Green leaves and seeds alcoholic extract controls Sporobulus indicus germination in laboratory conditions

High seed production makes Sporobolus indicus var. pyramidalis a difficult to control invasive grassland plant. The objective of the present study was to investigate the bioactivity of Cyperus rotundus, Phyllanthus tenellus and Ricinus communis green leaf extracts and of Carica papaya seeds on S. indicus germination without breaking dormancy, simulating the field conditions. The ethanolic extract bioactivity of C. rotundus, P. tenellus, R. communis green leaves and C. papaya seeds, at concentrations of 25, 50 and 75% in S. indicus germination was evaluated. Carotenoids, flavonoids, soluble phenolic compounds and total tannins were quantified in the extracts. The chemical component concentrations varied between alcoholic extracts. The P. tenellus extracts at all dilutions and those of R. communis and C. papaya at 75% completely suppressed S. indicus seed germination at five and ten days which can be attributed to their high tannin concentration, total phenolic compounds and flavonoids.

(Phyllanthaceae), Ricinus communis L. (Euphorbiaceae) and Carica papaya L. (Caricaceae) seeds have toxicological properties. Gallic acid, chlorogenic acid, 3,4-dihydroxybenzaldehyde, p-hydroxybenzoic acid, catechol, tannic acid, ricinine are some of the allelochemical phenolic compounds found in these species 15 . But the allelopathic potential of these plants on seeds weeds needs to be better studied 10 . Aqueous extracts have been studied [16][17][18] , but many non-polar bioactive substances cannot be dissolved by water at room temperature, unlike organic solvents 13 . Polar solvents such as methanol, ethanol, acetone, or acetonitrile give much high extraction efficiencies 14 .
Phenolic compounds, originated to protect plants from oxidative damage, are also involved in plant allelopathy inducing changes in membrane permeability, inhibition of nutrient uptake, cell division, stretching and submicroscopic structure, altering enzyme activity, respiration, and synthesis of hormones and proteins 14 . Studies on the performance of phenolic compounds such as allelopaths can provide data to development sustainable methods of agriculture, forestry, natural resources and conservation of the environment.
The objective of the present study was to evaluate the bioactivity of alcoholic extracts of C. rotundus, P. tenellus, R. communis green leaves and C. papaya seeds with on the S. indicus var. pyramidalis germination without breaking dormancy, simulating the field conditions.

Discussion
The variation in the concentration of chemical components between the alcoholic extracts confirms their wide occurrence and diversity in plants 19,20 21 and thirty-two other herbs 22 . This makes plants from different habitats in Sardinia, Italy 23 of the same species growing in different conditions 24 have chemical composition variation as reported for Myrtus communis L. (Myrtaceae). The variation in the concentration of the chemical components between the alcoholic extracts is due to their proportions in the solvent/solute (dilution) which determines the effectiveness of the plant extracts and the isolated compounds 25 . In addition to dilution, the solvent may also alter the chemical composition of the extracts, as reported for Origanum vulgare L. (Lamiaceae) 26 , Anthocleista grandiflora Gilg. (Gentianaceae) and Combretum erythrophyllum Burch. (Combretaceae) in wich compound quantity and diversity varied according to the extractors and their concentration 25 .
Flavonoids, a group of phenolic compounds resulting from secondary metabolism, are widely found in plants 27 and their higher amount in the 75% C. rotundus extract agrees with that reported for the rhizome extract of this plant [28][29][30][31] . However, abiotic and biotic stress 26 and changes in seasonal dynamics can affect compound content and when it is higher in the plant it will also be in the extract, as reported for Dryopteris erythrosora (DC Eaton) Kuntze (Dryopteridaceae) with the transport of flavonoids from the leaves to the stem in the growing season, comprising summer (26.9 °C) and early autumn (16.9 °C) in Shanghai, China 32 . The highest total tannin levels (another phenolic compound group) from the P. tenellus extract could be a response to the stressful environment in which this invasive species was collected 33 , area with stones, few soil and water deficit, in the microregion of Campina Grande, Paraíba, Brazil, with few soil and water deficits. The tannin accumulation, in this case, has an antioxidative function 34 and agrees with the phytochemical profile of the methanolic solution www.nature.com/scientificreports www.nature.com/scientificreports/ (80%) of the whole P. tenellus plant 35 . The highest phenolic compound content and total carotenoids in the C. papaya L. seed extract is due to its function in sanity and resistance to pests and diseases, as a strategy for seed survival 36 , mainly against oxidative stress 37 . These compunds act in response to environmental stress conditions protecting against injuries, as reported in the identification of the phenolic profile of papaya fruits 36,38 . Secondary products of metabolism such as flavonoids, tannins, phenolic compounds and carotenoids 39 , may act in inhibiting germination 40 reducing tissue growth or causing death by increasing cell membrane permeability, as reported for Cucumis sativus L. (Cucurbitaceae) 41 , Lactuca sativa L. (Asteraceae) 14 , Phaseolus vulgaris L. (Fabaceae) 42 resulting in the inhibition of radicular elongation and ultra structural changes and cell division.
The suppression of S. indicus germination by C. papaya, P. tenellus and R. communis extracts may be due to their high tannin concentration and total phenolic compounds (derived from the acetate and shikimic acid route or their combination) 39 . These compounds bind strongly to proteins by hydrogen bonds and hydrophobic interaction, deactivating them and blocking germination metabolism 43,44 or preventing the access of free oxygen to the embryo and the release of carbon dioxide 45 . This was reported for Sorghum bicolor L. Moench. (Poaceae) which tannin content was correlated with its germination. The highest flavonoid concentration in C. rotundus, R. communis and C. papaya extracts at 75%, also explains the allelopathic effect on S. indicus germination. In addition, the flavonoids are compounds with high antioxidant power 46 suppressing germination by inhibiting the indole-acidase oxidase (IAA oxidase), gibberellic acid (GA 3 ) and indo-3-acetic acid (IAA) 47 . The allelopathic effect of Dittrichia viscosa (L.) W. Greuter extracts was attributed to flavonoids 48 and, even at low concentrations (0.1-1.0%), those of Ocimum gratissimum L. (Lamiaceae) inhibited the germination and growth of corn and beans 49 . The suppressive germination effect by the C. papaya extract at 75% may also be due to caricacin 50 , which suppresses cell division and phytohormone production and increases the permeability of membranes, inhibiting germination 43,51 . The absence of toxicity of the R. communis extract at 25 and 50% can be explained by their adsorption by allelopathic active compounds such as sugars and other S. indicus seed carbohydrates, whereas this was not sufficient at 75% concentration due to the high concentration. High sugar concentrations as well as of other carbohydrates, such as glucose and fructose, maltose, sucrose, raffinose, myo-inositol and galactinol have been reported for Poa annua L. 52 53 from the same S. indicus family. Ricin, a highly toxic R. communis heterodimeric protein is composed of polypeptide chains with an affinity for cell surface carbohydrates 54-57 becomes inert when adsorbed by them thereby not influencing germination 18,58,59 . Lectin, a N-acetylgalactosamine present in seeds, including those of the Poaceae family 60 is another protein class with reversible carbohydrate binding capacity that can adsorb ricin and other allelopathic compounds, deactivating the R. communis extract atby inhibiting the germination of the invasive plant S. indicus var. pyramidalis.
conclusion Phyllanthus tenellus alcohol extracts at all R. communis concentrations and C. papaya, at 75%, suppressed the germination of S. indicus var. pyramidalis. These extracts have the potential to manage this plant in organic and agroecological production systems.

