International Journal of Food Mu'robtolagy, 7 (1988) 245 - 256 245 Elsevier

JFM 00MS5

Ecological determinants of mould growth in stored grain

N. Magan ~ and J. Lacey 2 ICranfield Institute of Technology, Cranfteld, Bedford, U.K. and

"Institute of Arable Crops Research, Rothamsted Experimental Statton, Harpenden, llertfordshire, U.K.

(;rain entering store carries a microflora of 'field' and 'storage' fungi. Field fungi require readily available water and therfore seldom develop in store. By contrast, storage fungi, especially Aspergilht~ spp., are able to grow at low water activities (a~, 0.70-0.75) enabling them to initiate grain spoilage. The ability of storage fungi to germinate, grow and sporulate in stored grain is dependent on the availability of water in the substrate, temperature and the intergranular gas composition. These factors may interact to have a profound influence on the initiation of spoilage of stored grain by fungi. An understanding of the ecological determinants of mould growth may help to develop improved and safer metht~ls of grain storage.

Key words: Water activity; Temperature: (;as composition; Germination: Sporulation: (;rain; Spoilage fungi

Introduction

Bulk grains are man-made ecological systems in which living, respiring grain interacts with both micro-organisms and the env i ronment (Sinha, 1973). Transpor ta - t ion and storage of bulk grains requires that both physical and microbial deteriora-

t ion is minimised, and part icular care is necessary when grain is moved through different climatic zones. Insects and mites can also cause serious deter iorat ion and often interact with micro-organisms (Mills, 1983; see Dunkel , this volume).

Mould growth and spoilage of stored grain are de termined p redominan t ly by water content (or more precisely the availabil i ty of water), the range of con tamina t - ing fungi and how they interact with temperature and gas composit ion. Fungal activity can cause rapid deter iorat ion of grain, sometimes with spontaneous heating. It also leads to losses of dry matter, nutr i t ive value, and germinabi l i ty and to the product ion of mycotoxins.

A detailed under s t and ing of the way in which availabil i ty of water in the grain substrate, temperature and gas composi t ion of the in tergranular a tmosphere interact

Correspondence address: N. Magan, Biotechnology Centre, Cranfield Institute of Technology, Cranfield, Bedford MK43 OAL, U.K.

0168-1605/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

246

in their effect on the germination, growth and sporulation of fungi responsible for spoilage is important in determining how well grain will store.

Field and storage fungi

The fungi colonizing grain have been classified into two groups, known as field and storage fungi (Christensen and Kaufmann, 1969). Field fungi characteristically colonize the ripening grain and include Ahernaria, Cladosporium and Fusarium spp., but they seldom develop further in store. By contrast, storage fungi are present in low numbers before harvest but develop rapidly in store when conditions are suitable and are predominantly species of Aspergillus and Penicillium. At harvest, storage fungi may be present at low levels, but much is added during harvesting, drying and when grain is placed in contaminated stores (Lacey, 1971; Flannigan, 1978). An intermediate group has been proposed for fungi such as Fusarium spp., which can sometimes develop in moist grain in store (Pelhate, 1968). Unfortunately, this division is inappropriate in the humid tropics where Aspergillus and Penicillium species, especially A. flavus and A. niger can occur prior to harvest and form mycotoxins (Williams and McDonald, 1983; Hill et al., 1985).

Water activity and water potential

Although water content is easily determined, it gives little indication of the availability of water for microbial growth in the substrate and allows few compari- sons since the relationship between water content and availability differs with product. Water availability in hygroscopic materials can be measured as equilibrium relative humidity (ERH), water activity (aw) or water potential (q,). ERH is the relative humidity of the intergranular air in equilibrium with water in the grain substrate; a w is the ratio of vapour pressure of water over the substrate to that over pure water at the same temperature and pressure; and ~ is the sum of osmotic, matric and turgor potentials. ERH and aw are the same numerically except that ERH is expressed as a percentage (%) and aw as a decimal fraction of one. Water potential is measured in pascals (Pa). The relationship between a w and water potential is given by the equation:

d / = ( R T / V ) l oge aw

where R is the ideal gas constant, T is absolute temperature and V the volume of one mole of water (Griffin, 1981; Papendick and Mulla, 1986). Although water potential takes temperature into account and aw does not, both change with temperature at constant water content. An advantage of water potential is that the osmotic, matric and turgor components of the total water availability can be expressed separately, but in grain osmotic potential is the major, if not the only component. Water activity is used predominantly in the agricultural and food

