Influence of leaf extract of Jatropha curcas on initial growth of cauliflower ( Brassica oleracea var . botrytis )

This paper aimed at evaluating the initial growth of cauliflower (Brassica oleracea var. Botrytis) subjected to various levels of aqueous extract of leaves of Jatropha (Jatropha curcas) according to the following parameters: stem diameter, stem length, root length, leaf length and leaf area, root dry mass, leaf dry mass, root fresh mass, and leaves fresh mass. The experimental design was randomly blocks with six concentrations (0, 5, 10, 20, 40, and 80%) and six replications. The study was conducted at the experimental campus of the State University of West Paraná, UNIOESTE, campus Cascavel. The statistical analysis of the results showed a significant effect of concentrations of aqueous extract of J. curcas on the initial growth of cauliflower. Except for root growth, there was a positive effect dependent on the increased concentration of crude aqueous extract.


INTRODUCTION
Cauliflower is an inflorescence-type vegetable that belongs to the species Brassica oleracea var.botrytis L., whose delicate and tender texture requires care and attention in its preparation (Filgueira, 2000).It is cultivated in the Middle East since antiquity.However, only from the twelfth century this culture began to expand to other parts of the world, being grown in Brazil in the States of São Paulo, Rio de Janeiro, Rio Grande do Sul, Minas Gerais, Paraná, and Santa Catarina.In Paraná, it is among the leading vegetables produced in the Metropolitan Region of Curitiba (Hasse, 2005;May et al., 2007).
China is the country with the title of world's largest producer of cauliflower, followed by India, Spain, Italy, and France (FAO, 2009).It is a vegetable containing appreciable amount of vitamins, free of fats and cholesterol and with low levels of sodium and calories (May et al., 2007).
According to Filgueira (2008), with the development of genetic studies, there was the development of hybrid cauliflower which has suitable production conditions in hotter climates, a factor that promoted the cultivation throughout the year, since it is a plant which generally requires low temperatures for cultivation (Blanco et al., 1997).With regard to Jatropha (Jatropha curcas L.), it is a perennial and monoecious species, belonging to the family Euphorbiaceae, the same of castor (Ricinus sp.), cassava (Manhiot sp.) and rubber (Hevea spp.).It is believed that, Jatropha is a native of Central America; however spontaneous vegetation in various regions of Brazil (Heller, 1996;Beltrão, 2005).Its leaves have high amount of nutrients which when extracted, can serve as a substrate for other cultures.Nutrients accumulated by the leaves include: N, Ca, K, Mg, P, S, Mn, Fe, B, Zn, and Cu.
Previous studies have shown that, the root and stem *Corresponding author.E-mail: wcezar85@hotmail.com.Tel: 554599278808.extracts of J. curcas have allelopathic effects on some crop species (Abugre et al., 2011;Rejila and Vijayakumar, 2011), also being observed phytotoxicity of its residues in soil (Wang et al., 2009).Igbnosa et al. (2009) suggests that, leaves of J. curcas exert greater allelopathic effect on germination, length of radical, and plumule of plants.It also states that, higher amounts of allelochemicals can be found in leaves than in roots of J. curcas.This is corroborated by the findings of Maharjan et al. (2007), where the preliminary screening showed that, the leaf extract had the strongest allelopathic effect on seed germination.Tefera (2002) also found that, the impact of allelopathic leaf extract was more powerful than other vegetative parts.
With respect to the effects of a plant (or microorganisms) on another plant, defined the term allelopathy from the Greek: allelon means from one to another and pathós means suffering, as the chemical interaction that occurs between organisms from the release of substances derived from the secondary metabolism of plants, so-called allelochemicals.According to the IAS (2010), this process can be defined as the studies involving any process with secondary metabolites which are produced by plants, algae, bacteria or fungi, which may affect the growth and development of agricultural production and biological systems.When released, the allelochemicals may influence the growth and development of surrounding biological systems (Razavi, 2011) and can even be used to control undesirable plants (Appleton and Berrier, 2009) in agriculture, in rotating systems such as Helianthus annuus that when grown before soybean (Glycine max (L.) Merr.) reduces the amount of weed species (Pasqualeto et al., 2007), terpenes, and phenols (Corsato et al., 2010) that can combat herbivorous, insects, and fungi, besides influencing the growth of other plants (Taiz and Zeiger, 2009).
Regarding bioassays, these procedures are used to evaluate the allelopathic potential of a given species through global parameters such as germination, growth, and development of seedlings or adult plants (Souza-Filho and Alves, 2002), where tests that add experimental procedures that characterize the allelopathic property of certain crops serve to contribute to the studies of dynamics between species, development of management and production strategies (Souza-Filho et al., 2010).
Therefore, considering the above, this study aimed at determining the effects of irrigating cauliflower crop using leaf extract of J. curcas in different proportions, as well as evaluating the development of the plant according to the following parameters: stem diameter, stem length, root length, leaf length and leaf area, root dry mass, leaf dry mass, root fresh mass, and leaf fresh mass.

