Scholarly article on topic 'Phytotoxicity of volatiles from fresh and dry leaves of two Asteraceae shrubs: Evaluation of seasonal effects'

Phytotoxicity of volatiles from fresh and dry leaves of two Asteraceae shrubs: Evaluation of seasonal effects Academic research paper on "Biological sciences"

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South African Journal of Botany
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{Allelopathy / " Baccharis patens " / Germination / Growth / " Heterothalamus psiadioides " / Seasonality}

Abstract of research paper on Biological sciences, author of scientific article — E.R. Silva, G.E. Overbeck, G.L.G. Soares

Abstract In South Brazilian grasslands, the shrubs Heterothalamus psiadioides Less and Baccharis patens Baker show a dominance pattern that may be a consequence of allelopathy. In this study, the phytotoxic effects of H. psiadioides and B. patens and the seasonal variation in these effects were assessed. The effects of the volatiles released directly from fresh and dry leaves of the both species were evaluated on germination and growth of the target plants lettuce and onion in four seasons. The volatiles from H. psiadioides and B. patens inhibited the germination rate and speed of germination and reduced the shoot and root length of the target plants, with fresh leaves showing greater phytotoxicity. These parameters were more affected by H. psiadioides than by B. patens in all seasons and both plants showed a decrease in phytotoxicity in summer. It can be concluded that both shrubs possess allelopathic potential, with H. psiadioides showing stronger effects. Interseasonal variation of phytotoxicity should be taken into account in studies of potentially allelopathic plants.

Academic research paper on topic "Phytotoxicity of volatiles from fresh and dry leaves of two Asteraceae shrubs: Evaluation of seasonal effects"

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South African Journal of Botany

journal homepage: www.elsevier.com/locate/sajb

Phytotoxicity of volatiles from fresh and dry leaves of two Asteraceae shrubs: Evaluation of seasonal effects

CrossMark

E.R. Silva

G.E. Overbeck a,c, G.L.G. Soares

a Programa de Pós-Graduafao em Ecología, Instituto de Biociencias, Universidade Federal do Rio Grande do Sul (UFRGS), Brazil

b Laboratório de Ecologia Química e Quimiotaxonomia (LEQTAX), Departamento de Botánica, Instituto de Biociencias, Universidade Federal do Rio Grande do Sul, Brazil c Laboratório de Estudos em Vegetafao Campestre, Departamento de Botánica, Instituto de Biociencias, Universidade Federal do Rio Grande do Sul, Brazil

ARTICLE INFO

Article history:

Received 23 August 2013

Received in revised form 16 February 2014

Accepted 13 March 2014

Available online xxxx

Edited by TR Marks

Keywords: Allelopathy Baccharis patens Germination Growth

Heterothalamus psiadioides Seasonality

ABSTRACT

In South Brazilian grasslands, the shrubs Heterothalamus psiadioides Less and Baccharis patens Baker show a dominance pattern that may be a consequence of allelopathy. In this study, the phytotoxic effects of H. psiadioides and B. patens and the seasonal variation in these effects were assessed. The effects of the volatiles released directly from fresh and dry leaves of the both species were evaluated on germination and growth of the target plants lettuce and onion in four seasons. The volatiles from H. psiadioides and B. patens inhibited the germination rate and speed of germination and reduced the shoot and root length of the target plants, with fresh leaves showing greater phyto-toxicity. These parameters were more affected by H. psiadioides than by B. patens in all seasons and both plants showed a decrease in phytotoxicity in summer. It can be concluded that both shrubs possess allelopathic potential, with H. psiadioides showing stronger effects. Interseasonal variation of phytotoxicity should be taken into account in studies of potentially allelopathic plants.