247

industries while water potential is used in other areas including soil microbiology. There is pressure for all disciplines to use water potential as a measure of water availability (Ayres and Boddy, 1986) but the jusitification for this must be which measure has the greatest relevance to microbial growth. Water activity will be used in this paper as it is widely understood in the grain trade. For more detailed information on water potential the reader is referred to Griffin (1981) and Papen- dick and Mulla (1986).

Experimental systems used to study ecology of grain fungi

Culture studies

Culture media used to study the water relations of field and storage fungi have included gelatin, malt gelatin, malt agar, invert sugar media and wheat extract agar (Snow, 1949; Pelhate, 1968; Pitt and Christian, 1968; Ayerst, 1969; Magan and Lacey, 1984a). While early experimental systems were reviewed by Scott (1957) more recently Pelhate (1968), Ayerst (1969) and Magan and Lacey (1985) used small sealed Petri dish or test tube systems that could be examined without removing the cultures to avoid disturbing the environment. Relative humidity of media were controlled by equilibration for periods from 1-3 days to up to a month over saturated salt solutions, or by direct addition of solutes to media, e.g., NaCI, to modify a , down to 0.80 or fructose + glucose or glycerol down to 0.70 a,,. The direct modification of media by addition of solutes rather than by equilibration over saturated salt solutions is more rapid and more accurately controls aw but may introduce solute-specific effects. The reactions of fungi to different a,~ and tempera- ture combinations can be assessed either by the proportion of spores germinated, linear growth, or spore production.

Grain ~vstetra"

Natural, irradiated and autoclaved grain have all been used in studies of the ecology of grain fungi. Grain modified to the required a w calculated from water sorption curves and with or without inoculation with single or mixed inocula has been stored in Dewar flasks, laboratory silos, glass jars and, more recently, in bags of microporous film. The microporous film has advantages over other containers in that it allows exchange of air and moisture with the atmosphere of humidified environmental chambers and thus closer control than with other methods of controlling the environment in which grain is stored. Autoclaved grain has often been used but it differs from natural grain in its a , J w a t e r content relationship and its nutritional status. Gamma irradiation sterilizes grain without destroying germinability so probably causes fewer changes than autoclaving. A dose of 12 kGy is sufficient to sterilize maize without harming germination (Cuero et al., 1985) but doses for other grains have still to be determined.

248

Growth of fungi in experiments with grain can be assessed using a variety of methods but none is ideal. Methods requiring isolation in culture allow assessment only of those fungi which will grow on the culture medium and at the incubation temperature used. Dilution plating of grain washings or comminuted material allows assessment of the numbers of propagules of different species but favours heavily sporing, fast growing species and underestimates those that produce few spores or grow slowly. The method gives little idea of the extent of colonization within the grain. Direct plating of whole grains enables the proportion carrying a particular fungus either on the surface or, after surface sterilization, within the tissues to be assessed, but again fast-growing fungi are favoured. Assay of adenosine triphos- phate assesses microbial activity in grain while chitin or ergosterol analysis de- termines the amount of fungal biomass. None of these methods allows identification of different species. However, in the future, immunological assays may allow particular fungi or their metabolic products, e.g., mycotoxins, to be quantified and enable a better estimate of the biomass of different species colonizing grain under different conditions.

