[advances in food research] advances in food research volume 15 volume 15 || ionizing radiation for...

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IONIZING RADIATION FOR CONTROL OF POSTHARVEST DISEASES OF FRUITS AND VEGETABLES I. Introduction .................................................. 147 11. Fungicidal and Fungistatic Efiects of Radiation .............. 149 A. Radiobiology ............................................. 149 R. Factors Affecting Dose Requirer.ients ....................... 151 C. Mutations ................................................ 158 159 A. Iii Vztro Studies ................................... 160 B. 1~ Viwo Studies ....... .................... 166 IV. The Nature and Causes of ases ................ 166 V. Disease-Control Investigations ................................ 170 A. General ................................................... 170 B. Irradiation-Induced Susceptibility to Infection ................ 180 VI. Protective Packa ......................... 182 VII. Research Needs ........................................... 183 References .................................................... 184 111. Techniques for Postharvest Disease Radiation Studies ............ I. INTRODUCTION The bactericidal and fungicidal properties of ionizing radiation have been studied from a time soon after Roentgen’s (1898) dis- covery of X-rays, in 1895, and Becquerel’s (1896) discovery of radioactivity. Notable early reports include those of Errera (1896) on the effects of X-rays on Phycom~ces; Dauphin (1904) on the influence of radium on the growth and development of certain lower fungi; and Minck (1896) and Rieder (1898) on the bactericidal properties of X-rays. Many of the earliest re- searchers reported no damage to the irradiated organism, but Rieder reported decided damage to bacteria. The early investiga- tions on the biological effects of ionizing radiation have been reviewed in detail by Duggar (1936) for bacteria and by Smith (1936) for fungi. The possibility of utilizing ionizing radiation for disease con- trol was pointed up by the successful use of X-rays to cure 147

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Page 1: [Advances in Food Research] Advances in Food Research Volume 15 Volume 15 || Ionizing Radiation for Control of Postharvest Diseases of Fruits and Vegetables

IONIZING RADIATION FOR CONTROL OF POSTHARVEST DISEASES OF FRUITS AND VEGETABLES

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147 11. Fungicidal and Fungistatic Efiects of Radiation . . . . . . . . . . . . . . 149

A. Radiobiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149 R. Factors Affecting Dose Requirer.ients . . . . . . . . . . . . . . . . . . . . . . . 151 C. Mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158

159 A. I i i Vztro Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160 B. 1~ Viwo Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . 166

IV. The Nature and Causes of ases . . . . . . . . . . . . . . . . 166 V. Disease-Control Investigations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170

A. General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170 B. Irradiation-Induced Susceptibility to Infection . . . . . . . . . . . . . . . . 180

VI. Protective Packa . . . . . . . . . . . . . . . . . . . . . . . . . 182 VII. Research Needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184

111. Techniques for Postharvest Disease Radiation Studies . . . . . . . . . . . .

I. INTRODUCTION

The bactericidal and fungicidal properties of ionizing radiation have been studied from a time soon after Roentgen’s (1898) dis- covery of X-rays, in 1895, and Becquerel’s (1896) discovery of radioactivity. Notable early reports include those of Errera (1896) on the effects of X-rays on P h y c o m ~ c e s ; Dauphin (1904) on the influence of radium on the growth and development of certain lower fungi; and Minck (1896) and Rieder (1898) on the bactericidal properties of X-rays. Many of the earliest re- searchers reported no damage to the irradiated organism, but Rieder reported decided damage to bacteria. The early investiga- tions on the biological effects of ionizing radiation have been reviewed in detail by Duggar (1936) for bacteria and by Smith (1936) for fungi.

The possibility of utilizing ionizing radiation for disease con- trol was pointed up by the successful use of X-rays to cure

147

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148 N. F. SOMMER AND R. J. FORTLAGE

dermatophytoses (Jungling, 1919 ; Levy, 1913 ; Melchoir, 1916 ; Sardemann, 1914). Similarly, Heyderdahl (1926) cured actino- mycosis with gamma rays from radium. These demonstrations that parasites could be selectively treated in host tissue suggested the possible use of ionizing radiation to eliminate certain plant parasites in seed-borne diseases. Pichler and Wober (1922) re- ported that X-rays killed Ustilugo t ~ i t i c i (Pers.) Rostr. in wheat seeds, and C. mtda (Jens.) Rostr. in barley seeds. In addition, those researchers reported elimination of the wart disease fungus, Svnchyt?*ium endobioticum (Schilb.) Percival, from potato tubers. From later studies, however, Tascher (1933) concluded that radi- ation doses sufficient to kill fungi in seeds would injure em- bryos, resulting in abnormal germination and seedling growth. More recently, Lo (1964) reached similar conclusions.

Studies have been conducted on the effect of X-radiation on the crown gall disease and its causal organism, Ag~obacteiiuni tzime- facieizs (E. F. Sm. and Town.) Conn. (Levin and Levine, 1917; Rivera, 1929 ; Waggoner and Dimond, 1952a).

The possible application of ionizing radiation to disease control is evidently severely limited by the susceptibility of host plant meristems to damage by relatively low doses. Treatments sufficient to inactivate pathogens would generally be expected to result in abnormal or inhibited growth (Dimond, 1951 ; Hellmers, 1959 ; Schwinghamer, 1957 ; Waggoner, 1956 ; Waggoner and Dimond, 1952b). Thus, deeply penetrating ionizing radiation does not ap- pear promising for therapy of plants or plant parts destined for later growth. Consequently, such treatments are evidently ruled out for seed grains and propagules such its bulbs, tubers, and cuttings. Much more promising subjects for irradiation are har- vested plant parts destined for consumption. Although the com- modities a re alive, cell division has often virtually ceased. In other cases, cell division may occur after harvest but abnormal or reduced growth from irradiation may not be objectionable. Where the effects on growth can be ignored, relatively high doses can frequently be tolerated by the host.

The present capabilities for source construction and design suggest that radiation could economically be used to treat food if benefits were sufficient. An area of particular interest in this regard is the possible use of ionizing radiation as a fungicidal treatment for the control of postharvest diseases of fruits and vegetables (Droge, 1963). Here, radiation treatments appear to have important potential advantages. Not the least among them

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RADIATION FOR DISEASE CONTROL I45

is the complete absence of chemical residues. Furthermore, radia- tion provides a fungicidal treatment uniquely different from chemical applications. The extreme penetration, particularly of gamma rays, permits the host to be considered essentially trans- parent to the rays. Therefore, organisms deep within the host tissue niay be treated as readily as if they were on the surface. Because of this feature, pathogenic organisms may be treated within host tissue to provide a therapeutic effect in a manner ordinarily not possible with chemical fungicides.

With radiation as with chemicals, control of postharvest dis- eases requires a thorough understanding of the diseases to be controlled. Particularly important are the time, place, and manner of infection; the influence of temperature and other environmental conditions on disease development ; the propensity for spread by contact ; and the physiological length of the postharvest life of the fruit o r vegetable. Extended storage following radiation may affect host injury responses (Maxie and Abdel-Kader, 1965) as well as change the relative importance of various diseases. The effectiveness of radiation treatments may therefore be determined in large measure by factors relating to disease etiology. Equally important are those facets of radiation biology which affect the fungicidal and fungistatic consequences of radiation. Therefore, these topics are considered at this time to lay a foundation for discussions of recent investigations in the area of irradiation of fresh fruits and vegetables.

1 1 . FUNGICIDAL AND FUNGISTATIC EFFECTS OF RADIATION

A. RADIOBIOLOGY Ionizing radiation is a form of energy which, if absorbed,

acts upon living cells to produce injuries. The absorption of energy ionizes and electronically excites molecules in a way that pro- duces molecular changes. The action is termed “direct” if the damage occurs in the molecule in which the energy has been absorbed, and is called “indirect” if it results from highIy reactive free radicals formed in water and reacting with cell constituents. The injury may directly damage genetic material, producing mu- tations which may or may not prove lethal. Molecular change may cause so-called biochemical lesions which are developed or in- tensified by metabolic processes of the cell. Biochemical lesions may affect vital genetic syntheses to result in a mutation which, if sufficiently severe, may result in cell death. On the other hand,

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150 N. F. SOMMER AND R. J. FORTLAGE

biochemical lesions may affect nongenetic processes and result in an altered physiology which may also prove lethal to the cell. The fundamentals of radiation biology are treated adequately in a number of excellent reports. Particular attention is called to those of Bacq and Alexander (1961a), Errera and Forssberg (19611, Harris (1961), Jenkinson (1963), Kimball (1957), Lea (1955), Romani (1965), Platzman (1952), Setlow e t al. (1961), and Spear (1953). Fungi were given special attention in the reports of Beck and Roschenthaler (1960), Pomper and Atwood (1955), and Tatum (1950). Certain aspects of radiation effects are of particular, and sometimes unique, importance to studies of the use of ionizing radiation in the pathology of harvested fruits and vegetables. These are the considerations which receive primary attention here.

Ionizing radiation may, in pathogens, cause morphological ab- normalities as well as genetic mutations and altered physiology (Atwood and Pittenger, 1955a ; Brace, 1950 ; Burns, 1955 ; John- son, 1932). The morphological effects on fungi are especially striking immediately after germination. When irradiated spores are plated on a medium conducive to prompt and rapid germina- tion, the germ tubes produced often have diameters much larger than normal (Berk, 1952a, 1953; Brace, 1950; Dimond and Dugger, 1940 ; Luyet, 1932). Frequently grotesque swellings occur at various places in the mycelium. Commonly, the germ tube may grow only a short distance and then round up at the end t o produce a monster germinant resembling a “dumbbell.” Occurring just as commonly, however, is extensive germ tube development, including branching. The amount of germ tube development is evidently inversely related to dose. Characteristically, cross walls are almost totally lacking in species that normally form regular and prominent walls in the germ tubes. Extensive growth may occur, but a point invariably seems to be reached at which all further development stops and death follows. The absorption of energy to cause lethal damage may be separated from death by many hours. If irradiated spores are stored in a n environment not conducive to germination, the time between energy absorption and death may be extended to a number of days. During that time respiration, the production of certain enzymes, and other metabolic functions are known to proceed at a normal or acceler- ated rate (Sommer e t aZ., 1963b, 1965a). The stimulation of me- tabolism or germination and growth (Buchwald and Wheldon, 1939 ; Vasudeva e t aZ., 1959) following lethal or sublethal irradia-

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RADIATION FOR DISEASE CONTROL 151

tion must presumably require an explanation based upon cell injury .

