Similarly, Ageratum conyzoides and Melia azedarach as medicinal plants promoted the growth and yield of rice and greatly reduced the growth of weed species . Batish et al.  also observed significant adverse effects of Tagetes minuta on the emergence and growth of the rice field weeds. Application of Anisomeles indica as an allelopathic plant for controlling weed species in wheat field inhibited the emergence and growth of weeds as same as chemical herbicide . Tinospora tuberculata , Malay name Batawali, belongs to the family Menispermaceae, and is a traditional medicinal plant . It is a plant that grows in primary rainforests in all parts of Malaysia, Thailand and Indonesia. There are a few studies that dealt with allelopathic activity of T. tuberculata . They observed the inhibitory activity of T. tuberculata against the germination and initial growth of crops and weed species. The current study was performed to  determine the allelopathic potential of T. tuberculata leaf methanol extract on the growth and development of the rice  and two common rice weeds, barnyard grass  and weedy rice , at three-leaf stage under hydroponic conditions; and  evaluate the role of phytotoxic compounds on the allelopathic activity of extract obtained from leaves of T. tuberculata.T. tuberculata plants growing in the Herbal Garden of University Putra Malaysia, Selangor, Malaysia, was used. T. tuberculata leaves were cleaned several times with tap water and air-dried for 3 weeks, then ground to a fine powder in a laboratory blender and stored in a refrigerator at 4°C until use.

Commercial seeds of barnyardgrass  and weedy rice  were purchased from Herbiseed company.Barnyardgrass seed coats were loosed by hand, one by one to improve the germination percentage. The seeds of rice  were manually collected in the rice field, Universiti Putra Malaysia. Rice and weedy rice seeds were surface sterilized in a 1:10  dilution of commercial hypochlorite bleach for 10 min and rinsed several times with distilled water. The rice seeds were cleaned, air-dried and all seeds stored in darkened,mobile vertical grow tables airtight containers at –18°C prior to their use. The germination of the seeds was randomly checked and was ~86–95%. Syringic acid , trans-ferulic Acid , trans-cinnamic acid , p-anisic acid , chlorogenic acid , vanillic acid , coumarin , gallic acid , caffeic acid  and benzoic acid  were supplied from Chemtron Biotechnology Sdn. Bhd . Vitexin, isovitexin , orientin  and isoorientin  were obtained from Sigma-Aldrich . Methanol and acetic acid of HPLC grade were obtained from Friendemann . Water was twice distilled and all chemicals used were HPLC grade.The experiment was conducted to determine the allelopathicpotential of the leaf methanol extract on the biomass and transpiration  of rice and rice weed seedlings  at the three-leave stage . Leaf methanol extract of T. tuberculata was prepared according to the methods described by Aslani et al. . Briefly, leaves of T. tuberculata were soaked in 1 000 mL of 80%  aqueous methanol, and shaken at room temperature  for 48 h. The solution was filtered through four layers of cheese cloth and then centrifuged at 3 000 r min–1 for 1 h. The solution was filtered again through a 0.2-µ, 15-mm syringe filters . The collected filtrate was evaporated to dryness under vacuum at 40°C using a rotary evaporator. Stock extract derived from leaves of T. tuberculata were diluted appropriately with 25% Hoagland’s solution  to obtain three different concentrations of hydroponic culture .

