Phytotoxic effect of Sesbania virgata(Cav.)Pers.on seeds of agronomic and forestry species

Phytotoxic effect of Sesbania virgata(Cav.)Pers.on seeds of agronomic and forestry species

Vera Lygia El Id•Ba´rbara Vale da Costa•Daiane Salete Broch Mignoni•Marina Belloni Veronesi•Kelly Simo˜es•Marcia Regina Braga•Nelson Augusto dos Santos Junior

Sesbania virgata(Cav.)Pers.is a pioneer species native to South America able to release allelochemicals thataffectgermination and developmentof other plant species.The aim of this work was to evaluate the allelopathic effect of S.virgata on the germination and development of co-occurring species from gallery forest and on agronomic species.Two forest native species,Enterolobium contortisiliquum(Vell.)Morong and Sapindus saponaria L.,and two agronomic species(these as control), Oryza sativa L.and Solanum lycopersicum L.were used in the lab and greenhouse assays with seeds and leaf extracts of S.virgata.Agronomic species were more affected than native species when co-germinated with seeds of S.virgata.The germination percentage and speed germination index of the agronomic species were reduced in both in vitro and greenhouse assays.In the same assays,the seeds of native species showed no significantdifferences in the parameters mentioned.However,the initial growth of the four species assayed was affected,with reduction in plant length and shoot diameter followed by significant reduction in plantlet shoot and root weights.In the assays with irrigation of S.virgata leaf extracts,no significant inhibitory effects on germination were observed for allspecies.Height and shoot diameter of the native species were not affected by the leaf extracts,nor were fresh and dry weights.However,these parameters increased in the agronomic species as they were irrigated with leaf extracts. Based on our data we conclude that seed leachates of S. virgata affect germination and seedling development of other species,suggesting that its invasive behavior is due mainly to seed allelochemicals.Although with less pronounced effects on native species,allelopathy of S.virgata mightalso influence Interspecific competition in its natural environment.

Allelochemicals·Allelopathy·Catechin· Fabaceae·Gallery forest

Introduction

Sesbania virgata(Cav.)Pers(Fabaceae-Faboideae)is a fast-growing pioneer shrub,native to South America, which is found in the south,southeast,and midwestregions of Brazil(Pottand Pott1994).Itproduces many seeds with long-term viability that are dispersed within indehiscent legume fruits that float in the water(Pott and Pott 1994; Simo˜es et al.2008).This shrub is a promising species for the restoration of degraded areas and to revegetate Gallery forests due its hardiness,tolerance of low fertility soils,and capacity to compete with herbaceous plants(Coutinho et al.2005;Zanandrea et al.2010).

Sesbania virgata is also tolerant to long periods of soil inundation,being able to survive under flooded soil during 56 days without damage(Zanandrea et al.2010).It has been described as an invasive species in flooded and damp soil,especially in irrigated rice fields(Kissmann and Groth 1999).In northeast Brazil,S.virgata was almost unknownfor about two decades,but recently started to colonize riverbanks and man-made water reservoirs,showing an invasive behavior,and causing serious impacts on ecosystems due the formation of a massive dominant population that suppressed the natural regeneration of native plants(Souza et al.2011).

According to recentreportsaboutplantinvasiveness,some features are necessary to allow species to become potentially invasive:rapid relative growth rate;large production ofsmall, readily dispersed seeds;extended longevity in the soil,and high rate of germination;flowering and fruiting more prolonged;high reproductive potential;pioneering;absence of natural enemies;and the production of phytotoxins that mediate plant–plant interspecific interference reducing the establishmentof competing species.S.virgata presents some of these characteristics,including the production and the release of phytotoxins(Simo˜es etal.2008).

Due to their invasive behavior and demonstrated ability to release phytotoxins from their seeds,Sesbania species may fitthe seed-related allelopathic invasiveness pattern.It has been reported that the seeds of Sesbania spp.contain toxic compounds that restrict the growth of other plants (Buta 1983;Powelletal.1990;Van Staden and Grobbelaar 1995;Ceballos et al.1998;Simo˜es etal.2008).S.punicea (Cav)Benth.is described as a noxious weed in the Republic of South Africa,invading abandoned fields and out-competing natural vegetation,and forming dense populations.This behavior seems to be associated with the presence of the potentalkaloid sesbanimide in its seeds that can inhibit the seedling growth of several species(Gorst-Allman et al.1984;Van Staden and Grobbelaar 1995).

