Evaluation of physiological quality of lettuce and rocket salad seeds in the presence of purple nuts edget extract

Purple nuts edge (Cyperus rotundus L.) is responsible for great losses in crop production areas. Purple nuts edge tubers possess substances with allelopathic effects on certain crops. The aim of this study was to evaluate the physiological quality of lettuce and rocket salad seeds grown on different substrates in the presence of purple nuts edge extract. A completely randomized experimental design was used with a 3×5 factorial scheme (three substrates × five doses of purple nuts edge extract) and four replicates of 50 seeds. The extract was diluted to four different concentrations (25, 50, 75 and 100%), and distilled water was used as a control treatment (0%). Seeds were sown in three different substrates: Germitest paper, sand or soil. The first and second germination count, the total germination and the germination speed index were evaluated. Lettuce germination was affected by the application of purple nuts edge extract to the tested substrates. Lower germination percentages were observed for sand than for the remaining tested substrates. The application of purple nuts edge extract did not affect the evaluated germination parameters for rocket salad seeds. The germination of lettuce and rocket salad seeds was higher with soil as substrate.


INTRODUCTION
Olericulture plays an important role in Brazilian agriculture and the economy.One of the main obstacles to achieving high productivity in vegetable production is the high incidence of weeds, especially those that are difficult to control.Searching for new management alternatives and for plants with allelopathic effects on other crops is important for producers to develop strategies that may increase production.*Corresponding author.E-mail: cleiton.benett@gmail.comAuthor(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Purple nuts edge (Cyperus rotundus L.) is an herbaceous perennial weed that reproduces both through seeds and vegetatively through bulbs, tubers and subterranean rhizomes and is considered one of the most important invasive plants in the world (Francineuma et al., 2005).It is responsible for extensive damage in production areas, especially due to its high photosynthetic efficiency and capacity to directly compete with crops for water, light and nutrients (Catunda et al., 2006).
Purple nuts edge can emerge and develop intensively at the beginning of the crop cycle.Although it is considered a small plant, it can substantially decrease the production of several crops due to the detrimental effects of its extensive subterranean structure and release of allelopathic substances (Novo et al., 2006;Durigan, 1991).Purple nuts edge is present in half of the agricultural soils in Brazil, regardless of the type of soil, climate and crops (Durigan et al., 2005).
Several plants possess allelopathic compounds.These chemical substances can interfere with seed germination and the development of other plants and can distinguish plants from the organisms that are beneficial or harmful to them (Mairesse et al., 2007).Purple nuts edge tubers possess substances with allelopathic effects on some crop species as well as insecticidal and repellent effects on arthropods.Furthermore, these substances act in synergy with indole acetic acid (IAA) and may be used to induce rooting on cuttings (Durigan et al., 2005;Quayyum et al., 2000;Costa, 1994).
Purple nuts edge presents high levels of indolebutyric acid (IBA), a plant hormone used in root formation and the improvement of bud formation on cuttings (Lorenzi, 2000).Exogenous application of IBA has been used to stimulate rooting of billets in several species (Alves Neto and Cruz-Silva, 2008).Purple nuts edge aqueous extracts have also been used to induce rooting and growth of Manihot esculenta cuttings (Mahmoud et al., 2009).However, purple nuts edge extracts have been observed to interfere with seed germination in some species (Muniz et al., 2007).Bolzan (2003) evaluated the germination of corn, bean and lettuce seeds and bud germination of sugarcane billets in the presence of purple nuts edge leaf and bulb extracts, and they observed decreased germination in lettuce seeds treated with leaf or bulb extract.
Studying the influence of different substrates on the germination of species of interest is advisable because the physiological response of seeds varies with different substrates (Stockman et al., 2007).Factors such as the water holding capacity, degree of pathogen infestation and aeration may vary between different substrates and directly influence seed germination.The main function of the substrate is to supply support and maintain adequate conditions for germination and seedling development (Moraes et al., 2007;Cunha et al., 2006).
It is therefore important to evaluate the allelopathic potential of purple nuts edge (Cyperus rotundus L.) extracts on seed germination.The aim of this study was to evaluate the physiological quality of lettuce and rocket salad seeds sown on different substrates in the presence of purple nuts edge extract.

