STORAGE OF MOIST GRAIN WITH ADDITION OF A BIOCONTROL AGENT, PICHIA ANOMALA ISOLATE

Abstract: A process is disclosed for reliable airtight storage and preservation of moist grain by using, as grain, a grain cultivar resisting infection by storage spoilage fungi and effecting the storage in the presence of an antifungally effective amount of a biocontrol agent having the ability to suppress or inhibit storage spoilage fungi while reinforcing the effect of using a grain cultivar resisting infection by such fungi. The preferred biocontrol agent is a Pichia anomala isolate. A biocontrol composition for use in said process is also disclosed, the composition preferably containing a Pichia anomala isolate.

Description: STORAGE OF MOIST GRAIN WITH ADDITION OF A BIOCONTROL AGENT, PICHIA ANOMALA ISOLATE Field of the Invention The present invention relates to a process for airtight storage of moist grain in the presence of a biocontrol agent being a microorganism suppressing storage spoilage fungi to control postharvest mold diseases. Background of the Invention Mold growth in stored grain not only decreases the nutritional value of the grain but also can lead to the formation of mycotoxins and allergenic spores. The storage stability of grains is usually ensuro-d through the removal of water. By reducing the water activity to below 0.60, the growth of spoilage organisms is prevented (Lacey and Magan 1991). However, the drying of grains in temperate climates is expensive. For moist grains used as animal feed, airtight storage provides a less costly alternative. By maintaining an atmosphere low in oxygen and high in carbon dioxide, fungal growth is inhibited, and grains can usually be successfully stored at water contents of 20-40%, corresponding to a water activity of 0.90-1.0 (Lacey and Magan 1991). In reality, however, oxygen tensions high enough to allow growth do occur in airtight systems, and considerable mold infestations can develop, especially if the surrounding temperature rises. Peni- cillium species are important spoilage organisms in airtight-stored grains. Penicillium roqueforti can grow at extremely low oxygen tensions, at least at a water activity of 0.98 (Magan and Lacey 1984). Contamination with Penicillium roqueforti is of special concern, as this organism has been found to produce the mycotoxin roquefortin C in grains (Haggblom 1990). Thus, additional measures to control mold growth in grain storage systems are needed. The possible use of antagonistic microorganisms in the biological control of postharvest mold diseases of fruits and vegetables has been reviewed by Wilson and Wisniewski (1989) and Droby et al. (1991). Certain osmotolerant yeast species, in particular Pichia guillerimondii, are very efficient in controlling wound pathogens, such as Penicillium rots of apples and citrus, when applied on the peel of the fruit (Wilson and Chalutz 1989; Droby et al. 1989; Chalutz 1990; McLaughlin et al. 1990). In an unpublished Swedish study on the microbiology of airtight-stored grains, mold spoilage was found to be greatly reduced in airtight 2 silos in which the yeast Pichia anomala (Hansen) Kurtzman was a dominating species (S. Lindgren, personal communication). This finding indicated that this yeast interacts competitively or antagonistically with molds. Pichia anomala (syn. Hansenula anomala) has also been frequently isolated in other studies on airtight- stored grains and from berries, rubber, brewer's and baker's yeasts, ragi, beer, soft drinks, fruit juices, maple syrup, condensed milk, sauerkraut, salt beans, molasses, miso, man and animals (Barnett et al, 1990; Lacey and Magan 1991). Bjόrnberg and Schnύrer (1993) undertook a study to investigate the possibilities of using a yeast to suppress the growth of common grain spoilage molds. They evaluated the antagonistic effects of Pichia anomala against the molds Penicillium roqueforti and Aspergillus candidus on agar plates. The organisms were cocultured under controlled conditions in vitro and the authors found that Pichia anomala suppressed the growth in vitro of said two important spoilage molds, even during periods of unrestricted air supply. Furthermore, the degree of inhibition of spoilage fungi was related to the initial concentration of Pichia anomala cells. Inhibition was most pronounced at temperatures suboptimal for the growth and sporulation of Penicillium roqueforti and Aspergillus candidus, i.e. below 15°C and above 30°C. In a further study, Petersson and Schnϋrer (1995) determined the antago- istic activity of Pichia anomala, Pichia guilliermondii and Saccharomyces cerevisiae on agar against 17 mold species. It was found that Pichia anomala in particular may be used for the biocontrol of mold growth in moist wheat stored under airtight conditions. This paper discloses that Pichia anomala reduces growth on agar plates for all of the tested mold species in a dose-dependent manner, the different mold species, however, showing different sensitivity of the yeast species investigated. Pichia anomala had the strongest antagonistic activity in wheat, with 10 and 10 CFU/g completely inhibiting the growth of Penicillium roqueforti. Also in this study it was established that the best effect was obtained at temperatures suboptimal for the growth of the mold species. The use of antagonistic microorganisms in the biological control of fungal diseases of e.g. grain, fruit and vegetables is also apparent from the patent literature, examples of which are given below. US 5,670,368 and its divisional US 5,413,783 (McLaughlin et al.) relate to biological control of plant pathogens on agricultural commodities, such as fruits, vegetables, cereals etc. by use of an antagonistic microorganism selected from Pichia guilliermondii and Hanseniasporum uvarum. 3 In US 4,820,531 (Tomes) there is disclosed a method of preserving hay quality in stored high moisture hay by treating fresh hay with the microorganism Bacillus pumilus or mutants thereof. US 4,981,705 (Tomes) is directed to a method of preserving silage by treating the silage with the microorganism Propionibacterium jensenii or genetic equivalents thereof. A method for the preparation or preservation of an animal feed-stuff composition is disclosed in US 5,371,011, comprising depositing onto a forage the antifungal bacterial strain Serratia rubidaea. In US 4,842,871 Hill describes a method of preserving an agricultural product by treating the same with the microorganism Lactobacillus plantarum. The agricultural product may be silage, baled hay or high-moisture grain. The use of Pichia guilliermondii for the biological control of postharvest rots in fruits is disclosed by Wilson et al. in US 5,041,384. Wilson et al. also disclose a method of using isolates of Candida oleophila for the control of postharvest diseases in fruit (US 5,425,941). A method for the treatment or prevention of microbial disease in fruit is also disclosed in US 5,525,132 (Shanmuganathan), the method involving the use of one or more yeast strains. However, none of the prior art references discloses or even indicates the combination of features being characteristic of the present invention. Problem to be solved by the Invention In temperate climates, such as Sweden, cereals grains are commonly harvested, at a water content of 20%, corresponding to a water activity of 0.9 (N. Jonsson, Swedish Inst. Agr. Eng., pers. comm.). Mold growth in cereal grains is often prevented by drying, but in a temperate climate drying procedures are costly and energy demanding. Feed grains can be stored at high-moisture content in airtight silos, where the growth of micro-organisms is prevented by low oxygen and high carbon dioxide levels, caused by respiration of the epiphytic microflora and the grain itself (Lacey and Magan 1991). Difficulties in maintaining a low partial pressure of oxygen and high carbon dioxide level make the storage system sensitive to air exchange between the silo and the external atmosphere. Imperfect sealing, daily temperature fluctuations and the frequent removal of grain all contribute to air leakage to the silo atmosphere. Addition of a biocontrol agent effective in inhibiting mold growth, such as the antagonistic yeast Pichia anomala, would enhance the reliability of airtight systems for storing high- 4 moisture grains. The object of the invention is thus to enhance the reliability of airtight systems for storing high-moisture grains and to counteract the effects of the above air leakage problems. This object is achieved by the present invention. Summary of the Invention The present invention is based upon the surprising discovery that growth of mold species belonging to the Penicillium roqueforti group when storing high- moisture grain with restricted air access is dependent on the grain cultivar used, i.e. there are cultivars with in-born resistance to spoilage fungi during these conditions. Thus, the present invention provides a process for reliable airtight storage and preservation of moist grain, the process being characterized by the combination of using, as grain, a grain cultivar resisting infection by storage spoilage fungi and effecting the storage in the presence of an antifungally effective amount of a biocontrol agent suppressing or inhibiting storage spoilage fungi and reinforcing the effect of using said resisting grain cultivar. The invention also provides a biocontrol composition for