Cupuaçu (Theobroma grandiflorum Schum.) tem a produção de frutos bastante afetada, quando atacada pela doença vassoura de bruxa, causada pelo fungo Crinipellis perniciosa (Stahel) Singer. Com o intuito de se estudar as alterações bioquímicas em plantas infectadas, folhas sadias e doentes foram analisadas quanto ao conteúdo de açúcares solúveis, amido, proteinas, clorofila, fenóis solúveis totais e taninos. De modo geral, os maiores teores desses compostos foram encontrados em folhas sadias

CONCEIÇÃO, H. E. O. da

MAZZAFERA, P.

ROCHA NETO, O. G. da

STEIN, R. L. B.

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COMMUNICATIONBIOCHEMICAL COMPOSITION OF Theobromagrandiflorum LEAVES INFECTED BY Crinipellisperniciosa1Heráclito Eugênio Oliveira da Conceição4, Paulo Mazzafera2,3, OlintoGomes da Rocha Neto4 and Ruth Linda Benchimol Stein4Laboratório de Ecofisiologia, Embrapa-CPATU, C.P. 48, Belém, PA,66095-100, Brazil.ABSTRACT- Cupuaçu (Theobroma grandiflorumSchum.) has fruit production severely affected by thedevelopment of the witches’ broom disease, caused bythe fungus Crinipellis perniciosa (Stahel) Singer. Inorder to investigate the biochemical alterations ininfected  plants, healthy and infected  leaves  werecompared regarding to the contents of soluble sugars,starch, proteins, chlorophyll, soluble phenolics andtannins. In general, healthy leaves showed the highestcontents of the analyzed compounds.Additional index terms: chlorophyll, cupuaçu, fungi,phenolic compounds, plant pathogen interaction,protein, soluble sugars, starch, tannin, witches’ broom.COMPOSIÇÃO BIOQUÍMICA E ENZIMASOXIDATIVAS EM FOLHAS DE CUPUAÇUINFECTADAS POR Crinipellis perniciosaRESUMO- Cupuaçu (Theobroma grandiflorumSchum.) tem a produção de frutos bastante afetada,quando atacada pela doença vassoura de bruxa,causada pelo fungo Crinipellis perniciosa (Stahel)Singer. Com o intuito de se estudar as alteraçõesbioquímicas em plantas infectadas, folhas sadias edoentes foram analisadas quanto ao conteúdo deaçúcares solúveis, amido, proteinas, clorofila, fenóissolúveis totais e taninos. De modo geral, os maioresteores desses compostos foram encontrados emfolhas sadias.Temos adicionais para indexação: açúcaressolúveis,  amido, clorofila, fenóis, fungo, interaçãoplanta-patógeno, proteína, Theobroma grandiflorum,vassoura de bruxa.INTRODUCTIONToday, witches’ broom represents the major problemlimiting cocoa (Theobroma cacao) production in Brazil.The basidiomycete fungus Crinipellis  perniciosa(Stahel) Sing. is the causal agent of this disease andit is also found infecting other species of Theobromaand Herrania, endemic in the forests of the Amazonbasin (Baker & Holliday, 1957). The life cycle of thepathogen can be divided into two well-defined phases,parasitic and saprophytic. The basidiosporesgerminate and penetrate unhardened tissues, mainlythrough stomata, fallen/collapsed multicellulartrichomes and wounds, causing hypertrophy andhyperplasia of stems, flowers and pods. The myceliumgrows only intercellularly and proliferation occursexclusively in active growing tissues. the funguspersists as a saprophyte, producing basidiocarps forlong periods (Evans, 1980; Calle et al., 1982; Frias etal., 1991). At present, a few cocoa cultivars displayingpartial resistance to C. perniciosa are known. So far,information on the biochemical basis of the resistancein these cultivars are scanty. Recent findings haveindicated that condensed tannins might be involved inthe resistance of cocoa to witches’ broom (Brownleeet al, 1990).Cupuaçu (Theobroma grandiflorum Schum.) is anindigenous tree of the Amazon basin and the pulpcovering the seeds is appreciated for its pronouncedaroma and flavor. The tree is not economicallyexploited on a large scale and the fruits are harvestedfrom native trees growing in the forest by small localfarmers. The pulp is mostly used for consumption asjuice.1Received 30/12/1996 and accepted 08/06/1997. Thiswork received financial support from JapanInternational Cooperation Agency and Embrapa-CPATU.2Departamento de Fisiologia Vegetal, IB, UNICAMP,C.P. 6109, Campinas, SP, 13083-970, Brazil.3CNPq fellow.4Laboratório de Ecofisiologia, Embrapa-CPATU, C.P. 48,Belém, PA, 66095-100, Brazil.Biochemical composition of Theobroma grandiflorum ... 