Allelochemicals are released after the start of seed imbibition and can contribute to the invasive behavior of some species(Ndakidemi and Dakora 2003).Phenolic metabolites are released quickly during the water absorption process and are found around the germinated seed.For instance,in S.drummondi(+)-catechin was found in great quantity around the germinating seeds whereas(-)-catechin predominated in S.vesicaria(Ceballos et al.1998).

The flavonoid catechin was characterized as the major phytotoxin leached by seeds of S.virgata,being released at the beginning ofgermination and inhibiting the rootgrowth of rice and Arabidopsis plantlets(Simo˜es et al.2008).The allelopathic effects of catechin exudation has been well demonstrated and discussed in Centaurea maculosa,an Asteraceae native from Eurasia that showed devastating effecton native grasses of the United States.Its roots exude a racemic mixture of(+)and(-)-catechin,which appears to be released in pulses of high concentration,conferring advantages to C.maculosa at critical periods of development(Bais et al.2002;Perry et al.2007).Recently,the aggressiveness of C.maculosa and other weeds has being elucidated by the‘Novel Weapon’hypothesis(He et al. 2009;Weidenhamer and Callaway 2010).This hypothesis suggests that plants that have co-evolved with species that secrete phytotoxins,in general,tend to develop resistance to these metabolites,which may not occur with other communities that are more susceptible to them.Thus, invasive species have greater competitive advantage in a new region when compared to their native region(Weidenhamer and Callaway 2010).

Little is known about the phytotoxicity of catechin and other metabolites on native species that co-occur with S. virgata in their natural environment.Additionally,is not known whether catechin can be found in the leaves of S. virgata.Thus,the present work investigated the allelopathic effect of S.virgata Cav.Pers on forest tree seeds of late successional stages that co-occur in riparian environments and on agronomic species.

Materials and methods

Biological material

Seedsand leavesof Sesbania virgata Cav.Perswere harvested from ten natural populations growing in Lavras MG,Brazil. Seeds of Enterolobium contortisiliquum(Vell.)Morong and Sapindus saponaria L.,species thatoccurs with S.virgata in its environment,were collected from various matrices growing in the Botanical Garden of Sa˜o Paulo,SP,Brazil.Rice seeds(Oryza sativa L.var.Ourominas)were kindly provided by EMBRAPA‘Arroz e Feija˜o’,Santo Antonio de Goia´s, GO,Braziland tomato(Solanum lycopersicum L.var.Santa Cruz Kada)Isla®seeds were purchased in the local market. These agronomic species were selected for the assays as control,since S.virgata has been described as an invasive species of irrigated rice fields,and tomato,which although is notinvolved in naturalallelopathic associations with S.virgata,is very sensitive to low allelochemical concentrations (Lara-Nu´n˜ez etal.2006,2009).

Co-germination in vitro and greenhouse assays

In laboratory assay,one seed of each species was co-germinated with 0(control),5 or 10 seeds of S.virgata inside 9 cm Petri dishes containing filter paper moistened with 5 mL of distilled water.Plates were maintained at 25°C and a photoperiod of 12 h in a germination chamber (BOD).Germination was evaluated daily,until the 10th day,by registering the protrusion of the primary root (≥5 mm).The experiment was set in four replicates.Each replicate was formed by four Petri dishes and each one contained a seed of each assayed species.

In greenhouse co-germination assay,the test species were sown in 150 cm3plastic pots(one seed per pot)containing sterilized commercial substrate MecPlant®.The tubes were arranged on desks inside a glasshouse with daily irrigation.Each species was co-germinated with 0 (control),5 or 10 seeds of S.virgata using four replications per treatment(each consisting of four plastic pots).Germination was evaluated daily by registering plant emergence above the substrate up to 20 days.At the end of the analysis,the germination percentage(%,G)and germination speed index(GSI)were calculated according Maguire (1962).Fourteen days after germination,S.virgata seedlings were removed,and the test species were maintained under the same experimental conditions for 30 more days. At the end of the experiment(50 days),plant height,shoot diameter,and root and shoot fresh and dry weights were evaluated.

