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BARBOSA, G. S.

FALCÃO, L. L.

GANDER, E. S.

MARCELLINO, L. H.

RAMOS, A. de R.

English

Repository Open Access to Scientific Information from Embrapa

ARTICLE IN PRESSMicrobiological Research 162 (2007) 238—2430944-5013/$ - sdoi:10.1016/j.CorrespondE-mail addrwww.elsevier.de/micresNeem (Azadirachta indica a. Juss) components:Candidates for the control of Crinipellis perniciosaand Phytophthora ssp.Alessandra de Rezende Ramosa, Loeni Lüdke Falcãob, Guilherme SalvianoBarbosab, Lucilia Helena Marcellinob, Eugen Silvano Ganderb,aUniversidade Federal do Pará, Centro de Ciências Biológicas, Depto. Genética, Rua Augusto Corrêa 1, Guamá, 66075-110 Belém-PA, BrazilbEmbrapa Recursos Genéticos e Biotecnologia, Parque Estac-ão Biológica – PqEB, Final W5 Norte, 70770-900 Brası́lia-DF,BrazilAccepted 6 June 2006KEYWORDSWitches’ broom;Pod rot;Crinipellis;Phytophthora;Neem tree;Biological controlee front matter & 2006micres.2006.06.004ing author. Tel.: +55 61ess: egander@cenargenSummaryWitches’ broom and pod rot are the two most devastating diseases of cocoa in SouthAmerica and Africa, respectively. Their control by means of phytosanitation andchemical fungicides is labor-intensive, costly and, in many cases, environmentallyundesirable. Therefore efforts are made in order to identify alternative,environmentally safe and cost-efficient methods for the control of these pathogens.Promising candidates are components of the neem tree (Azadirachta indica), thathave been used for centuries in Asia as insecticides, fungicides, anticonceptionals inpopular medicine. Here we report about tests on the effect of various concentrationsof extracts from neem leaves on growth of mycelia of Crinipellis and Phytophthoraand on germination of spores of Crinipellis. We show a 35% growth reduction ofmycelia of Phytophthora on neem leaf extract media, whereas growth of mycelia ofCrinipellis was not affected, even at the highest concentration of neem leaf extractsused (35%). However, the most dramatic effect of neem leaf extracts is observed onCrinipellis spore germination, here the extracts (20–35%) reduced germinationalmost completely. Based on these results, we believe that the neem tree might be asource for the production, on small and medium scale, of an effective and cheapformulation for the control of Crinipellis and Phytophthora.& 2006 Elsevier GmbH. All rights reserved.Elsevier GmbH. All rights reserved.3448 4699; fax: +55 61 3340 3658..embrapa.br (E. Silvano Gander).ARTICLE IN PRESSNeem (Azadirachta indica a. Juss) 239IntroductionWitches’ broom and pod rot are the two mostdevastating diseases affecting cocoa production.The first disease is caused by the basidiomyceteCrinipellis perniciosa [(Stahel) Singer, 1943] andaffects cocoa plantations specifically in SouthAmerica while pod rot, caused by Phytophthorassp., is ubiquitous and affects cocoa plantations inAfrica as well.Due to the action of Crinipellis, cocoa productionin Brazil decreased about 50% from 1985 to 2000(FAO 2002) and in Africa, pod rot caused productionlosses to amount to 50–80% (ICCO site). Control ofwitches’ broom has, so far, been tried throughmeans of phytosanitation, signifying the removal ofaffected shoots, flower cushions and pods from thecocoa tree. In order to increase its efficiency, thismethod is used in combination with chemicalmethods, mostly involving copper compounds. Thelatter are also used for the control of Phy-tophthora-induced diseases. Both methods, how-ever, are labor-intensive and expensive orenvironmentally undesirable and the results ob-tained so far are below expectations.For these reasons and out of growing concernabout the general degradation of the environment,more attention has been given, in recent years, topossibilities of biological control of these diseases.In the case of Crinipellis and Phytophthora moder-ate successes have been obtained by using antag-onistic fungi of the genus Trichoderma (Holmes2004). Methods that involve natural organic pro-ducts are alternatives to the use of life antagonisticorganisms in order to control these diseases. Withinthis scope neem, Azadirachta indica (A. Juss) is apromising candidate. This tree, originally culti-vated in India, belongs to the Meliaceae family andoccupies a prominent