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The influence of gamma irradiation and sodium hypochlorite sterilization on maize seed microflora and germination
Food Microbiology, 1986, 3, 107-113
The influence of gamma irradiation and sodium hypochlorite sterilization on maize seed microflora and germination R. G. Cuero 1, J. E. Smith 1 and J. Lacey 2
1 Division of Applied Microbiology, Department of Bioscience and Biotechnology, University of Strathclyde, Glasgow, Scotland UK 2 Rothamstead Experimental Station, Harpenden, Herts, UK Received 26 November 1985 An effective irradiation method for the removal of contaminating micro-organisms of maize seeds without the loss of germination capacity is described. Doses of gamma irradiation up to 1800 Krad were applied to maize seeds previously adjusted to 15 or 22% water content. Micro-organisms differed in their resistance to irradiation with Fusarium spp. and yeast being the most resistant. A dose of 1 200 Krad eliminated all micro-organisms without adversely affecting seed germination (gnotobiotic seeds). Sodium hypochlorite treatment did not completely eradicate the maize microflora and also reduced germination.
Introduction Naturally grown cereal seeds will have variable levels of surface microbial contamination together with some internal contamination. When cereals are used as components of foods/feeds or beverages or when used for laboratory experimentation with pure microbial cultures it may be necessary to kill all contaminating micro-organisms. The most widely used methods involve some form of heat sterilization which although destroying all micro-organisms will also drastically change the physicochemical properties of the seeds and result in complete loss of seed viability (Pixton 1968). Heat treatment of seeds may increase water activity predisposing the substrate to more microbial contamination and possible mycotoxin production (Harwig and Chen 1974). Surface decontamination with chemicals, in particular sodium hypo chlorite, has been widely practised at laboratory levels. However, such methods seldom totally eliminate 0740-0020/86/020107 + 07 $02.00/0
surface micro-organisms and have little effect on internally located microorganisms (Flannigan 1977, Sauer and Burroughs 1980). The susceptibility of micro-organisms to radiation has been well-documented (Dunn et al. 1948, Bridges 1956, Yen et al. 1956, Kiss and Farkas 1977) and many studies have demonstrated the effectiveness of ionization radiation for preserving food, including grain, from microbial growth and insect infestation (IAEA-FAO 1970, 1978). Gamma irradiation of grain reduces fungal growth without greatly affecting respiration and chemical composition of the grain or producing off-flavours (Metta and Connor 1959, Kiss and Farkas 1977). The aim of the present investigation was to extend the work of Yen et al. (1956) and Kiss and Farkas (1977) with maize seeds, using a wider radiation dosage and variable water content of the seeds, and to attempt to obtain microorganism-free seeds which would still be © 1986 AcademicPress Inc. (London) Limited
108
R.G. Cuero et al.
viable a n d able to g e r m i n a t e . T h i s p r e s e n t s t u d y is p a r t of a l a r g e r p r o g r a m concerned w i t h the i n t e r a c t i o n b e t w e e n toxigenic moulds and o t h e r microo r g a n i s m s on a n d w i t h i n living cereal seeds.
Methods and Materials Maize samples North American hybrid maize seeds (Pioneer 3369a) were obtained from the Southern Regional Research Centre of the USDA, New Orleans, USA. The original water content of the samples was between 11 and 13%. Whole seeds were selected and 100 g samples adjusted to 15 or 22% water content respectively (Pixton and Warburton 1971). Samples were stored for two weeks in hermetically-sealed flasks at 5°C to equilibrate. The water activity was determined by an electronic dewpoint meter (Protometer DP 680).
Sterilization procedure Seed samples were treated with sodium hypochlorite or gamma irradiation.
Sodium hypochlorite treatment. Seed samples were immersed briefly in 75% v/v ethyl alcohol, transferred to 2% aqueous sodium hypochlorite for 2 min, washed in sterile distilled water and then surface dried on sterile filter papers (Jarvis 1978).
Gamma irradiation treatment. Gamma irrad-
before and after treatment with 2% aqueous sodium hypochlorite or gamma irradiation. The viability of the seeds was determined by placing 100 sound whole seeds per each moisture content on 50 sterile moist filter papers contained in sterile petri dishes (2 seeds/petri dish) in a Fison Environmental Cabinet, Model 280E/MU/R-IND(D)/1982 at 25°C, 90% RH (de Temple 1961, Christensen 1974). The percentage of seed viability was determined after 5 days.
