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J. Phytopathology 132, 319—327 (1991) © 1991 Paul Parey Scientific Publishers, Berlin and Hamburg ISSN 0931-1785 Agricultural Research Organization, The Volcani Genter, Bet Dagan, Israel; and Department of Plant Pathology, University of Galifornia, Riverside, GA, U.S.A. Identification of an Antifungal Compound in Unripe Avocado Fruits and its Possible Involvement in the Quiescent Infections of Colletotrichum gloeosporioides^ D. PRUSKY, I. KoBiLER, Y. FiSHMAN, J. J. SiMs, S. L. MIDLAND and N . T. KEEN Authors' addresses: D. PRUSKY, I. KoBlLER, Y. FISHMAN, Depanment of Fruit and Vegetable Storage, A.R.O., The Volcani Center, P.O. Box 6, Bet Dagan 50250 (Israel). J. J. SiMS, S. L. MIDLAND and N. T. KEEN, Department of Plant Pathology, University of California, Riverside, CA 95251 (U.S.A.). With 3 figures Received October 9, 1990; accepted November 19, 1990 Abstract A new antifungai compound was isolated from peel and flesh of unripe avocado fruits and identified as l-acetoxy-2,4-dihydroxy-n-heptadeca-16-ene. The maximal concentration of the anti- fungal monoene in unripe fruits was about 800 ^g g"' fr.wt. During ripening the monoene decreased to 40 jUg • g"' fr.wt., concomitantly with the appearance of disease symptoms. The concentration of the previously described antifungal diene, l-acetoxy-2-hydroxy-4-oxo-heneicosa-12,15-diene (PRUSKY et al. 1982), in avocado peel was 1,600 ^g • g~' fr.wt. in unripe fruits, decreasing during ripening to 120 jug g"^ fr.wt. At 750 fig ml"' the inhibition of germ tube elongation of germinated conidia by the antifungal monoene and the antifungal diene was 15 % and 44 %, respectively. A 1 : 1 mixture of both antifungal compounds in concentrations ranging from 50 to 750 ^g ml"', showed synergistic activity and increased the percent of inhibited germ tubes of germinated conidia up to 15 % over the sum of activities of the separate compounds. The results are discussed in relation to the hypothesis that the antifungal diene and the antifungai monoene are involved in the quiescence of the germinated appressoria of Colletotrichum gloeosporioides in unripe avocado fruits. ' Contribution from the ARO, No. 3105-E, 1990 series. U.S. Copyright Clearance Center Code Statement: 0931-1785/91/3204-0319$02.50/0

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Page 1: Identification of an Antifungal Compound in Unripe Avocado Fruits and its Possible Involvement in the Quiescent Infections of Colletotrichum gloeosporioides

J. Phytopathology 132, 319—327 (1991)© 1991 Paul Parey Scientific Publishers, Berlin and HamburgISSN 0931-1785

Agricultural Research Organization, The Volcani Genter, Bet Dagan, Israel; andDepartment of Plant Pathology, University of Galifornia, Riverside, GA, U.S.A.

Identification of an Antifungal Compoundin Unripe Avocado Fruits and its Possible Involvement

in the Quiescent Infections of Colletotrichum gloeosporioides^

D. PRUSKY, I. KoBiLER, Y. FiSHMAN, J. J. SiMs, S. L. MIDLAND and N . T. KEEN

Authors' addresses: D. PRUSKY, I. KoBlLER, Y. FISHMAN, Depanment of Fruit and VegetableStorage, A.R.O., The Volcani Center, P.O. Box 6, Bet Dagan 50250 (Israel). J. J. SiMS, S. L.MIDLAND and N. T. KEEN, Department of Plant Pathology, University of California, Riverside, CA

95251 (U.S.A.).

