Allelopathic eff ects of orange ( Citrus sinensis L . ) peel essential oil

(Allelopathic eff ects of orange (Citrus sinensis L.) peel essential oil). Crop weeds are the main problem in agriculture, causing a worldwide annual loss of about US$95 billion. Th e principal method for control is the use of synthetic herbicides. Th e continued use of these products increases crop costs, reduces crop quality, and leaves toxic residues in the environment, which are a threat to human and livestock health. Th erefore, there is a demand for environmentally friendly methods of weed control. Th e use of allelopathic compounds from crop residues is an alternative. Orange is one of the biggest crops in the world, and its cultivation generates large amounts of residues. Th ere is strong evidence of bioactivity in orange peel essential oil. Th erefore, the objective in this work was to verify the allelopathic proprieties of this oil. We extracted the oil from the peels of recently discarded oranges using water vapor fl ow with a Clevenger extractor, and tested it against the growth of Euphorbia heterophylla L. and Ipomoea grandifolia (Dammer) O’Donell seedlings when placed in contact with the oil vapor. Th e results were both quantitative and qualitative in the inhibition of the seedlings.


Introduction
Crop weeds are the main problem in agriculture, causing a worldwide an annual loss of about US$95 billion (FAO 2009).Th e principal method to control weeds is the use of synthetic herbicides (Hong et al. 2004).Although these products have brought unquestionable gains to agriculture, their use increases production cost, reduces crop quality (resulting in less healthy plants and more expensive food) and, contaminates the environment, which is a threat to human and livestock health (Silva et al. 2005).Furthermore, the continued use of herbicides has led to a selection of resistant weed biotypes, on which a commercial dose of herbicide has a limited eff ect (Gelmini et al. 2001).Th erefore, there is a growing demand to replace synthetic herbicides with environmentally friendly weed control.
Th e possibility of weed control using allelochemicals is widely recognized (Rice 1995, Souza et al. 2002, Piccolo et al. 2007, Dayan et al. 2009).Allelochemicals are compounds from the secondary metabolism of plants, and present several advantages over herbicides, such as less toxicity, a shorter half life and higher water-solubility (Duke et al. 2000).Furthermore, because they have a lower specifi c action mechanism, they are less likely to select resistant biotypes (Reigosa et al. 2001, Durán-Serantes et al. 2002).
A main shortcoming of the commercial use of allelochemicals is their production at a large scale, because most donor plants are non-cultivated, poorly-studied species.One possible solution is to determine the allelochemical compound and then artifi cially synthesize it; however, this is far beyond the reality of most farmers.Furthermore, the synthetic versions of natural compounds are not accepted in organic agriculture in most countries.Another solution, which has been successfully tested, is the use of crop residues as allelochemical sources (Rizvi et al. 1999).Crop residues are likely to be used at a commercial scale because they are available in large amounts.
Brazil is one of the world's largest orange (Citrus sinensis L.) producers.Th erefore, residues from this crop might be an enormous source of allelochemicals.Th e orange peel, frequently discarded aft er juice extraction, is rich in essential oil.Essential oil of several species is able to inhibit the germination and growth of other species (Duke et al. 2002), and the orange peel essential oil seems to be highly bioactive (Sharma & Tripathi 2006, Ali et al. 2007, Tsai 2008).In preliminary assays, we detected strong inhibitory eff ects of orange peel essential oil on the germination and initial growth of lettuce (Lactuca sativa L.).
Th e objective of this work was to determine the allelopathic proprieties of orange peel essential oil on two of the most common weeds in Brazil: Euphorbia heterophylla L. and Ipomoea grandifolia (Dammer) O'Donell.Due to its high bioactivity and to the preliminary assay results, we predicted that the orange peel essential oil would be able to inhibit these species.

Essential oil extraction
We extracted the essential oil using water vapor fl ow, with a Clevenger type extractor.We used the crumbled peels of 14 oranges recently rejected from a juice store and about two liters of distilled water.We kept the oil in a freezer until the experiment started.

Bioassays
For the bioassay, we used plastic boxes (8x13x5 cm) with a double layer of fi lter paper (80 gm -², 205 μm), 10 mL of distilled water, and 10 weed seedlings (pre-germinated for 24h in distilled water).Inside the box we also placed an 8 mL beaker with 0.7 mL of essential oil, so only the oil vapor reached the seedlings (Fig 1).In an incubator (28 °C, with 12 h/12 h light regime at 83.16 μmol m -2 s -1 ), we allowed the seedlings to grow for fi ve days.Aft er that, we measured the root and shoot lengths of each seedling.For control we used the same procedure, but with an empty 8 mL beaker.
We also performed a qualitative analysis of the seedlings, by searching for any malformation or injury.

