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Verbindungen in infizienen Orangen war mit Wirt-Pathogen-Wechselwirkungen sowie Schalen- lasionen gekoppelt. U.S. CopynghiCl.ai^nce cancer Cod.
J, Phytopathology 128, 306—314 (1990) © 1990 Paul Parey Scientific Publishers, Berlin and Hamburg ISSN 0931-1783

Department of Fruit and Vegetable Storage, Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel

Volatile Production Induced by PenicilHum digitatum in Orange Fruit and in Culture EDNA PESIS and ROSA MARINANSKY Authors' address: E. PESIS and R. MARINANSKT, Department of Fruit and Vegetable Storage, Agricultural Research Organization, The Volcani Center, P.O. Box 6, Bet Dagan 50250, Israel. With 5 figures Received August 21, 1989; accepted October 9, 1989

Abstract Production of volatiles was stimulated by the infection of Penicilhum digitatum in 'Shamouti' oranges. During 7 days of disease development, there was a progressive increase in the emanations of the anaerobic metabohtes acetaldehyde, ethanol and ethyl acetare. There was also an increase in other volatiles including metbanol, acetone, and ethylene, accompanied by a high production of CO2. The production of acetaJdehyde, ethanol and COi in cultures of P. digitatum was highest on the first day after inocuiation, and decreased as the fungus developed. Ethyl acetate and ethylene increased when Sporulation had already begun. Neither acetone nor methanol was found in P. digitatum iti vitro. The increase of the voiatiles in the infected oranges ivas due to the hosr-pathogen interaction and peel lesion.

Zusammenfassung Die Produktion von fluchtigen Verbindungen verursacht durch PenicilHum digitatum in Orangen und in Kultur Nach einer Infektion mit Pemdllium dtgitatum an 'Shamouti'-Orangen wurde die Produktion von fluchtigen Verbindungen angeregt. Wahrend der ersten 7 Tage der Krankheitsentwicklung wurde eine progressive Steigerung der Ausstromung der anaeroben Metaboliten Acetaldehyd, Athanol und Athylacetat beobacbtet. Ermitteit wurde aufierdem eine Vemiehrung anderer fluchtiger Verbindungen wie Methanol, Aceton und Athylen, gekoppeh mit einer erhohten COj-Produktion. Die Bildung von Acetaldehyd, Athanol und CO? in P. digitatum-Kuhuren war am ersten Tag nach der Inokulation am hochsten, mit der weiteren Entwicklung des Pilzes wurde sie niedriger. Die Athylacetat- und Athylenproduktion verstarkte sich, nachdem die Konidienbildung schon angesetzt hatte. Weder Aceton noch Methanol wurde in vitro in P. digitatum festgestellt. Die Zunahme von fluchtigen Verbindungen in infizienen Orangen war mit Wirt-Pathogen-Wechselwirkungen sowie Schalenlasionen gekoppelt. U.S. CopynghiCl.ai^nce cancer Cod. Sutcmem: 093 1-1 7 S 5 / 9 0 / 2 8 0 4 - 0 3 0 6 $ 0 2 . 5 0 / 0

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One of the most important pathogenic fungi of harvested citrus fruit is Penicillium digitatum, the causal agent of green mold (GuTTHR 1977). It has been found that microorganism infections of various plant organs and fruits result in enhanced ethylene production and respiration rate. An increase in ethylene and CO2 production was reported by ScHiFFMANN-NADEL (1977) in lemons infected by various citrus pathogens, including P. digitatum. Changes in the respiratory rate and ethylene evolution were also reported by ZAUBERMAN and BARKAI-GOLAN (1975) in 'Valencia' oranges infected by Diplodia natalensis. Orange fruit consists of a great variety of volatile compounds that contribute to the aroma and the flavour, among them hydrocarbons, aldehydes, ketones, alcohols and esters (PINO et al. 1986a, b). NORMAN (1977) indicated that the flavour of intact 'Valencia' oranges was adversely affected when the fruit absorbed volatiles from oranges inoculated with decay organisms, including P. digitatum. The production of various volatile metabolites including aldehydes, alcohols and esters which emanated from Fusanum oxysporum in vitro, increased during the development of the culture; these volatiles exhibited varying levels of sporostatic effect (ROBINSON and GARRETT 1969). The present research involved a study of the volatile production by P. digitatum in vitro, and by 'Shamouti' oranges infected by P. digitatum, in respect to disease development.

Materials and Methods Plant and fungal material 'Shamouti' oranges were harvested in mid season from local orchards and inoculated 3 days after harvest whh the fungus P. digitatum. Inoculation was done on two sides of the fruit by introducing the appropriate inoculum into a wound in the pee! at a depth of 3 mm. Inocula were taken from postharv'est P. digitatum decay of oranges. Uninoculated fruit served as the control.