use in the claimed process, the composition being characterized by comprising at least one biocontrol agent suppressing or inhibiting storage spoilage fungi, preferably a Pichia anomala isolate. Description of the Drawings Fig. 1 Effects of increasing initial levels of Pichia anomala J121 (•) on the growth and sporulation of Penicillium roqueforti J5 (A) after 14 days of co-culture in winter wheat (cv. Kosack) (a), spring wheat (cv. Dragon) (b), rye (cv. Motto (c), barley (cv. Golf) (d), and oats (cv. Svea) (e). Inoculated level of Penicillium roqueforti J5 is denoted by (Δ). Inoculated grains (a w 0.95) were incubated with a restricted air supply at 25°C for 14 days. Data are given as log CFU/gram of grain (± SD, n=3). Fig. 2 Effects of increasing initial levels of Pichia anomala J121 on the growth and sporulation of the three isolates of Penicillium roqueforti J5 (•), J5A (■), and J282 (A), in co-culture on winter wheat (cv. Kosack, a w 0.95) at 25°C after 14 days. The dotted line represents the mean value of inoculated levels of Peni- 5 cillium roqueforti. Data are given as log CFU/gram of winter wheat. The coefficient of variation for log-transformed CFU values did not exceed 6% for any data point (n=3). Fig. 3 Effects of increasing initial levels of the three isolates of Pichia anomala J121 (•), J281 (A), and CBS5759 (■) on the growth of Penicillium roqueforti J5, in co-culture on winter wheat (cv. Kosack, a w 0.95) at 25°C after 14 days. The dotted line represents the mean value of inoculated levels of Penicillium roqueforti J5. Data are given as log CFU/gram of winter wheat. The coefficient of variation for log-transformed CFU values did not exceed 6% for any data point (n03). Fig. 4 Growth and sporulation of the Penicillium roqueforti-grovφ in glass tubes with a restricted air supply containing a) barley cv. Golf, b) rye cv. Motto, c) spring wheat cv. Dragon (lot A), d) and winter wheat cv. Kosack (a^ 0.96) incubated at 25°C for 14 days. Data are given as log mold CFU/gram cereal grain on day 0 (n= l, empty bars) and day 14 (filled bars). Indicated standard deviations were calculated on log-transformed values (n=3). Fig. 5 Growht or Penicillium roqueforti in wheat removed from the silos and exposed to air after 10 months storage. Wheat was stored with a full air supply at 20°C. Non-inoculated (•), inoculated with 10 3 CFU/g Penicillium roqueforti (■), 10 CFU/g Penicillium roqueforti and 10 CFU/g Pichia anomala (A), or 10 CFU/g Penicillium roqueforti and 10 CFU/g Pichia anomala (Y). Detailed description of the Invention The present invention relates to a process for reliable airtight storage and preservation of moist grain, the process being characterized by the combination of using, as grain, a grain cultivar resisting infection by storage spoilage fungi and effecting the storage in the presence of an antifungally effective amount of a biocontrol agent suppressing or inhibiting storage spoilage fungi and reinforcing the effect of using said resisting grain cultivar. The invention is based upon the surprising discovery that growth of mold 6 species, when storing high-moisture grain at conditions of low oxygen and high carbon dioxide concentrations, is dependent on the grain cultivar used, i.e. there are cultivars having an in-born resistance to spoilage fungi during these conditions. The grain cultivar used is preferably a feed grain cultivar including cereal grain cultivars. Any grain cultivar resisting infection by spoilage fungi can be used. In order to determine whether a grain cultivar resists infection by spoilage fungi, especially Penicillium roqueforti, the skilled man can easily effect that determination by using the procedures proposed in this specification under the headline "II. Cereal grain cultivars having in-born resistance to infection by spoilage fungi", "Materials and Methods". By "cereal grain" is meant, throughout the specification and claims, wheat, rye, barley, oats, rye wheat (triticale), corn (maize), sorghum, millet, rice etc. The most preferred cereal grain cultivars are rye cv. Motto, spring wheat cv. Dragon and the barley cultivars Maud and Mentor. At present, yeasts appear to be the most promising biocontrol agents as alternatives to chemical fungicides in postharvest storage. This is due to that yeasts do not produce mycotoxins or allergenic spores, the vast majority is not pathogenic to man or animal, many yeast strains can grow at low water availabil- ity and on very different substrates at low partial pressures of oxygen. As biocontrol agent, any yeast strain having the ability to suppress the growth of grain spoilage molds and reinforce the effect of using a resistant grain cultivar can be used in the process of the invention, provided it meets the following three selection criteria: (1) it should be naturally present in grains; (2) it should show good growth at low water activity; and (3) it should grow well at low temperature. All of these criteria are met by Pichia anomala, whose suitability has been further supported by observations indicating that it is dominating in mold-free airtight-stored grains. The ability of a yeast strain to suppress the growth of common grain spoilage molds can be determined by the inhibition experiments described by Bjornberg and Schnύrer (1993), page 624, or by Petersson and Schnύrer (1995), page 1028. The use of a Pichia anomala strain as a biocontrol agent is a preferred embodiment of the invention. Using Pichia anomala as a biocontrol agent in cereals could be counted as an extra addition of a common member of the microbial flora in cereals. This is advantageous in getting public acceptance for this biotechnical approach in feed storage. Generally, Pichia anomala has variable growth at both 35 °C and body temperature(Barnett et al, 1990). Human infections caused by Pichia anomala are extremely rare. Particularly useful strains are Pichia anomala J121, Pichia anomala J281 and Pichia anomala CBS5759. 5 The strain Pichia anomala J121 is described in Bjornberg and Schnύrer (1993) and in Petersson and Schnύrer (1995). Said strain has been deposited with the Centraalbureau voor Schimmelcultures (CBS), Baarn, The Netherlands on February 27, 1998 under accession number CBS 100487. A characterization of Pichia anomala J121 is given below. Growth temperature 25 °C Morphology: Pink colonies — Budding cells + Lemon-shaped cells Buds on stalks — Splitting cells — Filamentous + Pseudohyphae + Septate hyphae Arthroconidia — Ballistoconidia — Symmetric ballistoconidia Ascospores: +, hat-shaped Fermentation: D-Glucose + Maltese + Lactose — D-Galactose + Sucrose + Raffinose + Growth: D-Glucose + Maltose + Glycerol + D-Galactose + α,α-Trehalose + Erythritol + L-Sorbose — Methyl α-D-glucoside + D-Glucitol + D-Glucosam ne — Cellobiose — D-Mannitol -L. D-Ribose — Melibiose — myo-lnositol O -Xylose + Lactose — 2-Keto-D-gluconate L-Arabinose — Raffinose + D-Gluconate L-Rhamnose — Melezitose + D-Glucuronate Sucrose -r DL-Lactate + Nitrate Ethylamine + L-Lysine + Cadaverine + 0.01 % Cycloheximide Acetic acid r.roduction ~ at: 35 °C 37 °C + 40 °C 8 Pichia anomala J121 has the ability to grow at pH values from about 2 to about 11 and the ability to grow at temperatures from about 6°C to about 37°C. A particular property of Pichia anomala J121 is its ability to grow at lactate concentrations up to 0.5 M at pH values between about 4 and about 6. In the process of the invention, the presence of the biocontrol agent may be due to its natural occurence in the grain or due to application to the grain of the biocontrol agent, e.g. in the form of a biocontrol composition. The anti-fungal yeast strains according to the present invention may be prepared in any required quantity by fermenting a sample of said strain under suitable conditions in an appropriate medium. Such conditions and media are well lαiown in the art. An example is given below under the headline "I. Pichia anomala as a biocontrol agent of Penicillium roqueforti in high-moisture grain under airtight conditions", "Materials and Methods", "Inocula". The yeast strain may be obtained from the fermentation liquor by concent- ration or ultra-filtration, optionally followed by the addition of any formulating agents such as surface active agents, solid or liquid carriers or diluents, dispersing agents etc. Thus, according to a further aspect of the present invention there is provided a biocontrol composition comprising at least one biocontrol agent suppressing or inhibiting storage spoilage fungi and reinforcing the effect of using a grain cultivar resisting infection by spoilage fungi, preferably comprising a yeast strain and in particular any of the above-mentioned preferred Pichia anomala isolates. In the practice of the process of the invention, the biocontrol composition can be applied as a liquid or a particulate solid. When it is to be applied as a liquid, the carrier will be water whilst when it is to be applied as a solid, the carrier will be a solid material. Example of solid carriers include cereals such as ground barley and wheat, ground corn cobs and other materials such as clay, chalk, magnesite, limestone and talc. Any further material added to the biocontrol composition of the invention includes nutrients, growth factors, antioxidants and materials intended to reduce dusting tendencies of the additives, improve adhesion to the grain to be stored or enhancing selectively