In considering control, the relation of radiation dose to the ability of fungus spores to germinate is of little importance (Uber and Goddard, 1934). Much greater significance must be attached t o loss of the ability t o form a colony capable of indeterminate growth. In fungi the capacity for indefinite growth is lost a t a much lower dose than the ability to germinate (Beraha et al., 1959a,b). In experiments with Rhixopzts stolonifer, a dose of 500 Krad reduced the surviving fraction (i.e., ability t o form a colony) to less than 1% but hardly affected the ability to germinate (though many germ tubes produced were abnormal). Reducing germination to nea.r 1% required a dose of 1500 Krad. An even more resistant process than germination is the ability of the spore to swell prior to emergence of the germ tube (Sommer ef al., 1963a).

When colonies are irradiated at a dose insufficient to inactivate permanently, growth is halted temporarily (Beraha et al., 1959a,b; Nelson et al., 1959), to be resumed after a delay which can amount to several days. The basis for the delay is not well understood. It is likely, however, that most of the mycelium has been irreversibly injured. Only certain portions of hypha may, with time, recover. Essentially normal growth then occurs from localized areas.

B. FACTORS AFFECTING DOSE REQUIREMENTS

1. Genetic

The use of radiation in postharvest pathology obviously de- pends on the radiation sensitivity of the fungus compared with the ability of the host t o withstand the treatment with little or no obvious injury or deleterious side effects. The apparent sen- sitivity of the fungus is determined by a number of factors. One factor is the inherent resistance of a fungus to inactivation by ionizing radiation. The resistance varies widely between species, is evidently of a complex nature, and is genetically controlled. The genetics of fungi are reviewed by Fincham and Day (1963), while radiation genetics have been discussed by Wolff (1961).

In fungi it has been demonstrated, particularly with yeast, that diploid cells are more radiation-resistant than haploid cells of the same species (Fig. 1 ) (Beam, 1955; Latarjet and Ephrussi, 1949 ; Mortimer, 1954 ; Mortimer, 1961 ; Zirkle and Tobias, 1953).

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152 N. F. SOMMER A N D R. J. FORTLAGE

It appears tha t the greater radiation resistance of the diploid condition is a direct result of the presence of a second set of chromosomes. The nuclear redundancy of the diploid condition presumably provides protection against recessive lethal mutations. Data f rom higher plants suggest tha t the fur ther chromosome duplication of polyploids does not impart added radiation resist- ance (Sparrow and Miksche, 1961). Similarly, in fungi, Morti- mer (1961) found tha t hexaploid yeast cells were less resistant than triploids.

I 1

\

IC- L --L -1- 4 S 12 16 2 0 24%,000r

FIG. 1. Survival of haploid and diploid yeast cells a f t e r X-radiation. I . IIaploid. 11. Diploid. Redrawn from La ta r j e t and Ephrussi (1949).

The nuclei of fungus spores a re usually haploid, but, commonly, two or more nuclei may be present within a single-celled spore. Here, multinuclearity imparts a resistance analogous to the efiect of ploidy. In experiments with hrewospo?.a spores, Norman (1951 ) found uninucleate microconidia to be less radioresistant than conidia containing from one to several nuclei but averaging about two (Fig. 2 ) . Similarly, Atwood and Pittenger (195513) found multinucleate Sezwospo?*n ascopores more resistant than micro- conidia. The results suggest tha t the spore can survive if one ilucleus escapes inactivation. In the case of multicellular spores the presence of the several cells in a spore would presumably impart resistance: as long as lethal injury was escaped by any one constituent cell, the spore would retain the ability to form a colony.

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RADIATION FOR DISEASE CONTROL 153

Although the relation between nuclear conditions and radiation resistance is unmistakably important, damage to the cytoplasm is also important (Kimball, 1957). Furthermore, nuclear damage may not be all genetic damage.

I./ 0 10 2 0 3 0 40

DOSE IN QUANTA CM* x FIG. 2 . Survival of uninucleate microconidia and multinucleate (av. 2.27)

conidia of .Vcztrospoi.a o‘assa. Redrawn from Norman, (1951).

2. Popu la t ion S i z e

The size or amount of fungus “tissue” present affects the ap- parent sensitivity of a species. With filamentous fungi, direct determination of population size of mycelium is difF.cult in vitro and may be impossible in vivo. Problems of technique in studies involving mycelium are discussed in a later section. Obviously, however, the minimum dose required to inactivate with a high degree of probability all “cells” in a population is related to the number ol‘ cells in the population (Couey and Bramlage, 1965).

In studying the inactivation of mycelium, so-called “lethal doses” depend upon the size of the colonies. Presumably “lethal dose” values would always differ if the amount of mycelium was not the same. For example, a number of “lethal doses” have been reported for B o t r v t i s cinerea. Skou (1960) reported the “lethal dose” to be greater than 470 Krad. Beraha e t al. (1960) reported that the approximate doses lethal to young mycelium in vitro varied from 0.95 to 2.03 Krad. In the host, a comparable effect required 2.74 to 4.56 Krad. Saravacos e t al. (1962) reported a

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154 N. F. SOMMER AND R. J. FORTLAGE

“lethal dose” of 500 Krad for the fungus in culture. Kljajic (1960) reported that doses of 600-1,000 Krad were needed for a “lethal” effect.

Sommer e t al. (1964a,b) studied the doses required to inactivate every “cell” in widely varying sizes of populations of B. cineyea conidia and young mycelial “cells.” The young mycelium was ob- tained by germinating conidia until the resulting germ tubes had three to five cross walls. Figure 3 shows the doses which are capable of inactivating every “cell” within 80% of the popula- tions when populations of several different sizes are compared.

Careful consideration of population sizes is of great importance in interpreting the results of radiation treatments for the control of postharvest diseases in fruits and vegetables. If colonies are large, even radiation-sensitive fungi may require relatively large doses. For example, among Pviirius fruit decay fungi, Moiiilinia f i ircticola conidia a re relatively radiation-sensitive whereas Rki- :opus stolonife?. spores are much more resistant (Fig. 3 ) . Yet, because of population differences, &I. fructicola may appear the

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D 0

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- 400 w in (=& 300r

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7

O l I --- L- PA--

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POPULATIONS

FIG. 3. Approximate doses required to inactivate every cell in 80‘r (colony inactivation dose 80) of various sized populations of postharvest disease fung i . Data a r e for spores in vitro. 1) Trichoderma viridc. 2 ) P h o ~ o p s i s citn’. 3 ) Penicillium i ta l icum. 4) Pcnicillium e x p a m u m . 5) Pcxicilliltm digifatzLm. 6 ) Gcotr.idiunz canrlitlum. 7 ) Moiiilinia f ruc t ico la . 8) Botry t i s ci i icrea. 9) Di- p lod ia izatalc nsis. 10) Rhixopus stolonifer. ll) Alternaria citri. 1 2 ) Clado- .iporicim h c r b a m m . Redrawn from Sommer c t al., (1964a,b).

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RADIATION FOR DISEASE CONTROL 15.5

more resistant when fruit is irradiated at a given dose. The reason is that the R. stolonifer population may consist of only a few spores contaminating harvest or handling wounds whereas M . f m c t i c o l a may be present in well-established colonies resulting from infections occurring in the orchard before harvest.

3. Fungicidal or Fungistatic Effect Required

The degree of the fungicidal effect sought greatly influences the size of the radiation dose required. A relatively small dose frequently suffices if complete inactivation of lesions is not re- quired. At doses insufficient to inactivate lesions, growth is halted temporarily, with the length of the delay related to the size of the dose (Beraha et al., 1959a,b; Nelson et al., 1959). In the first place, fungus “cells” given sublethal doses are evidently injured sufficiently for a time lag to result before growth is resumed. Secondly, most of the constituent mycelium of 8 colony may be inactivated, so that resumption of colony growth is dependent on the activity of localized areas of a few hyphae. Such a temporary halt in lesion growth may provide a good and sufficient control in commodities that normally have only a short physiological life, such as strawberry or sweet cherry fruits. With longer-lived hosts a highel. proportion of fungal lesions will likely require inactiva- tion.

4. Dose-Modifying Factors

Experimental conditions before, during, or after irradiation may influence the results of experiments designed to determine the effectiveness of radiation as a fungicidal treatment. Some factors may magnify the damaging qualities of the irradiation, whereas others may alter the ability of the living cell to with- stand injury.

a. Clxy.c/en E f f e c t . Perhaps the most striking dose-modifying variable is the presence or absence of oxygen. Oxygen materially enhances the effectiveness of a given radiation dose (Bridges and Horne, 1959 ; Laser, 1954 ; Stapleton and Hollaender, 1952). The increased lethality is evidently the consequence of the formation of toxic peroxides in the presence of oxygen. In tests with several postharvest disease fungi the dose required to reduce survivors to the 1% level was only about 60% as great in the presence of oxygen as in anoxia (Sommer et nl., 1 9 6 4 ~ ) .

