Some physiological effects of gamma irradiation on lemon fruit

Radiation Botany, 1964, Vol. 4, pp. 405 to 41 I. Pergamon Press Ltd. Printed in Great Britain.

SOME P H Y S I O L O G I C A L E F F E C T S OF G A M M A I R R A D I A T I O N ON L E M O N FRUIT* E. C. MAXIE, I. L. EAKS t and N. F. SOMMER University of California, Davis, California

(Received 5 February 1964) A b s t r a c t - - G a m m a irradiation of lemon fruit at doses as low as I00 krads results in delayed but severe injury. After 30-40 days at 15°C (59°F) the fruits develop cavities along the segment walls. This injury apparently is not caused by dehydration even though irradiated fruits lose more water than do unirradiated fruits. Doses of 200-400 krads cause a dramatic loss in ascorbie and citric acids in juice extracted from lemon fruit after 40 days at 15°C. Radiation injury resembling the "Black Button" disease of citrus appears about two weeks after treatment, and Alternaria rot develops in the dying tissues. Auxin and kinetin treatments following irradiation did not protect the buttons from the injury. R~sum~----L'irradiation de citrons par les rayons ~" ~ des doses aussi faibles que 100 krads provoque des alterations tardives mais importantes. Apr~ 30 ~ 40 jours b. 15°C (59°F), les fruits se creusent au niveau des parois des loges. Ces ldsions ne proviennent apparemment pas de la ddshydratation, bien que la perte en eau soit plus grande chez les fruits irradi~es que chez les nonirradids. Des doses de 200 ~t 400 krads entrainent une dnorme diminution des acides ascorbique et citrique dans le jus de citron conserv~ 40 jours ~ 15°C. Le dommage dot aux radiations, semblable ~ la maladie "Black Button" du citron, apparalt environ 2 semaines apr~s le traitement. Des moisissures (AlternaHa) se ddveloppent dans les tissus n6cros6s. Des traitements ~ l'auxine et ~t la kin6tine apr~s irradiation n'ont aucun effet protecteur. Zusaxmme~ffass~g--Gammabestrahlung von Citrusfriichten mit Dosen v o n loo krad Rihrt zu einer versp~itet auftretenden, aber schweren Sch~idigung. Nach 30-40 Tagen bei 15°C entwickeln sich an den Segmentw~inden der Frtichte H6hlungen. Diese Sch~idigung wird offensichtlich nicht durch Austrocknung verursacht, obwohl bestrahlte Frtichte mehr Wasser verlieren, als unbestrahlte. Dosen yon 200-400 krad f'dhren zu einem starken Verlust yon Ascorbins~iure und Zitronens/iure im Saft yon Zitronen, die nach der Bestrahlung 40 Tage bei 15° gela gert wurden. Eine Strahlensch~idigung, die der "Black Button" Krankheit (Schw~irzung der Knospen) yon Citrusgew~ichsen ~ihnlet, zeigt sich ca. 2 Wochen nach der Bestrahlung. In den absterbenden Geweben entwickelt sich Alternaria-F/iule. Auxin- und Kinetinbehandlungim Anschluss an die Bestrahlung bedeutete keinen Schutz fiir die Knospen. INTRODUCTION LEMON f r u i t s i n coo1 storage are subject to decay ti'om a n u m b e r of pathogens, the most c o m m o n b e i n g Penicillium italicum W e h m e r , Penicillium d~gitatum Sacc., a n d Alternaria citri Ellis & Pierce.

L e m o n fruits are subject to chilling i n j u r y if stored at t e m p e r a t u r e s below 15°C(1). T h e r e q u i r e m e n t of l e m o n fruits for a relatively high storage t e m p e r a t u r e contributes to their susceptibility to decay. T h e p r o p o r t i o n of citrus fruits

*Research supported by the U.S. Atomic Energy Commission. Contract No. AT( 11-1)-34, Project Agreement No. 80. tAddress: Department of Plant Biochemistry, University of California, Riverside. 4O5

406

SOME PHYSIOLOGICAL EFFECTS OF GAMMA IRRADIATION ON LEMON F R U I T

showing decay in cool storage can be reduced by washes and by various fungicidest 18), but these treatments do not control well established infections in mechanical wounds. A fungicidal or fungistatic agent capable of penetrating the flesh of lemons without harming the fruit would be beneficial in controlling decay. B E R A H A et al. (~) reported that lemons and oranges inoculated with P. di¢itatum and P. italicum and subsequently stored at 55 and 75°F had an increased shelf life if given y-irradiation at doses of 150-200 krep. They noted injury to the fruit in the form of softening and browning of the rind from doses of about 200 krep. No objective measurements were made of the intensity of the injury. I f ionizing radiation has a role in extending the marketable life of lemons, it would most likely be in protecting the fruit from decay during longterm storage. B E R A H A et al.(2) did not study the long-term effects of y-irradiation on lemon fruits. The objectives of this study were to: (1) evaluate the effect of y-irradiation on the keeping quality of lemon fruits in short- and long-term cool storage; and (2) measure quantitatively the effects of y-irradiation on some physiological and chemical properties of lemon fruits. MATERIALS AND METHODS