space in Asian (and recentlyin Euro-American) popular medicine as well as incorporal hygiene. Among its many attributedproperties range antimicrobial activities and ther-apeutical effects in cases of digestive disorders, ofdiabetes, of high cholesterol and in cases of breast-and gastrointestinal cancer and it has been shownthat components of neem oil could, due to theirspermicidal activity, be used as contraceptives(SaiRam et al. 2000; Sinha et al. 1984). Scientificdata to support these claims, however, are onlyrecently beginning to emerge (see for instanceBramachari, 2004; Singh and Singh, 2003). Theconsensus is that the general antimicrobial activityof neem extracts is mainly due to azadirachtin, amolecule belonging to the class of tetranortriter-penoids (Salehzadeh et al., 2003). While theantimicrobial activity of neem-derived products isuncontested (Pai et al. 2004; SaiRam et al. 2000),only few reports are available on neem’s actionagainst fungi, mostly belonging to the phylumAscomycota (Bhonde et al. 1999; Mossini et al.2004). Since we are interested in alternativesallowing for the control of basidiomycetae andoomycetae, pathogenic to cocoa, we tested ex-tracts from neem and, as a control, cocoa leaveswith respect to their activity on growth of myceliaof C. perniciosa and Phytophthora spp. and withrespect to inhibitory effect on Crinipellis sporegermination.Material and methodsLeaf extractsLeaves were obtained from neem and cocoatrees, both grown at ‘‘Embrapa Recursos Genéticose Biotecnologia’’ in Brası́lia, Brazil. The leaves werecollected, washed in distilled water and ground topowder in liquid N2. Subsequently water was addedin order to obtain a 40% (w/v) extract. Since theseextracts, in particular those from cocoa leaves, areextremely rich in mucilage, the extracts werefiltered through two layers of household towelsand then stored at 20 1C until use.Culture mediaMycelia were grown and germination of sporeswas tested in solid ‘‘YG’’ medium (0.4%yeast extract, 2.5% glucose, 2% agar). For thetests, leaf extracts from neem and cocoawere added to YG medium to a final concentrationof 3, 5, 10, 15, 25 and 35% (v/v). Subsequently themedia were distributed into Petri dishes (+:90mm).Mycelia and sporesSpores of Crinipellis were obtained from basidio-carps grown at the farm ‘‘Almirante Cacau’’ inItabuna, Bahia, Brazil and kept as suspension in 22%glycerol. The concentration of spores was deter-mined by counting aliquots in a Neubauer chamberand their viability was determined by counting thegerminated spores after 4 h on 1% agar in H2O. Inorder to obtain monosporic mycelia, the sporeswere germinated on solid ‘‘YG’’ medium and amycelium originating from a single spore was thenisolated and grown at room temperature until 100%confluent.ARTICLE IN PRESSA. de Rezende Ramos et al.240Mycelia from Phytophtora were obtained fromthe farm ‘‘Almirante Cacau’’, where they wereproduced from fungi collected from infected cocoafruits.Effect of extracts from neem and cocoaleaves on spore germinationSpores from C. perniciosa were diluted to aconcentration of 700 viable spores/ml in H2O.Hundred microliter of this solution were platedout on 2% agar in ‘‘YG’’ containing leaf extracts inthe concentrations described above. The disheswere kept at 26 1C and a photoperiod of 14D/10Nand the germinated spores were counted after 7days. Each concentration was done in triplicate andthe standard deviation determined according to theunbiased formula.Effect of extracts from neem and cocoaleaves on mycelial growthMycelial discs (+: 8.0mm) from Crinipellis andPhytophthora were deposited on 2% agar plates in‘‘YG’’ containing 3, 5, 10, 15, 25 and 35% of neemor cocoa leaf extracts. The dishes were kept at26 1C and a photoperiod of 14D/10N and thediameter of the mycelia was determined at 2 daysintervals during ten days. Each concentration wasdone in triplicate.Results and discussionHere we report the effect of leaf extracts fromneem and cocoa on mycelial growth of Crinipellisand Phytophthora and on spore germination ofCrinipellis. These organisms are the causativeagents of witches’ broom and pod rot in cocoa.Both diseases are responsible for disastrous eco-nomic and ecologic damages, the latter becausethe uncontrolled use of cupric fungicides andthe deforestation brought about by