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5-1969
Effects of Gamma Radiation in Concurrence with Certain Environmental Conditions on Lethal and Physio-chemical Responses of Penicillium expansum L.
Tsong-Wen Chou Utah State University
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Recommended Citation Chou, Tsong-Wen, "Effects of Gamma Radiation in Concurrence with Certain Environmental Conditions on Lethal and Physio-chemical Responses of Penicillium expansum L." (1969). All Graduate Theses and Dissertations. 4852. https://digitalcommons.usu.edu/etd/4852
This Dissertation is brought to you for free and open access by the Graduate Studies at DigitalCommons@USU. It has been accepted for inclusion in All Graduate Theses and Dissertations by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected]. EFFECTS OF GAMMA RADIATION IN CONCURRENCE WITH CERTAIN
ENVIBONMENTAL CONDITIONS ON LETHAL AND PHYSIO-CHEMICAL
RESPONSES OF PENICILLWM EXPANSUM L.
by
Tsong-Wen Chou
A dissertation submitted in partial fulfillment of the requirements for the degree
of
DOCTOR OF PHILOSOPHY
in
Food Science and Technology
UTAH STATE UNIVERSITY; Logan, Utah
1969 ii
ACKNOWLEDGMENTS
I wish to express my gratitude to Professor D. K. Salunkhe for
his valuable guidance and assistance throughout this investigation.
I am thankful to Professor B. M. Baker, Professor J. M. Newhold,
Professor F . J. Post and Professor H. Van Orden for serving as members
of my supervisory and examining committee and also for their suggestions
during the course of my work.
Appreciation is expressed to Professor J. M. Newhold for the use
of the gamma irradiator and Professor J. 0. Jensen for the use of the radio-
active counter.
I am grateful to Professor L. E. Olson, Dr. J. Y. Do, Dr. M. H.
Yu, . and all the wonderful people in Food Processing Laboratory, who helped me in many ways.
I am also grateful to my grandfather Pe Chou, my parents Yin-Tsai and I-Fan for their encouragement.
To my wife, Fei-Mei, I express appreciation for her patience, encouragement, and help in accomplishing this dissertation.
Tsong-Wen Chou iii
TABLE OF CONTENTS
Page
INTRODUCTION
REVIEW OF LITERATURE 4
Use of Ioniz ing Radiations in Post-harvest Storage of Fruits 4 Mechanism of Action of Ionizing Radiation ...... 6 Dose Response Curves of Microorganisms to Ionizing Radiation. 8 Factors Influencing Radiation Destruction of Microorganisms 9 Effect of Plating Media ...... 9 Effect of Age of Culture ...... 10 Recovery of Microorganisms by Post-irradiation Treatments 11 Effect of Heat Treatments ...... 12 Radiosensitizing Action of Chemicals ...... 12 Effect of Irradiated Media on the Growth of Microorganisms 13 Morphological Changes of Irradiated Fungi 14 Mutations Induced by the Irradiation 15 Pectolytic Enzymes of the Irradiated Fungi 16 Nuc leic Acids of the Irradiated Microorganisms 17 Studies on Gamma Irradiation of E_• .expansum 19
MATERIALS AND METHODS 20
Radiation Facility ...... 20 Microorganism and Culture 20 ;(. Preparation of Spore Suspension 22 Germination of Irradiated Conidia 22 Colony Formation of Irradiated Conidia 22 Age of Spores 23 Delayed Plating 24 Magnetic Treatment 24 Heat Treatment 24 Chemical Sensitizers 25 Colony Grow th from Irradiated Myce Lia 25 Media Irradia tion ...... 26 Cu lture for Pectolytic Enzyme and Nuc leic Acid Analysis 27 Assay of Pectinesterase ...... 27 iv
TABLE OF CONTENTS (Continued)
Page
"\ Assay of Polygalacturonase ...... 28 Assay of Tissue Maceration Enzyme ...... 28 Separation of RNA and DNA Fractions from Mycelium 29 Determination of Nucleic Acids ...... · .... · · · · · 30 14 Culture for RadiCl!l.ctive Nucleic Acid from Thymine-2-c . . 30 Determination of Labeled Nucleic Acids . 32 Statistical Analysis 33
RESULTS AND DISCUSSION 34
Conditions Influencing the Effects of Gamma Radiation on E_. expansum ...... 34
Germination of irradiated conidia 34 Colony form ation of irradiated conidia on different medi a ...... 34 Effect of age of spores 40 Recovery of delayed plating 42 Effect of magnetic treatment 44 Effect of he a t tre atment 44 Effect of sensiti zing chemicals 46 Growth of irradiated myce lia 48 Growth of the fungus on irr adiated media 51
Effects of Gamma Radiation on Morphological and Selected Biochemical Changes in E_. expansum 53
Morphological changes 53 Fecto lyti c en zymes of the broth culture from irradiat-ed conidia ...... 56 Nucleic acid content- of mycelium from irradiated conidia ...... 61 Nucleic acids from irradiated mycelium 64
SUMMARY AND CONCLUSIONS 69
Conditions Influencing the Effect of Gamma Radiation on E_. expansum ...... 69 v
TABLE OF CONTENTS (Continued)
Page
Changes in the Fungus Induced by the Irradiation ...... 71 General Conc lus ions ...... 73
LITERATURE CITED 74
APPENDIX 83
VITA 88 vi
LIST OF TABLES
Table Page
1. Survivals of gamma-irradiated conidi a of _P.. expansum on supplemented Czapek solution agar and potato dextrose agar ...... 39
2. Survival of.!'.· eJSPansum conidia after heat treatment and gamma-irradiation ...... 45
3. Survival of_!:. expansum conidia irradiated in combination with chemicals ...... 47
4. Growth of_!".. expansum colonies from g amma-irradiated mycel!al discs on different media ...... 50
5. Growth of_!:. expansum colonies grown on gamma-irradiated agar media 52
6. Dry weights of_!:. expansum grown in ga mma-irradiated broth medi a , after 5 days of incubation ...... 53
7. Radioactive counts and DNA, RNA contents of gamma irradiated mycelia labeled with c14-thymine. The mycelia were hiJ.tvested from shaken 50 ml Czapek Dox broth culture ...... 67
8 . The formulae of the culture media 84
9. Analysis of variance and means for colony diameter of_!".. expansum grown on the irradiated apple juice agar after 5 days of incubation ...... 85
10. Analysis of variance and means for colony diameter of _£. expansum grown on the irradiated Czapek solution agar after 5 days of incubation ...... 85 '- 11. Analysis of variance and means for colony diameter of _!:. expansum grown on the irradiated potato dextrose agar after 5 days of incubation ...... 86 vii
LIST OF TABLES (Continued)
Table Page
12. Analysis of variance and means for colony diameter of E_. expansum grown on irradiated Sabouraud dextrose agar after 5 days of incubation ...... 86
13 . Ana lysis of variance and means for dry weight of E_. expansum grown in the irradiated Czapek Dox broth 87
14. Analysis of variance and means for dry weight of E_. expansum grown in the irradiated potato dextrose broth ...... 87 viii
LIST OF FIGURES
Figure Page
l. Gamm a Irradiator (American Nuclear Corp ., Oak Ridge, Tennessee) ...... 21
2. The schematic diagram of procedure for separation of RNA and DNA from mycelium ...... 31
3. Germination percentage curve of gamma-irradiated conidia of _E. expansum on Czapek solution agar ...... 35
4 . Survival curves of gamma-irradiated conidia of _E. expansum pl ated on different media ...... 37
5. Effect of conidia age on survival curves of gamma-irradiated conidia of E· expansum plated on three different media 41
6. Recovery of 100 Krad gamma-irradiated _E. expansum conidia of 1 week o Id cu lture, he Id in suspension at 23°C and 1 °c before plating ...... 43
7. Recovery of 100 Krad gamma-irradiated _E. expansum conidi a of 6 month old culture, held in suspension at 23°c and 1 °c before plating ...... 43
8 . Growth of gamma-irradiated _E. expansum colo nies on Czapek solution agar. Irr ad iated after 1 day of incubation ...... 49
9. Growth of gamma-irradiated _E. expansum colonies on potato dextrose agar. Irradiated after 1 day of incubation ...... 49
10. Co loni es of _E. expansum formed from gamma-irradiated conidia on plating media ...... 55
11. Mutants isolated from the colonies of gamma-irradiated conidia of E· expansum ...... 57 ix
LIST OF FIGURES (Continued)
Figure Page
12. Activities of pectolytic enzymes in pectin Czapek Dox broth culture inoculated from gamma-irradiated conidia of E· expansum ...... 59
13 . Ultraviolet absorption spectra of RNA of E· expansum mycelium and yeast RNA ...... 62
14. Nucleic acid contents of E- expansum mycelia from gamma-irradiated conidia incubated in shaken Czapek Dox broth ...... 63
15. Relation of RNA content and dry weight of mycelia grown from gamma-irradiated conidia of E· expansum 65 x
ABSTRACT
Effects of Gamma Radiation in Concurrence with Certain
Environmental Conditions on Lethal and
Physio-chemical Responses of
Penicillium expansum L.
by
Tsong-Wen Chou, Doctor of Philosophy
Utah State University, 1969
Major Professor : Dr. D. K. Salunkhe Department: Food Science and Technology
!!! vitro investigations were conducted to study the leth a l effect of gamma radiation on the fruit spoilage fungus Penicillium expansum under certain environmental conditions, and to study some physio-chemical changes in the funguli! induced by the radiation which may be related to the death of the fungus or its ability to invade fruits.
The radiation sensitivity of]:. expansum conidia was influ enced by such factors as nutritional condition of the post-irradiation media, age of the spores, delayed plating, heat treatment, and chem ical sensiti zers. Higher survival was obtained when irradiated conidia were plated on Czapek so lution agar (sugar-salts medium) than on potato dextrose agar; yeast extract added in Czapek's medium hastened colony growth but reduced survival. The 6-month - old conidia were more radiation sensitive than the 1-week-old conidia. Recovery xi
from the radiation injuries was observed when irradiated spores were held
in the suspension for several days at 23°c. No recovery was found at 1°c.
Heat treatment (58°C; 4 minutes) before or after irradiation increased the
radiation effect. Difolatan (10 ppm), iodoacetic acid (50 ppm), and second ary
butylamine (500 ppm) were effec tive radiation sensitizers to]:. expansum. The
co lony growths were inhibited for various periods following irradiation of the
mycelia. The duration of inhibition was influenced by the dose app lied and the nutrient of the medium tested. Irradi ation of the Czapek solution agar slowed the initial growth of unirradiated fungus. No such effects were found with the other media tested. From these results, it can be concluded that under proper cond itions, the fungus can be effectively inactivated by low doses of gamma radiation and thus retain the quality of fresh fruit with a minimum irradiation cost.
Abnormal colonies and mutants were induc ed by the radiation. Some mutants were no more radiation resistant than the normal strain. The culture grown from irr adiated conidia produced more of pectolytic enzyme than did the control after 3 days of post-irradiation incubation. The enzyme production was stimulated by the irradiation.
Nucleic aci d content of the mycelia grown from irradi ated spores was related lo the growth stage of the fungus and not directly influenced by the irradiation. The slowed growth rate of the irradiated fungus indirectly influ enced the nucleic acid content. When isotope labeled mycelia were irradiated , no radi at ion induced degradation of nucleic acid was detected in the fungus xii during 8 hours of post-irradiation incubation, either by chemical or isotopic analysis.
(101 pages) INTRODUCTION
Many millions of tons of fresh fruits and vegetables are lost each year in the world as a result of post-harvest spoilage. In the United States alone about a quarter of a billion dollars worth of fruits and vegetables was estimated to be lost annually after harvest and before reaching the consumer
(U. S. Department of Agriculture, 1965) . Major post-harvest deterioration is the result of the action of the microflora present on them . One of the common types of microbial spoilage in horticultural crops is caused by
Penicillium species. Penicillium expa nsum is the most prevalent and destruc tive fungus commonly found in fruits such as apples, cherries, grapes, peaches, etc . (Raper and Thom, 1949).
Recently, the use of ionizing radiation as a method of food preservation has received considerable attention. Application of radiation to food may be achieved by either complete sterilization of a ll microorganisms by employing high doses or reduction of spoilage microbes by low dose pasteurization. Com plete sterilization of fresh fruits and vegetables is not feasible, for being living matter, they are susceptible to radiation injury. Quality attributes such as appearance, color, aroma, flavor, texture and some nutritive values will be damaged by the treatment with sterilizing doses. Therefore, the use of ionizing radiation in the post-harvest decay control depends upon the radiation sensitivity of causal microflora compared with the radiation tolerance of the 2 host. For this purpose, studies elucidating the physiological and biochemical changes occurring in the microorganism following radiation are necessary to provide a basis for practical app lic ation in the field of food preservation.
Gamma irradiation of fresh fruits to inhibit or destroy the microflora and thus prolong their storage life has been reviewed by Salunkhe (1961) , and
Sommer and Fortlage (1966). The applicat ion of gamma radiation to inhibit the growth of E_. expansum on apple (Ber aha~~-, 1957; 1961) and i!! vitro study of the fungus (Beraha ~ ~. ,, 1960, Sommer ~ ~-, 1964) have been reported.
However, on ly meager information is available concerning effects of gamma radiation on E_. expansum particularly with regard to i!! vitro study of letha lit y and the accompany ing physiological changes caused by irradiation .
The primary objectives of this investigation were to study the lethal effects of gamma radiation on E_. expansum as influenced by environmental factors; and to s tud y the changes in the fungus induced by the radiation, which may be related to the death of the fungus or its ab ilit y to at tack fruits. The surviva l curves were obtained for irradiated spores from germination percent ages and colony formation. Some factors affecti ng lethality of irradi ated conidia such as plating media, age of spores, delayed plating, magnetic treat ment, heat treatment and chemical sensitizers were investigated to provide inform ation concerning feasibl e conditions to control the fungus at the pasteur i za tion doses of irradiation . Inhibition of growth of irradiated myce li a on different media was determined. Since fruits will be irradiated for pasteur iza tion , an expe riment was conducted to find out the effects of irradiat ed media 3 on the growth of fungus. The changes in characteristics of the fungus which resulted from Irradiation, such as morphology, activity of pectin-hydrolyzing e nzymes, and RNA and DNA contents were also investigated. Pectolytic enzymes are the main agents of the fungus to invade its host fruits. Any change in the enz yme activity could change its pathogenicity . Since the nucleic acids play an important role in the process of heredity and protein synthesis, there fore, an attempt was made to relate the nucleic acid contents and the death of the cells . 4
REVIEW OF LITERATURE
Use of Ionizing Radiations in Post-harvest
Storage of Fruits
The existence of ionizing radiation was recognized when artificially
produced radiation called X-ray was discovered by Roentgen in 1895 and natural
radioactivity was discovered by Becquerel in 1896 . It was found later by several
investigators that there are three kinds of ionizing radiation from radioactive
element, namely alpha and beta particles, and gamma rays or electromagnetic
waves . These are referred to as "ioni zing" radiations because they are capable of ionizing the materials they pass through. Of these ionizing radiations, alpha par ticles are not very useful in food processing because of the lack of sufficient penetrating ability. Only cathode rays (analogous to the beta ray, artificially accelerated electrons with higher energy), X-rays and gamma rays (similar in nature but of different origin) are of major interest in food processing.