Fungi or bacteria parasitizing fruit or vegetable tissue are

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156 N. F. SOMMER AND R. J. FORTLAGE

~ i ~ ~ i a l l y in a n aerobic environment, but, under certain conditions, low oxygen tensions may develop. Fo r example, if the f ru i t s to be irradiated a re sealed in plastic bags or other containers, the res- piration o i f ru i t and pathogen may modify the atmosphere by depleting oxygen while carbon dioxide accumulates. Similarly, Sungi prepared for irradiation iti v i t w may consume oxygen to the extent tha t near-anaerobic conditions may result unless guarded against.

A related factor is the presence of ozone produced by the i r - radiation of oxygen. Evidently concentrations toxic to host and parasite can accumulate, particularly if the irradiation is per- formed in a closed container. Rapid a i r movement through the irradiation chamber would presumably prevent accumulation of a n ozone atmosphere around the f ru i t or vegetable host. A more difficult measure, however, would be the prevention of ozone ac- cumulation within host tissues, since these tissues may contain as high as 20% a i r space (Maxie and Abdel-Kader, 1965).

b. P?-otection. A radiation dose is commonly less effective i t i

civo than in v i t m . The lower lethality is believed to be the conse- quence of the chemical protection afforded by host constituents. Many chemicals a re known to exert a protecting effect against radiation damage (Bacq and Alexander, 1961b ; Hollaender, 1960 ; Riley, 1955). In some cases the protective effect results f rom the compound’s removal of oxygen or f rom the promotion of oxygen removal by the cell by oxidation. Other compounds, however, a re able to exert a protection greater than found by removal of oxy- gen and a re believed to specifically protect radio-sensitive sites or promote recovery (Stapleton, 1960).

c. R a t e of Applicat ion of Radiation. The rate of application of a given dose or fractionation of the dose into two or more por- tions separated by time influences the biological effect achieved. Sometimes the greater biological effects of rapidly applied doses have been suggested to result from fewer opportunities for re- pair (Whiting, 1960).

Beraha e t al. (1959d) have studied the effects of the rate of application of the gamma-radiation dose in connection with cer- tain postharvest diseases of fruits. Within the range of 137-182 Krad, a high flux of 7 Krad/min. controlled Pythium deba?*yanz~nr Hesse inoculated in potato tubers better than the same dose ap- plied at 3 Krad min. In later studies (Beraha, 1964), Pexicilliu?~?, italicurn Weh. inoculated in oranges was controlled for 12 days by 137 Krad applied at 40 Kradlmin or 182 Krad applied at 20

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RADIATION FOR DISEASE CONTROL 1,5:

Krad min (Fig. 4 ) . Similar advantages of a high dose ra te were reported fo r Moriiliiiiu f ructicola (Wint.) Honey in peaches, and Rotqjfis cinema Pers. ex Fr. in pears.

Absorption of the dose by the host may reduce the dose a t critical sites, modifying the expected radiation effect. However, this is a problem of dosimetry and dose distribution rather than a dose-modifying effect.

5

0 40 Kradlmin 3 Krad/rnin i % 4

a 3 i

L 2

a u

z 0

W lL

z - I

0 56812 5 6 8 12 5 6 8 12 5 6 8 12 5 6 8 12

DAYS AFTER IRRADIATION 1-0-1 /--0.9-I I-1.25-1 1--1.57-1 1-1.82- I

GAMMA DOSE ( X lo5 R o d )

FIG. 4. Effect of the rate of application on the effect of radiation doses. Average infection grade of blue mold (Penicillium italicurn) on oranges a t 75°F. following gamma irradiation with different doses delivered a t 40 Krad/ min. and 3 Kradlmin. Redrawn from Reraha, (1964).

d. RecoveTy. I n v i f ro studies have demonstrated that , under certain conditions, the postirradiation environment can influence the dose effect, evidently by permitting the operation of recovery mechanisms. Inactivation as a response to irradiation has been reported to be reduced by holding bacteria in suspension before plating (Roberts and Aldous, 1949) ; incubation at suboptimal (Latarjet, 1943, 1934; Stapleton e t ul., 1953) o r supraoptimal (Stein and Meutzner, 1950; Anderson, 1951; Buzzell, 1956) tem- perature ; or starvation (Alper and Gillies, 1958). Alper and Gillies (1958) have suggested tha t a common feature of these procedures is their ability to slow metabolism and growth af te r irradiation. Repair of genetic radiation damage has been discussed by Sobels (1963).

Experiments with sporangiospores of the fungus R h i x o p m

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158 N. F. SOMMER AND R. J. FORTLAGE

stdot l i fer (Ehr. ex Fr.) Lind. have shown that a portion of the potentially lethal irradiation injuries can be restored to the non- lethal condition if germination is delayed for a time. The recovery involves metabolism, with the required energy supplied either by oxidative respiration or, if a glucose substrate is present, by anaerobic fermentation. When the germination of spores was in- hibited either by a lack of a medium suitable for germination or by anaerobiosis, the number of repaired spores was greatest a t a temperature near optimum for growth (Sonimer et al., 1963a, 1964c, 196Sa,b ; Sommer and Creasy, 1964).

The significance of the recovery mechanism as a factor niodify- ing the dose required for disease control has not been completely evaluated. Certainly recovery could influence the results of it/ vitt.0 studies of sensitivity to radiation. Whether the recovery mechanism is likely to operate in v i m is less certain. The number of recovered spores is greatest when growth has been prevented or slowed. The condition most likely to halt fungus growth in the host is cold storage, but the low temperature would be ex- pected to limit metabolic recovery drastically. Modification of the atmosphere to provide an elevated carbon dioxide level or a de- pressed oxygen content, or both, is sometimes employed in fruit storage or transit. Here, growth would be slowed while other conditions might permit metabolic repair. However, modified at- mospheres are used most often in combination with low tempera- tures, which would likely negate the possibility for extensive repair.

e. Preiwacliatio?z Conditions. Preirradiation conditions evi- dently may influence the dose effect as well as the environment during or after irradiation (Stapleton, 1960). The reasons f o r the different sensitivities have not been established. It has been suggested that, under certain preirradiation conditions, protective chemicals may be produced; that the number of nuclei per cell may be increased; or that after growth on a rich medium, nietn- bolic reserves may enhance the potentialities for repair.

C. MUTATIONS Irradiation treatments result in many mutants among surviv-

ing pathogens (Berk, 195213 ; Catcheside, 1948 ; Diller e t al., 1946 ; Hollaender and Emmons, 1946 ; Stapleton and Martin, 1949). Mutants are usually less vigorous than the original species. Of particular concern in connection with fruit and vegetable irradia- tion treatments are mutants which may exhibit increased radio-

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RADIATION FOR DISEASE CONTROL 159

resistance or become more vigorous pathogens as a result of mu- tation.

Variation to radioresistance has been observed in a number of studies (Clark and Frady, 1959; Hill and Simson, 1961; Witkin, 1947). Greenberg (1964) has studied the genetics of radiation resistance in Esciicrichia coli (Migula) Cast. & Chaf. There ap- pears little doubt tha t radioresistant s t ra ins of phytopathogenic fungi could appear a f te r a radiation technology is adopted. If their original level of pathogenicity should be maintained, such mutants might become troublesome. The extent of the potential problem cannot yet be adequately evaluated.

Most mutants appear to be less pathogenic than their parents (Beraha e t al., 1964) . Even a mutant with a n in vit7.o growth rate of about three times normal did not have greater patho- genicity (Buddenhagen, 1958). On the other hand, Flor (1958) and Schwinghamer ( 1959) observed mutation to wider virulence in the flax rust organism, M e l a v i p s o m Zini (Ehrenb.) Lev. Simi- larly, Day (1957) observed mutation to virulence in Cladospo~ iun i fulvzrm Cooke tested on tomato plants resistant to the original isolate.

The occasional occurrence of increased pathogenicity f rom niu- tation has usually been demonstrated in plants having a resistance introduced by a breeding program. Commonly, such plants a re resistant to certain fungus s t ra ins but not others. Furthermore, the parasitism exhibited by rust fungi is a highly advanced type which depends upon a delicate physiological balance between host and pathogen (Gaumann, 1950). In nature, new fungus races may appear as a result of mutation o r recombination, making breeding for resistance a continuing program. By contrast, f ru i t and vegetable rot disease organisms exhibit a primitive type of parasitism (Gaumann, 1950) in which the physiological balance between host and parasite is relatively less important. Although there appears to be no reason to believe tha t increased patho- genicity could not appear among postharvest disease mutants, the increased pathogenicity is likely to be much less dramatic than in the case of rusts.

Ill. TECHNIQUES FOR POSTHARVEST DISEASE RADIATION STUDIES

I t appears logical tha t studies of the fungicidal effect of radia- tion must be based on critical investigations of the radiation biology of the fungi involved. Attempts to determine the effec-

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160 rZ. F. SOMMER A N D R. J. FORTLAGE

tiveness of radiation by merely irradiating commercially har- \ ested commodities a re usually unsuccessful. At best, only quali- fied conclusions can be drawn. Often the results a re misleading.

A. Z I I V i t r o STUDIES

1. Eapei*ii?iciital M c t h o d

A number of different approaches have been used in studying the dose response of fungi t o irradiation. Since all approaches have various limitations or defects, the method must be selected with care.

Fo r in v i t w studies, the methods usually used with yeasts or bacteria frequently require modification because of the filamen- tous habit of growth of most postharvest decay species. Tech- niques used in studying chemical control of decay must be modi- fied to take into account the great penetration of the rays along \vith the frequent inability to achieve 100% inactivation a t doses tha t the host can withstand.

