Allelopathic effect of leaf extract of Poplar deltoidus M. in seed germination, root characters and physiology of rice seedlings

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1 2017 RELS ISSN: Res. Environ. Life Sci. 10(8) (2017) Allelopathic effect of leaf extract of Poplar deltoidus M. in seed germination, root characters and physiology of rice seedlings Kamla Dhyani*, Namrata Singh, Alok Shukla and Shweta Sahni Shri Guru Ram Rai Institute of Technology and Sciences, Patel Nagar Dehradun, Uttrakhand Dept. of Plant Physiology (Govind Ballabh Pant University of Agricultural Sciences and Technology) Pant Nagar, Uttrakhand, India * (Received: October 28, 2016; Revised received: April 19, 2017; Accepted: April 24, 2017) Abstract: In the present investigation an attempt to be made to analyse the potential allelopathic effect of Poplar deltoidus leaf extract (green and senescenced leaves) at various concentrations viz. 0, 1, 2, 5 and 10 %, leaf extract of Poplar deltoidus effected positively and negatively to the seed germination, radical growth, shoot length, chlorophyll and proline content in Oryza sativa L. The results revealed that the both leaves extracts of Poplar caused a general phytotoxic effect on seed germination and seedling growth of O. Sativa at some concentration like 10% and 5% and some stimulatory effect on some lower concentrations (1% and 2%). Leaf aqueous extract treatment had non significant effect on Germination percentage (85% in 10% extract treatment and 100% germination in 1%, 2% and control). Aquous leaf extract significantly reduced the root length, shoot length and dry weight at higher concentration viz. 10% and 5% treatments, maximum reduction in root length and shoot length was found in 10% extract while increased root length was achieved by in 2% treatments which is 10.33cm. The physiological parameter like chlorophyll content, biomass accumulation and proline is significantly effected by allelochemicals released by Poplar leaves extract. Key words: Allelochemicals, Phytotoxic, Biomass, Extract, Poplar, Chlorophyll Introduction Agroforestry systems provides several benefits to the farmers and poplar (Populus deltoidus) bocoming famous amongst the farmers as a important agroforestry crop, The different trees releases the chemical substances (allelochemicals) in the soil. These allelochemicals inhibit or promote the germination, growth and yield of crop plants and the phenomena is called as allelopathy. Allelochemicals plays a major role in the basic metabolism of the plants and affect numerous physiological and biochemical processes in the plants. Allelopathy in crop production is related to soil sickness, autotoxicity, yield decline, predeposition of plants to diseases, reduced nitrification, nutrient uptake and weed interference with crops (Chaturvedi et al., 2012). However, the effects of these chemicals on other plants are concentration dependent and also influences by other compounds present in the extract. Poplar (Populus deltoids M.) in Boundary plantation is getting popular among the farmers of India. It is a deciduous tree, its cultivation in agroforestry system adds organic matter through leaf litter, it improves physico-chemical properties of the soil however several allelochemicals have been isolated from its leaves thus, adverse effects of its litter and foliage extracts on crops. Tree leaves are the major source of allelochemicals so, The trees are planted in the cultivated fields for fodder, fuel and timber in agroforestry systems. However, farmers losses, perhaps due to wrong selection of tree species with arable crops, which results in negative allelopathic interactions. hense laboratory studies was carried out for the comparative assessment of allelopathic effects of aquous extract of leaves ( Green and senescenced leaves) at different concentaration of the Poplar deltoidus on seed germination behaviour and seedling growth of Rice (Oryza sativa). Materials and Methods Study area: This study was conducted at the Division of Life Sciences of the Shri Guru Ram Rai Institute of Technology and Sciences, Dehradun (Uttrakhand), All studies were done at Dehradun, India [53 24" (N) latitude and 34 27" (E) longitude] from March 2016 to June 2016 in Laboratary condition. Plant materials: The plant materials that were utilized in this study are the green and senescenced leaves of Poplar deltoidus which was collected from a agriculture field of Kanpur district (U.P). The seeds of the test crops (Oryza sativa.l) were collected from GBPU&T (Govind Ballabh Pant University of Agricultural Sciences & Technology,Pantnagar,Uttrakhand). Germination experiment: The green and senescenced leaves of poplar were brought into the laboratory and leaves of the fresh plant were cut into small pieces, shade dried and then ground separately with help of electronic grinder and made fine powder. Research in Environment and Life Sciences 712 August, 2017

