Allelopathic eff ects of Ipomoea cairica ( L . ) Sweet on crop weeds

(Allelopathic eff ects of Ipomoea cairica (L.) Sweet on crop weeds). Identifi cation of species with allelopathic potential has been a target of researches aiming to use them to control crop weeds. Ipomoea cairica is considered a weed with allelopathic potential, which has already been reported. Th e goal of this study was to evaluate the allelopathic properties of leaf extracts from Ipomoea cairica on the germination and early development of four of the worst crop weeds in Brazil: Bidens pilosa L., Echinochloa crus-galli (L.) Beauv., Euphorbia heterophylla L. and Ipomoea grandifolia (Dammer) O ́Donel. We tested the eff ects of leaf extracts, in four concentrations, on the germination and early development of these species. Th e extracts negatively aff ected the germination, early development and the morphology of all target species, and the phytotoxic eff ect was higher as the concentration of the extracts increased. Th e infl uence of the I. cairica extracts on germination, in addition to their eff ects on seedling development, made them more eff ective.


Introduction
Studies about the allelopathic process have recently focused on its application in agriculture, with the goal of using allelochemicals as herbicides to develop more sustainable practices (Chon et al. 2003).Using natural products, allelochemicals and plant extracts cannot provide the same level of weed control when compared to synthetic herbicides, but they can be worthwhile when only a small quantity of herbicide can be used (Khanh et al. 2006).Excessive use of synthetic herbicides can cause pollution of soil/water and injuries to humans (Ahn et al. 2008), and natural compounds have some advantages over synthetic compounds, such as absence of halogen molecules and a shorter half-life (Duke et al. 2000).Moreover, most natural products with biological activity are partially water soluble and are bioactive at lower concentrations (Vyvyan 2002).
Annual global crop losses because of weed species amount to around 95 billion dollars in food production (FAO 2009), and weeds are among the main components of the agroecosystem that interfere in crop production (Kuva et al. 2008).Th ey compete for resources, reducing production, and can deposit a high quantity of seeds in the soil, perpetuating the problem during future plantings (Vyvyan 2002).Bidens pilosa L. (hairy beggarticks), Echinochloa crus-galli (L.) Beauv.(barnyardgrass), Euphorbia heterophylla L. (wild poinsettia) and Ipomoea grandifolia (Dammer) O´Donel (morning glory) are among the worst weeds in several crops in Brazil and many other countries (Kissmann & Groth 1992;Norris et al. 2001).
Aquatic macrophytes have been the target of allelopathic studies showing a great number of promising species (Ahn et al. 2008).We previously verified the allelopathic potential of 25 aquatic macrophyte species and Ipomoea cairica (L.) Sweet was among those with the highest inhibition of lettuce germination.Allelopathy is pointed out as a common characteristic of weeds, which favors them in the environment (Wu et al. 2006).Ipomoea cairica is a perennial species of the Convolvulaceae that is widely distributed in tropical regions, considered noxious, invasive and becomes monodominant in invaded regions (Llamas 2003;Ma et al. 2009).Th e chemical nature of its secondary compounds was recently examined and two compounds, 3-3´-5-Trihidroxi-4´-7-dimethoxyfl anove and 3-3´-5-Trihidroxi-4´-7-dimethoxyflavone-3-Osulphate, were identified as being responsible for its allelopathic properties on radish (Raphanus sativus L.), cucumber (Cucumis sativus L.), Chinese cabbage (Brassica pekinensis (Lou.)Rupr.) and a weed (Ligularia virgaurea (Maxim.)Mattf), making it a possible candidate for the development of new herbicides based on natural products (Ma et al. 2009).However, its allelochemical eff ect on other crop weeds still needs to be elucidated.
Th e aim of this study was to evaluate the allelopathic activity of aqueous leaf extracts, made from Ipomoea cairica, on the germination and early development of four crop weeds: B. pilosa, E. crus-galli, E. heterophylla and I. grandifolia.Aqueous extracts can present a great variety of active substances from the secondary metabolism.Th us, we expect I. cairica to present allelopathic potential on other species of agronomical interest.

Collection and extract preparation
We collected adult leaves with no signs of herbivory or diseases from fl owering individuals of I. cairica in the Massaguaçu River estuary (23º37'20''S and 45º21'25''O), Caraguatatuba, Sao Paulo.We dried the leaves at 45ºC in a forced circulation chamber until mass stabilization, ground the material in a mill and stored it at -10ºC in plastic bags until use.We prepared a 10% (p/v) aqueous extract with the dry powdered leaves and distilled water, and stirred the extract with a magnetic stirrer for 5 minutes at room temperature (22ºC).Aft er storing the extract at 6ºC for 12 hours we fi ltered it using a vacuum pump coupled to a Büchner funnel covered with fi lter paper (3 μm) (Ribeiro et al., 2009 -modifi ed).We diluted the resulting solution (10%) in distilled water to obtain solutions at concentrations of 7.5; 5; 2.5 and 1.25%, and tested their eff ects on the germination of B. pilosa, E. crus-galli, E. heterophylla and I. grandifolia.We had previously scarifi ed the seeds of I. grandifolia with pure sulphuric acid (H 2 SO 4 PA) for 4 minutes to break dormancy, and then washed them in fl owing water and dried them on fi lter paper.