Some studies have been made in which fungus populations have been represented by all or par t s of mycelial colonies or by uniform loops of a spore suspension (Beraha e t ul., 1960; Nelson et nl . , 1959; Saravacos e t ul., 1962; Skou, 1960) . A common defect of these methods is the use of only one fungus population of a size which may be poorly defined. F o r example, colonies of different species might be grown on agar media in Petr i dishes. Uniform- sized pieces of aga r bearing fungus mycelium a re then removed and irradiated with various doses, and each fragment is then placed on media in a Pe t r i dish or tube slant. The dose at which nearly all, or a certain fraction, fails t o grow fur ther is deter- mined. This method is easy and rapid, but the relative sensiii:+ ties between species or conditions can usually only be approsi- mated. Since population sizes influence the probability of inactivation, accurate determinations a re needed. However, here there is usually no way of determining sizes except by measure- ments, such as diameters. Since the density of the mycelial mat l a r i e s with the species, the same size of block may not provide comparable mycelia. The same defect may occur when the same species is grown under different conditions of medium or environ- ment. Furthermore, the medium constituents would presumably influence the dose response by exerting a protective action during irradiation. The degree of protection might vary with the medium used. The medium constituents may change with age or as a

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consequence of fungus growth. If the medium is present during irradiation, the risk is added that changes in constituents might affect the physiology of the fungus after irradiation. The greatest obstacle of all, however, appears to be the absence of any con- venient method of estimating population sizes accurately.

Whole colonies in viti.0 can be irradiated with a cellophane technique, however. The medium in the Petri dish is covered with sterile cellophane approximately 1 mil thick. Commercial cello- phane may contain undesirable waterproofing additives which can be removed by soaking overnight in acetone followed by thorough washing in running t ap water. The fungus inoculum is placed on the cellophane near the center of the dish. After the desired growth, the cellophane bearing the colony may be removed for irradiation. The irradiated cellophane has not appeared to affect the fungus dose response, but for the most critical studies the fungus colony may be aseptically scraped from the cellophane before irradiation. After irradiation, the fungus colony, with or without cellophane, is placed on fresh medium in a Petri dish. To ensure that all surviving fungus cells will be in contact with the medium, the irradiated colony is then covered with a thin layer of medium cooled to ca. 45°C before i t is poured.

An obvious disadvantage of the cellophane technique is the inability to directly quantitate the fungus “tissue” using the same colonies as those irradiated. An indirect quantiation is possible, however, by relating the dry weight, protein, or nucleic acid content of similar colonies as discussed in a later section. The conditions of medium o r environment during growth must be standardized.

If a fungus sporulates abundantly, spores are the most con- venient fungus structure to use for dose-response studies. The spores are discrete individuals which can usually be easily quan- titated by counting procedures. The ease of obtaining reliable quantitative data is another reason that spores are the structures of preference when experimental requirements permit their use.

A number of limitations must be considered, however, when spores a re used. In some cases, sufficient numbers cannot be ob- tained. In other cases the spores are large and of indefinite multi- cellularity, causing difficulties in quantitating on a cellular basis. In still other cases, spores of more than one type may be present in a culture, making i t difficult or impossible to obtain uniform spore suspensions. Furthermore, the objects to be inactivated in fruit are more likely to be mycelia than spores.

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2 . EJ . 11 w s s i i i g Ex p e r i me ?i ta 1 Data

(1 . Ilosc I:cJspo?isc Crrwcs. Studies of the radiation sensitil ity of bacteria and yeast species have generally involved comparisons of the shape and slope of the curve established by plotting thc (lose against the log percent survival. Fungus spores can be studied in a similar fashion (Fig. 5 ) . Exponential survival can be explained either on the basis of a single hit to kill or a popu- lation distributed exponentially with respect to resistance to radiation, with the first possibility appearing the more plausible (Lea, 1955) . Most often, however, fungus curves are sigmoidal.

D O S E (K rad )

F I G . 5. Approximate dose response curves f o r spores of pos tharves t disease fungi . 1) Triclioderma viradc. 2 ) Phomopsis citri. 3) Penicillium italiczcm. 4) Pc?iicillium rxpaasum. 5) Penicil l ium cligitatum. 6 ) Geotr ichum candidurn. 7 ) .IZ<iitilinia fructzcola. 8) Botrytis c iner ta . 9 ) Dzplodia natalenszs . 10) Rh?zo- pzcs stolonifer. 11) Altelnaria czti i . 12) Cladospoi iuv i hcrborum. Redl awn f r o m Sommer c t ul . , (1664a,b).

A common reason for sigmoidal inactivation in fungus spores is a multinucleate or multicellular spore condition. Plat ing errors due to clumping would also result in a sigmoidal curve (Snyder, 1947). Atwood and Norman (1949) and Kimball (1953) have discussed the interpretation of multi-hit survival curves. Since the survival of discrete individuals is determined, this technique is not adapted to studies with mycelium, where cells may be ill defined. Furthermore, the technique appears to be limited to ~ T L

~ i t r o studies.

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0. Eutl-Poiirt Am1usi.s. To determine the doses required to in- activate a large percentage of each size of fungus population, a n “end-point’’ technique appears ideally adapted. Studies of the control of f ru i t disease organisms by irradiation frequently in- volve the probability of inactivating fungus populations of differ- ent sizes in host lesions by different doses. Such a technique has the advantage of being adapted to spores, mycelium, o r sclerotia. Certain types of studies can be made i?i vivo as well as i ? z vi t?o.

The “end-point” technique should permit infection levels in “orchard-run” f ru i t to be estimated and compared in terms of end-point results in experiments involving known populations. The proportion of infections inactivated by various doses would be related to population sizes behaving similarly in controlled experiments, and the field infections could be expressed in terms of “spore equivalents” or some other index of population size, a s discussed later.

In studies involving the inactivation of bacterial spores in canned foods, Schmidt e t al. (1962) and Schmidt and Nank (1960) used an end-point technique with results expressed as D values. Survival in different experiments were compared by the D values, calculated a s follows :

Radiation dose (megarad) D = - - - -~

where JI = the total inoculum; tha t is, the spores per container times the number of containers, and S Z= the number of containers not sterile, assuming one survivor per container.

D values could presumably be calculated in experiments in which known numbers of fungus spores constitute the inoculum. The technique is applicable to certain in vitro or in vivo experi- ments. However, D value calculations depend on the ability t o determine cell numbers in the inoculum, a procedure usually dif- ficult or impossible with mycelia or sclerotia.

In a variation of the end-point technique, Sommer et nl. ( 1964a,b) experimentally determined the doses required to inac- tivate every fungus cell, i?i vivo and in vitro, in populations of inocula varying in number f rom ca. 10 to 10‘. cells o r spores. Twenty replicates were used for each population size and for each dose. Fo r each population size, doses were selected so that, ideally, the lowest dose would inactivate no populations, inter- mediate doses would halt some replicates, and the highest dose would halt almost all.

Log M - Log s

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164 N. F. SOMMER AND R. J. FORTLAGE

With each inoculum size, the inactivation of populations was plotted to establish preliminary inactivation curves. Doses inac- tivating 50, 80, and 90% (or any other level) could be approx- imated by interpolation (Fig. 6 ) . The doses inactivating 80% (and sometimes 50 and 90%) of the populations were then plotted against the exponent of the population (Fig. 3 ) . With the populations and doses used, the points were experimentally determined to lie sufficiently near a straight line to permit ready interpolation between widely separated points.

DOSE (Krod)

FIG. 6. Plot of doses required to inactivate every cell in various sized popula- tions of conidia of Monilinia fructicola when each population and every dose was provided with 20 replicates. Doses inactivating 80% of the populations (C.I.D.,o) were determined by interpolation and constitute line 7 in Figure 2. Data from Somnier ct a l . , (1964a).

For i y b vit?*o end-point studies, portions of each suspension, representing the desired population, were pipetted into test tubes containing potato-dextrose agar slants immediately after irradia- tion. The incubation provided was at least two weeks a t room temperature.

For in vivo studies with spores, a portion of a suspension coil- taining the desired number of individuals was injected into each fruit with a hypodermic syringe. Fruits containing locules, such as the tomato, can be readily inoculated by inserting the needle through the locule wall. With other commodities, a hole about 5 mm deep and 5 mm in diameter was made with a cork borer. One-tenth or one-hundredth ml of a spore suspension of a con-

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centration that delivers the required number of spores was placed in the hole, which was immediately covered with cellulose acetate tape. To ensure isolation and to prevent contamination, each fruit was placed in a paper cup of appropriate size, which was tightly covered with cellulose acetate film. The atmosphere surrounding certain fruits was tested by gas chromatography to detect the possible development of conditions of low oxygen or high carbon dioxide.

Young mycelial cells of certain species have been obtained by placing the desired number of spores in a suitable liquid medium and incubating on a culture shaker until germ tubes of about four to six cells were present (Sommer e t al., 1964a,b). The germinated spores were collected on sterile filter paper, re- susnended in water in the presence of a surface-tension-reducing agent, and agitated briefly in a Waring Blendor t o disperse clumps of germinated spores. Average numbers of cells per germ tube and absence of tube breakage from blending were verified microscopically. Suspensions in appropriate aliquots were dis- pensed by pipette.

With mature mycelia and sclerotia, cellular units cannot be readily counted to provide a cellular basis for population sizes. In the Plrycomycetes the mycelium is nearly devoid of the de- liminating cross walls which permit discrimination of regular units of hypha for a “cell” count. Yet, careful studies of the radiation biology of pathogens must include the all-important my- celium as well as spores. The technique used should not only permit determination of the size of fungus populations but enable the preparation of comparable populations whenever required.

For i~ vi tro studies, fungus colonies can be grown on cello- phane. Colonies to be irradiated can be related to dry weights of colonies of similar size. The sensitivity to irradiation of several colony sizes could be determined by the end-point technique. The use of dry weight, however, poses some problems arising from the heterogeneity of the mycelium. Young mycelium near the edge of the advancing colony is metabolically active and is filled with protoplasm, while the hyphal walls are very thin. In older my- celium, in contrast, the walls may be much thicker and the proto- plasm may contain large vacuoles. In some cases, individual hyphae may become devoid of protoplasm. Therefore, on a dry- weight basis young mycelium might be expected to be much more resistant than old mycelium, and more resistant than spores if the latter have thick walls. In studies with B. cinerea, young

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mycelium was more resistant than either conidia o r old mycelium when dry weights were compared (Sommer e t al., 1965a). On a cellular basis, however, young mycelium (germ tubes) and conidia were nearly equal in sensitivity (Sommer, 1964a,b).

