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UV-induced resistance to postharvest diseases of citrus fruit
J. Photochem. Photobiol. B: Biol., 15 (1992) 367-374
367
News and Views
UV-induced resistance to postharvest diseases of citrus fruit Edo Chalutz+ and Samir Droby Agricultuml Research Organization, The Volcani Center, Bet Dugan 50250 (kmel)
Charles
L. Wilson
and Michael
E. Wisniewski
Appalachian Fruit Research Center, USDA, ARS, Keameysville, WV 25430 (USA)
Postharvest losses of fruits and vegetables due to disease are estimated at 25% of the harvested crop. Most of the losses are due to rots caused by micro-organisms [l]. Fungicides are the primary means of controlling postharvest diseases. However, they have recently come under scrutiny as posing potential health risks when applied to foods, according to a report by the US National Research Council [2]. In 1991, the European parliament considered imposing a total ban on postharvest treatments of fruits and vegetables with pesticides as soon as the practice will become feasible t31. As a result of these developments, several key fungicides have recently been withdrawn from the market. Even when acceptable fungicides are applied for the control of postharvest diseases, pathogens often develop resistance to them [4]. All these developments have weakened our ability to control postharvest diseases. Thus an urgent need exists for new and effective means of controlling postharvest diseases that pose less risk to human health and the environment. Several recent publications by Stevens and his associates have suggested that W irradiation might offer such an innovative means. They have reported that irradiation at 254 nm (a) reduced postharvest decay of black rot and bacterial soft rot of onion, (b) reduced Rhizopus and Fusarium storage rots of sweet potatoes and (c) extended the shelflife of these and other commodities [5-7]. In 1990, Stevens and coworkers were joined by the authors of this publication to conduct coordinated studies of the effect of low-dose UV irradiation on postharvest rots of several fruit commodities. Preliminary results from this collaboration were published recently [g, 91. In this report we summarize results which indicate that the effect of W in reducing the citrus green mould disease, the most important postharvest rot of citrus fruit, is an induced resistance phenomenon rather than a germicidal effect. Other related results are also included. To characterize the effect of W on the resistance of citrus to the green mould disease caused by the fungus Penicillium digitatum Sacc., we have employed two parallel approaches. One was to test the response of W-treated fruit to artificial inoculations, with emphasis on inoculations at various times after the treatment. The other was to ‘Author to whom correspondence should be addressed.
Elsevier Sequoia
368
NEWS AND VIEWS
record biochemical changes in the tissue and the possible accumulation of fungal inhibitory materials. The UV treatment was applied by exposing freshly harvested grapefruits to a germicidal low-pressure vapour UV lamp emitting a wave energy with a peak at 254 nm (General Electric (G3OTS) lamp; diameter, 2.5 cm; length, 88 cm; nominal power output, 30 W; O-36 A, maximum intensity perpendicular to the bare tube, 2.66 mW cm-‘). The fruit was placed, stem side up, at a distance of 10 cm from the light source and treated with a dose of (0.8-16.0 x lo3 Jm-* by varying the length of the exposure time. After the treatment the fruit was kept in the dark at room temperature for 24 h (or for other lengths of time as indicated) and then inoculated through freshly cut surface wounds, with a spore suspension of Penicilliumdigitatum(lo4 spores ml-‘). Incubation was at 24 “C and high humidity. Per cent infection of the inoculated wound was determined after several days. Phenylalanine ammonia-lyase (PAL) activity was determined by the method outlined by Lisker et al. [lo]. Grapefruits exposed to the UV treatments, and subsequently inoculated as described, exhibited a reduced incidence of decay: the number of inoculated wounds which developed decay decreased from 90 or 100% in control fruit not exposed to the W treatment to approximately 20% in fruits exposed to 4.0X lb or 5.0X lo3 Jm-‘. In fruits treated with UV doses of 16 X 103 J m-2 or higher, percent infection increased to 30% or more, along with the appearance of phytotoxic blemishes on the fruit peel. The response of grapefruit to inoculations at various times after the W treatment is shown in Fig. 1. Percent infection was high (95%) when fruits were inoculated 5 h after exposure to the W treatment and decreased to 30% and 25% when fruit was inoculated at 24 h and 48 h after the treatment respectively. Percent infection
loo
.-
fim 0
3
0
60.-
2
E
be
40.20..
