09'71-4693/94US $ 5.00 Allelopathy Journal 23 (l): 151-166 (2009) InternationaLAlleLooathv Foundation 2009 Tables:3,Fig: I Role of phenolic acids in expressionof barley (H ordeum vulg ar e) autotoxicity o. orrESLArr',M. BEN-HAMMR"I*I##_GHORBEL2, M.EL GAZZEH2
Laboratoirede Physiologiede la ProductionV6g6tale, Ecole Sup6rieured'Agriculture du Kef (ESAK), Kef, Tunisia. E. Mail: [email protected]
(Receivedin revised form: October 25.2008)
ABSTRACT
The role of phenolic acids in autotoxicity of four barley (Hordeunt vuLgare L.) varieties was investigated using radicle growth bioassays and analytical tecliniques. Total phenolic content of barley plant components variecl within ancl between varieties during the 1999-2002 gtowing seasons.Inhibition of barley radicle growth was positively correlated with total phenolics depending on growing season and variety. Only total phenolic content of barley stems contributed significantly to barley autotoxicity. Concentrationsof five phenolic acids differed in all plant components,among barley varietiesand growing seasons.Ferulic acid and vanillic acid occurred least and most frequently in barley plant tissues, respectively;p- hydroxybenzoicacid, syringic acid, and p-coumaric acids were positively associated with barley autotoxicity (r 2 0.31). Inhibition was significantly correlatedwith total phenolics,although other allelochemicalscould also contributeto barley autotoxicity. Variationsin total phenolicsand phenolicacid compositionover growlng seasonsmay indicate a strong impact of climatic conditions on phenolic accumulation in barley plants.
Key words: Autotoxicity, barley,phenolic acids, total phenolics.
INTRODUCTION
Phenolic compoundsform a large group of naturally occurring and chemically diverse substanceswidely distributed among plants.Characterized by the presenceof an aromaticring with one or more hydroxyl groups,phenolics include alkaloids,flavonoids, terpenoids,and glycosides(1). The most important groups of phenolics are flavonoids, phenolic acids and polyphenols, commonly known as tannins (lg). phenolics are secondarymetabolites, primarily synthesizedthrough the shikimate metabolic pathway (27). Phenolicacids contribute to the allelopathicexpression in numerous crops,inciuding sorghum (sorghum bicolor L. Moench) (4), whear (Triticum aestivumL.) (3,2g,31),oat \AvenasativaL.) (3), and rtce(Oryza sativaL.) (24). Phenolicacids as potential allelochemicals have been identified in numerouscrop and weed species.For example, wheat and barley residuestypically releaseferulic acid (25). Eight phenolic acids (trans-cinnamic, salicylic, f'erulic, chlorogenic, p-hydroxybenzoic,protocatechuic, p-coumaric, vanillic) were identified in spring barley
'Correspondence t author,r Facultddes Sciencesde Tunis, Tunisie, USDA-ARS, Cropping SystemsResearch Unit and Department of Soil, Environmental & Atmosphelic Sciences, 302 Natuial ResourcesBuildine. Universityof Missouri, Columbia,MO 6521I USA. 158 Oueslatiet al and oat tissues(13). Potentialallelochemicals including benzoicacids (p-hydroxybenzoic, vanillic, protocatechuic)and cinnamic acids (coumaric,caffeic, ferulic, chlorogenic)were detected in aqueous extracts of 30 barley varieties (33). Seven phenolic acids (p-hydroxybenzoic,vanillic, cls-p-coumaric,syringic, cis-ferulic, trctns-p-coumaric,trans- ferulic) were exudedby 17 day-old wheatseedlings into agar growth medium (29). Other allelochemicalsincluding hydroxamic acid2,4-dihydroxy-7-methoxy- I ,4-benzoxazin-3-I were also detected(30). Wheat accessionsdiffer significantly in the productionof phenolic acids;those with high levels of total phenolicacids in shoot and root tissuesare generally the most allelopathic(28,31). Highly allelopathicaccessions exuded high concentrations of allelochemicalsinto the growth medium (29). Sorghum root exudates containing p-hydroxybenzoic,vanillic and syringic acidsmay enhanceoverall allelopathicpotential in the field when residueson the soil surfaceor are incorporatedby tillage (4). Increased nutrient availability to two winter wheat cultivars lowered the concentrationof phenolic compoundsin the plants (tl). However, water stressincreased phenolic accumulationin maize plant tissues(23). Allelopathy is strongly coupled with a variety of crop environmentalstresses such as high temperatureand insect damagethat enhancethe potentialfor crop interferencethrough increased production of allelochemicals (8). In barley, phenolic-basedallelochemical content was influenced more by growth conditions than by variety (14). Temperatureand nitrate availability increasegramine content in leaves,which contributesto self-defencemechanisms in barley varietiesthat exhibit resistanceto aphid (Aphis spp.) attack (10, 7, 20). Secondarymetabolites such as gramine and hordenin play a role in barley allelopathicpotential and its defenceagainst fungal pathogens (Drechslera teres) and armyworm (Mythimttt convecta) larvae. Gramine, an indole protoalkaloid was identified in leavesof two sub-species(H' vulgare ssp.vulgare; H. vulgare ssp.spontaneum) of barley (32). Gramine is also releasedthrough roots and may aid barley in overcoming competitive effects of weeds such as white mustard (B rass ic a hirta Moench) (20). The presentwork investigatedthe potentialrole of phenolicsin the expressionof barley auto-toxicity for two reasons:(i) phenolics as allelochemicalsoccur in several cerealcrops and (ii) the concentrationand effectivenessof phenolic compoundsin barley tissuesmay be influencedby changesof environmentalfactors. Therefore, we focussedon the contribution of phenolic acid content to the allelopathic potential of barley, and investigated the presence and role of five phenolic acids for assessingdifferential allelopaihic potential among plant componentsof four barley varieties during three growingseasons in Tunisia.