2 The leachates from the leaves were prepared by immersing 100 g leaves in 1.0 L double distilled water in a flask for 24 h at room temperature (20±2 C) (Djanaguiraman et al., 2002). The precipitates so formed were recovered through centrifugation (2000 rpm) for 30 min.the extracts were then filtered with double layer of muslin cloth followed by Whatman filter paper No.1. and stored at 5 C. Stock extracts (green leaf and senescenced leaves) were diluted with sterile distilled water to give final concentrations of 10, 5, 2, 1 and control.petri-dishes were thoroughly washed and oven dried. Seed germination and seeling growth: Seeds were then soaked for five minutes separately in 5% sodium hypochlorite to prevent fungal infection. Thereafter they were rinsed for about five minutes in running tap water. Ten of the seeds were placed in each of the clean oven dried Petri-dish which had been lined with a Whatman No. 1 filter 0paper. The filter paper in each of the Petridishes allocated to the control was moistened with ten millilitres of distilled water while that of the Petri-dishes allocated to the other treatments were moistened with ten millilitres of the appropriate concentration of the extracts. The Petri-dishes were incubated at room temperature for two weeks. Emergence of one millimetre of the radicle was used as the criterion for germination. Measurements of germination, plumule and radicle lengths, fresh and dry weights were carried out using standard methods. Treatments were arranged in a completely randomized design with five replications. Germination was determined by counting the number of germinated seeds at 24-h intervals over a 7-days. The rate of germination was calculated by dividing the number of germinating seed each day by the number of days and summing the values (Maguire, 1962). Germination was deemed to occur only after the radicle had protruded beyond the seed coat by at least 1 mm. After7-days. Radicle and plumule lengths of O. sativa seedlings were measured. After measuring the radicle and plumule lengths, the dry weights of seedlings were determined by drying the plant material in an oven at 60 C for 24 h prior to weighing. At the end of the experiments 14-dayes of emergence the plumules tissue were used to determine their contents of chlorophyll. Statistical analysis: The data obtained were analysed by factorial Analysis of Variance (ANOVA) to determine significant (P < 0.05) Estimation of chlorophyll content of seedlings: Chlorophyll content was estimated according to methods given by Hiscox and Israelstam (1979). In a test tube containing 10 ml Dimethyl sulphoxide (DMSO), 50 mg of finely chopped fresh leaves were added. The tube containing leaf pieces and DMSO was then incubated at 65 O for 3hrs in an oven. The content was shaken once or twice during the incubation time. After 3hrs, absorbance was determined at 663 and 645nm using a spectrophotometer against pure DMSO blank. The amount of chlorophyll a, chlorophyll b total chlorophyll (mg -1 fresh weight of leaf) was calculated with the help of following formula: Total chlorophyll (mg/g) = [20.2 (A 645 ) (A 663 )] (V/1000 W) Proline estimation: Determination of free proline was done by Bates et al(1973)-250 mg fresh leaf was homogenized in 5 ml 3% aqueous sulphosalicylic acid and the homogenate was centrifuged at 8000 rpm for 10 min and 2 ml of filterate was used for proline estimation. The proline was calculated and expressed on fresh weight basis. (µg proline ml - 1 x ml toluene x 5) (115.5 x g sample) Results and Discussion Certain phenolic compounds were reported to functions µ moles proline g -1 fr. wt. = as allelochemicals and are toxic to the tested plants. Phenolic acids and volatile oils released from the leaves, bark and roots of certain Eucalyptus species have deleterious effects on other plant species (Sasikumar et al., 2002; Florentine and Fox, 2003). There are a lot of common practical example of allelopathy on the studied crop. In the present investigation allelopathic effect of green as well as senecsenced leaf aquous extract of Populus deltoidus on seed germination and seedling growth of Oryza sativa had been studied to compare the effect of both type of leaves. Different results are found in different parameters like germination percentage, vigour index, chlorophyll content etc. Germination percentage: The data of germination percentage of O. Sativa grains is presented in Table-1. The % germination of rice grains significantly reduced by aqueous extract of poplar leaves which produced 87, 100, 100 and 100% seed germination at 10, 5, 2 and 1%, respectively (Fig. 1). Whereas, the inhibition of Poplar on germination of rice is negligible respectively (Fig. 2). It was observed that the 1% treatments with aqueous leaf extract of poplar green as well as senescenced leaves plants induced germination % in rice seedlings. Similar results were shown by leela et al (2014) while working in seed germination of rice in leaf extract of casuriana leaf and found induction of germination at Table-1: Effect of Green leaf and senescenced leaf aquous extract on Germination%, Mean Germination Time and speed of germination Treatment Parameters of germination Germination % Mean germination time Speed of germination Concentration T1 T2 T1 T2 T1 T2 10% % % % Control ns ns * * ns ns Research in Environment and Life Sciences 713 August, 2017