the growth of the yeast strain used as biocontrol agent. The relative proportions of carrier and yeast strain included in the biocon- trol composition for use in storage of grain depend upon the particular yeast strain used and its activity and viability. The concentration of living cells, as 9 defined by the number of Colony Forming Units (CFU), may be used as a guide for determining application rates. As a guide, the yeast cells may be applied as a liquid suspension or solid additive in concentrations in the range from about 10 CFU to about 10 CFU per gram of grain stored, depending upon the nature and condition of the grain. Preferably, the concentration is in the range from about 10 4 to about 10 CFU per gram of grain. In the preparation of the biocontrol composition of the invention, the yeast strain is usually dried by suitable means, e.g. freeze dried or spray dried, or a living culture is resuspended in water, and is then mixed with other components by conventional blending/mixing procedures. In a further aspect of the invention, there is provided a method for reliable airtight storage and preservation of moist grain, the process being characterized by applying to the grain a biocontrol composition comprising a biocontrol agent suppressing υr inhibiting the growth of storage spoilage fungi, the grain used being a grain cultivar resisting infection by storage spoilage fungi. The biocontrol agent is preferably a Pichia anomala isolate as defined above. The biocontrol composition may be the yeast cells as such or be a formulation containing, in addition to the yeast cells, formulation agents as defined above. The application of the biocontrol composition of the invention may be effected by mixing, dusting, injecting, rubbing, spraying, brushing, dipping or rolling. The airtight storage may be effected in silos having restricted air access or in airtight sacs of plastic, rubber or similar materials. In the pilot plant experiments referred to below, Pichia anomala cells were applied by spraying onto the grains in a feed blender to get an even distribution. In full-scale, the yeast may be added by insertion of a spray jet in the transport screw when loading the silo. The subsequent mixing will distribute the yeast cells evenly on the grains. The inoculum was added contained in the growth medium comprising a minor part of the nutrients. It is also possible to add the cells of Pichia anomala washed in tap water. Commercial Utilization and Advantages The results presented in the Experimental Section to follow indicate the feasibility of large-scale application of Pichia anomala as a biocontrol agent against postharvest spoilage of airtight high-moisture feed grain. The yeast inhibits growth of the Penicillium roqueforti group, the most important spoilage 10 molds in airtight storage, challenged inoculated to a very high level during silo storage as well as delays mold growth when grain removed from the silos is exposed to air. The advantages obtained with the different aspects of the present invention are: the invention improves preservation of the grain; the invention improves the biological value of the grain stored; the invention permits grain to be stored at higher moisture content saving energy costs; - the invention provides a grain with better keeping qualities even after conclusion of the airtight storage; the invention exerts biocontrol mainly through competition for nutrients and oxygen; Pichia anomala is growing vigorously on cheap growth media using simple technique, important factors for commercial utilisation of this yeast on a large scale. The invention will be further illustrated by the Experimental Section to follow below. Experimental Section In the experimental support of the invention we evaluated the use of Pichia anomala as a biocontrol agent during storage of wheat (winter and spring forms), rye, barley, and oats, which are the most commonly grown cereals in Sweden. These cereals can all be used as feed grains and can be stored under airtight conditions without previous drying. To mimic a malfunctioning airtight system for storing high-moisture grain, glass tubes were filled with inoculated grain (water activity 0.95) and sealed with a butyl rubber stopper. To simulate air leakage a needle was inserted through each butyl rubber stopper. Three isolates of Pichia anomala were compared as biocontrol agents in high-moisture winter 11 wheat subjected to a restricted air supply. In addition, the sensitivity of three Penicillium roqueforti isolates to Pichia anomala were evaluated in winter wheat. I. Pichia anomala as a biocontrol agent of Penicillium roqueforti in high-moisture grain under airtight conditions Materials and methods Fungal isolates Pichia anomala (Hansen) Kurtzman J121 with previously shown antagonistic effect (Petersson and Schnύrer 1995) was originally isolated from airtight- stored wheat, as was our recent isolate Pichia anomala J281. For comparison we used the Pichia anomala strain CBS5759 from Centraalbureau voor Schimmelcultures (CBS), Baarn, The Netherlands. Penicillium roqueforti Thorn J5 and Penicillium roqueforti J5A, originally isolated from airtight-stored grains, and Penicillium roqueforti J282 recently isolated from silage were gifts from Dr. P. Haggblom, National Veterinary Institute, Uppsala, Sweden. All fungi used are available from the fungal collection of the Department of Microbiology, Swedish University of Agricultural Sciences, SLU, Uppsala, Sweden. The yeasts were identified with the ID32C test (Biomerieux, Marcy l'Etoile, France) and through morphological studies (Kreger-van Rij 1984; Kurtzman 1984). The mold isolates were identified as Penicillium roqueforti through morphological studies (Pitt 1991; Samson et al. 1995). The fungal cultures were maintained on slants of malt extract agar (MEA, Oxoid, Basingstoke, UK) at 2°C. Cereal grains Winter wheat (cv. Kosack), spring wheat (cv. Dragon), rye (cv. Motto), barley (cv. Golf), and oats (cv. Svea), all of seed quality, were bought from the Lantmannen Cooperatives, Uppsala, Sweden. Inocula Yeast suspensions were prepared by inoculating 100 mL of a yeast-extract malt-extract sucrose broth (0.2 yeast extract (BBL, Meyland, France), 1.5 g malt extract (Oxoid, Basingstoke, UK), 1.0 g sucrose (BDH, Poole, England) in 1.0 litre distilled water) with a loopful of cells from a culture stored on MEA at 2°C. After incubation on a rotary shaker (100 rpm) at 25°C for 48 h, the number of yeast cells was determined using a hemocytometer. The spore suspension of molds was 12 prepared by collecting spores from 7-day-old colonies (grown on MEA at 25°C) in peptone water (2 g Phytone™ peptone (BBL, Becton Dickson and Co., Cockeysville, USA) per litre distilled water) with 0.015% Tween 80 added to assist dispersal of conidia. The spore concentration was determined with a hemocytometer. Inhibition of Penicillium roqueforti J5 inoculated to wheat, rye, barley, and oats by the yeast Pichia anomala J121 Non-sterile cereal grains stored at a water content of 10% at 20°C were moistened with tap water to obtain a water activity of 0.95 (ca. 22% water content). To equilibrate the moisture content the grains were stored 3-5 days at 2°C with frequent mixing. Water activity (a w ) was measured at 25°C with a Novasina TH-2/RTD-33/BS equipped with a thermoconstanter (Defensor AG, formerly Novasina AG, Pfaffikon, Switzerland). Grains with a w 0.95 were inoculated with the mold spore suspension to about 3 x 10 mold spores per gram. The spores were applied by adding the suspension as drops onto the grain and mixing to obtain an even spore distribu- tion. The yeast Pichia anomala was similarly inoculated to reach 0, 10 , 10 , 10 , 10 , 10 and 10 cells per gram grain in separate tests. The inoculations, < 1 mL fungal suspension per 100 gram of cereal grains, did not measurable affect the water activity of the grains. Thick-walled glass tubes (27 mL) were filled with approximately 17-g portions of inoculated grain and sealed with a butyl rubber stopper. To simulate air leakage, a needle (diam 0.6 mm) was inserted through each butyl rubber stopper. The tubes were incubated at 25°C. According to earlier findings these conditions (a w 0.95, 25°C, 3 x 10 mold spores per gram) represents an environment favouring Penicillium roqueforti growth, and were choosen in order to subject the antagonistic activity of Pichia anomala to a very rigid test (Petersson and Schnύrer, 1995). On day 14 three tubes from each treatment were opened, and the contents were diluted tenfold with peptone water, soaked for 0.5 h and homogenised for 2 min at normal speed in a Stomacher 400 (Colworth, UK). Yeast growth was quantified on MEA supplemented with 100 ppm chloramphenicol (C-0378 Sigma Chemical Co. St. Louis; Mo. USA) (MEAC plates). To avoid growth inhibition by antagonistic yeast on agar plates intended for mold colony forming units (CFU) counting, MEACC (MEAC supplemented with 10 ppm cycloheximide C-7698 Sigma Chemical Co., St. Louis; Mo, USA) was used for surface plating 0.1 mL aliquots. The chloramphenicol is a bacteriostatic, broad-spectrum antibiotic, and 13 cycloheximide inhibits growth of Pichia anomala (Bjornberg and Schnύrer 1993). Lactic acid bacteria and the total number of aerobic bacteria were counted on LBS (BBL, Meyland, France) and plate count agar (Oxoid, Basingstoke, UK), respectively, both supplemented with 0.1% Delvocid ® (Gist-Brocades, Delft, The Nether- lands). Delvocid is a broad-spectrum fungicide with natamycin as the active agent. Only the control parts of the experiments, i.e. tubes not inoculated with yeasts, were analysed for bacteria. Inhibition of three isolates of Penicillium roqueforti using Pichia anomala J121 To assess isolate-related variation in sensitivity to the antagonistic yeast Pichia anomala J121 in airtight-stored high-moisture winter wheat, three isolates of Penicillium roqueforti (J5, J5A, and J282) were used in three separate experiments, performed as described above. Inhibition of Penicillium roqueforti J5 using three isolates of Pichia anomala Three isolates of Pichia anomala were compared in terms of their antagonistic activity against Penicillium roqueforti J5 in airtight-stored high-moisture winter wheat. Pichia anomala isolates J121, J281, and CBS5759 were used in three separate experiments performed as described above. All inhibition experiments were run in triplicate and repeated twice. RESULTS Inhibition of Penicillium roqueforti J5 inoculated to wheat, rye, barley, and oats by the yeast Pichia anomala J121 On winter wheat (cv. Kosack) did Pichia anomala reduce the growth of Penicillium roqueforti in a dose-dependent manner (Fig. la). Also, on spring wheat (cv. Dragon) Penicillium roqueforti was reduced in a dose-dependent manner, although its growth was not as vigorous as on winter wheat (Fig. lb). On rye (cv. Motto) Penicillium roqueforti did not grow, regardless of whether or not the grain had been inoculated with Pichia anomala (Fig. lc). In fact, a weak decline in Penicillium roqueforti CFU (compared with inoculated amount) was even observed on rye without inoculated Pichia anomala. Penicillium roqueforti grew well in barley (cv. Svea), increasing from 5 x 10 to 10 CFU/g, which was comparable 14 to its growth on winter wheat (Fig. la and d). Pichia anomala reduced the growth of Penicillium roqueforti in barley to 10 CFU/g at 10 inoculated CFU/g of Pichia anomala and totally inhibited growth at 10 5 CFU/g (Fig. Id). The growth of Penicillium roqueforti on oats was comparable to that on winter wheat and barley (Fig. le). Pichia anomala totally inhibited the growth of Penicillium roqueforti on oats at 10 4 CFU/g. Pichia anomala grew to the same level on four of the five tested cereals, i.e. to about 5 x 10 CFU/g, when inoculated to densities higher than 10 cells per gram, the exception being spring wheat (cv. Dragon) where it reached about 1 x 10 9 CFU/g (Fig. lb). At inoculation levels below 10 3 CFU/g the growth of Pichia anomala was most pronounced in spring wheat and rye (Figs, lb and c). After 14 days of incubation, lactic acid bacteria were found at levels between 10 2 and 10 3 CFU per gram in all cereals (data not shown). The total aerobic count of bacteria was 10 CFU per gram in rye and 10 CFU per gram in the other cereals (data not shown). Inhibition of three isolates of Penicilium roqueforti using Pichia anomala J121 Without addition of yeast Penicillium roqueforti J5, J5A, and J282 all grew to the same extent on winter wheat, i.e. from 5 x 10 3 to 10 6 CFU/g (Fig. 2). Penicillium roqueforti J5, J5A, and J282 were very similar in terms of their sensitivity to the antagonistic yeast Pichia anomala J121. Growth was distinctly affected by an initial level of 10 4 CFU/g and totally inhibited by 10 5 CFU/g (Fig. 2). Inhibition of Penicillium roqueforti J5 using three isolates of Pichia anomala Pichia anomala J121, J281, and CBS5759 all reduced growth of Penicillium roqueforti J5 on winter wheat (cultivar Kosack) (Fig. 3). Growth of Penicillium roqueforti was totally inhibited by an initial level of 10 CFU of Pichia anomala per gram. In the experiment with Pichia anomala J121, higher mold counts were found at both 0 and low yeast inoculations ( < 10 3 CFU/g) than the other two experiments (Fig. 3). 15 From the results above the following conclusions can be drawn. Pichia anomala inhibits the growth and sporulation (measured as CFU) of Penicillium roqueforti on winter wheat, barley, and oats in a dose-dependent manner. The antagonistic activity of Pichia anomala in cereal grains stored in a tube with a restricted air supply was not dependent on a cereal variety. Pichia anomala grew strongly on all the five tested cereals. For some reason, the grain infection with Penicillium roqueforti was clearly lower on rye, and to some extent also on spring wheat. Differences in micro- environment on the grains owing to the presence of husks, competing micro- organisms, nutrient availability, or antifungal compounds can all be expected to affect the growth of both the inoculated yeast and mold. The availability of nutrients on cereal grain probably differs between cultivars, and is also dependent on the microbial flora and earlier handling of the grain. We have observed n bacterial loads between 10 and 5 x 10 CFU per gram and yeast loads between 10 2 CFU (detection level) and 5 x 10 5 CFU per gram in wheat stored at 10% water content (unpublished data). If these differences are caused by microbial growth on the grains, and not only by chance contamination, considerable differences in consumed nutrients on the grains might be expected. However, the bacterial load on the cereals does not explain the lack of growth of Penicillium roqueforti in rye and weak growth on spring wheat compared with winter wheat, oats and barley. The count of aerobic bacteria after the incubation period was clearly lower in rye than in the other cereals. Still, the level reached by Pichia anomala on rye, on which Penicillium roqueforti did not grow, was at least as high as that reached on the other cereals. There could, however, have been antagonistic micro-organisms other than Pichia anomala in the rye but no bacteria or yeast were identified or characterised. The long-term storage and culture of micro-organisms can alter their properties. Consequently, a strain of Pichia anomala recently isolated from winter wheat was evaluated. Also, one of the three Penicillium roqueforti used was recently isolated from silage. The antagonistic activity of Pichia anomala against Penicillium roqueforti in our model system was not isolate-specific. Further, the three Penicillium roqueforti isolates were equally suppressed by Pichia anomala J121 on high-moisture winter wheat stored with a restricted air supply. The ability of Pichia anomala J121 to effectively reduce the growth of Penicillium roqueforti reflects the stability of this antagonistic activity, even after years of 16 storage under laboratory conditions. In conclusion, Pichia anomala reduced growth of Penicillium roqueforti in high-moisture winter wheat, oats, and barley in a container with a restricted air supply. This finding suggests that Pichia anomala could be used to improve reliability in airtight-storage of feed grains. Since Penicillium roqueforti did not grow on rye, it was not possible to evaluate mold inhibiting activity of Pichia anomala on rye. However, the surprising discovery that Penicillium roqueforti did not grow on rye further initiated us to investigate the growth of Penicillium roqueforti and a number of different mold species on different grain cultivars. The surprising results then obtained are discussed in detail below. II. Cereal grain cultivars having in-born resistance to infection by spoilage fungi The species Penicillium roqueforti comprises several taxa and has recently been suggested to be divided into three different species named Penicillium roqueforti, Penicillium carneum, and Penicillium panum based on i.a. different secondary metabolite profiles and nucleic acid sequence differences (Boysen et al., 1996). The three species, Penicillium roqueforti, Penicillium carneum, and Penicillium panum, are morphologically very similar and, for practical purposes, can be considered as subspecies or as a single species (Pitt and Hocking, 1997). Today nothing is known about differences in natural habitat for the three species. All of them probably have the potential to spoil food as well as feed, especially in silage and airtight stored high moisture grain. Below will follow an account of our investigations as regards the infection ability of Penicillium carneum, Penicillium paneum and Penicillium roqueforti during airtight storage of high-moisture winter wheat, spring wheat, rye, and barley. Also, the postharvest pathogenicity of the Penicillium roqueforti, i.e. Penicillium roqueforti, Penicillium carneum and Penicillium paneum, towards different cultivars and lots of spring wheat and cultivars of barley are compared. Materials and methods Fungi studied Penicillium roqueforti Thorn J5 and Penicillium roqueforti J5A were gifts from Dr. P. Haggblom (Swedish Veterinarian Institute, Uppsala, Sweden). Penicillium carneum IBT14042, Penicillium carneum IBT6889, Penicillium 17 paneum IBT13929, Penicillium paneum IBT13321, Penicillium roqueforti IBT18689, and Penicillium roqueforti IBT14088 were gifts from Dr. J. Frisvad, Biotechnological Institute, Lyngby, Denmark. Species identities were confirmed based on morphology and RAPD profiles (Boysen et al., 1996). The fungal cultures were maintained on slants of malt extract agar (MEA; Oxoid, Basingstoke, UK) at 2°C. All fungi used were deposited in the fungal collection at the Department of Microbiology, Swedish University of Agricultural Sciences, SLU, Uppsala, Sweden. Cereals studied Winter