MATERIALS AND METHODS
Four local varieties('Manel', 'Martin', 'Esp6rance','Rihane') of barley were cropped in a randomized complete block design (RCBD) with 4 replications over 3 g.o*ing seasons(1999/00, 2000/01, 2001/02) at the Experimental Station of Ecole Sup6rieured'Agriculture du Kef (ESAK) located in the semi-arid zone of Tunisia, on a clay soil with a pH of 8.1 and 2Vo organicmatter. Mature plants of barley, free of disease symptomsand insect infestation,were randomly collectedfrom plots. Roots were washed Role of phenolicacids in expressionof barley autolo.xicity 159
with tap water to remove the soil and whole plantswere storedfor one week in the dark at <5"C until extraction.
Extraction of Plant Tissues Barley plants were gently washedwith distilled water, blotted betweentwo paper towels, and then separatedinto roots, leaves,stems, and grain. Except for grain, all plant componentswere choppedinto 1-cm long piecesand dried at 50"C for 24 h, then extracted following the proceduredescribed by Ben-Hammoudaet al. (4).
Bioassays To evaluatethe inhibitory potential of 4 barley varieties, 'Manel' was chosenas the assayspecie due to its high sensitivityto allelochemicals(6). Water-extractsof the 4 varieties were tested by radicle growth bioassayfollowing the proceduredescribed by Ben-Hammoudaet al. (5). Radicle growth inhibition was calculatedas: [(Control - Treatment)/ControlX 1001.
Determination of Total Phenolics The Folin-Denis method was usedfor total phenol analysis(2), with tannic acid as the standard.Folin-Denis reagentis a mixture of l0 g of sodium tungstate,2 g of phosphomolybdicacid and 5 ml of phosphoricacid in 75 ml of distilled water rhat was refluxed for 2 h, cooled, and diluted to 100 ml wrth distilled water. The assayfor total phenolic content followed the procedureof Makkar (22): saturatedsodium carbonatewas preparedby adding40 g of sodium carbonateto 150 ml of distilled water,dissolving fbr I h in the dark and adjusting to 200 ml; tannic acid standard solution was preparedby dissolving50 mg of tannicacid in 100 ml of distilledwarer. Aliquots of 0, 20, 40, 60, g0 and 100 ;rl of standardtannic acid solution were dispensedinto tubes containing 0.5 ml Folin-Denis reagent and 2.5 ml saturatedsodium carbonate solution. Standardswere diluted to 4 ml with distilled water and quickly shaken,incubated in the dark at room temperaturefor 35 min, and absorbancedetermined spectrophotometrically at750 nm (2). For barley plant extracts, 0.5 ml Folin-Denis reagent and 2.5 ml saturated sodium carbonatewere combined with I ml of the water extract.Absorbance was determinedand the total phenolic content was estimatedby using the standardcurve derived with the tannic acid standards.Total phenolic content was expressed as trrg of tannic acid equivalents.For barley water extracts,tannic acid equivalents were multiplied by 20, basedon an extractionratio of l:20 (w/w), to yield unitsin pg of tannicacid equivalents per g of barley tissue.