With 3 figures

Received October 9, 1990; accepted November 19, 1990

Abstract

A new antifungai compound was isolated from peel and flesh of unripe avocado fruits andidentified as l-acetoxy-2,4-dihydroxy-n-heptadeca-16-ene. The maximal concentration of the anti-fungal monoene in unripe fruits was about 800 ^g • g"' fr.wt. During ripening the monoene decreasedto 40 jUg • g"' fr.wt., concomitantly with the appearance of disease symptoms. The concentration ofthe previously described antifungal diene, l-acetoxy-2-hydroxy-4-oxo-heneicosa-12,15-diene(PRUSKY et al. 1982), in avocado peel was 1,600 ^g • g~' fr.wt. in unripe fruits, decreasing duringripening to 120 jug • g"̂ fr.wt. At 750 fig • ml"' the inhibition of germ tube elongation of germinatedconidia by the antifungal monoene and the antifungal diene was 15 % and 44 %, respectively. A 1 : 1mixture of both antifungal compounds in concentrations ranging from 50 to 750 ̂ g • ml"', showedsynergistic activity and increased the percent of inhibited germ tubes of germinated conidia up to15 % over the sum of activities of the separate compounds.

The results are discussed in relation to the hypothesis that the antifungal diene and theantifungai monoene are involved in the quiescence of the germinated appressoria of Colletotrichumgloeosporioides in unripe avocado fruits.

' Contribution from the ARO, No. 3105-E, 1990 series.

U.S. Copyright Clearance Center Code Statement: 0931-1785/91/3204-0319$02.50/0

Page 2: Identification of an Antifungal Compound in Unripe Avocado Fruits and its Possible Involvement in the Quiescent Infections of Colletotrichum gloeosporioides

320 PRUSKY, KOBILER, FISHMAN, SIMS, MIDLAND and KEEN

Zusammenfassung

Identifizierung einer anti-pilzlichen Substanz in unreifen Avocadofruchtenund deren mogliche Verwicklung bei Latentinfektionen

mit Colletotrichum gloeosporioides

Isohert wurde eine neue anti-pilzliche Substanz aus Schale und Fruchtfleisch unreifer Avocado-friichte, die als l-acetoxy-2,4-dihydroxy-n-heptadeca-16-ene identifiziert wurde. Die maximale Kon-zentration des Fungizidmonoens in unreifen Friichten betrug ca. 800 fi^ • g"' Frischgewicht (fr.wt.).Wahrend des Reifens verringerte sich der Monoengehalt auf 40 ̂ g • g"' fr.wt. und gleichzeitigerschienen die Befallssymptome. Die Konzentration des schon beschriebenen Fungiziddien,l-acetoxy-2-hydroxy-4-oxo-heneicosa-12,15-dien (PRUSKYeJ al. 1982), in der Avocadoschale betrug1600 jUg • g~' fr.wt. bei unreifen Friichten, die bei reifen Friichten nur 120 /ig • g~̂ Bei einerKonzentration von 750 /xg • g"' wurde die Hemmung der Keimschlauchverlangerung gekeimterKonidien durch das Monoen und das Dien um 15 % bzw. 44 % ermittelt. Eine 1 : 1-Mischung beiderSubstanzen in einer Konzentrationsreihe von 50 bis 750 ̂ g • ml"' zeigte eine synergistische Aktivitat,wodurch der Prozentsatz der gehemmten Keimschlauche von gekeimten Konidien bis zu 15 % hoherlag als die Summe der Aktivitaten der einzelnen Substanzen.

Die Ergebnisse werden diskutiert in Zusammenhang mit der Hypothese, dafi das Dien und dasMonoen mit der Latenz der gekeimten Colletotrichum gloeosporioides-Appressorien in unreifenAvocadofruchten gekoppelt sind.