Bactericidal and fungicidal properties of ionizing radiation were studied soon after the discovery of radioactivity . The early investigations of the biological effects of ionizing radiation on bacteria were reviewed by Duggar
(1936) and on fungi by Smith (1936) . Between 1896 and 1898, several investi gations failed to prove bactericidal activity of X-rays. Rieder, in 1968, reported decided lethal X-ray effects upon bacteria. The following year, Pacinotti and 5
Pore e I Li succeeded In killing several species of bacteria by exposure to uranium radiation (Siu and Mandela , 1957) . Pichler and Wober in 1922 reported th at X-ray treatm e nt killed smut fungi which had infecte d whea t and barl ey seeds (Siu and Mande ls, 1957) . Th e first paper which attempted to develop a practical technology for perishable commodities was giv en by
Brasch and Huber (1947).
The explosion of atomic bombs at Hiroshim a and Nagasak i in 1945 was a hum an traged y. Afte r the war peopl e sought pe aceful us e s of atomic energY, which became poss ibl e with the advances in man's knowledg e of the atom. From about 1950, waste fission products were beginning to accum ulate in the nucl ear reactor oper ation ; resea rch in utilizing these rad ioactive products bec ame popular. The potenti a l us e of gamma ray emitting isotopes in preservation of foods appeared promising . Sinc e then numerous res ea rch pape rs r e late d to this area have bee n publish ed.
Des rosi e r and Rosenstock (1960) r ev iewed some of the technical problems involved in the irradiation of foods, including fruits ; effects on microorg anisms were also disc us sed . An ex cellent review concerning effects of irradiation on the quality attr ibutes of fruits has bee n pres ented by Max ie and Abtlel-Ka der (1966) . Invest igations related to post-harv est disease control off ruits were reviewed by Clarke (1959), Salunkhe (1961) , and Sommer and Fortlage (1966).
Many inv estiga tors agreed that since fresh fruits and vegetables are li ving matter , complete sterilization is not feasible . Undesir ab le damage such 6 as changes in color, a roma , tex tur e, and nutritiv e va lue would result from too high r ad iation doses. In relation to post-harvest disease control, the rad iation dose applied would be limited by the tolera nce of host crops . It has been found that radia tion tol erance of fruits was influ enced by species, var ieties, and stage of ripeness. Sa lunkh e (1961) stated that th e radiation dose required for pasteurization a lso de pends upon the numbers and kinds of microbi a l types p1·esent on a given product prior to irr ad iation . However, in gene ra l, he found th at a dose of 200 to 30 0 Kr ad could extend significantl y th e storage life of fruits without much loss in quality. Desrosier and Rosenstock (1960) concluded that s ur fa ce contami nation of organisms would be drastically reduced or completely e limin ated by the dose of 100 to 200 Krad. Maxie and Sommer (1964) concluded that tex tu ra l changes would like ly be the factor limiting the appli cat ion of ir rad iation as a practical tec hnolo gy for fruits, and 225 Krad would be the maximum dos e that most fruits could tolerate without unacceptab le suscep tibilit y to transit injur y.
Mechanism of Action of Ioni zing Radiation
Th e knowledge of the effec ts of ioni zing radiation in living s ystems is still fragm ent ary . Some of the recent reports pertaining to studi es of radio biological mech anisms were by Bacq and Alex ander (1961) , Errer a and Forsberg
(1961), Harr is (1961) , Jenkinson (1963), Lea (19 55), Romani (1966), and
Tischer and Kurtz (1957).
Ioni zi ng radiations in passing through matter lost their energy by transferring it to electrons, thus producing electric a lly excited or ioni ze d 7 molecules. Chemical effects follow and in most events resulted in the formation of free radicals which may combine with each other or react with other chemical species of the medium. Molecular changes caused by abs orbed energy produce injuries to living cells. The direct act ion theory states that damage is caused by direct impact of an ionizing particle or wave at a sensitive site in the ce ll responsible for the radiation effect. The indirect action theory states that ionization produces chemical changes, such as formation of highly reactive free radicals from water and that these react with ce LLconstituents, resu ltin g in cell injuries . The indir ect theory allows for somewhat greater flexibility and appears better able to accommoda te such observations as effects of chemica ls , of changes in oxygen co ncentr ation, etc. Pamper (1965), and
Tischer and Kurtz (1957) exp lained by combination of the two theories which reta ins the feature of the direct targ et theory of a sensitive site and the indfr ee t theory of radiation induced chemical eve nts , which in turn affect sensitive sites.
The injury may be a direct damage to genetic material, producing mutations which may or may not prove leth al. Molecular changes may cause biochemical lesions which are developed or intensified by metabolic processes of th<> ce ll. Biochemical les ions may affect vital genetic synthesis resulting in a mutation, or may affec t nongenetic processes and result in an altered physi ology. Both results, if sufficiently severe, m ay also prove let hal to the cell
(Sommer and Fortlage, 1966). 8
Dose Response Curves of Microorganisms
to Ionizing Radiation
Survival curves of microorganisms have been obtained to relate
the number of survivors and the radiation doses. The shape of the dose
survivor curve on a semilogarithmic paper may be either linear or sigmoid,
depending upon the strain and species of organism studied, growth phase of
the cells, nature of the radiation, and other factors (Rayman and Byrne, 1957).
Many bacteria and haploid strains of yeast are inactivated exponentially
while polyploid yeast and some fungal spores are inactivated sigmoidally. The
exponential curves of inactivation by ionizing radiation are similar to those of
thermal death curves of bacteria, and are in favor of the target theory that a
hit on sensitive target site may cause the death of the cell (Lea, 1955). The
sigmo ida l curves may be explained by the theory that a number of hits are
required to cause the death of the cell, or there cou Id be a number of sites
in the cell that must be inactivated (Atwood and Norman, 1949). The mitotic
stage, the ploidy and the number of nuclei per cell may play an important role
in survival curve response.
There is a lso a third type of survival curve which is a convex shape.
The inactivation of haploid yeast cells was logarithmic in part, but then
"tailed" off as though a portion of the population were radio-resistant. The resistant fraction was shown to consist of cells in the process of budding
(Beam~~- , 1954). 9
Factors Influencing Radiation Destruction
of Microorganisms
According to Desrosier and Rosenstock (1960), the extent to which an
organism is sensitive to radiation is dependent upon the following general
factors : presence of oxygen , presence of water, sporulation , species, and
different strains of the species , population of organisms, atmosphere in which
they are irradiated, physical state of the medium, hydrogen ion concentration,
composition of medium, age of culture, type of radiation, and post-irradiation
treatment.
There are many factors influencing the effects of ioni zing radiation on
fungus. More thorough understanding of these factors is very important in post
harvest disease control.
Effect of Plating Media
The survival of some irradiated bacteria is influenced by the post
irradi ation medium composition . Stapleton~ _:g.(1955) have shown that sensi
tivity of Escherichia coli B/r cell to ionizing radiation depended on the type of
medium used for growth prior to irradiation as well as type of plating medium
employed after irradiation. Cells cultured in nutrient broth resulted in a smaller surviving fraction when plated on a simple synthetic medium than when plated on a complete medium . However, greater survival was observed when cells were grown in minimal liquid medium before irradiation and plated afterwards on a minimal agar medium. 10
Alper and Gillies (1958b) r ep orted that different plating media gave rise to widely variable viable counts, and the surviving fraction was smallest on media which were optim al for the growth of unirradiated organisms. The y concluded that inhibition of colony formation was due in part to an injury which lea ds to imb a lanc e in the synthetic process of the ce LL, so that "recovery" or
"restoration" ma y be brought about by sub-optimal conditions of growth.
Adler and Engel (1961) reported that the X-ray sensitivity of~- coli
B/r was altered by post-irradiation plating conditions and was sm a ller for cells plated on a synthetic minimal medium than on a complete medium.
Lee and Sinnhuber (196 5) showed that vi ab le counts of~ · coli Bon glucose-salts agar was higher than on peptone agar although the organism was grown in pepton e broth.
Myasnik and Korogodin (1968) reported that survival of irradiated
~ · coli B pl ated on various media differed greatly. High correlation was found between the survival of irr adiated cells and the viability of nonirradiated cells plated on the different media of various temperatures. Thefr conclusion " was that variation in the Leve ls of survival of irr adiated cells may be due to a selective effect of the medium . However, they suggested that both recovery effects and selective effects may be "at work."
Effect of Age of Culture
Radio sensitivity of cells changes with age of culture. Stapleton
(1955) showed that resistance of~- coli increased in the lag phase of growth 11
to reach a maximum at the end of this phase, and increased sensitivity to
irradiation during the logarithmic phase, followed by a gradual recovery of
resistance in the stationary phase.
Fungal conidia of the strains of 6-month-old Aspergilus flavus and
Penicillium viridicatum cultures were more susceptible to irradiation than
3-week-old conidia (Malla.
Recovery of Microorganisms by Post-irradiation Treatments
It has been show n that survival of microorganisms was influenced by
post-irradiation treatment. The means used to enhance recovery of irradiated
bacteria are incubation at sub-optimal (Stapleton.
(Anderson, 1951) temperature, holding in suspension before plating (Roberts
and Aldous, 1949), and starvation (Alper and Gillies, 1958a). Alper and
Gillies (1958b, 1960) have suggested that a common feature of these procedures
is their ability to slow down metabolism after irradiation, and thus preventing death due to "u.nbalanced growth." With yeast, a great increase in viability was observed when plating was delayed (Patrick and Haynes, 1964; Bacchetti
.'!.!~·, 1966).
Beraha .
Exper im ents with sporangiospores of the fungus Rhizopus stolonifer have shown that a portion of the potentially let hal ir radiati on injuri es was 12
restored to the nonlethal condition when germination was del ayed for one or
two days. Th e recovery invo lved metabolism , wit h the requir ed ene rg y
sup pli ed either by oxidative resp ir ation or , if a glucose substrate was present,
by anaerobic fermentation. When the germination of spores was inhibited
e ither by a lack of a medium suitable for germination or by anaerob iosis, the
number of repaired spores was greatest at a te mpe r ature ne ar optimum for
gr owth (Somm er and Fortlage, 1966) .
E ffect of Heat Tr ea tm e nts
Kempe (1955) and Morgan and Reed (1964) found after ionizing irr adi
a tion , bacter ia became mor e se ns itiv e to heat treatment, but there was no effect
of prior hea ting on radiation inac tiv ation. Huber~ .,il. (1953), Kan~ .,il. (1957)
and Duggan~ .,il. (1963), on the other hand , observed that he ating before exposure
in creased se nsitivit y of bacteria to irradiation.
Somm er~ ~· (1967) obs erve d radiation-h ea t synergistic effec t on
Monilinia fructi co la, Botrytis c ine r ea, Cladosporium he rbarum , Rhizopus
stolonifer and Pen icillium expansum. With B_. stolonifer, the max imum
e ffec t occurr ed when irradi ation was applied first. In a ll oth er sp ec ie s studied,
th e reverse sequence resulted in the greatest inac ti vat ion.
Radios ensiti zing Action of Chemica ls
While som e chemicals are known to protect microorganisms from the lethal effects of radiation, som e chemica ls are reported to reduc e r adiation 13 resistance of spoilage organisms . Vi tamin K and related compounds (El 5 T abey Sheha ta, 1961; Noaman tl !!_!., 1964 ; and Silverman tl !!_!., 1963) , iodo aceta mide and iodoac e tic acid (Dean and Alexander, 1962 ; and Lee tl!!.!·,
196 3) and p-aminoph enol (Noam an tl !!_!., 1964) have been found to increase radio leth a lit y of bacteria. Vitamin K (El - T abey Shehata, 1961 ; and Silv erman 5 tl!!.! · , 1963), and ma le ic ac id (Kig atl!!_! . , 1955) sensitized yeast.
Georgopoulos tl !!_!. (1966) reported that iodo ace tamid e was a very e ffect ive radiosensitizer for spores of Penicillium it a li cum, Aur eobasidium pullul'ans , Rhi zopus nigric ans and Botrytis cinerea; and 1-naphtol reduced resistance of b_. pullulans and Can did a tropicalis to cons iderab ly low leve ls .
The combined action of diphenyl and radiation was reported to effective ly inhibit the growth of the citrus decay fungi Diplodi a nata lensis, Penicillium digitatum, Pe nicillum italicum and Tri chod erma viride !!!_v itro (Barkai-Golan and Ka han , 1967).
The radio-sensit izing e ffec t of chem ic a ls is highl y desirable for radiation past e uri za tion of fruits and vegetables where high radiation doses may cause damage to host product.
Effe ct of Irradiated Med ia on the Grow th
of Microorganisms
Fields tl !!_!. (1960) conducted a study on fungal growth on irradi ate d food components to rela te incr ease d susc ep tability of irradiated food to fungal deca y. The growth of Aspergillus ory zae was found to be significantly better 14 on irradiated glucose-asparagine and glucose when these materials were 4 present in growth media. Inhibition of growth at highest dosage (392 x 10 rads) was observed when the fungus was grown on irradiated starch . Fields
(1959) also found that irradiated Czapek Dox broth inhibited the growth of fi. oryzae .
Frey and Pollard (1966) reported that an irradiated medium caused a halt in~- coli cell growth. Growth was restored by catalase. Irr adiated medium also caused a decrease in the incorporation of thymine, pro line and valine into unirradiated cells of~ - coli. Reduction of ~ -galactosidase formation was observed, but there was no degradation of DNA (Pollard _Etl!!!_., 1964).
Morphological Changes of Irradiated Fungi
Sommer and Fortlage (1966) summari ze d the effect of ionizng radiation on morphology of pathogenic fungi. When irradiated spores were plated on a medium for germi na tion, the diameter of the germ tubes was often much larger than normal. Frequently grotesque swe !lings occurred _at various places in the mycelium. The germ tubes often grew only a shor t distance and then rounded up at the end to produce an abnor ma l germinant resembling a "dumbbell;" or grew extensively and branch ed, then stopped development, after which death follow ed. The amount of germ tube deve lopment is evidently inversely related to the dose applied. Characteristically, cross walls were almo st totally lacking in species that normally form regular and prominent wa-lls in the germ tubes. 15
The absor ption of energy causing lethal damage cou Id be separated
from death by ma ny hours. If irr adiated spores were stored in an environ
ment not conducive to germination, the time between energ y absorption and
death may be extended to a number of days . During that time respiration ,
the production of certai n e nzymes, and other metabolic functions are known
to proceed at a normal or an accelerated rate.
The ability of the irradiated spore of the fungus to from a colo ny
capab le of indefinite grow th is lost at a much lower dose than the abi lity to
germinate (Beraha _tl ~ · , 1959a , 1959b; and Som~er ~ ~·, 1963a) .