MATERIALS AND METHODS
This work was carried out during the autumn of the year 2013 at the State University of West Paraná, UNIOESTE campus Cascavel, PR, Brazil.The local climate is considered subtropical mesothermal and super humid with mean annual temperature of 19°C, mean rainfall of 60 mm per month distributed over all months of the year (Brazil, 1999).Before mounting and evaluation of experiments, the bench, hands and materials were disinfected using bactericide solution (Lysoform) and fungicide (Nystatin), both at 10%.Seeds of cauliflower were acquired in suitable trade.
To carry out this research expanded polystyrene trays with 200 cells was used, with a 10 × 20 cell division, each with 14 cm 2 and 5.5 cm deep.

Extraction and extract preparation
As a source of irrigation was employed, the use of leaf extract of J. curcas from the experimental field of UNIOESTE campus Cascavel.400 g of leaves were crushed in 2 L of distilled water using a blender.Then, suspensions were prepared containing specific amounts (weight g) of crushed plant species and a specific volume (ml) of distilled water according to the desired concentration, which are: 0, 5, 10, 20, 40, and 80% dilution.The solution was filtered through four layers of gauze at 15 × 15 cm 2 .Subsequently, the extracts were stored in specific sprayers and packed under refrigeration at 10°C throughout the experimental period in order to preserve their characteristics.

Planting of seeds and extract application
Each cell was filled with commercial extract and two seeds were inserted at 1 cm depth of each cell.The treatment was performed as follows: thirteen lines were used, the first boundary line (untreated), followed by the treatment with line 0% of extract (control), boundary line, line with 5% of extract, boundary line, line with 10% of extract, boundary line, line with 20% of extract, boundary line, line with 40% of extract, boundary line, line with 80% of extract followed by the final line of boundary.
Sowing occurred on the 28 of March, 2013.The treatment was performed from the first day of sowing, occurring in random blocks with 10 cells for each treatment.Six extract doses with the aforementioned dilutions were evaluated, and each dose was administered in three equal portions a day, totaling 100 ml of substrate by treatment.day - .After four weeks of planting, the harvesting of plants, preserving the root of each one was carried out.The root systems were air dried and weighed.To a caliper Tramontina brand was used to measure the stem diameter, stem length, root length, and leaf length.In determining the leaf area by point's method, transparency sheets containing digitized points with graph paper in 1 cm 2 square equispaced each was used, and were then counted points met by the contour of each sheet.Thus, the leaf area was estimated by the number of points completed (Peixoto and Peixoto, 2009).For analyses of fresh and dry mass of leaves and roots, paper envelopes of 10 cm 2 were used, where leaves and roots of 6 plants of each treatment were inserted, separately.The envelopes were weighed on analytical balance, Marte, AY220 model, year of manufacture 2011 and taken to oven at 65°C for 24 h, which were subsequently weighed again.

