Phytotoxic Extracts as Possible Additive in Subsurface Irrigation Drip for Organic Agriculture

V.Romanucci,A.Ladhari,G.De Tommaso,A.De Marco,C.Di Marino,G.Di Fabio,A.Zarrelli†

1Department of Chemical Sciences,University of Naples,Via Cintia 4,80126,Italy

2Department of Biology,University of Naples,Via Cintia 4,80126,Italy

Keywords Hydroalcoholic extracts Lactuca sativa Lycopersicon esculentum Allium cepa Phytotoxicity Anti-radical activity

Abstract The subsurface drip irrigation(SDI)system is a micro-irrigation technique applied below the soil surface through drip lines buried at a depth depending on the characteristics of the soil and on the plants to be irrigated.SDI distributes precise amounts of water directly to the root area,with the possibility of leaving the soil surface dry and less subject to weeds.This system reduces the use of water,herbicides,and environmental pollution.Furthermore,SDI allows the use of urban wastewater,advantageous from the environmentalpoint of view since it reduces the consumptionof ground water and energy costs required for its pumping.In addition,it reduces the use of chemical fertilizers through the enhancement of organic fertilizer content in the waste.However,there are issues related to the use of SDI systems,such as the elimination or reduction of roots that wrap the dripper thus blocking the water flow.It has been hypothesized that it would be useful to add a pure or blended phytotoxic mixture to plastic during the production of drippers,whose herbicidal action dissolves gradually with the passage of water.Five species of plants have been selected in this study:Vetch villosa,Brassica juncea,Secale cereale,Juncus effusus,and Vallisneria natans.The phytotoxicity has been tested in vivo on Lactuca sativa,Lycopersicon esculentum,and Allium cepa.The plants showed the same behavior but the aerial biomass of V.natans resulted the most active ones.The phytotoxicity of the hydroalcoholic extract of each plant was evaluated on the same test organisms,with peak inhibitions up to 60,70,and 80%at concentrations ranging from 10-4to 10-7M.In general,the most active hydroalcoholic infusion was that of V.villosa.Finally,after some chromatographic steps and LC/GC-MS analyses,the most abundant metabolites of the hydroalcoholic extracts were identified.

1 Introduction

The recent forums promoted by international organizations have defined the third millennium as that of the thirst for water(G20 Agriculture Ministers’Action Plan,2017).It is estimated that in the world there is only 0.3%of renewable freshwater with a very differentiated availability,while the promises of genetic engineering of more productive plants will serve little if there is not enough water to feed them.Thecontinuous increase in population and the need to improve the standard of living feed and exacerbate the conflict between different competing uses of freshwater resources:civil,industrial,and agricultural.The agricultural activities are the largest user of the water resource(on average two thirds of global consumption)and efforts are being made to find solutions to the growing water scarcity.Nowadays,irrigation of fruit and vegetable crops in modern agriculture is increasingly carried out with subsurface drip irrigation(SDI)systems(Camp,1988).

The SDI system is a micro-irrigation technique applied below the soil surface through drip lines buried at a depth depending on the characteristics of the soil and the plants to be irrigated.SDI distributes precise amounts of water directly to the root area,with the possibility of leaving the soil surface dry and less subject to weeds.In the last decades all the experimental tests have demonstrated the validity of the SDI in terms of greater productivity of the crops,water saving due to the lack of evaporation loss,and facilitating the mechanization of the various cultivation operations.The genesis of this technique is to be found in soil aridity increased by global climate change pushing employees in the primary sector to resort to systems allowing water savings.SDI systems reduce the use of water,herbicides,and environmental pollution.Furthermore,SDI allows the use of urban wastewater,advantageous from the environmental point of view since it reduces the consumption of ground water and energy costs required for its pumping.In addition,it reduces the use of chemical fertilizers through the enhancement of organic fertilizer content in the waste(Selvaratnam et al.,2016).The current knowledge on the advantages and mechanisms of operation of micro-irrigation,integrated with new agronomic and technological studies,has made possible the widespread diffusion of sub-irrigation,which has proved to be applicable to an increasing number of crops.The main obstacle to the diffusion of this technique in the last decades is due to the intrusion of root hairs that,looking for water and nutrients,are able to penetrate from the emission points by clogging the delivery devices.

In this study,it has been hypothesized that it would be useful to add a pure or blended phytotoxic mixture to plastic during the production of drippers,whose herbicidal action dissolves gradually with the passage of water.

The specific aims of the study were:1)identify from the selected Mediterranean plants the most phytotoxic hydroalcoholic extract on germination and seedling growth of target species;2)evaluate the insertion of the most active extract or one of its components in the production cycle of drippers;3)evaluate the possible release of the extract or of its pure component to the passage of water.