When co lonies were irradi ated at a dose insufficient to inactivate
permanently, growth was ha lted temporarily (Beraha _tl ~-, 1959a , b ; and
Nelso n tl ~ - , 1959), to be resum ed after a delay which amou nt s to severa l
days . It is like ly that most of the myc e li a have been irreversibly injuried .
Only certain portions of hypha may, with time, recover. Essen ti a lly normal
grow th th en occurs from loc a li zed ar ea (Somm er and Fortlage, 1966).
Mutations Indu ced by the Irr ad iat ion
Radiation is an agent known for its ability to cause geneti c cha nges
in biological material. Mutation production usu a lly incre ases when dose incre ases. Irr adi atio n tre at ment resulted in many mutants among surviving pathogens. Genetic changes appea r ed in man y ways, either morphologically or physiologic a lly.
Barron (1962) in his studies of par ase xua l cycle of E_. ex pansum found that ther e were morphologi cal mutant s and nutritiona l mut ants produced 16
by radiation treatment. The morphological mutants were related to the
pigmentation of conidia such as white, brown, olive and pale blue . Most of the nutritional mutants were satisfied by either a mino ac id or vitam in supple
ment 1 and only a few responded to the nucleic acid supplement. Although an
increase in dose resulted in an increase both in the numbers of abnorma l colonies and mutants, the nutrition a l mutants in the normal-appearing
survivors from the gamma-irradiated stocks never exceeded 1 percent .
A radiation resistant mutant was observed in §_. co Ii and stud ied extens ivel y (Greenberg, 1964) . Radiation resistant strains of pathogenic fungi ma y appear and cause some trouble when radiation pasteuri za tion is wide ly app lied , because ; they may not be killed effective ly at normal pasteurization doses. Most mutants appeared to be less pathogenic than their parents
(Ber aha fil.~ - , 1964). Even a mutant with a gre ater i.!!vitro growth rate did not have greater pathogenicity (Buddenhagen , 1958).
Pectolytic Enzymes of Irr adia ted Fungi
Decay in plant tissu e is assoc iated with, and possibly dependent upon, maceration by hydrolysis of the insoluble pect ate of the middle lamel la .
~- expansum was found to produce highly active pectic enzymes in rotted app le tissue or culture filtrate (Cole and Wood, 1961).
The abi lity of spores of Rhizopus stolonifer to produce pectolytic enzymes was found to be more radiation resistant than the potential for colony formation or the ability to germinate. Spores made incapable of 17 forming colonies by irradiation continued to produce pectolytic enzymes after a 6-day period following irradiation tr eatme nt (Sommer!!.!:!!! · , 1963b).
Nucleic Acids of Irradiated Microorganisms
DNA, taking an important part in the synthesis of proteins and in the process of heredity, is tqe candidate for principal target of lethal radiation effec ts in most unicellular systems. Irradiation of DNA in !'queous solution resulted in the changes in viscosity , sedimentation, and light-scattering , indicating degradation of DNA molecules (Gino za, 1967) . J The damage of DNA could be as follows: (A) Structural damage result- ing in major distortion of the DNA structure a nd change in its topology. Most often breakage of phosphodiester bonds with resulting single- or double-ch ain scission, or breakage of covalent linkage of the complementary strands, bind- ing to protein or other macromolecul ar ce ll components, and other changes affecting large regions of DNA. (B) Change in information caused most often by more subtle radio-chemical modification of individual DNA bases. This modification could lead to (1) inconsequenti a l changes, (2) major missense or fraudulent information and (3) complete nonsense (Szybalski, 1967).
It is generally recognized that the amo unt of the degradation of DNA required for a genetic effect is so little that it is probably undetectable by norm a l chemical analysis (Desrosier and Rosenstock, 1960 and Jenkinson ,
1963) .
One of the observations on r adi ation induced DNA degradation in 18
the microorg anism cell was made by Stuy (1961) . He observed considerable
DNA degradation in Haemophilis influenzae following X-irradiation. The
cause was considered to be the activation of cellular deoxyribonuclease . Since
the br ea kdown was fol lowed by an increased synthesis of DNA, he concluded
that it is not an important cause of cell death. Drakulic and Kos (1963),
Miletic ~ gl. (1963) demonstrated radiation induced DNA degradation in a number of strains of £e.·coli. The effect of oxygen in the pre-irradiation medium on the degradation process was demonstrated by Pollard and Achey
(1966) . Shaffer and McGrath (1965) found th at the amount of degradation among various cells in the population was simil ar, not restricted to those cells destined to die. Cells heated 70° C for 10 minutes before showed no DNA degradation after irradiation (Pollard and Achey, 1966). The degradative loss of DNA is temperature dependent, which suggests that an enzymatic process is associated with degrad a tion (McGrath ~gl ., 1966; and Achey and
Pollard, 1967). The amount of DNA degrad ation was proportional to the dose up to a certain maximum . With regard to the relation between dose and number of survivors, it could be argued that the phenomenon of DNA degrad ation is central to the killing of bacteria. On the other hand, it could be argued that
DNA degradation is a protective mechanism in which the cell discards a genome that is now useless because of radiation damage and preserves a genome that is intact (Achey and Pollard, 1967) .
DNA synthesis was inhibtted after exposure of bacteria to X-rays
(Doudney , 1956 and Miletic ~gl . , 1961), RNA synthesis was depressed in 19 irradiated cells of~ · coli Bir (Frampton , 1964) and part of the RNA in an irradiated culture of~· coli B was beginning to breakdown after 30 minutes of incubation (Petevsky and Mi leti'c, 1966).
Studies on Gamma Irradiation of E· expansum
The application of gamma radiation to Penicillium species on apricots, peaches , pears, strawberries and sweet cherries to depress their growth was successfully demonstrated by Cooper and Salunkhe (1963) , Dhaliwal and Salunkhe
(1963), and Salunkhe (1961). Penicillium species were found more sensitive to radiation than other species of mold.
Beraha tl_ !!.!, (1957) reported that E· expansum inoculated into apples and incubated for 24 or 96 hours prior to irradiation was suppressed for 10 days at 70-75° F by 200 Krad . Beraha tl_ !!.!· (1961) reported that 50 Krad did not reduce E· expansum rot whereas 100 Krad reduced day-old infections and
200 Krad was required to check decay in 4-day old infections.
An.!.!! vitro study of gamma sensitivities of E· expansum along with other decaypattiogenswas reported by Beraha tl_ !!.!· (1960) . The dose response of the selected Prunus fruit decay fungi to inactivation of their colony-forming potential by gamma irradiation was studied by Sommer tl !!.!· (1964).
Sigmoidal inactivation at the low doses was observed for those fungi. Conidia of E· expansum were the most sensitive among the fungi tested. Synergistic effects of combined gamma radiation and heat treatment on colony-forming ability of E· expansum were reported by Sommer tl !!.!· (1967). 20
MATERIALS AND METHODS
Radiation Fac:ility
The irradiator used as a radiation source (American Nuclear Corp. 37 Oak Ridge, Tennessee, Figure 1) was loaded with 1306 . 8 curies of Cs 1 , and was arranged in such a manner as to give an even flux of gamma radiation dose in the 30 x 29 x 10 cm irradiation chamber. The dose rate as measured by Bausch and Lomb glass rod was 345 roentgen/ per minute in January , 1966 (Sharma , 1968) , and the spore suspension absorbed approxi mately 20 Kract2 per hour. The irradiation temperature was about 20 to 24° C.
Microorganism and Culture
A strain of Penicillium expansum, isolated from a blue mold rot in apple, was selected for the experiments. The stock culture was maintained on potato dextrose agar slants. All media were prepared from Difeo dehydrated products (formula in Appendix).
1The roentgen (r) is the quantity of electromagnetic radiation which produces an electrostatic unit of charge of either sign per cubic centimeter of dry air at o° C and at standard pressure. , Solely for exposure doses. 2 The rad is the quantity of ionizing radiation which results in the absorptiOJl of 100 ergs per gram of irradiated material at the point of interest. Solely for absorbed doses and independent of the kind of ionizing radiation used. The energy of cs 137 gamma ray is 0. 66 Mev, and at this energy level water absorbs 0. 97 rad from 1 roentgen of the exposure dose. 21
Figure 1. Gamma Irradiator (American Nuclear Corp., Oak Ridge, Tennessee). Radiation source: cs 137. Irradiatio n c hamber: 30 x 29 x 10 cm. Dos e rate was 345 roentgen per minute in January, 1966. 22
Preparation of Spore Suspension
Conidia harvested from cultures grown on Czapek solution agar for
1-2 weeks were suspended in sterile distilled water and filtered aseptically
through a layer of glass wool (about l cm thick) to eliminate aggregated
spores and hyphae . The suspension was, except when otherwise stated , 6 then adjusted to 10 spores per ml with a Levy and Levy-Hauser improved
Neubauer Ruling counting chamber .
Germination of Irradiated Conidia
Pyrex test tubes (1. 2 x 10 cm) , covered with aluminum foil (0. 001 cm
thick), each containing 4 ml of spore suspension were irradiated at different
doses, from 20 to 350 Krad. Following irradi ation, 2 ml of spore suspension
was poured onto apreparedC zapek solution agar petri dish. When the spores
had settled on the agar surface, the surplus supernatant was discarded . The
plates were incubated for 20 to 40 hours at 23°c, then 5 randomly selected
areas on each plate were observed under the low power of a microscope.
Sufficient fields were counted in each area to give 100 spores and the relative
germination percentage among these spores.
Colony Formation of Irradiated Conidia
To determine the effect of treatment on the ability of conidia to produce colonies , test tubes containing l ml of spore suspension were irradiated at various dose leve ls , then diluted with a known volume of sterile distilled 23
water and plated within 2 hours after irradiation on the following media:
Czapek solution agar (pH 5. O with NCI (CA)) , potato dextrose agar (PDA) ,
Sabouraud dextrose agar (SDA), Sabouraud ma ltose aga r (SMA), and
nutrient agar (NA), Colonies were counted after 2 days of incub ation at
23°C, except CA, where coloni es were counted after 3 days of incubation.
In one experiment , O. 4 percent of Difeo potato extract or Difeo yea st extract
was added to CA. and colony counts were made. The platings were also
made on CA and PDA for the comparison.
Since only a small fraction of spores formed colonies after the irradi- ation, the s urv ival was expressed as a fraction, and calculated as
N Surviving fraction = ~ where
N = number of colonies per ml of irradi ate d spore suspension
No= number of colonies per ml of unirradiated control.
Age of Spores
To determine the effect of the age of spores on their response to irradiation, a CA culture was kept for 6 months at 23° C. Then the spore suspensions from this culture and also of 1- to 2-week-o ld conidia were irradiated at several doses, and the survivals were calculated as above.
CA, PDA and SDA were used for plating. 24
Delayed Plating
To study the recovery of irradiated spores during the post-irradi ation holding,irradiated spore suspensions in test tubes received doses of
100 Krad, were held at 23°c and 1°c for up to 6 days in dark incubator or refrigerator, and subsequent plate counts were made on CA, PDA and SDA.
Magnetic Treatment
The test tubes containing irradiated spore suspension were placed in an electromagnet generated magnetic field, having a distance between the two poles of 16 cm and a magnetic strength of 400 gauss. After 1 to 3 days.exposure, the tubes were taken out and plate counts were made on PDA.
Heat Treatment
To study the synergistic action of heat, some spore suspensions were heat treated before or after 40 Krad irradiation. Test tubes each containing
1 ml of spore suspension were placed in a hot water bath, allowed 1 minute to rise to 58°C, held at that temperature for 4 minutes, then cooled to room temperature in a 10°c water bath. The interval between the heat treatment and the irradiation was less than 30 minutes. Plate counts were made on
PDA. 25
Chemical Sensiti zers
3 To determine the sensitizing action of chemicals on spore survival,
0. 5 ml of the chemical solution, mostly fungicides, was added into a test tube 6 cont aining 10 spores in 0. 5 ml suspension. The suspension mixture was
irr adi a ted al 50 Krad, and the colonies were counted on PDA.
Colony Growth From Irradiated Mycelia
For the colony irradiation, three spots on each of duplicate plates of CA and PDA were inoculated with a loopful of spore suspen!lion . After incub ation at 23°c for 24 hours, the pl ates were exposed to gamma rays at sever a l dose levels, and were again incubated at 23°C. The colony diameters were measured periodically and comp ared to the control.
For the mycelial disc irradiation, a spore suspension was poured on a prep ared CA plate and incub ated at 23°C for 2 days. Discs of myceli a with 5 mm diameter and 1 mm thick were aseptically cut with a cork borer
3 The manufacturers of the chemicals were: Captan, Stauffer Chemical Co., New York, N. Y.; DAC 2787, Diamond Alkali Co., Cleveland, Ohio ; DHA-S and Dowicide A, The Dow Chemical Co., Midland, Michigan; Difolatan and Phaltan, California Chemical Co. , Richmond, Calif. ; iodoacetic acid, Sigma Chemical Co., St. Louis, Missouri; maleic acid, 1-naphthol, and vitamin K5, Eastman Organic Chemicals, Rochester, N. J.; Mycostatin, E. R. Squibb and Sons, New Brunswick, N. J.; Myprozine, American Cyanamide Co., Pr inc eton, N. J.; Neomyc,in, S. B. Penick and Co., New York, N. Y.; potassium sorbate, Union Carbide Chemicals Co. , New York, N. Y. ; secondary butylamine, Pennsalt Chemical Corp . , Phi ladelphia, Pa.; sodium benzoate, Braun-Kni,cht-Heimann Co., San Francisco, Calif. ; sodium propionate, Fisher Scientific Co., Fair Lawn, N. J.; U-2069 , the Upjohn Co. , Kalamazoo, Michigan . 26 from these plates . Discs were placed in sterile petri dishes and then irradiated. Subsequently, two discs were placed on each of triplicate plates of various agar media and were incubated at 23°c . The media used were
CA, PDA, SDA, and apple juice agar (apple juice , 1 part ; water, 2 parts; agar, 1. 5 percent) '(AJA). The colony diameters were measured periodically .
Media Irradiation
To determine the effects of irradiated agar media on the fungal growth, petri dishes containing 15 ml of agar medium were exposed to gamma rays at
100 and 200 Krad. AJA, CA, PDA , and SDA were irradiated. Conidia were then inoculated on the media, 2 loopfuls on each plate, and the colony growth was measured periodically.
To determine the effect of irradiated broth media , Czapek Dox broth
(CDB)) and potato dextrose broth (potato extract 0. 4 percent, dextrose 2 percent
(PDB) in separate 125 ml beakers covered with aluminum foil, each containing
80 ml , were irradiated at 20, 50, 100, 200 and 400 Krad. Twenty ml aliquots of the irradiated broth was poured into sterile 250 ml Erlenmeyer flask, 1 ml 6 of conidia suspension containing 2 x 10 spores was pipetted into each flask, and incubated at 23°c for 5 days. Mycelia from the flasks was filtered, dried at go0 c, and weighed. 27
Culture for Pectolytic Enzyme and
Nucleic Acid Analysis
The medium used for the pectolytic enzyme study was 0. 5 percent pectin (Sunkist Pectin N, F., Sunkist Growers, Inc ., Ontario, California) in
CDB, adjusted to pH 5. 0 with HCl befor~ autoclaving. Pyrex glass tubes each 6 containing 2 ml of spore suspension, 2 x 10 spores per ml, were irradiated at 50 and 100 Krad. The contents of each tube were then transferred to 50 ml of CDB medium in a 500 ml Erlenmeyer flask . The flasks in triplicate were shaken on a reciprocal shaker operating at 180 osci nations per minute with a stroke of 3. 8 cm, at 23°c, for up to 7 days. Triplicate flasks were removed from the shaker each day, and the liquid cultures were filtered so that the fi !tr ates could be employed for enzyme activity assays. After filtration, the mycelia were plunged into boiling methanol to inactivate their nuclease, freeze dried, and used to determine nucleic acids.