In vivo experiments Individual fruits were weighed and placed in 2-Iiter glass lars and kept at 22°C. The lars were closed every day for 1 h and head-space gas samples were taken for the measurement of CO:, ethylene, acetaldehyde, ethanol, ethyl acetate, methanol and acetone. These measurements look place during 7 days which covered the incubation period and all the stages of rot development until final deterioration. The data are means of five measurements from five different iars calculated on the basis of fresh weight of each fruit.

Measurements of volatites Carbon dioxide production was detected with a thermal conductivity detector, a Poropak Q column, and He as carrier. Ethylene was detected with a flame lonization detector with alumina column and N; as a carrier. The five different volatiles: acetaldehyde, ethanol, ethyl acetate, methanol and acetone, were detected with a flame ionization detector, carrier gas N^, with iwo different columns; (i) 20% Carbowax 20 M, detector at lSO^C, injection temperature IIO^C, and oven temperature 80°C; and (ii) Poropak Q, detector at 200'^C, injection temperature 110°C, and oven temperature 180°C. Volatiles' identification was on the basis of retention time in two different columns against known gas standards.

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In vitro experiments Samples of P. digitatum grown in potato dextrose agar (PDA) medium were suspended in 10 ml sterile 'water. This suspension, contaming 3 • 10' spores/ml, was divided into 50 ml Erlenmeyer flasks, each one containing 10 ml PDA. One ml of suspension was placed in each flask. Fungi developed on PDA in the flasks for 7 days at 22 °C. The flasks were closed with a rubber cap for 3 h every day, and head-space gas samples 'were taken. Measurement of CO;, ethylene and the other volatiles was done in the same way as with the fruit. Values of the volatile production are expressed on the basis of fresh weight of the fungal mycelium as described by CUALUrzetal. (1978). All results are the average of five measuremenrs done on five different Erlenmever flasks.

Results Volatile production in infected oranges Infected oranges produced several volatiles during development of the disease, among them volatiies that stem from the anaerobic metabolism. Acetaldehyde, which is the first metabolite in the anaerobic pathway, was low in comparison with other volatiles. It increased rapidly in the infected oranges, reaching a maximum of 0.86 mg • kg"' • h"' on the 5th day after inoculation and thereafter decreasing (Fig. 1 A). Fthanol, which is the second metabohte in the anaerobic pathway, showed a concomitant increase with acetaldehyde, but at 12 times the level (Fig. 1 B). Ethyl acetate production in infected oranges was the highest of all the volatiles, except CO2. Ethyl acetate production was almost zero until the 5th day, when a highly progressive increase started, reaching a maximum of 35 mg • kg"' • h"' on the 7th day, 3.5 times higher than the maximum level of ethanol (Fig. l C ) . In addition to the anaerobic metabolites, in infected oranges a significant amount of acetone and methanol was found; the two reached similar levels. Methanol was the first volatile to emanate after infection. It increased sharply, reaching a maximum of 2.1 mg • kg'' • h"' on the 3rd day and then decreasing progressively until the 7th day, when no methanol production was detected (Fig. 2A). Acetone evolved linearly, reaching similar levels only on the 7th day (Fig.2B). The CO2 levels were 10 to 100 times higher than the levels of all other volatiles. The CO2 evolution in the infected oranges increased rapidly from the 3rd to the 7th day after inoculation (Fig. 3). Ethylene production showed the same trend as the COi, but reached lower levels than the other volatiles (90 ^1 • kg-' ' h-' - 112 fig • kg-' • h-') (Fig. 3). AH the volatiles' levels found in uninfected fruits were very low and did not change during the testing period. Volatile production in PenicilHum digitatum in vitro On the first day after inoculation there was a high production of anaerobic metabolites. Already 8 h after inoculation, acetaldehyde production was at its maximum, which was relatively low (20 ^g • g"' • h~'), decreasmg afterwards until the last test day when the values were close to zero (Fig. 4). After 24 h ethanol

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Fig. 1. Acetaidchyde, ethanol and ethyl acetate evolution in 'Shamouri' oranges infected by Pemdllium digitatum during storage at 22 °C. A. Acetaidehydt. B. Ethanol. C. Ethyl acetate. Results presented are the means ± SE of 5 replications