Avocado anthracnose caused by Colletotrichum gloeosporioides Penz. is themost serious postharvest disease of avocado pears (BINYAMINI and SCHIFFMANN-NADEL 1972). The fungus infects the fruit in the orchard and remains quiescentuntil fruit ripening. At this stage the growth of the fungus is renewed resulting indecay development (BINYAMINI and SCHIFFMANN-NADEL 1972). SWINBURNE (1983)suggested that quiescent infections may be the result of antifungal compounds,preformed or induced, present in unripe but not in ripening fruits. Severalcompounds with antifungal and antibacterial activity were found in unripeavocado fruits (SIVANATHAN and ADIKARAM 1989, KASHMAN et al. 1969, 1970).SiVANATHAN and ADIKARAM (1989) reported the presence of four compounds within vitro antifungal activity against C. gloeosporioides. These compounds were notfully chemically identified. In previous work, PRUSKY et al. (1982) isolated theantifungal compound l-acetoxy-2-hydroxy-4-oxo-heneicosa-12,15-diene andsuggested that this compound was related to the quiescent infections of C. gloeo-sporioides in unripe avocado fruits. The presence of an additional antifungalcompound in unripe avocado fruits was initially reported by PRUSKY (1988). Wereport here the full chemical elucidation of a second antifungal compound and itspossible role in quiescent infections of C. gloeosporioides in unripe avocado fruits.

Materials and Methods

General

Avocado fruits of cultivars 'Fuerte', 'Ettinger' and 'Hass' were obtained from an orchard inRehovot, Israel. Firmness, an inverse parameter of ripening, was measured by recording the force (kg)required to penetrate the fruit skin and flesh with a conic probe (5 mm diameter and 4 mm length)(PRUSKY et al. 1990 a). The average of two determinations on each of five fruits was reported. Fruitwas considered ripe when firmness reached < 1 kg.

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Effect of Antifungal Compound on Quiescence 321

A single spore isolate of C. gloeosporioides from decayed avocado fruits was used in allexperiments (PRUSKY et al. 1982). It was maintained by inoculation of avocado fruits every 10 days oron Petri dishes with potato dextrose agar and incubated at 25 °C. Freshly harvested fruits wereinoculated by placing a 10 /̂ l drop of a spore suspension (9 10* spores ml"') at three different spotsalong the longitudinal axis of each of five fruits. These fruits were incubated at 20 °C in high humidity.Disease expression was defined as the occurrence of darkening over an area 5 mm in diameter.

Extraction of the active confound

Fruit peel tissue 1—2 mm thick, and flesh sampled 3—4 mm under the peel, were homogenizedfor 2 min in 95 % ethanol (20 : 100 w/v) with a Waring Blender operated at full speed. The filteredresidue was concentrated ten times in vacuo at 40 °C and the partitioned twice with dichloromethane.The organic layers were pooled, dried with anhydrous MgSO4, concentrated in vacuo, and used ascrude extract.

Isolation, purification and quantitative analysis

Isolation of large amounts of the active compound was done by flash chromatography on acolumn of siUca gel (10—15 fim, Merck) in a Buchner funnel with a fritted disk (PRUSKYet al. 1982).Inactive material was eluted with hexane containing 25 % dichloromethane followed by 100 %dichloromethane. The active fraction was then eluted with 25 % ethyl acetate in dichloromethane,dried in vacuo, and dissolved in 2 ml methanol. The compound was purified further by highperformance liquid chromatography (HPLC) on a Varian model 5000 fitted with a 25 X 1 cm Merck7jum RP-18 column eluted with 10% water in methanol. The biologically active peak wasrechromatographed on a Waters model 600 A HPLC fitted with a 50 x 1.2 cm Whatman lO/xmPartisil M9 column eluted with 8 % isopropanol in hexanes. The HPLC columns were monitoredwith a differential refractive index detector for the collection of the appropriate peaks.