Fig. 1. Effect of time of inoculation after UV treatment, on infection of grapefruit by Penicillium digitatum: top section, percent infected wounds; bottom section, rot diameter of infected sites; 0, control; 0, 8.0 x 103 J m-‘. Bars indicate the standard deviation.
NEWS AND VIEWS
369
of wounds increased to 40% when the fruit was inoculated at 72 h after the W treatment. Wounds of the untreated control fruit exhibited a high percent of infection regardless of the time at which the fruit was inoculated (Fig. 1, top section). A similar effect of the W treatment was observed on the reduction of rot diameter of the infected wounds (Fig. 1, bottom section). In addition, those wounds which developed infections on W-treated fruit exhibited a repressed growth of surface mycelium with reduced sporulation. This phenomenon lasted several days, after which time the normal growth characteristics of the rot developed. These observations suggested that the W treatment of the fruit induced processes in the peel that prevent and/or restrict the growth of the fungal pathogen. The process seemed to develop gradually with time after treatment and reached a maximum response at 24-48 h after treatment. Indeed, the activity of PAL in the peel of the W-treated grapefruit also increased within 24 h after treatment and remained high at 48 h, compared with the low and unchanged level of PAL activity in the peel of control fruit (Fig. 2). We have observed that the resistance of wounds to infections was not restricted to the site of the W treatment. It was exhibited in peel regions not directly exposed to the W irradiation. This observation suggests that a resistance induction signal may be transferred within the fruit. A similar phenomenon of an apparent signal being transferred from one part of the fruit peel to another has been described previously
WI. Additional evidence for the presence of fungus-inhibiting materials in grapefruit peel following the W treatment of the fruit has recently been obtained. When spores of Penidlium digitatum were placed on cut surfaces of the W-treated tissue after the treatment, percent germination and elongation of the germ-tube were markedly inhibited - compared with those of spores placed on cut surfaces of non-treated control fruit (Table 1). Considered together, these data strongly suggest that the W treatment of grapefruit induces the synthesis in the peel tissue of materials which inhibit fungal growth and disease development. The effect of W irradiation on plant growth has been studied extensively. Of particular interest within the context of this report is information on the W-mediated induction of the shikimic acid pathway in higher plants [ll-141. Products of this pathway, such as flavonoids, phenols and lignins, have been shown to play a role in
0
10
Time
20
so
after treatment
4.0
(hr)
Fig. 2. Effect of UV treatment on PAL activity in grapefruit peel: 0, control; 0, 8.0X1@ J m -2. Bars indicate the standard deviation.
