Trans. Br, mycol. Soc. 74 (1) 195-217 (1980)

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NOTES AND BRIEF ARTICLES

FUNGAL DETERIORATION OF GUNPOWDER

J. LACEY

Rothamsted Experimental Station, Harpenden, Herts AL5 2JQ

Gunpowder is made from potassium nitrate (75 %),sulphur (10 %) and charcoal (15 %), the last being,ideally, a high-grade wood charcoal, particularlythat made from alder (Alnus glutinosa (L.)Gaertn.), As such, it seems an unlikely substratefor mould growth. However, when a batch of gun-powder was examined after 2 years' storage,scattered fungus colonies were observed. The gun-powder had been stored in boxes lined with tarredKraft paper in a brick-built, mounded magazinein Kent, maintained at 21-25°C but lackinghumidity control. The colonies were up to l'5 emdiam and most frequent near the outside of thebulk. Near the centre of the colonies, grains ofgunpowder were covered with a weft of myceliumand were coloured green by the abundant spores.Viewed under a stereo-microscope, sporing headsresembling Aspergillus were observed and thesewere found to have the microscopic features of theA. restrictus group.

The fungus was isolated on 2 %malt 10% NaCland Czapek 20 % sucrose agars and comparedwith descriptions of the Aspergillus restrictus group(Raper & Fennell, 1965). Extremely slow growthoccurred on Czapek sucrose agar but malt saltagar supported good growth and sporulation. Theconidiophores expanded terminally into vesicles,9-17 pm diam, which carried a dense single layerof phialides 7-11 pm long over the upper half totwo-thirds of the surface (Fig. 1). Conidial headswere at first radiate but later formed loose columnsof tangled spore chains. Conidia were nearlycylindrical and smooth at first but rounded asthey aged, becoming ovate to subglobose, 3'6-6'7 x 3'4-4'9 pm, mean 5'2 x 4'0 pm with markedlyroughened walls. These characteristics corres-ponded most closely to those of Aspergillus peni-cillioides Speg,

To test their ability to grow on potassiumnitrate, an isolate of A. penicillioides was inoculatedinto flasks containing Czapek or water agars con-taining concentrations of KNOs. Water agarsupported no visible growth with any level ofKNOs' Also, no growth was visible on Czapek agarflasks containing less than 5 % KNOs' Additionof 9-18 %KNOs allowed small colonies to develop

but no sporulation, while above 20 % growth wasgood and abundant spores were produced. Mediumcontaining 38 and 50%KNOs was saturated fromthe start while that containing 20 % becamesaturated as it dried during incubation, crystalsforming in the agar.

Thus A. penicillioides could grow on saturatedKNOs solution provided a source of othernutrients was available. To test whether otherconstituents of gunpowder could provide thesenutrients, the above test was repeated in part withautoclaved gunpowder charcoal placed on theagar surface of some flasks with or without thefurther addition of 10% sulphur to the medium.Again the fungus grew well on flasks of Czapekagar saturated with KNOs but no growth wasvisible on those of water agar following 1 month'sincubation, whether or not charcoal and sulphurwere added. However, microscope mounts pre-pared from the agar surface in these flasks showedthat spores had germinated in all flasks examined,producing short lengths of hyphae. Some micro-colonies had formed short conidiophores 50-100pm long with small terminal vesicles from 3 pm,barely wider than the conidiophore stalk, to12 pm diam. The smaller conidiophores, whichwere the more common, carried only two or threephialides, 7-10 pm long at their tips (Figs 2, 3).Only rarely was a well-differentiated conidiophorefound with more than five phialides (Figs 4, 5),

Members of the A. restrictus group are wellknown as xerophilic fungi causing deterioration oforganic substrates at low water activities, parti-cularly of cereal grains, textiles and even opticalequipment. The water activity of saturated KNOssolution is 0'93 (Rockland, 1960). Growth ofisolates of the A. restrictus group is limited by aminimum water activity of 0'15-0'77 and a maxi-mum of about 0'97, with an optimum of about 0'91(Snow, 1949; Pelhate, 1968; Pitt & Christian,1968). Thus, the water activity of medium con-taining little or no KNOs was too great to permitgrowth of A. penicillioides while that of the satu-rated solution was close to its optimum. Whetherthe growth found on unsupplemented mediumwould be sufficient eventually to yield the amount

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Notes and briefarticles

3 4

Fig. 1. Conidiophores of A. penicillioides from culture on malt salt agar ( x 425).

Figs 2-5. Conidiophores of A. penicillioidesfrom culture on water agarsaturated with potassium nitrate with added charcoal.

Fig. 2. Diminutive conidiophore (x 850).

Fig. 3. Microcolony of A. penicillioidesbearing several diminutive conidiophores (x 425).

Fig. 4. Larger conidiophore (x 850).

Fig. 5. Conidiophore developing among potassium nitrate crystals and charcoal fragments (x 425).

of colonization observed on z-year-old gunpowderis not known. It is not obvious otherwise where thenutrients to support this growth originated unlessthe gunpowder was contaminated before or duringstorage or the fungus could utilize carbon sourcesin the charcoal. Such charcoal is permitted tocontain up to 10 % of organic matter but this isusually in the form of wood tar residues that maybe difficult for many fungi to utilize. This isthought to be the first reported instance of gun-powder deterioration by fungi.

