Rev. bras. farmacogn. vol.25 número2

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w w w . s b f g n o s i a . o r g . b r / r e v i s t a

Original

Article

Autotoxicity

in

Pogostemon

cablin

and

their

allelochemicals

Yan

Xu,

You-Gen

Wu

∗

_,

_Ying

_Chen,

_Jun-Feng

_Zhang,

_Xi-Qiang

_Song,

_Guo-Peng

_Zhu,

_Xin-Wen

_Hu

KeyLaboratoryofProtection,DevelopmentandUtilizationofTropicalCropGermplasmResourcesofMinistryofEducation,CollegeofHorticultureandLandscape, HainanUniversity,Haikou570228,PRChina

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received18September2014 Accepted24February2015 Availableonline21March2015

Keywords: Autotoxicity Allelochemicals Enzymaticactivity Pogostemoncablin Rhizospheresoil

a

b

s

t

r

a

c

t

TheeffectsofallelochemicalsandaqueousextractsfromdifferentPogostemoncablin(Blanco)Benth., Lamiaceae,partsand rhizospheresoilongrowthparameters, leafmembraneperoxidation andleaf antioxidantenzymeswereinvestigatedinpatchouli.P.cablinseedlingswereincubatedinsolutions con-tainingallelochemicalsandaqueousextractsfromdifferentpatchoulipartsanditsrhizospheresoilat severalconcentrations.Firstly,thegrowthparametersweresignificantlyreducedbythehighest con-centrationofleaves,rootsandstemsextracts(p<0.05).Ascomparedtothecontrol,plantheightwas reducedby99.8%inthetreatmentwithleavesextracts(1:10).Themalondialdehydecontentincreased greatlywhenpatchouliseedlingsweresubjecttodifferentconcentrationsofleaves,rootsandstems extracts;meanwhile,thesuperoxidedismutaseandperoxidaseactivitiesshowedanincreasetrendat thelowconcentration,followedbyadeclinephaseatthehighconcentrationofrootsandleavesextracts (1:10).What’smore,leavesandrootsextractshadamorenegativeeffectonpatchouligrowththanstems extractsatthesameconcentrations.Secondly,thetotalfreshmass,rootlengthandplantheightwere greatlyreducedbythehigheststrengthofsoilextracts.Theirdecrementswere22.7,74.9,and33.1%, respectively.Thirdly,growthparametersandenzymaticactivitiesvariedconsiderablywiththekindsof allelochemicalsandwiththedifferentconcentrations.Plantheight,rootlengthandtotalfreshweightof patchouliweregreatlyreducedbyp-hydroxybenzoicacid(200␮M),andtheirdecrementswere77.0,42.0

and70.0%,respectively.Finally,threeusefulmeasuresonreducingtheautotoxicityduringthesustainable patchouliproductionwereproposed.

Introduction

Successivecultureofthesamecroponthesamelandforyears causessoilsicknessorreplantinginjuries,resultinginreductionin bothcropyieldandquality.Thisphenomenonisevidencedin agri-culturalcroppingsystemespeciallyintheproductionofmedicinal crops(vandeVoordeet al.,2012).Itleadstotheresurgenceof diseasepest,exhaustionofsoilfertility,anddevelopingchemical interferenceintherhizospherereferringtoallelopathy.The con-tinuousmonocultureproblemsinsomecropssuchaswatermelon havebeeneffectivelyovercomethroughspecialcontrolmeasures, however,insomecrops,ithasnotyetbeencompletelyresolved, especiallyinsomemedicinalplantssuchasginsengandPogostemon cablin(Liuetal.,2006).

P.cablin (Blanco) Benth.,Lamiaceae(Patchouli),from south-eastAsia iscultivated extensively inIndonesia,thePhilippines,

∗ Correspondingauthor.

E-mail:ccyyccii@163.com(Y.-G.Wu).

Malaysia,China,andBrazil(Miyazawaetal., 2000;Singhetal.,

2002;Wuetal.,2008).TheaerialpartofP.cablinhasbeenused

for the treatment of thecommon cold, headache, fever, vomi-ting,indigestion anddiarrhea aswellas anantifungal agentin themedicinalmaterialsofChinaanditssurroundingregion(China

Pharmacopoeia Committee, 2010).It is a herbaceous perennial

plant withoilglands producing an essential oil(patchoulioil), whichiscommonlyusedtogiveabaseandlastingcharacterto a fragrance in theperfume industry. Patchouli was introduced into Chinafor perfumeand medicinalpurposes asearlyas the LiangDynastyorpotentiallybefore(Wuetal.,2007).Currently, patchouliiswidespreadinsouthernChina,includingGuangdong (Guangzhou,Zhaoqing,Zhanjiang,etc.)andHainan(Wanningand Haikou)Province,dividedintoPaixiang(cultivatedinGuangzhou), Zhaoxiang(cultivatedinZhaoqing),Zhanxiang(cultivatedin Zhan-jiang)andNanxiang (cultivatedinHainan)(Wu etal.,2010).In recentyears,themarketdemandforP.cablinhasforcedfarmers toplanttheminplacesoutsidetheabovefourcities.However,the

