liter glass beakers kept under outdoor conditions retained a lush green color for up to 20-30 days, Roots and margins of some of the fronds showing death and rotting (data not presented) indicate that root dysfunction induced a reduction in water supply to the fronds and sustained evapotranspiratory loss of water, probably contributing to the relatively rapid desiccation and death of the treated plants. The visual effects of LP treatment on the fronds were accompanied by a drastic loss of membrane integrity in the roots, as evidenced by excessive loss of solutes even in a relatively short period. There was a concomitant loss of dehydrogenase activity in the roots and loss of chlorophyll a, b, and total chlorophyll contents in the fronds when compared with untreated controls, Cellular membranes play important roles in living systems and are essential for the maintenance of structure and function. In addition to serving as a permeability barrier, they play vital roles in the compartmentalization of cellular components. Furthermore, cooperative enzymes of a metabolic pathway may be linked together in association with, or integrated into, membrane structures. Massive loss of membrane integrity due to allelochemicals leaching out of the LP and into the medium appears to be one of the main factors resulting in inhibition of salvinia. Likewise, loss of dehydrogenase activity in roots may indicate loss of respiration (Mackay, 1972). Damage to membranes, loss of dehydrogenase activity in roots, and reduced chlorophyll contents in the fronds indicate that the allelochemicals may have acted by affecting the macromolecules--proteins, lipids, and nucleic acids. Altelochemicals other than p-hydroxybenzoic acid and parthenin were either slightly inhibitory or did not inhibit the growth of salvinia, p-Hydroxybenzoic acid was a more effective allelochemical than parthenin in inhibiting and killing salvinia plants, as the former was lethal at 50 ppm and the latter at 100 ppm. The maximum levels of phenolic acids in the medium at 0.75% LP, the lethal dose, 24 and 72 hr after suspending the plant residue, were about 75 and 107 ppm, respectively (Pandey et al., 1993a,b). The level of phenolic acids did not increase further, rather it decreased subsequently (data not presented). Water-soluble compounds involved in altelopathy in LP include caffeic acid, vanillic acid, ferulic acid, and parthenin (Kanchan and Jayachandra, 1980). Thus, p-hydroxybenzoic acid is unlikely to be present at such a high concentration. Hence, other allelochemicals, including parthenin, appear to be mainly responsible for inhibition of salvinia by parthenin leaf residue. Consistent with earlier findings with water hyacinth (Pandey et al., 1993a,b), the aquatic weed salvinia also showed relatively more sensitivity to the inhibitory effect of allelochemicals when compared with wheat as a reference material. There is a strong possibility that salvinia could be biologically managed
INHIBITIONOF SALVINIABY PARTHENIUM I
3121
o r c o n t r o l l e d and r e p l a c e d in a natural e c o s y s t e m by a s t r o n g l y allelopathic terrestrial w e e d h a v i n g a l l e l o c h e m i c a l s w i t h s t r o n g i n h i b i t o r y activity s i m i l a r to p - h y d r o x y b e n z o i c acid and p a r t h e n i n .
Acknowledgments--The author thanks Mr. A,P. Singh and Mr. Sebstin for technical assistance, Mr. S. Dhagat for help in preparing the figures, and Dr. M.A,K. Lodhi for his critical review of an earlier manuscript.