Since lethal radiation injury involves the protoplast, protein analyses probably provide a more fundamental basis for com- parison than dry weights. However, if the lethality is a conse- quence of injuries to the genetic apparatus within the nucleus, protein content would presumably provide a valid comparison only if nuclear numbers were uniform within the protoplasm. Analyses of deoxyribonucleic acid would presumably provide the best basis for comparison.

B. I N VIVO STUDIES

F u r i u rivo experiments, mycelial inoculum evidently cannot be used conveniently, with the limited exception tha t germinated spores provide young mycelium in the form of germ tubes. How- ever, it appears t ha t fungus colonies growing in f ru i t could be sized indirectly by comparing the radiation dose required for in- activation of the lesion with the spore population size similarly affected by the same dose. The in vivo mycelial colony sizes would then be expressed as “spore equivalents.” Reasonably uniform colonies would be required. A high degree of uniformity could presumably be achieved by giving careful attention to the number of spores constituting the initial inoculum, the incubation tempera- ture , and time af te r inoculation.

The end-point technique is particularly well adapted to studies of colony inactivation. It appears to be equally usuable where only lesion delay, not inactivation, is measured.

IV. THE NATURE AND CAUSES OF POSTHARVEST DISEASES

Fresh f ru i t s and vegetables a re living plant par t s subject t o infection by parasites at all stages of their growth, maturation, and senescence. Defense mechanisms protect living frui ts and vegetables f rom disease. Only organisms capable of avoiding or overcoming host defenses can be successful pathogens. However, a f te r death occurs, a much wider range of fungi and bacteria a re capable of colonizing the tissue.

Most causal agents of postharvest diseases a re facultative para- sites. Some species commonly exist as saprophytes, becoming parasitic only under certain conditions. With some important ex-

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ceptions the agents of postharvest diseases a re filamentous fungi f rom the Phycowzycetes, Ascom ycetes, Basidiomycetes, and Deu- teromycetes. Probably the most important exception to the near monopoly of postharvest diseases by filamentous fungi is the bac- terium Erzuinia carotovora (Jones) Holland, the cause of sof t ro t in a wide range of hosts, primarily vegetables (Smith and Fried- man, 1953).

Species of bacteria and fungi which may be in contact with f rui ts and vegetables probably number in the thousands. Yet, very few are capable of invading living tissue. Even a list of the relatively few species known to be sometimes pathogenic is de- ceptively long. The most important fungi and bacteria which are pathologically associated with f ru i t s and vegetables are well known and have been carefully studied (Anderson, 1956; Klotz, 1961 ; Walker, 1952 ; Wiant and Bratley, 1948). Most postharvest pathogens are of minor importance, because of their rar i ty , slow growth, o r control by refrigeration. As a case in point, California s t rawberry f ru i t s are usually attacked by only about five or six species of fungi. Only two of these, Botryt is cinerea Pers. ex Fr. and Rhixopus StoZozife?. (Ehrenb. ex Fr . ) Lind., commonly develop extensively a f te r harvest. Of these two, R. stolonifer is held in check by temperatures below about 10°C. (Brooks and Cooley, 1921 ; Muller, 1956) . Therefore, modern refrigeration methods, which also extend the physiological life of the fruit, effectively control this fungus. B. cinerea growth, in contrast, is slowed but not entirely stopped by lowering the temperature to about 0°C. Refrigeration is consequently only partially effective in control- ling the gray mold disease caused by B. cinerea. As a matter of fact, if' the fungus has already colonized the f ru i t extensively, as is common with strawberries, rot development may be rapid despite refrigeration. Hence, studies of s t rawberry irradiation are, in large measure, studies of the radiation biology of B. cinprcn; other species a re usually of minor importance or nonpathogenic.

The reason some microorganisms can attack the living tissue of f rui ts and vegetables while others cannot, is a complex prob- lem which extensive investigations have not yet, in most cases, explained. In some cases the living tissue simply may not meet all the requirements of a good medium for the growth of certain fungi. That this explanation is not generally applicable is easily demonstrated by the fact tha t media made from cooked or dried frui ts o r vegetables a re frequently nearly ideal for a wide range

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of saprophytic organisms tha t a re entirely incapable of attacking the living host tissue. In some cases the lack of pathogenicity may be the consequence of a failure to synthesize necessary en- zymes and toxins. However, much of the host-pathogen specificity depends upon host resistance. The basis for the resistance may be mechanical, physiological, or biochemical.

Resistance by exclusion of organisms is provided by the epider- mis of the f ru i t or vegetable. Overlying the epidermal cells is a barr ier layer of relatively inert cutin, which is difficult to pene- t ra te . Additionally, the cuticle barr ier provides a surface un- favorable for spore germination and growth because of the lack of water and nutrients. However, small openings (stomata or lenticels) through the epidermis permit the entrance of some fungi which could not otherwise penetrate the cuticle. Many pathogens a re strictly wound parasites which gain entrance only a f te r the epidermis has been cut or broken, usually during harvest or handling. Air- or water-borne spores may be lodged in the wound, or objects t ha t produce the wound may be contaminated by spores of the pathogen. Wounds a re thus a favorite means of entrance even by species whose germinating spores are capable of penetrating the epidermis. Often, however, even wounds may serve as infection courts for only limited periods. F o r example, f ru i t tisdue immediately adjacent to wounds may rapidly become desiccated, no longer providing a good infection site. JITounds may heal, a s in the suberization of potato tubers. In some husts, such a s sweet potato roots, wounds may be walled off h\- the differentiation of a cork periderm.

Most organisms placed directly within a susceptible 71 (>rind

a re unable to parasitize the tissue. Although fungus spores may germinate, growth is only limited. In some cases, host mettibolic products a re toxic to certain fungi, resulting in a physiological or biochemical resistance. Toxic products may result f rom host- parasite interaction. F o r a fuller coverage of disease resihtance and susceptibility, some excellent discussions a re available (Allen, 1959 ; Barnett, 1959 ; Butler and Jones, 1949 ; Cruickshank, 1963 ; Farkas and KirBly, 1962; Giiumann, 1950; Horsfall and Dimond, 1957 ; Tomiyama, 1963).

For the majority of postharvest fungus pathogens, the se- quence of events leading to infection and f ru i t or vegetable rot is essentially a s follows: The first step in the infection process is spore germination. Spores on the host surface swell by an uptake of water, which requires energy for a t least par t of the

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process. The protrusion and growth of a germ tube follows. In species capable of direct penetration, the tip of the germ tube may form an appressorium from which a very fine infection peg grows to penetrate the host cuticle and epidermis. Entrance may similarly be achieved through stomata, lenticels, o r wounds. Once within the tissue, mycelia branch repeatedly. Bacteria that cause postharvest diseases have no means of pene- trating the plant epidermis and must usually depend upon me- chanical or insect wounds. Sometimes, however, entrance through natural openings may be an important factor. Once inside the host, postharvest disease pathogens commonly kill cells in ad- vance of actual contact. Fungus hyphae kill and degrade host tissues by the production of toxins and enzymes (Barnum, 1924; Braun and Pringle, 1959; Cole, 1956; Cole and Wood, 1961a,b; Ludwig, 1960 ; McCalla and Haskins, 1964 ; Norkrans, 1963 ; Tomiyama, 1963; Wood, 1959). The cycle is completed when the fungus produces spores on the surface of the rotting fruit and these spores a re released and disseminated, usually by air , in- sects, or water (Butler and Jones, 1949 ; Gaumann, 1950).

Once a fruit or vegetable is intensively rotted, other fruits in contact with i t may be invaded by pathogens capable of con- tact infection. Contact infections occur by mycelium growing from the rotted into healthy fruits or vegetables, with the healthy epidermis presumably being penetrated without any need for wounds. Some fungus pathogens have a well developed capacity to grow thus from frui t to fruit , producing a “nest” of decaying fruits held together by the intertwining mycelium. Thus, one infected fruit in a container may lead to eventual loss of the entire contents.

Environmental conditions play an important role in post- harvest diseases. Spore germination requires very high humidity or free water. Disease spread could be much reduced by lowering the humidity, but the drying environment would be objection- able because of weight loss and shrivel of the fruit or vegetable. Furthermore, the humidity may be much higher at the com- modity surface than in the air of the storage room. If i t is in a fresh Ivound, the spore may be bathed in liquid regardless of the humidity of the air. Temperature, the second major environ- mental factor, influences both spore germination and growth. If f iuits are cooled to a temperature unfavorable to fungus growth, rot stops. For example, if f rui t temperatures are re- duced to O’C, all rotting ceases except that caused by a few

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cold-tolerant fungus species (Brooks and Cooley, 1921 ; Muller, 1956 ; McClure, 1958; Smith and McClure, 1960).

Particularly dificult to control are those postharvest diseases resulting from infection that may occur in the field o r orchard. Since fungus lesions are present in fruits a t harvest, protective chemical fungicides are of no avail. A good example is the gray mold disease of strawberries. Field infections by B. ciiierea normally occur during blossoming, when senescing or dying floral parts are invaded by fungus spores. The fungus spreads from the floral parts to the developing fruit receptacle. There the fungus may remain relatively quiescent until the fruit starts to ripen (Powelson, 1960). Pickers attempt to reject rotting fruits a t harvest, but some fungus lesions are difficult t o see, so rotting berries become randomly distributed among healthy fruits in the container. Then large losses may result during transit, because of the ability of the fungus to grow at low temperatures and because of vigorous contact spread and “nesting.” Protective chemical fungicidal sprays in the field have been relatively ineffective. Postharvest treatment with chemicals may not affect the fungus, because i t is growing within the fruit tissue .