Microbial examination The surface and internal microflora before and after sterilization treatment was determined by placing 30 seeds on potato dextrose and malt extract agars (5 seeds/plate) (180 seeds total) and incubating at 25°C for 10 days. Microbial colonies (moulds, yeasts and bacteria) were counted (Jarvis 1978). The surface and internal microflora of samples was also assessed using a dilution plate method (Lacey et al. 1980). The primary dilution was prepared with a Colworth Stomacher (Shorpe and Jackson 1972) to detect propagules of seed-borne fungi, especially Fusarium species that survived irradiation. Organisms were identified to genus level using standard monographs (Breed et al. 1959, Barnett 1960, Raper and Thorn 1949, Raper and Fennell 1965, Onions 1966, Lodder 1970, Booth 1971).
Results Seed germination
iation was carried out at 25°C, in a 6°Cc T r e a t m e n t of m a i z e seeds w i t h 2% source at the Scottish Research Reactor Cen- sodium h y p o c h l o r i t e solution r e s u l t e d in tre of the National Engineering Laboratory, a m a r k e d decrease in g e r m i n a t i o n capEast Kilbride, Scotland, UK. Doses of 30, 120, 240, 420, 480, 600, 1200 or 1800 Krad were acity (c. 25%) a t 15 a n d 22% m o i s t u r e used. All samples were irradiated in concentrations) (Table 1). polyethylene bags and kept at 25°C for G a m m a i r r a d i a t i o n t r e a t m e n t s up to approximately 30 h until used. Radiation 600 K r a d m o s t l y i m p r o v e d t h e f r e q u e n c y doses were measured with a Perspex Dose of g e r m i n a t i o n w h e n c o m p a r e d to unMeter (IAEA 1977). All experiments were repeated three times t r e a t e d controls (Table 1). T h e r e w a s no and in each 2 x 50 g replicates of grain g e r m i n a t i o n a f t e r t r e a t m e n t w i t h 1800 received each dose. To determine the long K r a d . term effects of irradiation, microbiological examination was repeated after 30 days Influence of sodium hypochlorite on storage at 5°C. maize microflora
Determination of seed viability Seed viability was determined by incubation of 300 seeds (three experiments, two replicates, 50 seeds/rep) on sterile wet filter paper
T h e p e r c e n t a g e of seeds c o n t a m i n a t e d with different o r g a n i s m s at different w a t e r contents is s h o w n in T a b l e 2. T r e a t m e n t w i t h sodium hypochlorite
109
Gamma irradiation on maize seed
Table 1. The germination of maize seeds at 15 and 22% moisture content after sodium hypochlorite or g a m m a irradiation treatment. Germination Treatment
(%)a
15% MC
22% MC
Sodium hypochlorite
59
55
G a m m a Irradiation (Krad) from 30 to 360 420 480 540 600 1200 1800
95 90 95 86 86 75 0
92 90 95 83 82 72 0
Untreated
79
73
• On wet filter paper, mean of three experiments, after 5 days at 25°C, 90% RH. b Results are derived from three experiments, each experiment with a total of 100 seeds (2 seeds/wet filter paper), over 5 days at 25°C and 90% RH.
Table 2. Effect of s o d i u m hypochlorite on the percentage of seeds contaminated with different organisms at different water contents. 15% MC
Treatment
Frequency (%)a Colony type
22% MC
070b
Frequency (%) Colony type
Sodium hypochlorite
17
Fusarium spp. Aspergillus spp. Penicillium spp. Saccharornyces spp. Trichoderma spp.
62 18 10 5 5
20
Untreated
93
Fusarium spp.
40 18 17 15 5 5
94
Phycomycetes Aspergillus spp. Penicillium spp. Saccharomyces spp. Bacillus spp.
Fusarium spp. AspergiUus spp. Penicillium spp. Saccharomyces spp. Trichoderma spp. Cladosporium spp. Fusariurn spp. Phycomycetes
Aspergillus spp. PeniciUiurn spp. Saccharomyces spp. Bacillus spp.
%
71 9 5 10 3 2 40 20 15 13 7 5
• Frequency of isolation from 180 seeds (two replicates, three experiments, 30 seeds per experiment, two seeds per plate. b Relative abundance of different colony types. Incubation at 25°C for days.
110
R.G. Cuero et al.
