Phytotoxic effects of Euphorbia dracunculoides : a weed of rainfed chickpea-chickpea cropping system

Phytotoxic effect occurs when plants release chemicals that inhibit neighoubouring plants. Phytotoxic effects of aqueous extracts of different parts of Euphorbia dracunculoides L. (green spurge) at two concentrations, and its infested soil were investigated on germination and seedling growth of chickpea (Cicer arietinum L.). The fruit extract at 1:20 (w/v) concentration caused maximum reduction (12%) in germination of chickpea seeds while leaf extract at 1:10 (w/v) concentration resulted in maximum mean germination time value and minimum germination index of chickpea seeds. All the traits of chickpea seedling growth including emergence were adversely affected by the aqueous extracts at both concentrations. Further, the inhibition of chickpea seedling growth was more pronounced with 1:10 (w/v) concentration whereas the lower concentration (1:20 w/v) showed stimulatory effect on shoot length, seedling vigor index and chlorophyll contents of chickpea seedlings. The leaf extract at 1:10 (w/v) concentration proved most harmful to seedling growth and chlorophyll contents (76% reduction) of chickpea. Soil beneath the E. dracunculoides plants significantly reduced emergence (23%), seedling vigor index (55%) and chlorophyll content (19%) of chickpea but a significant increase in N (6%), P (16%) and K (4%) contents of chickpea seedlings was recorded. Thus it can be concluded that E. dracunculoides contains compounds in its tissues which may cause phytotoxic effects on chickpea under field conditions. Additional key words: Cicer arietinum; emergence; germination; green spurge; phytotoxicity; seedling growth.


Introduction
Allelopathy interactions are based primarily on the production of secondary chemicals by the plants that produce a wide array of biochemical compounds affecting the growth and development of plants grown in the neighborhood (Shaukat et al., 2003;Macias et al., 2004).The chemicals involved in allelopathic interactions are present in virtually all plant parts and in most tissues including leaves, stems, fruits, roots, rhizomes, buds and seeds ( Weston and Duke, 2003;Narwal, 2004).Different weed species differ widely in their ability to produce allelopathic effects (Hamayun et al., 2005).Different parts of same weed also differ in their ability to produce allelopathic effects so that some parts exhibit more suppressive activity against germination or growth than others (Tanveer et al., 2008).A number of studies have shown that residues from several weed species release phytotoxins into the soil, thus affecting the growth of crop plants (Batish et al., 2007a,b).
Chickpea (Cicer arietinum L.) is a dry pulse crop of the family Fabaceae and is one of the important conventional crop in rainfed chickpea-chickpea cropping system of Pakistan.Being drought tolerant, 70% of the rainfed agriculture is occupied by this crop.The chickpea yield is lower in Pakistan as compared to maximum potential of cultivars.One of the limiting factors of high yield is presence of weeds in field.In Pakistan, weeds reduce the yield by 24-63% in chickpea (Tanveer et al., 1998) because of slow growth rate and limited leaf area development at early stages of crop growth and establishment ( Solh and Pala, 1990).
Euphorbia dracunculoides L. (Green Spurge: family Euphorbiaceae) is a much branched annual winter weed.In Pakistan it is grown in October-November in rainfed (less than 200 mm yr -1 ) areas of chickpeachickpea cropping system and matures in April.Other species of Euphorbia, namely E. helioscopia, E. hirta and E. granulata have also been reported in Pakistan.World over there is no published data on E. dracunculoides being phytotoxic, although several other species of Euphorbia have been demonstrated to be phytotoxic (Alsaadawi et al., 1990;Tanveer et al., 2010).Thus the present study was undertaken to determine the phytotoxic effect of E. dracunculoides on the germination and seedling growth of chickpea crop which was found to be frequently infested by this weed.