P.cablinproducedintheseareascannotbeassuredforqualityas itisgrowninnon-optimalproductionareasandunderdifferent

http://dx.doi.org/10.1016/j.bjp.2015.02.003

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Y.Xuetal./RevistaBrasileiradeFarmacognosia25(2015)117–123

environmentalconditions(Wuetal.,2008).Ontheotherhand,in ordertoaddressthecontinuousmonocultureproblems,farmers tendedtoincreasefertilizerinputstoenhancecropyield.Therehas alsobeenarapidincreaseinpesticideuse,leadingtoexacerbated soilenvironment withexcessivepesticideresidues.Therefore, it hasbecomeamatterofprioritytostudythemechanismofthe continuousmonoculture problemsand providea rational crop-pingsystemforP.cablinproduction.However,fewstudieshave

focusedonconsecutivemonocultureproblemandautotoxicityof

P.cablin,anditremainsunknowninthecasehowaqueousextracts ofpatchouliplantandrhizospheresoilandtheirallelochemicals havesomeeffectsonthegrowthandantioxidantenzymesinP. cablin.

Thepresent studywasconductedtounderstandautotoxicity onthegrowthanddevelopmentand physiological–biochemical changes of P. cablin. Our objectives were to: (i) compare and determine the effects of aqueous extracts made from roots, stems and leaves on the growth and antioxidant enzymes in

P. cablin; (ii) examine the effects of extracts taken from soil conditionedbyP. cablin; (iii) test theinhibitory effects of alle-lochemicals on patchouli seedling performance. This study of autotoxicity in commonly grown P. cablin was a preliminary assay which would provide some suggestions on reducing the autotoxicityandfacilitatingthemaintenanceofpatchouli produc-tion.

Materialsandmethods

Preparationofpatchouliplantsextracts

Toobtain leaf,stem and root material of Pogostemon cablin

(Blanco) Benth., Lamiaceae, for the extract preparation, 200 seedlingsofpatchouliwereplantedinanewfield(pH4.5–5.5)in HainanUniversity.Shadeclothswereusedtopreventtheseedlings fromglaringsunandkeepthemgrowwellinthenative environ-ment.Theseedlingshadtobewateredtwiceaweekinorderto keepthesoilmoisturecontentat50–60%.Aftersevenmonthsthe plantswereharvested.Foreachplantallleafandstemmaterials wereclippedandcutintopiecesofapproximately1cmtobeused forextractions.Therootswerecutoff fromtheplant,rinsedin demineralisedwaterfor20s,andcutinto1cmpieces.Then oven-driedat55◦_C_for₇₂_h,_powdered_and_used_for_extraction₍_Omezzine

andHaouala,2013).

Onehundredgrams outof eachof thedriedmaterialswere soakedin1000mldistilled waterat roomtemperature for24h togivea concentration10%.Sampleswerethen centrifugedfor 20minat 4000×g and filtered.Crudeaqueousextracts of

pro-gressivelyincreasingconcentrationwerepreparedusing1.0,2.0, 4.0 and 10g of oven-dried leaf, stem and root per 100ml of water.

Preparationofrhizospheresoilextracts

Rhizospheresoilwascollectedinthesamefieldwhichseedlings ofP.cablinhadbeengrownin.Thesoilsurroundingtherootwas collectedandsievedthroughameshof2mmtoseparaterootsfrom soil.

Rhizosphere soil materials (50g) were soaked in 100ml demineralisedwater.Crudeaqueousextractsofsoilsamplesafter thesameconditionabovewereputintorotaryevaporatorsand con-densedintopaste(−0.08MPa,<56◦C).Thepastewasusedinthree

concentrations:pure(highstrength),diluted1:1with demineral-isedwater(mediumstrength),ordiluted1:19withdemineralised water(lowstrength)(vandeVoordeetal.,2012).

PreparationofP.cablinseedlingsforthreeautotoxicity experiments

P.cablinseedlingswerecultivatedin1/2MSmedium.Whenthe seedlingswereinthe8-leafstage,theyweredividedintotwoparts. Onepartoftheplantgrowninpots1.0(l)filledwithnewsterile fieldsoil(20minat110◦_C,_during_two_consecutive_days)_was_placed inthefieldenvironment(vandeVoordeetal.,2012).Theother grownhydroponicallyin ahalf-strength Enshinutrientsolution

(YuandMatsui,1994)wasplacedinagreenhouse;air

temper-aturewasmaintainedbetween18and28◦_C_and_relative_humidity rangedbetween80%and 95%.Theseseedlingswereusedforall autotoxicityexperiments.

Autotoxicitytestswithpatchouliplantsextracts

Eachseedlingthatwasputinthefieldreceived100mlleaf,stem, rootextractswhichweredilutedin1:10,1:25,1:50and1:100(dry weight:distilledwater)everythreedays(Yuetal.,2003).Control plantsreceived100mlofdemineralisedwater.Therewerethree replicatesfor each treatmentresultingin 117vials (3replicate pots×3extracttypes×4concentrations×3seedlings+9control

seedlings).

Autotoxicitytestswithrhizospheresoilextracts

AccordingtovandeVoordeetal.(2012),thesoilextract treat-mentsusingpure(highstrength),1:1(mediumstrength),1:19(low strength)(soilpaste:distilledwater)werethesameasabove.There werethreereplicatesforthesetreatmentsresultingin36vials(3 replicatepots×1extracttype×3concentrations×3seedlings+9

water control seedlings). During the experiment each seedling received100mlofsoilextractseverythreedays.After21daysall seedlingswereharvestedasdescribedabove.