Eureka lemon fruits of commercial maturity, unwashed and unwaxed, were obtained from a packing house near Riverside, California. The fruits were placed in fibreboard boxes and transported to Davis, California, in an insulated box. The fruit temperature during transit was never less than 15°C nor more than 21°C. For cool storage, the fruits were held at 15°C at a relative humidity of about 93 per cent. Respiration rates at 20°C were measured by the method of CLAYPOOL and KEEFER (5)on four replicates of ten fruit per treatment. Samples of internal atmospheres from a large number of fruits were extracted by the method of FREEBAIRN (7)afterthe fruitwas punctured as described by EAKS and LUDI(6). Ten fruitsper sample were placed beneath a large funnel with a serum cap fitted over the spout and the funnel completely filled with, and submerged in, a saturated solution of sodium chloride inside a pressure cooker. The cooker was sealed, and a vacuum of

2 cm of mercury was pulled on the system for one min. The extracted gases filled the spout of the funnel. A gas-tight syringe fitted with a 2-in. needle was used to remove a gas sample from the funnel for analysis. Samples withdrawn from individual fruits with a 2½-in. hypodermic needle affixed to a gas-tight syringe gave essentially the same analytical results as mass fruit samples. Ethylene was measured by gas chromatography with an Aerograph A-600 B Hy-Fy flame ionization unit fitted with a 5 ft. × ~ in. column packed with 60/80-mesh alumina. The identity of ethylene was confirmed by pretreating samples of the gas with mercuric perchlorate, brominated charcoal, and aqueous potassium hydroxide(3). Ethylene is removed by the first two systems but not by the last. Ethylene trapped in mercuric perchlorate was released by the method of YouNc et al.(16) and analyzed by gas chror~atography, as above. Carbon dioxide and oxygen were measured by gas chromatography with an Aerograph A-90-P thermal-conductivity unit fitted with a 6 x ~- in. column of silica gel and a 15ft x k in. column packed with 90 percent 13X and I0 percent 5X molecular sieve according to the method of LuH and CHAUDRY(10). Juice for analysis was extracted from fruit halves by a Sunkist power reamer. Ascorbic acid, expressed as mg/ml juice, was measured by the method of LOEFFLER and PONTING(9) on ten fruit in each of four replicates. Weight loss was measured on four replicates of 20 fruits per treatment. The fruits were shielded from direct air movement and randomly arranged on a bench in a controlled-temperature room at 15°C and about 93 percent relative humidity. Each fruit was numbered so that the replicates could be reassembled for weighing. Irriadiation was done in the M a r k I I Experimental Food Irradiator(n) with air passing through the chamber at a rate of 6,000 ml/min to preclude depletion of oxygen and the accumulation of carbon dioxide in the atmosphere. The dose rate was approximately 300 krads/hr. Dosimetry was by the methods described by ROMANI el a/.(x~) RESULTS

The rate of carbon dioxide evolution by lemon fi'uit 18-24 hr after irradiation showed a marked

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FiG. 2. Internal appearance ofirradiatedlemons after 40 days at l5°C.

R.B.f.p. 406

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FIo. 5. Effect of 200 krads of v-radiation on buttons of lemon fi'uits after 40 days at 15°C. Dark areas on irradiated fruit caused by Alternaria rot.

E. C. MAXIE, I. L. EAKS and N. F. SOMMER

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Fro. 1. Effect of y-irradiation on the respiration of Eureka lemons at 20°C. stimulation (Fig. 1). Fruits subjected to 50 or more krads showed an immediate increase in respiratory rate on the first day following treatment. It was noted that the green color disappeared more rapidly from fruits subjected to 50 and I00 krads than from control fruits. This phenomenon is the subject of another study, currently in progress. Fruits subjected to 400, 600, and 800 krads developed a bronze color on the fifth, fourth, and third days. Fruits subjected to 1,000 krads developed severe bronze color between the second and third days after treatment. At the time the injury symptoms appeared on these fruits the respiratory rate began a marked decline, reaching the level of the control fruit by the seventh day after treatment. Examination of the fruit at the end of the experiment revealed severe tissue breakdown and a translucent appearance of the juice vesicles. Figure 2 shows the internal condition of fruits stored 40 days at 15°C following treatment with 0, 50, 100, and 300 krads. Large cavities