MATERIALS AND METHODS
The experiment was performed at the Federal Institute of Goiás (Instituto Federal Goiano-IF Goiano), Campus Urutaí, located at Highway Geraldo Silva Nascimento, km 2.5, Rural Zone of the municipality of Urutaí, Goiás (GO), latitude 17° 27' 49'' S and longitude 48° 12' 06'' W, with an average altitude of 744 m.A completely randomized experimental design was used for both experiments with a 3×5 factorial scheme.The factors tested included three substrates (germitest paper, soil and sand) and five doses of purple nuts edge extract (0, 25, 50, 75 and 100%).Four replicates of 100 seeds of each species (curly lettuce and rocket salad) were used.The extract was diluted to four different concentrations, where the 100% concentration consisted of sieved crude extract and distilled water was used for the control treatment (0%).
Lettuce and rocket salad seeds sold for commercial vegetable production were used.The germination tests were performed in BOD germinators at 25°C with a constant photoperiod.Purple nuts edge (C.rotundus) tubers were obtained from plants collected from a homogeneous area of the Institute and used for the preparation of an aqueous extract.
The extract was prepared using purple nuts edge tubers at a concentration of 100% according to the method of Bolzan (2003).A pure extract stock solution was obtained using 100 g of tubers per litre of distilled water, and this solution was then diluted to the different tested extract concentrations.The tubers were washed in running water and homogenized in distilled water using a liquefier, and the resulting solution was filtered.
The substrates were placed in gerbox-type acrylic boxes and moistened with purple nuts edge extract at the different tested concentrations.The field capacity of sand and soil was measured to determine the amount of water required to moisten the substrate.The germitest paper was moistened with 2.5 times the weight of the paper.Evaluations were performed 4 and 7 days after the beginning of the experiment according to the Rules for Seed Analysis (Seed Analysis Rule -SAR) (Brazil, 2009) for the tested species.Germination was quantified by measuring the formation of normal seedlings, radicle and shoot.
The first and second counts of germination were performed four and seven days following germination, respectively, and expressed as the percentage germination according to the method of Brazil (2009).Total germination was determined by adding the values for the first and second counts of germination.The speed of germination index was determined by daily counting of germinated seeds according to Maguire (1962).The data for the different substrates were subjected to a variance analysis (F test) followed by Tukey's test at p<0.05.Regression analyses were performed for the data for the extract doses.All statistical analyses were performed using Sanest software.

Lettuce experiment
A significant interaction between the type of substrate and dose of purple nuts edge extract was observed for all analyzed parameters, with the extract addition decreasing and/or inhibiting the germination of lettuce seeds.The decrease in germination was proportional to the increase in the extract concentration.For the first count of germination, a significant interaction was observed between the extract dose and the soil and paper substrates (Figure 1).A negative relationship between the germination percentage and the extract dose was observed for both soil and paper.This relationship was best fitted by a linear regression equation for the soil, with germination linearly was decreased with increasing extract doses, and by a quadratic regression equation for paper, with a maximum germination value of 26.9 at 43.7% extract.Andrade et al. (2009) reported similar results for the germination of mustard (Brassica campestres L.) seeds subjected to different concentrations of purple nuts edge dry leaf extract.This finding is in agreement with those obtained by Castro et al. (1983), who evaluated the allelopathic effect of purple nuts edge tuber extracts in tomato plants and observed that germination decreased with the application of 50% extract and was completely inhibited by the crude extract.On contrary, Andrade et al. evaluated the germination of lettuce and tomato seeds subjected to nuts edge dry leaf extract and observed no significant differences in germination percentage with different extract concentrations.The aqueous extract of purple nuts edge tubers therefore presents a higher capacity to inhibit or decrease of germination of lettuce seeds than the dry leaf extract.In addition, lettuce may be one of the most sensitive crops to the substances present in purple nuts edge extracts.Lettuce has been used as test plant in some studies of the effects of purple nuts edge extract because it is one of the most sensitive species to this extract (Blum, 1999).
Changes in the activity of endo-β-mannanase and peroxidase, two very important enzymes for the germination process, have been observed in lettuce seeds subjected to different concentrations of purple nuts edge extract (Veiga et al., 2007).The substances present in the extract may therefore inhibit these enzymes and consequently decrease the percentage of germination.Similar to the findings of this present study were reported by Muniz et al. (2007), who also observed germination of lettuce seeds was decreased with increasing concentrations of purple nuts edge extract.
The results for the second count of germination (Figure 2), seven days following germination, were similar to those observed for the first count, with decreasing germination with increasing doses of purple nuts edge extract.The relationship between germination and extract doses was again best fitted by a linear regression equation for the soil, whereas for paper and sand, it was best fitted by a quadratic regression equation with minimum values of 79.4 and 72.7%, respectively.This may be related to the average germination time and the substrate structure and aeration because the main function of the substrate is to supply anchorage for plant support.At the same time, the substrate regulates the water and air supply to roots, contributing to the suitable development of seedlings (Minami and Puchala, 2000;Taveira, 1996).A significant interaction between the substrate type and the purple nuts edge extract dose was observed for the total germination of lettuce (Figure 3).A negative relationship between the total germination and dose of extract was observed for all tested substrates.This relationship was best fitted by a quadratic regression equation for sand, with minimum germination values at 72.9% extract, and by a linear regression equation for soil and paper.This result shows the potential of purple nuts edge extract to decrease seed germination, especially for lettuce.Gusman et al. (2012) evaluated the allelopathic effect of certain pharmaceutically important plant species on several different crops, including lettuce, and observed a decrease in lettuce germination proportional to increasing extract concentrations, similar to the effect observed in this present study.Periotto et al. (2004) observed the sensitivity of lettuce and radish germination to different concentrations of aqueous extracts of Andira humilis Mart.ex Benth.Aqueous, methanol and hexanol extracts of neem (Azadirachta indica A. Juss.) decreased the percentage germination of lettuce seeds (França et al., 2008).However, Pinus elliottii ethanol extracts had no allelopathic effect on lettuce, whereas Eucalyptus citriodora ethanol extracts significantly decreased its speed of germination (Ferreira et al., 2007).
The speed of germination index for lettuce seeds was negatively affected by the purple nuts edge aqueous extract, decreasing as increasing extract concentrations were applied to the substrate (Figure 4).This relationship was best fitted by linear regression equations for soil and paper and by a quadratic regression equation for sand, with a minimum speed of germination at 73% extract.This result is in accordance with the results of Gusman et al. (2012), who tested purple nuts edge aqueous extracts on tomato and rocket salad seeds and observed a decreased speed of germination index starting at 30% extract concentration when compared to the control treatment.