© 2014 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction

Allelopathy can be defined as any direct or indirect harmful effect by one plant on another through production and release of chemical compounds (allelochemicals), usually secondary metabolites (Andel, 2006; Rice, 1974). In many ecosystems, some plants form dominant stands and this pattern may be related to the release of allelochemicals, reducing or preventing the establishment of other species near them. The shrubs Heterothalamus psiadioides Less and Baccharis patens Baker (Asteraceae) grow in grasslands and shrublands in southern Brazil and in Uruguay (Deble et al., 2005), where they often establish in dense populations, forming shrublands (Setubal and Boldrini, 2010), thus indicating the possibility of allelopathic interactions with nearby species. However, it has not been investigated which factors are responsible for these patterns, and almost nothing is known about the allelo-pathic potential of these species; there is only a study reporting the phytotoxic effects of H. psiadioides essential oil (Schmidt-Silva, 2012).

The most promising way to assess the phytotoxicity of H. psiadioides and B. patens should be to evaluate their volatiles, as the phytotoxic effects of other aromatic shrubs have been mainly related to volatiles

* Corresponding author at: Programa de Pos-Gradua^ao em Ecologia, Universidade Federal do Rio Grande do Sul, Av. Bento Gon^alves 9500, 91501970 Porto Alegre, RS, Brazil. Tel.: +55 51 3308 7576; fax: +55 51 3308 7755. E-mail address: anesilva.bio@hotmail.com (E.R. Silva).

release (Ibanez and Zoppolo, 2011; Muller et al., 1964). Besides, recent studies revealed that the volatile essential oil of H. psiadioides is able to affect cell division in target species (Schmidt-Silva et al., 2011). To better assess allelopathic potential of plants, it is advantageous to use methods where emission of volatiles occurs in a way similar to processes actually taking place in the plant community. In this sense, volatiles from senescent leaves of plants may affect germination and development of other species when they are on the soil or incorporated into it (Huang et al., 2013); therefore, to evaluate volatiles released directly from dry leaves of plants seems to be an interesting approach. Nevertheless, the development of neighboring species may be also affected by volatiles from leaves that are still on the plant (Muller et al., 1964). The use of fresh leaves thus could be better to characterize potentially phytotoxic plants, as studies reported that the release of phytotoxic substances decreases during the decomposing process (Huang et al., 2013; Wilt et al., 1993).

The majority of studies consider only one point of time for testing the allelopathic potential of a plant. However, the production of secondary metabolites may vary during the year (Frizzo et al., 2008; Helmig et al., 2013) and this should be reflected by seasonal changes in plant phyto-toxicity. This seasonal variation in defensive chemistry is mainly related to the environmental conditions that plants are growing in: biotic and abiotic factors such as the presence of herbivores (Karban, 2007) and pathogens (Heil and Bostock, 2002), temperature (Lur et al., 2009) and precipitation (Gray et al., 2003) vary during the year, and are able to

http://dx.doi.org/10.1016/j.sajb.2014.03.006

0254-6299/© 2014 SAAB. Published by Elsevier B.V. All rights reserved.

change secondary metabolites synthesis. Assessing this variation is required to characterize the potential phytotoxicity of a plant species and to interpret its effects.

In this study, we aimed to evaluate the phytotoxic effects of H. psiadioides and B. patens and the seasonal variation of these effects, through a method that assesses the activity of volatiles released directly from fresh and dry leaves of the plants. We tested the following hypotheses: (1) Considering that both species form dense stands, both H. psiadioides and B. patens present phytotoxicity, affecting germination and growth of other plants; (2) as the release of phytotoxic volatiles must decrease with leaves senescence, the fresh leaves of the plants show stronger effects than the dry leaves; and (3) as allelochemicals production may vary during the year, the phytotoxic effects of H. psiadioides and B. patens change seasonally.

2. Material and methods

2.1. Plant collection

Leaves of H. psiadioides and B. patens were collected in natural grassland vegetation at Morro Santana (30° 03' S, 51° 07' W), a hill in the city of Porto Alegre, Rio Grande do Sul State, Brazil, where they occur with high frequency and abundance values (details on the grassland vegetation on Santana hill: Overbeck et al., 2006). Voucher specimens of H. psiadioides (register number 175007) and B. patens (register number 192266) were deposited in the herbarium ICN of the Federal University of Rio Grande do Sul.