40-
15%
22%
~
~
35
15%
22%
15%
22%
30 15%
-
22%
15%
22%
i5% 22%
25
l0
t,I
No tmotmenl ~
le
30Krod
=4 ~
120Krod ~
Je
N-- 2 4 0 Kind -i~ 14-- 4 2 0 K i n d --I,4 k i 6 O O K K x I ~ t,,--1200 K r o d - ~ IfradlahOn t re,alrnent
Fig. 1. Effect or irradiation treatment on the frequency of isolation of different colony types from maize of different water constants. (~: Aspergillus sp.; l : Bacillus sp.; []: Fusarium sp.; []: Penicillium sp.; []: Phycomycetes sp.; [-]: Saccharomyces sp. ).
gave much reduced counts at both water contents. In particular, the Phycomycete fungi and Bacillus spp. were eliminated and Fusarium spp. much reduced. Aspergillus, Penicillium and Saccharomyces spp. were less affected by sodium hypochlorite treatment while limited numbers of Trichoderma cladosporium and spp. appeared in treated samples.
Influence of irradiation on maize microflora The frequency of isolation of different organisms from maize of different water contents treated with different dosages of irradiation is shown in Fig. 1. Increasing dosages of irradiation from 30 Krad resulted in decreasing frequencies of micro-organisms up to 1200 Krad where no micro-organisms survived the treat-
ment. At 120 and 240 Krad, microorganisms were more susceptible at 22% moisture content than at 15%. The degree of susceptibility of microorganisms to irradiation differed between organisms. The aspergilli and penicilli were particularly susceptible and were eliminated by 120 Krad. The Phycomycetes spp. occurred up to 120 Krad but were eliminated beyond this level. Bacillus spp. showed significantly higher frequencies at levels of irradiation up to 420 Krad but were eliminated at 600 Krad. Yeast and Fusarium spp. were particularly resistant to irradiation and were only eliminated by 1200 Krad. When irradiated seeds (up to 600 Krad) were stored at 5°C for 30 days and then monitored for microbial frequencies there were slight increases in Fusarium
Gamma irradiation on maize seed
111
(3%), Penicillium (2.5%) and yeast (2%) tion (Bottomley et al. 1950, Prentice when compared with analysis immedi- 1962, Harrison and Perry 1976). Thus, in ately following irradiation. However, the present study the reduction in microthere was no microbial recovery from bial activity resulting from radiation seeds receiving dosages of 1200 and 1800 treatment could have stimulated germiKrad. nation. Sodium hypochlorite markedly reDiscussion duced germination at both moisture The present study established in maize levels and probably caused a toxic effect that a specific radiation dosage (1200 on the germination process. Although Krad) can eliminate all seed borne sodium hypochlorite reduced the nummicro-organisms while retaining the bers of contaminating micro-organisms ability of the majority of the seeds to as has previously been shown for many germinate; it also showed specific dif- types of seeds (Flannigan 1969, Burferential effects of the irradiation dosage roughs and Sauer 1971, Christensen and on each micro-organism type. Previously Mirocha 1976, Mislivec et al. 1983) the Webb et al. (1959) found that doses up to composition of the microflora was 1000 Krad were necessary to inactivate strongly influenced. In particular, moulds in ground maize while Kiss and Fusarium spp. formed a higher proFarkas (1977) found that doses as low as portion of the population while the 0.75 Krad retarded fungal growth in phycomycete fungi were totally eradimaize cobs. In wheat Aspergillus, Rhizo- cated. Micro-organisms varied in their pus and Absidia spp. were eliminated by sensitivity to gamma irradiation. In parexposure to 300 Krad and Fusarium spp. ticular, the Phycomycetes, Penicilium by 1000 Krad (Mohyuddin and Skoropad and Aspergillus spp. were much more 1970). However, none of these studies sensitive than Fusarium, Saccharoconsidered the effect of gamma irradia- myces spp. and Bacillus spp. (cf. Webb et tion on seed viability. Although Yen et al. 1959, Mohyuddin and Skoropad 1970, al. (1956) obtained microbial-free wheat Ito et al 1973, Hartung et al. 1973). seeds by irradiation the resultant seeds However, the present results on sensiwere non-viable. Doses of irradiation tivity of some toxigenic fungi to irradiafrom 30 to 480 Krad appeared to stimu- tion is in agreement with Ingram (1975). late germination of maize seeds. Stimu- Sensitivity to irradiation may also be lation of germination by irradiation influenced by water content. Thus, in the could be the result of breaking dormancy present study, most micro-organisms either through alterations of the outer were more sensitive to 240 and 420 Krad layers of the seeds where dormancy at 22% moisture level than at 15%, a factors are assumed to reside or by result in contrast to previous findings degradation of germination inhibitors (Webb et al. 1959, Mohyuddin and Skothrough slow metabolite changes (Amen ropad 1970, Ito et al. 1972, Kiss and 1964, McLeod 1967, Black 1970). Also, Farkas 1977). The present results may radiolytic products such as hydrogen arise from the long equilibrium period at peroxide, could contribute to breaking of 5°C prior to irradiation which could have dormancy (Heydecker 1969). Seed micro- generated more radio-sensitive preorganisms could also adversely affect germination structures in micro-organseed germination. Several studies have isms, particularly fungi. demonstrated the adverse effects of The utility of irradiation procedures microbial colonization on seed germina- for producing gnotobiotic seeds could
112
R.G. Cuero et al.
have important uses not only in food and Acknowledgements plant biotechnology but also in studies The authors wish to thank the staffofthe on the microbial ecology of seeds. Scottish Reactor Centre, East Kilbride for carrying out radiation exposures.