Qualitative and Quantitative Analysis of Phenolic Acids Water extractsof barley plantsused to estimatetotal phenolicswere analyzedfor p-hydroxybenzoic(PoH), vanillic (vAN), syringic (sYR), p-coumaric (pco), and ferulic (FER), which are associatedwith allelochemicalcontents of several cereal crop plants including wheat (28,31) and sorghum (4). Prior to analysis,water exrractswere filtered through a0.45-1tmsterile membrane.The filtered crude water extractswere analyzedfor phenolicacids using a Shin-packcLC (M-ODS) HPLC system,wirh two pumpsoperating at a O.'7ml/min flow rate and a UV detectorset at 280 nm. Separationof phenolic acids was performedon a ClS reversed-phasecolumn (4.6 x 250 mm). The mobile phasewas r60 Oueslatiet al
phenolicacids 0.1% phosphoricacid in'707oacetonitrile. Quantification of the individual was managedby a calibratedcomputerized package'
Data Analysis were Analyses of variance were conducted on bioassay results and means (o). Regressionof separatedusing Fisher's protectedLSD at 0.05 level of probability phenolic acids was l.udi"l" growtL; inhibition on total phenolic content and individual qualitative variables' carried out with plant component,variety and growing seasonas conductedto reach When necessary,data transformationof the independentvariable was an acceptablelevel of probability.
RESULTSAND DISCUSSION
the Extractsof all plant componentsof four testedvarieties substantially reduced Across all three seedlingradicle growth comparedto water control (without plant extract)' variety (across gro*ini ,"uronf the stem component(across varieties) and the 'Martin' barley varieties sourcesof water-extracts)showid highest inhibitory activity than other of (Table l). Total phenoliccontent was iignificantly(o: 0.05)higher in all components the 'Martin' variety and was highestin leaf and stem(Table l)' (assayvafiety) by water- Table 1. Growth inhibition(7o) of seedlingradicle of 'Manel' barley The exrracrsprepared from plantcomponents (5 g tissue/100ml) of four barleyvarieties growing respectivetotal-phenolic contents of eachplant component avelaged ovel three seasonsare presinted tbr comparisonwith respectto growthinhibition
Extract Parameter Barley variety rance' Means 48.50b 56.03 Roots RGI " 62.43a" 64.63a 48.57b 4'7.s9 TPCh 31.5'/c '75.25a 59.68b 23.84c 58.57a 58.94 Leaves RGI 58.60a 63.40a 55.17a TPC 441.80a 443.47a 279'95c 384.11b 387.33 '72.33 62.20c 67.13 Stems RGI a 6'1.57b 68.83b TPC 159.'73b 182.88a 130.61c 126.13c 149.84 38.23c 54.81 Seeds RGI 52'83b 60.47a 67.70a t0.67 TPC 12.91b 20.11a 3.84c 5.81c 51.88 Means RGI 61.55 64.02 60.07 TPC 161.50 r80.43 118.52 134.97 ontents(pg of tannic acid equivalent/g : dvZfueswithin rowsiollowed by thesame letter do not significantlydiffer at a 0 05 "ili*".ribasedon Fisher'sleast significance test. (Y) During rhe second growing season(2000/01) radicle inhibition correlated of variety and with total pheriolics(TP) (ri.42; plO.fO) as (TP + 0.25)001,independent of plant plant componentas sourceof the water-extract.Within variety and independently 'Rihane'' and growing season,Y correlated(r = 0.56; p<0.06) with TP only for growth "o*pon"niAllelochemicais other than phenolic acids may also be involved in potential suppression.Two alkaloids (hordenine,gramine) responsible for the allelopathic Role of phenolic acids in expressionof barley autotoxicity l6l of barley (I2, 19) may have causedthe growth inhibition. Sorgholeone(p-benzoquinone), l-tryptophan, DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one)and DIBOA (2,4-dihydroxy-1,4-benzoxazin-3-one)play roles in allelopathy of sorghum (9), oat (15, i6), wheat (30) and maize (Zea mays L.) (17). The autotoxic potential of barley is apparentlyassociated with concentrationsof specificphenolic acids rather than with total phenoliccontent. This is very similar to the findings of Ben-Hammoudaet al. (4), in which sorghumallelopathy to wheatwas relatedto specificphenolic acids. Multiple regressionof Y on TP (Xr = TP) as the quantitative variable and the source of phenolics (Xz = roots, X: = leaves, X+ = stems, Xs = seeds)as qualitative variablesshowed that stems were the sole significant variable in the best fitting model. : Significant parametersin the correspondingequation were: p6 56.6 and Ba= 11.1 (p < 0.05). Although leaves containedthe highest total phenolic content, stem extracts were most inhibitory to radicle growth (Table l). These results suggestthat inhibition was associatedwith phenolic composition rather than the concentration of total phenolic compounds.Barley varietiesalso varied in total phenolic content acrossyears, indicating the influence of variable growing conditions experiencedduring each growing season (Table2). Phenolic acid contentsof plant componentsin all test varietiesacross 3-growing