Autotoxicitytestswithallelochemicals

Accordingtopreviousresearch,eightallelochemicalssuchas dibutylphthalate,benzoicacid,cinnamicacid,malonicacid, vanil-licacid,salicylicacid,p-hydroxybenzoicacidandtetradecanoicacid

wereisolatedandidentifiedfrompatchouliplantsandtheir rhizo-spheresoil(Wuetal.,2013).Theseallelochemicalsatconcentration of0 (control),50,100, 200␮M,describedbyAsaduzzaman and

Asao(2012),werecombinedwithahalf-Enshinutrientsolution

(EC2.0dSm−1_)._The_inhibitions_of_the_test_solution_were_assayed_by theireffectsonP.cablinseedlings.Eachtreatmentwasreplicated

threetimes.Testsolutionswereaddedto200mlflaskswrapped withblackpolyethylenetoavoiddirectlightontherootsoftest plants.Theselectedplantsweretransplantedtoeachflaskwith urethanefoamassupport.WeplantedtheP.cablinplantsinsuch

awaythatrootswereinsertedintothenutrientsolutioninside theflaskkeepingtheshootoutside(AsaduzzamanandAsao,2012). Urethanefoamblockswereusedforholdingtheplantstightand uprightattheneckoftheflask.Theplantedflaskwasplacedina greenhouseat25◦_C_with_a_light_intensity_of_74–81_␮_mol_s−1_m−2 and16hphotoperiod.Tominimizetheeffectofaerationandthe microbialdegradationoforganiccompoundsonthebioassaywe renewedthetestsolutionsintheplantedflaskateverythreedays.

Lipidperoxidationandenzymeanalyses

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peroxidationwasdeterminedin0.5gleaffreshweightby mea-suringtheamountofmalondialdehyde(MDA),aproductoflipid peroxidation, bythethiobarbituricacidreaction (Gossett etal., 1994).Leaves of the seedlings were collected, weighed (0.5g), immediatelyfrozeninliquidnitrogenandstoredat−25◦_C_until extraction.Frozentissuesweregroundwithmortarwithpestle, suspended in 0.5ml 0.1mM Tris at pH 8. Extracts were cen-trifugedat12,000×g for20min(4◦_C)_and_the_supernatant_was usedforthedeterminationofenzymeactivity.Enzymeactivities were measuredat 25◦_C _using _a _{spectrophotometer} _(Shimadzu UV-2100,Japan).Superoxidedismutase(SOD)activitywas mea-suredaccordingtoMadamanchietal.(1994).Crudeextractwas added to a reaction solution (3ml) containing 50mM phos-phatebuffer(NaH2PO4/Na2HPO4)atpH7.8,0.1mMEDTA,13mM methionine, 2␮M riboflavinand 75␮M 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenyl-formazan(MTT).Thereactionwasstartedby exposingthemixturetocoolwhitefluorescentlightata photo-syntheticphotonfluxof50␮molm−2s−1for15min.Thenthelight wasswitchedoff,thetubeswerestirredandthebluecolorwas measuredat560nm.Catalase(CAT)activitywasassayedina reac-tionsolution(3ml)composedof50mMphosphatebuffer,pH7.0, towhich 30% (w/v)H2O2 wasadded. Thereaction wasstarted byaddingthereactionsolutionto10␮lofcrudeextractandthe activitywasfollowedbymonitoringthedecreaseinabsorbanceat 240nmasaconsequenceofH2O2consumption.Peroxidase(POD) activitywasassayedinareactionsolution(3ml)containing50mM phosphatebufferatpH7.0,1%guaiacol,0.4%H2O2and10␮lcrude extract.Increaseintheabsorbanceduetooxidationofguaiacolwas measuredat420nm(Yuetal.,2003).

Statisticalanalysis

Allexperimentswereconductedusingarandomizedcomplete blockdesignwiththreereplications. Dataweresubjectedtoan analysisofvariancebyusinggenerallinearmodelandthemeans comparedusingDuncan’smultiplerangetest(Duncan)methodat 5%levelusingSPSS(version19.0).

Resultsanddiscussion

Autotoxicitytestswithpatchouliplantsextracts

Theautotoxicityeffectsoftheleaves,rootsandstemsextracts wereassayedforgrowthparametersofP.cablin(Blanco)Benth., Lamiaceae,atseveralconcentrations(Fig.1).Plantheightandroot lengthweretwocommonindexesobservedintheautotoxic exper-iment(ChonandKim,2002;Gattietal.,2010).Inleaves,rootsand stemsextracts,plantheightandrootlengthofpatchouliseedlings handledbythehighconcentration(1:10)wereshorterthanthat oftheremainingconcentrations.Ourresultsshowedthataqueous extractsmadefrompatchouliplanthadsomeautotoxicpotential torestraintheseedlinggrowth,especiallyontheplantheightand rootlength(Fig.1).Moreover,differentconcentrationsofaqueous extractshaddiverseinhibitioneffectsonthegrowth.Thisshowed thattheextractconcentrationusedbytheautotoxicexperiment wasanimportantdeterminantoftheautotoxiceffect.