wheat (lat. Triticum aestivum) cv. Kosack, spring wheat (lat. Triticum aestivum) cv. Dragon (referred to as A), rye (lat. Secale cereale) cv. Motto, and barley (lat. Hordeum vulgare, spring form) cv. Golf were all bought from Lantmannen Cooperative, Uppsala, Sweden. Barley cv. Baronesse, Kinnan, Maud and Mentor were gifts from Jδrgen Lόhde (Svalof Weibull AB, Svalov, Sweden). Baronesse and Kinnan are used as feed grain while Maud and Mentor are malt barley cultivars. Spring wheat cv. Alve, Curry, Dragon (referred to as B), Thasos, and Tjalve were gifts from Jan Persson (Svalof Weibull AB, Svalov, Sweden). Four extra lots of spring wheat cv. Dragon from Svalof Weibull AB were used to investigate variations in resistance to growth of Penicillium roqueforti (J5) among lots of the same variety. The lots are being referred to as C (P151703 -95), D (M. Elit 38621), E ( > 96), and F (-97). All lots represent the same genotype of spring wheat cv. Dragon; however, each one was grown under different conditions. Inocula preparation The conidial suspensions of the molds were prepared by collecting spores from 7-day old colonies (grown on malt extract agar (MEA) [Oxoid, Basingstoke, England] at 25°C) in peptone water (2 g Phytone™ peptone [BBL, Becton Dickinson and Company, Cockeysville] per litre distilled water) with 0.015% Tween 80 added to assist dispersal of conidia. The spore concentration was determined using a hemocytometer. Infection ability of Penicillium roqueforti, Penicillium carneum, and Penicillium paneum in barley, rye, and wheat Non-sterile cereal grain, previously stored at a water content of 10% at 18 20°C, were moistened with tap water to obtain the water activity (a-^) of 0.96 (about 25% water). To equilibrate the moisture content the grains were stored for 2-4 days at 2°C with frequent mixing. Water activity was measured at 22-24°C using a CX-2 AquaLab equipment (Decagon Devices, Inc., Washington, USA). Grain (a w 0.96) was inoculated with mold spore suspension to reach about 10 mold spores per gram. The spores were applied by adding the suspension as drops onto the grain, followed by careful mixing to obtain an even spore distribution among the grains. Thick-walled glass tubes (27 ml) were filled with approximately 17-g portions of inoculated grain and sealed with a butyl rubber stopper. To simulate air leakage, a needle (diam. 0.6 mm) was inserted through each butyl rubber stopper. The tubes were incubated at 25°C. On day 14 three tubes from each treatment were opened and the contents were diluted tenfold with peptone water, soaked for 0.5 h and homogenized for 2 min at normal speed in a Stomacher 400 (Colworth, UK). To avoid growth inhibition by bacteria on agar plates intended for mold CFU counting, 0.1 ml aliquots were surface plated on malt extract agar, supplemented with 100 ppm chloramphenicol (C-0378 Sigma Chemical Co. St. Louis; Mo. USA) (MEAC). The chloramphenicol is a broad- spectrum bacteriostatic substance. Lactic acid bacteria and total number of aerobic bacteria were counted on M.R.S. (de Man, Rougosa, Sharpe; Oxoid, Unipath Ltd., Basingstoke, Hampshire, England) and plate count agar (Tryptone glucose yeast agar; Oxoid, Unipath Ltd., Basingstoke, Hampshire, England), respectively, both supplemented with 0.1% Delvocid ® (Gist-Brocades, Delft, The Netherlands). Delvocid is a broad-spectrum fungicide containing natamycin as active agent. RESULTS Infection ability of Penicillium roqueforti, Penicillium carneum, and Penicillium paneum in barley, rye, and wheat In barley (cv. Golf), all eight mold isolates in the P. roqueforti-group increased from the inoculation level of 5 x 10 3 to 4 x 10 5 CFU/g (Fig. 4a). The total count of molds other than those belonging to the added P. roqueforti-group declined during the incubation period from 1 x 10 to below the detection limit. The amount of yeast was below the detection limit on day 0 as well as day 14. The amount of aerobic bacteria in barley was 1 x 10 CFU/g both at the start and aftar 14 days of incubation, regardless of the species of mold species added. The 19 lactic acid bacteria increased in number from below the detection limit, i.e. 10 CFU/g, to 8 x 10 3 CFU/g in all treatments. In rye (cv. Motto) none of the eight mold isolates in the P. roqueforti-growp o grew (Fig. 4b). Instead, the average number of mold CFU decreased from 5 x 10 CFU/g to 3 x 10 2 CFU/g between day 0 and day 14. The total count of molds other than those belonging to the added P. roqueforti-grovφ declined from 1 x 10 to below the detection limit. The amount of yeast in the samples was below the detection limit on day 0 as well as day 14. The aerobic bacteria in rye declined 7 f from 1 x 10 to 2 x 10 regardless of the species of mold added. Lactic acid bacteria grew from below the detection limit to 5 x 10 CFU/g. In spring wheat cv. Dragon (A), all isolates showed poor growth. The average CFU increased from 4 x 10 to 7 x 10 per gram (Fig. 4c). Numbers of P. paneum and P. carneum increased slightly, but significantly more, compared with isolates of P. roqueforti (P < 0.01). The total count of molds other than those 9 * belonging to the added P. roqueforti-grovψ increased from 2 x 10 to 3 x 10 CFU/g. The concentration of yeast increased during the incubation period from 2 x 10 to 3 x 10 CFU/g. The aerobic bacteria in spring wheat increased in 7 7 number from 1 x 10 to 4 x 10 during the 14 days of incubation. The lactic acid o bacteria increased from below the detection limit to 1 x 10 CFU/g. In winter wheat (cv. Kosack) all three species in the P. roqueforti-groxip increased in number to about 5 x 10 CFU/g (Fig. 4d), from a detected inoculum level ranging from 2 x 10 to 9 x 10"* CFU/g. The total count of other molds increased from below the detection limit to 4 x 10 CFU/g. The yeast in winter wheat remained at the day-0 level of 8 x 10 CFU/g throughout the incubation. c 7 ** The aerobic bacteria increased in number from 6 x 10 to 6 x 10 CFU/g during the incubation regardless of the mold species added. The lactic acid bacteria increased in number from below the detection limit to 3 x 10 CFU/g. Growth of Penicillium roqueforti, Penicillium carneum, and Penicillium paneum in various cultivars of barley and spring wheat Barley For all strains of P. roqueforti, P. paneum and P. carneum CFU levels increased significantly (P < 0.001) in the feed cultivars Baronesse and Kinnan, but not in the malt cultivars Maud and Mentor (Table 1). In the cultivars Maud and Mentor, P. paneum and P. carneum increased to somewhat higher CFU values 20 than those reached by P. roqueforti (Table 1). The amount of aerobic bacteria on day 0 was 1.8 x 10 CFU/g in cv. Baronesse, ' 4.6 x 10 5 CFU/g in Kinnan, 1.8 x 10 7 CFU/g in Maud, and 3.7 x 10 6 CFU/g in Mentor. After 14 days of incubation the aerobic bacteria had grown to 3.9 x 10 7 CFU/g in cv. Baronesse and to 4.7 x 10 6 CFU/g in cv. Kinnan. In the malt cultivars Maud and Mentor there was no detectable growth of aerobic bacteria. In the feed cultivars Baronesse and Kinnan the aerobic bacteria grew significantly more in grain inoculated with P. roqueforti than in that inoculated with P. paneum or P. carneum (P < 0.001, data not shown). Numbers of lactic acid bacteria increased to 10 CFU/g in barley cultivar Kinnan in combination with P. roqueforti, but were not detectable with the other inoculated Penicillium spp. or in the other cultivars. The initial amount of yeast was 1.5 x 10 4 CFU/g in barley cv. Baronesse, 1.2 x 10 5 CFU/g in cv. Maud, 2.6 x 10 4 CFU/g in cv. Mentor, and 5.5 x 10 3 CFU/g in cv. Kinnan. Numbers of yeast colony forming units decreased during the experiment to 9 x 10 CFU/g in cv. Baronesse, 1.5 x 10 CFU/g in cv. Mentor, and to less than 10 3 CFU/g in cv. Maud and cv. Mentor. Spring wheat In the spring wheat cultivars Alve, Curry, Tjalve, and Thasos all strains of P. roqueforti, P. paneum and P. carneum increased from an inoculated level of about 10 4 CFU/g to 10 6 CFU/g during 14 days (P < 0.001). However, in the two lots of cultivar Dragon (A and B) there was no significant increase in numbers of CFU for any of the eight fungal isolates tested (Table 2). Aerobic bacteria increased in the spring wheat cultivars from about 10 to 10 7 CFU per gram in cv. Alve and Tjalve and from 10 7 to 10 8 CFU per gram in cv. Curry, Dragon (lot A), and Thasos during the 14 days of incubation, regardless of the mold species inoculated. Lactic acid bacteria grew during the incubation in spring wheat cv. Curry to 10 CFU/g, but were not detectable in the other cultivars. Yeast grew in spring wheat cultivar Dragon (lot A) to 6 x 10 CFU/g during the incubation, but were not detectable in the other varieties. Six different lots of spring wheat cv. Dragon were inoculated to about log 4.6 CFU/g with P. roqueforti J5 to evaluate the resistance of the cultivar to the fungus under conditions where the air supply is restricted. In lot A or F P. roqueforti J5 did not significant increase in CFU, whereas weakly significant (P < 0.05) growth was observed in B and significant growth (P < 0.01) in lot C. 21 Contrary, there was a highly significant (P < 0.001) increase of P. roqueforti J5 in lot D and E and in the control grain winter wheat cv. Kosack, (Table 3). In general, the Penicillium roqueforti-group, i.e. Penicillium roqueforti, Penicillium carneum, and Penicillium paneum, did not differ in their ability to grow in the tested cereal