Modulation of natural resistance of avocado fruits to Colletotrichum gloeosporioides by CO2 treatment

Physiological and Molecular Plant Pathology (1991) 39, 3 2 5-334

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Modulation of natural resistance of avocado fruits to Colletotrichum gloeosporioides by C0 2 treatment D . PRUSKY, R . A . PLUMBLEY+, and I . KOBILER

Department of Fruit and Vegetable Storage, ARO, The Volcani Center, P .O . Box 6, Bet Dagan 50250, Israel ! .4c(epted,for publication June 1991)

The level of the antifungal compound, 1-acetoxy-2-hydroxy-4-oxo-heneicosa-12,15-diene, in the peel of avocado fruits decreased during ripening . Fruits exposed to a high CO 2 concentration showed an initial increase in the diene level immediately after removal from CO 2 . The concentration of the diene then significantly decreased, before a second increase in concentration . Exposure of avocado fruits to different concentrations of CO 2 (11, 16 and 30 %) for different lengths of time (3, 10 and 24 h), enhanced the initial increase in diene concentration . Only 30 ;b CO 2 applied for 24 h enhanced a significant second increase in diene level and delayed symptom development caused by Colletotrichum gloeosporiodes. Fruit exposure to lower CO 2 concentrations 16 and 110/ ) or short periods (3 and 10 h) caused no significant second increase and no significant effect on symptom development . Fruits exposed for 24 h to 30 ;o CO 2 enhanced the concentration of the phenol epicatechin in a double-peak pattern similar to that exhibited by the diene . It is suggested that CO 2 treatments induce the enhancement of diene levels and consequently cause a delay in symptom development .

INTRODUCTION Postharvest decay is the principal problem encountered during storage of avocado fruits [9] . The use of controlled atmospheres is a possible way to inhibit decay development during storage [1] . Spalding & Reeder reported that storage of avocado fruits under a controlled atmosphere of 2 % 0 2 + 10 °;o CO 2 for 3-4 weeks at 7 °C, prevented the development of anthracnose [14] . Fungal growth was not affected by these conditions, and it was suggested that the inhibition of decay development was the result of an effect on the fruit rather than on the fungus . Much of the research on controlled atmospheres has been empirical, involving optimum levels of CO 2 or 0 2 for storage of a given commodity [1] . The mode of action of elevated CO 2 on the physiology of fruits is still largely unknown, as is the basis for its effect on decay development . Prusky et al . [9] have described the presence of a preformed antifungal compound, l-acetoxy-2-hydroxy-4-oxo-heneicosa-12,15-diene, in the peel and flesh of unripe avocado fruits . This compound has been suggested as the basis of resistance to decay in unripe fruit [8, 9] . The diene was considered to be a pre-infectional, static inhibitor Contribution from the Agricultural Research Organization, Bet Dagan, Israel . No . 3004-E, 1990 series. tPresent address ; Natural Resources Institute, Central Avenue, Chatham Maritime, Chatham ME4 4'I'B, U . K . Abbreviations used in text : PAL, phenylalanine ammonia lyase .

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15] . Recent results, however, have indicated that the concentration of the preformed diene can be enhanced by challenge inoculation with Colletotrichum gloeostporioides (7 Karni el al . [3] showed that despite an initial reduction in the concentration of the diene in freshly harvested fruits, a subsequent rapid increase in the concentration occurred to regain the initial level . This suggests a rapid turnover of the compound . The present work describes the modulation of the levels of the preformed diene as a novel way of increasing resistance to postharvest diseases . Results suggest that CO 2 treatments enhance the level of the diene and delay symptom development . The regulation of the diene increase by CO 2 is thought to be due to an effect on epicatechin, 1

1 .

a phenol that inhibits the activity of the enzyme lipoxygenase in the fruit peel [11 ] . This results in the accumulation of the diene, thus preventing symptom development .