Plant Extraction

Freshly harvested leaves of S.virgata from the matrices that produce catechin were crushed using a rotator knife mixer and extracted in distilled water(1 g per 5 mL), vacuum filtered,and freeze-dried.The residue was suspended again in distilled water to 0.1,0.5,and 1.0%.The presence of catechin was confirmed after partition of the extracts with ethyl acetate(Simo˜es et al.2008)and subsequentanalysis by HPLC.Aliquots of the extracts(20μL) were analyzed on a C18 column in a Varian High Performance Liquid Chromatographer using a linear gradient of water and methanolacidified with 1%acetic acid(from 20 to 100%methanol)with a flow of 0.8 mL min-1.(+)-Catechin Sigma-Aldrich was used as standard.

Quantification of catechin

To quantify the levels of catechin,100 mg of S.virgata leaf extracts obtained as described above were submitted to partition with ethyl acetate.Organic fractions were dried and derivatized for analysis in a gas chromatograph coupled to a mass spectrometer(GC 6890 and MS 5973 N, Agilent Technologies,USA).The dried extracts were suspended in 150μL of pyridine and 50μL of BSTFA and subjected to stirring and heating at 75°C for 1 h.The samples were then transferred to glass vials,which were sealed,and automatically injected into the GC/MS system containing a HP5 column of 0.25 mm thickness and 30 m length(Supelco,Bellfonte,USA).The sample injection temperature was 230,250°C was the temperature of interface,and ion source temperature was 150°C.Helium was utilized as the carrier gas ata flow rate of 1 mL·min-1. The analysis followed the program:5 min isothermal heating at 70°C,followed by a ramp heating at 5°C per minute until 310°C,and a final minute of heating at 310°C.The chromatograms and mass spectra generated were analyzed using the Chemstation software(Agilent Technologies,USA)and detected peaks were compared with standards of catechin(Sigma-Aldrich)and the NIST mass spectral library.The catechin contents were determined using a catechin standard curve according the same procedure as described for the samples.Initially,for the quantification of the extracts,we used 10 different natural populations(M1-M10).After this process,to produce the extracts,we chose only those that showed catechin.

Germination and plant growth assays with S.virgata extracts

The extract assays were performed as described above for the co-germination experiments(each treatment with four repetitions and each one consisting of four recipients).In the in vitro assays,seeds of S.virgata were replaced by the aqueous plant extracts at 0,0.1,0.5 and 1%.In these experiments,seeds of the testspecies were moistened daily with 5 mL of the extracts and of distilled water(control). In greenhouse,seeds of test species were sown in 150 cm3plastic pots containing sterilized commercial substrate MecPlant®.Germination was daily evaluated by registering plant emergence above the substrate up to 20 days. Germination was monitored and germination percentage and germination speed index calculated as already described in the co-germination assay.After that,each plantlet received 7 mL of S.virgata extracts and of distilled water (control)four days a week.Test species were maintained under the same experimental conditions as above for 3 months when plant height,shoot diameter,and root and shoot fresh and dry weights were evaluated.

Design and statistical analysis

All assays were installed in completely randomized design (CRD).The statistical analysis was performed using SISVAR 5.1.(Ferreira 2011).Data were subjected to variance analysis(ANOVA)and significant differences between means were identified by the Tukey test at5%probability.

Results

Seeds of S.lycopersicum co-germinated with 5 or 10 seeds of S.virgata showed reduced germination percentage and germination speed index in both in vitro and greenhouse assays(Table 1).These effects were stronger for in vitro assays in which germination was reduced to ca.50%ofthe control when compared to greenhouse experiment(reduction of 15–20%).A delay of one third in germination speed was observed in tomato seeds germinated with S. virgata in Petridishes whereas in the greenhouse assay thereduction was ca.50%.The germination of O.sativa was also reduced but only by 10%for in vitro assays and by 35–40%in greenhouse assay.No significantdifferences in the germination speed index were observed between cogerminated and control rice seeds in both experiments (Table 1).