Germination bioassay
We placed the seeds of thirty target species in Petri dishes (9 cm diameter) containing a double layer of fi lter paper (3 μm) that was moistened with 5 mL of solution, or distilled water for control.We sealed all of the Petri dishes with PVC fi lm, closed the lid and placed them in a DBO incubator (28ºC and light-dark cycle of 12h-12h).We counted the seeds at 12h intervals and considered seeds to be germinated when there was a protrusion of one part of the embryo through the seed coat (Borghetti & Ferreira 2004).Germinated seeds were removed to avoid recount.Aft er 10 days we calculated the percentage, average time and informational entropy of germination (Labouriau 1983).

Early development bioassay
We placed ten seedlings of the target species, previously germinated (2-4 mm radicle), in sterilized plastic boxes (8x13x5 cm) containing a double layer of fi lter paper (3 μm) moistened with 8 mL of solution or distilled water for control.We closed all plastic box lids and placed them in a DBO incubator (28ºC and 12h-12h light-dark cycle).Aft er fi ve days, we measured the length of radicles and aerial parts (hypocotyl+cotyledon) of each plant using a caliper and calculated the percentage of dead seedlings.We also analysed the morphology of the seedlings.

Osmotic potential and pH
We measured the osmotic potentials (Micro-Osmette, model: 5004 automatic osmometer) and pH of extracts and set a bioassay of germination and early development with the target species subjected to treatments of polyethylene glycol 6000 in concentrations of corresponding osmotic potentials.

Data analyses
Each target species received fi ve treatments (witness, 2.5, 5, 7.5 and 10% solutions) in a completely randomized block design, with fi ve replicas, for the germination and early development bioassays.Aft er a normality test (Kolmogorov-Smirnov: p<0.05) we analysed the percentage of germination, informational entropy and percentage of dead seedlings using the Kruskall-Wallis test and Dunn's post test (p<0.05),and the average time of germination and early development by ANOVA and Tukey's post test (p<0.05).

