Study of Seed Germination and Enzyme Metabolism During Allelopathic Interaction of Trianthema portulacastrum L . and fenugreek seeds

Absract In the fields, it is commonly observed that fenugreek plants are not growing to their maturity in the vicinity of Trianthema portlacastrum L. In this context, present study was conducted in laboratory to observe allelopathic interaction between Trianthema and fenugreek. Aqueous extracts of seed, stem and leaf of Trianthema were made. Treatment of these aqueous extracts was given to fenugreek seeds. Germination study was performed by using petriplates. All the experiments were performed in triplicates. Pre-treatment of aqueous extracts of seed, stem and leaf of Trianthema. had caused decline in germination percentage, fresh weight, radical and coleoptile length of fenugreek seeds when compared to untreated seeds. At the same time, mean germination time was found to be more in pre-treated seeds compared to control seeds. After 24 hrs of treatment, enzymatic study was performed. Enzymes from antioxidant metabolism such as peroxidase, ascorbate peroxidase, superoxide dismutase and activity of DPPH was reduced in treated seeds than control seeds. Activity of catalase was increased after treatment of leachates. All the data is graphically presented with the standard deviation. Exogenous treatment of aqueous extracts of Trianthema has caused variation in saponin content of fenugreek seeds. This observation was recorded by using finger prints of HPTLC. Finger prints showed that content of saponin was also declined in treated seeds as compared to control. All these observations indicate the strong negative influence of Trianthema on growth and metabolism of fenugreek. Current Agriculture Research Journal


Introduction
Trianthema portulacastrum L. is a herbaceous plant, growing annually.This plant commonly found in moist area, which spreads on ground not more than 4-6 ft. 6In India, it is commonly grown as a wild vegetable.This edible plant is vital source of fibres, proteins, riboflavin, potassium, sodium and iron. 13t is also considered as a weed which has many active compounds such as alkaloid, flavonoids, tannins etc.These compounds are also called as allelochemicals, which may stimulate or supress the growth of neighbouring plants. 26Study of these allelochemicals and its effects on interaction of plants is called as allelopathy.This is now upcoming branch of biology which involves study of allelochemicals and its influence on plant distribution, building of plant biodiversity and its conservation, evolution of mix crop system in nature and community formation. 33By exuding these allelochemicals and sharing same ecological conditions such as moisture, nutrients, light, space and air these weeds pose the growth and development of neighbouring plants. 34Depend upon the type of weed species, its causative effect on seed germination and seedling growth is varied. 8Plant parts of weed are also equally potential to exert their impression on other plants. 4,1rianthema is a leafy vegetable with the active wild components.Many workers observed noteworthy observations during allelopathic interaction of this plant.Yield of many economically important plants like cotton, maize, wheat, sorghum, sunflower and mungbean is suppressed due to presence of Trianthema. 2,25method of intercropping is followed during cultivation of fenugreek and Trianthema.A huge area in Vasai (Maharashtra, India) is under this mix cultivation.But in field, this notorious plant had caused noticeable damage to the fenugreek yield.Considering this on field observations, present study is conducted.During this study, allelopathic effect of Trianthema. on fenugreek seed germination, seedling growth and on some enzymes of antioxidant metabolism is evaluated.

Material and Methods
Fresh and matured plants of Trianthema.were collected from local field of Vasai.Plant parts (seeds, stems and leaves) were separated and sundried for 15 days.Leachates of seed, stem and leaf of Trianthema.were prepared by following ratio 1/15 (w/v).These leachates were prepared in distilled water and kept for 24 hr.Afterwards, these extracts were filtered through Whatman filter paper No.1.Authorised fenugreek seeds were procured from the market.According to ratio 1/15 (w/v) fenugreek seeds were soaked for 24 hr in seed, stem and leaf leachates of Trianthema.
For germination studies, pretreated seeds of fenugreek were kept in three sets of petriplates for 96 hr.Ten millilitre distilled water was poured in each petriplate.Germination count was taken for each day and radical, coleoptile length was measured at 96 hr.
Mean germination time and germination index (GI) were calculated by following the method of 5

Mean germination time = Σ Dn \ Σn
Where n is the number of seeds, which were germinated on day D, while D is the number of days, counted from the beginning of germination.

Graph no 1: Effect of seed, stem and leaf leachates of Trianthema on germination percentage of fenugreek
Method 9 was employed for studying enzyme peroxidase.Activity of catalase was determined by following method of 17 as described by. 27Method of 29 was adopted for determining activity of ascorbate peroxidase.Enzyme super oxide dismutase was determined by using method. 11Soluble proteins were estimated by the method. 16Activity of DPPH was estimated using the method described by. 28Content of saponin was studied using HPTLC finger printing with slight modification in method. 3

