i JOURNAL OF AGRICULTURAL SCIENCE IN FINLAND Maataloustieteellinen A ikakauskirja Voi 58: 197—208, 1986 Urea treatment of barley grain. Effect on storage properties and fungal growth ASKO HANNUKKALA' and PEKKA HUHTANEN Department of Plant Pathology, University of Helsinki Department ofAnimal Husbandry, University of Helsinki SF-00710 HELSINKI, Finland Abstract. Barley harvested at three different moisture contents (22, 25 and 32 %) was treated with varying levels of urea (1.0, 1.5, 2.0 and 2.5 % on fresh weight basis) and stored aerobically in experimental silos of 400 liters. The chemical composition of barley was analysed before treatment and after 12 months of storage. Fungal contamination of grain samples was analysed. Application of 1.5 % or more urea effectively preserved the grain for 12 months. At the highest moisture content, 1.0 %of urea did not prevent deterioration during storage. The average recovery of dry matter was 97.1 %, of nitrogen 93.9 Vo. Ammonia was released from hydrol- ysis of urea. The degree of hydrolysis increased with increasing moisture content of barley. Raising the levels of urea from 1.0 to 2.5 % significantly increased the quantity but tended to decrease the proportion of urea hydrolysed. Representatives of 33 genera of fungi and unidentified actinomycetes were present in the grain lots examined. Most fungi were found only occasionally and their incidence declined towards the end of storage. The grain stored at the highest moisture level (32 %) contained more fungi and less actinomycetes than other seed lots. All urea treatments reduced the number of fungi on seeds. Urea concentration of 2 % or more eliminated Fusarium- and Aspergillus- species on all moisture levels. Scopulariopsis brevicaulis (Sacc.) Bain, and other species of Scopulariopsis were the most commonly encountered fungi after urea treatment. The total number of Scopulariopsis ssp. exceeded 100 % and the amount remained high for 40 weeks of storage until a rapid decline towards the end of storage. Urea treatment also favoured the occurrence of actinomycetes. Index words: barley, urea, preservation, fungi, Fusarium, Scopulariopsis Introduction Grain achieves a physiological maturity at a moisture content of 30—40 %. Thereafter, Present address: Agricultural Research Centre Depart- ment of Plant Pathology SF-31600 JOKIOINEN Fin- land. the major change in grain is a loss of moisture (Krall 1972). In Finland, the moisture con- tent of cereal grains at harvesting time usual- ly exceeds 20 %. The grain must be dried to a moisure content of 15 % or less to prevent nutritional losses and microbial activity. 197 https://www.c-info.fi/en/info/?token=8BhzXFEl6GO5Vi6j.KwWEWLTdDWFY8EnWYGMHvw.598ajalA46qKHHYnof47cIUw-mqcXHcgFl2HPHzDe0Aa71JVf5L1J8YOx5tcz40-IT2rWj9nEjYhDG9DkI7ixM0M94KL5ncAfL-5wzDaqLpAaS6lD_Pw_4kK9xol-GgB5GdcAX21CuYF_9zAxD8i_PrwE5AcAuJSiuC1uW9_KmdcvEDIbf8yxAi_vfc6AzHe Today, more than 90 °7o of the total grain yield is dried, the rest either being treated with propionic acid or ensiled. Propionic acid has for long been known to be an effective preservative for high moisture grain. The nutritional value for ruminants and swine appears to be equal to that of dried grain (Jones et al. 1974). The preservative effect of propionic and other volatile fatty acids is based on their fungicidal properties. The efficiency of ammonia as a preserva- tive of high moisture corn has been reported by BoTHASTCt al. (1973, 1975) and Britt and Huber (1976). It has eliminated molds and yeasts in initially highly contaminated high moisture corn (Bothast et al. 1975). Both liquid and gaseous anhydrous ammonia has been used (Montgomery et al. 1980). Schmidt et al. (1978) and orskov et al. (1979) reported that moist urea-treated grain can be preserved for several months. The pre- servative effect of urea is also based on am- monia. In moist feeds, microbially produced urease hydrolyzes urea to ammonia and car- bon dioxide. Urea offers certain advantages over ammonia; it is easier to handle and nit- rogen losses are smaller. The long term pre- servative effect of urea has been better than that of ammonia al. 1978). The objective of the present study was to quantify the effect of different levels of urea treatments on some storage properties and the viability of certain fungal propagules on barley grain preserved at three moisture con- tents. Materials and methods The barley lots in the present study were harvested on August 24 and 30 and Septem- ber 4, 1984. The intension was to harvest at the moisture