For quantitative analysis of the antifungal monoene in the crude extracts at different periodsafter fruit harvesting, the extracts were dissolved in 5 ml hexane and 0.5 ml aliquots purified by flashchromatography as described before on a 6 ml capacity "Speed Silica gel" column (PRUSKY et al.1990 b). The 1.2-cm-diameter column was filled with 1.45 g of 40—63 fim silica to a depth of 2.2 cmand pre-equilibrated with 4 ml hexane. Determination of the antifungal monoene was done by HPLCanalysis of 50 jul aliquots of the active fraction in ethanol (PRUSKY et al. 1982). Calculations were basedon comparison of HPLC peak area with that obtained from a standard.

Purification and quantification of the antifungal diene of avocado fruits were conductedaccording to the methods of PRUSKY et al. (1982, 1983, 1990 a).

Identification of the antifungal compound

The highly purified compound obtained from the normal phase HPLC column was examinedby IR and NMR spectroscopy and Mass Spectrometry. Infrared spectra were acquired with a Perkin-Elmer model 202 spectrophotometer, proton and carbon NMR data with a JEOL-200FX Fouriertransform instrument, and mass spectra on a VG-7070-EHF spectrometer.

Bioassay for antifungal activity

Pure antifungal compounds were dissolved initially in 100 % ethanol. The final assay solutioncontained 0.05 % polysorbate 20 (Tween 20, ICI) and 0.0015 % dimethyl sulfoxide in 5 % ethanol.Spore suspensions of C. gloeosporioides of 10'* per ml of assay solution were added to the assaymixture together with antifungal compounds and vortexed slightly. Thirty jul of the mixture wasspotted on 13-mm-diameter Millipore filters (HAWP, 0.45/xm pore size, Tamar, Jerusalem) andplaced on glass slides, which were incubated at 26 °C for 16 h.

When the biological activity was tested in crude extracts, the relative concentration of chemicalpresent in fruit peel was reconstituted by dissolving the crude extraas from 10 g fr.wt. in 7.6 mlethanol (avocado peel contains 76 % water) (PRUSKY et al. 1982). Forty n\ of extract in ethanol wasspotted onto Millipore filters and dried. Forty (il of a conidial suspension in the assay solutiondescribed above, was apphed on the filters.

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322 PRUSKY, KOBILER, FISHMAN, SIMS, MIDLAND and KEEN

Results

Purification and identification

The active fraction obtained by flash chromatography of crude extracts waspurified further by HPLG on the RP-18 column. The active peak appeared at9 min at a flow of 3 ml min"^ This material was repurified on a Partisil M9column, where it showed a single symmetrical peak at a retention time of 20 minat a flow rate of 5 ml min"^

The purified antifungal compound was characterized by its spectroscopicproperties. Proton nmr data indicated a long hydrocarbon chain, a terminalolefin, an acetate, and two other electronegative substituents: (200 MHz, CDGI3)5.81 ddt (IH, J = 16.5, 10.1, 6.8 Hz), 4.98 dq (IH, J = 16.5, 1.7), 4.93 dt (IH,J = 10.1, 1.2), 4.08 m (3H), 3.88 brm (IH), 3.4 brs (IH), 2.7 brs (IH), 2.10 s(3H), 2.04 dtd (2H, J = 6.8, 6.8, 1.2), 1.59 t (2H, J = 4.3), 1.26 brs (2OH).Infrared absorbances at 2.9 and 8.7 jU and DaO-exchangeability of pmr signals at2.7 and 3.4 pointed toward the presence of two OH groups. These features wereconfirmed by carbon nmr; the multiplicities of the carbon peaks were determinedby the INEPT sequence (MORRIS and FREEMAN 1979, PEGG et al. 1981): (50 MHz,CDCI3) 171.2 s (G-O), 139.3 d (G16), 114.1 t (G17), 72.4 d (G2 or G4), 70.7 d(C2 or G4), 68.5 t (Gl), 39.1 t (G3 or G5), 38.2 t (G3 or C5), 33.8 t (C15), 29.6 t(overlapping G8—G13), 29.1 t {C7 or G14), 28.9 t {C7 or G14), 25.3 t (C6),20.9 q (GH3G=O). All seven aliphatic hydrocarbon peaks were methylenes. Ofthe oxygenated carbons, two were methines and the third was a methylene group.The oxygenated methylene chemical shift of 68.5 in cmr and 4.08 in pmr indicatedthat this carbon carried the acetate. The position of the two remaining hydroxylgroups and the exact length of the chain were elucidated by mass spectrometryand finally proved by pmr decoupling experiments in the presence of lanthanideshift reagent.