Result and Discussion
Changes in plants at morphological, biochemical, physiological and molecular level occurred due to environmental conditions.Allelopathy influences other neighbouring plants to change their growth pattern and metabolism.Such changes are also reported in present study.Germination percentage (Graph 1), fresh weight (Graph 2) and mean germination time of fenugreek seeds (Graph 3) is affected after treatment with leachates of Trianthema.More significant reduction in above parameters was noticed in case of leaf leachate treated fenugreek seeds.Seeds of rice were showing similar germination and fresh weight pattern when pre-treated with root, shoot, leaf, seed and whole plant extract of plant Trianthema. 14Many secondary metabolites such as phenols, flavonoids are present in this plant which are responsible for germination retardation.Activity of protease is vital for seed to hydrolyse the proteins, which crucial for imbibition and for water uptake.Soluble phenolic compounds are largely present in leaf leachate of Trianthema, which may be responsible for disturbances in seed protease activity.This ultimately leads in more decrease in seed germination of fenugreek seeds pre-treated with leaf leachate compared to seed and stem leachate.
Leachates of Trianthema had caused reduction in coleoptile and radical length of fenugreek seeds (Graph 4 and 5). 10  Stressful environmental situations are responsible for limiting the agricultural productivity, as plants are unable to achieve their maximum genetic strength. 18,7ensitive cell organelles of plants react quickly to such environmental changes. 30Stressful conditions caused increase in ROS in plants.These enhanced ROS are scavenged through many enzymatic and non-enzymatic antioxidants.In present study, activity of antioxidant enzyme, peroxidase is decreased in pre -treated fenugreek seeds compared to control (Graph 6).Similar reports are mentioned 24 in Phalaris after treatment with extract of Eucalyptus leaf.Allelopathic effect of Trianthema had caused enhancement in catalase activity of fenugreek seeds (Graph 7).Such kind of enhancement in activity was noticed 19  In case of fenugreek seeds, as other enzymes are unable to show their antioxidant scavenging activity, accumulation of H 2 O 2 may be caused in the cell.This harmful compound is efficiently catalysed to H 2 O in peroxisome by increasing the catalase activity in fenugreek.Seed, stem and leaf extract of Trianthema had caused decline in enzymatic activity of ascorbate peroxidase in fenugreek seeds (Graph 8).Leaf extract of Trianthema had major effect than seed and stem extract.Activity of ascorbate peroxidase in germinating wheat seeds was also hampered by treatment with dried shoot extract of Achillea santolina L. 20 In this relation, 15 noticed that a decline in activity of enzyme catalase and enhancement in APX activity in leaves of cucumber seedlings when prone to stress.Similarly, to compensate an increased activity of catalase in fenugreek seeds after allelopathic stress, activity of  Cu and Cd had caused stressful effect on medicinal plant Gynura procumbens (Lour.)Merr.Saponin, Phenolic and flavonoid contents of this medicinal plant were reduced after treating with these metals. 22ccording to, 23 phenolic compounds such as caffeic acid, ferulic acid, M-coumeric acid, P-coumeric acid, syringic acid, venillic acid; and caffeic acid, gallic acid, 4-Hydroxy-3-Methoxybenzoic acid, P-coumeric acid, syringic acid present in Trianthema which are responsible for negative allelopathic effect on plants.

Conclusion
Present study showed that germination ability and seedling growth was badly affected in fenugreek due to treatment of Trianthema.Antioxidant enzymes such as peroxidase, ascorbate peroxidase, superoxide dismutase and total antioxidant such as DPPH was also reduced after treatment of Trianthema.Leaf lechates of Trianthema were found to be more hazardous than other plant parts.Most vital content of fenugreek i.e. saponin was also affected due to treatment of Trianthema.Similar kind of observations were noted in fields of Vasai (Dist.Palghar).
reported decline in germination Graph no.2: Effect of seed, stem and leaf leachates of Trianthema on fresh weight of fenugreek seeds Graph no 3: Effect of seed, stem, leaf leachates of Trianthema on mean germination time of fenugreek percentage, plumule and radical length of rice and cowpea, when treated with increasing concentration of Acacia auriculiformis leaf leachates.Allelopathic compounds present in water soluble form are reported to reduce the activity of GA and IAA. 32This inhibition in hormonal activity results in decline in cell division and reduction in growth of a plant.Graph no.4: Effect of seed, stem and leaf leachates of Trianthema on radicle growth of fenugreek seeds Graph no.5: Effect of seed, stem and leaf leachates of Trianthema on coleoptiles growth of fenugreek seeds Graph no.6: Effect of seed, stem and leaf leachates of Trianthema on enzyme peroxidase of fenugreek in Black gram seeds after treatment with Graph no.7: Effect of seed, stem and leaf leachates of Trianthem on enzyme catalase of fenugreek seeds Graph no.8: Effect of seed, stem and leaf leachates of Trianthema on enzyme ascorbate peroxidase of fenugreek seeds Graph no.9: Effect of seed, stem and leaf leachates of Trianthema on enzyme superoxide dismutase of fenugreek seeds lower concentration of leaf extract of Tectona grandis.

Graph no. 10 :
Effect of seed, stem and leaf leachates of Trianthema on DPPH activity of fenugreek seeds Plate no.1: Allelopahic influence of leaves, stem and seeds of Trianthema on saponin content of fenugreek seeds