Interaction of water activity and temperature with germination, growth and sporula- tion of stored grain fungi

Germination

Fungai spores usually contaminate the surface of grain and water availability and temperature are crucial factors in determining their rate of germination and which species develop. These factors can be quantified in several ways, including (a) the minimum aw for germination (b) minimum and maximum temperature allowing germination (c) lag time for germination and (d) rate of germ tube elongation.

Storage fungi usually germinate at lower a,~ than field fungi. Thus, conidia of Alternaria and Cladosporium spp. require more than 0.85 a,, while those of Eurotium rubrum (Aspergillus ruber) can germinate at 0.70 a,~ and A. restrictus at 0.72 a,,. (Pitt, 1975; Magan and Lacey, 1984a). The minimum a,,. for germination and growth is markedly influenced by temperature, nutrition and the solute used in culture media to control a,~.. The xerophilic Penicillium spp. from corn can germinate with 0.81-0.83 aw at 16°C, 0.81 a,,. at 23°C and 0.83--0.86 at 3 0 o ( ̀ (Mislivec and Tuite, 1970). The minimum a,~ allowing germination usually occurs at the temperature allowing opt imum growth but this aw may sometimes be too low to allow subsequent growth.

The lag time before germination occurs increases with decreasing a~. At levels of high water availability (> 0.98 aw) this can range from a few hours to days, while at very low aw it can extend for months or years (Scott, 1957; Pitt, 1975). A. echinulatus (a member of the Aspergillus glaucus group) germinated only after 2 years at 0.64-0.66 aw and aleuriospores of Monascus bispor~" after 120 days at 0.61 aw (Pitt and Christian, 1968). Spore age and nutrition can also affect the lag time for germination (Snow, 1949).

249 3,0[ 280i • 0.90

,.-, 240 o 0.83

~ 2 0 0 ~ , o.8o

, , , 0 . 7 8

r,~ 80 ______----II 0.76

40 j l

I I i

2 4 6 8 10 1'2 T I M E [ D A Y S ]

Fig. 1. Effect of water activity (aw) on germ tube extension (p.m) of Eurotium repens at 30°C on wheat extract agar. • O, 0.90 aw; o o . 0.83 aw; • A. 0.80 aw; zx zx, 0.78 aw;

• m, 0.76 a w.

Rate of germ tube e longat ion at cons tan t t empera tu re usual ly decreases with decreas ing aw ( e . g . E . repens, Fig. 1) but increases to a m a x i m u m before sharp ly dec l in ing with increas ing t empera tu re at cons tan t a,~ (e.g. Penicillium brevicompac- tum, Fig. 2).

Growth

Knowing how aw and t empera tu re in teract to affect growth is par t i cu la r ly impor t an t in unde r s t and ing the ecology of indiv idual fungi and their in ter - re la t ion- ships. Not all fungi grow max ima l ly with m a x i m u m water avai labi l i ty (Fig. 3). Some species, e.g. Eurotium repens, E. amstelodami, Aspergillus niger and A. versicolor, grow best between 0 .90-0 .95 aw at o p t i m u m tempera tu res (Ayers t , 1969; Magan and Lacey, 1984a). Some Penicillium species also grow bet te r close to 0.98 a,~ than at 1.00 a w (Pit t and Hocking, 1977; Hock ing and Pitt, 1979).

Isolates of indiv idual fungal species differ l i t t le in the a w / t e m p e r a t u r e ranges pe rmi t t ing their growth and Pitt (1979) has been able to use growth rates at d i f ferent t empera tu res and aw as t axonomic cr i ter ia for Penicillium species. The

250

3 2 0

280

240

.=. -I-200

160 . - I

o0

~ 1 2 0

Q~

~ 8o

40

• 25 / /

/ / /

/-

oY~J ~ ' i ' ~ ' i ' lb ' 1'2

TIME [DAYS]

Fig. 2. Effect o f t e m p e r a t u r e o n ge rm tube ex tens ion ( v m ) of Pentctlltum brevtcompactum at 0.90 wa te r

ac t iv i ty o n w h e a t ex t r ac t aga r . • • , 25; o o , 20; • • , 30; ,', zx, 1 0 ° C .