levels of 22, 27 and 32 °7o before treatment, but the difference between the two lowest moisture contents was only about 3 %. At all moisture contents barley was treated with 1.0, 1.5, 2.0 and 2.5 % of urea on fresh weight basis and placed in 400-1 experimental glass fibre silos. The silos were covered with plastic and insulated with a 10 cm layer of glass wool to avoid losses of heat produced and left at ambient temperatures for one year. Urea was delivered in a water solution (1:1) to the boot end of a 0.15 x 5 m grain auger. The grain temperatures were recorded by placing thermocouples in the centre of each silo. Samples for pH measurements and for anal- yses of fungal contamination were taken be- fore treatment, 3 days after treatment, then weekly for 10 weeks and subsequently at about monthly intervals. Germination and presence of fungi and actinomycetes were examined by a slightly modified blotter test (de Tempe 1963). Seeds were placed on 14cm Petri dishes on moist filter paper, 50 seeds per dish. Each dish was moistened with 10 ml of distilled water. The Petri dishes were kept in plastic bags in diffuse day light at room temperature (20 °C). After an incubation period of 14—18 days, the numberof germinated seeds was cal- culated. Each individual seed was studied under a stereo microscope and the fungi and actinomycetes showing growth were identified directly or after isolation on PDA (potato dextrose agar, Difco). The presence of any fungus on individual seed was recorded. No attempts were made to estimate the vigour of growth on individual seeds. A sample of 200 seeds of each moisture content and urea treatment was examined at each sampling time. The total number of seeds examined was 43800. The chemical composition of barley was determined before the treatment and after unloading the silos. Feed analyses were made according to standard procedures. Total nit- rogen (N) and soluble N were determined in fresh samples which were stored frozen. Am- monia N, sugars and volatile fatty acids (VFA) were analysed by the methods described by Huhtanen (1984). Urea was determined as described in the Technical Bulletin 27 of the Ministry of Agriculture, Fisheries and Food (Anon 1973). In calculation of dry matter (DM) losses, 89 % of VFA was assumed to be lost in oven 198 drying at 100°C (Porter et al. 1984). Also the weight loss due to hydrolysis of urea to ammonia and carbon dioxide and N lost as ammonia in oven drying were taken into ac- count. Analyses of variance were made ac- cording to Snedecor and Cochran (1967). Differences between urea levels were further partitioned into linear, quadratic and cubic effects. The counts of fungi were studied by the G-test for independence (Sokal and Rohlf 1969). The results are expressed as per- centages of examined seeds. Results and discussion Preservation and chemical composition Urea treated barley kernels were brown in colour. The colour was deeper at high mois- ture levels. Visible mold growth was apparent only in the silo of the lowest urea level and highest moisture content. Urea treatment increased the crude protein content from the initial 108 to 190 g/kg DM. The urea level had no effect on ether extract, crude fibre or sugar contents (Table 1). The sugar content decreased from the initial 31.6 to 20.6 g/kg DM. No lactic acid was recovered in the treated barley. Acetic acid fermentation increased with the urea level (P < 0.01). Similar low concentrations of acetic acid in urea-treated grain have been observed by Schmidt et al. (1982). Certain apparently significant effects of moisture contents on chemical composition of grain indicate merely the diversity of grain at the time of preserva- tion caused by different dates of harvest. pH rose from the initial 6.5 above 8.7 within 3 weeks in the silos which were pre- served at a moisture content of 22 or 25 % and within three days in thosepreserved at the moisture content of 32 %. The average DM recovery from 12 silos was 97.1 % (SE 0.7 %). It was not affected either by the level of urea or moisture content. Similar DM losses in urea treated grain have Table I. Effect of urea level on chemical composition (g/kg DM), pH and dry matter (DM) and nitrogen (N) recoveries. Statistical Urea level Moisture content SEM significance 1.0 1.5 2.0 2.5 22 25 32 Urea Mois- Urea Mois- ture . „ ture Dry matter (g/kg) 722 725 726 721 768 743 659 3.6 3.1 NS NS *** In dry matter Ash 28 28 27 27 30 25 28 0.2 0.2 * NS *** Crude protein 142 159 171 190 171 157 169 2.2 2.8 *** NS Ether extract 19.4 19.4 19.3 