Meanwhile,leavesandrootsextractshadamorenegativeeffect onplantheightandrootlengththanstemsextractsatthesame con-centrations.Ascomparedtothecontrol,plantheightwasreduced by99.8%inthetreatmentswiththe1:10oftheleavesextracts. Whenrootsandleavesextractswerecompared,rootlengthwas morereducedbyleavesextractsthanbyrootsextracts.Thefresh masswasreducedsignificantlyby1:10ofleaves,rootsandstems extracts(p<0.05).Theresultsdescribedaboveshowedthatas com-pared to theroots extractsand stems extracts,leaves extracts showedamorepotentinhibitoryeffectonrootlengthatthesame concentration.AhmedandWardle(1994),whostudiedthe allelo-pathiceffectsofragwortonotherspecies,alsofoundthatextracts fromshootshadthestrongestallelopathiceffects onother pas-turespecies,andthisappearedageneralobservationinstudieson allelochemicaleffects(LipinskaandHarkot,2007).Otherseveral studiesalsoshowedthatextractsmadefromleafmaterialinhibited plantgrowthmorethanrootsextracts.Ourresultwasagreement withtheselistedinliterature(Macelet al.,2005;Thodenetal., 2009).Theresultsdemonstratedthatthepartofplantextractused wasanotherimportantdeterminantoftheautotoxiceffect.

Control 1:100 1:50 1:25 1: 10 _Control _1:100 _1:50 _1:25 _{1: 10} Control 1:100 1:50 1:25 1:10 bAdBeA bB

bA dB _bB

bA bA bBcA

cA aA aAaA 15 10 5 0 –5 –10 –15 –20 1–1 Plant growth

Concentration bA cdB bcA

bA aA –10 –5 0 5 1–2 Concentration Concentration

bA bA bA

cB aA 0 –5 –10 1–3 5 Plant growth Plant growth cA bA bB aA cB –10 –5 0 5 Plant growth 2-2

Control 1:100 1:50 1:25 1:10 Concentration Plant growth –10 –5 0 5

Control 1:100 1:50 1:25 1:10 bB bB bB bB cA cA bA bA bB bB bB Concentration

Control 1:100 1:50 1:25 1:10 15 10 5 0 –5 –10 –15 Plant growth 2–1 aB aB aA aA Concentration bB bB aA cB cA 2-3 Concentration

1:100 1:50 1:25 1:10 Control cB cC aA aAbC bC bA

aA _bC aC

aA aA aB_aB cA –5 0 5 10 3–1 Plant growth

1:100 1:50 1:25 1:10 Control cA aB bB aC bcB –5 –2.5 0 2.5 5 3–2 Concentration

1:100 1:50 1:25 1:10 Control bA aA aB bC bC Concentration 2.5 5 0 –2.5 –5 3–3 Plant growth Plant growth

No. of leaves per plant Root length (cm)

Max.leaf length (cm)

Plant height (cm) Total fresh mass (g)

Root length (cm) Max.leaf length (cm)

Plant height (cm) No. of leaves per plant Total fresh mass (g)

Total fresh mass (g) No. of leaves per plant

Root length (cm) Max.leaf length (cm)

Plant height (cm)

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Table1

EffectsofaqueousextractsfromdifferentpatchoulipartsandrhizospheresoilonleafmembraneperoxidationandantioxidantenzymesofPogostemoncablinseedlings.

Treatment Conc.(w/v) SOD(␮g−1FW) POD(␮molg−1FW) MDA(␮molg−1FWh−1)

Leafextracts 0(control) 18.4±0.283aA 0.8±0.028aA 1.2±0.019aA 1:100 21.1±0.424bA 0.7±0.014aA 1.6±0.020cB 1:50 27.1±0.141cA 1.0±0.039bB 1.4±0.008bB 1:25 31.5±0.566eA 1.9±0.098dC 1.8±0.021dC 1:10 29.6±0.283dA 1.2±0.042cB 3.1±0.084eB Stemextracts 0(control) 18.4±0.283aA 0.8±0.028aA 1.2±0.019aA 1:100 23.1±1.414bB 0.9±0.084abC 1.7±0.009bC 1:50 29.9±1.131cB 1.0±0.141abB 1.3±0.010aA 1:25 31.3±0.566cA 1.4±0.042bcA 1.7±0.006bB 1:10 42.3±1.137dB 1.8±0.056cC 2.2±0.019cA Rootextracts 0(control) 18.4±0.283aA 0.8±0.028bA 1.2±0.019aA 1:100 38.9±1.136cC 0.8±0.015bB 1.2±0.013aA 1:50 34.2±1.141bC 0.3±0.070aA 1.4±0.006aB 1:25 70.6±0.567eB 1.6±0.027cB 1.3±0.041aA 1:10 42.6±0.424dB 1.0±0.033bA 5.5±0.084bC Soilextracts 0(control) 18.4±0.283ab 0.8±0.028a 1.2±0.019a

1:19 17.1±0.707a 1.0±0.012b 1.7±0.004b 1:1 19.6±0.682b 0.9±0.009ab 1.5±0.004b Pure 30.7±0.363c 1.6±0.036c 3.3±0.169c

Thecapitalletterswerewithinthesameconcentrationscomparingthevariousexperiments.Thelowercaseletterswerewithinthesameexperimentcomparingthevarious concentrationsbyDuncan’stest(p<0.05).