Statistical analysis
Data analysis was performed using the software ORIGIN 8.5, producing graphics and fit of curves.Student's t test was used, the software version 7.6 beta ASSISTAT from the regression analysis at 5% probability of error.

RESULTS
Figure 1 shows that, the effect on growth of stem diameter by the extract of J. curcas does not have a linear behavior.Although the F-test analysis with 5% significance has provided that the control value is different from those obtained with the treatments of 5, 20, 40, and 80% extract, the level of 10% extract was found to be not significant due to its high standard deviation value.
Figure 1 also shows the effect of the extract on the stem length, the linear fit whose Pearson coefficient is 0.899, the line equation obtained is Y = 1.814 + 0.013 X.According to the values obtained, the extract concentration of 80% caused on average of 1.3 cm increase compared to the control sample.With respect to leaf length (Figure 2), its behavior was growing as the level of J. curcas extract was increased.The t test indicated difference values between the last two samples and control with 0.05 significance.In the same figure we have the behavior of leaf area with increasing extract concentration.According to the observed values, there is increased mean value of leaf area from 1.0 to 1.86 cm 2 (86% higher).The t test indicated a significant difference between the last two samples and control at 0.05 significance.
Figure 3 shows that, the treatment with different concentrations of extract leads to a significant increase in the fresh mass of leaves and roots.The fitted lines were Y = 0.3712 + 0.0046 X to the leaves and Y = 0.0820 + 0.0010 X to the roots.According to these settings there is 12.4% increase in the mass of leaves and 12.1% for mass of roots for each 10% increase of extract used.
Figure 4 shows a linear increase of dry mass of leaves and roots up to 40% extract of J. curcas used.The point in 80% had no proportional increased to the previous points, indicating a tendency to stabilize its value at the extract concentration of 80%.Using the values, up to 40% of extract was obtained the line equation of Y = 0.0282 +0.00039 X to the leaf (DL) and Y = 0.0101 +0.00012 X to the root (DR).The Pearson coefficients were greater than 0.96 for treatments used.
By substituting the mass value obtained at 80% in the line, equation is obtained that the extract concentration required to produce the greatest gain would be 46.4% to leaves and 49.2% to the root, that is, extracts around 50% would cause approximately 64.7% increase in the mass of leaves and about 66.7% increase in the mass of roots compared to the mass in the control.
For root length, the values of this parameter do not differ markedly from each other.The Student's analysis indicated that, means with 5, 10, 20, and 80% extract did not differ from control sample and, the sample with 40% extract of J. curcas has a very close value to the limit to accept the null hypothesis.Considering the result, the graph for root length was not exposed.Data with regard to this parameter can be found in the Table 1.
Table 1 shows in summary the means results and their standard deviations for the parameters studied and the percentage of variation for samples of 40 and 80% compared to the control sample.Table 1 summarizes the results obtained using treatment with the extract of J. curcas.In general, there was increase in the diameter values of the stem, stem length, leaf length, and leaf area as the extract concentration also increased.The t test at  0.05 significance level confirmed that the control value (concentration 0%) differs from the values obtained with 40 and 80% of extract for quantities mentioned in the previous paragraph.Only the variation in root length was not significant according to the t test.Increases compared to control (in percentage) are presented in the last two rows of Table 1, being possible to verify a sharp increase of leaves characteristics, increasing their area by 87% approximately.Significant values were also observed for stem diameter and stem length, but less pronounced as those found for the leaf.