Table 1 Germination(%,G) and germination speed index (GSI)evaluated after cogermination with 0(C,control), 5 or 10 seeds of Sesbania virgata(Sv)

Fig.1 Height(bars)and diameter(geometric points)of A Oryza sativa,B Solanum lycopersicum,C Enterolobium contortisiliquum, and D Sapindus saponaria measured 50 days after co-germination with Sesbania virgata(Sv)seeds.0 Sv corresponds to control,5 Sv to five seeds of S.virgata,and 10 Sv refers to ten seeds of S.virgata. The values are means(±SD)of four replicates.Capital letters compare height and lowercase letters compare diameter(of each species)among treatments by Tukey 5%

Seeds of native species were less sensitive to the effects of S.virgata seeds.In general,no significant differences were observed in germination percentage or germination speed index of E.contortisiliquum or S.saponaria in in vitro or greenhouse assays(Table 1).A low inhibitory effect in the speed of germination was observed only when E.contortisiliquum was sown together with 10 seeds of S. virgata.

Fig.2 Fresh weight of A Oryza sativa,B Solanum lycopersicum, C Enterolobium contortisiliquum,and D Sapindus saponaria measured 50 days after co-germination with Sesbania virgata(Sv)seeds. RFW is root fresh weight;(SFW is shoot fresh weight.0 Sv corresponds to control,5 Sv to five seeds of S.virgata,and 10 Sv refers to ten seeds of S.virgata.The values are means(±SD)of four replicates.Capital letters compare root fresh weight and lowercase letters compare shootfresh weight(of each species)among treatments by Tukey 5%

Co-germination with seeds of S.virgata also affected initial growth of the four species assayed(Fig.1).Plant length and shoot diameter were reduced to 50%in tomato and rice seedlings that co-germinated with S.virgata in greenhouse conditions(Fig.1).The growth of E.contortisiliquum was less inhibited by S.virgata,with no reduction in the shoot diameter and only 20%diminution in plantheight(Fig.1),while S.saponaria showed a decrease of more than 50%in both parameters(Fig.1).These inhibitory effects on growth were confirmed by the fresh and dry weight determinations,which showed significant reduction of plantlet shoot and root weights for all species (Figs.2,3).

Fig.3 Dry weight of A Oryza sativa,B Solanum lycopersicum, C Enterolobium contortisiliquum,and D Sapindus saponaria measured 50 days after co-germination with Sesbania virgata(Sv)seeds. RDW is root dry weight;SDWis shoot dry weight.0 Sv corresponds to control,5 Sv to five seeds of S.virgata,and 10 Sv refers to ten seeds of S.virgata.The values are means(±SD)of four replicates. The values are means(±SD)of four replicates.Capital letters compare root dry weight and lowercase letters compare shoot dry weight(of each species)among treatments by Tukey 5%

Fig.4 GC-MS quantification of catechin in the leaf extracts of 10 different natural populations of S.virgata(matrices M1 to 10) growing in Lavras(MG,Brazil)

Although catechin has been detected in the S.virgata leaf extracts(Fig.4),no effects on germination or germination speed index were observed(Table 2).Height and shoot diameter of the native species were notaffected by the leaf extracts regardless the increasing concentration(Fig.5). The same was observed for fresh and dry weights(Figs.6, 7).In contrast,a stimulating effecton plantheightand fresh weight was seen on agronomic species when the highest extract doses were provided(Figs.5,6).Increased dry weightwas observed when O.sativa plantlets were irrigated with 0.5 and 1.0%of S.virgata leaf extracts(Fig.7).

Discussion

Seeds of S.virgata affected germination and growth of S. lycopersicum and O.sativa in both in vitro and greenhouse assays.These findings indicate the allelopathic potentialof S.virgata that may be related to its invasive behavior in irrigated rice fields and also in suppressing other natural species in invaded areas.Indeed,in a previous study with S. virgata,Simo˜es et al.(2008)reported that metabolites released by its seeds inhibited the growth of O.sativa and also of Arabidopsis thaliana.These effects were attributed to the presence of(+)-catechin in the seed coat that is leached during imbibition of the S.virgata seeds.Our data show that the inhibitory action was the same regardless of the increase in the numberof S.virgata seeds co-germinated.