References Amen, R. D. (1964) The concept of seed dormancy. WaUerstein Lab. Commun. 27, 7-18. Barnett, H. L. (1960)Illustratedgenera of imperfect fungi. Burgess Publishing Co. Minnesota. Black, M. (1970) Seed germination and dormancy. Sci. Prog. (London) 58, 379--393. Booth, C. (1971) The genus Fasarium, Kew, Commonwealth Mycological Institute. Bottomley, R. A., Christensen, C. M. and Cedes, W. F. (1950) The influence of various temperature, humidity and oxygen concentrations on mold growth and biochemical changes in stored yellow corn. Cer. Chem. vol. 4, July 261-276. Breed, R. S., Murray, E. G. D. and Smith, M. R. (1959) Bergey's Manual: Determinative Bacteriology Baltimore, The Williams and Wilkins Co. Bridges, A. E., Olivo, J. P. and Chandler, V. L. (1956) Relative resistance of micro-organisms to cathode rays. II. Yeasts and Molds. Appl. Microbiol. 4, 147-149. Burroughs, R. and Sauer, D. B. (1971) Growth of fungi in sorghum grain stored at high moisture contents. Phytopathology. 61,767-772. Christensen, C. M. (1974) Storage of cereal grains and their products. American Association of Cereal Chemists Inc., St Paul, Minnessota. Christensen, C. M. and Mirocha, C. J. (1976) Relation of relative humidity to the invasion of rough rice by Aspergillus parasiticus. Phytopathology. 66, 204--205. de Temple (1961) Routine methods for determining the health condition of seeds in the seed testing station. Proc. Seed Test Assoc. 26, 27-60. Dunn, C. G., Campbell, W. L., Fram, H. and Hutchins, A. (1948) Biochemical and photo-chemical effects of high energy, electrostatically produced roentgen rays and cathode rays. J. Appl. Physics 19, 605-616. Flannigan, B. (1969) Microflora of dried barley grain. Trans. Br. Mycol. Soc. 53, 371-379. Flannigan, B. (1977) Enumeration of fungi and assay for ability to degrade structural and storage components of grain. In Biodeterioration Investigation Techniques. Walters, A. H. (Ed.). London, Applied Science Publishers Ltd. Hartung, T. E., BuUerman, L. B., Arnold, R. G. and Heidelbargh, D. (1973) Application of low dose irradiation to a fresh bread system for space flights. J. Food Sci. 38, 129-132. Harwig, J. and Chen, Y. K. (1964). Some conditions favouring production of ochratoxin A and citrinin by Penicillium viridicatum in wheat and barley. Can. J. Plant Sci. 54, 17-22. Heydecker, W. (1969) The vigour of seeds: a review. Proc. Int. Seed Testing Assoc. 34,201-219. IAEA-FAO (1970) Training manual on food irradiation technology and techniques. Technical Reports. Series No. 114, IAEA, Vienna. IAEA (1977) Manual of food irradiation dosimetry. Technical Reports Series, No. 178. IAEA, Vienna. IAEA-FAO (1978) Food preservation by irradiation. Vol. I. Proceedings of a Symposium, Wageningen, 21-25 Nov. 1977. IAEA, Vienna. Ingrain, M. (1975) Technical Report series. International Project in the Field of Food Irradiation. IFPR 33, 14. Ito, H., Iizuka, H. and Sato, T. (1973) Identificationof osmophilic Aspergillus spp. isolated from rice and their radiosensitivity.Agr. Biol. Chem. 37, 789-798. Jarvis, B. (1978) Methods for detecting fungi in foods and beverages. In Food and beverage mycology. Beauchat, L. R. (Ed.) Wesport, Connecticut, Avi Publishing Co., Inc. Kiss, I. and Farkas, J. (1977) The storage of wheat and corn of high moisture content as affected by ionizing radiation. Acta Alimentaria 6, 193--214. Lacey, J., Hill, S. T. and Edwards, M. A. (1980) Micro-organisms in stored grains: their enumeration and significance. Trop. Stored Prod. Inf. 39. Lodder, J. (1970) The yeasts: a taxonomy study. 2nd Rev. North Holland Publishers. Macleod, A. M. (1967) The physiology of malting: a review. J. Int. Brew. (London) 73, 146-162.