developed along the segment walls in the irradiated fruit. The injury was first noted in fruits subjected to 200 and 300 krads in the third week after treatment. The injury was minor in fruits subjected to 50 krads. However, a third of the fruits subjected to 100 krads showed severe injury. The possibility that separation of the tissues occurred when the fruits were cut was explored by freezing fruits in a dry ice-acetone solution. T h e cavities were clearly defined in the frozen fruit. When lemon fruits were held for 24 hr in an anaerobic condition and irradiated, the severity of the cavities was less than for fruits in air, though still sizable. Figure 3 shows the carbon dioxide, oxygen, and ethylene composition of the internal atmosphere extracted from lemon fruits after 40 days at 15°C following irradiation with a range of doses. To retard moisture loss, these fruits were dipped in a 0.5 percent solution of Sunkist Water Wax immediately after irradiation, thus being coated with a substance that m a y have restricted

408

SOME PHYSIOLOGICAL EFFECTS OF GAMMA I R R A D I A T I O N ON L E M O N F R U I T

gas exchange(s) . T h e percent of carbon dioxide in the internal atmosphere increased with increasing dose of ,(-rays. T h e oxygen concentration was slightly less in the irradiated than in the control fruits. T h e concentration of ethylene in the internal atmosphere of the irradiated fruit increased markedly with dose. There was a possibility that dehydration contributed to the development of the cavities in irradiated lemon fruits. Figure 4 shows the effect ofT-irradiation at 0, 100, and 200 krads on weight loss by lemon fruits held at 15°C for 30 days following irradiation. T h e irradiated fruit lost more weight than the unirradiated fruit, both waxed and unwaxed. Cavities, however, were of equal severity in all the irradiated fruits. W h e n fruits subjected to 100 krads or more were reamed for juice the membranes of the segment walls disintegrated completely. I n unirradiated fruit, these membranes remained relatively intact although flattened against the rind.

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E. C. MAXIE, I. L. EAKS and N. F. SOMMER The flesh of fruits subjected to 100, 200, and 300 krads developed an orange tint about three days after treatment. This color gradually changed to a bronze shade after one month at 15°C. Immediately after treatment, the flesh of fruits subjected to 800 and 1,000 krads was translucent and appeared devoid of yellow pigments. Fruits subjected to 300 krads or more were lost to severe radiation injury within three weeks at 15°C. Fruits subjected to 100 and 200 krads were normal in flavor and odor immediately after treatment, whereas severe off-flavors and -odors were noted in all fruits subjected to 300 krads or more. After three weeks at 15°C, fruit subjected to 200 krads developed severe off-flavor and -odor. No off-flavor or -odor developed in fruits subjected to 100 krads. Figure 5 shows the effect of 200 krads of y-rays on the buttons (calyx) of lemon fruits after five weeks at 15°C following treatment. The first evidence of radiation injury to the buttons appears approximately two weeks after treatment, when the green color turns to yellow. This is followed by dehydration and apparent death of the tissues, between the third and fourth weeks. The buttons turned black, and many were infected with AlternaHa citH. The infection spreads quickly, as shown in Fig. 5. Unirradiated lemons in cool storage may develop a condition known as "Black Button" whose symptoms are similar to the radiation injury described above. This disorder is apparently hormonal in nature and can be controlled

409

by dipping the fruit in a solution of 500 ppm of 2,4-dichlorophenoxyacetic acid(iS). Table 1 shows the results of an experiment in which the auxin failed to prevent radiation-induced black buttons. For this reason it is likely that the fruits treated with auxin would have developed Alternaria rot if held for a considerable time in cool storage. Kinetin (6-furfurylamino purine) is effective in retarding senescence and related phenomena in some plant tissues(S). Leomon fruits irradiated at 100 and 200 krads were dipped in Kinetin solutions of 10, 100, and 1,000 ppm, but no reduction in radiation injury of buttons was detected. Figure 6 shows the effect of y-irradiation on the ascorbic acid content of lemon juice from fruits held at 15°C for 40 days following treatment. One day after treatment, the loss in ascorbic acid in irradiated fruits was negligible. At 40 days, however, there was a marked reduction in ascorbic acid in fruits subjected to 200, 300, and 400 krads. Figure 7 shows the effect of various levels of y-irradiation on the citric acid level in the juice after the fruit was held at 15°C for 40 days following treatment. At doses of 200 krads and higher, there is a dramatic loss in citric acid.