V. DISEASE-CONTROL INVESTIGATIONS

A. GENERAL Although a major motive for irradiating fruits and vegetables

would be postharvest disease control, not a single disease ap- pears to have been studied in depth. Similarly, little study has been made of the radiation biology of postharvest pathogens in relation to the diseases they cause.

There are many reports of the reduction or delay of decay, but, all too frequently, no mention is made of the disease o r organism involved. The reader is often uninformed as t o the diseases being controlled, the circumstances leading to infection, whether infection occurred before or after harvest or before or after irradiation, the extent of the infections at the time of irradiation, or the extent of control sought. In some studies the more serious diseases must have been entirely missing, for the organisms reported were relatively innocuous. In most cases the experiments were based upon natural infections, which can yield meaningful results only if the nature and inci- dence of the diseases are representative of usual conditions. If

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the various important diseases a re not present in the frequency required to provide a good test, experiments of this type can yield results which lead to overly optimistic or pessimistic con- clusions as to the fungicidal value of radiation. In relation to disease control, the most important contribution of studies of this type is the determination of the maximum doses tolerated by the host species or variety. Such a dose will presumably be the one generally used for disease control. It appears abundantly clear that the optimum dose desired for pathogen control will almost invariably be higher than the host can tolerate. Thus, the radiation dose used will be determined by the host, not by the pathogen. Different host species and varieties within a species differ in radiation tolerance. Additionally, the tolerance may be influenced by the stage of ripeness at the time of t reat- ment. Since adverse effects may appear days or weeks a f te r i r - radiation, the dose tolerated may be influenced by the presence or absence of extended storage. Generally, f ru i t s destined for storage a re limited to a lower radiation dose (Maxie and Abdel- Kader, 1965).

Many f ru i t and vegetable species have been subjected to irradi- ation treatments, primarily to test the effect on the host ra ther than to study the relation of the t reatment to disease control. In some studies, the objectives of radiation were not to control disease but to control specific host responses such as sprouting or ripening. Commodities which have received the most attention or which appear to be the most likely candidates for irradiation will be briefly considered at this juncture. Fo r other discussions of the s ta tus of investigations of ionizing radiation for f ru i t and vegetable postharvest disease control, consult surveys by Clarke (1959), Dupaigne (1964), Salunkhe (1961), and Willison (1963). Attention is also called to reports by DeZeeuw (1961), Heeney ~t al . (1964), Maxie e t al. (1965) , Rubin e t al. (1959), Saravacos and Macris (1963), Tamburino (1959), Truelsen (1963), and Workman c.t a l . (1960).

1. B e w i e s

The s t rawberry appears to be the f ru i t most likely to benefit in the near future f rom irradiation as a fungicidal treatment. This is particularly t rue in California, where benefits are likely to be maximum because of special circumstances : relatively long producing periods in a single location (6 to 8 months) ; the high market value of the f ru i t ; the presence of a destructive disease

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which cannot be adequately controlled by other pre- or post- harvest treatments ; long distances to market (up to 3,000 miles by rail or 6,000 miles by air) ; and important benefits to be derived from only a few days' delay in disease development.

Probably the most important disease, by far , is gray mold caused by Botiytis cinerea Pers. ex F r . Because small lesions are already present a t harvest (Powelson, 1960), decay may proceed without the delay that would be caused by the time necessary for infection and colonization. Under refrigerated transit condi- tions the growth of the fungus is only slowed, not stopped, by the low temperatures. Furthermore, the fungus can spread vigorously from fruit to fruit by contact. Spread by conidia is relatively unimportant in harvested strawberries, because of the time required for infections to develop at low temperatures and because of the short postharvest life of the host.

Nelson e t al. (1959) reported that strawberries could not be sterilized at noninjurious doses. However, i~ vivo and i?i vitr.0 studies determined that the growth of mycelial colonies of B. cixei-ea could be halted by a dose of 200 Krep (Fig. 7 ) . At 5'C, a normal rate of growth resumed after 10 to 14 days. Contact infection and nesting were prevented for a similar period.

"- I0 1 4 - 7 2 - 26 -Yo DAYS AFTER RADIATION

FIG. 7. The effect of ionizing radiation (electrons) on the subsequent growth of B o t t y t i s cimel-a in pure culture at 3-4"C. Redrawn from Nelson, e t 111.

(1959).

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The other major disease of strawberry fruits is “leak,” usually caused by Rhixopus stolonifer (Ehr. ex Fr . ) Lind. al- though other mucoraceous species may sometimes be associated with the disease. R. stolonifer commonly infects wounds inflicted during harvesting and handling. Like B. cinerea, R. stolonif er may also infect by contact from fruit to fruit, producing an extremely rapid growth throughout the contents of a fruit container. For rapid development, however, temperatures must be warm. At temperatures near the optimum for fungus growth, i.e. 25-27”C, all strawberries within a container may be reduced to a watery residue within one or two days. Unlike B. cinerea, R. stolonifer does not grow at temperatures below about 10°C. Modern handling practices provide for quick cooling and main- tenance a t ca. 5°C or less from the grower to the housewife’s refrigerator. Under these conditions the disease cannot occur.

The possibility of using radiation to extend the marketing period in the absence of refrigeration appears extremely unlikely for strawberries. At elevated temperatures the physiological life of the fruit is extremely short, while the irradiation induced delay in fungus growth is minimal (Maxie e t al., 1964). More- over, even in the unlikely event that R. stolonifer could be completely controlled by radiation, the possibility of postirradia- tion infections must be considered because of the explosive nature of the growth of this pathogen at near-optimal tempera- tures. Prevention of postirradiation infections would likely re- quire a sophisticated (and costly) packaging program.

A third fungus species, Cladospo?.ium ke?.Dal-um Lk. ex Fr., is common in strawberries. It is not only capable of growing a t refrigerated temperatures but is radiation-resistant as well (Figs. 3, 5) . Fortunately, its growth is slow and it is evidently only weakly pathogenic. Fruit rots caused by Phzjtophthom sp. and Rhizoctoxia sp. seldom develop vigorously after harvest.

Skou (1964a) has shown that Aureobasidium pulluluns (de By.) Arnaud is radiation-resistant and might be a limiting factor with irradiated fruits and vegetables. An evaluation of this pos- sibility requires further information regarding its pathogenicity in irradiated strawberries and other fruits.

2. Stone Fruits

In America, cherry, peach, nectarine, plum, and apricot fruits are destroyed by two major and several minor postharvest diseases (Rose e t al., 1937). Brown rot, incited by Monilinia

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fruct icola (Wint.) Honey and closely related species, and K k i z o - pus stolonife,. (Ehr . ex F r . ) Lind., sometimes associated with other mucoraceous species, result in widespread losses. I n ex- tended cold storage, g ray mold caused by B. c i n e ~ e a and blue mold caused by Penicillium expamum Lk. ex Thom. may cause important damage. Gray mold has been discussed in connection with strawberries, and blue mold is discussed with pome f ru i t diseases. Cludosporium her?mmm Lk. ex Fr. is of particular im- portance in sweet cherries, and this or a closely related species of Cludosporizini may produce a serious rot of other stone f ru i t s if they a re held in cold storage for extended periods.

The brown rot disease is destructive both before and af te r harvest. Particularly during periods of wet weather, f ru i t rot in orchards may be extensive even if a comprehensive protective spray program is followed. Because of the spread in the orchard, small lesions may be present at harvest. Since the mycelium of lesions is internal, postharvest sprays or dips a re usually entirely ineffective in eradicating the fungus. Chemical t reat- ments a re sometimes applied to reduce the occurrence of in- fections a f te r harvest, however. Although small lesions may be present a t harvest, the contamination of harvest and handling wounds by spores constitutes an ever-present and important means of infection.

Growth of M . f m c t i c o l a may be extremely slow at , or halted by, temperatures below 5°C. The brown rot disease cannot de- velop under refrigeration. Ho.vever, stone f ru i t s are frequently harvested while still firm in order to limit t ransi t and handling injuries and to provide added time for marketing. When these f ru i t s are removed from refrigeration to permit final ripening, the brown rot disease is then free to develop.

If, as appears likely, the maximum permissible radiation dose for stone f ru i t s is 200 to 250 Krad (Maxie and Abdel-Kader, 1965), i t would appear unlikely tha t a large proportion of large, Lvell established colonies could he inactivated. This conclusion is borne out by a failure to inactivate a high proportion of infec- tion sites when inoculated f ru i t was permitted to incubate at room temperatures for 48 hours before irradiation or when massive inoculum was used (Sommer e t aZ., 1964a). At best, only a delay in the fur ther development of well established lesions could be expected. On the other hand, irradiation soon af te r harvest should inactivate a large proportion of very small lesions. An even higher proportion of contaminated harvest and

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handling wounds should be inactivated. I t appears almost cer- tain that i t will be highly important to accomplish cooling and irradiation as quickly as possible after harvest.

Rhizopus rot may be, under certain conditions, the most destructive postharvest disease of peaches ( Wiant and Bratley, 1948) and may attack other stone fruits as well. Particularly seriously attacked are fruits that are shipped without adequate refrigeration or fruits in which cooling has been delayed. Disease development seldom occurs at 10°C. or below (Brooks and Cooley, 1921). If fruits are shipped ripe and are refrigerated from grower to home refrigerator, Rkixopus rot should not be a problem. If fruits are harvested and marketed only partially ripe, as is common with peaches and nectarines, the disease may develop during the final ripening period. In general, how- ever, it has been noted that the incidence of Rhixopus rot is less if fruits are promptly cooled and held under refrigeration for a few days (McClure, 1958; Pierson e t al., 1958). Smith and McClure (1960) reported that, under certain conditions, merely holding inoculated fruit at O'C. for 5 days might reduce the incidence of Rhixopas rot by nearly 50%. The reason for this cold-induced reduction in disease development is not known.