Five species of plants were selected in this study:Vetch villosa Roth.(Ercoli et al.,2007),Brassica juncea L.(Ercoli et al.,2007),Secale cereale L.(Ercoli et al.,2007),Juncus effusus L.(Ervin et al.,2000)and Vallisneria natans L.(Wu et al.,2000).The phytotoxicity was tested in vivo on Lactuca sativa,Lycopersicon esculentum,and Allium cepa.

2 Materials and methods

2.1 Plant material

In this study,V.villosa,B.juncea,S.cereale,J.effusus and V.natans, five species from Mediterranean plant communities,were collected during May 2015,near the city of Naples(Southern Italy).These plants were easily available in large quantities and from geographically accessible locations.

2.2 Extraction and isolation

Each selected plant(roots+aerial parts)was air dried and grinded to fine powder(200 g),and then macerated with methanol/water 1:9(v/v;2 L)for 3 days.The prepared extracts were filtered and stored at 4◦C until their use(DellaGreca et al.,2007).

Fig.1 Separation procedures of compounds1-4 of V.villosa.

2.3 In vitro phytotoxicity bioassay

The phytotoxicity of the crude hydroalcoholic extracts and the 1:1 and 1:3 dilutions was tested in vitro on Lactuca sativa L.,Lycopersicon esculentum L.,and Allium cepa L.(Macias et al.,2000;Ladhari et al.,2013).The seeds of the target species were surface sterilized with sodium hypochlorite solution(0.4%,v/v)for 3 min and soaked in sterile distilled water for 30 min.The test solutions(10-4M)were prepared using 10 mM MES buffer(2-N-morpholino-ethanesulfonic acid,pH 6).Then,25 sterilized seeds of target species were separately placed on the filter paper(Whatman No.1)in 5 cm Petri dishes.After adding the seeds and 4.5 mL of the test solution,the Petri dishes were sealed with Para film®to ensure closed-system models.The seeds were placed in a growth chamber(KBW Binder 240)at 25◦C,in the dark(5 days for L.sativa and L.esculentum,and 7 days for A.cepa).After growth,the plants were frozen at-20◦C to avoid subsequent growth until measurement.Effects of the hydroalcoholic extracts from the 5 selected plants on the germination and growth of L.sativa,L.esculentum and A.cepa were tested.Treatments were arranged in a completely randomized design with three replications and data were transformed to percent of control for analysis.

2.4 Isolation of the phytotoxic compounds

The hydroalcoholic extract of V.villosa,the most active among those tested,was suspended in water and partitioned between ethyl acetate(EA)and water(W)(Fig.1).

The EA fraction was fractionated into neutral and acidic fractions using an aqueous 2N NaOH solution.The neutral fraction was washed with water and concentrated in vacuo(EAN fraction)while the acidic fraction was first acidified with aqueous 2N HCl to neutral pH and then extracted with ethyl acetate(EAA fraction),according to Fig.1.The neutral fraction was subjected to silica gel column chromatography using gradient solvent systems.The fractions were injected into the GC-MS,and the structure of compound 1(Fig.1)was measured for comparison with the reported data in the literature and in the database.

A volume equal to one tenth of the W fraction was partially lyophilized(320 mg)and filtered using a Sep-Pak RP-18 by eluting with water,methanol and acetonitrile(WW,WM,WA,respectively).The fractions were dissolved in 0.8 mL of MeOH, filtered through a 0.45 mm filter and injected into the LC-MS,identifying compound 2 from the WW fraction and compounds 3 and 4 from WM fraction(Fig.1).

2.5 Identification of the phytotoxic compounds

2.5.1 GC-MS/MS and LC-MS/MS analysis

Approximately 1 mg of EAN fraction was dissolved in 0.8 mL of acetone and subjected to a triple quadrupole gas chromatograph mass spectrometer(GC-MS).The analyses were performed with the following temperature program:40◦C for 5 min,220◦C at 5◦C/min,and 220◦C for 10 min,and the injection volume was 1 µL(DellaGreca et al.,2011;Zarrelli et al.,2011).

Approximately 1 mg of WW and WM fractions were dissolved in 0.8 mL of acetone and subjected to LCMS analysis.The most appropriate precursor ion,daughter ion,cone voltage,and collision energy were adjusted according to each analysis.

2.6 Spectrophotometric determination of cyanamide

2.6.1 Preparation of the standard cyanamide solution

Standard solution of cyanamide(2.5 mM as final concentration)was obtained dissolving pure cyanamide in 0.1 M HCl,freshly prepared and the pH was brought to a final value of 7.0.

The calibration line obtained allowed to determine a molar extinction coefficient of 2950 cm-1M-1,in good agreement with the values reported in the literature(Yoshifumi et al.,2009;Nieman et al.,1976).