Rocket salad experiment
No significant interactions between the extract dose and type of substrate were observed for any of the parameters evaluated for rocket salad seeds.
The average values for the first and second counts of germination, the total germination, and the speed of germination index for rocket salad seeds are presented in Table 1.A significant difference between the first and second counts of germination was observed with paper and soil as substrates (Table 1).Paper allowed the germination of rocket salad seeds but did not provide conditions that supplied vigour to the resulting seedlings.However, a significant positive effect of soil on the germination of rocket salad seeds was observed at the second count of germination.
No significant differences in the total germination and the speed of germination index were observed between treatments (Table 1).This result indicated that the purple nuts edge aqueous extract and the tested substrates did not affect seedling development, indicating a possible higher tolerance of rocket salad to purple nuts edge.Gusman et al. (2012) tested the allelopathic effect of several different extracts on certain crops and observed that although purple nuts edge was the only species  Averages followed by the same letter within the same column were not significantly different at p<0.05.
lacking an allelopathic effect on the germination of rocket salad and tomato seeds, it had a pronounced effect on their speed of germination indices, which corroborates the results of this present study.The germination potential is a widely used parameter in studies of allelopathic effects.However, this parameter only considers the final results and does not reveal other factors intrinsic to the germination process, such as delays, and it ignores periods of inactive germination that occur during the experiment (Chiapusio et al., 1997).In most cases, extracts have been observed to have significant effects on the average germination time and the germination entropy but not the germination capacity when compared to controls (Ferreira and Áquila, 2000).This observation is in accordance with the present results for rocket salad.

Conclusion
Lettuce germination was affected by the application of purple nuts edge extract to the tested substrates.Lower germination percentages were observed for sand than for the remaining tested substrates.Purple nuts edge extract did not affect the evaluated parameters for rocket salad seeds.A higher germination of lettuce and rocket salad seeds was observed with soil as the substrate.

Figure 1 .
Figure 1.Interaction between the substrate and dose of purple nuts edge extract for the first count of germination (1 st C) of lettuce seeds.Urutaí-GO, 2014.** = significant at p<0.01.

Figure 2 .
Figure 2. Interaction between the substrate and the purple nuts edge extract dose for the second count of germination (2 nd C) of lettuce seeds.. * = p<0.01(Urutaí-GO, 2014).

Table 1 .
First count (1 st C) and second count (2 nd C) of germination, total germination (TG), and speed of germination index (SGI) with different substrates and concentrations of purple nuts edge extract in rocket salad seeds(Ipameri-GO, 2015).