Enzymeactivitieswereafactorofreactiononplantgrowth.A summaryoftheantioxidantenzymeactivityaffectedbyaqueous extractsofpatchouliplantisgiveninTable1.BothSODactivityand PODactivityintheleafgreatlyincreasedwhenpatchouliseedlings weresubjecttodifferentconcentrationsofstemsextracts. Nev-ertheless, both of them showed an increase trend at the low concentration,followedbyadeclinephaseatthehigh concentra-tionofrootsandleavesextracts(1:10).Thisresultwasconsistent withprevious studieson cucumber (Yu et al., 2003). A stimu-lation of the POD and SOD activities has beendocumented in cucumber under different concentrations of roots extracts (Yu etal., 2003).In contrast,however, decreasedactivities ofthese antioxidant enzymes in Evernia prunastriL. had been reported

(Deltoroet al.,1999).CATactivity,however,wastoolowtobe

detectedinthepresentstudy.Theconcentrationofrootsextracts (1:25)increasedSODactivityby284.2%andPODactivityby99.5% (p<0.05).Additionally, increasesinMDAwerealsoobserved in

theseedlingsincubatedinallsolutionswiththeleaves,rootsand stemsextracts.Accordingtotheseresults, higherconcentration levelsofaqueousextractsmighthaveexceededtherateof detox-ificationwhichthenresultedinpotentinhibitoryeffectonplant growthanddramaticallydecliningonPODactivityandSOD activ-ity.

Autotoxicitytestswithrhizospheresoilextracts

The more wasallelochemicals accumulation in thesoil, the strongerwastheinhibitioneffectonplantperformance(vande

Voordeetal.,2012).Foraqueousextractsmadefromrhizosphere

soil,thehighstrength(pure)treatmenthadthemostautotoxic effectsongrowthparametersofP.cablininalltreatments(Fig.2).

Thetotalfreshmasswasgreatlyreducedbythemediumstrength (1:1)and highstrength (pure)soilextracts,and thedecrement was19.6% and 22.7%, respectively (p<0.05). The high strength

(pure) soil extract also inhibited the root length significantly. Theseresults,ontheonehand,demonstratedthatsomeautotoxins existedinthesoilextractsthatcauseinhibitiononP.cablinplants.

Ontheotherhand,theautotoxiceffectsofP.cablinwere dosage-dependent,beingstrongestforthemostconcentratedextracts.This wasinlinewithstudiesonallelopathiceffectsofotherplantspecies

(DorningandCipollini,2006).

In addition, relatively lighter growth reduction on P. cablin

was detected in low strength (1:19) soil extracts than in low

concentration (1:100) of the leaves, stems and roots extracts. Thisphenomenonwasinteresting.Wespeculatedthatsoilbiota mightreducetheecologicalconsequencesofreleasedplant chem-icals.SimilarresultshavealsobeenfoundbyInderjitandPutten

(2010). Anotherinteresting phenomenon we had foundin our

experimentwasthatP.cablinseedlingsgrewbetterinsterile sub-stratetestedbyaqueousextractsmadefromthesoilwhichhad nevercultivated P.cablinbefore. Thereasonwespeculatedwas probability that P. cablin had grown in sterile substrate which hadnomicrobialcommunities;inturnthebeneficial microorgan-ismmightexistinthatsoilextractsmentionedabove.Therefore, inthesoil–microorganism–plant system,beneficial microorgan-ismcouldmore likely promotetheperformance of plants.This result was consistent with previous studies by Acosta et al.

(2010).

Thechanges of SOD activity,PODactivityand MDAcontent affectedbyaqueousextractsof rhizospheresoilarereportedin

Table1.The highstrength (pure)soil extract greatlyincreased

theSODandPODactivityandMDAcontent,andtheircontents were 30.7␮g−1 FW, 1.6␮molg−1 FW and 3.3␮molg−1FWh−1, respectively.Theirchangesaffectedbyaqueousextractsof rhizo-spheresoilwerepartlyattributedtoallelopathiceffects(Yuetal., 2003).Sowespeculatedthatthereweresomeallelochemicalsin aqueousextractsofrhizospheresoil.Theseallelochemicalscould damagecell membranesthrough directinteraction witha con-stituentofthemembraneorasaresultofanimpairmentofsome metabolicfunction necessarytothemaintenance of membrane function.

Autotoxicitytestswithallelochemicals

Thedifferencesthateight kindsofallelochemicalshad some phytotoxicity on patchouli seedling growth were observed in our experiments (Fig. 3). Growth parameters varied consider-ably with the kinds of allelochemicals and with the extract concentration. Plant height, root lengthand total fresh weight ofpatchouliseedingweregreatlyreducedbyp-hydroxybenzoic

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–10 20

Plant growth

Root length (cm) Max. leaf length (cm)

Plant height (cm)

Control 1:19 (Low strength) 1:1 (Medium strength) Pure (High strength) Concentration Concentration Concentration

Control 1:19 (Low strength)

1:1 (Medium strength)

Pure (High strength)

Control 1:19 (Low strength) 1:1 (Medium strength) Pure (High strength) 10 0 b b b b b b

c a c a

a a

b b

a a a

20 15 10 5 0 –5 20 15 10 5 0 –10 –5

No. of leaves per plant Total fresh mass (g)

a b b

Plant growth Plant growth

Fig.2.Theeffectsofaqueousextractsfromrhizospheresoilongrowthparameters.