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Metta, V. Ch. and Connor, J. (1959) Biological value of g a m m a irradiated corn protein and wheat gluten. Agric. Food Chem. 7, Feb. 131-133. Mislivic, P. B., Bruce, V. R. and Ginson, R. (1983) Incidence of toxigenic and other molds in green coffee beans. J. Food Protect. 46, 969-973. Mohyuddin, M. and Skoropad, P. (1970) Effect of S°Co g a m m a irradiation on the survival of some samples of each of three differentgrains of wheat. Can. J. Bot. 48, 217-219. Onions, A. H. (1966) Description of pathogenic fungi and bacteria. Set 10, No. 91-100. C M I Surrey, England. Pixton, S. W. (1968) The effectof heat treatment on the moisture content, relative humidity equilibrium relationship of Manitoba wheat. J. Stored Prod. Res. 4, 267-280. Pixton, S. W. and Warburton, S. (1971) Moisture content relative humditity equilibrium at different temperatures of some oilseeds of economic importance. J. Stored Prod Res. 7, 261-269. Prentice, N. (1962). Partial purification of a metabolite produced by Fusarium moniliforme which inhibitsthe utilisationof oxygen by germinating barley. Physiol. Plan. 15, 693--699. Raper, K. B. and Fennell, D. I. (1965) The genus Aspergillus. Baltimore, The Williams and Wilkins Co. Raper, K. B. and Thorn, C. (1949) A manual of the PeniciUia. London, Bailliere,Tindall and Cox.
Sauer, D. B. and Burroughs, R. (1980) Fungal growth, aflatoxin production and moisture equilibration in mixtures of wet and dry corn. Phytopathology. 70, 516-521. Shorpe, A. N. and Jackson, A. K. (1972) Stomaching: a new concept in bacteriologicalsample preparation. Appl. Microbiol. 124, 175-178. Webb, B.D., Thiers, H. D. and Richardson, L. R. (1959) Studies in feed spoilage. Inhibition of mold growth by g a m m a radiation. AppL Microbiol., 7, 329-33. Yen, Y., Milner, M. and Ward, H. T. (1956) Treatment of wheat with ionizing radiation. II. Effect on respiration and other indices of storage deterioration.Food Technol. 10,411--415.
The influence of gamma irradiation and sodium hypochlorite sterilization on maize seed microflora and germination R. G. Cuero 1, J. E. Smith 1 and J. Lacey 2
1 Division of Applied Microbiology, Department of Bioscience and Biotechnology, University of Strathclyde, Glasgow, Scotland UK 2 Rothamstead Experimental Station, Harpenden, Herts, UK Received 26 November 1985 An effective irradiation method for the removal of contaminating micro-organisms of maize seeds without the loss of germination capacity is described. Doses of gamma irradiation up to 1800 Krad were applied to maize seeds previously adjusted to 15 or 22% water content. Micro-organisms differed in their resistance to irradiation with Fusarium spp. and yeast being the most resistant. A dose of 1 200 Krad eliminated all micro-organisms without adversely affecting seed germination (gnotobiotic seeds). Sodium hypochlorite treatment did not completely eradicate the maize microflora and also reduced germination.
Introduction Naturally grown cereal seeds will have variable levels of surface microbial contamination together with some internal contamination. When cereals are used as components of foods/feeds or beverages or when used for laboratory experimentation with pure microbial cultures it may be necessary to kill all contaminating micro-organisms. The most widely used methods involve some form of heat sterilization which although destroying all micro-organisms will also drastically change the physicochemical properties of the seeds and result in complete loss of seed viability (Pixton 1968). Heat treatment of seeds may increase water activity predisposing the substrate to more microbial contamination and possible mycotoxin production (Harwig and Chen 1974). Surface decontamination with chemicals, in particular sodium hypo chlorite, has been widely practised at laboratory levels. However, such methods seldom totally eliminate 0740-0020/86/020107 + 07 $02.00/0
surface micro-organisms and have little effect on internally located microorganisms (Flannigan 1977, Sauer and Burroughs 1980). The susceptibility of micro-organisms to radiation has been well-documented (Dunn et al. 1948, Bridges 1956, Yen et al. 1956, Kiss and Farkas 1977) and many studies have demonstrated the effectiveness of ionization radiation for preserving food, including grain, from microbial growth and insect infestation (IAEA-FAO 1970, 1978). Gamma irradiation of grain reduces fungal growth without greatly affecting respiration and chemical composition of the grain or producing off-flavours (Metta and Connor 1959, Kiss and Farkas 1977). The aim of the present investigation was to extend the work of Yen et al. (1956) and Kiss and Farkas (1977) with maize seeds, using a wider radiation dosage and variable water content of the seeds, and to attempt to obtain microorganism-free seeds which would still be © 1986 AcademicPress Inc. (London) Limited
108
R.G. Cuero et al.