As say of Pectinesterase
Four ml of a culture filtrate at about pH 5. 0 and 2 ml of 1 N NaCl, were added to 10 ml of 1 percent pectin (Sunkist, NF) solution and adjusted the pH to
5. 0 at 24°C. The pH of the mixture was kept at 5. 0 by continually adding
0. 1 N NaOH. Enzyme activity was measured based upon the amount of NaOH used in 1 hour after addi ng the enzyme preparation (Cole and Wood, 1961). 28
Assay of Polygalacturonase
Polygalacturonate-splitting enzymes were assayed using the vjs ·cosity
reduction method. One ml of the culture filtrate was added to 5 ml of 1 per-
cent pectin solution containing 0. 1 M citrate buffer at pH 5. 0. The reaction
was carried oul in a pyrex test tube (18 x 150 mm) covered with aluminum
foil at 30°c for 30 minutes . The reaction was stopped by placing the tube
inaboilingwaterbath for 4 minutes, and relative viscosity was measured
in a Cannon-Fenske viscometer at 30°c.
Percentage reduction in viscosity was calculated as
T - T O T _ Tt x 100, where T , Tt and Tw are flow times in seconds for the 0 O W
control, the test mixture and water respectively. The control was a reaction
mixture containing boiled enzyme. "Pectinase" (Nutritional Biochemicals
Cor p ., Cleveland, Ohio) was employed as the standard, using 1 mg of the
powder per ml of the solution as the enzyme unit. A straight line is obtained
when known enzyme concentration is plotted against percentage reduction in
viscosity on logarithmic-probability paper (Cappellini, 1966) .
Assay of Tissue Maceration Enzyme
Assay of tissue maceration enzyme was based on a modified pressure
tester method (Sommer _tl!!_l., 1963b). Tissue discs, 1. 0 mm thick and 17 mm
diameter from the pith of large potato tubers, were washed in dis ti lied water,
0 drained, immersed in 2 or more vo lumes of acetone, and he ld overnight at o c . 29
Before use, the slices were drained and soaked in a large volume of tap
0 water for l to 2 hours at room temper a ture (20-25 C) .
For each test, a total of 10 discs were immersed in a mixture of
10 ml of enzyme so lution (culture filtrate which was appropr iate ly diluted
with distilled water) and 2 ml of 0. l M citrate buffer at pH 5. 0 contained in
petri dishes (15 x 60 mm). After 30 minutes with occasiona l shaking at 24°C,
discs were positioned over a hole (0. 25 in . diameter), and a Chatillon fruit
pressure tester (John Chati llon and Sons, New York, N. Y.) with a 1/ 8 inch
tip and a scale range of Oto 500 g was pressed against the slice until the tip
passed through the tissue . The measurements were completed within 2 minutes
after the end of the reaction.
The relation between enzyme activity and the strength of the potato tissue discs are expressed as follows: s 0 log (8 - 1) = log a+ k log (E) where (E) is enzyme concentration; S is the strength of the blank after soaking 0 in the buffer in the absence of the enzyme; Sis strength of macerated tissu e, s and a and Kare constants. When ( 1) is plotted against (E) on logarithmic 8 ° - paper, a straight line is obtained (Mcclendon and Somers, 1960). "Pectinase" was again emp loyed as standard, with l mg per ml of the enzyme considered a unit.
Separation of RNA and DNA Fractions from Mycelium
The preparation of the RNA and DNA fractions for nucleic ac id 30
determinations is illustrated in Figure 2. It is a modification of the Schmidt
and Thannhauser's method (Schmidt and Thannhauser, 1945; Hutchison and
Munro , 1961 ; Mizuno, 1965; and Munro and Fleck, 1966).
Determination of Nucleic Acids
RNA was measured by the ultraviolet absorpt ion. The amount of RNA
was calculated from the difference between the absorptio ns at 260 mpand
290 mp (Minagawa~i!!-, 1959) and expressed as percent dry weight. Yeast
RNA (Nutritional Biochemicals Corp., Cleveland, Ohio) was hydrolyzed by
0. 3 N KOH and used as a standard.
DNA was determined by an improved diphenylamine method (Gi Les
and Meyers, 1965). About 1. 5 percent of sulfuric acid was added to the
diphenylamine reagent, because the samp le was in 5 percent perchloric acid
solution. Sperm deoxyribonucleic acid (Nutritional Biochemicals Corp.,
Cleveland, Ohio),hydro ly zed in 5 percent perchloric acid solution at so0 c for
20 minutes, was employed as a standard. DNA conte nt was expressed as percent dry weight.
Culture for Radioactive Nucleic Acid 14 from Thymine-2-C
7 Three ml of spore suspension containing 10 spores was inoculated 14 into 100 ml of CDB containing 2 J'c of thymine-2-c (New England Nuclear
Corp., Boston, Massachusetts) in a 500 ml Erlenmeyer flask . After 31
Dry myce Ii um (20-40 mg) I 2 ml of 5% TCA (Trichloroacetic acid) Centrifugation (6000 rpm, 0°C)
Residue Supernatant I 2 ml of 5% TCA at o 0 c Centrifugation
Residue Supernatant Washing and centrifugation 3 ml of 2% Na acetate in 90% ethanol at OoC 3 ml of 95% ethanol at ooe 3 ml of ethanol-ether (1, 1) tw ice, 20-25°C
Residue Supernatant Air drying 2 ml of 0.3 N KOH at 37°C for 10-16 hrs. Cool to 0°C 0.1 ml of 6 N HCI and 0.2 ml of 60"!., PCA (Perchloric acid) Cool to 0°C lCentrifugation Residue Supernatant J 1.7 ml of 5% PCA Centrifugation
Residue Supernatant 2 ml of 5% PCA Heat to 80°C for 20 min . RNA fraction Cool to 0°C 1Centrifugation
Residue Supernatant J 2 ml of 5% PCA Centrifugation
Supernatant
DNA
Figure 2. The schematic di ag r am of procedure for separation of RNA and DNA from mycelium. 32
incubation on a shaker for one and one-half days, 10 flasks were removed,
and the mycelia were collected . They were then filtered by suction and
was hed with unlabeled medium. The filtered mycelia were resuspended in
200 ml of unlabeled medium in a Waring blendor (30 seconds at low speed
twice), then made up to original volume for irradiation. All of the procedures
were done aseptically. Fifty ml of culture was poured into each of several
sterile 125 ml beakers covered with aluminum foil, and half of the group was
irradiated with 100 Krad dose. The irradiated cultures were collected, mixed
well, and a 50 ml aliquot was poured into a sterile 500 ml Erlenmeyer flask,
for incubation on the shaker at 23°C. Two treated and two control flasks were
removed every 2 hours, and the mycelia were filtered. They were then plunged
into boiling methanol, cooled, and freeze dried after removal of the methanol.
Determination of Labeled Nucleic Acids
14 It was found that thymine-2-c was not incorporated directly into
DNA as expected before the experiment, but instead, converted to other pyrimidine derivatives, and, as the result, the radioactivity was also found in
RNA fraction . Therefore DNA degradation could not be determined by count ing the radioactivity of whole cells. It was necessary to isolate the DNA fraction from the mycelium before counting. Approximately 30 mg of the freeze dried mycelium was ground in a Ten Broeck type tissue grinder with approxi
0 mately 3 ml of 5 percent trichloroacetic acid at o c, then poured into a centrifuge tube. The grinder was washed twice with 1 ml of the acid solution, 33 and the washings were mixed with mycelium suspension . Isolation of the
RNA and the DNA fractions and chemical determination of nucleic acids were the same as described before. One ml of the RNA or DNA fraction was pipetted into a centrifuge tube, and perchloric acid was neutralized and
0 precipitated with 50 percent KOH solution, cooled to o c, and then cen trifuged. The precipitate was washed with 0. 2 ml of distilled water and centrifuged again. The two supernatants were combined and dried in a stain less steel cupped planchet (3 cm diameter) under an infra-red lamp. Radio activity was counted with a thin-window gas-flow counter.
Statistical Analysis
In order to evaluate the effect of irradiated media on growth of the fungus, the data on colony diameter and mycelial dry weight were statistically analyzed for determination of the least significant differences (LSD) described by Li (1964). 34
RESULTS AND DISCUSSION
Conditions Influencing the Effects of Gamma Radiation
on_!:. expansum
Germination of irradiated conid ia
The average percentage of spore germination on CA was plotted against
irradiation dose on semilogarithmic paper in Figure 3. Almost 100 percent of
the unirradiated conidia germinated on this medium, and germination percentage
was reduced when irradiation dosage increased. About 62 percent germinated
after 100 Krad, 32 percent after 200 Krad, and 17 percent after 300 Krad doses
of gamma irradiation. The dose-germination relationship curve was sigmoid with
a small shoulder at the dose lower th an 50 Krad. This implies that more than a
single hit is required to prevent an individu a l spore from germination. This
result is in disagreement with that of Beraha .
.£. italicum (1959b), when their raw data were plotted on semilogarithmic paper,
a lmost a straight line was resulted. This difference may be due to the different
test conditions, since their spores were irradiated in Tochinai 's or Czapek's
media and plated on 0. 5 percent water agar.
Colony formation of irradiated conidia on different media
Dose-response. When the unirradiated conidia were plated on the
media, all spores grew to visible colonies afte r 2 days of incubation; except 35
100 90 80 w 70 C> <( 60 I- z 50 w u °'w 40 D.. z 0 30 j:: <( z ~ 20 °'w C>
10 0 50 100 150 200 250 300 350 DOSE (Krad)
Figu re 3 . Ge rmin a tion percentage curve of gamma-ir radi ated conid ia of .J?..expansum on Czapek solution aga r . 36
3 days on CA. Most colonies from the irradiated spores were visible after
2 days of incubation, butsomenew surviving colonies appeared after several more days incubation on all the media. Hence, the colony counts were made on each plate on several successive days , to ascertain all the colonies.
The survivals on the several plating media (CA, NA, SDA, SMA, and
PDA), average of 4 replications, were plotted against irradiation doses in
Figure 4. Although a high percentage of spores germinated after exposure to the higher doses, most of them failed to grow further to produce colonies. At
100 Krad, while 62 percent of spores germinated, only 1. 5 percent formed colonies on CA; less than 1 percent formed colonies on PDA. 'The germination percentage of the irradiated conidia seems not important for the control of the fungus, since only a small number of those exposed would grow further to produce rots in their hosts. This is in agreement with the work of Beraha ~ ~
(1959a, b) and Sommer~~- (1963a) done on other fungi.
£. expansum was cited as a radiation sel'lsi tive species by Sommer~~
( 1964, 1967). The data obtained here confirmed their results. As the conidia of Penicillium species are uninucleate and unicellular, they have less resistance to gamma radiation than those of multicellular-spored fungi as pointed out by
Cooper and Salunkhe (1963). The dose-survival curve of spores observed in the present work was also in the sigmoid form, indicating that lethality resulted from a number of events taking place in a spore cell. This sigmoid curve was not likely obtained with the aggregated spores since the spore suspension was filtered through a glass wool layer before the irradiation. 37
• Czapeksolution agar o Nutrientagar x Sabourauddextrose agar • Sabouraudmaltose agar o Potato dextroseagar
10-l
z 0 ..... u <{ "'..... zCl 10-2 > > :::>"' VI
25 50 75 100 125 150 DOSE (Krad) F igure 4. Surv iv a l curves of gamma irradi ated conidi a of_!'.. exp ansum plated on different medi a. 38
Effect of plating medium. Almost equal numbers of colonies formed
from unirradiated conidia on each of 5 plating media (CA, NA, SDA, SMA and
PDA). Survival from the irradiated conidia, however, varied among the
media . The survival was the highest on CA and the lowest on PDA. Survivals
on ' the other three media (NA, SDA, and SMA) were between those two media.
The differences were magnified when the irradiation doses were increased.
It was expected that more colonies would grow on a natural medium than on a
synthetic medium, because some auxotrophic mutant might have been induced
by radiation, and might survive on a natural medium. Since CA contains
only sucrose and inorganic salt, whereas ~DA and NA conta in complex
organic products, the difference in survival on different media does not seem
to be mainly due to a nutritional requirement .
In order to study the effect of nutrient on the survival of irradiated
spores, 0. 4 percent of the potato extract or yeast extract was added to CA,
and colony counts were made. The platings (6 replications) were also made on CA and PDA for comparison (Table 1). Colony growth was faster than
CA when the extra nutrients were added to this medium . The growth rate on the medium containing potato extract was the same as on the PDA; and the growth rate on the medium containing yeast extract was faster. Survival on CA medium
containing yeast extract was much higher than on the medium containing potato extract. On the latter medium, survival was slightly higher but close to that observed on PDA. However, survival on CA was the highest. The growth rate increase cau,sed by the additional nutrient resulted in the lower survival. 39
Table 1. Survivals of gamma-irradiated conidia of .f.. expansum on supple mented C zapek solution agar and potato dextrose agar
Surviving fraction Growth r ate 50 K rad 100 K rad Medium rank (x 10- 2) (x 10- 3)
Potato dextrose agar 2 6.4 3.2
Czapek solution agar 3 14. 1 10.5
Czapek + potato extract 2 8.9 4.3
C zapek + yeast extract 12.1 8.1
In summ ary, the effects of plating media were : (1) the survival was the highest on CA (sucrose-salts medium) ; (2) the incubation time required for the spore to grow into a visible colony on CA was slower than on the other media ; and (3) addition of yeast extract or potato extract to this medium resulted in faster growth but less colony formation. These facts support the view th at a slower met abolism during germination may allow part of the spores to repair the radiation injury . Faster growth condition may cause unbal anced metabolism induced by the radi a tion to le ad to death (Alper and Gillies , 1958b, 1960 ;
Sommer _!tl .!!l,, 1963a).
Yeast extract, rich in kinds of amino acids, nucleotide derivatives, and vitamins, resulted in high er survival than the potato extract even though growth of colony was faster on the yeast extract containing CA. The reason may be that some factors in the yeast extr act stimulate repair mechanisms in the irradiated cells, as ~urned by Stapleton _!tl.!!l· (1953). Another possibility is that some factors in the potato extract may inhibit the repair mechanism . 40
Components of the growth medium for irradiated spores Is apparent ly an important factor deciding their survival.