MATERIALS AND METHODS Avocado fruits [Persea americana Miller var . drymifolia (Schlect and Cham . Blake] of cv . Fuerte were obtained from an orchard at Ayanot, Israel . Fruits of different maturity were harvested at different periods after fruit set [7] . A single spore isolate of C . gloeosporioides from decayed avocado fruits was used to inoculate fruits in all experiments [9] . Spot inoculation was carried out as described previously [8] . Darkening of the peel in a 5-mm diameter or greater zone was taken as a symptom of disease . The effect of the different treatments on disease development was determined by spot inoculation at six points, three each on opposite sides along the longitudinal axis of the fruits . Fifteen fruits were inoculated per treatment (total of 90 inoculation spots/ treatment) and experiments were repeated four times . Firmness of avocado fruits, an inverse parameter of ripening, was recorded as the force (kg) required to penetrate the fruit skin and flesh with a 5-mm diameter, 4-mm long conical probe [9] . CO 2 treatment Approximately 1-2 h after harvest, fruits were placed in 15-1 sealed glass jars through which was passed a stream of air containing 11, 16 or 30%0 CO 2 at a flow rate of 100 ml min -1 for 24 h . CO 2 and air were obtained from commercial high pressure cylinders . The CO 2 concentrations were obtained by quantitating the flow of pure CO 2 through needle valves to give the appropriate mixture of gases . The outlet of the jars passed to the outside of the storeroom, which was maintained at 20 °C . Control fruits were stored in similar jars under a stream of air . In some experiments, where the effect of high CO 2 was tested under low 02 (0 . 75 % O 2 ), the 30 °,/° CO 2 treatment was applied in a mixture with pure N 2 from commercial cylinders . In some experiments fruits treated with 30 % CO 2 were removed to normal atmosphere at different time intervals (3, 10 and 24 h) after initiation of the CO 2 treatment . Throughout the experiments fruits were stored at 20 ° C . Diene and epicatechin concentrations were assessed on removal from the jars and at daily intervals until the fruits were fully ripe . Each day three fruits from each treatment were sampled, the degree of firmness determined, and then the peel of each fruit was extracted separately to determine diene concentration . Each experiment was repeated at least four times during three growing seasons . Upon completion of the CO 2 treatment fruits were inoculated as previously described



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4 5 6 7 8 9 10 Days after harvest Fto . 1 . Diene levels and symptom development of Colletotrichum gloeosporioides in avocado fruit peel cv . Fuerte exposed to different concentrations of CO 2 at 20 °C . Freshly harvested fruits were sealed in flow-through jars, treated under a stream of CO 2 in air (100 ml min - ') for 24 h and then transferred to normal atmospheric conditions ; A- -A 11 % ; E- ---E 16% ; 0 -- O 30 % CO 2 . Percentage of 02 in each treatment was 20 . 19 and 15 %, respectively . Control fruits were treated with air for the same period of time (s- -•) . The mean of three extractions is shown . The LSD is indicated by bars . After treatment the fruits were point-inoculated with a spore suspension ofC. gloeosporioides (approx . 108 spores or l ) . 'The fruits were incubated overnight at 25 °C and then transferred to 20 °C until disease symptoms were visible . Bars indicate SE oh the mean . 0

I

2

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CO 2 and 0 2 concentrations were monitored by GC using a thermal conductivity detector after passage through a Porapak (,molecular sieve column . Helium served as the carrier gas . Extraction and quantitative analysis of the diene and epicatechin

The diene was extracted from the fruit peel according to the modified method described by Prusky et al . [7] and analysed by HPLC, using 50 p1 samples . Epicatechin was extracted from 10 g of avocado peel, with modifications to shorten the purification process, according to the method described by Prusky et al . [11 ] . The organic fraction was dissolved in 8 ml of 25 % ethyl acetate in dichloromethane and a 2 ml sample was further purified by flash chromatography . A " Spe-ed Silica gel" column (Laboratory Impex Ltd, U .K .), of 1 . 2 cm diameter and 6 ml capacity, was filled with 1 . 45 g of 40-63 tm of silica to 2 . 2 cm depth and equilibrated with 6 ml of dichloromethane . Inactive material was eluted with 8 ml of 30 % ethyl acetate in dichloromethane . The active fraction was then eluted by washing the column twice with 6 ml of ethyl acetate, after which it was dried under a flow of nitrogen . Epicatechin was determined by HPLC analysis of 10Itl samples taken from the concentrated epicatechin fraction dissolved in 400 gl of methanol : water (80 :20, v/v) . 2t-2