responses of fungi to a,,. and temperature, can be summarized effectively in two-dimensional diagrams with a,~ and temperature as axes, clearly indicating the optimal and limiting conditions for growth and germination of each species (Fig. 4). While field fungi require at least 0.90 aw for growth some Aspergillus and Penicil- lium spp. have minima of 0.72 and 0.82 a,, respectively. The limits for growth are

3 r

0 7 0 0.80 0 9 0 1.0 Water ac t i v i t y ( aw)

Fig. 3. L inea r g r o w t h ( m m / d a y ) o f Aspergill~ am,*telodami (o ( *

o , 30 o C) a n d Pentcilltum pceum e , 25 o C) at d i f fe ren t wa te r act ivi t ies ( f rom M a g a n a n d Lacey, 1984a).

251

1.00

0 .90

0 .80

0 .70

1.00

]= 0 .90

:~ 0 .80

u 0 .70

1.oo

0.90

0.80

0.70

Al te rna r ia a l te rna ta ~ usarlum culmorum

Aspergi l lus candidus Eurotium repens

Penici l l ium aurant io gr iseum P. plceum

, /

, i i i

10 20 30 40 10 20 30 40

Temoerature (oc)

Fig. 4. Effect of water activity and temperature on growth rate of two field fungi. AspergtlhL~ and Penicdlium spp.. The numbers on the isopleths are growth rates in m m / d a y (adapted from Magan and

t.acey, 1984a)

usually assumed to be defined by the isopleth indicating growth rates of 0.1 m m / d a y although slower growth rates can be determined if longer incubation periods are used. Also, aw/ tempera ture responses may be modifed by changes in available nutrients and gas composition.

Some workers have found a significant correlation between the reciprocal of germination lag time with linear growth rate for A. restrict~ and A. l~,ersicolor over the ranges of a,~ and temperatures permitting growth (Ayerst, 1969: Smith and 11i11, 1982). However, growth conditions determined in laboratory studies may not ahvays indicate environmental conditions at which some species occur most abundantly in stored grain. For example, P. aurantiogriseum and P. t,iridicatum were found to be most abundant at 0 ° C and 1.00 a,~ and Eurotium (Aspergillus glaucus group) spp. at 30°C and 0.70 a~ in stored grain although in vitro culture studies show opt imum growth at 25°C and 1.00 a~, and at 30°C and 0.90 a~, respectively (Hill. 1979: Hill and Lacey, 1983). More information is perhaps required on the ability of fungi to grow at marginal a~ and temperature where a slight change in substrate water availability can allow the initiation of germination and subsequent growth.

252

Sporulation

The effects of a,, on sporulation of grain fungi have been little studied although the minimum a,, for anamorph and teleomorph formation of some Aspergillus species are known (Snow, 1949: Mislivec and Tuite. 1970; Pitt, 1975: Magan and Lacey, 1984a). Sporulation at low a~, is important in enabling fungi to complete life-cycles, to survive adverse conditions and to be spread by insects and mites. Depending on species, sporulation may occur at the minimum a,~ for growth or at a slightly higher a~, (Magan and Lacey, 1984a).

For Penicillium spp. isolated from corn, the minimum a,, for anamorph forma- tion changes with temperature (Mislivec and Tuite, 1970). For example, P. brevi- compacturn and P. aurantiogriseum produce conidia to a minimum of 0.86 a,, at 16 and 30°C but to 0.83 a,~ at 23°C. The lag times before conidiophores were formed were also similarly changed. P. citrinum, P. chrysogenum, P. frequentans and P. expansum behaved very similarly.

Production of cleistothecia and the development of ascospores are often much slower than conidiophore production and are often less tolerant of low a,,.. For instance, Emericella (Aspergilha~') nidulans produces conidia down to 0.85 a,, but ascosporcs to 0.995 a,~ only (Snow, 1949).