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production reached its maximum level, which was much higher than that of acetaidehyde. However, already on the second day there was a sharp decrease in ethanol emanation, and levels remained low until the 5th day, when values were negligible {Fig. 4). On the other hand, ethyl acetate emanation was very low during the first 3 days. On the 4th day it reached a significant peak of 110 ;Ug • g"' • h"^ coinciding with the sporulation stage, and thereafter gradually decreased (Fig. 4). Neither acetone nor methanol production was detected in

vitro. Carhon dioxide production in vitro was the highest of all the volatiles. It showed the same trend as ethanol, reachmg its maximum on the first day after moculation and sharpjy decreasing afterwards to ten times lower values (Fig. 5), Maximum production of CO2 on the first day was 60 times higher than that of ethanol. At that stage the mycelium had just begun to develop. Ethylene production was very low during the first 3 days after inoculation. A iarge increase

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Fig. 2. Methanol and acetone evolution in 'Shamouti' oranges infected by Penicilhum digitatum during storage at 22 °C. A. Methanol. B. Acetone. Results presented are the means ± SE of 5 replications

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Fig. 3. Carbon dioxide and eihylene evolution in 'Shamouti' oranges infected by PenidlUum digitatum during storage at 22°C. Results presented are the means ± SE of 5 replications

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Volatile Production Induced by Penicillium digitatum Fig. 4. Acetaidehyde, ethanol and ethyl acetate evolution in cuhures of Pemcillmm digitatum during 7 days of growth at 12°C. Results presented are the means ± SE of 5 repKcations

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in ethylene production was found on the 4th day, when sporulation had begun, and continued to increase during the incubation period. However, the levels of ethylene evolved were the lowest of all the volatiles (0.7 u\ • g"' • h"' =

Discussion Acetaidehyde and ethanol are anaerobic metabolites that reach their maximum production when the orange is quite covered by the fungus and sporulation begins (Figs 1 A, 1 B). It is probable that at this stage the fruit is under anaerobic conditions that lead to a higher production of acetaidehyde and ethanol. Citrus fruit under the anaerobic conditions of a CO2 or N2 atmosphere showed increased production of acetaidehyde and ethanol (NORMAN and CRAFT 1971, PESIS and AvissAR 1989). It seems that acetaidehyde and ethanol are produced mostly by the

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Fig. 5. Carbon dioxide and ethylene evolution in cultures of Pcmcillium dtgitatum during 7 days of growth at 2 2 X . Results presented are the means ± SE of 5 rephcations

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fruii tissue and not by the fungus, as production of these two metabohtes at this stage in vitro in very low (Fig. 4). The maximum acetaldehyde and ethanol production by P. digitatum in vitro occurred 8 and 24 h after inoculation, respectively. At this stage the spores were probably surrounded by water, while during their development, when they were under aerobic condition, there was a significant decrease in the acetaldehyde and ethano! production. In fermentation processes, fungi under continuous anaerobic conditions will continue to produce ethanol for longer periods (ABOUZIED and REDDY 1986). Similar behaviour was found in germinating seeds: during the first day of germination, when the seed is surrounded by water, there is increased production of acetaldehyde and ethanol, but after 24 h the seed overcomes the anaerobiosis, and acetaldehyde and ethanol production drop to zero (PESIS and N G 1984 a). The large amounts of ethanol and ethyl acetate in both the infected fruit and in vitro, compared with the quantity of acetaldehyde found, suggest a rapid conversion from acetaldehyde to ethano! and ethyl acetate. The conversion from acetaldehyde to ethanol is apparently performed by the enzyme alcohol dehydrogenase (PESIS and N G 1984b). Acetaldehyde transformation to ethyl acetate could be explained by its conversion via acetyl CoA, which is the precursor of ethyl acetate in plant tissue {CossiNS 1978). In infected oranges, acetaldehyde decrease (Fig. lA) is accompanied by a rise in ethyl acetate production (Fig. 1 C) that reaches its maximum on the seventh day. NORMAN and CR.M-T(1971) showed that emanation of ethyl acetate under nitrogen atmosphere appeared later than that of other anaerobic volatiles. This lag in ethyl acetate production in vitro and in vivo may be due to a need for time to be synthesized from acetyl CoA and ethanol. Ethyl acetate emanations in vitro and in vivo occur when sporulation has already occurred. In the infected fruit, ethyl acetate is probably the result of an interaction between host and pathogen. It is possible that the peel lesion after infection causes increased emanation of ethyl acetate. Ethyl acetate, acetone and methanol were found in grapefruit and orange juice, in which the concentration of ethyl acetate is higher than that of the other volatiles (PiNOff al 1986a, b). Acetone and methanol production was found only in infected oranges and not in vitro., suggesting that these products emanate from the fruit when it is lesioned by P. digitatum. In juice of lemon fruit rotted by Phytophthora citrophthora it was found a marked rise in ethanol and methanol (SCHIFFMANN-NADEL 1977). Acetone and methanol concentrations in the head-space of infected fruit are similar (Figs 2A, 2B). However maximum production of methanol occurred on the 3rd day, while acetone started to increase from the 5th day. Also in oranges under anaerobic conditions of Ni atmosphere, methanol emanation was the first to appear of all the volatiles (NORMAN and CRAFT 1971). The respiration pattern and ethylene production found m P. digitatuminfected oranges (Fig. 3) were in agreement with the results presented in previous papers about lemons infected by P. digitatum (SCHIFFMANN-NADEL 1977) and oranges infected by Diplodia natalensis (ZAUBERMAN and BARKAI-GOLAN 1975). Ethylene evolution in the fruit (Fig. 3) had the same pattern as in vitro (Fig. 5), increasing at the same time in vivo and in vitro when sporulation had already occurred. This indicates that most of the increase in ethylene production by