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Calculations of the diene and epicatechin concentrations were based on the comparison of the HPLC peak areas with those of the standards [7, 11 Average values of three different extractions are presented . 1 .

RESULTS E#ect of 24 h treatments with different concentrations of CO 2 on the diene level and symptom development Levels of the diene in the peel of untreated fruits decreased from approx . 1000 ftg g _ . t g_t fresh weight 1 day after harvest to 220 µg fresh weight in fruits showing decay symptoms (Fig . 1) . After removal of fruits from a 24 h treatment at different concentrations of CO 2 (11, 16 and 30%), an increase in the concentration of the antifungal diene was observed . This increase, in fruits treated with 1 1 °~;, CO 2 , was followed by a continuous decrease in diene level, but 7 days after harvest the concentration of the diene remained higher than in the untreated fruits . When fruits were treated with 16 o o or 30 % CO 2 , a significant decrease in the diene level occurred between the third and fifth day after harvest . This was followed by a second increase in diene concentration (Fig . 1) . The concentration of the diene reached a level of 825 µg g - ' fresh weight and 1200 µg g -1 fresh weight 5 days after treatment with 16 °(, and 30 % C02, respectively, while in the control fruits the diene level was 490 µg g -t fresh weight . Symptoms of disease by C . gloeosporioides appeared first in untreated fruits . At increasing concentrations of CO 2 symptom development was slower . If the freshly harvested fruits were exposed to 30 % CO 2 for 24 h starting on the third day after harvest, no effect was observed on the enhancement of the antifungal diene concentration compared with control fruits (result not presented) .

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FIG . 2 . Diene levels in the peel of avocado cv . Fuerte exposed for 24 h to 30 0 o C0 2 +0 - 75 1) ;, 0 2 at 20 °C . Fruits were then transferred (arrow) to atmospheric conditions (0- -0) . Control fruits were exposed to normal atmospheric conditions during the same period (0 0 1 . The mean of three extractions is presented . The LSD is indicated by a bar .



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FIG . 3 . Symptom development by Colletotrichum gloeosporioides (0--0), fruit firmness (0---0) and diene leves (0, 0) on avocado fruits cv . Fuerte harvested at different maturity stages and exposed for 24 h to 30°)) C02+ 15 0 0 02 at 20 °C . 0, 0, C02-treated ; 0, 0, control fruits . Fruits were harvested at (a) 180, (b) 210, (c) 240 and (d) 300 days after fruit set .

Exposure of fruit to 30 0/ C0 2 +0-75 0/ 0,instead of 300/ % C02 + 15 % 0, as described in Fig . 1, produced a similar change in the diene pattern (Fig . 2) . The concentration in the treated fruits was 1120 Itg g- ' fresh weight compared with 850 pg gi fresh weight in control fruits . Subsequently the diene concentration declined, followed by the second increase to give a level in treated fruit that was double that present in the controls . Three days later the levels of the diene decreased and were similar to untreated fruits .

Effect of 24 h of 30 % CO 2 treatment on avocado fruits of different maturity When fruits were harvested 180, 210 and 240 days after fruit set and exposed for 24 h to 30 0/,0 CO 2 , symptom development by C . gloeosporioides was significantly delayed



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compared to untreated fruits (Fig . 3 ; . Concentration of the diene in the CO 2 treated fruits, on the last 3 days of the experiment, was always significantly higher than in untreated fruits (Fig . 3 Softening of the fruits treated by CO,, was delayed by I day at the most (Fig . 3) . Fruits harvested 300 days after fruit set (very mature fruits, ; and treated with CO 2 showed a smaller effect on symptom development and diene level and no effect on fruit firmness .