19.0 21.5 18.8 17.6 0.47 0.41 NS NS * Crude fibre 73 65 71 65 68 71 67 3.5 3.0 NS NS NS NFE 1 764 768 760 763 749 771 771 3.6 3.1 NS NS ** Sugars 21.1 20.4 20.4 20.6 22.7 17.3 21.8 0.9 0.8 NS NS •* Acetic acid 0.95 1.16 2.45 2.92 2.08 1.00 2.84 0.29 0.26 ** NS ** Propionic acid 0.24 0.16 0.14 0.12 0.10 0.38 0.08 0.07 NS NS ** Butyric acid + + Isovaleric acid 0.02 0.02 0.01 + 0.04 0.01 0.01 NS NS NS pH 8.70 8.85 8.90 8.87 8.81 8.81 8.87 0.03 0.03 * * NS DM recovery (%) 94.3 98.1 98.1 97.9 97.5 97.3 96.3 1.4 1.2 NS NS NS N recovery (%) 94.1 95.6 93.4 92.6 96.1 94.5 91.2 1.1 1.0 NS NS NS 1 NFE = nitrogen free extracts. SEM = standard error of means. Statistical signicicance: NS non-significant, * (P < 0.05), ** (P < 0.01), *** (P < 0.001). L = linear trend of urea level, Q = quadratic trend of urea level; no significant cubic effect. 199 been reported also by Schmidt et al. (1978) and Mowat et al. (1981). In contrast, Pep- linski et al. (1978) recorded a DM loss of 14 °7o during long-term storage of ammo- mated high moisture corn, suggesting the bacterial activity to be the major cause for los- ses. Schmidt et al. (1978) observed the long- term preservative effect of urea to be better than that of ammonia when both were applied at the same level of N. In the present study, DM losses were sligthly smaller than when barley was ensiled at the moisture content of 55—60 % (Huhtanen 1984). The N losses slightly exceeded the DM los- ses, increasing with increasing moisture level (P > 0.05). The average loss of urea N, 27.2 °/o (SE 2.9 %) (Table 2), was lower than that reported by Schmidt et al. (1978). On the other hand, orskovc7 al. (1979) found only small changes in the N content of urea-pre- served barley after 5 months of storage. The reason for differences in N losses of urea- treated grain might be the treatment of the sample before N analyses. In the present study, the fresh samples contained on the average 6.2 % more N than samples dried at 50 °C in vacuum. The difference in N con- tent of fresh and dried samples was very closely related to the ammonia concentration (r 0.943). Montgomery et al. (1980) found much higher N losses in ammonia-treated corn. Lower N losses during application and storage of urea-treated grain could explain the better long-term preservative effect of urea com- pared to ammonia. A higher proportion of added urea was hydrolysed to ammonia at lower application and higher moisture levels (Table 2). Similar effects have also been found in urea-treated wheat (Schmidt et al. 1978) and straw (Williams et al. 1984). However, raising the levels of urea applied significantly increased the quantity of urea hydrolysed. In the present study, the proportion of re- sidual urea was much higher than that re- ported by Schmidt et al. (1978), probably be- cause of the different ambient temperature or different urease activity of native microbiota. The proportion of ammonia N and soluble N of total N increased with increasing urea level (P < 0.001) and moisture content (P < 0.001). The urea level had no effect on the true protein concentration or solubility of barley N. Schadereit et al. (1982) observed no dif- ferences in amino acid composition of urea- treated and dried wheat. The initial soluble N content of 3.65 g/kg DM was slightly lower than that of 4.29 g/kg DM after one year of Table 2. Effect of urea treatment of barley on different N fractions (g/kg DM). Statistical Urea level Moisture content SEM significance 1.0 1.5 2.0 2.5 22 25 32 Urea Mois- Urea Mois- ture , « ture Total N 22.8 25.4 27.4 30.4 27.4 25.1 27.0 0.52 0.45 *** NS * Protein N 14.9 14.9 14.9 14.9 16.0 14.3 14.4 0.10 0.09 NS NS *** Residual urea N 1.14 2.30 2.93 4.55 4.52 2.43 1.24 0.86 0.75 * NS %of addition 17.8 25.3 24.4 29.2 39.8 21.5 11.3 6.3 5.5 NS NS * Ammonia N 4.28 5.81 7.12 8.72 4.83 6.20 8.43 0.50 0.42 ** NS ** %of total N 18.8 22.8 26.1 28.8 17.4 24.3 30.7 1.3 1.2 ** NS *** % of urea N addition 54.5 50.3 45.6 43.8 40.6 47.6 57.8 0.83 0.72 *** NS *** N loss of urea N 27.4 24.1 29.9 27.0 19.7 30.9 30.9 5.4 5.9 NS NS NS Soluble N 9.6 12.0 14.7 17.4 13.4 12.2 14.9 0.22 0.22 *** NS *** % or total N 42.2 47.2 53.5 58.3 48.3 48.0 54.6 0.63 0.55 *** NS *** Soluble Nof barley' 4.203.87 4.624.47 4.083.57 5.220.04 0.03 NS NS * 1 Soluble N ammonia N urea N. For statistical significance; see Table 1. 