2.2. Germination and growth assays

The effect of the volatiles from H. psiadioides and B. patens was evaluated on the germination and growth of the target species lettuce (Lactuca sativa L. cv. Grand Rapids) and onion (Allium cepa L. cv. Baia Periforme). Both target plants were used in the assays because of the relevance of determining the potential selectivity of the phytotoxic compounds on dicotyledons and monocotyledons (Lotina-Hennsen et al., 2006). Two separate experiments were conducted: the first experiment was designed to test for differences between effects of fresh leaves and dry leaves of both species and the second experiment to evaluate the difference in magnitude of the effects across different seasons.

For the first experiment, dry leaves of H. psiadioides and B. patens were obtained by collecting fresh leaves and leaving them exposed to natural temperature conditions in the laboratory until they were dried, for about ten days. For the treatment with fresh material, leaves were collected and immediately used in the assays. The control group consisted of absence of leaves.

The treatments were arranged in a completely random design with four repetitions. Each repetition corresponded to 50 diaspores of lettuce or onion, obtained from commercial dealers and sown in a layer of filter paper (11 cm2, 0.16 mm thick) moistened with 7 mL of distilled water, inside germination boxes of 11 cm x 11 cm x 4 cm. Heterothalamus psiadioides and B. patens leaves (5 g) were slightly chopped, wrapped in cloth (tulle) and attached with double-sided tape to the inner face of the box lid without direct contact with the seeds, but allowing the compounds to volatilize into the airspace within the box. The leaves were attached in the corners of the box lid to avoid interfering in light supply for the seeds. The boxes were sealed with plastic film to minimize loss of the volatiles and were incubated in a germination room at an average temperature of 21 °C, with a daily light/dark cycle of 12 h/12 h. The illumination was provided by white fluorescent lamps (20 W), with an irradiance of48 ^mol m-2 s-1.

For the germination assay, the diaspores were sown and the treatments were added. The germinated diaspores were recorded every 12 and 24 h, for lettuce and onion, respectively. Speed of accumulated germination (AS) was calculated using the following formula: AS = [N1/1 + N2/2 + ... + Nn / n], where N1, N2, and Nn are the

cumulative numbers of seeds that germinate by times 1, 2.....n

(Anjum and Bajwa, 2005). The final germination percentage of the diaspores (germination rate) was calculated after 96 h (lettuce) and 240 h (onion). For growth experiments, the lettuce and onion diaspores were previously germinated (24 h for lettuce and 96 h for onion) and the treatments were added. The seedlings remained exposed to the volatiles for 72 h. After this period, the length of shoot and root (cm) of 20 seedlings per repetition was measured.

The experiment on differences of effects of the volatiles on germination and growth of the two target species across seasons was conducted at four different times of the year, in winter, spring, summer and autumn (from June 2012 to May 2013), with fresh leaves of H. psiadioides and B. patens. Only fresh leaves of the plants were used because they were hypothesized to show stronger effects than dry leaves and should allow for a better characterization in the variation in phytotoxic effects of the plants. Methods followed those described above for the first experiment.

2.3. Statistical analysis

The parameters analyzed (germination rate, speed of germination, length of shoot and length of root) in the experiment with fresh and dry leaves of H. psiadioides and B. patens were submitted to one-way PERMANOVA univariate analysis, comparing all the treatments by randomization tests with 10,000 permutations and including pairwise comparison. In the second experiment, the seasonal variation in these parameters was evaluated, through two-way PERMANOVA that tested the interaction between treatments and seasons, also using randomization tests. Still, to test for differences between treatments specifically in each season, groups were compared through one-way PERMANOVA, including pairwise comparison. PERMANOVA enabled a conventional ANOVA approach to be conducted without transformation on the raw data, since it is not based on assumptions of normality and homogeneity of variances (Anderson, 2001). The analysis used Euclidean distances and considered a significance level of p < 0.05. The tests were performed using the software MULTIV (Pillar, 2009).