135R. Bras. Fisiol. Veg., 9(2):135-138, 1997.Cupuaçu is also infected by witches’ broom. Vieira(1942) considered that, compared to cacao, theproduction of fruits in this plant is more affected by thedisease, since fruit set occurs only in the flushes.However, the isolates of C. perniciosa infecting cacaodo not infect cupuaçu (Bastos, 1986), showingspecificity for the host. So far, to our knowledge, thereis no cultivar of cupuaçu displaying resistance towitches’ broom nor any report on the physiologicalbehavior of this plant when infected by the pathogen.Most of the information available on pathogen-plantinteractions relates to cacao.Thus, this papper is concerned with the interactionbetween cupuaçu and C. perniciosa, where infectedand healthy leaves were compared with regard tosome chemical constituents.MATERIALS AND METHODSCupuaçu has alternate leaves. The first and second,the fourth and the eighth leaves were collected frominfected and healthy flushes of 12 year old cupuaçuplants growing at Centro de Pesquisa Agroflorestal daAmazônia Oriental, Belém (CPATU-Embrapa). Sincethere was no way to control the infection of theseplants, for each leaf type (healthy or infected) fifteen totwenty leaves were collected from several branches offive plants and mixed. Then, they were divided in twosamples, and three replicates were taken from eachone. After harvesting, the leaves were kept on ice untilthey were taken to the laboratory, where they weremeasured in length and width, for determination of leafarea, weighed and immediately used for theextractions. Ten leaves with the same physiologicalage were used to obtain the fresh and dry matter.Segments of five healthy and infected stems,between the first and eighth leaves, were collectedfrom the same cupuaçu trees. After removal of theleaves, they were weighed and their volumes wereobtained by recording the displacement volume of amixture (80 ml of 50% aqueous ethanolic solution in a100 ml graduated cylinder. The dry matter wasobtained after one week at 70oC.Approximately 1g of leaves were covered with 25 mlof ethanol 80% and disrupted in a Virtis “45”homogeneizer (Virtis Company, New York). A secondextraction was made with the same volume of ethanoland the combined extracts transferred to 250 mlErlenmeyer flasks.After covering with aluminum foil, the flasks weremaintained in a water bath (80oC) for one hour, withoccasional agitation. After cooling the debris werefiltered off and the filtrate was used for thedetermination of soluble sugars according to Dubois etal. (1956). Sucrose was used as standard.The starch content was measured in the recovereddebris after overnight digestion with 10ml of 35%perchloric acid. The extracts were centrifuged at 1500g and the glucose residues were quantified (Dubois etal, 1956). Glucose was used as standard.The    same    ethanolic extract used for thedetermination of soluble sugars was also used forphenolic (Swain & Hillis, 1959) and chlorophylldeterminations (Arnon, 1949). For phenolics, phenolwas used as standard.Tannins were twice extracted with boiling distilledwater (1g/ 2 X 25ml). The leaves were disrupted in aVirtis homogenizer and incubated for one hour in aboiling water-bath. The debris were filtered off andtannin concentration was measured by theprecipitation of methylene blue in phosphate buffer(Okuda et al., 1985).Soluble proteins were determined only in the firstand second and fourth leaves. The main vein wasremoved and the leaf blades were ground in a mortarwith cold 250 mM sodium phosphate buffer, pH 7.0,containing 5% ascorbic acid. The extracts werecentrifuged at 1500 g, 4oC and the supernatantrecovered for protein determination (Bradford, 1976).Bovine serum albumin was used as standard.RESULTS AND DISCUSSIONSurprisingly, the fourth infected leaf differedsignificantly from the others, showing in infectedplants a lower accumulation of dry mass (Fig. 1B)associated with a faster leaf expansion (Fig. 1A), and,as a result, the dry mass per leaf area for this leaf wasthree