Effect of pre-irradiation media. When conidia harvested from PDA culture instead of from CA were irradiated, repeated results showed that the radio-sensitivity of spores was not changed. Furthermore, the order of survival on three media, CA> SDA > PDA was not changed . This differed from the result of Stapleton~.!!!· (1955) that survival of!,_. coli cells on differ ent media was influenced by the pre-irradiation medium used. Conidia are the resting reproductive cells which differ from the vegetable ce lls .
Effect of age of spores
Radio sensitivities of spore suspensions of 6-month -old culture and
1-week-old culture were compared. The average of 3 replications of the survival against radiation d<>13esare shown in Figure 5. The order of survival on three media (CA, SDA, and PDA) was the same, but 6-month-old spores were more susceptible than 1- to 2-week-o ld spores to irradiation damage.
The differences became larg er when the radiation dose was increased. All the survival curves were in sigmoid shape . The older spores were more easily inactivated by the radiation. This is in agreement with Malla~.!!.\, (1967).
Although fungal spores are resting reproductive cells, their respiration does not completely cease. Certain metabolic processes function at very low rates until the spores finally lose their viability during senescence. Asexual spores such as conidia are more short-lived than sexual spores (C=hrane,
1958; Sussman and Halvorson, 1966). The relatively high radio sensitivity of 41
1 to 2 weekold conidia • Czapeksolution agar o Sabourauddextrose agar o Potatodextrose agar 6 monthold conidia 10-1 • Czapeksolution agar o Sabourauddextrose agar o Potatodextrose agar
z 0 10-2 j:: u <( a,: ..... (!) z > a,:> :::, 10-3 "'
10-4
50 100 150 DOSE (Krad)
Figure 5. E ffect of conidi a age on survival curves of gamm a- irr adiated conidi a of R,. exp ansum plated on three different media . 42
6-month-old conidla may be due to metabolic destruction of Internal substances
that are protective to the radiation sensitive site of the cell or due to lessening
the ability to produce certain enzymes, including "repair enzymes, 11 which are
needed for the spore to retain its viabi lity .
Recovery of delayed plating
More colonies developed from irradiated conidia when the plating was
delayed for several days than when plated immediately after the irradiation
and were influenced by the temperature at which the spores were he Id.
The survival of 100 Krad irradiated conidia on three plating media
(CA, SDA and PDA), average of 3 replications, were plotted against the days
of holding before plating, as in Figure 6 (1- to 2-week -old conidia) and Figure 7
(6-month-old conidia). Survival increased with time of holding at 23°c. After
6 days of holding, survivors were severa l times more numerous than when
plated immediately after the irradiation. Both 1-week-old conidia and 6-month
old conidia demonstrated the same tendency of increase in survival; the final
survival rati\) of 6-month-old spores was still lower than that of the 1-week-old spores.
Sommer~ .!!J.(1963a) showed that the manifestation of irradiation injuries by sporan'giospores of Rhizopus stolonifer can be reduced if germination is delayed for 1 or 2 days. The same repair mechanism may be operating in the irradiated E_. expansum conidia. Difference in the survival between the three plating media was smaller after 6 days of holding; the presumed lethal damage caused by faster and unbalanced growth on the PDA may be reduced 43
N I 0 ::::. 3.0 ~~~::Z ------~23 °C z 0 / /' • Czapeksolution agar t u o Sabourauddextrose agar <( 2.0 c,:: o Potatodextrose agar ..... C) ·<./.~~ ...... 7 z ...... 0 1°C Q.l'I~ 11110111111111111111111111 0 u111u 1 11 11 n•O > 1.0 1111 1111...... 0 11111110 ...... 0, c,::> ::::> VI
0 3 6 DAYS AFTER IRRADIATION Figure 6. Recovery of 100 Krad gamma-irradiated.!:_. expansum conidia of 1-week-old culture, he ld in suspension at 23°C and 1°c before plating.
N I 0 z- • Czapeksolution agar 0 o Sabourauddextrose agar i==2 .0 u o Potatodextrose agar <( c,:: ..... C) z 1.0 >
c,::> ::::> VI 0 3 6 DAYS AFTER IRRADIATION Figure 7. Recovery of 100 Krad gamma-irradiated.!:_. expansum conidia of 6-month-o ld culture, he ld in suspens ion at 23°C and 1°c before plating. 44
by the recovery action of del ayed plating. However, the delay effect was not observed when the spore suspension was held at 1°c. This temperature dependence may be becau s e enzymes were related to the repair mechanism .
These repair enzymes are active in the dark, since the spore suspension was held in the dark incubator .
The recovery of the spores after irradiation cou Id reduce the effective- ness of radiation for post-harvest disease control. The temperature dependence of recovery is in favor of the combination of the radiation and the low temper- ature storage of host fruits. Additional studies are necessary on the conditions for avoidi~ recovery for the effective use of ~amma radiation.
Effect of magnetic treatment
When the irradiated spores were held in suspension and exposed to a magnetic fie ld for 1 to 4 days before plating, there was an increase compared to the control in the number of colonies formed due to the recovery of spores .
But no direct effect of the magnetic force on colony formation of both unirradi- ated and irradiated spores was found . Magnetic force was reported to inhibit the growth of bacteria (Gerencser ~ !!:!,, 1962), but 400 gauss of flux density used in this study may not be strong enough to a lter the survival of the irradiated spores .
Effect of heat treatment
From the data of the survivals for radiation only and he at only in . - 3 Table 2, survival of about 1. 8 x 10 would be elfpeCtfd if two treatments 45
Table 2 . Survival of£ . expansum conidia after heat treatment and gamma irradiation
Treatment Surviving fraction -1 Irradiation only (40 Krad) 1. 69 x 10 -2 Heat only (58°C, 4 min) 1. 08 x 10
-3 Irradiation-Heat 1.08 x 10 -3 Heat ~Irradiation 0. 75 x 10
were additive in effect; actual results were lower than the expected value. The radiation inactivation of conidia was slightly enhanced by the heat treatment.
The heat-radiation combination produced lower survival than the radiation- heat combination. This concurs with the results of Sommer f! ~ - (1967). Heat treatment prior to the irradiation may cause denaturation of some cell com- ponents such as DNA and cell protein to become more sensitive to the radiation injury, or may cause inactivation of repair enzymes and inhibit the recovery.
The interval between the irradiation and the heat treatment was less than half an hour. It is unlikely, from Figure 6, that repair process took place during this period. Radiation treatment may cause damage to some cell com- ponents and reduce the heat resist,t:mce of the survival spores.
Post-harvest diseases could be effectively controlled by a proper combination of heating and radiation. The studies elucidating sensitivity of the fungus, as well as the to lerence of fruits, to the combination treatment are nece s sary. 46
Effect of sensitizing chemicals
The survivals of conidia irr ad iated in combination with chemicals are tabulated in Table 3. The fungal spores remained in contact with the chemicals for abo ut 3 to 4 hours because of the low dose rate of the availab le radiation source. Many of the 18 chemicals tested, which are known as fungicides, did not reduce colony formation of either irradiated or unirradiated conidia of
R_. expansum under the test conditions. Some chemicals demonstrated fungicidal actio n but no sensiti zing effect. Difolatin, iodoacetic acid, and secondary butylamine had significant sensitizing effects. About half of unirradiated spores lost their ability to form colonies at 10 ppm of Difolatan a lone , but when irradiated in co mbin ation with the chemical survival was reduced to less than 0. 1 percent of th e irradiated control. A treatment with 50 ppm of iodoacetic · acid produced the same results . Fungicidal and sensitizing actions were a lso demonstrated by secondary butylamine at 250 and 500 ppm.
The 1-naphthol, Neomycin, and vitamin K treatment a lso slightly increased 5 the inhibiting effect of radiation, but their sensitizing effects were not so great as the aforemen tioned chemicals.
Sensitizing actions of iodoacetic acid and 1-naphthol has been reported on fungi and yeasts (Georgopoulos ~ill· , 1966), and of vitamin K on bacteria 5 and yea sts (Silverman~~·, 1963). The effective fungicidal actio n and the high sensitizing effect of Difol ata n (N-(1, 1, 2, 2-tetrachloroethy ls ulfenyl) - cis-
64-cyclohexene-l, 2-dicarboximide) on the test fungus are noteworthy .
Secondary buty lamine was found to be effective in control of Penicillium _decay 47 Table 3. Survival of ~- expansum conidia irradiated in combination with chemicals
Irradiated 50 Krad Concentration Unirradiated relative survival Chemical ppm percent survival ratio
Control 0 100 1. 00 Cap tan 500 90 .95 DAC 2787 500 79 .90 DHA-S 500 50 . 71 Difolatan 5 58 . 15 10 51 . 001 Dowicide A 250 84 . 86 500 23 .28 Iodoac e tic acid 50 48 . 001 Maleic acid 500 92 .94 Mycostatin 500 85 .91 Myprozine 500 82 1. 09 1-Naphthol 50 88 .69 Neomycin 500 72 . 56 Phaltan 500 84 . 89 Potassium sorba te 500 99 1. 03 Sec. Butylamine 250 65 . 11 500 15 . 01 Sodium benzoate 500 77 . 78 Sodium propionate 500 86 . 82 U-2069 500 90 .91 Vitamin K 30 58 . 29 5 48
of citrus fruits (Eckert and Kolbezen, 1962) , and a lso is a promising
sensiti zer.
The fungus could be eff ec tively inactiv ated by combining these chemi
ca ls with low doses of gamma irradiation. Adequate combination for the
practical use and the mechanism of sensiti zing ac tion of these chemicals need
to be investigated.
Grow th of irradiated myceli a
The average diameters of 12 colonies that were irradiated 1 day after
inoculation on CA and on PDA . were plotted aga inst day s of incub at ion in
F igures 8 and 9, respectively. Grow th was retarded by the irr adiation, but
eve ntuall y the colonies started to grow at about the same growth rate as the
control. Some cells in the colonies survived eve n a fter being irradiated at
500 ! were increas ed. The colony growth (ave rag e of 18 colonies) after irradi a ted myceli a l discs . were inocul ated on unirr adiate d media are shown in Table 4. The growth rates of unirradi ated colonies ar e in the following order: AJA> PDA > CA and SDA. Colonies from the irradiated myceli al discs did not grow for a time, but then st arted to grow. The periods of delay we re directly proportional to the radiation dose applied. The recovery of colonies from the radiation injuries was faster on SDA and on AJA than on PDA and CA. Periods of delay were also affected by components of the media ; some components inay stimulate or inhibit the recover y of the ce ll. 49 E v 3.0 "'w ....w :E < 2.0 c z>- _,0 1.0 0 u 3 4 5 6 DAYS OF INCUBATION Figure 8. Growth of gamma-i rr adiated_!:. expansum colonies on Czapek so lution agar. Irradiated after 1 day of incubation. E ~ 3.0 "'w ....w :E < 2.0 c >-z g 1.0 0 u 2 3 4 5 6 DAYS OF INCUBATION Figu re 9. Growth of gamma-irradiated_!:. expansum co lonies on pot ato dextrose aga r . Irr adiated after 1 day of incu bation. 50 Table 4. Growth of E· expansum colonies from gamma-irradiated mycelial discs on different media Days of incubation Dose 2 3 4 5 6 7 Medium Krad Diameter in cm Apple juice agar 0 l. 18 2.32 3. 59 4.93 6.15 7.32 80 0. 95 2. 02 3. 23 4 . 53 5.77 6.98 160 0. 68 l. 58 2.79 3.99 5. 26 6.49 Czapek solution agar 0 0.94 l. 63 2.15 2.63 2.92 3.24 3.47 80 0.66 l. 29 l. 93 2.41 2. 76 3. 01 3.25 160 0.56 0. 98 1 62 2.13 2.55 2.88 3.02 Potato dextrose agar 0 0.8 6 l. 57 2 . 30 3. 07 3.69 4 . 35 5.02 80 0. 62 1. 22 1. 85 2. 53 3.23 3.85 4.45 160 0. 54 o. 98 1. 55 2.21 2.85 3.50 4. 09 Subouraud dextrose agar 0 0. 89 1. 45 1. 99 2.49 2.89 3. 18 3. 45 80 0. 75 1. 34 1. 85 2 . 37 2.78 3. 08 3. 35 160 0. 65 1. 12 1. 65 2.21 2.64 2.96 3.20 51 The temporal halt of the colony growth would be important in the storage of some fruits such as cherries, strawberries, etc. since their relatively short storage life would be lengthened by the irradiation. Growth of the fungus on irradiated media The diameters of the colonies grown on the irradiated media, average of 8 replications, are shown in Table 5. There were no significant differences between the growth of the fungus on the control and the irradiated media of AJA, PDA, and SDA. But growth of the fungus was slower on the irradiated CA than the unirradiated medium for the first 3 to 4 days of incubation. The micro scopic observations at 24 hours of incubation showed the mycelia were longer and more branched on the control medium than on the irradiated medium. The development of colonies on the irradiated medium was slower than that of the control up to 3 or 4 days. Thereafter the growth rates were the same as that of control. Some unknown components induced in the medium by the irradiation may inhibit the fungus at the initial growth stage. The dry weights of mycelia after 5 days incubation in irradiated broth media are shown in Table 6. The variance within treatment is small in PDA but very large in CDB. However, the data failed to show significant differences among the treatments. Aspergillus oryzae was inhibited on CDB, either irradiated in liquid form or powder form (Fields, 1959). The effect of irradiated CDB on the growth of E_. expansum, if any, was concealed by the large variance of the mycelial dry weight. However irradiated CA plate did influence the initial 52 Table 5. Growth of£. expansum colonies grown on gamma-irradiated agar media Days of incubation Dose 3 5 7 9 Medium Krad Diameter in cm Apple juice agar 0 1. 94 4.36 6. 79 100 1. 93 4 . 31 6.81 200 1. 88 4 . 30 6.74 C zap ek solution agar 0 0.99 2 . 47 3.46 3 . 94 100 0. 88 2.34 3 . 31 3.81 200 0. 74 2.20 3.30 3.78 Pot a to dextros e agar 0 1. 08 2 . 55 3. 91 4.91 100 1. 01 2 . 51 3 . 91 4 . 89 200 0.99 2. 59 3 . 97 4 . 96 Sabouraud dextrose agar 0 1. 08 2 . 42 3 . 29 3.70 100 1. 08 2.45 3.32 3 . 71 200 1. 10 2.38 3 . 33 3.75 LSD 0. 05 for apple juice agar, Czapek solution agar, potato dextrose agar and Sabouraud dextrose agar at 5th day are 0. 138, 0. 102, 0. 098 , and 0. 085 respectively. LSD . 01 for C;zapek solution agar is 0.139. 53 Table 6. Dry weights of E,. expansum grown in gamma-irradiated broth media, after 5 days of incubation Medium Dose Czapek Dox Potato dextrose Krad mg/20 ml mg /20 ml 0 38.9 40.3 20 31. 7 40.8 50 36.0 40. 6 100 31. 6 39. 2 200 33. 5 40. 2 400 27.5 41. 0 LSf) 0. 05 for Czapek dox broth and potato dextrose agar are 15. 9 and 3. 