Effects of water activity, gas composition and temperature on growth of grain fungi

Changes in the concentrations of (-)2 and (702 in the intergranular atmosphere of grain bulks can be important in modifying fungal colonization during storage. Damp grain and its colonizing microflora respire and, if air exchange is restricted, CO, can accumulate and 02 decline sufficiently to inhibit fungal growth. This characteristic has been used to store damp grain in sealed and unsealed systems and also in the controlled atmosphere storage of grain (Shejbal, 1980: Hyde and Burrell, 1982) where CO 2, 02 and N: concentrations are manipulated artificially. Low temperature systems have also been developed utilizing SO 2 or NHs to inhibit microbial growth (Eckhoff et al., 1979: 1983). However, the inhibitory effects of gases on fungal growth, particularly at low a,~, have seldom been studied.

Fungi are usually considered to be obligate aerobes but the partial pressure of 02 necessary for their growth is often over-estimated. In maize, for example, fungal colonization was less affected by decreasing 02 from 21 to 1% than a,~ from 1.00 to (I.70 (Bottomley et al., 1950). Aspergillus and Penicillium spp. appeared to be relatively tolerant of low O z. However, with the exception of work by Magan and Lacey (1984b), the effect of gas compostition on growth and sporulation of spoilage fungi has been tested without considering its interactions with a,~ and temperature.

The effect of decreased O~ concentration on germination of spoilage fungi suggests that anaerobiosis for up to 3 months may decrease germination of A. t'ersicolor and P. roquefortii conidia from 75 and 90~ to 10 and 0%. respectively (Richard Molard et al.. 1980). In general, the delay before germination occurs appears to be negatively correlated with both a,,. and O, concentration (Magan and

253

Lacey, 1984b). At 0.98 aw, decreasing 02 concentration increased the lag phase for growth initiation only slightly. However below 1.0% 02, with 0.80-0.85 a,,, the increase in lag phase was much more marked when temperature was decreased from 23 to 14°C than at 0.98 aw (Magan and Lacey, 1984b).

Growth of Aspergillus spp. was affected only by atmospheres containing < 5% 02 at 0.95 aw (Miller and Golding, 1949). However, the concentration required for 50% inhibition of growth can change markedly with a,,. and temperature. Arbab (1976) showed that Penicillium spp. differ in their tolerances of adverse gas compositions. At 0.92 a~, P. funiculosurn and P. oxalicum were more sensitive to different high CO 2 and low O 2 mixtures than P. aurantiogriseum and P. t~iridica- turn. Sporulation was also decreased by such gas mixtures compared to air. Fusariurn rnoniliforme could tolerate 60% 02 at 23°C but its tolerance of large 02 concentra- tions was decreased by lowering temperature to 15.5°C or by changing a,~ from 0.99 to 0.94 at 23°C (Tuite et al., 1967). F. moniliforme could grow slowly with 90% CO 2 and 2% 02. Growth of A. versicolor was unaffected by 2% 02 and that of A. flavus inhibited only by less than 1% 02 (Landers et al., 1967). Large concentrations of CO 2 are necessary to prevent spore germination and growth of Aspergillus and Penicilliurn spp. For instance, 5% of conidia of P. aurantiogriseurn (P. rnartensii) germinated when CO 2 concentration was increased to 60% provided oxygen was adequate (Lillehoj et al., 1972). The temperature range permitting germination also decreased as CO 2 concentration was increased.

Water activity may interact with gas compostition especially in increasing lag phase before spore germination occurs (Magan and Lacey, 1984b). For example, the lag phase for some Aspergillm" and Penicilliurn spp. at 0.90 a,, was 16 to 18 days with 15% CO 2 and 21% oxygen compared to 4 days with 0.03% CO 2 and 21% 02. P. roquefortii was found to have a CO 2 concentration at 50% growth inhibition of > 15% at 0.98-0.95 aw at 23°C but only 5% at 0.90 aw (Magan and Lacey, 1984b). However, Clarke and Hill (1981) found P. roquefortii in sealed silos containing barley grain at 0.87-0.80 a,~ in which CO 2 concentration had, at some stage, reached 90%. By contrast, P. roquefortii was also found to be common in sealed drums of barley with a,, close to 1.00 (Hill and Lacey, 1983).