Seeds were added to moist germination paper and 7-dayold seedlings were subsequently added to 8 mL amber vials containing 7.5 mL of 25% Hoagland solution. The seedlings were placed in the glasshouse for 14 days. Five seedlings of uniform size per pot of each plant species were chosen and inserted carefully into holes in a styrofoam float. The seedlings were grown individually 2 cm apart and transplanted into a plastic pot  with 100 mL of 25% Hoagland’s solution amended with methanol leaf extract at 3.12, 6.25 and 12.5 g L–1. Hoagland’s solution without leaf extract was used as the control. The solution was replenished as needed with the appropriate solution to avoid exposure concentration changes. After 14 days, the plants were harvested; the root lengths and plant heights, biomass, transpiration volume and chlorophyll and carotenoid content were measured. The fresh leaves  of tested plants were taken, mixed and subsequently pulverized with liquid nitrogen to obtain a freeze dried mixture of old and young leaves. The freeze dried plant material of each sample  was extracted with aqueous 80% acetone and kept in the freezer at −10°C for 24 h. The suspension was filtered through a Whatman filter paper No. 1. Chlorophyll and carotenoid contents were determined spectrophotometrically using spectronic Genesys 20 spectrophotometer  at 3 wavelengths: 663 nm for chlorophyll a, 647 nm for chlorophyll b and 470 nm for carotenoids. Calculations were completed using Lichtenthaler’s equation  and expressed as mg g–1 fresh weight . The results of Experiment 1 showed that the amounts of tested index of rice, weedy rice and barnyardgrass seedlings grown under hydroponic culture were significantly affected when the leaf methanol extract of T. tuberculata in different concentrations  were added to the Hoagland’s solution. However, inhibition was more severe when these species were treated with higher concentrations.This suggested that allelopathic substances decreased stomatal conductance, leaf transpiration and net photosynthesis .

The concentrations required for 50% growth inhibition  of the root , shoot  and biomass  of target plants were calculated . It shows the leaf methanol extract had various degrees effects depending on target plant species and each tested index . The differences were evident from the rank values of each plant species and each tested index. Table 1 indicates that barnyardgrass was more sensitive followed by weedy rice and rice. Indeed, for half inhibition of biomass, root and shoot elongation of rice, the concentration of methanol extracts must be doubled to have the same effect registered on barnyardgrass. The present observations revealed that the photosynthetic pigments exhibited a significant decline in rice, barnyardgrass and weedy rice seedlings. Reduction of chlorophyll and carotenoid content in the presence of allelopathic plant extracts has been reported by Sodaeizadeh et al.  and Lokajová et al. . Zhou and Yu  opined that the inhibition of chlorophyll accumulation by allelochemicals might be due to the blockage of the biosynthetic pathway of chlorophyll or stimulation of chlorophyll degradation or both processes. A greater reduction was observed in chlorophyll a content compared to chlorophyll b in all target plants. These results are in accordance with the findings of Elisante et al.  and Lokajová et al. . Yang et al.  opined that probably two different enzymes located in the chloroplast of higher plants. Siddique and Ismail  demonstrated that chlorophyll content is closely related to plant dry matter production, reduction in leaf chlorophyll content would cause decreased photosynthesis and hence total plant growth.

Therefore, the present observations revealed that methanol extract may diminish the accumulation of dry matter in tested species through the decrease of chlorophyll levels and photosynthesis rate . Identification of the inhibitor components is important to determine the phytotoxic interference mechanism on receiver plants. The UFLC analyses showed T. tuberculata leaves are capable of producing numbers of compounds into the environment which may affect the growth of other plants in the vicinity. Verdeguer et al.  suggested that the suppressive effect of the extracts is dependent on the chemical composition. The chemical characterization of different fractions of Calamintha nepeta showed the magnitude of allelopathic activity depends on the presence of five phenolics  in varied amounts . Patterson  reported vanillic, ferulic, gallic and p-coumaric acids inhibited chlorophyll content, stomatal conductance, net photosynthetic rate and assimilation rate of soybean. The pure compound bioassay revealed that the components and their combination were biologically active by inhibiting germination and seedling growth of barnyardgrass. It shows that they are probably responsible for the allelopathic activity of T. tuberculata. Benzoic acid was found in the leaf methanol extract at the highest concentration , while the amount of chlorogenic acid was 427.8 mg kg–1 DW. On the other hand, benzoic acid and chlorogenic acid showed the highest and weakest allelopathic activities compared to the others . Therefore, the magnitude of inhibitory activity of the methanol leaf extract can be explained by the level of benzoic acid as most effective compound and orientin as one of the weakest allelopathic compounds. Therefore, based on the concentration and growth inhibitory effect of each tested compound, it seems that benzoic acid, among the other tested compounds, is the most effective in T. tuberculata allelopathic activity. Kato-Noguchi et al.  believed that benzoic acid could be a main contributor to allelopathic activity of Ginkgo biloba leaf extract. The mixture of all tested compounds was moderate inhibitory when compared with the allelopathic activity of individual compounds .