Material and methods
Seven months old plants of E. dracunculoides were uprooted at maturity in May 2008 and dried at room temperature for one week.Average dry weight was 18 g plant -1 .Plant parts i.e. roots, stems, leaves and fruits were separated and small pieces were made with a scissor.Dried plant parts as well as whole plants were immersed in tap water separately in the ratio of 1:10 and 1:20 (w/v) at room temperature for 24 hours.The aqueous extracts of each were obtained by filtering water through sieve and then through Minisart © nonpyrogenic, 0.45 µm filters.The pH, osmotic potential and electrical conductivity of these extracts ranged from 8.26 to 9.07, -0.48 to -0.78 MPa and 3.08 to 9.45 ms m -1 , respectively.
Two experiments were carried out with whole plant, stem, leaf, fruit and root extracts of E. dracunculoides using distilled water as a control in the laboratory.A third experiment was conducted with soil collected from E. dracunculoides infested field and an adjacent field without this weed.In experiments I, II treatments were arranged in a completely randomized design (CRD) with factorial arrangement in 9 cm diameter Petri dishes.Experiment III was done in CRD.There were four replications in each experiment.

Experiment I: Phytotoxic effects of aqueous extracts of different parts of E. dracunculoides on germination of chickpea
In this experiment 25 seeds of chickpea were placed on filter papers in Petri dishes, and 5 mL distilled water or plant extract was added to each Petri dish according to the treatment and concentration at the start of the experiment.The Petri dishes were placed at an average room temperature of 20.5°C.Extract or water was added according to need during the course of experiment.The experiment was repeated twice.
Germination was determined by counting and removing germinated seeds daily upto 10 days.Seeds were considered germinated when radical length was over 2 mm.Germination was observed daily according to AOSA (1990).Mean germination time (MGT) was calculated as per equation of Ellis and Roberts (1981): where n is the number of seeds that had germinated on day D, and D is the number of days counted from the beginning of germination.The germination index (GI) was calculated as per AOSA (1983) by using the following formula: No. of germinated seeds No. of germinated seeds GI = -------------+ … + -------------Days of firts count Days of final count Seedling vigour index (SVI) was calculated according to Abdul-baki and Anderson (1973) as:

Experiment II: Phytotoxic effects of aqueous extracts of different parts of E. dracunculoides on seedling growth of chickpea
In this experiment ten seeds of chickpea were sown in sand filled Petri dishes, and 12 mL of extract or distilled water was added in Petri dishes according to the treatment and concentration.Petri dishes were placed at an average room temperature of 19°C for 15 days.During this period Petri dishes were observed daily and distilled water or extract was added in each Petri dish according to requirement.After 15 days seedlings were up rooted and washed with water.The length of roots and shoots was measured.One gram of fresh leaves was collected from each replication and leaf area was measured using leaf area meter.Roots and shoots were dried in an oven at 70°C till constant weight was obtained.

Experiment III: Phytotoxic effects of E. dracunculoides infested soil on seedling growth of chickpea
Soil was collected to a depth of 10 cm from plot where E. dracunculoides had been growing for the last three years (test) and also from fields free of this weed (control).Soil was dried at room temperature and sieved through 2 mm mesh.Ten grams of test and same amount of control soil was uniformly spread in 9 cm diameter Petri dishes, separately.Ten seeds of chickpea were placed uniformly on soil.Seeds were covered with the same soil as spread underneath.Soil was adequately moistened with distilled water.Petri dishes were placed at an average room temperature of 19°C for 15 days.During this period Petri dishes were observed daily and equal amount of water was added in each Petri dish according to requirement.After 15 days seedlings were up rooted and washed with water.The length of roots and shoots was measured.Roots and shoots were dried in an oven at 70°C till constant weight was obtained.
A modified protocol (Arnon, 1949;Mochizuki et al., 2001) was used to measure chlorophyll content.Fresh leaves of chickpea weighing 300 g were collected from each replication and grinded with a pestle and mortar.The powder was transferred to a Falcon tube and 20 mL of 80% acetone was added and thoroughly mixed.The absorbance (A) of chlorophyll content was measured at wavelengths of 645 and 663 nm using a spectrophotometer.The total chlorophyll concentration was calculated as follows: Total chlorophyll (mg g -1 ) = [(8.02× A663) + + (20.20 × A645)] × V/1,000 × W where V = volume of the extract (mL); W = weight of fresh leaves (g).