grain. Among the tested cereals, i.e. barley (four cultivars), rye, winter wheat, and spring wheat (five cultivars), only rye cv. Motto and two lots of spring wheat cv. Dragon resisted infection by the Penicillium roqueforti-group . The lack of general differences in infection ability of Penicillium roqueforti, Penicillium carneum, and Penicillium paneum in the tested cereals does neither contradict nor support the split of Penicillium roqueforti into three species. Neither is it possible to predict, out of the presented results, if one of the species is more likely than the others to be the major spoilage fungi in airtight storage of high-moisture grains. The microbial flora in the grains can protect against invading fungi. Fungi can reduce growth of other fungi by parasitism, antibiosis, or nutrient competition. The yeast in all barley cultivars tested was reduced in number during the incubation period. However, in spring wheat yeast numbers after the incubation period were higher in cv. Dragon than in other cultivars. The yeast might have contributed to the resistance to fungal growth of spring wheat cv. Dragon. The bacterial load might also inhibit fungal invasion by consuming nutrients or excreating antibiotic substances. In the feed barley cultivars both aerobic bacteria and mold grew well, whereas in the malt cultivars there were less growth of both aerobic bacteria and mold. Thus, the bacterial growth was not inhibiting the mold growth. In conclusion, barley cv. Maud and Mentor, rye cv. Motto, and two lots of spring wheat, cv. Dragon were resistant to the Penicillium roqueforti-group. The resistance is assumed to be dependent on both pre- and post-harvest treatments. All three species in the Penicillium roqueforti-group, i.e. Penicillium carneum, Penicillium paneum, and Penicillium roqueforti, uniformly infected the tested cereals. Consequently, all three species can spoil airtight stored high-moisture grains. The selection of less suspectible cultivars with in-born resistance to infection by spoilage fungi will be of great importance in assuring better microbial quality of the final feed or foodstuff, while minimizing the use of agrochemi- cals. 22 III. Pilot plant experiments Petersson and Schnύrer (1995) showed that the strong inhibitory effect of Pichia anomala on Penicillium roqueforti on agar plates was also evident on moist wheat kernels stored at 25°C, even though this is close to the optimum tempera- ture for this mold (Bjornberg and Schnύrer 1993). However, antagonists that show promise under laboratory conditions do not necessarily perform as well in a scaled up-trial. For example, interactions may vary, depending on substrate type or other environmental conditions (Magan and Lacey 1985). Used as a biocontrol agent, a micro-organism has to compete with other fungi to establish an active population in the new environment. The ability to rapidly build up a population is assumed to be a prerequisite for successfully competing with spoilage microorganisms or pathogens. In Sweden, cereal grain are commonly harvested at a water content of 20- 22%, corresponding to water activity of 0.9. Mold growth in cereal grain is often prevented through high-temperature drying, though it is costly and energy demanding in a temperate climate. On farms where cereals are grown using conventional cultivation methods, about 60% of the total quantity of fuel used in plant husbandry operations is consumed in connection with the high-temperature drying of grain (Pick et al. 1989). Airtight storage, which is an alternative method of storing feed grain, relies on perfect sealing in combination with the anaerobic conditions caused by respiration of the grain itself and the adhering microorganisms. Airtight storage only requires about 2% of the energy consumed in high-temperature drying (Pick et al., 1989). However, air leakage during airtight storage can readily lead to a deterioration of the conditions required for preserva- tion. Changes in internal silo temperature (mostly due to solar radiation) and atmospheric pressure, together with an increasing gas volume as the silo empties, lead to air movements through the relief valves and shell perforations, or through the emptying auger tube. In practice, grain is commonly not taken out from the silos every day. Instead, larger batches are stored with full access to air for several days. Measures for preventing mold spoilage and mycotoxin production would be advantageous during this short-term storage as well. To evaluate the practical potential of Pichia anomala as mold biocontrol agents we constructed 0.2 m "pilot scale silos" for airtight storage of moist grain. High-moisture wheat with various inoculum concentrations of Pichia anomala and 23 Penicillium roqueforti were stored in the silos, and sampled twice a week. Concentrations of oxygen and carbon dioxide were monitored and the microbial quality of the grains were followed during 13 months of storage. The mold suppressing ability of Pichia anomala during aerobic storage of wheat removed from the silos was evaluated after 10 and 12 months of airtight storage. Also, the survival of Pichia anomala in high-moisture wheat was evaluated at different temperatures during 15 months of airtight storage. Silos The silos each contained 0.21 m grain corresponding to 160 kg. To equalize differences between internal and external air pressure each silo was equipped with both a breather bag and a 2-way pressure relief valve made of an U-tube filled with a non-freezing liquid. Except when grain was removed through the unloading spouts, no leakage could occur through the relief valve until the pressure exceeded ± 60 mm static water gauge. This pressure differential could not build up until the breather bag had been either filled with 14 liters of air or 7 liters of air had been drained from it. Gas analysing equipment The concentrations of C0 2 and 0 2 in the silos were measured by an infrared monitor (accuracy ± 0.5%) and a galvanic cell (accuracy ± 1%) (GA 90, Geotechnical Instruments Limited, Leamington Spa, Warwickshire, England), respectively. Pressure variations were continously registered with a differential pressure meter (Model 600D, Innovex, Hopkins, MN, USA) in one of the silos. The temperature of the wheat was measured on each sampling occasion with an infrared thermometer (Model C-600, Linear Laboratories, Mountain View, CA, USA). Inhibition experiments Yeast and mold spore suspensions were prepared as described above. Conventionally grown winter wheat (cv. Kosack) was harvested at a water content of 24% corresponding to ^ 0.94. Water activity was measured at 22-24°C with an Aqua Lab CX-2 (Decagon Devices Inc., Pullman, Washington, USA). Water activity in the wheat was measured on samples after 0, 8, 10 and 57 weeks. 24 Inoculation Freshly harvested wheat grain was inoculated with the Penicillium roqueforti spore suspension to about 3 x 10 3 mold spores per gram. The spores were applied by adding the suspension as drops onto the grains during mixing in a feed mixer to obtain an even spore distribution on the grains. The yeast was similarly inoculated to reach 0, 10 and 10 cells per gram wheat. In the control treatment only tap water was added. The same volume of inoculum was used in all experiments. The silos were filled with approximately 160-kg of inoculated wheat, and each experiment was run in triplicate. After sealing, the silos were placed in an outdoor shelter protected from rain and direct solar radiation during 13 months. Intentional air inlet After 54 weeks of storage, the silos were opened to obtain an elevated oxygen concentration in the silos. First the caps of unloading spouts (diameter 44 mm) were removed and after 3 days also the connections to the breather bags (diameter 8 mm) were removed until the oxygen concentration was about 18% before closing, i.e. 5 days. The concentrations of C0 2 and 0 2 were continuously monitored first in one silo with wheat inoculated with 10 CFU of Pichia anomala per gram and then in one of the εilos with vninoculated grains. The microbial flora was analysed one week after closing the silos. Sampling and quantification of microbial growth The silos were initially sampled after one month of storage and then twice a week. The wheat samples, 1 kg each, were removed through the unloading spouts. Samples were taken after 5, 5.5, 6, and 8 weeks, and monthly thereafter. After thorough mixing, aliquots of about 20 gram were diluted tenfold" with peptone water, soaked for 0.5 h and homogenised for 2 min at normal speed in a Stomacher 400 (Colworth, UK). Yeast growth was quantified on MEA supplemented with 100 ppm chloramphenicol (C-0378 Sigma Chemical Co. St. Louis; Mo, USA) (MEAC plates). To avoid growth inhibition by antagonistic yeast on agar plates intended for mold CFU counting, MEACC (MEAC supplemented with 10 ppm cycloheximide; C-7698 Sigma Chemical Co., St. Louis; Mo. USA) was used for surface plating of 0.1 ml aliquots. Cycloheximide at 10 ppm inhibits the growth of Pichia anomala (Bjornberg and Schnύrer, 1993). Lactic acid bacteria and the total number of aerobic bacteria were counted on MRS (Oxoid, Basing- 25 stoke, UK) and plate count agar (Oxoid, Basingstoke, UK), respectively, both supplemented with 0.1% Delvocid ® (Gist-Brocades, Delft, The Netherlands). Delvocid is a broad-spectrum fungicide containing natamycin as active agent. Aerobic