200 storage. At the highest moisture level the soluble N content tended to be higher than at the lower moisture level. The average temperatures of treated grain showed an initial rise for few days after treatment (Fig. 1). At the lowest urea level the temperature rise was slightly higher, probably indicating higher microbial activity. After I—2 weeks of the treatment the temperature began to rise again in the silos preserved at 22 and 25 % moisture contents. The reason for the rise in temperature might be the hydrolysis of urea to ammonia and carbon dioxide, which is a heat generating reaction. Also the chemical reaction of ammonia with grain (Bothast et al. 1975) might explain the rise temperature to some extent. In silos pre- served at 32 % moisture content there was no secondary rise in temperature, probably due to the higher rate of hydrolysis of urea and thus higher initial ammonia concentration. At all three moisture levels the temperature was highest at 1.0 % urea level. This urea level was probably insufficient to produce enough am- monia to eliminate microbial activity. After the initialrise the grain temperatures declined, thereafter generally reflecting ambient temper- atures except for the silo treated with 1.0 % of urea at 32 % moisture level. In this silo, the temperature rose later 5—7 °C above ambient temperature, and the growth of mould could be seen. The germination of untreated seed lots was low, about 20 %, since the examination was made soon after harvesting. Urea treatment stimulated germination for one week after treatment, but germination was inhibited rapidly after I—4 weeks following treatment Fig. I. Effect of urea level on temperature in barley preserved at moisture content of 22 °7o (a), 25 % (b) and 32 % (c). Urea level: 1.0 % • ; 1.5 % a ; 2.0 % ■ ; 2.5 % O. Fig. 2. Effect of four levels of urea application on the percentage of germination of barley seeds during 10 first weeks of storage. 201 202 (Fig. 2). The concentration of urea clearly affected the rate of inhibition of germination. The highest concentration inhibited germina- tion within two weeks, the lowest within 8 weeks. Fungi and actinomycetes on grain Representatives of 33 genera of fungi and unidentified species of actinomycetes mainly belonging to the genus Streptomyces were present in the seed lots. Bacteria other than actinomycetes were not analysed. Most of the fungi were field fungi (Christensen and Kaufmann 1965), mainly Acremonium, Alter- naria, Bipolaris, Cladosporium, Fusarium and Harzia (Table 3). These are frequent inhabi- tants of cereal grain (Malone and Muskett, 1964, Ylimäki 1981). Typical storage fungi, Aspergillus ssp. and Penicillium ssp. were Table 3. The occurrence of fungi and actinomycetes on untreated and urea-treated barley grain during 12months of storage. Fungus Untreated Time after urea application (months) Bram o—3 4—6 7—9 10—12 °/o of seeds contaminated (+ = less than 0.1 %) Absidia van Tieghem 0 + 000 Acremonium Link:Fr. 39.7 2.7 + + 0 Alternaria Nees:Fr. 83.3 2.8 0 + 0 Arthrinium Kunze:Fr. 0 + 000 Aspergillus Mich.iFr. 0 4.6 3.2 2.6 + Bipolaris Shoemaker 12.7 1.0 0 0 0 Botryotrichum Sacc. & March. 0 + + + + Ceralocyslis Ellis & Halst. 5.2 + 0 0 0 Chaetomium Kunze:Fr. 0 + 0 + + Chrysosporium Corda 0 0 0 + 0 Cladosporium Link:Fr. 54.8 +OOO Doralomyces Corda 0 + 000 Drechslera Ito + + 0 0 0 Epkoccum Link:Schlecht. 2.2 + 0 0 0 Fusarium Link:Fr. 18.7 9.6 1.6 + 2.5 Fusidium LinkiFr. 4.7 + 0 0 0 Gliocladium Corda + + + 00 Gonatobolrys Corda +OOOO Graphium Corda 0 + 0 + 0 Harzia Cost. 10.5 + 0 + 0 Humicola Traaen 0 0 0 + 0 Morlierella Coemans 0 + 000 Mucor Mich.:St.-Am. 2.2 + + + + Papulaspora Preuss 0 0 0 + 3.2 Penicillium Link:Fr. + 1.7 + + + Peziza L.:St.-Am. 0 0 0 1.3 0 Phoma Sacc. 0 + 0 0 + Scopulahopsis Bain. 0 67.4 97.3 89.1 35.8 Stilbum TodeiFr. 0 + 000 Trichocladium Harz 0 0 0 + 0 Trichoderma Pers.:Fr. 0 0 0 + + Trichothecium Link:Gray +OO + 0 Ulocladium Preuss + + 0 0 0 Verticillium Nees:Link + + 0 0 0 unidentified genera + + + + + Bacteria: actinomycetes 11.8 45.2 20.2 29.6 34.7 Table 4. Effect of urea concentration of the grain on the percentage of barley seeds contaminated with fungi and actinomycetes during 12 months of storage. Fungus Urea con- 1.0% 1.5 % 2.0 % 2.5 % Counts not independent centration %0 f seeds contaminated control' (+ = iess than 0.1 %) of urea (G-test, SOKAL and ROHLF 1969) Urea vs. Urea con- control 2 centrations 3 Acremonium spp. Allernaria spp. Aspergillus spp. Bipolaris sorokiniana Cladosporium spp. Fusarium spp. Penicillium spp. Scopulariopsis brevicaulis Scopulariopsis spp. Actinomycetes 39.73.5 3.90.8 0.3 *** 83.3 5.9 1.4 0.1 + **� O 11.21.6 1.20.5 12.71.6 0.60.1 + »** 54.80.2 + + + ••• 18.715.5 6.34.2 1.3 **• 0.23.3 0.80.5 0.3 NS O 71.372.2 67.965.3 **» O 20.127.7 32.937.2 11.852.3 42.433.2 33.0 *�* � *•* NS NS NS NS ** * * * Number of seeds examined 600 10 800 10 800 10 800 10 800 1 Control seeds were not stored; the effect of storage and urea-treatment cannot be partitioned. 