The development of Rhixopus rot in market areas may be as- sociated with poor temperature management, particularly de- layed cooling or elevated transit temperatures. Presumably, how- ever, much of the Rhixopus rot may be of local origin. In markets, the incidence of Rhixopus rot of peaches and nectar- ines is often limited to very ripe fruit held without refrigera- tion. Almost omnipresent with these ripe fruits are vinegar flies, D?,osophila melunoyaster Meig. One wonders if the flies a re not inoculating the fruit with spores of R. stolonifer during ovi- posit. Inoculation and spread of R. stolonif er by spore-contami- nated U . melanoguste?, has been demonstrated in ripe canning tomatoes (Butler and Bracker, 1963). Alternatively, extensive handling and resulting injuries, particularly at retail markets, pro- vide ample opportunity for inoculation and disease development if fruits are not refrigerated.

Whether Rhixopus rot can be controlled by irradiation is still uncertain. It is true that R. stolonifer is relatively radiation- resistant (Figs. 3 and 5 ) . However, if the irradiation is per- formed promptly, the fungus population to be inactivated may consist of only a few spores contaminating harvest wounds. In such case a modest dose should inactivate the fungus in a high

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proportion of inoculation sites. However, radiation would not al- leviate and might actually increase the possibilities for inocula- tion and infection in market areas.

Among stone fruits, C. he?-bamm causes an important rot of sweet cherries grown along the Pacific Coast (Rose et al., 1937). Infection and fungus growth occur in the orchard, particularly in fruits that have cracked or in doubles in which one of the fruits has aborted. The fungus normally grows relatively slowly. The rot develops gradually in infected fruits, and a spread to other fruits during transit and marketing appears to be only a minor problem even though growth can occur at low tempera- tures. The radiation resistance of this fungus, its slow growth, and the fact that well established infections exist at harvest sug- gest that the benefits from irradiation as a fungicidal treatment to control this disease would be minimal.

3 . Citrus Fruits

Postharvest losses in Citrus fruits a r e severe from Penicilliunt itulicum Whemer (Citrus blue mold) and Penicillium d i g i t a t u ~ Sacc. (Green mold), or mixtures of the two (Klotz, 1961). In- fections generally result from contamination of harvest and handling wounds. According to Klotz (1961), the postharvest development of blue mold is directly proportional to the con- centration of spores in the air and on the surfaces of eyuip- merit. Considerable spread by fruit-to-fruit contact occurs in the case of the blue mold disease. In addition to inducing decay, both species sporulate profusely and the colored spores may be deposited on other fruits, rendering them unsightly in appear- ance. Present control measures include the avoidance of in- juries ; placing in packages such volatile materials as biphenyl- or ammonia-emitting chemicals or nitrogen trichloride-forming chemicals which tend to prevent in-transit spread and sporula- tion; or fumigation in cars or storage by nitrogen trichloride or ammonia (Eckert and Kolbezen, 1963a,b, 1964; Eckert ct al., 1963; Harvey and Pentzer, 1953; Smith, 1962).

Citrus brown rot, caused by Phytophthora spp., occurs in the orchard when motile spores are splashed from the soil to lower- hanging fruits. Small or incipient infections may be present when fruits are picked. Sound fruits may be infected by washing in contaminated water. When Citrus brown rot is a problem it is controlled in the packing house by immersing all fruits for 2-4 minutes in water or fungicide solutions at 46 to 49°C. (Klotz, 1961).

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Stem-end fruit rots a r e caused by A l t e m m i a citri Ellis and Pierce ; Diapor.tlie citri (Faw.) Wolf ; Pleospom l re rbam~n (Pers.) Rab. ; Bot)yosphao, ia ribis Dug. ; and Diplodia .iiatalensis Pole- Evans. Extensive disease development is often preceded by low vitality caused by poor growing conditions or extremes of temper- ature before or after harvest.

Other fruit diseases which may sometimes be destructive in- clude gray mold, caused by B. ciwerea; Trichoderma rot, caused by Tr ic i ioderm lignorurn (Tode) Harz; cottony rot, caused by Sclerotirhia sclerotiorzm (Lib.) Mass. ; and sour rot, caused by Geotrichum candidum Lk. ex Pers. (Klotz, 1961).

Possible use of ionizing radiation to control Citrus fruit rots was investigated by Beraha et al. (1959a,c), who gave particular attention to the Penicillium blue and green molds. Under care- fully controlled conditions, a radiation dose of 150-200 Krep pro- tected inoculated fruits against rotting by P. digi tatum for about 12 days at 75°F. and 17 days at 55°F. Results were equal or better when P. italicurn was the organism involved. No de- tailed studies seem to have been made of other postharvest Citrus diseases. However, Sommer et al. (196413) studied the sensitivity of the more important Citrus f rui t decay fungi in vivo and in uitT.0 and reported dose-response and end-point studies, mainly of spores, under various conditions.

An increased incidence of Alternaria stem-end rot following irradiation was noted by Beraha et al. (1959a,c). It was found that A. citri could be isolated from the calyces of irradiated or unirradiated fruits, but decay developed only in the former. Investigations of Maxie et al. (1965) confirmed this greater inci- dence of Alternaria rot, apparently associated with irradiation- induced death of calyx tissue and possibly with destruction of auxin (Gordon and Weber, 1955; Skoog, 1934, 1935). Since A. citr.i is very radiation-resistant (Sommer e t al., 1964b), incipi- ent lesions known to sometimes be in calyx tissue (Bartholo- mew, 1923) may escape inactivation. If the calyx tissue re- mains alive and healthy, no rot occurs. If the calyx tissue dies, however, the fungus grows into the fruit proper. The usual means of delaying calyx senescence, i.e., plant growth regula- tors, were ineffectual in prolonging life following irradiation (Maxie e t al., 1965).

4. Pome Frui ts

Apples and pears held in modern cold storage suffer large losses from several diseases, both parasitic and nonparasitic. Of

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greatest importance among parasitic diseases of stored apples is the blue mold rot, caused by Penicillium expmrsum Lk. ex Thom. and possibly certain other species of Penicillium. The fungus is capable of saprophytic growth on a wide range of decaying matter. Since the fungus typically sporulates profusely, the spores can be considered almost omnipresent. Contamination of harvest and handling wounds is a major means of gaining entry into the fruit. Infection may also occur through natural open- ings, the lenticels (Anderson, 1956).

Although P. expansum can grow at temperatures as low as fruit can endure without danger of freezing, fungus develop- ment is slow near 0°C. Consequently, if fruit is cooled to the storage temperature without delay, several months may be re- quired before the disease becomes readily apparent. If cooling is delayed, however, the spores in contaminated wounds may germinate and form small colonies before growth is slowed by the cold. After such a start, the rot lesions appear much earlier in the storage period (Ramsey and Smith, 1953).

The most serious disease of pears in storage is usually gray mold, caused by Botrytis cinerea (see Strawberries for a more complete discussion). Apples a re attacked less seriously. An abundant source of inoculum is generally present since B. ci?lerea grows saprophytically on many rotting materials in orchards and around packing houses. Infection is frequently found in stems, which the fungus colonizes before rotting the fruit proper. Prompt cooling reduces the seriousness of the disease. The fungus can grow only very slowly a t 0”C, but losses may become serious in long-term storage. The seriousness of the disease is intensified by the “nesting” which results from contact infections, with the fungus growing from one rotting fruit to infect adjacent fruits. Contact spread is frequently prevented by wrapping individual fruits in tissue paper containing copper compounds (Ramsey and Smith, 1953).

A serious storage disease, Bull’s Eye rot, caused by GLoeospoi.- ium perennuns Zeller & Childs, is sometimes prevalent in the Pacific Northwest and certain other parts of the world. In humid regions, younger branches are infected, producing a canker disease in the orchard (Perennial Canker). Frui t infec- tions may occur at any time between petal fall and harvest. Rot does not become extensive, however, until later in storage (Sprague, 1958). Rots of similar appearance may be caused by Gloeosporium albunz Osterw., G. f ructigenuin Berk., and certain other fungi.

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Scald is the name given to physiological diseases of apples and pears which develop in storage. They have in common the irregular browning of the surface and immediate underlying fruit tissue. The cause of the development of scald diseases is not well understood.

Beraha et al. (1957) reported that Penicillizcm expamzcrri inoculated into Jonathan apples and incubated for 24 or 96 hours prior to irradiation was suppressed for 10 days at 70-75°F. by 200 Krep. In later work, Beraha et al. (1961) reported that 50 Krep did not reduce P. erpaiiswm rot whereas 100 Krep re- duced day-old infections and 200 Xrep was required to check decay in 4-day old infections. The sensitivity of P. ezpansunz as determined in vitT.0 and in hosts other than apples suggests that radiation could effectively control the blue mold disease. Such a conclusion is based on the assumption that disease lesions are not already present in the fruit when harvested and that the fruit can withstand a dose of about 200 Krad. Similarly, gray mold should be readily controlled if the source of infection is primarily the contamination of harvest and handling wounds. The effect of radiation on Bull’s Eye rot appears not to have been studied.

A reduction in the incidence of apple scald has been reported (Massey et al., 1964; Phillips et al., 1960; Phillips and Mac- Queen, 1961). Similarly, another physiological disease, called brown core or core flush, was reduced by irradiation in the same studies. In other studies, core flush was variable or was increased by irradiation (Anon., 1961).

Although pathogenic or nonpathogenic storage diseases of pome fruit might be controlled by radiation, apples and pears appear to be unlikely candidates for such treatment at this time. Interest in irradiation would appear to be limited by the avail- ability of reasonably satisfactory and cheap chemical treatments, on the one hand, and the possible development of delayed irradia- tion injury in storage, on the other.