REFERENCES ARNON, D.I. 1949, Copper enzymes in isolated chloroplast. Polyphenol oxidase in Beta vulgaris. Plant Physiol. 24:1-15. FoRNO, I.W., and HARLEY, K.L.S. 1979. The occurrence of Salvinia molesta in Brasil. Auat. Bot. 6:185-187. HARLEY, K.L.S., and MITCHELL, D,S. 1981. The biology of Australian weed. Salvinia molesta D.S. Mitchell, J. Austr. Inst. Agric. Sci. 47:67-76. JAIN, R., SINGH, M., and DEZMAN, D,J. 1989. Qualitative and quantitative characterization of phenolic compounds from lantana (Lantana camara) leaves. Weed Sci. 37:302-307. Joy, P.J. 1978. Ecology and control of salvinia (African Payal) the molesting weed of Kerala, Tech. Bull. Kerala Agric. Univ.. India 2:40 pp. KANCHAN, S.D. 1975, Growth inhibitors from Parthenium hysterophorus L. Curr. SoL (India) 44:358-359. KANCHAN, S.D., and JAYACHaNDRA. 1979a. Allelopathic effects of Parthenium hysterophorus L, I, Exudation of inhibitors through roots. Plant Soil 53:27-35, KANCIIAN, S.D., and JAYACHANDRA, 1979b. Allelopathic effects of Parthenium hysterophorus L. 111. Inhibitory effects of the weed residue. Plant Soil 53:37-47, KANCHAN, S.D., and JAYACHANDRA. 1980. Allelopathic effects of Parthenium hysterophorus L, IV. Identification of inhibitor's. Plant Soil 55:67-75. MACKAY, D.B. 1972, The measurement of viability, pp. 172-208, in E.H. Roberts (ed.). Viability of Seeds. Chapman and Hall. London. MERSlE, W., and SINGH. M. 1987. Allelopathic effect of parthenium (Parthenium hysterophorus L.) extract and residue on some agronomic crops and weeds. J. Chem. Ecol. 13:1739-1747. M~RSlE, W., and SINGH, M. 1988, Effect of phenolic acids and ragweed parthenium (Parthenium hysterophorus L,) extracts on tomato (Lycopersicon esculentum) growth and nutrient and chlorophyll content. Weed Sci. 36:278-281. MICttAEL, B.E., and KAUFMANr~,MR. t973. The osmotic potential of polyethylene glycol 6000, Plant Physiol. 51:914-9 t 6. PABLlCO. P.P., ESTORr'qNOS,J.R., CaSTIN, E.M., and MooDy, K. 1989. The occurrence and spread of Salvinia molesta in the Philippines. FAO Plant Prot. Bull. 37(3): 104-109. PANDEr, D.K., KAURAW, L.P., and BHAN, V.M. 1993a. Inhibitory effect of parthenium (Parthenium hysterophorus L.) residue on growth of water hyacinth (Eichhornia crassipes Mart Solms,) 1. Effect of leaf residue. J. Chem. Ecol. [9:2651-2662. PANDEY, D.K., KAURAW,L.P., and BHAN. V.M, 1993b. Inhibitory effect of parthenium (Parthenium hysterophorus L.) residue on growth of water hyacinth (Eichhornia crassipes Mart Solms.) I1. Relative effect of flower, leaf, stem, and root residue. J. Chem. Ecol. 19:2663-2670. PtCMAN, A.K. 1986. Biological activities of sesquiterpene lactones. Biochem. Svst, Ecol. 14:255281. P1CMAN, AK., TOWERS, G.H.N., and StJBBARaO, P.V. 1980. Coronopilin--another major sesquiterpene lactone in Parthenium hysterophorus. Phytodwmist~. 19:2206-2207.
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SHARMA, K.K.V., GIRl, G.S., and SUBRAMANYAM.K. 1976, Allelopathie potential of Parthenium hysterophorus L. on seed germination and dry matter production in Arachis hypogea Willd., Crotalaria juncea L. and Phaseolus mungo L. Trop. Ecol. 17:76-77. SWAIN. T., and HiLLtS, W.E. 1959. The phenolic constituents of Pn, nus domestica. 1. The quantitative analysis of phenolic constituents. J. SoL Food Agric. t0:63-68. TOWERS, G,H.N., MITCHELL, J.C., RODRIGUEZ, E,, BENNEa-F, F,D., and SUBBARAO, P.V. 1977. Biology and chemistry of Parthenium hysterophon~s L., a problem weed in India. J. Sci. hid. Res. 36:672-684.