Effect of the length of exposure offruits to 30 % CO 2 on diene levels and symptom development The concentration of the diene was immediately enhanced after removal of avocado fruits from 30°,0 CO 2 following 3, 10 or 24 h treatment (Fig . 4) . The 3 h treatment

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FIG . 4 . Diene levels in the peel of avocado fruits cv . Fuerte exposed to 30 °-0 CO 2 for different A, 3 h ; 0 -- 0, periods of time at 20 °C . Freshly harvested fruits were treated for : A 10 h ; and ]-- - El, 24 h under a stream of 30 ° o CO 2 + 15 °: 02 and then transferred (arrow) to atmospheric conditions . Control fruits ( • •) were treated with air under similar conditions for 24 h . The mean of three extractions is shown . The LSD is indicated by bars .

enhanced the diene level 17 h later by almost eight-fold . Exposure of fruits to 30o , CO 2 for 10 and 24 h enhanced a lower increase than the 3 h treatment . Subsequently, there was a significant decrease in concentration of the diene in all the treated fruits followed by a second increase in concentration . The duration of the second increase was dependent on the length of exposure of the fruits to CO 2 (Fig . 4) . One-hundred and forty hours after harvest, the level of the diene in the 24 h CO 2 treated fruits was approx . 2000 tg g- ' fresh weight while the concentration of the compound in the controls was 210 .tg g' fresh weight . Fruits treated for 10 h did not show such a significant second increase in the diene concentration while the 3 h treatment enhanced an early decrease of the diene . Symptom development by C . gloeosporioides was significantly delayed by the 24 h treatment while it was enhanced by the short 3 h treatment (Fig . 5) . The 10 h treatment did not affect symptom development compared with the control .



Induction of fruit resistance by CO 2

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6 0

9 10 8 Days after harvest

0

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FIG . 5 . Symptom development of Colletotrichum gloeosporioides on avocado fruits cv . Fuerte exposed to 30% C0 2 +15% 0 2 for different periods of time at 20 °C . A A, 3 h ; 0- 0, 10 h ; 0-- -E], 24 h ; and untreated controls. Fruit inoculation as described in Fig . 1 .

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FIG . 6 . Epicatechin concentration in the peel of avocado fruits cv . Fuerte exposed to 30 0 , CO 2 + 15 % 02 for 24 h at 20 °C . Freshly harvested fruits were treated as in Fig . I and then

transferred (arrow) to air (0 •) . Control fruits (0--- 0) were treated under normal atmospheric conditions for 24 h . The mean of three extractions is presented . Bars indicate SE of the mean .



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of C0 2 treatment on epicalechin concenlralion When avocado fruits were removed from a 24 h exposure to 30",, CO 2 , the concentration of epicatechin in the fruit peel increased to approx . 800 pg g ' fresh weight, from the initial level of 600 pg g ' Iresh weight (Fig . 6) . This was followed by a steady decrease and a second increase in epicatechin level by the fourth day after harvest . The level of epicatechin in untreated fruits during all the experiments was significantly lower than in the CO 0 -treated fruits . Effect