Water and activity and interactions between spoilage fungi on grain

In both temperate and tropical regions, grain carries a range of 'field" and "storage' fungi. These may be present as spores or mycelium and carried either superficially or growing within the outermost layers of the grain. Which fungi develop in store and come to dominate the grain ecosystem depends on how they interact with each other and how they respond to physical conditions within the grain. Interaction between mycelial hyphae inevitably occurs and can result in either intraspecific or interspecific interactions. Interactions between fungi and other grain microflora in stored grain ecosystems may also have a profound effect on the ability of the component fungi to produce mycotoxins (Cuero et al., 1987).

A range of interspecific interactions, ranging from mutual intermingling, through mutual inhibition to dominance by one species over another has been recognised.

254

T h e s e we re g iven n u m e r i c a l sco res by M a g a n a n d L a c e y (1984c) in a n a t t e m p t to

d e v e l o p a n in v i t r o s y s t e m for a s s a y i n g the ab i l i t y of d i f f e r e n t s p o i l a g e fung i to

d o m i n a t e u n d e r d i f f e r e n t e n v i r o n m e n t a l c o n d i t i o n s . A n u m e r i c a l I n d e x of D o m i -

n a n c e was d e r i v e d as a m e a s u r e o f the spec ies ab i l i t y to d o m i n a t e u n d e r a p a r t i c u l a r

se t o f e n v i r o n m e n t a l c o n d i t i o n s . T h e I n d e x o f D o m i n a n c e was f o u n d to d i f f e r w i t h

t e m p e r a t u r e a n d a ~ b u t was n o t d i r e c t l y r e l a t e d to g r o w t h ra te . P. brevicompactum a n d P. hordei a re s low g r o w i n g b u t we re the m o s t c o m p e t i t i v e Penicillium spp . a t

0 . 9 8 - 0 . 9 5 a~. A. candidus a n d A. nidulans were h i g h l y c o m p e t i t i v e o n l y at 3 0 ° C

w h i l e E. repens a n d A. versicolor were u n c o m p e t i t i v e r e g a r d l e s s o f a ~ a n d t e m p e r a -

t u r e a l t h o u g h t hey a re c o m m o n o n s t o r e d g ra ins .

W h e n these r e su l t s we re c o m p a r e d w i t h c o l o n i z a t i o n of a u t o c l a v e d g r a i n b y the

s a m e g r o u p s o f fung i d i f f e r e n t r e su l t s were f o u n d ( M a g a n a n d Lacey , 1985). A.

versicolor, w h i c h c o m p e t e d p o o r l y in cu l t u r e , w as d o m i n a n t a t all t e m p e r a t u r e s a n d

0.98 to 0 .90 a ~ in g ra in . E. repens a n d A. candidt~s" w e r e a l so p r e s e n t at 1 5 ° C a n d

A. nidulans a t 2 5 - 3 0 ° C a n d 0 . 9 0 - 0 . 9 5 a , , . By c o n t r a s t , d o m i n a n c e o f P. hordei in

g r a i n c o r r e s p o n d e d wel l w i t h I n d e x of D o m i n a n c e at s o m e b u t n o t all t e m p e r a t u r e s

a n d a w. F u r t h e r d i f f e r e n c e s m a y o c c u r w h e n n a t u r a l g r a i n is i n o c u l a t e d w i t h g r o u p s

o f s p o i l a g e fungi . P e r h a p s i r r a d i a t e d g r a i n , w h i c h r e t a i n s i ts g e r m i n a t i v e c a p a c i t y ,

w o u l d be a b e t t e r tes t s u b s t r a t e t h a n a u t o c l a v e d g r a i n for s t u d i e s to d e v e l o p

m e t h o d s for p r e d i c t i n g f u n g a l a c t i v i t y in s t o r e d g r a i n bu lks .