5, respectively . growth of the fungus. The nature and the formation of inhibiting substance(s) is not clear at this time . Since the fruits will be irradiated for pasteurization, the effect of irradiated substrate on the growth of fungi must be considered. More studies are needed to relate the changes in irradiated substrate and the fungal growth. Effects of Gamma Radiation on Morphological and Selected Biochemical Changes in R., expansum Morphological changes A large number of the irradiated conidia germinated when incubated on the agar media. But most of the germ tubes grew only a short distance, then 54 deve lopment stopped . The length of the dead germ tube differed from spore to spore; some grew extensive ly and some even branched, but the average length was reduced when the radiation dose increased . Most of the inactivated germ tubes appeared microscopically similar to the unirradiated fungus. Grotesque swelling of the spor es or mycelia was found in only a few, and the diamete!' of the swellings was not wider than twice its diameter of the unirradiated fungus. Cross walls were observed. When plates were made from the irradiated spores, colony growth was var iab le . Althoug h most of the colonies had appeared after 2 days of incubation, additional ones appeared after 3 days or 4 days of incubation. Some abno rmal colony types, such as a smaller size colony, a dense hyphal colony, a sparse sporulation colony, and co lonies of white or lighter green spores were noted on plates of gamma-irradiated conidia. A majority of the abnorma l co lonies were related to the color of the conidia. Examples of the plates are shown in Figure 10. Increasing the gamma radiation dose increased the number of abnorm a l co lonies . Strains with the white or light green conidia were stable mutants, since they retained their color after several conidial transfers. According to Barron (1962) , colors of the conidia of mutants of E_. ex pansum were governed by the genotype of the nucleus (such as white conidia or brown conidia) or by a diffusible component(s) from the parental cytoplasm (such as olive conidia). Table 1 shows higher survival rates on CA th an on yeast extract supplemented medium, indicating that auxotrophic mutants cons titu te a very low percentage among the survivors . 55 Figure 10. Colonie s of E_. expansum formed from gamma-irrad iated conidi a on plating media. CA: Czapek solution agar SDA: Sabouraud dextrose agar 1: Unirradiated conidia 2: Irradiated conidia, 150 Krad. 56 Two strains of mutants, with white spores and with light green spores are shown in Figure 11. When plated on PDA after 80 Krad irradiation, the survival for dark green normal, white and light green mutant strains were 2 2 2 0. 98 x 10- , 1.02 x 10- and 0.87 x 10- respectively. The two mutants were no more resistant to the gamma irradiation than the dark green wild type strain. When irradiated discs were inoculated on new media, the colony edges were uneven, as compared to the round circle of a normal colony, for the first 3 to 4 days of incubation. This could be due to difference in recovery rate of the injured cells in a colony or uneven distribution of viable cells in a colony. Brown colored substances were secreted around the part of the colony on the irradiated disc after a few days of incubation . This might be the metabolic or dissolution products of injured or dead cells, since this brown pigment was also observed around a normal colony if the incubation temper- ature was higher than optimum or if the colony was aged. Pecto lytic enzymes of the broth culture from irradiatAA conidia ]:. expansum produced active enzymes which macerated potato discs and hydrolyzed citrus pectin, but pectinesterase activity was low in the fungus in CDB either with or without 0. 5 percent pectin. It appears, there- fore, that the fungus produced polymethylgalacturonase, which can hydrolyze pectin directly without de-esterification of pectin to pectic acid by pectin- esterase. This concurs with the results of Cole and Wood (1961) that all 57 'Vii r;_\\.i : ~-) 1 1(!) ·) 2 Bil 3 1 11'_.._· -..=>.. J ii ~I ;· ··:--.. . .·· ) I' \, .. !, . ' . ···. 2 mil 3 F igure 11. Mutants isolated from the colonies of gamma-irradiated conldla of E_. expansum. CA: Czapek solution agar PDA: Potato dextrose agar 1: Normal (wild) strain 2: White conidia mutant 3: Light green conidia mutant 58 three group s of the enzymes were found in the juice obtained from E_. expansum rotted app les , but no pectinesterase was found in a synthetic medium culture; the culture filtrate hydrolyzed pectin but not polypectate. Broth culture of the fungus grow n from conidia irradiated at 50 and 100 Krad and their respective controls were filtered; and mycelial dry weight, polygalacturonase activity, and potato tissue maceration enzyme activity were determined. The results of three replications are shown in Figure 12. Growth of the fungus was in the stationary phase after 7 days of shaking . The mycelia of the irradiated conidia grew slower than the control. The mycelial growth was retarded by the radiation treatment. Excretion of a water soluble dark brown colored substance was observed after 3 days of incubation in the control culture and the next day in the 50 Krad irradiated culture. The polygalacturonase activity was measured by the loss in viscosity of pectin so lution . Activity of the control cultures incre ased proportionally to dry weight during the first 3 days of incubation, then decreased even though mycelial dry weights increased. The enzyme act ivity of the c~lture from irradiated spores was initially lower and proportional to the mycelial dry weight but became higher than the control after 3 days of incubation,even though its mycelial dry weight was lower than that of the control. Enzyme activities of irradiated samples peaked after 4 days of incubation. The 100 Krad irradiated cu lture was having higher activity than the 50 Krad irradi ate d culture, and both were higher than the control. Apparently the production of pectin hydrol yz ing enzymes of the fungus was stimulated by the radiation treatment of the conidia . 59 8 >- E ci:: ...... 0 C> 6 ....E < 4 ;:j .... w ::c u (!) >- - 2 :E w :\: 0 O Krad w 0.3 VI o---o 50 Krad <- z- 0 111111111 0 100 Krad o~ :::::,ci:: -~ c: 0. 2 ....::, u < >- .... !::: <> (!) j::: 0.1 ....>- - u-w I- < === :E 0 w < 5 :::::,w VI VI :E j::: >- zN w 0 3 4 5 6 7 DAYS OF INCUBATION Figu r e 12. Act ivi tie s of pecto lytic en zy mes in pect in Czap ek Dox brot h cultu r e in oculated from ga mm a-ir r adia ted co nidia of_!:. expansum. 60 The activity of the enzymes which could macerate the potato tissue exhibited the same tendency as that of polygalacturonase, Figure 12, except the peak of activity of the control and the irradiated culture was reached after 4 days of incubation. Enzyme activity was proportional to the mycelial dry weight in the first two days of incubation, then activities of the irradiated culture became higher than those of the control. The potato maceration enzyme macerates plant tissue by destruction of the middle lamella, the cementing material between cells. It includes several kinds of pectolytic enzymes and others such as cellulolytic enzymes (Wood, 1960). The cellulase activity was not found in E_. expansum (Cole and Wood, 1961), so the tissue maceration activity of this fungus is mainly related to the pectolytic enzymes. Sommer~ fil· (1963b) reported that sporangiospores of!!_. stolonifer can synthesize abundant enzymes capable of macerating plant tissue, even after the loss (through irradiation) of their ability to form colonies. Increase in enzymatic activity has frequently been seen in organisms soon after irradiation , and a suggested explanation is that the radiation broke down I internal barriers within the cell and released the enzyme (Alexander and Bacq, 1961 and Jenkinson, 1963). Pectolytic enzymes of E_. expansum in this experi- ment were, however, very low in the spores, and the stimulation of enzyme production was observed after more than two days of incubation. Some unknown substances may have been induced by the radiation, either in the nucleus or in the cytoplasm, and may be responsible for the stimulation of enzyme produc- tion; or some mutant may be induced among the survivors and produce large 61 amounts of the enzymes or excrete some compounds which stimulate the enzyme production. Since pectolytic enzymes are important agents in breaking down the host cell wall barrier, an enhanced enzyme activity could increase the fungal pathogenicity and cause practical handicaps when radiation is applied to control post-harvest diseases . An!!! vivo study of the enzyme production by irradi ate d fungus seems necess ary because of the difference in nature of _!:. expansum enzyme production on the rotted apple and in the synthetic medium as reported by Cole and Wood (1961) . Nucleic acid content of mycelium from irradiated conidi a The ultraviolet absorption curve of the RNA fraction obtained from myce lia was compared with that of yeast RNA hydrolyzate in Figure 13. The RNA content of the control mycelium in shaken CDB culture was higher in the initial stage due to the high protein synthesis activity of the ce ll division , and decre ased during incubation (Figure 14). The RNA content of the mycelium grown from the irradiated spores was slightly lower than the control in the initial stage and became higher after 1. 5 days and 2 days of incubation for 50 Krad and 100 Krad irradiated conidi a , respectively, and then decreased. The RNA content of the culture of irradiated spores remained higher than that of the control , then decreased to the same level of the control. Because RNA content of the fungus changed with the growth stage, when the RNA content of a dry weight of irradiated fungus was compared to the same dry weight of 62 0.7 0.6 >- 1- v, 0.5 z w _,C 0.4 u< 0.3 .::: Ill. 0 0.2 0.1 230 240 250 260 270 280 290 300 310 WAVE LENGTH (m,u) Figure 13. Ultr av iolet absorption sp ectr a of RNA of E_. exp ansum mycelium and yea st RNA. A: E_. expansum myceli a l RNA fr action in 5 percent perchloric acid . B: Yea st RNA (Nutrition a l Biochemic a l Corp., Clevel and, Ohio) hydroly zed with 0. 3 N KOH then measured in 5 percent per ch loric acid. 63 t- :I: 10 C> I I I I I I I iii 3: 8 L... - >- - ~ - ~ /" .... Cl:: E 6 - c ...... /,,...... --- ••••,,o Cl ....I o•- --a~·········...... <( E 4 - - ::::;- LU /9.o; ;"ct'' ····· u 2 ~ ,•' - .,,,,,,,,..; ,.,."...... >- • ., ,,,,,10'' :E 0 -~l::l!ft.,8, ..... 10 - ·-· O Krad - o--- o 50 Krad - -~ 01111111110 100 Krad <( 6 ~ z Cl:: 4 '-- - 2 '--- - -~ <( z c 0.15 '-- - ~ I I I I I I I 2 3 4 5 6 7 DAYS OF INCUBATION Figure 14. Nucleic acid co nte nts of 1:., expansum myceli a from gamma irr adi ated conidia incuba ted in s ha ke n Czapek Dox brot h. 64 the control, they were almost the same. This relation can be se en more easily from Figure 15. The RNA content was related to the growth stage of the fungus and was not directly influenced by the irradiation. The DNA content of the mycleia was about 0. 33 percent dr y weight in the beginning, decreased from 2 days to 4 days of incubation, then rem ained at about 0. 2 percent. The DNA content of the culture from irradiated spor es was also at the same level in the beginning, but higher than the contr ol at 2 to 4 days of incubation, then declined and reached the same level of the control afte r 5 days of incubation . The DNA content of mycelia from the Irradiated spores was almost the same as of the same dry weight growth stage of the con t rol , DNA contents of mycelia was also related to the growth stage of the fungus. The radiation treatment indirectly influenced the DNA and RNA contents by repress ing the fungus growth. Nucleic acids from irradiated mycelium 14 Radioactive thymine-2-c is widely used as a tracer for cell DNA 14 studies in many organisms. When a small amount of thymine - 2-c wa s added into CDB, and E· expansum grown, radioactivity was detected in th e mycelium . But later when the mycelium was analy zed, the radioactivity was found both in the RNA fraction and the DNA fraction, and not in the remainin g pro te in fraction. Radioactivity was much higher in RNA fraction than in DNA fr a ction. This result was similar to the finding of Fink and Fink (1962), who reported that Neurospora .£!.ill!! failed to utilize exogenous thymidine dire ctl y by phosphorylation and incorporation into DNA. But apparently the l'ungnB utilized 10 , • o Krad 0 0 50 Krad . 0 0 0 ,-0 0 100 Krad H - 0 - -- ~ 0 . 0 0 ~ u • 0 - 0 . ~ 6 -1<( 0 z 0 IX 4 I - , ~ - 0 2 0L.....---l~~...l.._~~....L...~~...... 1....~....L...~--L~~---'-~~~'-----'-~~~~--' 0.02 0.03 0.05 0.10 0.20 0.30 0.50 1.00 2.00 3.00 5.00 10.00 MYCELIAL DRY WEIGHT (mg/ml) Figu re 15. Rela tio n of RNA con tent and dr y weight of myceli a grown from gamma-irradiated con idi a of E_. expansum. "' 66 thymidine by converti ng it to uridine or some related RNA precursors, which can follow the metabolic pathways of the usual RNA precursors, and event ua l incorporation into DNA as a thymidylate residue. The radio activity was higher in RNA than in DNA because RNA content was higher in the cells of the fungus. As a result, the radioactivity determination had to be conducted after the RNA and DNA fractions were separated chemically. The results of the ana lysis of nucleic acid contents and radioactivity from the fungus of shaken broth cultures of irradiated mycelia are shown in Table 7. The growth of fungus was halted during and after irradiation while the fungus was transferred to a new non-labeled medium and when the shaking was temporarily stopped during the irradiation. Growth of the control was started after 4 hours of incubation and that of the irradiated mycelium after 8 hours of incubation . A !though slight decreases in DNA and RNA contents were observed in both control and irradiated mycelia, no radiation-induced degradation of DNA or RNA was detected by chemical analysis or by radio- active ceunting during the incubation. The breakdown of DNA and RNA was v / found in bacteria after irradiation (Stuy, 1960; Pecevsky and Mi letic, 1966; and Achey and Po ll ard, 1967) but has not been reported in fungi. In the lethally irradiated mouse leukemic cells, DNA degradation process was not detected from post-irradiation tissue culture (Watanllfe and Okada, 1968). The degrad ation in bacteria was explained as an enzymatic reaction induced by the radiation. It may be associated with the killing effect of the irradiation or the repair action of the injured cell. The dose (100 Krad) applied in this 14 Table 7. Radioactive counts and DNA, RNA contents of gamma-irradiated rrwcelia labeled with c -thymine. The mycelia were harvested from shaken 50 ml Czapek Dox broth culture. DNA fraction RNA fraction Incubation ti me Mycelial dry DNA Radio- RNA Radio- after irradiation Dose weight content a ctivity content activity hour Krad mg/50 ml % count / min. % count / min. 0 0 34.0 . 334 4040 6.62 29400 100 35 . 7 . 320 3870 6.43 27250 2 0 33.8 . 330 3585 6.52 30250 100 35. 2 . 325 4102 6. 