2 Sums of all urea treatments are tested against controls. 5 All four treatments are compared with each other. Zero counts were replaced with 10-50 to enable In trans- formations. present on less than 5 °7o of seeds. The most commonly encountered fungi after urea treatment were Scopulariopsis ssp. (Table 4). The indirect method for detecting fungi on seeds merely reflects the potential ability of certain fungal spores to germinate in adequate conditions. The activities of microbes in actual storage silos compared to those on Petri dis- hes must be interpreted with caution. The decline of certain fungi in the blotter test indicates the death of fungal propagules in storage silos. The detected fluctuations and increasement of some fungi is probably an ex- pression of these fungi having reproductive or metabolically active phases during storage. The grain stored at the highest moisture level (32 %) contained more fungi and less actinomycetes than other grain lots. Especially Fusarium ssp. occurred frequently in moist grain. High moisture at harvesting favoured the occurrence of Fusarium ssp. (Ylimäki 1981). High counts ofFusarium ssp. in moist grain are probably due to the late harvesting date of the seed lot. The number of fungal genera on grain declined after all urea treatments during storage. Only three of the initial 17 genera in untreated controls were present after 10 months of storage. The decline was much faster than that reported of untreated seeds heat-dried to a moisture content of 14 % (Christensen and Kaufmann 1965). However, the decline was not as rapid as reported for urea-treated hay by Hlödversson and Kas- Persson (1986) or ammonia-treated corn by Bothast ef a/. (1973, 1975) and Montgomery et al. (1980). High urea concentrations (2.0 and 2.5 %) accelerated the decline of fungi. After urea treatment there were 16 genera of fungi not detected in untreated seed lots. Most of them were found only occasionally during the first three months of storage, and their presence is apparently due to the greater number of seeds examined compared to the untreated controls. Scopulariopsis ssp. were the only fungi showing a rapid increase after urea treatments. In certain seed lots the total number of Scopulariopsis ssp. exceeded 203 100 %, one seed oftenbeing occupied by more than one species (Table 4). Urea and ammo- nia treatments have shown to increase the number of Scopulariopsis ssp. on different plant materials (Bothast et al. 1973, 1975, Montgomery et al. 1980, Hlödversson and Kaspersson 1986). The vigorous growth of Scopulariopsis ssp. on seeds may be due to underestimation of counts of certain other fungi not cabable of competing with Scopu- lariopsis. In addition, the counts of actinomy- cetes increased after urea treatments, espe- cially at the lowest urea concentration. Also ammonia has been observed to increase the number of actinomycetes on corn (Bothast et al. 1975). Aspergillus ssp. Aspergillus ssp. were not found in untreated seed lots. After urea treatment they were prevalent in seed lots of the highest and lowest moisture contents. At urea concentrations ex- ceeding 1.5 ®/o Aspergillus ssp. were found only occasionally. At the lowest urea concen- tration (1.0 %) the percentage of contami- nated seeds started to rise one week after treatment and reached the maximumof 27 % within six weeks, declining slowly towards the end of the storage period (Fig. 3). Aspergillus ssp. are frequently reported on stored grain and they have been found to be viable still after seven years of storage. Sev- eral species are known for their ability to pro- duce a wide variety of chemical substances including mycotoxins (Malone and Muskett 1964, RAPERand Fennel 1965, Domsch et al. 1980). The most dangerous species, A. flavus Link:Gray, was not found in the present study, and sufficient urea treatment seemed to reduce contamination of Aspergillus ssp. efficiently. Fusarium ssp. Fusarium species occurred most frequent- ly in seed lots of the highest moisture content and lowest urea concentration. Urea concen- tration of 2.5 % reduced the percentage of contaminated seeds to zero within4 weeks and 1.5—2.0 % within 7 weeks. However, there was a slight increase of contaminated seeds towards the end of storage (Fig. 4). The