Due to overlap of the oxygenated methines and methylene signals in theproton nmr spectrum it was necessary to add Eu(fod)3-d3o to achieve adequatespecificity in decouplings. Addition of one equivalent of lanthanide resolved theoxygenated methines and methylene into individual proton resonances. Irradia-tion of the methylene peaks originally at 4.08 caused the broad methine signalsoriginally at 4.08 to sharpen. When the peaks originally at 1.59 were irradiated,

H H H H 0 OH 0II II II 1 ii

A.

H H OH OH 0I I I I II

B . C ( )Fig. 1. The chemical structure of the antifungal diene (A) and the proposed structure

for the monoene (B) from avocado fruit

Page 5: Identification of an Antifungal Compound in Unripe Avocado Fruits and its Possible Involvement in the Quiescent Infections of Colletotrichum gloeosporioides

Effea of Antifungal Compound on Quiescence 323

the alcohol methines originally at 3.9 and 4.08 sharpened. The olefinic peaks wereabsolutely unaffected by the europium reagent; all coordination sites are on theother end of the chain.

Electron ionization mass spectra (20 e.v.) gave a small high mass ion at 310amu. Exact mass calculation, 310.2495, provided C19H34O3 (310.2508) as the onlyformula within experimental error. That this corresponded to an already dehy-drated ion, was shown by a chemical ionization (methane) peak at 329 due toprotonated C19H36O4. A major fragment ion observed at 147 amu indicated thepresence of CH3(C=O)OCH2-CH(OH)-CH2-CH=O+H. Only very small ionswere observed at 161 and 133 amu. Other significant e.i. fragments were at 268,255, 250, 237, 219, 211, 209, 137, 136, 135, 129, 121, 118, 109, 100, 97, 95, 87(100 %), 69 and 43 amu. The proposed structure is l-acetoxy-2,4-dihydroxy-n-heptadeca-16-ene (Fig. 1).

Biological activity of the purified antifungal monoene and antifungal dieneon C. gloeosporioides

The inhibition of the germ tube elongation of C. gloeosporioides by theantifungal monoene and the antifungal diene at 750 jUg • ml"̂ was 15 % and 44 %,respectively (Fig. 2). At 1000 jUg • ml"̂ the inhibition of germ tube elongation bythe monoene increased to 90 % while that of the antifungal diene was only 55 %.The biological activity of a mixture of equal concentrations of the antifungalcompounds at concentrations ranging between 50 and 750 jUg • ml~\ increased thepercent inhibition of germ tube elongation by 7—15 % over the sum of activitiesof both compounds alone.

100

Fig. 2. Effect of purified anti-fungal monoene (A—A),diene (O—O) and the mix-ture of both compounds( •—•) on the percent ofconidia of Colletotrichumgloeosporioides with inhibitedgerm tube elongation. Germtube length after 24 h on thecontrol was 180 ± 35 jumcompared with 37 ± 25 punfor the inhibited germ tubes

'0 200 400 600 800CONCENTRATION OF ANTIFUNG/O. CCM^KHJM)

1000

Page 6: Identification of an Antifungal Compound in Unripe Avocado Fruits and its Possible Involvement in the Quiescent Infections of Colletotrichum gloeosporioides

324 PRUSKY, KOBILER, FISHMAN, SIMS, MIDLAND and KEEN

I 2 3 4 5 6 7 8 9TIME AFTER HARVEST (Days)