impoverishedfarmers.The rationale of testing components of neem wasbased on its traditional use, in Asia, in popularmedicine against a broad spectrum of microbialdiseases. Recently these claims have been sup-ported by scientific facts, amongst which are somereports on neem seed extracts being antagonistic tofungi as described in: http://www.organicconsu-mers.org/patent/neemtree030905cfm.Mycelial growthThe effect of neem and cocoa leaf extractsIn the neem tree the occurrence of bioactivesubstances is not restricted exclusively to theseeds; bioactive terpenoids are also present inthe leaves. Therefore we tested aqueous neem leafextracts in the concentrations indicated in ‘‘Mate-rial and methods’’ on mycelia of Crinipellis andPhytophthora. Since there are no reports on theoccurrence of antimicrobial substances in cocoa,we included extracts of cocoa leaves as a control.The effect of neem leaf extracts on mycelialgrowth of Crinipellis (Fig. 1a) and Phytophthora(Fig. 1b) over a 10-day period are shown. In thecase of Crinipellis and at the concentrations used,the growth of mycelia is not affected at all, but inthe case of Phytophthora, a 35% inhibition isobserved at 35% extract present in the media. Withextracts from cocoa leaves (Figs. 2a and b), on theother hand, the picture is different: the presenceof up to 15% cocoa extract in the mediumstimulates mycelial growth slightly in Crinipellis(Fig. 2a) and for Phytophthora (Fig. 2b) inhibitoryeffects are observed at concentrations between25% and 35%.Germination of C. perniciosa sporesEffect of leaf extracts from neem and cocoaThe life cycle of C. perniciosa involves abiotrophic and saprotrophic phase and infectionstarts with the biotrophic stage when basidiosporesgerminate on meristems of the host plants. Theysubsequently form mycelia which invade the hosttissue and are responsible for broom formation.After the dead of the broom, the saprotrophicphase sets in and ends with the formation of newbasidiocarps producing the next spore generation.From this it follows that the crucial phase ininfection is the germination of the spores oncethey have settled on the host’s surface. Conse-quently an effective control of the pathogen couldbe obtained by inhibiting spore germination. There-fore we tested the effect of leaf extracts on sporegermination. The results shown in Fig. 3 documentthat extracts from neem leaves are inhibitinggermination at concentrations between 15% and35%. With leaf extracts of cocoa, on the otherhand, an amazing picture emerges: in the concen-tration range of 3–10% cocoa leaf extract, sporegermination is stimulated. As outlined in ‘‘Materialand methods’’, the spore concentration was deter-mined by counts in a Neubauer chamber and sporeviability was subsequently determined by countingARTICLE IN PRESS0246810Diameter (cm)0246810Diameter (cm)control 10%5%3% 15% 25% 35%Extract concentrationColumns correspond to 0, 2, 4, 6,8 and 10 days of mycelial growthcontrol 10%5%3% 15% 25% 35%Extract concentrationColumns correspond to 0, 2, 4, 6,8 and 10 days of mycelial growth(a)(b)Figure 2. Effect of Cocoa Leaf Extract on Growth of Mycelia. X-axis: Days of mycelial growth and extractconcentration; Y-axis: Diameter of mycelia (mean value of three experiments).0246810control 10%5%3% 15% 25% 35%Extract concentrationDiameter (cm)Columns correspond to 0, 2, 4, 6,8 and 10 days of mycelial growthcontrol 10%5%3% 15% 25% 35%Extract concentrationColumns correspond to 0, 2, 4, 6,8 and 10 days of mycelial growth0246810Diameter (cm)(a)(b)Figure 1. Effect of Neem Leaf Extract on Growth of Mycelia. X-axis: Days of mycelial growth and extract concentration;Y-axis: Diameter of mycelia (mean value of three experiments).Neem (Azadirachta indica a. Juss) 241ARTICLE IN PRESS0102030405060708090control3% 5% 10%15%25%35% 3% 5% 10%15%25%35%Germinated sporesNeem leaf extracts Cocoa leaf extractsFigure 3. Effect of Neem Leaf and Cocoa Leaf Extracts on Germination of Crinipellis Spores. X-axis: Concentrations ofextracts; Y-axis number of germinated spores, vertical bars: Standard deviation.A. de Rezende Ramos et al.242the percentage of spores capable to germinate. Forthe experiments, aliquots containing 70 viablespores, independent of the actual number of sporespresent, were plated out. The fact that, withextracts of cocoa leaves, the