pre- dominant species were F. avenaceum (Fr.) Sacc., F. culmorum (W. G. Sm.) Sacc. and F. poae (Pk.) Wr. Fusarium species are common inhabitants of Finnish grain especilly after rainy harvest season. (Uoti and Ylimäki 1974, Ylimäki 1981) They are destructive plant pathogens which can produce mycotoxins such as tri- chotocene (T-2 toxin, diacetoxyscirpenol), zearalenone and vomitoxin before harvesting and during storage (Joffe 1974, Neish et al. 1982) Estrogenically active zearalenone has been detected in undried seed samples con- taining Fusarium species, while dried samples containing Fusarium ssp. were free from me- Fig. 3. Occurrence of Aspergillus ssp. on barley seeds during storage at four levels of urea application. Fig. 4. Occurrence of Fusarium ssp. on barley seeds during storage at four levels urea application. 204 tabolites of fungi. Seed lots containing zea- ralenone were heavily contaminated (Ylimä- ki et al. 1979). In the present study, sufficient application of urea probably eliminated the risk of mycotoxin production by Fusarium ssp., while the percentage of contaminated seeds rapidly declined to less than 5 % (Fig. 4). On the other hand, ammonia treatment has been shown to inactivate zearalenone in grain (Muller 1983). Scopulariopsis ssp. Scopulariopsis ssp. were the most frequent fungi in all seed lots after urea treatments. Fungi were not present on untreated grain. S. brevicaulis was the first invader of the seeds. During the first two weeks of storage there was a rapid increase of S. brevicaulis (Sacc.) Bain in all urea-treated grains and the percent- age of contaminated seeds remained high for 40 weeks until a rapid decline towards the end of storage. The highest urea concentration appeared to delay the increase and fasten the decline of the fungus (Fig. 5). The percentage of Scopulariopsis ssp. other than S. brevicaulis began to increase after one week’s storage, reaching a peak within 18 to 40 weeks of storage, depending on the urea concentration: the higher the concentration, the higher the incidence of Scopulariopsis ssp. (Fig. 6). Scopulariopsis ssp. are distributed world- wide. They have been reported on plant debris, numerous organic materials and soils (Domsch et al. 1980). They are not typical seed contaminants (Malone and Muskett 1964, Ylimäki 1981). High counts of S. brevicaulis as well as other species have been reported on ammonia- treated corn (Bothast et al. 1973, 1975, Montgomery et al. 1980) and urea-treated hay (Hlödversson and Kaspersson 1986). The optimal growth is reported at pH 7—B and above. The fungi tolerate temperatures ranging from + 5 °C to 37 °C; they are rela- tively xerophilic and they can decompose, uti- lize and tolerate a wide range of different organic and inorganic compounds (Domsch et al. 1980). S. brevicaulis is encountered as a parasite causing onychomycosis in man, and dermatomycosis of feet and other parts of body (Rarer and Thom 1949, Onions 1966). Possible risks for human health caused by S. brevicaulis in urea-treated grain warrant further investigation. In conclusion, the results of the present study show that urea is an effective and cheap preservative of high moisture grain. In certain feeding conditions when the supply of rumen degradable nitrogen is inadequate, the addi- tional nitrogen may be useful for rumen mic- robes. Application of urea, 2.0 °7o or more on fresh weight basis, eliminated the growth of fungi, especially those producing mycotoxins. Acknowledgement. The authors are grateful to Prof. Eeva Tapio for critical reading of the manuscript and to Fig. 5. Occurrence of Scoputariopsis brevicaulis (Sacc.) Bain on barley seeds during storage at four levels of urea application. Fig. 6. Occurrence of Scopulariopsis ssp. other than S. brevicaulis on barley seeds during storage at four levels of urea application. 205 Mrs Hilkka Koponen for her practical knowledge and advice on mycological problems. We wish to thank Mrs Pirkko Korhonen and Mr Mikko Ranta for their technical assistance during the experiment. References Anon. 1973. The determination ofurea in feedingstuffs. The analysis of agricultural materials. Ministry of Agriculture, Fisheries and Food. Technical Bulletin 27. London. Britt, D.G. & Huber, J.T. 1976. Preservation of and animal performance of high moisture corn treated with ammonia and propionic acid. J. Dairy Sci. 59: 668—674. Bothast, R.J., Lancaster, E.B. & Hesseltine, C.W. 1973. Ammonia kills spoillagemolds in corn. J. Dairy Sci. 56: 242—245. —, Adams, G.H., Hatfield, E.E. & Lancaster, E.B. 1975. Preservation of high moisture corn; A micro- biological examination. J. Dairy Sci. 58: 386—391. Christensen, C.M. & Kaufmann, H.H. 1965. Deteriora- tion of stored grains by fungi. Ann. Rev. Phytopath. 