DISCUSSION A fungitoxic concentration of the diene is present in the peel of freshly harvested avocado fruits [9] . The diene decreased to subfungitoxic concentrations during fruit ripening permitting the development of disease symptoms caused by C . gloeosporioides . [9] . CO 2 treatment of freshly harvested fruits affected the diene level in a double-peak pattern . Diene levels significantly increased after the removal of fruits from the CO 2 atmosphere . This was followed by a decrease in diene concentration before a second increase in the diene level occurred ; under these conditions decay development was delayed . The effect of the CO 2 on the double-peak pattern of the diene level and symptom development could be most readily observed if : (i) the concentration of CO 2 was 30 °-o ; (ii) the length of exposure was 24 h : (iii) the treatment was applied during the first day after harvest ; and (iv) the treatment was applied on fruit harvested up to 240 days after fruit set . The concentration of 0 2 in the mixture was not critical . At lower CO 2 concentrations (11 and 16 0, 0 ), a shorter period of exposure (3 and 10 h) or very mature fruits (300 days after fruit set, results not shown), the first increase in diene level was present . However, no significant second increase in diene level occurred and there was no delay in symptom development . This effect of a high CO 2 level on avocado fruits is the first report that such an abiotic treatment can enhance the level of an antifungal compound . Previous work by Swinburne [16] on Bramley's Seedling apples showed that 5 °0 CO 2 had no effect on the level of antifungal benzoic acid and that 100 0 CO 2 caused a decrease in benzoic acid concentration after 10 days treatment . Most of the studies aimed at examining the effect of CO 2 in controlled atmospheres, were conducted under low 0 2 and consequently the specific effect of CO 2 could not be evaluated [1, 2] . In avocado fruits, treatment with 0. 75 °% 0 2 and 30 °/o CO 2 induced a similar double-peak increase to that observed after treatment with 15 °.0 0 2 and 30 °0 CO 2 , suggesting that high CO, and not low 0 2 specifically affects the sequence of events leading to diene increase . The decrease in concentration of the diene has been reported to depend on the lipoxygenase-catalysed oxidation of the compound [3] . This oxidation is regulated by the natural inhibitor, epicatechin . Those results suggested a close relation between epicatechin and diene levels [10] . Treatments with 30 °~o CO 2 for 24 h enhanced the epicatechin concentration which followed the same double-peak behaviour observed for the diene . The effect of CO 2 on the phenol metabolism was also found by Siriphanich & Kader [13] . Treatments of lettuce with 15 °.- o CO 2 for 10 days enhanced



Induction of fruit resistance by CO 2 333 the phenol content after plants were transferred to air . It was suggested that CO 2 treatment affected the normal metabolic balance of the plant cell and the activity of phenylalanine ammonia lyase (PAL), a key enzyme in the synthesis of phenolic compounds [12, 13] . In this work PAL activity was not examined, but we suggest that CO 2 stress enhances the epicatechin level, thus inhibiting the system involved in diene breakdown and resulting in an initial increase of the diene level [3, 10] . It is clear that avocado fruit can react to CO 2 treatment 3-7 days after treatment . It is not clear, however, if the second increase is the result of (a) an enhanced diene synthesis as a result of the presence of a high concentration of a precursor of the diene, possibly the CO 2 [5], or (b) the increase is a result of 'a delay in the ripening process normally occurring in untreated fruits . The direct relation between the duration of the 30 CO 2 treatment and the presence of the second increase of the diene, suggests the possibility that CO 2 is metabolised by the fruit tissue [5], resulting in enhanced diene synthesis . On the other hand, the CO 2 treatment only delayed softening by 1 day at the most . However, the pattern of changes in diene levels showed an enhancement in levels during fruit softening . This result clearly suggests that the levels of diene in 24 h CO2 treated avocado fruits are not affected by ripening changes . The question as to how CO 2 induced the epicatechin and diene levels should be further studied . Present results indicate that levels of the antifungal compound are affected by abiotic factors [3, 9, 11] . The increase in the soluble phenolic, epicatechin [4, 13], after 24 h of CO 2 treatment may represent an important factor in the inhibition of the lipoxygenasecatalysed oxidation of the antifungal diene and its enhanced concentration .