2.7 Statistical analysis

The phytotoxic essays were conducted in a complete randomized design with three replications and a two-way ANOVA was performed to analyze treatment differences followed by the post hoc test of Tukey.The statistical assays,performed by using the Systat SigmaPlot 12.2 software(Jandel Scientific,USA),were considered statistically significant for P＜0.05.

3 Results and discussions

3.1 Phytotoxic evaluation of plant extracts

Our first objective was to identify easily accessible terrestrial or aquatic plants from the Mediterranean area and evaluate their phytotoxicity on test organisms normally used for this purpose(DellaGreca et al.,2004,2011).Five plants were selected:V.villosa,B.juncea,S.cereale,J.effusus,and V.natans,infused with methanol/water 1:9 for three days at room temperature.The phytotoxicity of the obtained extracts and the corresponding diluted 1:1 and 1:3 was measured on the two dicotyledonousè L.sativa and L.esculentum and on the monocotyledonous A.cepa(Table 1).

Among the five aqueous infusions,V.villosa was the most active regardless of dilution degree and in relation to both germination and elongation of the root and hypocotyl(Table 1).In particular,the undiluted extract of V.villosa showed an anti-germination activity higher than 70%on all three test organisms,while the 1:1 and 1:3 diluted extracts showed an activity of 45-50%and～30%respectively.The other extracts showed a generally lower activity of 9-13%and,in particular,that of J.effusus was the least active(Table 1),especially on L.sativa(-25%).

Table 1 Effect (mean ± SD) of the hydroalcoholic extracts of V. villosa, B. juncea, S. cereale, J. effusus and V. natans, on the germination (A), root elongation (B)and shoot elongation (C) of L. sativa, L. esculentum and A. cepa, for the extracts as such, diluted 1:1 and diluted 1:3, respectively. Positive percentages represent stimulation while negative values represent inhibitions. The three extracts concentrations showed always statistically different effects. Different uppercase letters indicate a significant difference among the five types of extracts (extracts of V. villosa, B. juncea, S. cereale, J. effusus and V. natans). Statistically differences were performed by two-way ANOVA (P ＜ 0.05).

Regarding the root elongation,the undiluted hydroalcoholic extract V.villosa wasable toinhibit it uptoabout 90%,while those diluted 1:1 and 1:3 showed an inhibition of 60%and slightly less than 40%,respectively.The other extracts were less active about 9-21%and,among these,the less active was once again the extract of J.effusus,especially on A.cepa(30-35%)(Table 1).

A slightly different situation in the evaluation of shoot elongation was found(Table 1).In this case there were two extracts that were far more active than the others,namely V.villosa and B.juncea,with inhibition peaks higher than 85%for the undiluted extract.The other three were generally less active than 18-20%.However,the diluted 1:1 extract of V.villosa was 15%more active than B.juncea extract and 35%more than the others,while the diluted 1:3 extract showed an almost double activity of B.juncea extract and 2-5 times more than others(Table 1).

3.2 Isolation of major components of V.villosa extract of and their phytotoxic evaluation

Considering that the extract of V.villosa was the most active,it was decided to continue the investigation by evaluating its chemical composition,at least in the commercially and economically most abundant accessible components.The whole extract showed a composition strongly depending on the extraction conditions and so hardly reproducible.Moreover,its use in the production of the dippers was advised.The hydroalcoholic extract was partitioned between ethyl acetate and water(EA and W,respectively;Fig.1).In turn,the EA fraction was fractionated into neutral(EAN)and acidic(EAA)fractions that were then subjected to silica gel column chromatography using gradient solvent systems.The obtained fractions were injected into the GC-MS identifying the structure of compound 2-Methyl-1,4-benzoquinone(1).Only part of fraction W was reduced in volume and filtered on a Sep-Pak RP-18 by eluting with water,methanol and acetonitrile(WW,WM and WA,respectively).The fractions were dissolved in 0.8 mL of MeOH, filtered through a 0.45 mm filter and injected into the LC-MS identifying 2,6-Dimethyl-1,4-benzoquinone(2)from the fraction WW and Cyanamide(3)and Calcium cyanamide(4)from fraction WM(Fig.1).

The four identified compounds were tested to determine their phytotoxicity on the three test organisms already considered,at four different concentrations between 10-4and 10-7M(Table 2)(D’Abrosca et al.,2005;Fiorentino et al.,2007;Cutillo et al.,2004;DellaGreca et al.,2003).The phytotoxicity assays were carried out according to the previous protocol.In particular,the quantities necessary for the preparation of the test solution(10-4M)were taken from the compounds and subsequent concentrations(10-5-10-7M)were prepared by dilution:all compounds were dissolved in MES in the ratio of 5 L per mL of buffer.