Fig. 3. Fruit firmness (D—D), antifungal activity {M), concentration of the antifungal monoene(O—•) and concentration of the antifungal diene (A—A) in crude extracts from peel (O, A) and flesh(• , A) of avocado cv. 'Fuerte' at different stages after harvest. The arrows denote the appearance of

visible symptoms

Occurrence of the antifungal monoene and diene in avocado fruitsat different stages after ripening

The concentration of the antifungal monoene in the peel and flesh of avocadofruits one day after harvest was 450 and 700 jUg • g"̂ fr.wt., respectively. Two dayslater levels of the antifungal monoene decreased and by the 8th day after harvest,when symptoms of disease appeared, the amount of the antifungal monoene inthe peel and flesh was 120 and 40 /̂ g • g~̂ fr.wt., respectively. The concentrationof the antifungal diene in the same fruits was much higher: 1,500 and 1,600 jUg •g~̂ fr.wt. in the peel and flesh, respectively, decreasing 8 days later to 200 and20 |Ug • g~̂ fr.wt. (Fig. 3). Crude extracts of peel from unripe fruits inhibited germtube elongation of germinated conidia by 87 %. Inhibition decreased to 7 % insoft, symptom expressing fruits (Fig. 2).

Presence of the antifungal monoene in different cultivars of avocado

The compound was extracted from the peel of several unripe avocadocultivars in order to determine if it is generally present in avocado fruits. In cv.'Fuerte' the concentration in unripe fruits varied between 450 and 600 ^g • g~̂ ; incv. 'Hass' the concentration was 1,060/igg"^ fr.wt. and in cv. 'Ettinger'1,160 jUg • g~̂ fr.wt.

Discussion

The resistance of unripe avocado fruits to attack by C. gloeosporioides wasattributed to the presence of high concentrations of the antifungal l-acetoxy-2-hydroxy-4-oxo-heneicosa-12,15-diene prior to the infection of the fungus

Page 7: Identification of an Antifungal Compound in Unripe Avocado Fruits and its Possible Involvement in the Quiescent Infections of Colletotrichum gloeosporioides

Effect of Antifungal Compound on Quiescence 325

(PRUSKY et al. 1982). During fruit ripening, the concentration of the antifungaldiene decreased to subfungitoxic levels. The inhibition of germ tube elongationby crude extracts decreased concomitantly. SIVANATHAN and ADIKARAM (1989)sprayed fungal spores on TLC plates and detected the presence of four com-pounds with antifungal activity, viz., Av I, Av II, Av III and Av IV. Based onpartial chemical analysis, SIVANATHAN and ADIKARAM (1989) suggested that Av II,the most active compound, is similar to the antifungal diene, previously isolatedby PRUSKY et al. (1982). A second active compound, the Av IV, was not fullyidentified chemically and could be similar to the second compound reported byPRUSKY (1988). We have purified this second antifungal compound from avocadopeel and flesh, and identified it as l-acetoxy-2,4-dihydroxy-n-heptadeca-16-ene.This compound was previously described in avocado mesocarp as a cytokinininhibitor (BITNER et al. 1971), and was assigned the same structure we report here,based on incomplete spectral data. We have confirmed the presence of themonoene in cultivars 'Fuerte', 'Hass' and 'Ettinger'. Based on the partial chemicaldata presented by SIVANATHAN and ADIKARAM (1989), this compound might alsobe present in avocado cv. 'Green'.

Activity of the antifungal compound in the bioassay was dependent onconcentration and solubility. The antifungal monoene is less fungitoxic than theantifungal diene up to 750 jig • ml~^ At 500 fj,g • mt^, the inhibition of germ tubeelongation by the antifungal 16-ene was only 10 % compared with 35 % inhibi-tion by the antifungal diene. At 1,000 jUg • ml~\ the inhibition of germ tubeelongation by the antifungal monoene increased to 90 % but the biologicalactivity of the antifungal diene increased only up to 55 %. It was suggested inprevious work that the critical micellar concentration reached by the antifungaldiene at approx. 1,000 fig • ml~̂ (PRUSKY et al. 1982) may prevent the increase ineffective concentration of the compound beside its increase in absolute concentra-tion (SHINODA 1963). The antifungal activity of the diene was suggested to be theresult of increased polarity of the molecule resulting from the hydroxyl andacetate groups (PRUSKY et al. 1982). Those groups might be also involved in theantifungal activity of the monoene. Certain fatty acids and their derivatives havebeen demonstrated to be fungitoxic (HOFFMAN et al. 1939, PURITCH 1975, PURITCH