NPK concentration in seedlings (%)
Dried ground material (0.1 g) was placed in digestion tubes, 2 mL of conc.H 2 SO 4 and 1 mL of 35% hydrogen peroxide was added and incubated over night at room temperature.Then 1 mL of H 2 O 2 (35%) was poured and heated up to 350°C in digestion block until fumes were produced and continued to heat for another 30 min.Digestion tubes were removed from the block and cooled.Then 1 mL of H 2 O 2 was slowly added and tubes were placed back into the digestion block until fumes were produced for 20 min and material became colorless.The volume of extracts was made up to 50 mL with distilled water.Then it was filtered and used for determination of mineral elements (Wolf, 1982).
Potassium was determined with flame photometer.A graded series of standards (ranging from 2 to 12 mg L -1 ) of KCl was prepared and standard curve was drawn.The values of K from flame photometer were compared with standard curve and total quantities were computed.
The extracted material (5 mL) was mixed in 10 mL of Barton reagents and total volume was made as 50 mL.The samples were kept for half an hour and P contents were determined by spectrophotometer using standard curve.The Barton reagent was prepared as MGT= (Dn) ∑ n ∑ described by Ashraf et al. (1992): Solution A, 25 g of ammonium molybdate was dissolved in 400 mL of distilled water; Solution B, 1.25 g of ammonium metavenadate was dissolved in 300 mL of boiling water, then cooled and 250 mL of conc.HNO 3 was added and again cooled.Solution A and B were mixed and volume was made up to one liter.
For N determination, 5 mL of aliquot was taken in Kjeldhal flask and it was placed on the Kjeldhal ammonium distillation unit, 10 mL of 40% sodium hydroxide solution was added and the flask was immediately connected to the distillation apparatus.Five mL of 2% boric acid solution was taken and few drops of mixed indicator (bromocresol green + methylene red) in 100 mL conical flask.When the distillate was approximately 40-50 mL, the conical flask was removed and distillation was turned off.The distillate was cooled for few minutes and titrated with 0.01 N standard sulphuric acid (90% H 2 SO 4 ) up to pink end point.

Statistical analysis
The data collected were analyzed statistically using Fisher's analysis of variances technique and treatment means showing F-values significant were compared using least significant difference (LSD) at 0.05 probability level (Steel et al., 1997).

Experiment I
It is evident form data (Table 1) that E. dracunculoides extracts significantly influenced germination percentage, MGT and GI of chickpea seeds.The lowest germination (80.50%) was recorded in seeds treated with fruit extract and it was statistically similar to all other extracts except distilled water (control).A considerable reduction of 6-8% was noted with the application of different E. dracunculoides extracts over control.The highest germination (91%) was recorded in control which was statistically at par with whole plant extract.The statistically maximum MGT value was observed in leaf extract followed by whole plant and stem extracts as against the minimum MGT recorded in control.When chickpea seeds were exposed to leaf, stem, whole plant, fruit and root extracts, MGT was increased by 46, 29, 29, 22 and 18% over distilled water (control), respectively.The lowest GI was recorded in seeds subjected to leaf extract.Germination