3. Results

In the germination bioassays with fresh and dry leaves of the plants, the volatiles from fresh leaves of H. psiadioides caused a significant, though not always very strong, reduction on the speed of germination and germination rate of lettuce (37% and 7%, respectively, when compared to germination rate of the control treatment) and of onion (37% and 47%, respectively). The dry leaves of H. psiadioides only reduced the speed of germination of lettuce (39%). Fresh and dry leaves of B. patens did not cause any significant effect on lettuce and onion germination (Fig. 1A and B).

The effects of the volatiles from H. psiadioides and B. patens on seedlings growth are shown in Fig. 1C and D. The dry leaves of both shrubs caused lower inhibition on growth of the target plants in relation to the fresh leaves, as shown in the germination assays. The fresh leaves of H. psiadioides caused the greatest reduction on shoot and root length of the target plants (21% and 52% for lettuce and 43% and 46% for onion, respectively). The dry leaves of H. psiadioides reduced shoot and root length of lettuce (13% and 28%) and shoot length of onion (28%). B. patens fresh leaves also caused negative effects on the shoot and root length of lettuce (11% and 32% of reduction) and shoot of onion (13% of reduction). The dry leaves of B. patens only reduced lettuce root (15%) and onion shoot length (14%).

In relation to the experiment on differences of effects of the plants volatiles across seasons, the interaction between treatment and season was significant in almost all the parameters evaluated, except for the speed of germination and the root length of onion, i.e. differences among treatments vary according to the season (Table 1). In winter, H. psiadioides caused a pronounced inhibition in all the parameters

90 75 60 45 30 Ô 15 0

a aab ab

Co Bp Hp Lettuce

■ Dry leaves □ Fresh leaves

Co Bp Hp Onion

40 35 30 25

M 20 M 20

15 10 5 0

Co Bp Hp

Lettuce

■ Dry leaves □ Fresh leaves

a a ab ab

Co Bp Hp

■s 1

(3 0.5

Co Bp Hp

Lettuce

■ Dry leaves □ Fresh leaves

Co Bp Hp

is 2.1

■3 1.5

r 0.9 oot 0.6

£ 0.3

Co Bp Hp

Lettuce

■ Dry leaves □ Fresh leaves a

abc iP bcd Id

Co Bp Hp

Fig. 1. Germination rate (A), speed of germination (B), root length (C) and shoot length (D) of lettuce and onion exposed to the volatiles from B. patens (Bp) and H. psiadioides (Hp) and control group (Co). Data presented as mean and standard deviation. Treatments sharing the same letter do not differ significantly according to one-way PERMANOVA for the same target species, at p < 0.05 level.

and in spring and autumn almost all the parameters were also substantially affected by the plant volatiles. In summer, however, only the parameters related to seedling growth were considerably inhibited by H. psiadioides volatiles. The seasonal pattern in the effects of B. patens on the target plants was similar to the pattern observed for H. psiadioides: the inhibition by B. patens volatiles was pronounced in some parameters in winter, spring and autumn. Nevertheless, almost only the parameters related to seedling growth were considerably affected by the plant. In summer, the inhibition by B. patens was not pronounced for any parameter. Besides, the inhibition in almost all the parameters was greater when the target plants were exposed to H. psiadioides; the reduction by B. patens was only slightly greater on the speed of germination of lettuce in spring and summer, but in these cases the inhibition was not substantial and does not consist in considerable difference between the treatments.

4. Discussion

The results of the assays confirmed our hypothesis that both H. psiadioides and B. patens present phytotoxicity, affecting germination and growth of seedlings. A greater phytotoxic effect of H. psiadioides compared to B. patens was observed. The volatiles released from H. psiadioides negatively affected all the parameters evaluated in the germination and growth assays. Baccharis patens affected only the parameters related to growth (shoot and root length), which are usually more sensitive to phytotoxic effects than parameters related to germination (speed of germination and germination rate) (Chon and Nelson, 2010). Besides, the inhibition on the target plants was stronger when they were exposed to H. psiadioides leaves. The differences in the effects of H. psiadioides and B. patens are likely associated to the amount of phytotoxic volatiles released by them and to differences in their

Inhibition (%) of germination rate, speed of germination, root length and shoot length of lettuce and onion exposed to the volatiles from fresh leaves of H. psiadioides (Hp) and B. patens (Bp) in the four seasons of the year.