3; 69—84. Domsch, K.H., Gams, W. & Andersen, T. 1980. Com- pendium of soil fungi. 859 p. Academic Press. Lon- don, New York, Toronto, Sydney, San Francisco. Hlödversson, R. & Kaspersson, A. 1986. Nutrient los- ses during deterioration of hay in relation to changes in biochemical composition and microbial growth. Anim. Feed Sci. Techol. 15: 149—165. Huhtanen, P. 1984. Wood molasses as a preservative for high moisture barley. 1. Preservation and digestibility in pig. J. Agric. Sci. Finl. 56: 255—263. Joffe, A.Z. 1974. Growth and toxigenity of Fusaria of the Sporotrichiella section as related to environmental factors and culture substrates. Mycopathol. Mycol. Appi. 54: 35—46. Jones, G.M., Mowat, D.N., Elliot, J.I. & Moran, E.T., Jr. 1974. Organic acid preservation of high mois- ture corn and other grains and the nutritional value. A review. Can. J. Anim. Sci. 54: 499—517. Krall, J.L. 1972. High moisture barley harvesting, storing and feeding. Mont. Agric. Exp. Stn. Bull. 625. 45 p. Malone, J.P. & Muskett, A.E. 1964. Seed borne fungi. Seed Test. Ass. 29, 2; 179—384. Muller, H-M. Entgiftung von Mycotoxinen. Übers. Tierernährg. 11: 47—80. Montgomery, R.R., Nofsinoer, G.W. & Bothast, R.J. 1980. Preservation of highmoisture maize a com- parison of gaseous and liquid anhydrous ammonia with methylene-bis-propionate. 5; 337—345. Mowat, D.N. McCaughy, P. & McLeod, G.K. 1981. Ammonia or urea treatment of whole high moisture shelled corn. Can. J. Anim. Sci. 61: 703—711. Neish, G.A., Farnworth, E.R. & Cohen, H. 1982. Zearalenone and trichotecene production by some Fusarium species associated with Canadian grains. Can. J. PI. Pathol. 4: 191—194. Onions, A.H.S. 1966. Scopulariopsis brevicaulis. C. M. I. Descriptions of Pathogenic Fungi and Bacteria No 100. ORSKOV, E.R., Stewart, C.S. & Greenhaloh, J.F.D. 1979. The effect of sodium hydroxide and urea on some storage properties of moist grain. J. agric. Sci., Camb. 92: 185—188. Peplinski, A.J., Brekke, 0.L., Bothast, R.J. Black, L.T. 1978.High moisture corn an extended preser- vation trial with ammonia. Trans. Amer. Agric. Eng. 21: 773—781. Porter, M.G., Patterson, D.C., Steen, R.W. & Gor- don, F.J. 1984. Determination of dry matter and gross energy of grass silage. Proc 7th Silage Conf. Queen’s Univ. Belfast. (Ed. Gordon, F.J. & Unsworth, E.F.). Raper, K.B. & Fennel, D.l. 1965. The genus Aspergil- lus 686 p. Williams & Wilkins Co. Baltimore. & Thom, C. 1949. The Manual of Penicillia. 875 p. Williams & Wilkins Co. Baltimore. ScHADEREIT, R., SCHMIDT, L., WEISSBACH, F., HeNK, G. & Pohlmann, U. 1982. Harnstoff als Konservierungsmit- tel bei der Lagerung feuchter Futterstoffe. 5. Mit- teilung. Erfahrungen und Ergebnisse beim Einsatz von harnstoffkonserviertem Feuchweisen in der Broilerfut- terung. Arch. Tierernähr. 32; 119—128. Schmidt, L., Weissbach, F. & Cöster, H. 1982. Harn- stoff als Konservierungsmittel bei der Lagerung feuchter Futterstoffe. 3. Mitteilung. Verfutterung von harnstoffkonserviertem Feuchtgetreidean Mastrinder. Arch. Tierernähr. 32; 99—108. —, Weissbach, F. &Peters, G. 1978. Harnstoff als Kon- servierungsmittel bei der Lagerung Feuchter Futter- stoffe. 1. Mitteilung Konservierung von Feuctgetreide. Arch. Tierernähr. 28: 123—139. Sokal, R.R. & Rohlf, F.J. 1969. Biometry. The prin- ciples and practice of statistics in biological research. 776 p. W.H. Freeman & Co. San Francisco. Snedecor, G.W. & Cochran, W.G. 1967. Statistical methods. 593 p. 6th Ed. lowa State Univ. Press, Ames. Tempe, J. de 1963. The blotter method for seed health testing. Proc. Intern. Seed Test. Ass. 28, 1: 133—151. Uoti, J. & Ylimäki, A. 1974. The occurrence of Fusa- rium species in cereal grain in Finland. Ann. Agric. Fenn. 13: 5—17. Williams, P.E.V., Innes, G.M. & Brever, A. 1984. Am- monia treatment of straw via the hydrolysis of urea. 1. Effect of dry matter and urea concentrations on the rate of hydrolysis of urea. Anim. Feed Sci. Technol. 11: 103—113. 206 Ylimäki, A. 1981. The mycoflora of cereal seeds and some feedstuffs. Ann. Agric. Fenn. 20: 74—88. Finnish grain. Tech. Res. Centre Finl. Mater. Proc. Tech. 21, 28p. —, Koponen, H., Hintikka, E.-L., Nummi, M., Niku- Paavola, M.-L., Ilus, T. & Enari, T.