viable a n d able to g e r m i n a t e . T h i s p r e s e n t s t u d y is p a r t of a l a r g e r p r o g r a m concerned w i t h the i n t e r a c t i o n b e t w e e n toxigenic moulds and o t h e r microo r g a n i s m s on a n d w i t h i n living cereal seeds.
Methods and Materials Maize samples North American hybrid maize seeds (Pioneer 3369a) were obtained from the Southern Regional Research Centre of the USDA, New Orleans, USA. The original water content of the samples was between 11 and 13%. Whole seeds were selected and 100 g samples adjusted to 15 or 22% water content respectively (Pixton and Warburton 1971). Samples were stored for two weeks in hermetically-sealed flasks at 5°C to equilibrate. The water activity was determined by an electronic dewpoint meter (Protometer DP 680).
Sterilization procedure Seed samples were treated with sodium hypochlorite or gamma irradiation.
Sodium hypochlorite treatment. Seed samples were immersed briefly in 75% v/v ethyl alcohol, transferred to 2% aqueous sodium hypochlorite for 2 min, washed in sterile distilled water and then surface dried on sterile filter papers (Jarvis 1978).
Gamma irradiation treatment. Gamma irrad-
before and after treatment with 2% aqueous sodium hypochlorite or gamma irradiation. The viability of the seeds was determined by placing 100 sound whole seeds per each moisture content on 50 sterile moist filter papers contained in sterile petri dishes (2 seeds/petri dish) in a Fison Environmental Cabinet, Model 280E/MU/R-IND(D)/1982 at 25°C, 90% RH (de Temple 1961, Christensen 1974). The percentage of seed viability was determined after 5 days.
Microbial examination The surface and internal microflora before and after sterilization treatment was determined by placing 30 seeds on potato dextrose and malt extract agars (5 seeds/plate) (180 seeds total) and incubating at 25°C for 10 days. Microbial colonies (moulds, yeasts and bacteria) were counted (Jarvis 1978). The surface and internal microflora of samples was also assessed using a dilution plate method (Lacey et al. 1980). The primary dilution was prepared with a Colworth Stomacher (Shorpe and Jackson 1972) to detect propagules of seed-borne fungi, especially Fusarium species that survived irradiation. Organisms were identified to genus level using standard monographs (Breed et al. 1959, Barnett 1960, Raper and Thorn 1949, Raper and Fennell 1965, Onions 1966, Lodder 1970, Booth 1971).
Results Seed germination
iation was carried out at 25°C, in a 6°Cc T r e a t m e n t of m a i z e seeds w i t h 2% source at the Scottish Research Reactor Cen- sodium h y p o c h l o r i t e solution r e s u l t e d in tre of the National Engineering Laboratory, a m a r k e d decrease in g e r m i n a t i o n capEast Kilbride, Scotland, UK. Doses of 30, 120, 240, 420, 480, 600, 1200 or 1800 Krad were acity (c. 25%) a t 15 a n d 22% m o i s t u r e used. All samples were irradiated in concentrations) (Table 1). polyethylene bags and kept at 25°C for G a m m a i r r a d i a t i o n t r e a t m e n t s up to approximately 30 h until used. Radiation 600 K r a d m o s t l y i m p r o v e d t h e f r e q u e n c y doses were measured with a Perspex Dose of g e r m i n a t i o n w h e n c o m p a r e d to unMeter (IAEA 1977). All experiments were repeated three times t r e a t e d controls (Table 1). T h e r e w a s no and in each 2 x 50 g replicates of grain g e r m i n a t i o n a f t e r t r e a t m e n t w i t h 1800 received each dose. To determine the long K r a d . term effects of irradiation, microbiological examination was repeated after 30 days Influence of sodium hypochlorite on storage at 5°C. maize microflora
Determination of seed viability Seed viability was determined by incubation of 300 seeds (three experiments, two replicates, 50 seeds/rep) on sterile wet filter paper
T h e p e r c e n t a g e of seeds c o n t a m i n a t e d with different o r g a n i s m s at different w a t e r contents is s h o w n in T a b l e 2. T r e a t m e n t w i t h sodium hypochlorite
109
Gamma irradiation on maize seed
Table 1. The germination of maize seeds at 15 and 22% moisture content after sodium hypochlorite or g a m m a irradiation treatment. Germination Treatment
(%)a
15% MC
22% MC
Sodium hypochlorite
59
55
G a m m a Irradiation (Krad) from 30 to 360 420 480 540 600 1200 1800
95 90 95 86 86 75 0
92 90 95 83 82 72 0
Untreated
79
73
• On wet filter paper, mean of three experiments, after 5 days at 25°C, 90% RH. b Results are derived from three experiments, each experiment with a total of 100 seeds (2 seeds/wet filter paper), over 5 days at 25°C and 90% RH.