Season Treatment Germination assays — inhibition (%) Growth assays — inhibition (%)

Germination rate Speed of germination Shoot length Root length

Lettuce Onion Lettuce Onion Lettuce Onion Lettuce Onion

Winter Hp 7a 47a 37a 37a 21a 23a 52a 20a

Bp 0 15 02 08 11 13a 32a 14

Spring Hp 2 25a 04 31a 32a 43a 42a 46a

Bp 0 08 10 15 24 19a 19a 10

Summer Hp 0 10 01 10 03 21a 30a 21a

Bp 0 04 09 03 00 08 15 06

Autumn Hp 6 12 25a 07 11a 19a 33a 26a

Bp 2 11 21a 07 00 11a 23 14

Treatment P-value 0.009 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

Season 0.04 <0.001 <0.001 <0.001 <0.001 <0.001 0.02 <0.001

Treatment » season 0.01 <0.001 <0.001 0.5 <0.001 <0.001 <0.001 0.18

a Significant differences in relation to control (no treatment), according to PERMANOVA, with p < 0.05.

chemical composition. Most of the volatiles in these plants are in the essential oil of their leaves, which showed 1.92% and 0.2% yield for H. psiadioides and B. patens, respectively (unpublished data). Therefore, H. psiadioides is able to release a larger amount of volatiles than B. patens. The major compounds of H. psiadioides essential oil, obtained from the same population studied in this article, are monoterpenes hydrocarbons (77.31%), mainly (3-pinene (43.17%), A3-carene (13.73%) and limonene (6.32%) and although this oil affected germination and growth of other plants, (3-pinene, the major compound, did not show phytotoxicity (Schmidt-Silva, 2012). Therefore, the phytotoxic effects of H. psiadioides must result from interactive effects of several compounds, as assumed for the effects of plants allelochemicals in field conditions (Inderjit et al., 2011). The essential oils of several Baccharis species have already been characterized (Frizzo et al., 2008; Simoes-Pires et al., 2005), and although the species share most of the compounds, the major ones vary among them: while monoterpene hydrocarbons are the predominant fraction in some species, in others oxygenated sesquiterpenes or oxygenated monoterpenes are more significant. The chemical composition of B. patens has not been described up to now. Thus, further studies characterizing the volatile compounds of B. patens essential oil will shed light on the phytotoxic activity of this species.

The action of allelochemicals on germination and growth of plants is diverse and affects different physiological functions, as cell division, metabolic processes, photosynthesis and respiration. A mechanism related to modification in cell division was already reported for H. psiadioides: the essential oil of this plant caused a decrease in mitotic activity and an increase of chromosomal abnormalities in target plants (Schmidt-Silva et al., 2011). Besides, effects of allelochemicals on seed germination appear to be mediated mainly through a disruption of normal cellular metabolism. One of the effects can be in reserve mobilization, a process that usually takes place rapidly during early stages of seed germination and seems to be delayed or decreased by phytotoxicity (Gniazdowska and Bogatek, 2005). Seedlings growth is usually affected by disturbances of photosynthesis (Qian et al., 2009) and respiration (Hejl and Koster, 2004; Mushtaq et al., 2013), which are some of the most observed physiological effects of many allelochemicals.

