Characterization of the Allelopathic Potential of Sugarcane Leaves and Roots

Sugarcane cultivars that are currently planted are the result of genetic im-provement focused on increased crop yield. However, this selection and genetic alteration reduced the competitive potential of sugarcane, as well as its allelopathic capabilities. Many members of the Poaceae family are highly allelopathic. Thus, the objective of this study was to characterize the allelopathic potential of two sugarcane cultivars (CTC 2 and IAC 91109) by bioassay-guided fractionation, isolation, and identification of significant phytotoxins, including those that are lipophilic. For both leaves and roots, alpha-linolenic and linoleic acid were found to be the most phytotoxic compounds found with this approach. Both compounds were phytotoxic when applied in soil and caused light-independent cellular leakage of treated cucumber cotyledon discs. We conclude that some of the phytotoxic effects of sugarcane residues in soil are due to the combined action of alpha-linolenic and linoleic acid.


Introduction
Weed interference in sugarcane (Saccharum officinarum L.) can reduce crop yield by up to 97% [1]. However, interference is not a phenomenon caused only by weeds on crops, as the crop has the potential to limit the growth and development of weeds [2]. Allelopathy is one of the factors involved in interference between plants.
Allelopathy is the chemical inhibition of one plant by another, due to the release into the environment of phytotoxic compounds (allelochemicals) that inhibit germination and/or growth. Plants may adversely affect growth and development of each other through the production and release of allelochemicals into the environment [3]. The Poaceae family, to which sugarcane belongs, is among the most studied plant families regarding allelopathy, producing a wide variety of allelochemicals. A necessary step in proving allelopathy is isolating and identifying the putative allelochemicals involved [4]. Some compounds produced by sugarcane can adversely affect weed communities within sugarcane fields [5]. In phytosociological surveys, Kuva et al. [6] [7] and Ferriera et al. [8] reported differences in weed infestations between areas planted with different sugarcane cultivars. This may occur due to differences in the preservation of some of the ancestors' aggressive characteristics in some cultivars, such as allelopathic potential. Viator et al. [9] reported postharvest sugarcane residues to be phytotoxic, and Majeed et al. [10] found aqueous extracts of sugarcane to be inhibitory to wheat germination and growth. Aqueous leachate of sugarcane straw can inhibit the growth of both weed and crop species [11]- [16].
The effect in these papers was largely attributed to ferulic, syringic and vanillic acids.
These studies did not look for lipophilic allelochemicals, a common oversight in many allelopathy studies [4], even though Rice [3] listed fatty acids as one class of compounds with allelopathic potential. Fatty acids of rice were shown to reduce the growth of the weeds Heteranthera limosa and Echinochloa crus-galli [17] [18]. In Helianthus anuus and Helianthus tuberosus, C10 and C18 fatty acids were associated with the allelopathic properties of these species [19]. Indeed, simple fatty acids are sufficiently phytotoxic that crude preparations of fatty acids and the nine-carbon fatty acid, pelargonic acid are sold as herbicides for organic gardening [20]. Simple fatty acids such as alpha-linolenic and linoleic acid can alter the permeability of the plant plasma membrane and disrupt chloroplast membranes [21]. Others have found alpha-linolenic and linoleic acid to be among the phytotoxic compounds produced by southern cattail (Typha domingensis) [22]. Recent studies have found lipophilic allelochemicals that are secondary metabolites from plants such as the very potent phytotoxin sorgoleone, which is produced in small amounts by Sorghum species [23] and the less potent phytotoxin aplotaxene which is produced in large amounts by Carduus species [24].
Thus, to properly identify all of the allelochemicals products produced by a plant species requires determination of lipophilic, as well as water-soluble phytotoxins.
The intent of this work was to partially characterize the allelopathic potential of two sugarcane cultivars (CTC 2 and IAC 911099). Yamauti [25] found aqueous extracts of CT 2, but not of IAC 911099, to inhibit Lactuca sativa seedling growth in a soil-free bioassay. In this study, we isolated and identified phytotoxic com-pounds from leaves and roots of these cultivars with a bioassay-guided process that identifies the most significant phytotoxic compounds, regardless of lipophilicity.