Experiment II
Extract of different parts of Euphorbia dracunculoides and their concentrations produced a significant effect on emergence percentage of chickpea seeds (Fig. 1a).The lowest emergence was recorded in seeds treated with leaf extract at 1:10 (w/v) concentration followed by whole plant extract at the same concentration.The highest emergence was in stem extract at 1:20 (w/v) concentration which was statistically at par with fruit and whole plant extract at the same concentration and stem extract at 1:10 (w/v) concentration.Chickpea seed emergence was reduced by nearly 85, 44 and 19% when they were subjected to leaf, whole plant and root extract, respectively.In contrast, approximately 11% increase was observed with stem extract over distilled water.
All the traits of chickpea seedling growth were adversely affected by the application of E. dracunculoides extracts (Table 2).Both root and shoot length, their individual and collective dry weight were significantly (p ≤ 0.05) affected and the effects were more pronounced on root development than shoot growth.Chickpea seedlings grown in leaf extract produced minimum root and shoot length while it was maximum with distilled water.The application of leaf and whole plant extract depicted about 81 and 60% reduction in root length, respectively, whereas reduction in shoot length was 69 and 46%, respectively.Likewise the root and shoot dry weights individually and collectively were also severely affected by leaf and whole plant extract depicting 83 and 52% reduction in root dry weight, 68 and 40% reduction in shoot dry weight and 75 and 45% reduction in total dry weight over control, respectivly.
Euphorbia dracunculoides extracts produced a significant effect on SVI (Table 2).The statistically lowest SVI was recorded in seedlings subjected to leaf extract which was statistically at par with whole plant extract The statistically maximum SVI was recorded with control followed by leaf extract.Decrease in SVI was maximum (82%) with leaf extract followed by whole plant extract (63%) whereas reduction was minimum (24%) with stem extract.
The results indicated that extract prepared from E. dracunculoides leaves caused maximum suppression in chlorophyll contents of chickpea seedling followed by whole plant extract at both concentrations.The inhibitory effects increased with increasing concentration of extracts.Root extract at lower concentration (1:20) exhibited a 4% increase in chlorophyll contents over control while all other treatements showed reduction in chlorophyll (Fig. 1b).Chickpea seedlings treated with all extract of E. dracunculoides showed significant reduction (p ≤ 0.05) in leaf area (Fig. 1c).Higher reductions were observed with 1:10 compared to 1:20 concentrations.A maximum reduction (96%) in leaf area was observed in seedling with leaf extract which was not statistically different from fruit extract and whole plant extract.Results also revealed that the interaction between concentration and types of extracts was significant at p ≤ 0.05.

Experiment III
The soil collected from E. dracunculoides infested field significantly and negatively affected the emergence percentage, SVI and chlorophyll contents of chickpea seedlings, whereas NPK contents were positively influenced compared with the control soil (Table 3).Soil from E. dracunculoides grown field caused 55, 23 and 19% reduction in SVI, emergence percentage and chlorophyll contents, respectively.Contrarily an increase of 16, 6 and 4% was recorded in P, N and K contents of chickpea seedlings, respectively over seedling raised in control soil (Table 3).