NEWS AND VIEWS
370 TABLE 1
Inhibition of spore germination and germ-tube elongation of Penicillium digitatum by anti-fungal substances produced in the peel of grapefruit following UV irradiation Inoculation time” (h) 0
24
UV dose (xl@ J m-‘)
Germination (% + SD)b
Germ-tube length
0
0.8 1.6 4.8 8.0
73i-21 73*1a 70*2O 76&11 72rtlO
48*8 32*2 42k5 23i-4 21&3
0 0.8 1.6 4.8 8.0
78*14 6Ok16 44*20 35*13 47+16
62i-3 39&2 30*5 23*4 19*3
inoculation time is the time elapsed between W
(pm f SD)
treatment and inoculation. Inoculation was
done by placing a spore suspension on a slightly cut surface of the fruit peel. Spore germination and germ-tube length were determined 20 h after inoculation. bSD, standard deviation (%). ‘SD, standard deviation (pm). defence reactions of plants against pathogens 1121. Regulation of the shikimic acid pathway is affected by factors such as wounding, pathogenicity, ethylene and light, including UV light [15, 161. The induction of anti-fungal materials in citrus fruit has been demonstrated in the past. In an analysis of peel extracts from gamma-irradiated fruit, Riov [17] and later Dubery and coworkers [18, 191 identified the anti-fungal compounds 6,7-demethoxycoumarin (scoparone) and 4-(3-methyl-2-butenoxy) isonitrosoacetophenone. Scoparone has also been identified in UV-treated citrus [20]. However, whether or not this material is the one actually responsible for the UV-induced resistance of citrus fruits against the green mould disease remains to be demonstrated. Additional work is therefore needed not only to answer this question and possibly to identify other UV-induced anti-fungal compounds, but also to elucidate the nature of the signal that may be transmitted within the fruit. Nonetheless, the induced resistance achieved in harvested commodities by treatments with UV irradiation is a promising innovative approach as a potential alternative to chemical control of postharvest diseases of fruits and vegetables. The excellent technical assistance of L. &hen and B. Weiss is herewith acknowledged. This research was supported in part by grant IS-1908-90F from the US-Israel Binational Agricultural Research and Development Fund.
1 A. L. Snowdon, A Color Atlas of Post-harvest Diseases and Disorders of Fruits and Vegetables, Vol. 1, General Introduction and Fruits, CRC Press, Boca Raton, FL, 1990. 2 Regulating pesticides in foods - the Delaney paradox, Rep. Board of Agriculture, National Research Council, National Academy Press, Washington, DC, 1987. 3 Posthawest News InjI, 2 (1991) 3.
NEWS AND VIEWS
371
4 J. W. Eckert and J. M. Ogawa, The chemical control of postharvest diseases: subtropical and tropical fruits, Annu. Rev. Phytopathol., 23 (1985) 421454. 5 J. Y. Lu, C. Stevens, P. Yakubu and P. A. Loretan, Gamma, electron beam and UV radiation on control of storage rots and quality of Walla Walla onions, J. Food Process Preserv., 12 (1987) 53-62. 6 C. Stevens, V. A. Khan, J. Y. Lu and A. Y. Tang, The effect of ultraviolet radiation on resistance of sweetpotato to storage rots, Froc. 5th Znt. Congr. on Plant Pathology, Kyoto, 1988, p. 424. C. Stevens, V. A. Khan, A. W. Tang and J. Y. Lu, The effect of ultraviolet radiation on mold rots and nutrients of stored sweetpotatoes, J. Food Prot., 53 (1990) 223-226. J. Y. Lu, C. Stevens, V. A. Khan, M. Kabwe and C. L. Wilson, The effect of ultraviolet irradiation on shelf-life and ripening of peaches and apples, J. Food QuaI., 14 (1991) 299-305. C. Stevens, J. Y. Lu, V. A. Khan, C. L. Wilson, E. Chalutz and S. Droby, Ultraviolet light induced resistance against postharvest diseases in vegetables and fruits, in C. L. Wilson and E. Chalutz (eds.), Proc. Znt. Workshop on Biological Control of Postharvest Diseases of Fruits, 92, ARS ML, 1991, pp. 268-298. 10 N. Lisker, L. Cohen, E. Chalutz and Y. Fuchs, Fungal infection suppresses ethylene-induced phenylalanine ammonia-lyase activity in grapefruit, Physiol. Plant PathoL, 22 (1983) 331-338. 11 M. M. Caldwell, A. H. Teramura and M. Tevini, The changing solar ultraviolet climate and the ecological consequences for higher plants, Tre&s Ecol. Evd, 4 (1989) 363-367. 12 K. Halbrock and D. Scheel, Biochemical responses of plants to pathogens, in I. Chet (ed.), Innovative Approaches to Pkmt Disease Control, Wiley, New York, NY, 1987, pp. 229-254. metabolism and its regulation in disease, in J. A. Callow (ed.), 13 M. Legrand, Phenylpropanoid Biochemistry P&t Pathology, Wiley, New York, 1983, pp. 367-375. 