stability after long time airtight wheat storage After 10 and 12 months of storage 700 g grain from each silo were stored for 3 weeks at 20°C in Erlenmeyer-flasks sealed with cotton wool. Before sampling, the grains were thoroughly mixed. Microbial analysis was performed as described above. Growths of Penicillium roqueforti, Pichia anomala, aerobic bacteria and lactic acid bacteria were determined frequently during the 3 weeks as described above. RESULTS Environmental conditions The temperature of the sampled wheat closely followed the changes of the daily mean temperature of the outdoor air during the experiment. Differences in air pressure between the gas in the silos and external atmosphere were most of the time close to zero. The pressure inside the silos was low enough to cause a movement of air into the silos through the pressure relief valve on only seven occasions during the first two months and on one occasion during week 28, 29, 44 and 46, respectively. The water activity of the high-moisture wheat grain remained between 0.93 and 0.94 during the storage period (data not shown). Gas Analysis Already 5 hours after sealing no 0 2 was detected in the air inside the * silos and the concentration of C0 2 increased fast during the first week of the experi- ment. After two months the concentration of C0 2 had increased to its maximum, about 70%, in all silos. When the temperature decreased below zero the average concentration of C0 2 of the triplicates decreased and stabilized at between 50 and 60%. During the same time oxygen concentrations < 1% could be detected between samplings, remaining time only just after sampling. Microbial growth The inoculation with Pichia anomala intended to reach 10 and 10 CFU/g 26 resulted in 4 x 10 4 CFU/g and 5 x 10 5 CFU/g, respectively. Additionally, the grain had 1 x 10 CFU/g of Pichia anomala as a natural flora. After the first five weeks of storage, in closed silos, the yeast had grown to 6 x 10 CFU/g in the uninoculated grains, to 2 x 10 CFU/g in grains inoculated with Penicillium roqueforti, to 4 x 10 5 CFU/g when inoculated with 1 x 10 4 CFU of Pichia anomalalg, and to 9 x 10 CFU/g when inoculated with 5 x 10 CFU of Pichia anomala/ g. After eight weeks of storage, the amount of Pichia anomala in the two inoculated treatments was 4 x 10 CFU/g, while the treatments not inoculated with yeast contained 4 x 10 5 CFU/g grain. After 10 weeks of storage, the inocula- ted Pichia anomala in both treatments had grown to 7 x 10 CFU/g. Natural occurring Pichia anomala had grown from 1 x 10 4 CFU/g to about 6 x 10 5 CFU/g in the non-inoculated wheat. This difference remained during week 20 to 35, i.e. January to the end of April. During May the yeast in the non-inoculated grain n increased to the same level as in the inoculated grains, i.e. close to 10 CFU/g. q Penicillium roqueforti was inoculated to 3 x 10 CFU/g in order to represent an extremely high contamination level. Still, the inoculated level of Penicillium roqueforti was refound in the wheat only at one occasion during the year. The 9 inoculated amount was reduced to the detection limit, i.e. 10 CFU/g, during the 9 initial five weeks of closed silos. Also, at the end of the storage period about 10 CFU/g of Penicillium roqueforti was found. In grains not inoculated with either Pichia anomala or Penicillium roqueforti, endogenous Penicillium roqueforti was found at one sampling occasion, i.e. after 36 weeks of storage. At the start of the experiment about 10 CFU/g of lactic acid bacteria were found in all treatments except the non-inoculated one. Up to week 39 of storage, < 10 CFU/g were found in one of the silos of this treatment. On rare occasions, less than 10 CFU/g lactic acid bacteria were found in the other treatments in concentrations. Initially, the wheat contained 5 x 10 CFU/g of aerobic bacteria. During the five weeks of closed storage the amount decreased, but it recovered to 10 CFU/g in the non-inoculated treatment, to 10 CFU/g in the treatment with Penicillium roqueforti only, and to 10 CFU/g in both treatments with inoculated Pichia anomala. During weeks 20 to 35 (January to end of April) the amount of aerobic bacteria remained stable, followed by a decline in all treatments around week 40 (beginning of June). 27 Microbial stability after a simulated larger air leakage During the simulated air leakage after 54 weeks of storage, the oxygen concentration increased in all silos to ca. 18% in five days (data not shown). Once the silo with wheat inoculated with 10 CFU Pichia anomala per gram was closed, all oxygen was consumed within 20 h, whereas it took 46 h in the non- inoculated grain. One week after the planned air-leakage no increase in Penicillium roqueforti CFU was detected in any of the treatments. Aerobic stability after long-term airtight storage In wheat stored under airtight conditions for 10 months and then exposed to air, Penicillium roqueforti inoculated alone reached 10 CFU/g after 6 days, whereas it took 12 days to reach the same level in wheat inoculated with 10 CFU/g and 14 days in wheat inoculated with 10 6 CFU/g Pichia anomala (Fig. 5). Similar results were obtained with wheat after 12 months of airtight storage. Growth of Penicillium roqueforti, measured as colony forming units (CFU), in the high-moisture wheat was totally inhibited during the airtight storage in the silos. When the stored grains were taken out from the silos and exposed to air, growth of Penicillium roqueforti was delayed by inoculated Pichia anomala in a dose dependent manner. Since Penicillium roqueforti grew when grains were exposed to air it can be concluded that at least some of the inoculated fungal spores were viable. Thus, growth of Penicillium roqueforti in the silos was inhibited by unfavourable conditions. Probably both the atmosphere in the silos and the high levels of Pichia anomala in the grains contributed to the growth suppression of Penicillium roqueforti. Our earlier tests using Pichia anomala as a biocontrol agent in grains have been performed with 1-2 year old remoistened grain. Probably there are physiological differences between freshly harvested and remoistened grain. Still in this pilot scale tests the antagonistic effect of Pichia anomala was evident even when using freshly harvested grains. When exposed to air, grains stored airtight for 10-12 months were spoiled faster when not previously inoculated with Pichia anomala, despite the high level of endogenous Pichia anomala in the grains before inoculation. In grains taken from the silo after 10 months, inoculated Pichia anomala delayed growth of 28 inoculated Penicillium roqueforti to 5 x 10 CFU/gram with 6 days. During the same period Pichia anomala did not increase in number. Thus, the antagonistic activity of Pichia anomala is not strictly associated with detectable growth. Prevention of growth of Aspergillus flavus by Pichia guilliermondii in soya beans is dependent on active growth of the antagonistic yeast. Aspergillus flavus added when the extensive yeast growth during the first 2-3 days had decreased could compete with the yeast (Paster et al., 1993). Thus, in contrast with Pichia anomala, an active growth of Pichia guilliermondii affects the mold inhibition. In test tube experiments with 18 g high-moisture wheat, Penicillium q roqueforti growth is restricted by Pichia anomala inoculated to 10 CFU/gram and almost totally inhibited by 10 CFU/gram during 14 days of air leakage (Petersson and Schnύrer, 1995). In the present study with silos containing 160 kg wheat 10 4 CFU/gram and 10 5 CFU/gram were used to evaluate the amount of Pichia anomala needed to inhibit growth of Penicillium roqueforti during the 13 months of storage. In comparison, other antagonistic yeasts are often used at very high concentrations in experiments, e.g. 10 CFU/gram or even higher concentrations. The constructed silos were mimicing the function of full-scale airtight silos and were easy handled. However, the impenetrability for air might have been too high in the silos compared to a full-scale silo. When grain with a moisture content of 23% is stored in a completely sealed container, levels of carbon dioxide up to 95% have been measured. In practice, because of imperfect sealing etc, the concentration of carbon dioxide often increases only to 15-25% In this experiment the level of carbon dioxide in the silos stabilized at 50-60%. There were not as many air leakages through the air regulator as expected, probably due to protections from direct solar radiation at the silos and the capacity of the rubber lungs. Nevertheless, the silos were opened twice a week about 1 minute each" time. These openings did probably not cause considerable amounts of gas exchange, but equalised the pressure in the silos to the outside. However, not even the inlet oxygen at the end of the storage caused any growth of Penicillium roqueforti. The system seems to be robust in mold inhibiting ability, also at minor air leakages. 29 Table 1 Growth and sporulation measured as colony forming units (CFU) per gram of the Penicillium roqueforti- complex on different cultivars of barley (a w 0.96) incubated with restricted air supply in glass tubes at 25° C for 14 days. Values are given as logarithms and represent means of three tubes. Least significant difference (LSD) between day 0 and day 14 is 0.33 at P<0.05 (*), 0.43 at P<0.01(**), and 0.55 at P<0.001 (***). Barley P. ro queforή P. paneum P. car neum cultivar dav J5 J5A 18689 14088 13929 13321 14042 6889 Baronesse 0 4.6 4.3 4.4 4.2 4.3 4.2 4.5 4.6 14 5.3 5.3 5.5 5.7 5.5 5.5 5.8 5.9 sing. 1 *** *** *** *** *** *** *** Kinnan 0 4.5 4.4 4.2 4.4 4.4 4.2 4.5 4.3 14 5.8 5.8 6.3 6.1 5.5 5.9 6.1 6.1 sing. *** *** *** *** *** *** *** *** Maud 0 4.5 4.4 4.5 4.4 4.5 4.4 4.4 4.4 14 4.4 4.3 4.4 4.2 5.0 5.0 4.9 4.7 sing. r n,s-. 