This work was supported by grants from BARD, the Binational (US-Israel) Agricultural Research and Development Fund and AID-CDR, with additional funds from the Natural Resources and Environment Department, Overseas Development Administration, U .K . We acknowledge the excellent and skilful help of Mr J . Zuthi in setting up the system for the controlled CO 2 treatments . REFERENCES 1 . KADER, A. A . (1980) . Prevention of ripening in fruits by use of controlled atmospheres . Food Technology 34, 51 . 2 . KADER, A. A . (1985) . Modified atmospheres : an indexed reference list with emphasis on horticultural commodities. Postharvest Horticultural Series 3 . (Suppl . 4) . University of California, Davis, CA . 3 . KARNI, L ., PRUSKY, D ., KOBILER . I ., BAR-SHIRA, E . & KOBILER, D . (1989), Involvement of epicatechin in the regulation of lipoxygenase activity during activation of quiescent Colletotrichum gloeosporioides infections of ripening avocado fruits . Physiological and Molecular Plant Pathology 35, 367-374 . 4 . KE, D . & STALTVEIT, M . E . (1989) . Carbon dioxide-induced brown stain development as related to phenolic metabolism in iceberg lettuce . Journal of the American Society for Horticultural Science 114, 789-794 . 5 . PESIs, E . & BEN-ARIE, R . (1986) . Carbon dioxide assimilation during postharvest removal of astringency from persimmon fruit . Physiologia Plantarum 67, 644-648. 6 . PRUSKY, D . (1988) . The use of antioxidants to delay the onset of anthracnose and stem and decay in avocado fruits after harvest . Plant Disease 72, 381-384 . 7 . PRUSKY, D ., KARNI, L ., KOBILER, I . & PLUMBLEY, R . A . (199W . Induction of the preformed antifungal diene in unripe avocado fruits : effect of challenge inoculation by Colletotrichum gloeosporioides . Physiological and Molecular Plant Pathology 37, 425-435 . 8 . PRUSKY, D ., KEEN, N . T . & EAKS, 1 . (1983) . Further evidence for the involvement of a preformed antifungal compound in the latency of Colletotrichum gloeosporioides in unripe avocado fruits . Physiological Plant Pathology 22, 189-198 .



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9 . PRUSKY, D ., KEEN, N . '1' ., SIMS, J . J . & MIDLAND, S . i 1982 r . Possible involvement of an antifungal compound in latency of Colletotrichum gloeosporioides in unripe avocado fruits . Phvtopathology 72, 1578-1582 . 10 . PRUSKY, D ., KOBILER, 1 . & JACOBY, B . 1988 ; . Involvement of epicatechin in cultlvar susceptibility of avocado fruits to Colletotrichum gloeosporioides after harvest. Phvtopathologische Zeitschrift 123, 140-146 . 11 . PRCSKY, D ., KOBILER, I ., JACOBY, B ., Sims, J . J . & MIDLAND, S . L . ; 19851 . Inhibitors of avocado lipoxygenase : their possible relationship with the latency of

Plant Pathology 27,

Colletotrichum gloeosporioides . Physiological

269-279 .

12 . RHODES, J. M. & WOOLTORTON, L . S . C . 19781 . The biosynthesis of phenolic compounds in wounded plant storage tissue . In

Biochemistry of Wounded Plant Tissue .

Ed . by G . Kahl, pp . 243-286 . W . de

Gruyter, Berlin . 13 . SIRIPHANICH, J . & KADER, A. A . (1985, . Effects of CO 2 on total phenolics, phenylalanine ammonia lyase and polyphenol oxidase in lettuce tissue .

Journal of the American Society for Horticultural Science 110,

249-253 . 14. SPALDING, D . H . & REEDER, W . F . '1975) . Low-oxygen high carbon dioxide controlled atmosphere storage for control of anthracnose and chilling injury of avocados . Phytopathology 65, 458-460 . 15 . STOESSL, A . (1983' . Secondary plant metabolites in preinfectional and postinfectional resistance . In The Dynamics of Host Defence, Ed . by J . A. Bailey & B . J. Deverall, pp . 71-122 . Academic Press, London . 16 . SWINBURNE, T . R . (1973) . The resistance of immature Bramley's Seedling apples to rotting by Vectria galligena Bres . In Fungal Pathogenicity and the Plant Response, Ed . by R . J. W . Byrde & C . V . Cutting_, pp . 365-382 . Academic Press, London .