Results and discussion
Th e extracts of I. cairica negatively aff ected the germination of all crop weed species (Tab. 1) and the eff ect was higher as the concentrations of the extracts increased.In general, the average time of germination was the most sen-sible parameter, followed by percentage and informational entropy of germination.Th e average time of germination for B. pilosa was signifi cantly increased at all concentrations, and in E. crus-galli's, E. heterophylla's and I. grandifolia's for the 5% concentration.Regarding the percentage of germination, only the 10% extract signifi cantly decreased values for I. grandifolia, E. heterophylla and E. crus-galli; and a concentration of 7.5% was toxic to B. pilosa.Besides this, the synchrony of the germination was also aff ected.Some informational entropy of germination values of I. grandifolia's seeds were increased.Informational entropy of germination values of B. pilosa, E. crus-galli and I. grandifolia seeds could not be calculated under the eff ect of higher concentration extracts due to the absence of germination in some replicas.However, germination curves (Fig. 1) emphasize the inhibitory eff ects of the extracts on synchrony and distribution at the time of germination.We observed erratic curves for B. pilosa, platikurtic (5, 7.5 and 10%) for E. heterophylla and E. crus-galli, and kurtosis for I. grandifolia, suggesting an extension of germination in time.Generally, higher extract concentrations were the same as lower percentage values and higher values of average time and informational entropy of germination.
Th e extracts I. cairica also inhibited the early development of all target species (Fig. 2).Th ere was an increasing inhibitory eff ect that concurred with and increase in extract concentration, and the radicle was the most sensitive organ.In addition, about 80% of the B. pilosa and E. heterophylla seedlings, and 30% of I. grandifolia seedlings, died as an eff ect of the 10% extract.In most of the cases the seedlings were rotten, impeding measurement.Generally, aerial parts are less sensitive to allelochemicals (Pires & Oliveira 2001) and only exhibit less growth.For example, the development of aerial parts of seedlings of E. crus-galli was stimulated by the 2.5% extract and inhibition increased at higher concentrations.Past studies have oft en related stimulation to low concentrations and inhibition to increasing concentrations (hormesis).Several species have presented a hormetic response because of allelopathic substances (Calabrese & Baldwin 2003) as well as many types of herbicides (Duke et al. 2006).Hormetic eff ects can infl uence the occurrence of primary/secondary succession in biological systems, and this concept is important in the development of herbicides based on natural products, where the focus is on toxic dose responses (Calabrese & Baldwin 2003).Qualitative evaluation of seedlings is another method used to analyze the effects of allelopathic substances (Ferreira & Aquila 2000).We verifi ed abnormalities in the seedlings of at all target species and the radicular system was the most aff ected.It presented necrosis (Fig. 3c, 3d, 3e), was short and disproportionate compared to the aerial part (Fig. 4b, 4c), was atrophied (Fig.     4d, 4e and Fig. 5c, 5d, 5e) and defective (Fig. 5b and Fig. 6c, 6d, 6e).We also observed stimulation of the development of the lateral roots compared to the detriment of the primary root (Fig. 6b), suggesting hormonal disruption (Dayan et al. 2000).Many studies relate germination and inhibition of early development, and seedling abnormalities, to the eff ects of allelochemicals (Gatti et al. 2003;Maraschin-Silva & Aquila 2006;Ribeiro et al. 2009).However, changes in morphology and size of the roots and aerial parts can be the result of secondary eff ects and not because of the primary mechanism of action caused by a phytotoxic compound.For example, an evaluation of the eff ects of sorgoleone (a root exudate of Sorghum bicolor L.) on the early development of roots is not adequate because there is no direct relationship between this parameter and the mechanism of action of the allelochemical, which is the inhibition of the photosystem II (Dayan et al. 2000).
Generally it is assumed that the response of seeds and seedlings to plant extracts is due to allelopathy but osmotic potential can also have a negative effect on the target species (Astarita et al. 1996).The osmotic potential values of the leaf aqueous extracts of I. cairica varied from -0.13 to -0.43 MPa (Tab.2).We observed that the effects of the 7.5 and 10% extracts on the percentage of germination of I. grandifolia and the average time of germination of E. crus-galli are partially related to osmotic potential.Osmotic potential is also partially responsible for the inhibition caused by the 5, 7.5 and 10% extracts on the aerial parts of the seedlings of E. heterophylla and I. grandifolia, and for the inhibition verified for the radicles of E. crus-galli for all concentration of the extracts.Germination and early development can also be negatively affected in extreme acid or alkaline conditions (Souza Filho et al. 1996).We observed low acidity in the extracts, and pH values varied from 5.74 to 5.92 (Tab.2), which had no implications on germination and early development of the target species.
Th e eff ects of the I. cairica extracts on B. pilosa, E. crus-galli, E. heterophylla and I. grandifolia can benefi t crops infested with them.In the control of weed species, reduction in the number of individuals in one stage (e.g., germination) increases the options that can be used to manage the following stages (e.g., development).Th e effectiveness of a treatment can be greater than indicated in studies that deal with the factor in an indirect way (Mohler 2001).For example, a weed population can have a few individuals capable of germinating under the eff ects of an allelochemical.Th is reduced number of individuals also germinates sparsely over time and suff ers phytotoxic eff ects from the extracts during early development.Th e eff ects of the I. cairica extracts could be more eff ective if we considered them together, reducing competition for resources and space in the agricultural environment.Although our experiment is limited to the laboratory, the results show the sensitivity of another four important crop weeds in Brazil when exposed to aqueous extracts of Ipomoea cairica, and emphasizes the eff ects of the extracts on three diff erent processes, germination, early development and seedling morphology.

Figure 2 .
Figure 2. Eff ects of the aqueous extracts from the leaves of Ipomoea cairica (L.) Sweet on the early development of seedlings of Bidens pilosa L., Echinochloa crus-galli (L.) Beauv., Euphorbia heterophylla L. and Ipomoea grandifolia (Dammer) O´Donel aft er 5 days.Aerial part: length of hypocotyl+cotyledon.Values inside brackets: dead seedling percentage.Same capital letters are not statistically diff erent by ANOVA and Tukey's post test (p<0.05) in each part of the seedling and species.Same lower case letters are not statistically diff erent by Kruskal-Wallis' test and Dunn's post test (p<0.05) in each species.

Table 1 .
Eff ects of leaf aqueous extracts from Ipomoea cairica (L.) Sweet on the germination of Bidens pilosa L., Echinochloa crus-galli (L.) Beauv., Euphorbia heterophylla L. and Ipomoea grandifolia (Dammer) O´Donel, aft er 10 days of bioassay.Mean±standard deviation.G(%): germinability; Tm: average time of germination; E: informational entropy of germination.Mean values followed by the same capital letters are not statistically diff erent by ANOVA and Tukey's post test (p<0.05) in each species.Mean values followed by the same lower case letters are not statistically diff erent by Kruskal-Wallis' test and Dunn's post test (p<0.05) in each species.-:non-calculated values due to replicas with no germination.

Table 2 .
pH and osmotic potential of Ipomoea cairica (L.) Sweet extracts in diff erent concentrations.