rootlength(485.8%)and total freshweight (143.0%).Thesame changetrendalsohappenedonsalicylicacid. Ingeneral, allelo-chemicalsphytotoxicityonseedlinggrowthwasmuch stronger whenconcentrationincreased.Thisresultwasinagreementwith

that listed in literature (Pergo et al., 2008). In addition, some allelochemicals, such as dibutyl phthalate, had little influence on plant growth and development as compared to the con-trol. 2 1 0 –1 –2 –3 –4 –5

0 50 100 200

aA aB bC bC bA cB cB cD cA dA bA bA bA

dA cA aA dA bD

cE aCbE dE cE aD bD aD aC aE aC aB aB aF

0 50 100 200

aA bC bB cB cB cD cA dA bA bA bA

dA cA dA cA bD

bcE abC cE cE aD aD aC aD aB aC bE

0 50 100 200

aB aC bC bA bB cA cA bA bA dA

dA dA cA dA cE

bE aD bF aE aC aB aB aB aB aD bD aA

Concentration (μm)

bC aB

bA cD

aF

Concentration (μm)

cE bF aC bD 2 1 0 –1 –2 –3 –4 –5 –6 –7

Concentration (μm)

Growrh plant length (cm)

Growrh of root length (cm)

Variation of plant total fresh

weight (g) 10 0 2 4 6 8 –2 –4 –10 –6 –8

p-Hydroxybenzoic acid Salicylic acid Vanillic acid Myristic acid Malonic acid Dibutyl phthalate Benzoic acid Cinnamic acid

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Table2

EffectsofallelochemicalsisolatedfromPogostemoncablinplantandrhizospheresoilonleafmembraneperoxidationandantioxidantenzymesactivities.

Treatment(␮m/l) Conc.(␮M) SOD(Ug−1FW) POD(␮molg−1FW) MDA(␮molg−1FW)

Benzoicacid 0 38.5±0.016aA 1.6±0.028aA 2.3±0.054aA 50 40.9±0.578aF 2.6±0.073bE 3.5±0.067bC 100 59.5±1.635cE 5.4±0.099dD 4.1±0.044cE 200 50.9±0.641bD 3.3±0.045cC 10.2±0.155dG Cinnamicacid 0 38.5±0.013aA 1.6±0.021aA 2.3±0.057aA 50 69.7±0.565dG 7.5±0.136dF 4.2±0.073bD 100 60.3±0.144cE 3.8±0.052cC 5.5±0.141cF 200 57.1±0.219bE 3.4±0.067bC 7.7±0.168dE Myristicacid 0 38.5±0.016bA 1.6±0.028aA 2.3±0.054aA 50 37.3±0.282aD 2.3±0.024bD 3.0±0.029bB 100 40.0±0.158cB 2.3±0.019bA 3.2±0.093cB 200 40.5±0.221cA 3.0±0.148cB 3.9±0.185dA Dibutylphthalate 0 38.5±0.016bA 1.6±0.025bA 2.3±0.056aA 50 39.7±0.078cE 1.3±0.037aB 2.9±0.112bB 100 39.1±0.283aA 2.7±0.089cB 3.6±0.041cC 200 39.5±0.429dB 3.3±0.041dC 4.4±0.252dB Vanillicacid 0 38.5±0.013bA 1.6±0.022aA 2.3±0.055aA 50 39.5±0.631aC 1.8±0.055bC 5.1±0.168bE 100 40.9±0.492cC 2.3±0.090cA 6.0±0.124cG 200 50.9±0.356dD 5.5±0.013dE 7.2±0.217dD SalicylicAcid 0 38.5±0.018bA 1.6±0.026aA 2.3±0.059bA 50 26.4±0.352aA 2.6±0.076bE 7.1±0.093cG 100 52.6±1.554dD 6.2±0.182cF 1.8±0.065aA 200 43.4±0.144cC 1.4±0.069aA 8.1±0.251dF

p-Hydroxybenzoicacid 0 38.5±0.011aA 1.6±0.026aA 2.3±0.057aA 50 77.5±1.413dH 8.6±0.057dG 6.9±0.172bF 100 67.9±0.986cF 5.6±0.071cE 8.6±0.217dH 200 50.9±0.632bD 3.7±0.069bD 7.6±0.033cE Malonicacid 0 38.5±0.018bA 1.6±0.022bA 2.3±0.058bA 50 19.2±0.714aA 0.7±0.076aA 1.1±0.026aA 100 39.2±0.577cB 2.4±0.039cA 3.8±0.073cD 200 56.9±0.926dE 5.8±0.153dF 6.6±0.147dC

Thecapitalletterswerewithinthesameconcentrationscomparingthevarioustreatments.Thelowercaseletterswerewithinthesametreatmentcomparingthevarious concentrationsbyDuncan’stest(p<0.05).

EnzymeactivitiesandMDAcontentwerecarriedoutto evalu-atetheautotoxicitypotentialoftheseidentifiedallelochemicalsat severalconcentrations(Table2).Thetestedcinnamicacidsandp -hydroxybenzoicacidincreasedPODactivityby368.1–437.4%and SODactivityby81.9–101.6%,respectivelyattheconcentrationof 50␮M,andhadgreateffectsonMDAcontentinpatchoulileavesin mostcases.Amongthetestedacids,p-hydroxybenzoicacidshowed greatestactivityinincreasingSODactivity,followedbycinnamic acid,soastothePODactivities.Meanwhile,thehighestMDA con-tentwasobservedinthebenzoicacidtreatment,followedbythe salicylicacidandp-hydroxybenzoicacidtreatments.Theircontent was10.2, 8.1and7.6␮molg−1 FW,respectively.Fromthe fore-going,someallelochemicalsincreasedtheenzymaticactivitiesata lowconcentration(50␮M),butinhibitedthepatchouligrowthand developmentatthehighconcentration(200␮M).Similarresults havebeenalsofoundinsoybeanseedlingbyBaziramakengaetal.