The compounds tested were active and,generally,more than 4-hydroxybenzoic acid(HBA),the herbicide used as a reference,at all concentrations considered(DellaGreca et al.,2007).At the highest concentration(10-4M),compounds 1-4 were able to inhibit the germination of the three test organisms with percentages between 70 and 85%,at least 0-25%more than the reference herbicide(Table 2).Compound 3 was the most active,with a percentage of germination inhibition that rarely fell below 70%even at the concentration of 10-7 M,in particular with an activity around 80%on A.cepa,regardless of concentration tested.Its calcium salt(4)was slightly less active but still more than the compounds 1 and 2(Table 2).

Compound 3 was the most active also in inhibiting root elongation,never below 50%until the concentration of 10-7M,and with peaks up to 90%at the highest concentration on L.esculentum.Compounds 1,2 and 4 showed slightly lower activities,similar among them but the first two slightly more active than compound 4(Table 2).

The same trend was found also for the shoot elongation,the most active were cyanamide(3)and its calcium salt(4).Compounds 1 and 2 were much active at the two highest concentrations,but less than com-pounds 3 and 4,and much less at the lower concentrations.Often,at the concentration of 10-7M compounds 1 and 2 were even slightly stimulating the shoot elongation(Table 2).

Table 2 Effect (mean± SD) of the compounds 1-4 on the germination (A), root elongation (B) and shoot elongation (C) of L. sativa, L. esculentum and A. cepa. at four different concentrations between 10-4 and 10-7 M. Positive percentages represent stimulation while negative values represent inhibitions. Different lowercase letters indicate a significant difference among the four concentrations. Different uppercase letters indicate a significant difference among different compounds. Statistically differences were performed by two-way ANOVA (P＜0.05).

Table 3 Percentage of cyanamide released from drippers.

Cyanamide(1)was not only the most phytotoxic(Soltys et al.,2011),among the four compounds iso-lated and tested,but was the only molecule with the necessary requirements for its inclusion in the produc-tion process of plastic drippers.In order for the insertion to be feasible,the molecule had to be very soluble in water(850 g/L,compared to a request of at least 100 g/L,at room temperature),moderately soluble also in ethanol and acetone,with a low melting point,between 40 and 70◦C(44◦C)and a boiling point below to 300◦C(260◦C),a molecular weight below 200 uma,of natural origin,commercially available,cheap and,obviously,phytotoxic.

The pure substance was used to obtain two series of drippers with an average content of 2.5 and 1.25%by weight.

3.3 Determination of cyanamide released by drippers

Around 30 drippers,divided in three groups of 10,with an average cyanamide(1)content of 2.5%were considered.The drippers were washed with water and then with acetone,leaving them stirred for one mi-nute.The water and the acetone were recovered,dried,weighed and analyzed to quantify the possible con-tent of cyanamide,which was found to be absent.The drippers were dried,weighed,cut into small pieces and left in dark glass containers,capped with a te flon stopper,stirring with 50 mL of water,at room tem-perature,for 1-5 weeks.The same operations were repeated for 30 drippers with an average content of 1.25%cyanamide.

The amount of cyanamide released varies from 2.0%after one week to about 3%after 5 weeks(Table 3).The drippers with 2.5%of cyanamide released more than that with 1.25%.It should be noted that for both types of drippers,after the third week,there was a slowing in the rate of release of the test substance.In particular,at the fifth week the drippers with a lower%of cyanamide release the same amount of substance detected seven days before(Table 3).

4 Conclusions

Five plants of Mediterranean area,V.villosa,B.juncea,S.cereale,J.effusus,and V.natans,were infused with methanol/water 1:9 for three days at room temperature.The phytotoxicity of the obtained extracts and the corresponding diluted 1:1 and 1:3 was measured on the two dicotyledonous,L.sativa and L.esculentum,and on the monocotyledonous,A.cepa,in the concentration range of 10-4-10-7M.The study of the chem-ical composition of the major components of the most phytotoxic hydroalcoholic extract corresponding to V.villosa,allowed to identify four substances namely 2-Methyl-1,4-benzoquinone,2,6-Dimethyl-1,4-benzoquinone,Cyanamide and Calcium cyanamide.All compounds were tested for their ability to inhibit germination,root elongation and shoot elongation of the previous test organisms.Of the substances tested,cyanamide was the most phytotoxic and with chemical-physical characteristics suitable for being used in the production process of drippers for subirrigation.Finally,two series of drippers were made up with an average content of 2.5 and 1.25%of cyanamide,respectively,whose release at distance of 1,2,3,4 and 5 weeks was evaluated between 2 and 3%.The results indicate a possible use of this substance as a phytotoxic additive in the drippers slowly released with the passage of water.