Table 2. Effect of s o d i u m hypochlorite on the percentage of seeds contaminated with different organisms at different water contents. 15% MC
Treatment
Frequency (%)a Colony type
22% MC
070b
Frequency (%) Colony type
Sodium hypochlorite
17
Fusarium spp. Aspergillus spp. Penicillium spp. Saccharornyces spp. Trichoderma spp.
62 18 10 5 5
20
Untreated
93
Fusarium spp.
40 18 17 15 5 5
94
Phycomycetes Aspergillus spp. Penicillium spp. Saccharomyces spp. Bacillus spp.
Fusarium spp. AspergiUus spp. Penicillium spp. Saccharomyces spp. Trichoderma spp. Cladosporium spp. Fusariurn spp. Phycomycetes
Aspergillus spp. PeniciUiurn spp. Saccharomyces spp. Bacillus spp.
%
71 9 5 10 3 2 40 20 15 13 7 5
• Frequency of isolation from 180 seeds (two replicates, three experiments, 30 seeds per experiment, two seeds per plate. b Relative abundance of different colony types. Incubation at 25°C for days.
110
R.G. Cuero et al.
40-
15%
22%
~
~
35
15%
22%
15%
22%
30 15%
-
22%
15%
22%
i5% 22%
25
l0
t,I
No tmotmenl ~
le
30Krod
=4 ~
120Krod ~
Je
N-- 2 4 0 Kind -i~ 14-- 4 2 0 K i n d --I,4 k i 6 O O K K x I ~ t,,--1200 K r o d - ~ IfradlahOn t re,alrnent
Fig. 1. Effect or irradiation treatment on the frequency of isolation of different colony types from maize of different water constants. (~: Aspergillus sp.; l : Bacillus sp.; []: Fusarium sp.; []: Penicillium sp.; []: Phycomycetes sp.; [-]: Saccharomyces sp. ).
gave much reduced counts at both water contents. In particular, the Phycomycete fungi and Bacillus spp. were eliminated and Fusarium spp. much reduced. Aspergillus, Penicillium and Saccharomyces spp. were less affected by sodium hypochlorite treatment while limited numbers of Trichoderma cladosporium and spp. appeared in treated samples.
Influence of irradiation on maize microflora The frequency of isolation of different organisms from maize of different water contents treated with different dosages of irradiation is shown in Fig. 1. Increasing dosages of irradiation from 30 Krad resulted in decreasing frequencies of micro-organisms up to 1200 Krad where no micro-organisms survived the treat-
ment. At 120 and 240 Krad, microorganisms were more susceptible at 22% moisture content than at 15%. The degree of susceptibility of microorganisms to irradiation differed between organisms. The aspergilli and penicilli were particularly susceptible and were eliminated by 120 Krad. The Phycomycetes spp. occurred up to 120 Krad but were eliminated beyond this level. Bacillus spp. showed significantly higher frequencies at levels of irradiation up to 420 Krad but were eliminated at 600 Krad. Yeast and Fusarium spp. were particularly resistant to irradiation and were only eliminated by 1200 Krad. When irradiated seeds (up to 600 Krad) were stored at 5°C for 30 days and then monitored for microbial frequencies there were slight increases in Fusarium
Gamma irradiation on maize seed
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(3%), Penicillium (2.5%) and yeast (2%) tion (Bottomley et al. 1950, Prentice when compared with analysis immedi- 1962, Harrison and Perry 1976). Thus, in ately following irradiation. However, the present study the reduction in microthere was no microbial recovery from bial activity resulting from radiation seeds receiving dosages of 1200 and 1800 treatment could have stimulated germiKrad. nation. Sodium hypochlorite markedly reDiscussion duced germination at both moisture The present study established in maize levels and probably caused a toxic effect that a specific radiation dosage (1200 on the germination process. Although Krad) can eliminate all seed borne sodium hypochlorite reduced the nummicro-organisms while retaining the bers of contaminating micro-organisms ability of the majority of the seeds to as has previously been shown for many germinate; it also showed specific dif- types of seeds (Flannigan 1969, Burferential effects of the irradiation dosage roughs and Sauer 1971, Christensen and on each micro-organism type. Previously Mirocha 1976, Mislivec et al. 1983) the Webb et al. (1959) found that doses up to composition of