3 Dhyani et al. Fig. 1: Seedling Growth under different allelopathic effct of Poplar Leaves extract (Green leaves aquous extract in 10%, 5%, 2%, 1 % and Distilled water as control Left to right) Fig. 2: Seedling Growth under different allelopathic effct of Poplar Leaves extract (Senescenced leaves aquous extract in 10%, 5%, 2%, 1 % and Distilled water as control Left to right) Fig. 3: Comparative Allelopathic effect of Green (Left side) and Senescensed (Right side) leaves of extract (10% ) on seed germination of Rice Fig. 4: Comparative Allelopathic effect of Green and Senescensed leaves of extract (5% ) on seed germination of Rice Research in Environment and Life Sciences 714 August, 2017

4 Fig. 5: Comparative Allelopathic effect of Green and Senescensed leaves of extract (2%) on seed germination of Rice Fig. 6: Comparative Allelopathic effect of Green and Senescensed leaves of extract (1% ) on seed germination of Rice lower concentration (<2%). Uniyal et al. (2013) showed similar observations on other plant species while working on allelopathic effect of populus deltoidus (Leaves and bark extract) on seed germination of black gram, he also found stimulatory effect at lower concentration and inhibition at higher concentration treatments. Kaushal et al. (2003) also reported reduction in germination of crops with the aqueous extract of Grewia optiva and Populus deltoids at higher concentration. Stimulatory effect on germination of Oryza sativa by Alnus nepalensis, has also been reported by Kumar et al. (2006). Root length and shoot length: Data in Table (2) showed that the root and shoot length significantly (P<0.05) influenced by various aqueous extract concentrations as compared with the control treatment and the degree of inhibition increased with increasing extract concentration. Result related to root length (Table 2) showed that that root length is significantly effected by the treatments maximum root length was observed in 2% extract treatment which is cm in green leaf extract and 9.3 cm in senescent leaf experiment followed by 1% treatments (8.7cm int1 and 7.1 in T2) and control. Research in Environment and Life Sciences 715 August, 2017 In case of shoot growth maximum hight attained by the seedling in 1% treatments and minimum in 10% extract, inhibition in shoot growth is highest at higher concentration as compare to lower concentration, Nandal et al. (2005) reported that inhibition of shoot growth and germination in wheat varieties bf aquous extract of poplar leaves.vigour index was high in 1% solution similar results was found by, Sharma et al. (2000), Kumar et al. (2006). Uniyal et al. (2013) stimulative effect aquous leaf extract of poplar on wheat, lentil and chickpea has also been reported. It is found to be inhanced in 1% treatment as compare to control. But higher concentration 5% and 10% showed inhibitory effects on root and shoot growth. Nasrine et al. (2011) stated that the germination efficiency, plumule and radicle length of Bromus was completely inhibited at the highest concentration of aqueous extracts of the donor species level (10%) and it is about to zero at 50% extract. Negative impact of Grewia oppositifolia, Ficus roxburgii and Celtis australis of seedling growth of wheat has already been reported by Todaria et al. (2005). We can see that in higher concentration the leaf extract effect negetatively the root growth and shoot growth and in lower concentration it has

5 Table-2: Effect of Green leaf and Senescenced leaf aquous extract on vigour index, root length, shoot length Treatment Parameters on seedling growth Vigor index Root length (cm) Shoot length (cm) Concentration T1 T2 T1 T2 T1 T2 10% % % % Control * * * * * * Table-3: Effect of Green leaf and senescenced leaf aquous extract on, chlorophyll & proline content ((ì gm g -1 fr.wt) and Biomass of Rice seedlings Treatment Physiological parameters Chlorophyll content Proline content Biomass of plant (gm) (mg/gm fr wt) (µgm/gm fr wt) Concentration T1 T2 T1 T2 T1 T2 10% % % % Control S* S* ns ns * * stimulatory effect which is also reported by many other workers above mentioned. Plant dry weight: Plant dry weight is significantly effected by the both the extracts in 10 % and 5 % leaf extract treatments green leaves as well as the senescenced leaf extract but it was found increased in 1 % leaf extract treatment there is stimulatory effect on dry weight it may be due to presence of some minerals in lower concentaration as compare to allellochemicals. In 1 % treatment the growth of seedling was highest as compare to control and lowest in 10 % treatment. Reduction in plant dry weight is also reported by Leela (2014) during seed germination studies in wheat by casuriana leaf extract it was also reported that in lower concentration Plant dry weight was high. Plant dry weight is a important