seasons(1999-2002) are given in Table 2. Three phenolic acids, POH, VAN, SYR were always present and were detected in different amounts in stem. Concentrationsof individual phenolic acids varied widely in plant componentswithin and among barley varietiesand growing seasons(Table 2), which was problematic for statisticalanalyses. However, the data helped in deriving correlationsbetween the seedlinggrowth inhibition and phenolic content of plant componentextracts (Table 3). Generally,concentrations of phenolic acids were higher in 2000/01 than 1999/2000and 20001102seasons (Fig. la). This seemedto be relatedto the relatively dry seasonof 2000/01,as only 283 mm rain was received during the barley growing season(November-May). Independent of plant component, variety and growing season,the phenolic acids, POH, SYR, PCO, were positively correlatedto radicle growth inhibition (Table 3). Vanillic and o-coumaricacids were also implicated by Baghestani et al. (3) as possible allelochemicalsof barley. Inhibitory propertiesof phenolic acids originating from barley are similar to those from other cerealspecies, such as PCO in rice (24), POH, SYR, and PCO in sorghum(4) and in wheat(28,31). Phenolic acid content differed greatly among plant components within and between barley varieties across growing seasons(Fig. lb). Sorghum hybrids exhibit a similar pattern (4). Fluctuationsin phenolic acid contentsacross growing srasonsfor the same variety are partially due to variations in climatic conditions experiencedat the experimental sites where the test barley varieties were grown. This is in complete agreement with the results of Einhellig (8) who reported that the production of allelochemicalswas dependenton the degreeof environmentalstress. The large variability in seasonalphenolic content among a setof saheliansorghum genotypes was thoughtto be more due to climatic conditions than cropping and soil factors (26). The exudation of DIBOA by maize roots wa$ influencedby the duration of light irradiation (17). Similarly, hordenineproduction by barley leaveswas determinedmore by environmentalconditions than geneticfactors (21). t62 Ouesl.utiet al
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Table 3. Simple correlation coefficients arrcl regression equations between bariey radicle growth inhibition and individual phenolic acids extracted from plant components of fbur barley varieties. grown over three seasons
a Phenolic acid Correlation coefficient Begressionequation p-Hydroxybenzoic acid 0.31 Y = 0.lMlX -2|7881 Vanillic acid 0.21Ns Y=0.0989X-2.2238 Syringic acid 0.251 Y=0.0392X-0.8848 p-coumaric acid 0.30. Y=0.0179X-0.4611 Ferulic acid 0.12Ns Y=0.0482X -0.4628 u = g.0ipiooiuit-ity df 46;. Significantat tn- tevel Sipificant at the 0.09 probability level; Not sigpificant at the 0.05 probabilitylevel.
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Figure 1. Total phenolicscontents in plant components (a) in 4 barley varieties and (b) 3 growing seasons. t64 Oueslatiet al
control of this Autotoxic potentialwas not stableover time, indicatinglow genetic to the highest trait. In fact a relatively dry growing season(2000/01) that contributed in which a significant concentrationsof phenoiicsin barley tissues,was the only season phenoJic content' relationship occurred between radicle growth inhibition and total .Rihane, was the only variety to show barley autotoxicity and was significantly compounds play an correlated with total phenolic content, suggesting that phenolic production of phenolic essentialrole in the allelopathy of individual varieties. Since changes, varieties compounds appeared to be controlled largely by environmental barley/barleycropplng characterizedby relatively low autotoxicpotential can be used in residuesremain on the sequences,especially in conservationagriculture in which barley using this cereal soil surface as mulch, which assuresa net economic gain for farmers grain productionsYstem.
CONCLUSIONS
SYR, PCO) partially explains the expression Phenolic contents(primarily'most POH, (VAN) in barley tissuesdid not of barley autotoxicitY.The common phenolic acid with other individually cause barleY autotoxicity but may contribute synergistically phenolicacids.
ACKNOWLEDGMENTS
WewishtothankAgenceFrangaisedeD6veloppementandtheTunisian "Direct sowing" which Research-DevelopmentP.ogram- for co-sponsoringthe project potential of barley residues' supported the present *or[ o' phenolics and phytotoxic from the C'B'S (Centre Thanks are exrendedto Pr. sami sayAnt and Mr. Hedi ISSAOUI and to Dr" Hichem BEN de Biotechnologie, sfax) for assistancewith IIPLC analysis, de Tunis) tbr SALEM from INRAT (Institut National de Recherche Agronomique assistancewith the total phenolicanalyses'
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