52 33500 4 0 36 . 4 . 322 3094 6. 32 23680 100 33.3 . 327 3495 6 . 55 25380 6 0 43.8 . 288 2658 6. 00 30680 100 36 . 3 .313 2361 6 . 12 24790 8 0 49 . 6 . 299 3055 6.11 23850 100 40 . 6 . 286 3102 6 . 14 26980 __,a, 68 experiment reduced the colony forming ability of the conidia to 1 percent of the control. but had no detectable effect on the nucleic acid contents. The difference between this fungus and the bacterium K, coli may be explained on the basis of differences in enzyme activities and cell structures, such as nuclear membrane, nucleoprotein etc . . The fungal cells are more complicated than the bacteria cells. The nucleic acid of the fungal cells may be structurally more resistant to the degradation than ' that of the bacterial cell. Comparable enzyme may not be induced or activated, or they may not be able to penetrate to the nucleic acid site. 69 SUMMARY AND CONCLUSIONS The use of ionizing radiations is a promising and successful method for prolonging the shelf-life of certain foods by killing or inhibiting the spoi lage inducing microorganisms. For post-harvest storage of fresh fruits and vege tables, the doses employed will be limited by the radiation tolerance of the host. For this reason, research has been conducted to define adequate pasteurization conditions which effectively inactivate pathogenic microflora by low doses of gamma irradiation. Penicillium expansum is the most prevalent and most destructive fungus found in spoiled apples. It is also the causal agent of the "blue mold rot" of many other fruits such as cherries, grapes, and peaches. !!! vitro investigations were made to study some factors influencing inactiyation of E_. ex pansum at low doses of gamma radiation. and physio chemical changes in the fungus induced by the irradiation which may be related to the death of the fungus or its ability to invade fruits. Conditions Influencing the Effect of Gamma Radiation on E_. expansum _R. expansum is one of the radiation sensitive fungi. A !though quite a high percentage of spores germinated after irradiation, only a small number of them were capab le of forming co lonies; almost 99 percent were inact iv ated when irradiated at 100 Krad. Effect of irradiat ion on preventing colony 70 formation is so great that the germination percentage of irradiated spores is of no practical significance. The number of colonies formed from a given dose of radiation varied according to the kind of plating media. The nutritional condition influenced survival, but the medium richest in nutrient did not give maximum survival. From the results it was found that (1) survival of treated conidia was the highest in CA among the plating media, (2) ~rowth of the colonies was the slowest on CA, (3) additional nutrient such as yeast extract added to CA hastened co lony growth but reduced the number surviving, and (4) CA also provided the highest surviving when conidia from PDA were irradiated . It was co nc luded that slow metabolism during germination was the factor which allowed more spores to be repaired from the radiation injuries . The conditions which support faster growth may cause radiation induced unbalanced metabolism to lead to death. The recovery of the spores of E_. expa nsum was observed when the irradiated spore suspension was held at 23°c for several days before plating, probably by the same repair mechanism, The difference in degree of survival among three media became smaller after 6 days of holding. Recovery action is temperature dependent, since no increase in survival was observed after 6 days of holding at 1°c, indicating a possible enzymatic metabolism relation ship. The 6-month-o ld spores showed less resistance to irradiation than 1-w eek? old spores. The age of spore was one of the factors which influence 71 the sensitivity of fungus to the radiation. A magnetic field under a flux density of 400 gauss did not affect the survival of irradiated spores. The combination of heat treatment followed by radiation demonstrated a synergistic effect. Difolatan and secondary butylaminE, were found to be two promising radio ··sensitizers in addition to the already known iodoacetic acid, 1-naphthol, and vitamin K . The use of chemical sensitizers is a very promis 5 ing method for reducing the spoilage fungi in the host. When colonies or mycelial discs were irradiated, colony growth was inhibited. But recovery of colonies was observed even at the very high doses, probably due to a great number of cells present in a colony. Of all media tested, recovered colonies did grow again after a temporary halt of growth by the radiation. The duration of inhibition was increased when radiation dose was increased . De lay period was also dependent upon the nut ritio nal condition of the media on which colonies were grown. Irradiated CA retarded the initial growth of the fungus. Some of the components produced in the medium by radiation may inhibit the fungus. Chemica l changes caused by radiation treatment in the fruits should be con sidered as they may influence the fungal growth. Changes in the Fungus Induced by the Irradiation Gamma radiation treatment of the conidia resulted in death of a high percentage of the fungus. Abnormal colonies and stable mutants with white spores or light green spores were fouw! amorg the survivors. The 72 radio -resistance of their conidla was not higher than that of normal strain. Cultures of E_. expansum demonstrated strong polymethylgalacturonase and plant tissue maceration enzyme activities. The culture grown from irradiated conidia produced more of these enzymes than did the control conidia after 3 days of post-irradiation incubation despite lower mycelial growth. Evidently enzyme production was stimulated by irradiation. Some substances in the cell induced by irradiation may be responsible for this stimulation. Since the pectolytic enzymes are cons idered to be the main means of this pathogenic fungus to invade plants by softening their tissues, this stimulation could cause practical handicaps when radiation is applied to control post -h arvest diseases. A thorough study of this kind of stimulation, both!!! vitro and!!! vivo, is necessary. The nucleic acid content of the mycelium grown from the irradiated conidia were related to the growth stage of the fungus but not directly influenced by the irradiation. The growth of the fungus was slowed by irradiation of conidia and thus indirectly affected the nucleic acids content. 14 When thymine-2-c was used in broth medium, radioactivity was found both in the DNA and RNA fractions of the mycelium instead of being directly incorporated into DNA. No radiation induced nucleic aci d degradation was observed in fungus during the 8 hours of post-irradiation incubation, either by chemica l or isotopic analysis. 73 General Conclusions In general , it was found that sensitivity of.!:'_. expansum to gamma radiation was influenced by such factors as nutritional condition of the media, age of spores , delayed plating , heat treatment, chemical sensitizers, and pre- irradiation of the medium. If the condition is properly selected, this fungus could be effectively inactivated by low doses of gamma radiation and thus retain the quality of fresh fruit and reduce the irradiation cost . Since considerable numbers of irradiated conidia recovered from injuries under conditions which slowed down or delayed the germination, the conditions for prevention of recovery should be studied more extensively. Abnormal colonies and mutants were induced by the radiation. Some mutants were no more radiation resistant than the normal strain , but there is still a possibility that resistant mutants could be induced. Pectolytic enzyme production of the fungus was stimulated by the irradiation. Since the enzyme is an important agent in breaking down the host cell wall barrier, pathogenicity of the fungus is expected to be increased . The degradation of nucleic acids in irradiated mycelia was not detected by the analysis conducted; ' the changes may be too small to be measured. 74 LITERATURE CITED Achey, P. M. , and E. C. Pollard . 1967. Studies on the radiation-induced breakdown of deoxyribonucleic acid in Escherichia coli 15 T-L-. Radiation Research 31:47-62. Adler, H. I. , and M. S. Engel. 1961. Factors influencing the survival of bacteri a after exposure to ionizing radiation. Journal of Cellular and Comparative Ph}'siology 58 supplemental 1:95-106 . Alexander, P ., and Z. M. Bac q. 1961. The nature of the initial radiation dam age at sub-cellular level, p. 3-19 . .!ll R. J. C. Harris (ed . ). The initial effects of ioni zing radiations on cells . Academic Press, New York . Alper, T., and N. E. Gillies . 1958a . The dependence of the observed oxygen effect on the post-irradi at ion treatment of micro-organisms. Nature 181:961-963. Alper, T ., and N. E. Gillies. 1958b. "Restoration" of Escherichia coli strain B after irradi ation : its dependence on suboptimal growth conditions. Journal of General Microbiology 18:461-472. Alper, T. , and N. E. Gillies . 1960. The relationship between growth and survival after irradiation of Escherichia co Ii strain B and two resistant mutants . Journal of General Microbiology 22:113-128. Anderson, E . H. 1951. Heat reactivation of ultraviolet-inactivated bacteria. Journal of Bacteriology 61:389-394. Atwood, K. C ., and A . Norm an. 1949 . On the interpretation of multi-hit survival curves. Proceedings of the National Academy of Sciences, U. s. 35:696-709. Bacchetti, S. , M. Cassandro, R. Elli, R. Falchetti, and F. Mauro. 1966. Relationship between recovery from sublethal damage by dose fractionation and the restoration of viability after delayed plating in diploid Saccharomyces cerevisiae. Radiation Research 29: 295-315. Bac q, Z. M. , and P. Alexander. 1961. Fundamentals of Radiobiology (2nd ed.) . Pergamon Press, New York. 555 p. 75 Barkai-Golan, R., and R. S, Kahan . 1967 . Combined action of diphenyl and gamma radiation on the iQ vitro development of fungi pathogenic to citrus fruits. Phytopathology 57:696-698. Barron, G. I. 1962. The parasexual cycle and linkage relationships in the storage rot fungus Penicillium expan sum . Canadian Journal of Botany 40:1603-1613. Beam, C. A., R. K. Mortimer , R . G. Wolfe, and C. A. Tobias. 1954. The relation of radioresistance to budding in Saccharomyces cerevis iae . Archives of Biochemistry and Biophysics 49:110-122. Beraha, L., E. D. Garber , and (iJ. Strilmaes. 1964 . Genetics of phytopatho genic fungi. Virulence of color and nutritionally deficient mutants of Penicillium italicum and Penicillium digitatum. Canadian Journal of Botany 42 :429-436. Beraha, L., G. B. Ramsey , M. A. Smith, and W. R. Wright . 1957. Gamma radiation for possible control of post-harvest disease of apples, straw berries, grapes, and peaches. Phytopathology 47:4. (Abstract . ) Beraha, L., G. B. Ramsey, M. A. Smith, and W. R. Wright. 1959a . Factors influencing the use of gamma radiation to control decay of lemons and or anges. Phytopatho logy 49 :91-96. Beraha, L., G. B. Ramse y , M. A. Smith, and W. R. Wright. 1959b . Effects of gamma radiation on brown rot and Rhizopus rot of peaches and the causal organisms. Phytopathology 49:354-356. Beraha, L., G. B. Ramsey, M. A. Smith, W.R. Wright, and F. Heiligman. 1961. Gamma radiation in the control of decay in strawberries, grapes, and apples. Food Technology 15:94-98. Beraha , L., M. A. Smith , and W. R . Wright. 1960. Gamma radiation dose response of some decay pathogens. Phytopathology 50:474-476 . Brasch, A., and W. Huber. 1947. Ultrashort applica tion time of penetrating e lectrons : A tool for sterilization and preservation of food in the raw state . Science 105:112-117. Buddenhagen, I. W. 1958. Induced mutations and variabi lit y in Phytophthora cactorum. American Journal of Botany 45:355-365. Cappe llini , R. A. 1966. Growth and polygalacturonase produced by Rhizopus stolonifer. Phytopathology 56:734-737. 76 Clarke, I. D. 1959. Possible applications of ionizing radiations in the fruit, vegetable, and related industries . International Journal of Applied Radiation and Isotopes 6:175-181. Cochrane, V. W. 1958. Physiology of fungi. John Wiley & Sons, Inc., New York. 524 p. Cole, M., and R. K. S. Wood. 1961. Pectic enzymes and phenolic substances in apples rotted by fungi. Annals of Botany (N, S,) 25:435-452. Cooper, ·,G:~ -M. . ,, and D. K. Salunkhe. 1963. Effect of gamma-radiation, chemical, and packaging treatments on refrigerated life of straw berries and sweet cherries. Food Technology 17:801-804. Dean, C. J. , and P. Alexander. 1962. Sensitization of radioresistant bacteria to X-rays by iodoacetamide. Nature 196:1324-1326. Desrosier, N. W., and H. M. Rosenstock . 1960 . Radiation .technology in food, agriculture, and biology. Avi Publication Company, West port, Connecticut. 401 p. Dhaliwal, A. S. , and D. K. Salimkhe. 1963. Ionizing radiation and packaging effects on respiratory behavior, fungal growth, and storage-life of peaches, Prunus persica. Radiation Botany 3:75-83. Doudney, C. 0. 1956. Restoration from an X-ray induced block in deoxyribonucleic acid synthesis in Escherichia coli. Journal of Bacteriology 72 :488-493. DrakuJ(c, M., and E. Kos. 1963. Effect of some metabolites and anti metabolites on the breakdown of DNA and on the colony-forming ability of];_. coli B after gamma irradiation . I. Effect of citrate, glutamate, succinate, versene, and magnesium ions. Radiation Research 19 :429-438. Duggan, D. E., A. W. Anderson, and P. R. Elliker. 1963. Inactivation of the radiation-resistant spoilage bacterium Micrococcus radiodurans. II. Radiation inactivation rates as influenced by menstruum temperature, pre-irradiation heat treatment, and certain reducing agents. Applied Microbiology 11:413-417. Duggar, B. M. 1936. Effects of radiation on bacteria, p. 1119-1149. !!! B. M. Duggar (ed.). Biological effects of rad iation. Vol. II. McGraw-Hi LLBook ' Co. , New York. 77 Eckert, J . W., and M. J. Kolbezen . 1962. Control of penicillium decay of citrus fruits with 2-aminobutane. Nature 194:888-889 . El-Tabey Shehata, A. M. 1961. Effect of combined actio n of Ionizing radiation and chemical preservatives on microorganisms. I. Vitamin K as a sensitizing agent. Radiation Research 15: 5 78-85. Errera, M., and A. Forsberg . 1961. Mechanisms in radio-biology. Vol. I. General principle. Academic Press, New York. !:i34 p. Fields, M. L. 1959 . The effect of cathode rays on food spoilage fungi. PhD dissertation . Purdue University , Lafayette, Indiana . 160 p. University Microfilms, Ann Arbor , Michigan . Fields, M. L., G. R. Ammerman, and N. W. Desrosier. 1960 . A study of the growth of Aspergillus oryzae in irradiated components of foods. Food Technology 14:407-409. 3 14 Fink, R . M., and K. Fink . 