References

Arbab. A.K. (1976) Effect of modified atmospheres on the growth, sporulation and spore germination of selected storage and field Pencilha of corn kernels. M.Sc. Thesis, Purdue University.

Ayerst, G. (19691 The effects of moisture and temperature on growth and spore germination in some fungi. J. Stored Prod. Res. 5, 127-141.

Ayres, P. and Boddy, L. (19861 Water, Fungi and Plants. Cambridge University Press, Cambridge. Bottomley, R.A., Christensen, C.H. and Geddes, W.F. (19501 Grain storage studies IX: The influence of

various temperatures, humidities and oxygen concentrations on mould growth and bkx:hemical changes in stored yellow dent corn. Cereal Chem. 27, 271-296.

Christensen, C.M. and Kaufmann, H.H. (19691 Grain storage: The role of fungi in quality loss. University of Minnesota Press, Minneapolis, MN.

Clarke, J.H. and Hill, S.T. (1981) Mycoflora of moist barley during sealed storage in farm and laboratory silos. Trans. Br. Mycol. Soc. 77, 557-565.

Cuero, R.G., Smith. J.E. and Lacey, J. (1985) Influence of gamma irradiation and sodium hypochlorite sterilization on maize .seed microflora and germination. Food. Microbiol. 3, 107-113.

Cuero, R.G., Smith, J.E. and Lacey, J. (1987) Stimulation by Hyphoplchta burtonii and Bacillu.~" am~'loliquefaciens of aflatoxin production by Aspergilhts" flaous in irradiated maize and rice grains. Appl. Environ. Microbiol. 53. 1142-1146.

• ~khoff . S.R., van Cauwenberge, J.E., Bothast, R.J., Nofsinger, G.W. and Bagley, E.B. (19791 Sulphur dioxide-supplemented ambient air drying of high moisture corn. Trans. A.S.A.E. 23, 1028.

• ~khoff , S.R., Tuite, J.F., Foster, G.H., Kirleis, A.W. and Okos, M.R. (19831 Microbial growth inhibition by SO 2 and SO 2 plus ammonia treatments during slow drying of corn. Cereal Chem. 6(1, 185-188.

Flannigan, B. (19781 Primary contamination of barley and wheat grain by storage fungi. Trans. Br Mycol. Soc. 71, 37-42.

Griffin, D.M. (19811 Water and microbial stress. In: M. Alexander (Ed.) Advances in Microbial Ecology, Vol. 5, Plenum, pp 91-136.

255

Hill, R.A. (1979) Barley grain microflora with special reference to Penicillium spp., Ph.D. Thesis, University of Reading, U.K.

Hill, R.A. and Lacey, J. (1983) Factors determining microflora of stored barley grain. Ann. Appl. Biol. 102, 473-483.

Hill, R.A.. Wilson, D.M., McMillian, W.W., Widstrom, N.W., Cole, R.J., Sanders, T.H. and Blankenship, P.D. (1985) Fkzology of the Aspergillusflavus group and aflatoxin formation in maize and groundnut . In: J. Lacey (Ed.), Trichothecenes and Other Mycotoxins, John Wiley. Chichester, pp. 79-95.

Hcxzking, A.D. and Pitt, J.l. (1979) Water relations of some Penicillium spp. at 25 C. Trans. Br. Mycol. Soc. 73, 141-145.

Hyde, M.B. and Burrell, N.J. (1982) Controlled atmosphere storage. In: C.M. Christensen and H.H. Kaufmann (Eds.), Storage of Cereal Grains and Their Products, 3rd Edn., University of Minnesota Press, Minneapolis, MN, pp. 443-478.