Considering the differences on the phytotoxicity of fresh and dry leaves ofH. psiadioides and B. patens, our hypothesis that the fresh leaves of the plants present stronger effects than the dry leaves was confirmed. This indicates that in natural ecosystems the phytotoxicity of the shrubs leaves must decrease until they are incorporated into the soil. Besides, due to the low phytotoxicity of the dry leaves of H. psiadioides and B. patens, they would need to drop large amounts of leaves on the soil to affect germination of other species; as these plants are not deciduous, this is unlikely to occur. Thus, it seems difficult that the volatiles from these shrubs are able to affect the germination of other species in plant communities. On the other hand, the strong effects of the fresh leaves of the shrubs, mainly H. psiadioides, on growth of the target plants suggest that the volatiles may affect the development of other neighboring species when the leaves are still on the plants, as assumed for other aromatic shrubs which were reported to affect nearby species in field conditions (Ibanez and Zoppolo, 2011; Muller et al., 1964).

The variation in magnitude in the phytotoxic effects of B. patens and H. psiadioides according to the period that they were collected confirmed our hypothesis that the plants phytotoxicity changes seasonally. The plants showed generally lower phytotoxicity in summer than in the other seasons — a pattern that must be related to seasonal fluctuations of environmental parameters and trade-offs between primary and secondary metabolism. In summer, plants are exposed to higher temperatures and solar radiation, which were reported to have an inverse relation with secondary metabolite accumulation in plant tissues (Lur et al., 2009; Yu et al., 2003). Besides, plants growth increases in summer; studies revealed trade-offs between primary biological functions of plants, as growth, and resources allocation for chemical defense (Donaldson et al., 2006; Ztist et al., 2011). However, other factors,

such as interactions with pathogens (Heil and Bostock, 2002) and herbivores (Karban, 2007) or water (Gray et al., 2003) and nutrients (Tharayil et al., 2009) availability are also able to change secondary metabolite production and consequently the plants phytotoxicity. The variation in the phytotoxic effects of H. psiadioides and B. patens evidences the importance of conducting allelopathy studies along different seasons; if this variation is not considered, the results may not reflect the potential phytotoxicity of a plant species correctly or, on the other hand, the phytotoxic effects may be overestimated.

The method used in this study was effective to determine volatiles phytotoxicity even though only a small amount of plants leaves was used. The similar pattern of phytotoxicity observed for both shrubs at all times when the assays were conducted, i.e. H. psiadioides showing stronger effects than B. patens, indicates the efficacy of the technique. Besides, an advantage of the method is that it allows carrying out phy-totoxicity studies without interferences, as shown in other methods frequently used: extracts, for example, can inhibit test species through altering pH and osmotic potential (Wardle et al., 1998), masking the true phytotoxic effects of a plant. Assays with essential oils, one of the most used ways to characterize volatiles phytotoxicity (e.g. Pawlowski et al., 2012; Zahed et al., 2010), also show a constraint: the concentration of these substances is much greater than what would be naturally volatilized by plants. However, even though we can assume that our method should be more similar to natural emission of volatiles, it must be considered that the interactions between plants and allelochemicals in the plant community occur in a more dynamic ecological situation and that phytotoxic effects observed in laboratory conditions may not be relevant in nature. Many plants release compounds into the surrounding environment with minor consequences in their native habitat due to a long coevolutionary history with other plants (Thorpe et al., 2009). Besides, there are several biotic and abiotic factors that are able to change allelopathic activity (Inderjit et al., 2011).

5. Conclusions

The shrubs H. psiadioides and B. patens show phytotoxicity, which is greater for H. psiadioides. The phytotoxic effects of both species differ in magnitude across seasons, evidencing the relevance of considering this variation in allelopathy studies. The strong effects of the fresh leaves of these shrubs indicate that the volatiles may affect the development of other neighboring species when the leaves are still on the plants; thus, allelopathy is a possible mechanism involved in the dominance pattern shown by H. psiadioides and B. patens. However, additional studies under field conditions are needed to evaluate if these shrubs, through allelopathic interactions, are effectively able to affect other species in plant communities.

Acknowledgments

We thank the Coordena^ao de Aperfei^oamento de Pessoal de Nivel Superior (CAPES) for the MSc scholarship granted to the first author. We also thank MSc. Angela Pawlowski, Diana Carla Lazarotto and two anonymous reviewers for their valuable suggestions on the manuscript.

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