Plant Material and Extraction
Sugarcane mini-joints were used to produce seedlings of two cultivars (CTC 2 and IAC 911099) in sand. After sprouting and the beginning of root growth, the seedlings were transferred to soil (dystrophic Red Latosol of medium texture) in pots with a 5.0 L capacity.

Bioassay of Lactuca sativa and Agrostis stolonifera without Soil
Phytotoxicity-guided bioassays of fractions were performed with lettuce (Lactuca sativa) and creeping bentgrass (Agrostis stolonifera) in 24-well plates with hexane, DCM, and EtOH extracts from leaves and roots of the two cultivars with the method of Dayan et al. [26]. Phytotoxicity was assessed by qualitatively comparing seed germination and seedling growth in each well after seven days, using a rating scale of 0 to 5, where 0 indicates no effect and 5 indicates complete inhibition (no germination). The experiment was replicated. were bioassayed for phytotoxicity as described in Section 2.2.

Bioactivity-Guided Fractionation of Phytotoxicity
The fractions were analyzed by 1 H NMR, 13 C NMR, GC/MS, and GC/FID, and fatty acids present were identified as described below.

Gas Chromatography Coupled to Mass Spectrometry (GC-MS) for Compound Identification
The GC-MS analysis was performed on an

Gas Chromatography Flame Ionization (GC-FID) for Fatty Acid Quantitative Analysis and Identifications
GC-FID analysis was performed on a Varian CP-3800 GC instrument. The GC was equipped with as DB-23 column (Agilent Technologies) (60 m × 25 mm capillary column, 25 µm film thickness) operated using the following conditions: injector temperature: 270˚C; column temperature: 130˚C kept for 1 min, followed by 130˚C to 170˚C at 6.5˚C min −1 , followed by 170˚C to 215˚C at 2.8˚C•min −1 and kept for 12 min followed by 215˚C to 230˚C at 40˚C min −1 and kept for 3 min; injection volume: 1 µL (20:1 split); 3 mL•min −1 constant flow; and FID temperature of 300˚C. Fatty acid methyl esters were identified by injecting commercially available standards, purchased from Sigma Aldrich, and comparing retention times with unknown times. Fatty acids were quantified by performing percentage area calculations based on the combined total area of the FID.
Free fatty acids or fractions were converted to their corresponding fatty acid methyl esters using diazomethane and direct methylation prior to GC-FID analysis. For this, 1 mg of compound/fraction in 1 mL of diethyl ether was treated at room temperature overnight with a solution of diazomethane in diethyl ether.
The solvent and residual CH 2 N 2 were removed using N 2 , and the sample was resuspended in DCM for GC analysis.

Quantitative Bioactivity-Guided Fractionation of Phytotoxicity
The quantity of fatty acids in leaves and roots of the cultivars was determined following the method of Wang et al. [27] using direct methylation. An internal standard was prepared with tricosanoic acid (C23:0).

Soil Fatty Acid Bioassays
Two substrates were used in these bioassays. One substrate was s clayey, silty soil collected in a field that was never treated with herbicides at the USDA Ja- In each well, 0.2 mL of test solution and 0.6 g of soil were added. L. sativa and A. stolonifera were sown in the clayey, silty clay soil. L. sativa and Solanum lycopersicum were sown in the dystrophic Red Latosol of medium texture. The amount of seeds used in each cell was approximately five seeds of L. sativa, 10 mg (approximately 115 seeds) of A. stolonifera, and six seeds for S. lycopersicum. The plates were incubated at 26˚C in a Conviron growth chamber at 173 µmol•s −1 •m −2 photosynthetically active radiation. Then, 300 μL of distilled deionized water (DDI) were added on the first day and another 100 μL were added on the fourth day.
To maintain humidity, the trays were covered with plastic chambers. At seven days after sowing, root length and fresh mass were determined for L. sativa, and the average shoot length of A. stolonifera was determined. The results were subjected to analysis of variance by F test, and the means were compared by Tukey test at 5% probability.

Cellular Leakage Bioassay
The effects of linoleic acid and alpha-linolenic acid on cell leakage of cucumber cotyledon discs (Cucumis sativus L.) were determined by the method of Dayan The results were subjected to analysis of variance by F test, and the means were compared by the Tukey test at 5% probability.