(1995).

Tothebestofourknowledge,allelopathyusuallywastheresult ofthejointactionofseveralcompoundsreleasedbyadonorplant. Sinceeightallelochemicalswereonlyafractionofthecomplexroot exudates,itshouldbeborninmindthatthebioassayexperiment witheightallelochemicalswasonlyarepresentationofthe mech-anismsthattheseallelochemicalsmightcontributetothedifferent influenceofP.cablin.What’smore,someunknownmechanismson howtheautotoxinsactedonP.cablinalsoexisted.Extractsfrom P.cablintissuespotentiallyexhibitedsomeautotoxiceffects,soit wasunlikelythattheseautotoxiceffectsweretheonlycauseof stronggrowthreductioninsoilswherepatchouliplantpreviously hadbeengrown.Futurestudiesshouldfocusondisentanglingthe mechanismsthroughhowtheautotoxinsaffectedthepatchouli growth,andinteractionbetweensoilmicroorganismsand allelo-chemicals.

Usefulmeasuresonreducingtheautotoxicityincontinuous croppingofpatchouli

ResultsshowninFig.1,Figs.2and3revealedthat,somepositive effectsweredetectedongrowthparametersatlowconcentration, however,thegrowthindexesweregreatlyreduced bythehigh concentrationofallelochemicalsandaqueousextractsfrom dif-ferentpatchoulipartsandrhizospheresoil.Moreover,theresult inFig.1showsthatleavesextractshadmorenegativeeffecton growthindexesespeciallyrootlengththanstemsextractsandroots extractsatthesameconcentration.Thus,wepresumedthatmore autotoxinsmightbefromthepatchoulirottenleaves.According tothepreviousreport(Yaoetal.,2012), inordertopreventthe leavesfromfallingdowninthesoilanddecomposing,wecould taketwoefficientmeasuresthatpickedupthefallenleavesintime andharvestedthepatchouliplantin2–3daysaheadofpatchouli fullmaturity.

TheresultsinFig.3andTable2indicatedthatsome allelochem-icals,suchasp-hydroxybenzoicacidandcinnamicacid,hadmore negativeinhibitiononpatchouligrowthanddevelopment.Mostof theseallelochemicalsidentifiedinourexperimentswereoforganic acidtypes.Therefore,anotherusefulmeasurewascarriedoutto mixthesoilplantedbypatchouliforoneortwoyearswithalkaline substrateagainstthemonocultureproblem.

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weightofpatchouliweregreatlyreducedbyp-hydroxybenzoicacid (200␮M),andtheirdecrementswere77.0,42.0and70.0%, respec-tively.Threeusefulmeasures,namelypickingupthefallenleavesin time,harvestingthepatchouliplantin2–3daysaheadofpatchouli fullmaturityandmixingthesoilplantedbypatchouliforoneor twoyears withalkalinesubstrate,wereproposedtoreducethe autotoxicityduringthesustainablepatchouliproduction.

Authors’contribution

Allauthors contributed in collectingthe plant sample, run-ning the laboratorywork, analyzing the data and drafting the manuscript.Allauthorsparticipatedinrevisingthearticlecritically.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

ThisworkwassupportedinpartbygrantsfromtheNational NaturalScienceFoundationofChina(81360618and31360210), TheSpecializedFundfortheModernizationofTraditionalChinese MedicineofHainanProvince(ZY201413),TheStateKeySubject ofBotanyatHainanUniversity(071001),AcademicDiscipline Con-structionProjectPlanintheCentralandWesternRegionsofHainan University(ZXBJH-XK008).

References

Acosta,V.,Bell,C.W.,Morris,B.E.L.,Zak,J.,Allen,V.G.,2010.Long-termsoilmicrobial communityandenzymeactivityresponsestoanintegratedcropping-livestock systeminasemi-aridregion.Agric.Ecosyst.Environ.137,231–240.

Ahmed,M.,Wardle,D.A.,1994.Allelopathicpotentialofvegetativeandflowering ragwort(SeneciojacobaeaL.)plantsagainstassociatedpasturespecies.PlantSoil 164,61–68.

Asaduzzaman,M.,Asao,T.,2012.Autotoxicityinbeansandtheirallelochemicals. Sci.Hortic.134,26–31.

Baziramakenga,R.,Leroux,G.D.,Simard,R.R.,1995.Effectsofbenzoicandcinnamic acidsonmembranepermeabilityofsoybeanroots.J.Chem.Ecol.21,1273–1285. Chon,S.U.,Kim,J.D.,2002.Biologicalactivityandquantificationofsuspected

allelo-chemicalsfromAlfalfaplantparts.J.Argon.CropSci.188,281–285.

ChinaPharmacopoeiaCommittee,2010.PharmacopoeiaofthePeople’sRepublicof China.ChinaMed.Sci.Press,pp.342–373.