germination rate ofspores almost doubles means that cocoa extractscan restore germinative bioactivity. Actually thisinterpretation is supported by the other observa-tion i.e. that cocoa leaf extracts seem to stimulatemycelial growth (Fig. 2a) at a concentration of 15%.From an evolutionary point of view this makessense since the patosystem Crinipellis/cocoa mustbe extremely fine-tuned towards the needs of thepathogen.ConclusionsTaken our results together, we believe that theypoint to the potential of neem leaf extracts, asbioactive components useful for the control, aloneor in combination with traditional methods, ofwitches’ broom and of pod rot of cocoa. Bothdiseases have devastating effects on economy aswell as on ecology and the methods actually usedare not efficient or economically satisfying. Inparticular in Brazil, a secondary effect of witches’broom is of alarming ecological importance: cocoawas traditionally grown in the shadows of theAtlantic rain forest. Since Crinipellis abolishedalmost all cocoa trees and, with that, a fairly easyand risk-less income, the farmers started sellingthe rain forest trees. It is possible that the neemtree might represent a major component in astrategy that aims at the reforestation and simul-taneous revival of the cocoa plantations in areasaffected by witches’ broom or pod rot. Botanicallyit is a close relative of mahogany (Swieteniamahagoni) with very tough and termite-resistantwood. It is a tropical evergreen, grows fast up to30m and thrives even in poor and exhausted soil.These characteristics make it a candidate to beuseful in establishing environmental sustainabilityand economic profitability for small- and medium-holder cocoa producers. Not only would it providethe necessary shadow for the cocoa trees andcommercially high-value wood, but it also wouldprovide the ‘‘materia prima’’ for the preparation ofecologically safe and easy to produce formulationsfor the control of cocoa’s most important diseases.AcknowledgmentsThis study was supported by the BrazilianNational Research Council (CNPq). ARR was sup-ported by a grant from Coordination for theImprovement of Higher Education Personnel(CAPES). We thank Dr. Stephan Nielen for criticalreading of the manuscript.ReferencesBhonde, S.B., Deshpande, S.G., Sharma, R.N., 1999. Invitro evaluation on inhibitory nature of some Neemformulations against plant pathogenic fungi. Hindu-stan Antibiot. Bull. 41 (1–4), 22–24.Bramachari, G., 2004. Neem – an omnipotent plant: aretrospection. Chembiochem 5 (4), 408–421.FAO Production Yearbook, 2002. Vol.54, 2000, 260 p.Rome.Holmes, K.A., Schroer, H-J., Thomas, S.E., Evans, H.C.,Samuels, G.J., 2004. Taxonomy and biocontrol poten-tial of a new species of Trichoderma. Mycol Progr 3(2), 199–210.ICCO /http://www.icco.org/questions/pests.htmS.Mossini, S.A., de Oliveira, K.P., Kemmelmeier, C., 2004.Inhibition of patulin production by Penicillium ex-pansum cultured with neem (Azadirachta indica) leafextracts. Basic Microbiol. 44 (2), 106–113.Pai, M.R., Acharya, L.D., Udupa, N., 2004. Evaluation ofantiplaque activity of Azadirachta indica leaf extractARTICLE IN PRESSNeem (Azadirachta indica a. Juss) 243gel-a 6-week clinical study. J. Ethnopharmacol. 90 (1),99–103.SaiRam, M., Ilavazhagan, G., Sharna, S.K., Dhanraj, S.A.,Suresh, B., Parida, M.M., Devendra, K., Selvamurthy,W., 2000. Anti-microbial activity of a new vaginalcontraceptive from neem oil (Azadirachta indica). J.Ethnopharmacol. 71 (3), 377–382.Salehzadeh, A., Akhkha, A., Cushley, W., Adams, R.L.,Kusel, J.R., Strang, R.H., 2003. The antimitotic effectof the neem terpenoid azadirachtin on cultured insectcells. Insect Biochem. Mol. Biol. 33 (7), 681–689.Singh, U.P., Singh, D.P., 2003. Neem in human and plantdisease therapy. J. Herb. Pharmcother. 2 (3),13–28.Sinha, K.C., Riar, S.S., Tiwary, R.S., Dhawan, A.K.,Bardhan, J., Thomas, P., Kain, A.K., Jain, R.K.,1984. Neem oil as a vaginal contraceptive. Indian J.Med. Res. 79, 131–136.

Neem (Azadirachta indica a. Juss) components: candidates for the control of Crinipellis perniciosa and Phytophtora spp.

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Neem (Azadirachta indica a. Juss) components: candidates for the control of Crinipellis perniciosa and Phytophtora spp.

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