5. Bulb, Tuber, and Root Crops

The prevention of sprouting or growth by radiation treat- ments has been suggested as a means of maintaining quality dur- ing storage. Comparatively little work has been done with storage diseases of these crops. However, Beraha et al. (1959d) investigated some potato storage rots. Gamma-rays at 17.7 to 477.4 Krad did not prevent decay in Red Pontiac tubers previ- ously inoculated with the soft rot bacterium, Erzoinia ca?*otoz.om

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(Jones) Holland. Higher doses caused extensive discoloration and softening. Against the late blight tuber rot, caused by Phgtophthora iwfes tam (Mont.) de By., 45.64 Krads prevented tuber decay without injuring the tubers, but did not control natural infections of F u s a ~ i i c m sp. in the same tubers. In tubers inoculated with Pgtliiunz debaiyaiizim Hesse, doses of 137 Krad gave almost complete control under certain conditions, but a slight softening of the tubers occurred. The susceptibility of ir- radiated potato tubers to storage rots is discussed by Brownell e t al. (1957), Duncan et al. (1959), Hooker and Duncan (19591, and Waggoner (1955).

6. Tomatoes

Irradiation treatments have been reported to extend the shelf life of tomatoes by delaying ripening (Maxie and Abdel-Kader, 1965). No detailed studies seem to have been made on the use of radiation as a fungicidal treatment for postharvest diseases such as Alternaria rot (Altemaria tenuis Nees ex Corda) or Rhizopus rot (Rhixopus stolonifer) .

B. IRRADIATION-INDUCED SUSCEPTIBILITY TO INFECTIOK

Attention has frequently been called to an increase in suscep- tibility to infection and decay following irradiation. In some reports the changes followed high doses which must have re- sulted in near death or extreme injury to host tissue. Such massive, injury-inducing doses would seemingly impair host re- sistance in a manner analogous to that resulting from excessive heat or cold. From the standpoint of food irradiation, it is of primary importance to determine any increased susceptibility in hosts resulting from irradiation at near the highest dose that does not impair quality factors.

Susceptibility could be increased by a reduction in the physi- ological and biochemical resistance of the host tissue. The result would be more rapid growth of a parasite in host tissue. Further- more, some fungi that are normally weakly pathogenic might vigorously colonize tissue of lowered resistance. In addition, op- portunity for infection may be greater in irradiated hosts. Radiation-induced tissue softening might, under some condi- tions, render the fruit or vegetable more injury-prone during transit or handling. Particularly if the injury resulted in rup- ture of the epidermis, natural inoculation by contamination of the wound by the pathogen would likely result.

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Skou (1964b) suggested a n alternative possibility in which changes in cell wall pectins and increased permeability of host tissues would “provide the saprophytes with the same growth possibilities which the parasites obtain independently on un- treated material.” It is evidently assumed that the presence of exudates from host tissue would permit colonization of organisms on the surface, which would facilitate eventual penetration of the host epidermis. Cell wall changes would presumably further facilitate penetration by fungi. It would appear, however, that these manifestations of host injury a re accompanied by a seri- ously altered physiology which would also reduce the physiolog- ical and biochemical resistance of the host.

An example of radiation-induced susceptibility of a host to a weak pathogen is seen in the previously mentioned Alternuria stem-end disease of Citrus. Incipient infections in the caIyx evi- dently do not develop extensively while the host tissue remains healthy. If, on the other hand, the fruits have been subjected to adverse conditions, rotting may be extensive. Irradiation at 150-200 Krad is followed by senescence of the calyx accompanied by development of A . citri, which is now capable of growing from the calyx into the main body of the fruit. The fungus, which is radiation-resistant (Figs. 3 and 5) , presumably escaped inactivation. As might be expected, if spores of A. citri a re placed in knife wound of healthy Citrus fruits, few rot lesions develop. If, however, the fruits have been subjected to extended storage or have undergone poor growing conditions, fungus colonization and rot occur. Similarly, irradiated frui t a r e made susceptible to artificial inoculations.

Alternaria rot of tomato fruits, caused by Alternaria tenuis, seldom occurs in fruits of high vitality. The disease may appear, however, after storage at low temperature if incipient chilling injury has occurred (i.e., 0 4 ° C for several weeks). Also, after gamma irradiation of 300-400 Krad, the fruits seem more sus- ceptible to this weak pathogen.

The relatively low doses of gamma irradiation used t o inhibit the sprouting of potatoes have been reported to increase suscep- tibility to storage rot organisms. At least a portion of the in- creased susceptibility is evidently due to greater opportunity for pathogens to gain entrance via wounds. Irradiation has been re- ported to slow suberization and wound healing and thereby render wounds highly susceptible to infection for a longer period (Henriksen, 1960; Isleib, 1957; Sawyer and Dallyn, 1955, 1961).

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It has thus been amply demonstrated that irradiation may render fruits or vegetables more susceptible to subsequent infec- tion. The extent of the problem is yet unclear, for many observa- tions of increased disease incidence followed excessively high irradiation doses. More information is needed on the increased susceptibility following doses of 250 Krad or less. The diseases should preferably be studied individually, with carefully con- trolled inoculations.

VI. PROTECTIVE PACKAGING

The use of packaging to protect fruits or vegetables from postirradiation infection has been suggested by examples of in- creased postirradiation susceptibility and by the fact that irradi- ation does not leave a protective residue. Used most commonly have been bags or plastic films. Obviously, if rot organisms were completely eliminated by irradiation, a barrier would prevent any subsequent infection or rot. Seldom, however, will the high- est permissible dose even approach this ideal effect. If postirradi- ation rot results primarily from field infections that are not completely inactivated, as in Alternaria rot of Cit?*us, a protective film will be of little benefit. Similarly, the postirradiation gray mold rot of strawberries occurs primarily from lesions, estab- lished in the field before harvest, that are slowed by irradiation but not halted completely. Spores reaching susceptible infection sites may establish new infections, but the life of the strawberry, even under the best of conditions, is so short that new infec- tions originating from single spores are unlikely to be of con- sequence. Furthermore, protective packaging will not protect from contact infections and “nesting” unless fruits are packaged or wrapped individually. On the other hand, if rotting following irradiation occurs from postirradiation infections-not irradia- tion escapes-protective packaging might be helpful.

Any packaging material used must be sufficiently permeable to permit ready passage of oxygen and carbon dioxide. Other- wise, suboxidation or carbon dioxide injury to the fruit or vege- table may result from a respiration-induced atmosphere modified by reduction of oxygen or an accumulation of carbon dioxide or both. Furthermore, to be effective the barrier likely must be sealed. The use of ventilation holes in films to increase the gas exchange almost certainly destroys the protective effect. The area thus exposed is small, but the movement of bags and

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temperature changes would cause the air t o be "pumped" in and out of the bag, thereby permitting the entrance of air-borne con- tamination. Furthermore, if insects or other small animals are present, the openings permit them to enter and bring contaminat- ing organisms with them (Cooper and Salunkhe, 1963; Salunkhe c t al., 1959; Sommer and Luvisi, 1960).

VII. RESEARCH NEEDS

It is readily apparent at this time that ionizing radiation will not have universal or even, perhaps, widespread application for the control of postharvest diseases of fruits and vegetables. In many cases an adequate control can be achieved by chemical fungicides or by temperature management. In other cases the sensitivity of the host to the damaging effects of radiation (off- flavors, softening, tissue death) will not permit the application of adequate doses. Where diseases exist that cannot be adequately controlled by other means, however, radiation may permit re- duction of important losses. Furthermore, the great penetration of some rays provides a therapeutic effect not ordinarily possible with chemical fungicides. Pathogens in established lesions with- in host tissues can be controlled by this fungicidal treatment, whereas chemicals are usually only protective in nature. Conse- quently, those individual postharvest diseases which are most difficult to control by chemicals should receive particular atten- tion.

Various problems of radiation biology should receive more at- tention in relation to the control of postharvest diseases. A few of these are the following: the relation of high vs. low rates of application of a given dose ; comparisons of highly penetrating gamma-rays vs. the limited penetrating electrons with regard to the level of control and to adverse host responses; the radia- tion resistance and pathogenicity of survivors; the effect of at- mosphere modification on postirradiation disease expression ; the effect of irradiation on subsequent fruit transit injury and post- irradiation infection ; careful evaluation of the need for protective packaging; and the sensitization of pathogens by heat.

Probably the greatest need is for investigators interested in both radiation and pathology. The postharvest pathologist must be competent in mycology ; be thoroughly acquainted with post- harvest diseases ; and be knowledgeable about problems in- volved in the storage. transport, and marketing of fruits and

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vegetables. He must also be familiar with plant pathological techniques and be prepared to develop new methods as needed. Finally, an intense interest in radiobiology must be developed. If such a person is included in all research teams, faster progress can be expected.

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Heam, C. A. 1965. The influence of ploidy and division stage on anoxic pro- tection of Soccharonz! /ccs ccrt- is iuc against X-ray inactiviation. I ’ w c . S a t / . Acccd. Sei. I’.S. 11, 857-861.

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Beraha, I,. 1961. Influence of gamma radiation dose rate on decay of citrus, pears, peaches, and on Penicilliitm italicuin and Botyutis ciiicrea iir wit?,o.

Heraha, L., Ramsey, G. R., Smith, &I. A,, and Wright, W. R. 1957. Gamma radiation for possihle control of postharvest diseases of apples, straw- berries, grapes, and peaches. P / i ! / t o p a f h o l o g y 1 7 , 4.

I’ l l! / top,clf lrolog~ 31, 755-759.

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I3eraha, L., Ramsey, G. B., Smith, M. A., and Wright, W. R. 1959a. Factors influencing the use of gamma radiation to control decay of lemons and oranges. P h y t o p a t h o l o g y 49, 91-96.

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