1962. Relative retention of tt and c labels of nucleosides incorporated into nucleic acids of Neurospora. Journal of Biological Chemistry 237:2889-2891. Frampton, E. W. 1964 . Synthesis of ribonucleic acid by X-irradiated bacteri a. Journal of Bacteriology 87:1369-1376. Frey, H. E. , and E. C. Pollard. 1966. Ionizing radiation and bacteria: Nature of the effect of irradiated medium . Radiation Research 28:668-676. Georgopoulos, S. G., B. Macris, and E . Georgiadou. 1966. Reduction of radiation resistance in fruit spoilage fungi by chemicals. Phyto pathology 56 :230-234 . Gerencser, V . F ., M. F . Barnothy, and J . M. Barnothy. 1962. Inhibition of bacterial growth by magnetic fields. Nature 196:539-541. Giles, K. W., and A. Meyers. 1965 . An improved diphenylamine method for the estimation of deoxyribonucleic acid. Nature 206:93. Ginoza, W. 1967. The effects of ionizing radiation on nucleic acids of bacteriophages and bacterial cells. Annual Review of Microbiology 21:325-368 . Greenberg, J. 1964. A locus for radiation resistance in Escherichia coli. Genetics 49 :771-778. 78 Harris, R. J . C. 1961. The initial effects of ionizing radiation on ce !ls. Academic Press, New York. 367 p. Huber, W., A. Brasch, and A. Waly. 1953 . Effects of processing con ditions on organoleptic changes in foodstuffs sterilized with high intensity electrons. Food Technology 7:109-115. Hutchison, W. C., and H. N. Munro. 1961. The determination of nucleic acids in biological materials. The Analyst 86:768-813. Jenkinson, I. S. 1963. The impact of ionizing radiation on living matter at the cellular level. Food Technology in Australia 15:242-251. Kan, B. , S. A. Goldblith, and B . E. Proctor. 1957. Complementary effects of heat and ionizing radiation. Food Research 22:509-518. Kempe, L. L. 1955. Combined effects of heat and radiation in food sterilization. Applied Microbiology 3:346-352. Kiga, M., Y. Ando, and H. Koike . 1955. Enhancement of radiobiological effect by malonic and maleic acids. Science 122:331-332. Lea, D. E. 1955. Actions of radiations on living cells. Second ed. Cambridge University Press, Cambridge, England. 416 p. Lee, J . S., A. W. Anderson, and P. R . Elliker. 1963 . The radiation sensitizing effects of N-ethylmaleimide and iodoacetic acid on a r adi ation-resistant Micrococcus . Radiation Research 19 :593- 598. Lee, J. S., and R. 0 . Sinnhuber. 1965. Post-irradiation recovery of Escherichia coli. Nature 207:1212-1214 . Li, J. C. R. 1964. Statistical fnterference. Vol. I. Edwards Brothers, Inc. , Ann Arbor, Michigan. 658 p. Malla, D. S., J, F. Diehl, and D. K. Salunkhe. 1967 . !!! vitro suscepti bili~ of strains of Penicillium viridicatum and Aspergillus flavus to 1:5-irradiation. Experientia 23:492-495. Maxie, E. C., and A. Abdel-kader. 1966. Food irradiation-physio logy of fruits as related to feasibility of the technology. Advances in Foo d Research 15: 105-145 . Maxie, E. C., and N. F. Sommer. 1964. Status of gam ma irradiation as a technology for perishab le commoditi'es . Proceed ings of Fruit and Vegetab le Handli ng Conference, University of Ca lifor nia, Davis, California . Ma'rch, 1964. p. 96-100 . ' 79 McClendon, J . H., and G. F . Somers. 1960. The enzymatic maceration of plant tissues: Observations using a new method of measurement. American Journal of Botany 47:1-7. Mc(jrath, R. A. , R . W. Williams, and D. C. Swartze ndruber. 1966. Break down of DNA in X-irradiated Escherichia coli . Biophysical Journal 6:113-122. v Miletfc, B., Z. Kucan, and DJ. Novak. 1964 . Effect of repeated X-irradiation on the process of degrndation of deoxyribonucleic acid . Nature 202:106-107 . Milet i'c, B. ~ Z. Ktfcan, and L. Sabel. 1963 . The effect of X-irradiation on the degradation of DNA in closely-related strains of~- coli. Inter national Journal of Radiation Biology 7:141-144. Milet(c , B . , Z. Kucan, and L. Zajec. 1961. Synthesis of DNA in X-irradiated Escherichia coli B. Biochemical and Biophysical Research Communi cations 4:343-347. Minagawa, T., B. Wagnar, and B. Strauss. 1959 . The nucleic acid content of Neurospora ~- Archives of Biochemistry and Biophysics 80:442-445 . Mizuno, S. 1965. Isolation and determination of nucleic acids . I. Kagaku to Seibutsu 3:148-158 (J apanese). Morgan, B . H . , and J . M. Reed . 1964. Resistance of bacterial spores to gamm a irradiation. Food Research 19:357-366. Munro, H. N. , and A. Fleck . 1966. Recent developments in the measure ment of nucleic acids in biological materials. The Analyst 91:78-88. Myasnik, M. N., and V. I. Korogodin. 1968. On correlation of viability of irradiated and nonirradiated E100hericia co li B cultivated on various culture media. Radiat ion Research 34:661-668. Nelson, K. E., E. C. Maxie, and W. Eukel. 1959. Some studies of the use of ionizing radiation to contro l Botrytis rot in table grapes and strawberries. Phytopatho logy 49:475-480. Noaman, M. A., B. J. Silverman, N. S. Davis, and S. A. Goldblith. 1964. Radiosensitization of Streptococcus faecalis and Escherichia coli. Journal of Food Science 29:80-86. 80 Patrick, M. H . , and R. H. Haynes. 1964. Dark recovery phenomena in yeast. II. Conditions that modify the recovery process. Radiation Research 23:564-579 . Pec'evsky, I. , and B. Miletif. 1966 . Breakdown of ribonucleic acid in Escherichia coli B after X-irradiation. Nature 210 :325-326 . Pollard, E. C., and P. M. Achey . 1966 . The action of ionizing radiation on postirradiation synthesis and degradation of DNA: in Escherichia coli 15 T-L - . Radiation Research 27:419-433. Pollard, E. C ., M. J. Ebert, C. Miller, K. Kolacz, and T. F . Barone . 1964 . Ionizing radiation: Effect of irradiated medium on synthetic processes. Science 147:1045-1047. Pomper, S. 1965. The physical environment for fungal growth. IV . Effect 1 \ of radiation, p. 595-599. !!! G. C . Ainsworth and A. S. Sussman (eds.). The Fungi. An Advanced Treatise. Vol. I. A cademic Press, New York. Pomper, S., and K. C. Atwood . 1955. Radiation studies on fungi, p. 431- 453. !!! A. Hollaender (ed.). Radiation biology. Vol. II. McGraw Hill Book Co., New York. Raper, K. B., and C. Thom. 1949. A manual of the Penicillia. Williams & Wilkins Co . , Baltimore, Maryland. 875 p. Rayman, M. M. , and A. F. Byrne. 1957. Action of ionizing radiations on microorganisms, p . 208-224 . !!! The U. S. Army Quartermaster Corps. (ed.). Radiation preservation of food . u. s; Government Printing Office, Washington, D. C. Roberts, R . B . , and E. Aldous. 1949 . Recovery from ultraviolet irradiation in Escherichia coli. Journal of Bacteriology 57 :363-375. Romani, R . J. 1966. Radiobiological parameters in the irradiation of fruits and vegetables . Advances in Food Research 15:57-103. Salunkhe, D. K. 1961. Gamma radiation effects on fruits and vegetables . Economic Botany 15:28-56. Schmidt , G., and S. J. Thannhauser . 1945 . A method for the determination of desoxyribonucleic acid, ribonucleic acid, and phosphoproteins in animal tissues. Journal of Biological Chemistry 161:83-89. \..,.. 81 Shaffer, C. R., and R. A. McGrath. 1965 . The effects of X-irradiation on the DNA of Escherichia coli. Experimental Cell Research 39:604-608 . Sharma, R. K. 1968. Effects of gamma radiation and its interactions with sodium halides on rainbow trout. Unpublished PhD dissertation . Utah State University Library, Logan, Utah. 107 p. Silverman, G. J ., N. S. Davis, and S. A. Goldblith. 1963. Modification of radiolethality by Vitamin K5 and certain analogs in model systems and in foods. Journ a l of Food Science 28:687-691. Siu, R . G. H., and G. R. Mandels. 1957 . Beginning of radiobiology, p. 31-34. !!l The U. S. Army Quartermaster Corps. (ed.). Radiation Preservation of Food. U. S, Government Printing Office, Washington, D. C. Smith, E. C. 1936. The effects of radiation on fungi, p. 889-918. !!l B. M. Dugger (ed.). Biological Effects of Radiation. Vol. 2. McGraw-Hill Book Co. , New York. Sommer, N. F., M. T. Creasy, R. J. Romani, and E . C. Maxie. 1963a. Recovery of gamma irradiated Rhizopus stolonifer sporangiospores during autoinhibition of germination . Journal of Cellular and Comparative Physiology 61:93-98. Sommer, N. F., M. T. Creasy, R. J •. Rom ani, and E. ·c. Maxie. 1963b . Production of pectol ytic enzymes by Rhizopus stolonifer sporangio spores after "lethal" gamma irradiation. Applied Microbiology 11 :463-466. Sommer, N. F., and R. J. Fortlage . 1966 . Ionizing radiation for control of post-harvest disease of fruits and vegetables. Advances in Food Research 15:147-193. Sommer, N. F., R. J . Fortlage, P. M. Buckley, and E. C. Maxie. 1967. Radiation-heat synergism for inactivation of market disease fungi of stone fruits. Phytopathology 57:428-433. Sommer, N. F., E. C. Maxie, and R. J. Fortlage. 1964. Quantitative dose-response of Prunus fruit decay fungi to gamma irradiation . Radiation Botany 4:309-316 . Stapleton, G. E. 1955. Factors modifying sensitivity of bacteria to ionizing radiations . Bacteriological Reviews 19:26-32. 82 Stapleton, G. E . , D. Billen, and A. Hollaender. 1953 . Recovery of X-irradiated bacteria at sub-optimal incubation temperatures. Journ a l of Cellular and Comparative Physiology 41:345-358 . Stapleton, G. E., A. J. Sbarra, and A. Hollaender . 1955. Some nutritional a spects of bacterial recovery from ioni zing radiations. Journal of Bacteriology 70:7-14. Stuy, J. H. 1961. Studies on the r adiation inactivation of microorganisms. VII. Nature of the X-ray-induced breakdown of deoxyribonucleic acid in Haemophilus influenzae. Radiation Research 14:56-65. Sussman, A. S., and H. D. Halvorson. 1966 . Spores: Their dormancy and germination. Harper & Row, Publishers, New York. 354 p. Szybalski, W. 1967. Molecular events resulting in radiation injury, repair, and sensitization of DNA. Radiation Research Supplement 7:147- 159. Tischer, R. G., and G. W. Kurt z . 1957. Mechanism of action of ioni zing r adi ations on living matter , p. 208-224. In U. S. Army Quarter master Corps. (id.) . Radiation preservation of food . U. S. ~vern- · ment Printing Office, Washington, D. C. U. S. Department of Agriculture. 1965 . Losses in Agriculture. Agriculture Handbook No .. 291. U. s. Government Printing Office, Washington, D. C. Watanabe, I. , and S. Akada . 1968. Deoxyribonucleic acid synthesis during the first post-irradiation life cycle of lethally irradiated cultured mammalian cells (L5178Y). Radiation Research 35:202-212. Wood, R . K. S. 1960 . The pathogen, p. 233-272. !!! J. G. Horsfall and A. E. Dimond(eds.), Plant pathology. An Advanced Treatise . Vol. II . A cade mic Press, New York. 83 APPENDIX 84 Table 8. The formulae of the culture media Ingredients per liter Czapek Solution Agar (CA) Czapek Dox Broth (CDB) Sucrose 30 g Sucrose 30 g Sodium Nitrate 2 g Sodium Nitrate 3 g Dipotassium Phosphate 1 g Dipotassium Phosphate 1 g Magnesium Sulfate 0. 5 g Magnesium Sulfate 0. 5 g Potassium Chloride 0. 5 g Potassium Chloride 0. 5 g Ferrous Sulfate 0. 01 g Ferrous Sulfate 0. 01 g Agar . 15 g Nutrient Agar (NA) Potato Dextrose Agar (PDA) Beef extract 3 g Potatoes, Infusion from 200 g Peptone 5 g Dextrose 2 0 g Agar 15 g Agar 15 g Sabouraud Dextrose Agar (SDA) Sabouraud Maltose Agar (SMA) Neopeptone 10 g Neopeptone 10 g Dextrose 40 g Maltose 40 g ' Agar 15 g Agar 15 g 85 Table 9. Analysis of variance and means for colony diameter of E· expansum grown on the irradiated apple juice agar after 5 days of incubation Degrees of Sum of Mean Source of variation freedom squares squares F-test va lue Total 23 0.3846 Treatments 2 0.0152 0.00760 0. 432 Experimental erro ,r 21 0.3694 0. 01759 LSD. 05 0.138 Table 10. Analysis of variance and means for colony diameter of E· expansum grown on the irradiated Czapek solution agar after 5 days of incubation Degrees of Sum of Mean Source of variation freedom squares squares F-test value Total 23 0.4924 Treatments 2 0. 2890 0. 14450 14.919** Experimental error 21 0.2034 0.00968 LSD. 05 0.102 LSD. 01 0.139 **Significant at 1 percent Level 86 Table 11. Analysis of variance and means for colony diameter of E_. expansum grown on the irradiated potato dextrose agar after 5 days of incubation Degrees of Sum of Mean Source of variation freedom squares squares F-test value Total 23 0.2100 Treatments 2 0.0225 0. 01125 1. 260 Experimental error 21 0.1875 0. 00893 LSD. 05 0. 098 Table 12. Analysis of variance and means for colony diameter of E_. expansum grown on irradiated Sabouraud dextrose agar after 5 days of incubation Degrees of Sum of Mean Source of variation freedom squares squares F-test value Total 23 0.1546 Treatments 2 0. 0159 0.00795 1. 052 Experimental error 21 0.1387 0. 00660 LSD. 05 0.085 87 Table 13. Analysis of variance and means for dry weight of E, ex2ansum grown in the irradiated Czapek Dox broth Degrees of Sum of Mean Source of variation freedom squares squares F-test value Total 89 18,669.69 Replications 4 4,432.32 1, 108. 08 Treatments 5 1,167.39 233.48 0. 533 Experimental error 20 8,766 . 77 438.34 Sampling error 60 4,303.21 71. 72 LSD. 05 15.94 Table 14. Analysis of variance and means for dry weight of E_. ex2ansum grown in the irradiated potato dextrose broth Degrees of Sum of Mean Source of variation freedom squares squares F-test value Total 35 883.45 Replications 719 . 13 719. 130 Treatments 5 12. 21 2.442 0.427 Experimental error 5 28. 60 5.720 Sampling error 24 123.51 5.146 LSD. 05 3.55 88 VITA Tsong-Wen Chou Candidate for the Degree of Doctor of Philosophy Dissertation: Effects of Gamma Radiation in Concurrence with Certain Environmental Conditions on Lethal and Physio-chemical Responses of Penicillium Expansum L. Major Field: Food Science and Technology Biographical Information : Personal Data : Born at Tokyo, Japan, February 10, 1933, son of Yin-Tsai and I-Fan Chou; married Fei-Mei Lee, November 12, 1958; four children-Ri-Jen, Ri-Chee, Ri-Pen and Ri-Shea. Education: Attended elementary school in Peitou, Taiwan; gradu ated from Provincial Keelung High School, Kee lung, Taiwan in 1951; received the Bachelor of Science degree from National Taiwan University, Taipei, Taiwan, with a major in Agricultural Chemistry, in 1955; completed requirements for the Doctor of Philosophy degree, with a major in Food Science and Technology, at Utah State University, Logan, Utah in 1969. Professional Experience: 1964 to present, graduate Besearch Assistant, Food Science and Technology Program, Utah State Unive;sity ;'° 1959-1964 -:Jresearch Qhemist and Bacteriologist, Research Division, Wei-Chuan Foods Corp., Taipei, Taiwan; 1957-1959, Quality Control Chemist, Formosan Rubber Corp., Taipei, Tifwan; 1956-1957, teacher, Keelung High School, Keelung, Taiwan. Professional Organizations: Society of the Sigma XI, Instltute ·of Food Technologists, Chinese Agricultural Chemical Society, American Institute of Biological Science . 89 Honors and Awards: J. Fish Smith Scholarship awarded to the outstanding foreign student in College of Agriculture, Utah State University, 1967; The Society of the Sigma Xi Grants-in-Aid of Research Award, 1968.