14 T. M. Murphey and J. A. Huerta, Hydrogen peroxide formation in cultured rose cells in response to UV-C radiation, Physiol. Plant., 78 (1990) 247-253. 15 R. K. Hughes and A. G. Dickerson, The effect of ethylene on phenylalanine ammonia-lyase induction by fungal elicitors in Phuseolus wIga&, Physiol. Mol. Plant Pathol., 34 (1989) 361-378. ethylene production, phenolic metabolism and 16 D. Ke and M. E. Shaltveit, Wound-induced susceptibility of russet spotting in iceberg lettuce, Physiol. Plant., 76 (1989) 412-418. 17 J. Riov, 6,7-Dimethozycoumarin in the peel of gamma-irradiated grapefruit, Phytochenktry, 10 (1971) 1923. 18 I. A. Dubery and J. C. Schabort, 6,7-dimethozycoumarin: a stress metabolite with antifungal activity in gamma-irradiated citrus peel, S. Afi. J. Sci., 83 (1987) 440-441. 19 I. A. Dubery, C. W. Holzapfel, G. E. Kruger, J. C. Schabort and M. Van Dyk, Characterization of a gamma-radiation-induced antifungal stress metabolite in citrus peel, Phytochemisby, 27 (1988) 2769-2772. 20 Y. Arimoto and Y. Homma, Studies on citrus melanose and citrus stem-end rot by Diaphorthe citri (Faw.) Wolf. Part 9: Effect of light and temperature on the self-defense reaction of citrus plants, Ann. PhytopathoL Sot. Jpn., 54 (1988) 282-289.
Sunscreens retain their efficacy on human skin for up to 8 h after application Patricia P. Agin+ and Donald J. Levine Research and Development, Scherkg-Piough HealthCare Z%ducts, 3030 Jackson Avenue, PO Box 3n, Memphk, TN 3glSI (USA)
tAuthor
to whom correspondence
should be addressed.
367
News and Views
UV-induced resistance to postharvest diseases of citrus fruit Edo Chalutz+ and Samir Droby Agricultuml Research Organization, The Volcani Center, Bet Dugan 50250 (kmel)
Charles
L. Wilson
and Michael
E. Wisniewski
Appalachian Fruit Research Center, USDA, ARS, Keameysville, WV 25430 (USA)
Postharvest losses of fruits and vegetables due to disease are estimated at 25% of the harvested crop. Most of the losses are due to rots caused by micro-organisms [l]. Fungicides are the primary means of controlling postharvest diseases. However, they have recently come under scrutiny as posing potential health risks when applied to foods, according to a report by the US National Research Council [2]. In 1991, the European parliament considered imposing a total ban on postharvest treatments of fruits and vegetables with pesticides as soon as the practice will become feasible t31. As a result of these developments, several key fungicides have recently been withdrawn from the market. Even when acceptable fungicides are applied for the control of postharvest diseases, pathogens often develop resistance to them [4]. All these developments have weakened our ability to control postharvest diseases. Thus an urgent need exists for new and effective means of controlling postharvest diseases that pose less risk to human health and the environment. Several recent publications by Stevens and his associates have suggested that W irradiation might offer such an innovative means. They have reported that irradiation at 254 nm (a) reduced postharvest decay of black rot and bacterial soft rot of onion, (b) reduced Rhizopus and Fusarium storage rots of sweet potatoes and (c) extended the shelflife of these and other commodities [5-7]. In 1990, Stevens and coworkers were joined by the authors of this publication to conduct coordinated studies of the effect of low-dose UV irradiation on postharvest rots of several fruit commodities. Preliminary results from this collaboration were published recently [g, 91. In this report we summarize results which indicate that the effect of W in reducing the citrus green mould disease, the most important postharvest rot of citrus fruit, is an induced resistance phenomenon rather than a germicidal effect. Other related results are also included. To characterize the effect of W on the resistance of citrus to the green mould disease caused by the fungus Penicillium digitatum Sacc., we have employed two parallel approaches. One was to test the response of W-treated fruit to artificial inoculations, with emphasis on inoculations at various times after the treatment. The other was to ‘Author to whom correspondence should be addressed.