Fractionation-Guided Bioassay of Leaf and Root Extracts
The hexane and DCM crude extracts from the leaves and the DCM crude extract from the roots of CTC 2 were phytotoxic at 1 mg•mL −1 . All had a phytotoxicity score of 3 for A. stolonifera (Table 1) Three of them had a phytotoxic effect (fractions D, E, and F) ( Table 1).
The hexane and DCM crude extracts from the leaves of IAC 911099 were phytotoxic at 1 mg•mL −1 . All had a phytotoxicity score of 3 for A. stolonifera ( Table   2). The fractionation of DCM crude extracts from the leaves of IAC 911099 with column chromatography resulted in twenty distinct fractions. Six of them were phytotoxic (fractions G to L) ( Table 2).
The fractions were subjected to analysis by 1 H NMR, 13    Values denote toxicity at 1.0 mg/mL for L. sativa and A. stolonifera. 0 = no effect, 5 = maximum effect.

Identification of Compounds
In the analysis of fatty acid quantification, there was no significant difference for the concentration of linoleic acid between cultivars and leaves and roots. Regarding the concentration of alpha-linolenic acid, it was higher in leaves than in roots for both cultivars. For both CTC 2 and IAC 911099, there was a higher concentration of alpha-linolenic acid in leaves and a higher concentration of linoleic acid in roots ( Table 5 (Table 6).
For the bioassay of A. stolonifera in soil, there was a reduction in average   (Table 7). For linoleic acid, inhibition occurred at concentrations higher than 3.33 mM.  In a different soil, 0.33 mM alpha-linolenic acid in the soil reduced the shoot length of seedlings of L. sativa dramatically (Table 8). For root length and germination, reductions occurred only at the highest concentration (33.3 mM). Linoleic acid stimulated the growth of shoots at the lowest concentrations. Allelochemicals are known to stimulate plant growth at subtoxic concentrations [29] [30]. Viator et al. [9] found crude sugarcane residue extract to enhance sugarcane bud germination at low doses, but to be autotoxic at higher doses, but to be autotoxic at higher doses. Linoleic acid reduced growth only at the concentration of 33.3 mM. A concentration of 33.3 mM of alpha-linolenic acid in the soil reduced the shoot length of S. lycopersicum seedlings, and there was no statistical difference between it and the highest dose of atrazine (Table 9). For root length, only the highest concentration differed from that of the other treatments because, although the other concentrations differ from the control, they were equal to the relative control (acetone). The same effects also occurred for linoleic acid. However, there was a reduction in germination at the concentration of 33.3 mM. The differences in activity in the different soils, is not surprising, as Hiradate et al. [31] reported considerably variation in the phytotoxicity of the same allelochemical in different soils. The combined concentration of these two fatty acids in leaf material is close to 7 mM (Table 5). Our soil phytotoxicity studies (Tables 6-9) indicate that such a concentration would adversely affect the growth of some species in some soils. Such lipophilic compounds are more likely to adhere to soil particles and, thus, much less likely to leach from soil with rainfall, as would be expected with many water-soluble compounds.    (Figure 1 and Figure 2). The effects were not light dependent, as is the case for acifluorfen, a herbicide that causes phytotoxicity by causing the accumulation of the photodynamic compound protoporphyin IX [32]. Unlike acifluorfen, the two compounds caused leakage in darkness, and the effect seemed to be reduced by exposure to light. Thus, these compounds might cause phytotoxicity in soil, where there is little or no light.
Fatty acids are ubiquitous in plants. In sugarcane, palmitic and linoleic acids occur in culms and leaves and stearic and oleic acids occur in culm wax [33]. A DCM extract from sugarcane leaves containing fatty and phenolic acids showed a deleterious effect on the weed Calopogonium mucunoides [34]. It inhibited germination (35%), root growth (52.8%), and hypocotyls (47.1%). These previous data support those found for the sugarcane cultivars CTC 2 and IAC 911099. Fatty acids occurred in the fractions of leaves extracted using dichloromethane and in the fraction with hexane for CTC 2. Fatty acids also occurred in the DCM fraction of the roots of CTC 2. In wheat, there are three main phytochemical categories that can cause allelopathic effects, namely phenolic, hydroxamic, and short-chain fatty acids [35]. Fatty acids inhibit the growth of wheat seedlings [36] [37].