DISCUSSION

It is clear from the data presented that an evaluation of the effects of y-rays on lemon fruits immediately after treatment could be misleading. After one or two weeks, one might

Table 1. Effect of "t-rays and 2,4-dichlorophenoxyaceti¢ acid treatments on button condition of lemons after 5 weeks at 15°G*

Treatment Control; no 2,4-D Control + 500 ppm 2,4-D 200 krads; no 2,4-D 500 ppm 2,4-D; then 200 krads 200 krads; then 500 ppm 2,4-D

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410

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Fxo. 6. Effect of 7-irradiation (applied on second day) on ascorbic-acid content of lemon fruits stored at 15°C. conclude that radiation injury is minor at doses of 200 krads or less. However, lemons may occasionally be stored for as long as six months(x) ; so the long-term effects of radiation are important. In such periods, injury symptoms become severe, indicating a time-dose effect that must be considered in evaluating radiation as a protective treatment for lemons. The presence of ethylene in the internal atmospheres of irradiated l e m o n s ( F i g . 3) is indicative of radiation injury, since healthy fruit of this species has been shown to produce only small amounts of this gas(4). The development of cavities in irradiated lemons is apparently not a matter of water loss. The treated fruits do lose more weight than do the control fruits, but the amount is not enough to account for the magnitude of the injury. The development of Alternaria has precluded examination of irradiated fruit for a period longer than six weeks after treatment. However, it is probable that cavities ofunacceptable severity would occur

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E. C. MAXIE, I. L. EAKS and N. F. S O M M E R in fruits subjected to as tittle as 50 krads during a prolonged cool-storage period. SO~¢ER et al.(14) showed that the c o m m o n fruit decays of citrus are not inactivated b y doses o f 7-rays as high as 200 krads if the level of infection is high. T h e r e m a y be an inhibition of decay, but with time the fungi will resume growth. Considering the development of cavities in fruits subjected to as little as 50 krads, the loss of ascorbic and citric acids at 200 krads or more, the development of off-flavors and off-odors and the injury to the buttons followed by rapid development of Alternaria rot, we conclude t h a t -f-irradiation is of doubtful value as a supplement to cool storage for lemons.

1. 2.

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REFERENCES BAaWHOLOMEWE. T. and SmCLAm W. B. (1951) The lemon fruit. University of California Press. BERAHA L., RAMSEY G. B., SMITH M. A. and WmOHT W. R. (1959) Factors influencing the use of ,t-radiation to control decay of lemons and oranges. Phytopathology 49, 91-96. BuRo S. P. (1962) The physiology of ethylene formation. Ann. Rev. Plant Physiol. 13, 265-302. BuRo S. P. and Buxo E. A. (1962) Role of ethylene in fruit ripening. Plant Physiol. 37, 179-189. CLAYPOOLL. L. and I~EPER R. M. (1942) A colorimetric method for CO~ determination in respiration studies. Proc. Am. Soc. Hort. Sd. 49, 177-186.

411

6. EAKSI. L. and Lure W. A. (1960) Effects of temperature, washing and waxing on the composition of the internal atmosphere of orange fruits. Proc. Am. Soc. Hort. SSI. 76, 220-228. 7. Fm~EBAmNH. T. (1962 ) Personal communication. 8. LEOPOLD A. C. (1961) Senescence in plant development. Sdznce 134, 1727-1732. 9. LOEIU~LERH.J. and PONTINOJ.D. (1942) Ascorbic acid. Rapid determination in fresh, frozen, or dehydrated fruits and vegetables. Ind. Eng. Chem. 14, 846-849. 10. LUH B. S. and CHAtrORY M. S. (1961) Gas chromatography of COg, H, O~, and N~ in processed foods. Food Technol. 15, 52-54. 11. Romaa~ R. J., MAXm E. C., I-IazssE C. O. and Sore,mR N. F. (1962) Coe° 7-ray irradiator opens new doors to biological research at Davis. Calif. Agric. 16, I-4. 12. ROMANXR.J., ROBXNSONB.J., RAE H. L., MAXm E. C. and SO,#mR N. F. (1963) Fruit irradiation-physical methods. Radiation Botany 3, 345-350. 13. SINCLAmW. B. (1961) The Orange. University of California Press. 14. SOMMERN. F., MAXm E. C., FORTLAGER . J . and ECKERT J. W. (1963) Sensitivity of citrus fruit decay fungi to 7-irradiatlon. Radiation Botany 4~ 317-322. 15. STEWARTW. S. (1948) The effects of 2,4-D and 2,4,5-T on citrus fruit storage. Citrus Leaves 28 (I 1), 5-7; 24-27. 16. YOUNOR. E., PaATT H. K. and BXALEJ.B. (1952) Manometric determination of low concentrations of ethylene. Anal. Chem. 24, 551-555.