Elsevier Sequoia
368
NEWS AND VIEWS
record biochemical changes in the tissue and the possible accumulation of fungal inhibitory materials. The UV treatment was applied by exposing freshly harvested grapefruits to a germicidal low-pressure vapour UV lamp emitting a wave energy with a peak at 254 nm (General Electric (G3OTS) lamp; diameter, 2.5 cm; length, 88 cm; nominal power output, 30 W; O-36 A, maximum intensity perpendicular to the bare tube, 2.66 mW cm-‘). The fruit was placed, stem side up, at a distance of 10 cm from the light source and treated with a dose of (0.8-16.0 x lo3 Jm-* by varying the length of the exposure time. After the treatment the fruit was kept in the dark at room temperature for 24 h (or for other lengths of time as indicated) and then inoculated through freshly cut surface wounds, with a spore suspension of Penicilliumdigitatum(lo4 spores ml-‘). Incubation was at 24 “C and high humidity. Per cent infection of the inoculated wound was determined after several days. Phenylalanine ammonia-lyase (PAL) activity was determined by the method outlined by Lisker et al. [lo]. Grapefruits exposed to the UV treatments, and subsequently inoculated as described, exhibited a reduced incidence of decay: the number of inoculated wounds which developed decay decreased from 90 or 100% in control fruit not exposed to the W treatment to approximately 20% in fruits exposed to 4.0X lb or 5.0X lo3 Jm-‘. In fruits treated with UV doses of 16 X 103 J m-2 or higher, percent infection increased to 30% or more, along with the appearance of phytotoxic blemishes on the fruit peel. The response of grapefruit to inoculations at various times after the W treatment is shown in Fig. 1. Percent infection was high (95%) when fruits were inoculated 5 h after exposure to the W treatment and decreased to 30% and 25% when fruit was inoculated at 24 h and 48 h after the treatment respectively. Percent infection
loo
.-
fim 0
3
0
60.-
2
E
be
40.20..
Fig. 1. Effect of time of inoculation after UV treatment, on infection of grapefruit by Penicillium digitatum: top section, percent infected wounds; bottom section, rot diameter of infected sites; 0, control; 0, 8.0 x 103 J m-‘. Bars indicate the standard deviation.
NEWS AND VIEWS
369
of wounds increased to 40% when the fruit was inoculated at 72 h after the W treatment. Wounds of the untreated control fruit exhibited a high percent of infection regardless of the time at which the fruit was inoculated (Fig. 1, top section). A similar effect of the W treatment was observed on the reduction of rot diameter of the infected wounds (Fig. 1, bottom section). In addition, those wounds which developed infections on W-treated fruit exhibited a repressed growth of surface mycelium with reduced sporulation. This phenomenon lasted several days, after which time the normal growth characteristics of the rot developed. These observations suggested that the W treatment of the fruit induced processes in the peel that prevent and/or restrict the growth of the fungal pathogen. The process seemed to develop gradually with time after treatment and reached a maximum response at 24-48 h after treatment. Indeed, the activity of PAL in the peel of the W-treated grapefruit also increased within 24 h after treatment and remained high at 48 h, compared with the low and unchanged level of PAL activity in the peel of control fruit (Fig. 2). We have observed that the resistance of wounds to infections was not restricted to the site of the W treatment. It was exhibited in peel regions not directly exposed to the W irradiation. This observation suggests that a resistance induction signal may be transferred within the fruit. A similar phenomenon of an apparent signal being transferred from one part of the fruit peel to another has been described previously
WI. Additional evidence for the presence of fungus-inhibiting materials in grapefruit peel following the W treatment of the fruit has recently been obtained. When spores of Penidlium digitatum were placed on cut surfaces of the W-treated tissue after the treatment, percent germination and elongation of the germ-tube were markedly inhibited - compared with those of spores placed on cut surfaces of non-treated control fruit (Table 1). Considered together, these data strongly suggest that the W treatment of grapefruit induces the synthesis in the peel tissue of materials which inhibit fungal growth and disease development. The effect of W irradiation on plant growth has been studied extensively. Of particular interest within the context of this report is information on the W-mediated induction of the shikimic acid pathway in higher plants [ll-141. Products of this pathway, such as flavonoids, phenols and lignins, have been shown to play a role in
0
10
Time
20
so
after treatment
4.0
(hr)
Fig. 2. Effect of UV treatment on PAL activity in grapefruit peel: 0, control; 0, 8.0X1@ J m -2. Bars indicate the standard deviation.