Discussion
Inhibition of chickpea germination increased with increasing concentration of extracts of different parts of E. dracunculoides.Mishra et al. (2001) andXiangxiang et al. (2009) reported that increasing concentration of Asphodelus tenuifolius and Hemistepta lyrata extract adversely affected the germination of wheat, mustard, lentil and chickpea; wheat, sorghum, cucumber, rape and radish seeds, respectively.Maximum reduction in germination percentage of chickpea seeds with aqueous extract of fruit at 1:20 concentration could be due to more inhibitory effect of allelochemicals present in fruit.Delayed germination and less GI with leaf extract of E. dracunculoides at 1:10 (w/v) concentration may reflect the presence of allelochemicals in maximum concentration.Tanveer et al. (2008) reported similar results by using leaf leachates of Xanthium strumarium, which caused maximum reduction in germination, MGT and GI of wheat, barley, maize, rice, cotton and sunflower.Reduction in emergence percentage of chickpea seeds with leaf extracts could be due to the availability of inhibitory chemicals in higher concentration in leaf parts than others.Further, stimulatory effect observed with stem extract might be due to the lower concentration of allelochemicals in this extract because slight change in concentration may modify the functions of allelochemicals.Xingxiang et al. (2009)  H. lyrata extracts stimulated the growth of roots and hypocotyls of Sorghum vulgare and Cucumis sativus at lower concentrations, while it inhibited their growth at higher concentrations.These results are also in accordance with the findings of Ahmad et al. (2007) who reported that aqueous leaf extracts of Lantana camara caused strong inhibitory effect on germination of test crops, whereas the lower concentration showed stimulatory effect in some cases.The significant reduction in shoot and root length and dry weight of chickpea seedlings by different plant part extracts of E. dracunculoides at two concentrations could be due to the presence of water soluble inhibitors ( Kil and Yun, 1992).All the traits of chickpea seedling growth were more adversely affected by the application of E. dracunculoides leaf extract at 1:10 concentration.This indicated the availability of inhibitory chemicals in higher concentration in leaf parts than others (Shukla et al., 2003).Dongre et al. (2004) reported that greatest inhibition of seedling growth in black gram (Phaseolus mungo) cultivars (P-19 and PU-35) was caused by leaf extracts of Parthenium hysterophorus at different concentrations.Varying rate of inhibition of chickpea seedling growth with aqueous extracts of E. dracunculoides parts at both concentrations (1:10 and 1:20) indicate that inhibitory effects of E. dracunculoides differed with plant parts and extract concentration.These results are in line with those of Aziz et al. (2008) who observed that fruit extract of Gallium aparine was more inhibitory than root, stem and leaf extract on germination and seedling growth of wheat.
The highest reduction in SVI with leaf extracts at both concentrations is supported by Channappagoudar et al. (2005) who reported that leaf extracts of Commelina benghalensis and Cyperus rotundus had greater inhibitory effect on SVI of sorghum, wheat, green gram, soyabean, sunflower and groundnut.The chloro-phyll contents of chickpea seedlings were significantly reduced with different extracts.However, the detrimental effects were more conspicuous at 1:10 compared to 1:20 concentration showing that the responses were concentration dependent.It could be due to the presence of inhibitory chemicals in leaf parts of E. dracunculoides.These results are in line with the findings of Boby et al. (2002) who reported that aqueous extracts of Ageratum conyzoides, Benincasa hispida and Eleusine indica altered the chlorophyll contents of two upland rice cultivars, Lachit and Fapori ahu.Likewise, Batish et al. (2007a) observed a marked reduction in total chlorophyll contents of chickpea and pea plants with Chenopodium murale residues.The maximum reduction in leaf area of chickpea seedlings with leaf extracts could be due to the presence of water soluble inhibitors.Boby et al. (2002) reported that aqueous extracts of A. conyzoides, B. hispida and E. indica significantly reduced the leaf area of two upland rice cultivars, Lachit and Fapori ahu.
Decline in emergence percentage, chlorophyll contents and SVI of chickpea in E. dracunculoides infested soil could be due to bioactive molecules released from plant materials which may interact with the soil then taken up by chickpea plants.Similar results were observed by Batish et al. (2007a) on growth of chickpea seeds in soil infested with Chenopodium murale.Increase in NPK percentage could be due to the enrichment of soil infested with weed residues (Batish et al., 2007b).The higher concentration of NPK in chickpea seedlings could also be attributed to increased uptake of nutrients from the infested soil (Michelsen et al., 1995).Boby et al. (2002) reported that P content in Lachit and K content in Fapori ahu were increased by soil amendment with B. hispida.
Based on the present investigations, it could be concluded that aqueous extract of E. dracunculoides had  detrimental effect on seed germination and seedling growth of chickpea that increased with increasing concentration.Euphorbia dracunculoides contains compounds in its tissues which may cause phytotoxic effects under field conditions without depletion of available soil nutreints.Therefore, E. dracunculoides should be removed from fields at early stages to save chickpea from harmful effects of this weed.

Table 1 .
Phytotoxic effects of Euphorbia dracunculoides on chickpea 583 Phytotoxic effect of aqueous extracts of E. dracunculoides on germination of chickpea.Means sharing the same letter in a column did not differ significantly at 5% probability level.Figures in parenthesis show % increase (+) or decrease (-) over control

Table 2 .
observed that Phytotoxic effects of Euphorbia dracunculoides on chickpea 585 Phytotoxic effects of aqueous extracts of E. dracunculoides on seedling growth of chickpea.Means sharing the same letter in a column did not differ significantly at 5% probability level.Figures in parenthesis show % increase (+) or decrease (-) over control

Table 3 .
Phytotoxic effects of E. dracunculoides infested soil on seedling growth, chlorophyll, N, P, and K content of chickpea.Means sharing the same letter in a column did not differ significantly at 5% probability level.Figures in parenthesis show % increase (+) or decrease (-) over control soil