DISCUSSION
The stem development in the parameters length and diameter (Figure 1) found in this work differs from the results of Abugre and Sam (2010) who suggest that, the growth inhibition of seedlings of Zea mays exposed to high concentrations of root extract of J. curcas.In this work, the extracts of J. curcas provided positive development of cauliflower.
Despite their having been no increase in root length, mass gain was achieved, indicating a higher number of roots and in some cases, increase of its diameter.Bonamigo et al. (2009) found a positive effect of the application of aqueous extract of J. curcas for the mean root length of the seedlings of Brassica napus.These contradictory results are possibly related to the allelopathic properties of the extract of J. curcas.
The increased leaf length (Figure 2) obtained in this study does not agree with the results of Wang et al. (2009).The authors reported allelopathic effect of leaves of J. curcas in seedling development of Tagetes erecta L. According to the authors, the leaves caused decreased shoot length of the seedlings evaluated and when added the residues of J. curcas on the ground, there was increased toxicity, exerting greater phytotoxicity in seedlings root system.Regarding the increased leaf area found in this study (Figure 2), data diverge from those of Rejila and Vijayakumar (2011), where the aqueous extracts of J.curcas showed inhibitory effects on shoots of Capsicum annum L. (green pepper), this increasing effect with the increased levels of substrate.Similar effects to those in the work of Rajangam (1984), where the leaf area development of Oryza sativa and Z. mays were inhibited by Rhizophora apiculata, and in peanuts by bamboo (Eyini and Jayakumar, 1989).Data corroborate Palani and Dasthagir (1998), who reported leaf area reduction of cultivated peanut by aqueous extracts of leaves of Eucalyptus globulus.
The observed increase in the parameters from the previous paragraph and the fresh and dry mass of roots and leaves (Figures 3 and 4) found in this study may be related to the type of substances, functional group, chemical properties, and concentration in the medium in which the extracts of J. curcas leaves are acting (Goldfarb et al., 2009).Still, Reigosa et al. (2006) state that, the allelochemicals have the power to interfere with cell division, organic synthesis, hormonal interactions, nutrient uptake, protein synthesis, lipid metabolism changes, stomatal conductance, assimilation of CO 2 and electron transport in photosynthesis, as well as the content of chlorophyll in the plant.
Regarding the allelopathic power of J. curcas, in the study of Abugre et al. (2011), among all species evaluated, this was the one that most inhibited the okra germination (Abelmoschus esculentus (L.) Moench), with its root extracts having inhibited 53.3% of the seed lot and leaf extract 68.3%, which showed a stronger effect  (Abugre and Sam, 2010).
On the other hand, the use of products extracted from plants can be a strong ally to other methods of insect control, maintaining the environmental balance, leaving no chemical residue, no toxic effects on animals and humans, and reducing the negative effects caused by the uncontrolled application of organosynthetic insecticides (Machado et al, 2007).
Thus, even checking that the species J. curcas exercised positive activity on most of these parameters in the laboratory, it is necessary to carry out field studies to prove these effects and also its intensity (Corsato et al., 2010), once in addition to the aforementioned effects, there is possibility of allelochemicals transforming themselves in the environment due to the action of microorganisms, as well as other factors present in the upper layer in the soil, in substances with completely different chemical properties, which can be beneficial or detrimental to neighboring plants (Ferreira and Borghetti, 2004).

Conclusion
The conditions under which this experiment was conducted, there was no statistical effect on the root growth of cauliflower; however, the diameter, stem length, leaf length, leaf area, fresh and dry mass of leaves, and roots were influenced positively by the high concentrations of crude aqueous extract.According to the fitted values, the maximum value for mass gain would be achieved with levels of substrate of 46.4% to leaves and 49.2 to the roots.

Table 1 .
Mean results and standard deviations of the parameters evaluated on the initial growth of cauliflower.
keto-ƒβ-sitosterol, and ƒβsitosterol through analyses in leaf extracts.For this species, phenolic compounds are the main substances responsible for exercising allelopathic effect, whose high concentrations of leaf and root extracts were found to inhibit the growth of seedlings of beans (Phaseolus vulgaris L.), corn (Z.mays L.), tomato (Solanum lycopersicum L.) and okra (A.esculentus (L.) Moench)