NEWS AND VIEWS
370 TABLE 1
Inhibition of spore germination and germ-tube elongation of Penicillium digitatum by anti-fungal substances produced in the peel of grapefruit following UV irradiation Inoculation time” (h) 0
24
UV dose (xl@ J m-‘)
Germination (% + SD)b
Germ-tube length
0
0.8 1.6 4.8 8.0
73i-21 73*1a 70*2O 76&11 72rtlO
48*8 32*2 42k5 23i-4 21&3
0 0.8 1.6 4.8 8.0
78*14 6Ok16 44*20 35*13 47+16
62i-3 39&2 30*5 23*4 19*3
inoculation time is the time elapsed between W
(pm f SD)
treatment and inoculation. Inoculation was
done by placing a spore suspension on a slightly cut surface of the fruit peel. Spore germination and germ-tube length were determined 20 h after inoculation. bSD, standard deviation (%). ‘SD, standard deviation (pm). defence reactions of plants against pathogens 1121. Regulation of the shikimic acid pathway is affected by factors such as wounding, pathogenicity, ethylene and light, including UV light [15, 161. The induction of anti-fungal materials in citrus fruit has been demonstrated in the past. In an analysis of peel extracts from gamma-irradiated fruit, Riov [17] and later Dubery and coworkers [18, 191 identified the anti-fungal compounds 6,7-demethoxycoumarin (scoparone) and 4-(3-methyl-2-butenoxy) isonitrosoacetophenone. Scoparone has also been identified in UV-treated citrus [20]. However, whether or not this material is the one actually responsible for the UV-induced resistance of citrus fruits against the green mould disease remains to be demonstrated. Additional work is therefore needed not only to answer this question and possibly to identify other UV-induced anti-fungal compounds, but also to elucidate the nature of the signal that may be transmitted within the fruit. Nonetheless, the induced resistance achieved in harvested commodities by treatments with UV irradiation is a promising innovative approach as a potential alternative to chemical control of postharvest diseases of fruits and vegetables. The excellent technical assistance of L. &hen and B. Weiss is herewith acknowledged. This research was supported in part by grant IS-1908-90F from the US-Israel Binational Agricultural Research and Development Fund.
1 A. L. Snowdon, A Color Atlas of Post-harvest Diseases and Disorders of Fruits and Vegetables, Vol. 1, General Introduction and Fruits, CRC Press, Boca Raton, FL, 1990. 2 Regulating pesticides in foods - the Delaney paradox, Rep. Board of Agriculture, National Research Council, National Academy Press, Washington, DC, 1987. 3 Posthawest News InjI, 2 (1991) 3.