et al. 1981), with maximal activity at approx. 600 pig • ml""̂ (RIGLER and GREAT-

HOUSE 1940). The biological activity of the mixture of both compounds in a 1 : 1ratio up to a concentration of 750 ;Ug • ml"\ was synergistic and increased thepercent of germinated conidia, with inhibited germ tubes, up to 15 % over thesum of activities of both antifungal compounds. Bioassay of the mixture at1,000 jUg • ml~̂ did not increase the biological activity, suggesting that theantifungal diene solubiUty determines the toxicity of the extract.

Concentrations of the monoene in the peel varied in freshly harvested,unripe fruits, between 500 and 650 pLg • g"̂ fr.wt. (approx. 900 jUg • ml~ )̂ anddecreased to 40 jUg • g"̂ fr.wt. (60 jUg • ml~ )̂ when symptoms of disease wereobserved. Concentration of the antifungal diene were much higher in both thepeel and flesh. In freshly harvested fruits the concentrations reached up to1,500 jUg • g"̂ fr.wt. and decreased almost ten times on ripe fruits. Inhibition ofgerm tube elongation by peel crude extracts of freshly harvested fruits was 78 %

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326 PRUSKY, KOBILER, FISHMAN, SIMS, MIDLAND and KEEN

and decreased to 7 % in ripe fruits showing symptoms of disease. Since (i)biological activity of the pure antifungal monoene at the range of concentrationspresent in freshly harvested fruits was relatively low (12 % inhibition); (ii) thegeneral amount of the compound was half of that of the antifungal diene; and (iii)the decrease in biological activity of the crude extract closely related to thedecrease in the amount of the antifungal diene, this compound seems to be theone with the highest biological activity, while the monoene has a synergisticeffect.

The evidence presented in this paper supports the hypothesis that theantifungal monoene and the antifungal diene prevent C. gloeosporioides develop-ment in the peel of unripe avocado fruits and that their decreased concentrationduring ripening enables the renewed growth of the fungus: (i) crude extracts fromunripe fruits of cv. 'Fuerte' exhibited antifungal activity (PRUSKY et al. 1982);(ii) concentrations of the monoene decreased from fungitoxic concentrations,700 /Ug • g~' fr.wt. (approx. 850 fig • ml~ )̂ to subfungitoxic ones, 120 fig • g~^ fr.wt.(approx. 130 jUg • ml~^); (iii) the in vitro activity of the purified compounds againstC. gloeosporioides (ED50 of 870 fig • ml~̂ for the monoene) is consistent with theconcentrations observed in ripe and unripe fruits peel; (iv) all the tested avocadocultivars had high concentrations of the compound in unripe fruits, generallyranging from 900 to 1,500/ig • g~̂ fr.wt.; (v) there is a 7—15 % synergisticactivity by the mixture of both compounds. These factors are consistent with thehypothesis that sufficient amounts of both compounds present in unripe fruit, arethe cause of the quiescent infections of the fungus. The involvement of Av I andAv III in the total biological activity seems to be minor since their toxicity andamounts are lower than the antifungal diene and monoene (SIVANATHAN andADIKARAM 1989).

Mass spectral data were provided by Dr. RICH KONDRAT of the University of California,Riverside, Chemistry Department, Mass Spectrometry Center. This work was partially supported byBARD and Office of Science Advisor, U.S. Agency for International Development.

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