This indicates that the dose of ca.1.2 mg of catechin leached by 5 seeds(235μg per seed,Simo˜es et al.2008) was enough to cause the maximal effects detected.Under our experimental conditions,tomato seeds were more affected by co-germination with S.virgata than rice in in vitro assays while the inverse was seen when co-germination was performed under greenhouse conditions.

The comparison of catechin phytotoxicity in natural soil and in vitro conditions showed thatthis allellochemicalcan be modified depending on the soil dynamic(Inderjit et al. 2008).This could explain the differences in delayed germination observed in tomato and rice seeds between our in vitro and greenhouse conditions in the co-germinated experiments with S.virgata.

S.virgata has been described as an invading species in irrigated rice fields(Kissmann and Groth 1999).Therefore, it is reasonable to suppose that the effects of S.virgata seeds on rice germination and plantlet growth seen in this work could also occur in field,leading to reduced plant growth and productivity.

Rice has been extensively studied with respect to its allelopathy once many varieties were found to inhibit the growth of several plant species when they were grown together with these plants under field or/and laboratory conditions.Rice plants possibly release allelochemicals into the neighboring environment that are responsible for these effects(Kato-Noguchi 2012 and refs therein). Although the release of allelochemicals by the rice variety used in the present work has not been assessed,if it occurred,the released compounds did not affected seed germination and seedling growth of S.virgata.Recently, Kato-Noguchi and Ino(2013)reported that the allelochemical momilactone B exuded by rice,besides having a phytotoxic effect on barnyard grass,is also sensed by this species,increasing its own allelopathic activity.Whether this interplay between species could also occur between rice and S.virgata itis an interesting question that remains to be investigated.

Table 2 Germination(%,G)and germination speed index(GSI)evaluated after treatment with Sesbania virgata leaf aqueous extracts at0.1,0.5 and 1%

Fig.5 Height(bars)and diameter(geometric points)of A Oryza sativa,B Solanum lycopersicum,C Enterolobium contortisiliquum, and D Sapindus saponaria measured 110 days after irrigation with Sesbania virgata leaf aqueous extracts at 0.1,0.5 and 1%.Control corresponds to irrigation with distilled water.The values are means (±SD)of four replicates.Capital letters compare height and lowercase letters compare diameter(of each species)among treatments by Tukey 5%

When compared to agronomic species,the phytotoxic effect of S.virgata seeds on germination was less intense on co-occurring forest seeds.These results are in agreement with the novel weapons hypothesis that suggests that allelochemicals exuded by one species are relatively ineffective against their natural neighbors but may be highly inhibitory to newly encountered plants(Callaway and Ridenour 2004;Bais et al.2006).According to Gatti (2008),the process of co-evolution can be related to the ability of some plants to detoxify or metabolize allelochemicals and this could explain the lower sensitivity of E. contortisiliquum and S.saponaria seeds to seed leachates of S.virgata when compared to tomato and rice.However, recent evidence obtained from field studies with three invasive species in Europe indicated that germination suppression by allelopathic compounds from invasive species was similar to that of native plant communities(Del Fabbro et al.2013).According to Ferreira(2004), germination is less sensitive to allelochemicals than is the growth of seedlings and although germination may not be inhibited by phytotoxic substances,the presence of allelopathic compounds can lead to the appearance of abnormal plantlets.In fact,growth of S.saponaria was significantly affected by S.virgata while its germination was not inhibited.