-M. 1979. Mycoflora and occurrence of Fusarium toxins in Ms received November 18, 1986 SELOSTUS Urea rehuviljan säilöntäaineena. Säilöntäominaisuudet ja vaikutus homeiden kasvuun Asko Hannukkala 1 ja Pekka Huhtanen Helsingin yliopisto, kasvipatologian laitos, 00710 Helsinki Helsingin yliopisto, kotieläinlieleen laitos, 00710 Helsinki Tutkimuksessa selvitettiin urean annostelutason (1.0, 1.5, 2.0 ja 2.5 % tuorepainosta) vaikutusta eri kosteus- pitoisuuksissa (22, 25 ja 32 %) puidun ohran säilönnäs- sä. Urea lisättiin ohraan vesiliuoksena (1:1) hapotuslait- teella viljansiirtoruuvin alkupäähän ja ohra säilöttiin 400 litran lämpöeristettyihin koesiiloihin aerobisesti. Viljan kemiallinen koostumus analysoitiinennen säilöntää ja 12 kuukauden kuluttua säilönnästä. Ureatason ollessa 1.5 % tai korkeampi ohra säilyi laa- dultaan hyvänä koko varastointiajan. Ainoastaan 32 %:n kosteudessa 1.0 %:n ureatasolla säilötty ohra pilaantui 8 kuukauden varastoinnin jälkeen. Urean säilöntävaiku- tus perustuu ammoniakkiin, jota muodostuu urean hyd- rolysoituessa pääasiassa mikrobien tuottaman ureaasin vaikutuksesta. Hydrolysoituneen urean määrä lisääntyi viljan kosteuspitoisuuden ja annostelutason lisääntyessä. Toisaalta hydrolysoituneen urean osuus näytti kuitenkin laskevan annostelutason lisääntyessä. Ureakäsitellyn viljan lämpötila nousi säilönnän jälkeen 17—20 °C:sta 22 23 °C:seen. Keskimääräinen kuiva-ainetappio oli 2.9 % ja raakavalkuaistappio 6.1 %. Ureakäsittelyn vaikutus ohran kemialliseen koostumuk- seen oli vähäinen. Merkittävin muutosoli raakavalkuais- pitoisuuden nousu sekä ammoniakki- ja liukoisen typen osuuden lisääntyminen. Maitohappokäymistä ei todettu, mutta etikkahappoa muodostui jonkin verran (0.6— 3.5 g/kg kuiva-ainetta). Homesienten esiintyminen urealla käsitellyissä ohra- erissä selvitettiin idättämällä siemennäytteet petrimaljoissa kostutetulla suodatinpaperilla. Siementen itävyys todet- tiin 10 vrk:n idätysajan jälkeen. Siementen pinnalla esiin- tyneet homeet tunnistettiin n. 3 viikon idätysajankulut- tua stereo- ja valomikroskoopin avulla. Kullakin näyt- teenottokerralla tutkittiin 200 siemenen näyte-erä jokaista 1 Nykyinen osoite: Kasvitautiosasto, MTTK 31600 Jokioinen. eri kosteus- ja ureatasoa edustavasta ohrasiilosta. Yhteen- sä tutkimuksen kuluessa määritettiin 43800 siemenen ho- melajisto. Siemeneristä tavattiin yhteensä 33 sienisuvun edusta- jia sekä tarkemmin määrittämättömiä sädesieniä. Useim- mat sienisuvut esiintyivät näytteissä satunnaisesti ja nii- den määrä väheni varastoinnin aikana. Kosteimpana (32 %) varastoidussa ohrassa esiintyi enemmän homeita ja vähemmän sädesieniä kuin muissa sienierissä. Kaikki ureakäsittelyt vähensivät homesienten määrää siemenissä. Käsittelemättömissä siemenissä yleisimmät sienisuvut, Acremonium, Allernaria ja Cladosporium, tuhoutuivat siemenistä nopeasti ureakäsittelyn jälkeen. Punahomei- ta (Fusarium-\a]e']a), jotka varsinkin kosteana varastoi- dussa viljassa saattavat muodostaa haitallisia homemyrk- kyjä, esiintyi käsittelemättömissä siemenerissä varsin run- saasti. Niiden määrä oli suurin kosteimpana puidussa oh- raerässä. Alhaisimmat ureatasot eivät vähentäneet puna- homeiden määrää kovin nopeasti. Ureapitoisuuden ollessa vähintään 2 % myöskin punahomeet tuhoutuivat noin 2 viikon varastoinnin aikana. Tyypillisiä varastohomeita, AspergUlus-\n]e]n , sieme- nissä esiintyi vähän ja yli 1.5 %:n ureapitoisuus esti nii- den lisääntymisen varastoinnin aikana. Kaikki ureakäsit- telyt lisäsivät Scopulariopsis-sienlen, joita ei esiintynyt kä- sittelemättömissä siemenissä, määrän hyvin suureksi. Scopulariopsis-sieniä esiintyi toisinaan yli 100 <%:ssa sie- menistä, sillä samassakin siemenessä saattoi esiintyä useita eri lajeja. Scopulariopsis-sienet alkoivat lisääntyä hyvin nopeasti toisen varastointiviikon aikana ja niiden määrä saavutti huippunsa noin 4 viikon varastoinnin jälkeen. Sienten määrä pysyi hyvin suurena 35—40viikkoa, mut- ta väheni nopeasti viimeisen 10viikon aikana. Korkeim- mat ureapitoisuudet näyttivät nopeuttavan Scopulariopsis- sienlen vähenemistä varastoinnin lopussa. Yleisin laji oli S. brevicaulis, jonka tiedetään aiheuttavan ihmisille iho- tauteja. Myöskin sädesienten määrä lisääntyi ureakäsit- 207 telyn seurauksena kaikissa siemenerissä. Niillä tiedetään olevan osuutta homepölykeuhkon synnyssä. Tämän tutkimuksen perusteella urea soveltuu hyvin kostean viljan säilömään ja on lisäksi kustannuksiltaan edullinen. Käytettäessäheinää tai olkea karkearehuna ure- alla säilötyllä viljalla voi lisäksi olla merkitystä pötsimik- robiston typen lähteenä. Ureatason ylittäessä 2 % useim- pien homesienten, erityisesti homemyrkkyjä muodosta- vien, kasvu estyi lähes kokonaan. 208