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infected fruit was a contribution from the fungus, as shown by ACHILLA et al. (1985). It was demonstrated that the ethylene produced by the infected parts of the fruit originated mostly from glutamic acid» which is the precursor of ethylene in fungi (AGHILEA ef W. 1985). On the other hand,, the increase in COi in the infected fruit occurred during sporuiation (Fig. 3), while COi production in culture at that stage is relatively low (Fig. 5). It IS probable that the CO2 increase in infected oranges is due to the fungus-host interaction, as was shown previously (SCHIFFMANN-NADEL 1977, ZAUBERMAN and BARKAI-GOLAN 1975). The CO2 production in culture was concomitant with the ethanol increase: highest on the first day, when the mycelium mass was the smallest (Fig. 5). Most of the C O : production is due to aerobic respiration, but a small part of it can be related to anaerobic respiration as was determined by ethanol production (Fig. 4). In our work we studied the host-pathogen interaction and identified some of the volatiles emanated from P. digitatum in vitro and m vivo. NORMAN (1977) showed that the flavour of intact 'Valencia' oranges was adversely affected when the fruit absorbed voiatiies from oranges inoculated with decay organisms. Thus, it IS possible that the volatiles released from the host-pathogen relationship shown in this paper could influence the flavour of surrounding healthy fruit during storage. Supported by grant No. 1-900-85 from BARD, ihe US-Israel Binationa! A^ncuimral Research and Development Fund. Contribution from the ARO, The Volcani Center, Bet Dagan, Israel. No. 2744-E. 1989 series.

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production by phosphate in Penicilhum digitatum. PI. Cell Physiol. 19, 1S9—196. CossiNS. E. A., 1978: Ethanol metabolism in plants. In: HooK, D. D., and R. M. M. CRA^'FORD (Eds), Plant Life in Anaerobic Environment, pp. 169—202. Science Publishers Inc., Anti Arbor, MI. GUTTER, Y., 1977: Problems of decay in marketing citrus fruits: strategy and solutions around the world: Israel. Proc. Int. Soc. Citriculrure 1, 242—244. NORMAN, S. M . , 1977: The role of volatiles in storage of citrus fruits. Proc. Int. Soc. Citnculturc 1, 238—242. , and C. C. CRAIT, 1971: Production of ethanol, acetaldehyde, and methanol by intact oranges during and after nitrogen storage. J. Amer. Soc. Hort. Sci. 96, 464—467. PESJS, E., and I. AvisSAR, 1989: The post-harvest quahty of orange frtjits as affected by pre-storage treatments with acetaldehyde vapour or anaerobic conditions. J. Hon. Sci. 64, 107—113. , and T. J. N G , 1984a: The role of anaerobic respiration in germinating muskmelon seeds. I. in relation to seed lot quality. J. Exp. Bot. 35, 356—365. -——, and — —, 1984 b: The role of anaerobic respiration in germinating muskmelon seeds. 11. Effect of anoxia treatment and alcohol dehydrogenase activity. J. Exp. Bot. 35, 366—372. J. Phytopathology, Bd. 128,Heft4

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Valencia orange juice from Cuba. Acta Aliment. 15, 291—297. , R. ToRRlCELLA, and F. ORSI, 1986b; Correlation between sensory and gas-chromatographic measurements on grapefruit juice volatiles. Die Nahrung 30, 783—790. ROBINSON, P. M., and M. K. GARRETT, 1969; Identification of volatile sporostatic factors from cultures of Fusanum oxysporum. Trans. Br. Mycol, Soc. 52, 293—299, SCHIFFMANN'NADEL, M . , 1977: Chemical and physiological changes in citrus fruit during storage and their relation to tungal infection. Proc. Int. Soc. Citriculture 1, 311-^317. ZAUBERMAN, G . , and R. BARKAI-GOLAN, 1975; Changes in respiration and etbylene evolution induced by Diplodia natalensis in orange fruit. Phytopathology' 65, 216—217.