the microflora was 1000 Krad were necessary to inactivate strongly influenced. In particular, moulds in ground maize while Kiss and Fusarium spp. formed a higher proFarkas (1977) found that doses as low as portion of the population while the 0.75 Krad retarded fungal growth in phycomycete fungi were totally eradimaize cobs. In wheat Aspergillus, Rhizo- cated. Micro-organisms varied in their pus and Absidia spp. were eliminated by sensitivity to gamma irradiation. In parexposure to 300 Krad and Fusarium spp. ticular, the Phycomycetes, Penicilium by 1000 Krad (Mohyuddin and Skoropad and Aspergillus spp. were much more 1970). However, none of these studies sensitive than Fusarium, Saccharoconsidered the effect of gamma irradia- myces spp. and Bacillus spp. (cf. Webb et tion on seed viability. Although Yen et al. 1959, Mohyuddin and Skoropad 1970, al. (1956) obtained microbial-free wheat Ito et al 1973, Hartung et al. 1973). seeds by irradiation the resultant seeds However, the present results on sensiwere non-viable. Doses of irradiation tivity of some toxigenic fungi to irradiafrom 30 to 480 Krad appeared to stimu- tion is in agreement with Ingram (1975). late germination of maize seeds. Stimu- Sensitivity to irradiation may also be lation of germination by irradiation influenced by water content. Thus, in the could be the result of breaking dormancy present study, most micro-organisms either through alterations of the outer were more sensitive to 240 and 420 Krad layers of the seeds where dormancy at 22% moisture level than at 15%, a factors are assumed to reside or by result in contrast to previous findings degradation of germination inhibitors (Webb et al. 1959, Mohyuddin and Skothrough slow metabolite changes (Amen ropad 1970, Ito et al. 1972, Kiss and 1964, McLeod 1967, Black 1970). Also, Farkas 1977). The present results may radiolytic products such as hydrogen arise from the long equilibrium period at peroxide, could contribute to breaking of 5°C prior to irradiation which could have dormancy (Heydecker 1969). Seed micro- generated more radio-sensitive preorganisms could also adversely affect germination structures in micro-organseed germination. Several studies have isms, particularly fungi. demonstrated the adverse effects of The utility of irradiation procedures microbial colonization on seed germina- for producing gnotobiotic seeds could
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have important uses not only in food and Acknowledgements plant biotechnology but also in studies The authors wish to thank the staffofthe on the microbial ecology of seeds. Scottish Reactor Centre, East Kilbride for carrying out radiation exposures.
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Metta, V. Ch. and Connor, J. (1959) Biological value of g a m m a irradiated corn protein and wheat gluten. Agric. Food Chem. 7, Feb. 131-133. Mislivic, P. B., Bruce, V. R. and Ginson, R. (1983) Incidence of toxigenic and other molds in green coffee beans. J. Food Protect. 46, 969-973. Mohyuddin, M. and Skoropad, P. (1970) Effect of S°Co g a m m a irradiation on the survival of some samples of each of three differentgrains of wheat. Can. J. Bot. 48, 217-219. Onions, A. H. (1966) Description of pathogenic fungi and bacteria. Set 10, No. 91-100. C M I Surrey, England. Pixton, S. W. (1968) The effectof heat treatment on the moisture content, relative humidity equilibrium relationship of Manitoba wheat. J. Stored Prod. Res. 4, 267-280. Pixton, S. W. and Warburton, S. (1971) Moisture content relative humditity equilibrium at different temperatures of some oilseeds of economic importance. J. Stored Prod Res. 7, 261-269. Prentice, N. (1962). Partial purification of a metabolite produced by Fusarium moniliforme which inhibitsthe utilisationof oxygen by germinating barley. Physiol. Plan. 15, 693--699. Raper, K. B. and Fennell, D. I. (1965) The genus Aspergillus. Baltimore, The Williams and Wilkins Co. Raper, K. B. and Thorn, C. (1949) A manual of the PeniciUia. London, Bailliere,Tindall and Cox.
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