times lower than for the healthy one (Fig. 1C). Forthis reason, the contents of the chemical constituentsshown in Table 1 were expressed as kg.m-2.The percentage of dry mass was higher for healthytissues  (23.4%) when  compared to infected ones(17.8%). When the density was calculated using thefresh matter, no difference was observed (0.931leaves kg.L-1 for healthy and 0.972 kg.L-1 for infected).However,, using dry mass the healthy stems showedhigher density (0.218 kg.L-1) than diseased ones(0.174 kg.L-1), indicating that they accumulated moredry matter per volume.Cupuaçu leaves infected with C. perniciosa showedreduction in the contents of phenolic compounds andtannins (Table 1). This data, associated with theknowledge that infected cocoa tissues becomeswollen and the cells disorganized as a response toincreased phytohormone levels (Isaac, 1992), leadsus to speculate that in order to facilitate its growth inthe apoplast, in some way the fungus changes thehormonal balance in the leaves, affecting theaccumulation of cell wall constituents. In addition, it is136 Biochemical composition of Theobroma grandiflorum ...R. Bras. Fisiol. Veg., 9(2):135-138, 1997.known that increased levels of auxin may induce anincrease in elasticity of the cell wall due to chemicalalterations of its composition (Salisbury & Ross,1992). Since in plants lignin would account for 20-35%of the cell wall (Monties, 1989), acting therefore asstrong sink of phenolic acids, a low accumulationwould cause a lowered demand of these compoundsin cupuaçu leaves infected with C. perniciosa.TABLE 1- Chemical composition of healthy andinfected cupuaçu leaves.LeafpositionFirst + second Fourth EightSol.Sugars Healthy 5.34 a 1.12 a 1.00 aInfected 2.97 b 0.62 b 0.52 bStarch Healthy 4.08 a 0.72 a 0.30 aInfected 5.16 a 0.31 b 0.55 aProteins Healthy 0.84 a 0.21 a ndInfected 0.62 b 0.08 b ndChlorophyll Healthy 0.062 a 0.013 a 0.028 aInfected 0.026 b 0.005 b 0.020 bPhenolics Healthy 9.13 a 1.67 a 1.24 aInfected 4.83 b 0.86 b 0.89 aTannins Healthy 8.75 a 1.16 a 0.38 aInfected 3.21 b 0.64 b 0.40 aData are means of three replicates. Different lettersindicate statistical differences between healthy andinfected leaves (Duncan, P  0.05).Data expressed as X 10-2 kg.m-2.There is little information about the effects of fungalinfection on photosynthesis in plants and theconsequent effect on sugar content. Some studiesreported increased (Klecan & Buchanan, 1988) ordecreased (Manners, & Gay, 1982) starch levels insome plant/pathogen combinations, andphotosynthetic rates not necessarily accompany theincrease of sugars in infected tissues (Coghlan &Walters, 1992).Here, infected leaves showed a decrease in thecontents of soluble sugars, starch, and chlorophyll(Table 1). Healthy leaves almost always showed thehighest values for all the compounds analyzed.Similarly, the first/second leaves had the highestcontents. Soluble proteins also decreased in infectedleaves, which might be a consequence of the alteredCO2 assimilation.C. perniciosa grows exclusively in the intercellularspace (Frias et al., 1991). Therefore, the nutrientsused for its growth must be available in the apoplast. Ithas been shown in cocoa tissue culture andprotoplasts that the fungus causes leakage ofelectrolytes (Isaac, 1992), suggesting that nutrientsmay migrate from inside to outside the cells, asdemonstrated for other plants attacked by pathogens(Aked &Hall, 1993). Increase of extracellular invertaseactivity was also detected (Aked & Hall, 1993).Our results indicate a general metabolic disturbancein leaves of cupuaçu infected by the fungus. It seemslikely that due to a reduced chlorophyll content,photosynthesis is affected in these leaves and,consequently, the level of carbohydrates. However,considering that C. perniciosa grows in theintercellular space, further studies on the chemicalcomposition of leaf apoplastic washout, and invertaseactivity are necessary and should provide new insightson the physiology of this plant-pathogen interaction.Whith respect to the variations of phenolics andtannins, a more detailed study should be conducted,investigating the nature of these compounds, the lignincontent, and ultrastructural alterations of the cells indiseased stems and leaves.ACKNOWLEDGMENTSFIGURE 1- Leaf area - m2 (A), % dry mass (B) and drymass/leaf area - kg.m-2 (C) in the first/second, fourthand eighth healthy and infected leaves. Data aremeans of ten replicates and the asterisk indicatesstatistical difference (Duncan, P 0.05) betweenhealthy and infected leaves at the same leaf position.Biochemical composition of Theobroma grandiflorum ... 