It may indicate that interactions of concentration and combination of compounds determine the allelopathic activity of a mixture. Biological activity of collective compounds depends on several factors such as: individual chemical structure, antagonistic, agonistic,mobile vertical farm additive, sequential, or synergistic effects . Both antagonism and synergism were observed when three flavonoids isolated from Derris urucu combined in pairs, applied on the seed germination and development of root and hypocotyl of Mimosa pudica . In contrast, Al Harun et al.  observed litter-mediated soil leachate of Chrysanthemoides monilifera had stronger phytotoxicity effects against Isotoma axillaris relative to individual phenolic compounds due to synergistic contributions of other unidentified compounds. Therefore, it might be the allelopathy activity of T. tuberculata leaves depending on the concentration, combination and inhibitory activity of its bioactive compounds. Phenolic and flavonoid compounds have been found in a wide range of plants and often exert allelopathic responses . Sludge produced during the municipal wastewater treatment process contains numerous nutrients such as nitrogen, phosphorus and small amounts of potassium , organic matter  and other trace elements. Therefore, this sludge could be used as a nutritional amendment to improve soil physical characteristics, fertility, microbial diversity and enzyme activity. It has been shown that the agricultural utilization of sludge is one of the most economical and effective options for sludge treatment . However, sludge is also rich in organic matter, pathogenic bacteria, parasitic eggs, heavy metals and other toxic and harmful substances that was difficult to degrade biologically . Such sludge could not be used in raw or dried form for agricultural application and composting, leading to the secondary environmental pollution caused by improper disposal of sludge . Ozone is a functional disinfectant, oxidizer, and reducer when used to treat sludge . Furthermore, the sludge mineralization process during ozone treatments could decompose organic matter into inorganic materials . Significant research efforts have been devoted to the application of ozone in sludge treatment , which promote cell cracking and reduce the number of excess sludge, thus reducing the waste disposal of activated sludge  and decreasing the chemical oxygen demand , chromaticity, and turbidity in conventional activated sludge process with ozone treating partial backflow sludge.

Ozone has also been shown to be a superior disinfectant compared to conventional disinfectants such as chlorine and its derivatives . When microorganisms were exposed to ozone, their cell walls were destroyed and internal organic matter was oxidized. The ozone could directly disrupt viral and bacterial metabolism and reproduction by breaking down macromolecule polymers. Most studies have focused on ozone treatments on sewage sludge derived from the municipal wastewater treatment plants where wastewater was collected from industrial and domestic sources . Very little research has been focused on the agricultural utilization of solely domestic sludge, which avoids the environmental pollution introduced by industrial sludge. The sludge was collected from the digester of the Jizhuangzi Wastewater Treatment Plant in Tianjin, China. The water content of the sludge was 99.5%. The sewage treatment plant only deals with domestic sewage, and the sludge is not doped with industrial sources. After testing, the selected experimental sludge met the specific requirements of the control standard of pollutants in agricultural utilization of sludge . The sludge contains numerous nutrients, meeting the plant growth to a certain extent. Through ozone treatment, the sludge organic pollutants and micro pollutants which were detrimental to both plants and human health were degraded . The sludge represented a viable alternative nutrient and water source for plants. Therefore, hydroponics was used in this experiment, which was a widely used technique for growing plants without soil, and could eliminate all the problems associated with soil culture.