In Typha latifolia, a perennial monocot, there were unsaturated fatty acids, alpha-linolenic acid, and linoleic acid [22] [38]. Despite being common compounds in plants, they caused inhibition in algae similar as that of CuSO 4 (0.5 µmol). Alpha-linolenic acid caused the greatest algae inhibition.
There was an inhibitory effect on the growth of the weed Echinochloa crus-galli, due to 50 ppm fatty acids in rice husk extracts (Oryza sativa) from [39]. Fatty acids are among the compounds with the greatest herbicidal potential according to Macías [40]. In the present study, by observing the effects of alpha-linolenic and linoleic acids on both L. sativa and A. stolonifera, there was a reduction in the characteristics analyzed in relation to the control at concentrations near 10 −3 M. This means that, for inhibition to occur, the concentration should be higher than that indicated by Macías [40].
For L. sativa germinated in soil, there was a greater reduction in fresh mass compared to the control for the treatment with alpha-linolenic acid (88.4% at the highest concentration) than for the linoleic acid (59.6%) ( Table 6). This may have occurred due to the greater amount of double bonds in alpha-linolenic acid. Medium chain fatty acids (those between nine and 11 carbons) cause damage to bimolecular lipid membranes. It makes the membrane structure unstable and allows leakage of electrolytes from the cell, culminating in plant death. This damage caused to the plant membrane can occur due to hydrophobicity of fatty acids. Hydrophobicity increases as the carbon chain of fatty acids increases [43].
In the present study, the leakage of electrolytes from cucumber cells occurred with exposure to alpha-linolenic acid and linoleic acid at 330 and 1000 µM. Concentrations below these did not differ statistically from solvent control. However, even the highest concentrations of these fatty acids did not cause the level of Journal of Agricultural Chemistry and Environment cellular leakage that acifluorfen caused, which promotes loss of plasma membrane integrity after exposure of samples to light, due to production of high levels of reactive oxygen species generated by accumulation of the photodynamic compound protoporphyrin IX [28]. The importance of monitoring the plasma membrane stems from its role in the interface between the cell and the environment. If there is sufficient loss of the integrity of the lipid bilayer, the leakage of electrolytes will result cell death [28].
In a search for bioactive compounds from the plant Ligularia macrophylla, both linoleic and alpha-linolenic acids were isolated [44], but they had little or no phytotoxicity in a bioassay for which the highest concentration was 3.4-fold lower than the initial bioassay used in this paper. However, both compounds were antifungal.
Both linoleic and alpha-linolenic acids are ubiquitous compounds in plants, so one could argue that they are unlikely to influence other plant species as allelochemicals. However, as the father of toxicology, Paracelsus, deduced almost five centuries ago, "the poison is in the dose". We have found these compounds are phytotoxic at relatively high doses and that they retain their phytotoxicity in soil, something that is critical for an allelochemical. The simple phenolic acids previously reported as allelochemicals from sugarcane [11]- [16] are virtually inactive in most soils [45]. Our results are consistent with those of Luz et al. [46], who found the DCM and ethyl acetate fractions of sugarcane vinasse to be the most phytotoxic to lettuce seedling root growth. Many compounds touted as allelochemicals because of activity in soil-free bioassay have little or no activity in soil [4] [31] [45]. In some soils of sugarcane, there could be high enough combined concentrations of these compounds in soil to inhibit the growth of some weed species.

Conclusion
Alpha-linolenic acid and linoleic acid were found and identified as potential allelochemicals from leaves and roots of the sugarcane cultivars CTC 2 and IAC 911099. These compounds caused growth inhibition of Lactuca sativa, Agrostis stolonifera, and Solanum lycopersicum seedlings and cellular leakage of Cucumis sativus cotyledon discs in darkness. Furthermore, both compounds were active in inhibiting seedling growth in soil, a prerequisite for allelochemical activity.