Material and Methods
Raw material, preparation and characterization of extracts. Extracts were obtained from C. rotundus, P. tenellus and R. communis green leaves and C. papaya seeds by immersion in 70% ethyl alcohol for seven days 14 . The alcohol was extracted at 250 °C and the extract filtered and diluted in distilled water to obtain the concentrations of 25, 50 and 75% and their effects were compared with distilled water (control). The chemical composition of extracts at all concentrations was characterized. total tannins. The samples of alcoholic extracts were allowed to stand for 1 h in 40 mL of 50% methyl alcohol, centrifuged at 15,000 rpm for 15 min and the supernatant transferred to a 100 mL volumetric flask. A 70% acetone solution was added to the precipitate, which was kept standing for a further 1 h. The mixture was again centrifuged at 15,000 rpm for 15 min and the supernatant discarded. The precipitate was placed in a thermostatic bath at 100 °C for 3 h, cooled in an ice bath, filtered into a 50 ml volumetric flask and the volume filled with the extractive solution. The readings were made in 6 mL aliquots of butanol: HCl and 0.2 mL of 2 N:FeNH 4 (SO 4 ).12H 2 O per test tube. After stirring, these tubes were placed in a thermostatic bath at 100 °C for 50 min and cooled in an ice bath. The reading was performed in a UV-Vis spectrophotometer at 550 nm and the results expressed in mg of catechin g −1 .

Soluble phenolic compounds.
panicles collection. Panicles without evidence of herbivory and fungi and with mature seeds were collected from plants distributed in ten (10) farms with pastures infested by S. indicus var. pyramidalis in the state of Paraíba, northeastern Brazil. Mature seeds were randomly selected and naturally dried. The viability test was performed in duplicate in batches of 100 seeds of each property. The viability test was performed for 30 days 62 . However, seeds that did not germinate within 10 days were rotting 4 .
Bioassay. S. indicus germination was evaluated in triplicate with 100 seeds every 10 days in a germination box (Gerbox ® ) (11 × 11 × 3.5 cm) with two germination paper (Germitest ® ) moistened with 18 ml of the different extracts and distilled water in the control. Seed dormancy were not broken to simulate field conditions. The germination assays were done in a germination chamber at 20 °C with 14 h light per daily. Germination was evaluated daily by 10 days 4 . Seeds with radicle protrusion were considered germinated. The percentage of germination was obtained with the formula: % G = (N/A)*100, where: N = total number of seeds germinated; A = total number of seeds placed to germinate 63-67 . experimental design and statistical analysis. The experimental design was completely randomized with three replicates of 100 seeds. The germination rates was compared across independent samples by using non-parametric Kruskal Wallis H test. Further, Mann Whitney U test was used to compare the two germination rates.