physiological criteria which shows the plant health and dry matter accumulation in plants which always found to be possible in any favourable conditions of the growth. Chlorophyll content: Leaves chlorophyll content is an important physiological parameter which shows the quantity of chlorophyll in leaves, in the above experiment there is inhibitory effect on chlorophyll content in rice seedlings minimum chlorophyll content was recorded in 10 % leaf extract treatment which is (1.22 mg/gm fr wt) in green leaves extract treatment and (.90 mg/gm fr wt) in decomposed leaves treatment. In chlorophyll content there is significant difference in between both the treatment green leaves extract shows inhibitory effect on chlorophyll while senescenced leaves showed less effect on chlorophyll, chlorophyll content was found to be higher in senescenced leaf extract. Which is (3.06 mg/ Gm Fr Wt) in 1 % leaf extract treatment (Senescenced), and (2.17 mg/gm Fr Wt ) in green leaf extract treatment (1%) but it is nonsignificant as compare to control but minimum was observed in 10 % treatment in both the treatments. So we can say that allelochemicals released by poplar green as well as senescenced leaves influence the chlorophyll content of the seedling which is an important pigment for initial photosynthesis and growth. Chlorophyll content and photosynthesis are interrelated. Reduction in chlorophylls may decrease the photosynthesis and There by substantially decrease all the metabolites viz., total sugars, proteins and soluble amino acid. The reduction in chlorophyll contents observed in all the concentrations might be due to degradation of chlorophyll contents or reduction in their synthesis (a and, b) this finding also supported by (Tripathi et al. 2000). This finding is supported by many investigators (Kavitha et al, 2012; Gonzalez et al. 1997), who stated that a decrease in that a decrease in photosynthesis efficiency is a common effect of allelopathic phenolics. The treated plants were yellowish due to chlorophyll as compare to control. Due to the main pigment of a plant cell it retarde the synthesis of other metabolite it may be the cause of slow growth and less biomass of treated plants. Root characters: In the present seed germination experiment, It was observed that allelochemicals mostly effects the root growth and its morphology with the. Root morphology which is highly effected by these treatments a significant difference can be seen (Fig.1, 2, 3 and4) of the roots in different treatments. Root growth with more root hairs (Fig.5) was recorded highest in 1% leaf extract treatment and poor root hair development (Fig.1) was seen in 10 % treatment of leaf extract similar results were found by Majeed et al (2012), that there is a negative impact of allelochemicals on root cell division, absorption rate and physiological functions of root. Root is the main absorbing part of the plant which is extensive in 1 % treatment so other physiological Research in Environment and Life Sciences 716 August, 2017

6 characters like chlorophyll, dry weight and proline content was highest in 1% treatment as compare to control. Poplar deltoids which is an important agroforestry crop has inhibitory effect on germination and seedling growth of tested plants which is Rice (Oryzae. sativa) in this study as mainly due to the presence of allelochemicals in laboratory condition.the inhibitory effect of leaf leachates on seed germination is at higher concentration (>5 and 10 % ) and In lower concentration (< 2%) it stimulate the growth of young seedlings.by the present study it can be stated that inhibition is a concentration dependent phenomena and accumulated allelochemicals can harm the seed germination of Paddy crop. Acknowledgment This study was supported by Shri Guru Ram Rai Education Mission and Shri Guru Ram Rai Institute of Technology and Sciences, Dehradun. We are thankful to Prof Preety Kotiyal, Director SGRRITS, Dehradun for constructive suggestions and comments on the manuscript. We are also thankful for Prof. Alok Shukla, GBPUA&T, Pantanagar for correction on the manuscript. Special thanks to the Editor-in- Chief and two anonymous reviewers for their in sightful comments. We are grateful to Mr Bhadri and Manish Chaudhary for Lab assistance. References Bates,L.S., R.P. Waldron and I.D Teare: Rapid determination of free proline in water stress studies. Plant Soil, 39: (1973) Banga,A; A.P.S. Bana., S. Chaturvedi. R. Kaushal. S. Tiwari: Effects of leaf leachates of different tree species on germination and growth of various agricultural crops. Journal of Tree Science, 34: (2015) Chung, I.M., J.K Ahn, S.J. Yun: Assessment of allelopathic potential of barnyard grass (Echinochloa crus-galli) on rice (Oryza sativa L.) cultivars. Crop Protection, 20: (2001). Chaturvedi S., J. Pandey, V.C. Dhyani, S.K. Guru and R. Kausal: Phytotoxic potential of Eukalyptis leaf essential oil to control Parthenium hysterophorous. L. Allelopathy Journal, 29: (2012). Djanaguiraman, M., R. 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