Data analysis
Th e experimental design was entirely casualized.For both treatment and controls we assembled six replicates, and analyzed the data using the Mann-Whitney test (p<0.05).

Seedlings size and mortality
Th e contact with the essential oil vapor reduced the length of both the roots and shoots (and therefore, the total size) of E. heterophylla seedlings.Th e number of dead plants was also higher for those in the presence of oil.Ipomoea grandifolia seedlings were more resistant.Only shoots were aff ected, and no seedlings died.

Malformations
Besides the quantitative reduction of seedlings size, the essential oil also led to malformations.Euphorbia heterophylla seedlings exposed to the oil vapor suff ered cotyledon injuries, malformation of the secondary roots, and presented fragile shoots (Fig 2A).Ipomoea grandifolia seedlings also presented cotyledon and secondary root malformations (Fig 2B).Th e shoots of both species were red in the control and almost white in the treatment, showing pigmentation diff erences.

Discussion
Th e orange peel essential oil inhibited E. heterophylla seedlings.Besides reducing the average seedling size, the oil also led to several injuries and bad formations in both the roots and shoots.In several cases, injuries were severe enough to kill the seedling.Ipomoea grandifolia seedlings were less sensitive to the oil, and root size and total size of the seedling were not aff ected in this species.Nevertheless, the contact with the volatilized oil reduced shoot size, and led to morphological changes, particularly cotyledon malformations and a change in shoot color.Th erefore, quantitative analysis alone may underestimate the inhibitory eff ects of the orange peel essential oil on this species.
In the end, there was still oil in all of the beakers.Th is suggests that essential oil vapor saturation inside the plastic box can be reached with less than 0.7 mL of oil.Th erefore, it is reasonable to propose that similar inhibitory eff ects could be produced with a smaller amount of oil.Most allelopathic surveys with essential oils use emulsions of the oils in their assays.Furthermore, the target species varies (Alves et al. 2004;Liu et al. 2008) and thus, it is diffi cult to compare our results with others.Nevertheless, contact with the oil vapor inhibited E. heterophylla and Ipomoea grandifolia, which suggests that the orange peel essential oil has high inhibitory eff ects.
Th e bioactivity of the orange peel essential oil is well documented.Ali et al. (2007) detected anti-mitotic propri-  eties and Tsai (2008) showed that the oil can kill nematodes.Sharma &Tripathi (2006) documented fungicide proprieties.Our results corroborate this because we found strong inhibitory activities.
Th e main compound in the essential oil of citric fruits is limonene (Tsai 2008).In oranges, this monoterpene represents 84.2% of the essential oil in the peel.Th is high concentration has been suggested as the reason of the high bioactivity of orange peel essential oil (Tsai 2008).Limonene is also present in the essential oils of non-citric species (Agostini et al. 2005;Apel et al. 2005;Apel et al. 2006;Carvalho Filho et al. 2006;Silva et al. 2009), which are frequently associated with high inhibitory eff ects (Azirak & Karaman 2008;Silva et al. 2009;Souza Filho et al. 2009).To the best of our knowledge, there has been no allelopathic study that used isolated limonene, but it is reasonable to propose that this was the main compound responsible for the allelopathic eff ects found (because we used a purifi ed version of the orange peel essential oil, one might choose to call this a phytotoxic eff ect).
Th e citric industry is one of the world's largest agroindustries, and the juice manufacturing process creates enormous amount of residues (Tsai 2008).Although further research is required, particularly in applicable studies (Inderjit & Weston 2000), the present work showed that it is possible to control weeds using an allelochemical from citric crop residues.Th is is an environmentally friendly weed control, and helps dispose residues produced by the citric industry.

Table 1 .
Average size (cm) of shoots (ST), roots (RT), total size, and mortality (M, in number of deaths) of Euphorbia heterophylla L. and Ipomoea grandifolia (Dammer) O'Donell.in contact with orange (Citrus sinensis L.) peel essential oil.Values followed by * diff ers signifi cantly from the control by the Mann-Whitney test (p<0.05).