Deltoro,V.I.,Gimeno,C.,Calatayud,A.,Barreno,E.,1999.EffectsofSO2fumigations onphotosyntheticCO2gasexchange,chlorophyllafluorescenceemissionand antioxidantenzymesinlichensEverniaprunastriandRamalinafarinacea.Plant Physiol.105,648–654.

Dorning,M.,Cipollini,D.,2006.LeafandrootextractsoftheinvasiveshrubLonicera maackii,inhibitseedgerminationofthreeherbswithnoautotoxiceffects.Plant Ecol.184,287–296.

Pergo,É.M.,Abrahim,D.,daSilva,P.C.S.,Kern,K.A.,DaSilva,L.J.,Voll,E., Ishii-Iwamoto,E.L.,2008.BidenspilosaL.exhibitshighsensitivitytocoumarinin comparisonwiththreeotherweedspecies.J.Chem.Ecol.34,499–507. Gossett,D.R.,Millhollon,E.P.,Lucas,M.,1994.AntioxidantresponsetoNaClstress

insalt-tolerantandsalt-sensitivecultivarsofcotton.CropSci.34,706–714. Gatti,A.B.,Ferreira,A.G.,Arduin,M.,Perez,S.,2010.Allelopathiceffectsof

aque-ousextractsofArtistoluchiaesperanzaeO.KuntzeondevelopmentofSesamum indicumL.seedlings.ActaBot.Bras.24,454–461.

Inderjit,Putten,W.H.,2010.Impactsofsoilmicrobialcommunitiesonexoticplant invasions.TrendsEcol.Evol.25,512–519.

Liu,H.Y.,Wang,F.,Wang,Y.P.,Lu,C.T.,2006.Thecausesandcontrolofcontinuous croppingbarrierindihuang(RehmanniaglutinosaL.).ActaAgric.Boreali-Sin.21, 131–132.

Lipinska,H.,Harkot,W.,2007.Allelopathicactivityofgrasslandspecies.Allelopath. J.19,31–36.

Madamanchi,N.R.,Donahue,J.L.,Cramer,C.L.,Alscher,R.G.,Pedersen,K.,1994. Dif-ferentialresponseofCu,Znsuperoxidedismutasesintwopeacultivarsduring ashort-termexposuretosulfurdioxide.PlantMol.Biol.26,95–103. Miyazawa,M.,Okuno,Y.,Nakamura,S.I.,Kosaka,H.,2000.Antimutagenicactivityof

flavonoidsfromPogostemoncablin.J.Agric.FoodChem.48,642–647. Macel,M.,Bruinsma,M.,Dijkstra,S.M.,Ooijendijk,T.,Niemeyer,H.M.,Klinkhamer,

P.G.L.,2005.Differencesineffectsofpyrrolizidinealkaloidsonfivegeneralist insectherbivorespecies.J.Chem.Ecol.31,1493–1508.

Omezzine,F.,Haouala,R.,2013.EffectofTrigonellafoenum-graecumL.development stagesonsomephytochemicalscontentandallelopathicpotential.Sci.Hortic. 160,335–344.

Singh,M.,Sharma,S.,Ramesh,S.,2002.Herbage,oilyieldandoilqualityofpatchouli [Pogostemoncablin(Blanco)Benth.]influencedbyirrigation,organicmulchand nitrogenapplicationinsemi-aridtropicalclimate.Ind.CropProd.16,101–107. Thoden,T.C.,Boppré,M.,Hallmann,J.,2009.Effectsofpyrrolizidinealkaloidsonthe performanceofplant-parasiticandfree-livingnematodes.PestManag.Sci.65, 823–830.

vandeVoorde,T.F.,Ruijten,M.,vanderPutten,W.H.,Bezemer,T.M.,2012.Canthe negativeplant–soilfeedbackofJacobaeavulgarisbeexplainedbyautotoxicity? BasicAppl.Ecol.13,533–541.

Wu,Y.G.,Guo,Q.S.,Zheng,H.Q.,2007.Textualresearchonhistoryofintroduction andherbalmedicineofPogostemoncablin.J.Chin.Mater.Med.32,2114–2117. Wu,Y.G.,Guo,Q.S.,Zheng,H.Q.,2008.Studiesonresidualsoforganochlorine

pes-ticidesandheavymetalsinsoilofplantingbaseandPogostemoncablin.J.Chin. Mater.Med.33,1528–1532.

Wu,Y.G.,Guo,Q.S.,He,J.C.,2010.Geneticdiversityanalysisamongandwithin popu-lationsofPogostemoncablinfromChinawithISSRandSRAPmarkers.Biochem. Syst.Ecol.38,63–72.

Wu,Y.G.,Li,X.C.,Yang,D.M.,Hu,X.W.,Zhang,J.F.,2013.Isolationandidentification ofthewater-solublecomponentsofPogostemoncablin.Chem.Eng.III,71. Yao,R.Y.,Chen,X.F.,Meng,J.,Li,Z.F.,Yang,X.W.,2012.Developmentstrategiesfor

greencontrolofmedicinalplantsdiseasesinGAPproduction.J.Chin.Mater. Med.37,2242–2246.

Yu,J.Q.,Matsui,Y.,1994.Phytotoxicsubstancesinrootexudatesofcucumber (CucumissativusL.).J.Chem.Ecol.20,21–31.