NEWS AND VIEWS
371
4 J. W. Eckert and J. M. Ogawa, The chemical control of postharvest diseases: subtropical and tropical fruits, Annu. Rev. Phytopathol., 23 (1985) 421454. 5 J. Y. Lu, C. Stevens, P. Yakubu and P. A. Loretan, Gamma, electron beam and UV radiation on control of storage rots and quality of Walla Walla onions, J. Food Process Preserv., 12 (1987) 53-62. 6 C. Stevens, V. A. Khan, J. Y. Lu and A. Y. Tang, The effect of ultraviolet radiation on resistance of sweetpotato to storage rots, Froc. 5th Znt. Congr. on Plant Pathology, Kyoto, 1988, p. 424. C. Stevens, V. A. Khan, A. W. Tang and J. Y. Lu, The effect of ultraviolet radiation on mold rots and nutrients of stored sweetpotatoes, J. Food Prot., 53 (1990) 223-226. J. Y. Lu, C. Stevens, V. A. Khan, M. Kabwe and C. L. Wilson, The effect of ultraviolet irradiation on shelf-life and ripening of peaches and apples, J. Food QuaI., 14 (1991) 299-305. C. Stevens, J. Y. Lu, V. A. Khan, C. L. Wilson, E. Chalutz and S. Droby, Ultraviolet light induced resistance against postharvest diseases in vegetables and fruits, in C. L. Wilson and E. Chalutz (eds.), Proc. Znt. Workshop on Biological Control of Postharvest Diseases of Fruits, 92, ARS ML, 1991, pp. 268-298. 10 N. Lisker, L. Cohen, E. Chalutz and Y. Fuchs, Fungal infection suppresses ethylene-induced phenylalanine ammonia-lyase activity in grapefruit, Physiol. Plant PathoL, 22 (1983) 331-338. 11 M. M. Caldwell, A. H. Teramura and M. Tevini, The changing solar ultraviolet climate and the ecological consequences for higher plants, Tre&s Ecol. Evd, 4 (1989) 363-367. 12 K. Halbrock and D. Scheel, Biochemical responses of plants to pathogens, in I. Chet (ed.), Innovative Approaches to Pkmt Disease Control, Wiley, New York, NY, 1987, pp. 229-254. metabolism and its regulation in disease, in J. A. Callow (ed.), 13 M. Legrand, Phenylpropanoid Biochemistry P&t Pathology, Wiley, New York, 1983, pp. 367-375. 14 T. M. Murphey and J. A. Huerta, Hydrogen peroxide formation in cultured rose cells in response to UV-C radiation, Physiol. Plant., 78 (1990) 247-253. 15 R. K. Hughes and A. G. Dickerson, The effect of ethylene on phenylalanine ammonia-lyase induction by fungal elicitors in Phuseolus wIga&, Physiol. Mol. Plant Pathol., 34 (1989) 361-378. ethylene production, phenolic metabolism and 16 D. Ke and M. E. Shaltveit, Wound-induced susceptibility of russet spotting in iceberg lettuce, Physiol. Plant., 76 (1989) 412-418. 17 J. Riov, 6,7-Dimethozycoumarin in the peel of gamma-irradiated grapefruit, Phytochenktry, 10 (1971) 1923. 18 I. A. Dubery and J. C. Schabort, 6,7-dimethozycoumarin: a stress metabolite with antifungal activity in gamma-irradiated citrus peel, S. Afi. J. Sci., 83 (1987) 440-441. 19 I. A. Dubery, C. W. Holzapfel, G. E. Kruger, J. C. Schabort and M. Van Dyk, Characterization of a gamma-radiation-induced antifungal stress metabolite in citrus peel, Phytochemisby, 27 (1988) 2769-2772. 20 Y. Arimoto and Y. Homma, Studies on citrus melanose and citrus stem-end rot by Diaphorthe citri (Faw.) Wolf. Part 9: Effect of light and temperature on the self-defense reaction of citrus plants, Ann. PhytopathoL Sot. Jpn., 54 (1988) 282-289.
Sunscreens retain their efficacy on human skin for up to 8 h after application Patricia P. Agin+ and Donald J. Levine Research and Development, Scherkg-Piough HealthCare Z%ducts, 3030 Jackson Avenue, PO Box 3n, Memphk, TN 3glSI (USA)
tAuthor
to whom correspondence
should be addressed.