137R. Bras. Fisiol. Veg., 9(2):135-138, 1997.To Cleo Marcelo de Araújo Souza for his technicalassistance and Dr. Jorge Vega for helpful comments.REFERENCESAKED, J. & HALL, J.L. Effect of powdery mildew infectionof concentrations of apoplastic sugars in pea leaves.New Phytologist, 123:283-288, 1993.ARNON, D.I. Coper enzymes in isolated chloroplasts.Polyphenoloxidases in Beta vulgaris. PlantPhysiology, 24:1-15, 1949.BAKER, R.E.D. & HOLLIDAY, P. Witches’ broom diseaseof cacao (Marasmieus perniciosus Stathel). TheCommonwealth Mycological Institute, 1957.Phytopathological paper, 2o, 57.BASTOS, C.N. Comparação morfológica e patológica deisolados de Crinipellis perniciosa (Shahel) Singer.Belém, CEPLAC/DEPEA, 1986. p. 45-49. (InformeTécnico).BRADFORD, M.M. A rapid and sensitive method forquantification of microgram quantities of proteinutilizing the principle of protein-dye binding.Analytical Biochemistry, 72:248-251,1976.BROWNLEE, H.E.; McEUEN, A.R.; HEDGER, J.  &SCOTT, I.M. Antifungal effects of cocoa tannin on thewithches’ broom pathogen Crinipellis perniciosa.Physiological and Molecular Plant Pathology,36:39-48, 1990.CALLE, H.; COOK, A.A. & FERNAND, S.Y. Histology ofwitches’ broom caused in cacao by Crinipellisperniciosa. Phytopathology, 72:147-148, 1982.COGHLAN, S.E. & WALTERS, D.R. Photosynthesis ingreen-islands on powdery mildew-infected barleyleaves. Physiological and Molecular PlantPathology, 40:31-38, 1992.DUBOIS, M.K.; GILLER, K.A.;   HAMILTON, J.K.;RIBERS, P.A. & SMITH, J. Colorimetric method fordetermination of sugars and related substances.Analytical Chemistry, 28:350-366, 1956.EVANS, H.C. Pleomorphism in Crinipellis perniciosa,causal agent of witches’ broom disease of cocoa.Transactions of the British Mycology Society,74:515-523, 1980.FRIAS, G.A.; PURDY, L.H. & SCHMIDT, R.A. Infectionbiology of Crinipellis perniciosa on vegetative flushesof cacao. Plant Disease, 75:552-556, 1991.ISAAC, S. Interactions between the pathogen Crinipellisperniciosa and cocoa tissue. In: Tropical MycologySymposium, British Mycological Society, Universityof Liverpool, 6th-9th April, 1992. Proceedings.KLECAN, A.L. & BUCHANAN, B.B. Powdery mildewinfection alters 2,6-bisphosphate content andsucrose to starch ratio in leaves of wheat plantsactively forming grain. Physiological and MolecularPlant Pathology, 32:221-227, 1988.MANNERS, J.M.& GAY, J.L. Transport, translocation andmetabolism of 14C photosynthates at thehost-parasite interface of Pisum sativum andErysiphe pisi. New Phytologist, 91:221-244, 1982.MONTIES, B. Lignins. In: HARBORNE, J.B. ed. Methodsin Plant Biochemistry. v. 1. London, AcademicPress, 1989. p.113-157.OKUDA, T.; MORI, K. & HATANO, T. Relationship of thestructures of tannins to the binding activities withhemoglobin and metylene blue. Chemical andPharmacology Bulletim, 32:1424-1433, 1985.SALISBURY, F.B. & ROSS, C. Hormones and growthregulators: auxins an gibberelins. In: SALISBURY,F.B. & ROSS, C. ed. Plant Physiology. Belmont,Wadsworth, 1992. p.357-381.SWAIN, T. & HILLIS, W.E. The phenolic constituents ofPrunus domestica. I. The quantitative analysis ofphenolic constituents. Journal of the Science ofFood and Agriculture, 10:63-68, 1959.VIEIRA, J.T. “Lagartão” ou vassoura-de-bruxa. Brasília,DF, Ministério da Agricultura, 1942. (Boletim)138 Biochemical composition of Theobroma grandiflorum ...R. Bras. Fisiol. Veg., 9(2):135-138, 1997.

Biochemical composition of Theobroma grandiflorum leaves infected by Crinipellis perniciosa.

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Biochemical composition of Theobroma grandiflorum leaves infected by Crinipellis perniciosa.

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