Lacey, J. (1971) The microbiology of moist barley stored in unsealed silos. Ann. Appl. Biol. 69, 187-212. Landers, K.E., Davis, N.D. and Diener, D i . (1967) Influence of atmospheric gases on aflatoxin

production by Aspergillusflaott~" in peanuts. Phytopathology 57, 1086-1090. Lillehoj, E.B., Milburn, M.S. and Ciegler, A. (1972) Control of P. martenstt development by atmospheric

gases and temperature. Appl. Microbiol. 24, 198-201. Magan, N. and Lacey, J. (1984a) The effect of temperature and pH on the water relations of field and

storage fungi. Trans. Br. Mycol. Soc. 82, 71-81. Magan, N. and Lacey, J. (1984b) The effects of gas composition and water activity on growth of field and

storage fungi and their interactions. Trans. Br. Mycol. S~x:. 82, 305-314. Magan, N. and Lacey, J. (1984c) The effect of water activity, temperature and substrate on interactions

between field and storage fungi. Trans. Br. Mycol. Soc. 82, 83-93. Magan, N. and Lacey, J. (1985) Interactions between field and storage fungi on wheat grain. Trans. Br.

Mycol. Soc. 85, 29-37. Miller, D.D. and Golding, N.S. (1949) The gas requirements of moulds. V. The min imum oxygen

requirements for normal growth and germination of six mould cultures. J. Dairy Sci. 32, 191-210. Mills, J.T. (1983) Insect-fungus associations influencing seed deterioration. Phytopathology 73, 33(I-335. Mislivec, P.B. and Tuite, J.F. (1970) Temperature and relative humidity requirements of species of

Penictllium spp. isolated from yellow dent corn. Mycologia 62, 75-88. Papendick, R.I. and Mulla, D.J. (1986) Basic principles of cell and tissue water relations. In: P.G. Ayres

and L. Boddy (Eds.), Water, Fungi and Plants, Cambridge University Press, Cambridge, pp. 1-25. Pelhate, J. (1968) A study of water requirements in some storage fungi. Mycopathol. Mycol. Appl. 36.

117-128. Pitt, J.I. (1975) Xerophilic fungi and the spoilage of f{x-,ds of plant origin. In: R.B. Duckworth, (Ed.),

Water Relations of Foods, Academic Press, London, pp. 273-307. Pitt, J.l. (1979) The genus Penicillium and its teleomorphic states Eupenictlhum and Talaromyces.

Academic Press, London. Pitt, J.l. and Christian, J.H.B. (1968) Water relations in xerophilic fungi isolated from prunes. Appl.

Microbiol. 16, 1853-1858. Pitt, J.l. and Hocking, A.D. (1977) Influence of solutes and hydrogen ion concentration on the water

relations of some xerophilic fungi. J. Gen. Microbiol. 101, 35-40. Richard Molard, D., Cahagnier, B. and Poisson, J. (1980) Wet grain storages under modified atmo-

spheres: microbiological aspects. In: J. Shejbal (Ed.), Controlled Atmosphere Storage of Grain, Elsevier, Amsterdam, pp. 173-182.

Scott, W.J. (1957) Water relations of food spoilage micro-organisms. Adv. Food. Res. 7, 83-127. Shejbal, J. (1980) Controlled Atmosphere Storage of Grains. Elsevier, Amsterdam. Sinha, R.N. (1973) Inter-relationship of physical, chemical and biological variables in the deterioration of

stored grains. In: R.N. Sinha and W.E. Muir (Eds.), Grain storage: Part of a System, Avi Publishing Co., Westport, CN, pp. 15-48.

Snow, I). (1949) The germination of mould spores at controlled humidities. Ann. Appl. Biol. 36. 1-17. Smith, S.L. and Hill, S.T. (1982) Influence of temperature and water activity on germination and growth

of Aspergillus restrictus and A. t~erstcolor. Trans. Br. Mycol. Soc. 49, 558-560.

256

Tuite. J., Haugh, C.G., Isaac, G.W. and Huxsoll, C.C. (1967) Growth and effect of molds in stored high moisture corn. Trans. Am. Soc. Agric. Eng. 10, 730-737.

Williams, R.J. and McDonald, D. (1983) Grain molds in the tropics: problems and importance. Annu. Rev. Phytopath. 21, 153-178.