I am grateful to Mr R. G. Hall for bringing theproblem to my notice and supplying samples ofgunpowder and charcoal.

REFERENCES

PELHATE, J. (1968). A study of water requirements insome storage fungi. Mycopathologia et MycologiaApplicata 36,117-128.

PITT,J. 1.& CHRISTIAN, J. H. B. (1968). Water relationsof xerophilic fungi isolated from prunes. AppliedMicrobiology 16, 1853-1858.

Trans. Br, mycol. Soc. 74 (1), (1980). Printed in Great Britain

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Notes and brief articles 197RAPER, K. B. & FENNELL, D. I. (1965). The Genus SNOW, D. (1949). The germination of mould spores at

Aspergillus. Baltimore: Williams and Wilkins. controlled humidities. Annals of Applied Biology 36,ROCKLAND, L. B. (1960). Saturated salt solutions for 3-13.

static control of relative humidity between 5° and40°. Analytical Chemistry 3%, 1375-1376.

AFLATOXIN PRODUCTION ON WHEAT SEED STOREDIN AIR AND NITROGEN

A. A. FABBRI, C. FANELLI, M. SERAFINI, D. DI MAGGIO

Cattedra di Micologia, lstituto dell'Orto Botanico, Uniuersita di Roma,Largo Cristina di Svezia 2400165, Roma, Italy

AND R. PIRAZZI

Encc (Centro Sperimentazione Agricola e Forestale), Casalotti, Roma

The contamination by aflatoxins of foods andfeeds is a world problem, the economic andhealth consequences of which are so serious thatit has become necessary to apply new methodolo-gies to prevent, limit and eliminate their production(Austwick, 1975; Brekke et al., 1977). Modifiedatmospheres are one of the methods more widelyused to prevent the growth of fungi producingtoxins (Wilson & Jay, 1975; Epstein et al., 1970;Diener & Davis, 1969). High CO 2 or N2 atmos-pheres with very low O2 percentages have showngood results in limiting both production of toxinsand growth of insects in silos of stored seeds (Jay& Pearman, 1973; Pearson & Sorensen, 1970). InItaly the problem of seed storage is very important,as foodstuffs are stored in silos for a long timebefore being used. In this work we have examinedthe production of aflatoxins on soft wheat seedstored in a N 2-controlled atmosphere with lessthan 0'03 % oxygen. The wheat used for this testpresented moisture and temperature conditionsfavourable to the production of aflatoxins (Shih &Marth, 1974).

The soft wheat seed utilized for experimentswas sterilized by exposure to 60CO for 90 min at6296 rad min-I using Gammacell 220 (AtomicEnergy Ltd of Canada). After sterilization, seedswere moistened with sterile distilled water to avalue of 18'5 % moisture content measured with athermobalance (Buhler) and were inoculated with8 x 106 conidia of Aspergillus fiavus Link ex Fr.(ATCC 22548) 200 g-I of seed. The wheat wasput into 250 ern" cylindrical glass jars with a gasinlet in the upper part and an outlet in the lowerpart. The gas flowed from cylinders of N 2 (0'03 %O2) through a sterilizing filter (0'2 pm GelmanPreflow 200) and pressure regulators and micro-meter regulating valves, connected to flowmeters,

to regulate the flow to 4'5 ern" h-I. Triplicate jarsin series were incubated at 32° in a thermostaticallycontrolled incubator containing the same N 2

atmosphere. In a second series of jars a flow of airwas substituted for N 2• After 7, 14 and 21 daysincubation, seed in three jars kept in N2 and threein air were analysed for the production of afla-toxins. Samples (100 g) were prepared for extrac-tion and quantitative analysis of aflatoxins byhigh-pressure liquid chromatography (HPLC)according to the method of Pons (1976). Forpurification of aflatoxins by column chroma-tography we have partially used the methods ofAnon. (1975). Each lot of grain was homogenizedusing a Waring Blender and shaking for 30 minwith 275 em" chloroform:water (10:1, vjv). Theextracts were filtered through phase separationpaper (Whatman, 1PS), concentrated to 1 em" ona rotary evaporator and purified by elution througha chromatographic column (30 x 200 mm) con-taining 10 g of silica gel 60 (Merck) supported ona thin layer of anhydrous sodium sulphate. Theextracts in the column were eluted at a flow rate of20 em" min-I with 150 em" hexane followed by150 em" anhydrous ether. The adsorbed aflatoxinswere eluted with 150 em" chloroform:methanol(19:1, vjv). This fraction was concentrated byevaporation to dryness and resuspended in 100 pIof the HPLC elution solvent, water-saturatedchloroform : cycloexane : acetonitrile (50 : 15 : 2,v jv). For adjusting the retention time we usedelution solvent: ethanol (98'5:1'5, vjv). HPLCseparation was conducted with a Waters ALC-202instrument equipped with M-6000 pump, H6Kseptumless injector and a small particle silica gelcolumn (300 x 4 mm, stainless steel, packed withLichrosorb, 10 pm) operating at 1800 psi with anelution rate of 2 ern" min-I. Aflatoxins were

Trans. Br. mycol, Soc. 74 (1), (1980). Printed in Great Britain

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