Fig.6 Fresh weight of A Oryza sativa,B Solanum lycopersicum, C Enterolobium contortisiliquum,and D Sapindus saponaria measured 110 days after irrigation with Sesbania virgata leaf aqueous extracts at 0.1%,0.5%,e 1%.RFW is root fresh weight;SFW is shoot fresh weight.Control corresponds to irrigation with distilled water.The values are means(±SD)of four replicates.Capital letters compare rootfresh weightand lowercase letters compare shoot fresh weight(of each species)among treatments by Tukey 5%

Fig.7 Dry weight of A Oryza sativa,B Solanum lycopersicum, C Enterolobium contortisiliquum,and D Sapindus saponaria measured 110 days after irrigation with Sesbania virgata leaf aqueous extracts at 0.1%,0.5%e 1%.RDW is root dry weight;SDW is shoot dry weight.Control corresponds to irrigation with distilled water.The values are means(±SD)of four replicates.Capitalletters compare root dry weight and lowercase letters compare shoot dry weight(of each species)among treatments by Tukey 5%

In contrast,growth of E.contortisiliquum was only slightly affected by S.virgata seeds.Despite the differences in the sensitivity of native species to allelochemicals, the release of these compounds in the soil can inhibit the establishment of seedlings,influencing the intraspecific competition during forestregeneration.Allelopathic effects of leaf extracts are well documented for several plant species(e.g.Alrababah et al.2009;Braine et al.2012; Ayub et al.2013;Won et al.2013).Recently,Ayub et al. (2013)reported the phytotoxic effectof severallegume leaf extracts on germination and growth of maize,and found more pronounced effect in a sesbania species.Although S. virgata leaves also contain catechin(Fig.4),no inhibitory activity was observed when agronomic or native species were irrigated with their extracts under our experimental conditions.

In fact,a stimulating effect was observed in the agronomic species,with the increase in growth and fresh mass in rice and tomato.Itis wellknown thatlow concentrations of allelochemicals can often stimulate growth in target species(Rice 1984).Low doses of catechin(50 and 100μM)were found to enhance growth in Arabidopsis thaliana,possibly by mimicking or modulating the auxin (IAA)content(Rani et al.2011).

Catechin estimated in leaf extracts of S.virgata was ca. 18 ng mg-1a concentration much lower than thatfound in seed leachates(235μg per seed,Simo˜es et al.2008),and this could explain the stimulatory growth effects observed when the agronomic species were irrigated with 0.5 or 1.0%of leaf extracts.

The fact thateffects of S.virgata reported here were less intense on native species than on agronomic plants could contribute to reinforce the‘Novel Weapon’hypothesis (He etal.2009,among others).However,our data together with recentresults,which indicate that seed exsudates of S. virgata delay carbohydrate mobilization in seed of cooccurring species(Veronesi 2013),suggest that the allelochemicals of S.virgata also interfere with the biochemical processes taking place during germination of native species.This effectis consistentwith invasive behavior and suppression of natural vegetation recently reported for this species(Souza et al.2011)and may interfere with the performance of co-occurring species in natural conditions or in restoration programs.

In conclusion,our findings demonstrate that seed leachates of S.virgata have stronger effects on agronomic species than on native plants,and that the allelochemicals exuded affected germination and their effects lasted until the early development of plants.The absence of inhibitory activity in leaf extracts indicates that the invasive behavior of S.virgata is due mainly to allelochemicals in its seeds. Albeit with less pronounced effects on native species, allelopathy of S.virgata might also influence interspecific competition in its natural environment.

AcknowledgmentsThis work was supported by Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico(CNPq,Grant 474325/2009-1)and Fundac¸a˜o de Amparo a Pesquisa do Estado de Sa˜o Paulo(FAPESP,Grants 2005/04139-7 and 2012/16332-0).M.R. Braga acknowledges CNPq for the research fellowship.Thanks are due to Dr.Danilo C.Centeno,Universidade Federal do ABC,for the help with catechin quantification.

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29 November 2013/Accepted:26 February 2014/Published online:5 February 2015

ⒸNortheast Forestry University and Springer-Verlag Berlin Heidelberg 2015

The online version is available at http://www.springerlink.com

Corresponding editor:Zhu Hong

V.L.El Id·B.V.da Costa·N.A.dos Santos Junior(✉)

Seed Department,Institute of Botany,PO Box 68041,Sa˜o Paulo, SP 04045-972,Brazil e-mail:njunior@ibot.sp.gov.br

D.S.B.Mignoni·M.B.Veronesi·K.Simo˜es·M.R.Braga Plant Physiology and Biochemistry Department,Institute of Botany,PO Box 68041,Sa˜o Paulo,SP 04045-972,Brazil