2 ns ns ns ** *** ** ns Mentor υ 4.3 4.3 4.2 4.3 4.2 4.5 4.6 4.4 14 5.0 4.2 4.4 4.5 5.1 4.8 4.9 4.9 sins. *** ns ns ns *** ns us ** Note: 1. "sing." indicates significance level of the increase in CFU between values on day 14 and day O 2. "ns" indicate nonsignificant 30 Table 2 Growth and sporulation measured as colony forming units (CFU) per gram of the Penicillium roqueforti- complex on different cultivars of spring wheat (aw 0.96) incubated with restricted air supply in glass tubes at 25° C for 14 days. Dragon A and Dragon B represent independent samples of the same variety. Values are given as logarithms and represent means of three tubes. Least significant difference (LSD) between day 0 and day 14 is 0.75 at P<0.05 (*), 0.99 at P<0.01 (**), and 1.27 at P<0.001(***). Spring P. roi queforti P. paneum P. carneum wheat cultivar day J5 J5A 18689 14088 13929 13321 14042 6889 Alve 0 3.9 4.4 4.6 4.4 3.5 4.2 4.4 4.3 14 6.7 6.7 6.9 6.9 • 6.2 6.3 6.6 6.3 sign. 1 *** *** *** **# *** *** Curry 0 4.3 4.0 3.9 4.5 4.0 4.3 4.0 4.3 14 5.9 5.3 6.5 6.5 6.1 6.4 6.0 6.2 sign. *** *** **# *** *** *** Dragon A 0 4.6 4.5 4.5 4.5 4.3 4.3 5.0 4.5 14 4.8 4.3 4.9 4.2 4.8 5.2 5.1 5.0 sign. ns 2 ns ns ns ns ns ns ns Dragon B 0 4.3 4.3 3.8 4.6 4.0 4.3 4.0 4.3 14 3.8 4.2 3.8 4 2 3.6 4.0 3.7 3.9 sign. ns ns ns ns ns ns ns ns Thasos 0 3.8 4.0 3.9 3.9 4.0 4.0 4.0 4.3 14 6.2 5:6 6.1 5.6 6.0 6.4 6.1 5.9- sign. *** *** **# *** *** *** Tjalve 0 4.3 4.7 4.0 4.0 4.3 4.3 4.2 4.0 14 6.0 6.5 6.5 6.8 5.7 6.3 6.4 6.0 " sign. *** *** *** *** *** *** *** *** Note: 1. "sing." indicates significance level of the increase in CFU between values on day 14 and day O 2. "ris" indicate nonsignificant. 31 Table 3 Growth, measured as colony foπiiing units (CFU) per gram, of Penicillium roqueforti J5 on different batches of spring wheat (a w 0.96) cv. Dragon, and winter wheat cv. Kosack as a control, in glass tubes at 25° C for 14 days. All lots represents independent samples of the same cultivar. Values are given as logarithms and represents means of three tubes. The statistical significance of differences in CFU values between day 0 and 14 is indicated (n=3). Dragon Detected P. roqueforti J5 lot (mean value log CFU/g) day O day 14 A 4.6 47 πsl B 4.6 5.2 * C 4.3 5.4" D 4.6 6.3" * E 4.6 5.8" * F 4.3 4.5 ns Control: Kosack 4.6 6.6"' Note: 1. ns indicate nonsignificant, * (P<0.05), **(P<0.01), and ***(P<0.001) 32 REFERENCES Barnett, J.A., Payne, R.W. and Yarrow, D., 1990. Yeasts: characteristics and identification. Cambridge University Press, Cambridge. Bjornberg, A. och Schnϋrer J., 1993. Inhibition of the growth of grain-storage molds in vitro by the yeast Pichia anomala (Hansen) Kurtzman. Can. J. Microbiol. 39: 623-628. Boysen, M., Skouboe, P., Frisvad, J., and Rossen, L., 1996. Reclassification of the Penicillium roqueforti group into three species on the basis of molecular genetic and biochemical profiles. Microbiology, 142: 541-549. Chalutz, E. 1990. Postharvest biocontrol of green and blue mould and sour rot of citrus fruit by Debaryomyces hansenii. Plant. Dis. 74: 134-137. Droby, S., Chalutz, E., Wilson, C.L. and Wisniewski, M. 1989. Characterization of the biocontrol activity of Debaromyces hansenii in the control of Penicillium digitatum on grapefruit. Can. J, Microbiol. 35: 794-800. Droby, S., Chalutz, E. and Wilson, C.L. 1991. Antagonistic microorganisms as biological control agents of postharvest diseases of fruits and vegetables. Postharvest News and Information. Vol. 2, pp. 169-173. Haggblom, P. 1990. Isolation of roquefortin C from feed grain. Appl. Environ. Microbiol. 56: 2924-2926. Kreger-van Rij, N.J.W. (Ed.), 1984. The yeasts. A Taxonomic study. North- Holland Publishing Co., Amsterdam. Kurtzman, C.P., 1984. Synonomy of the yeast genera Hansenula and Pichia demonstrated through comparison of deoxyribonucleic acid relatedness. Antonie van Leeuwenhoek, 50: 209-217. Lacey, J., and Magan, N. 1991. Fungi in cereal grains; their occurrence and water and temperature relationships. In Cereal grain. Mycotoxins, fungi and 33 quality in drying and storage. Edited by J. Chelkowski, Elsevier, Amsterdam, pp. 77-118. Magan, N., and Lacey, J. 1984. Effects of gas composition and water activity on growth of field and storage fungi and their interactions. Trans. Br. Mycol. Soc. 82: 305-314. McLauglin, R.J., Wisniewski, M.E., Wilson, C.L., and Chalutz, E. 1990. Effect of inoculum concentration and salt solutions on biological control of postharvest diseases of apple with Candida sp. Phytopathology, 80: 456-461. Petersson, S. and Schnύrer, J. 1995. Biocontrol of mold in high-moisture wheat stored under airtight conditions by Pichia anomala, Pichia guillerimondii and Saccharomyces cerevisiae. Applied and Environmental Microbiology, 61: 1027- 1032. Pitt, J.I., 1991. A laboratory guide to common Penicillium species. CSIRO Food Research Laboratory, North Ryde, Australia. Pitt, J.I. and Hocking, A.D., 1997. Fungi and Food Spoilage, second Edition. Blackie Academic & Professional, London. Samson, R.A., van Reenen-Hoekstra, E.S., Frisvad, J.C., and Filtenborg, O., 1995. Introduction to food-borne fungi. Centraalbureau voor Schimmelculture, Institute of the Royal Netherlands Academy of Arts and Sciences, Baarn, The Netherlands. Wilson, C.L. and Chalutz, E. 1989. Postharvest biological control of Penicillium rots of citrus with antagonistic yeasts and bacteria. Sci. Hortic. (Amsterdam), 40: 105-112. Wilson, C.L., and Wisniewski, M.E. 1989. Biological control of postharvest diseases of fruits and vegetables: an emerging technology. Annu. Rev. Phyto- pathol. 27: 425-441. 33a Applicant s or agent's file International apphcatic o. reference number 990055-PC INDICATIONS RELATING TO A DEPOSITED MICROORGAtaSlVI (PCT Rule 13../.- ) A. The indications made below relate to the miciooigamsm referred to in the descπptio on page Z . line • § B. IDENTIFICATION OFDEPOSIT Furtherdeposits are identified on an additional sheet | | Name of depositary institution Ceπtralbureau voor Schimmelcultures Addiess of depositaiy institution (unhiding poital code and toitntn) Oosterstraat 1 P.O. box 273 NL-3740 AG Baarn The Netherlands Datcof deposit Accession Number 27 February 1998 CBS 100487 C. ADDITIONAL INDICATIONS (leaie blank if not applicable) Thisinformationiscontinuedonanadditionalsheet |_J D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MAOE(ιf the indication, aie nolfoi all deviated State.) E. SEPARATE FURNISHING OFINDICATIONSr/eαi'eΛ/α/ii-i πn/αyψ/itaft/e) The indications listed below will be submitted to the International Bureau later (ψecify the general naliiieo) the indication, eg., 'A ewon Nitmber of Depovt ') Forreceiving Office useonly For International Bureau use only [ | This sheet was received with the international application I I This sheet was received by the International Bureau on: Authorized officer Authorized officer Form PCT/RO/134 (July 1992)

Claims: 34 PATENT CLAIMS 1. A process for reliable airtight storage and preservation of moist grain, characterized by the combination of using, as grain, a grain cultivar resisting infection by storage spoilage fungi and effecting the storage in the presence of an antifungally effective amount of a biocontrol agent having the ability to suppress or inhibit storage spoilage fungi while reinforcing the effect of using a grain cultivar resisting infection by such fungi. 2. The process of claim 1, characterized in that the grain cultivar used is intended for use as animal feed. 3. The process of claim 2, characterized in that the grain cultivar used is a cereal grain cultivar. 4. The process of claim 3, characterized in that the cereal grain cultivar is selected from wheat, rye, barley, oats, rye wheat, corn, sorghum, millet and rice. 5. The process of claim 4, characterized in that the cereal grain cultivar used is rye cv. Motto, spring wheat cv. Dragon or barley cv. Maud or cv. Mentor. 6. The process of any of claims 1-5, characterized in that the biocontrol agent is a Pichia anomala isolate having the ability to suppress or inhibit storage spoilage fungi while reinforcing the effect of using a grain cultivar resisting infection by such fungi. 7. The process of claim 6, characterized in that the Pichia anomala isolate is Pichia anomala J121, deposited with the Centraalbureau voor Schimmelcultures (CBS) under accession number CBS 100487, Pichia anomala J281 or Pichia anomala CBS 5759. 8. The process of any of claims 1-7, characterized in that the storage spoilage fungi belong to the Penicillium species group. 9. The process of claim 8, characterized in that the Penicillium species are Penicillium roqueforti, Penicillium carneum, Penicillium panum and Penicillium 35 verrucosum. 10. The process of any of claims 1-9, characterized in that the presence of the biocontrol agent is due to its natural occurence in the grain and/or due to application to the grain of a biocontrol composition containing the biocontrol agent. 11. The process of claim 10, characterized in that the biocontrol composition is in liquid, semiliquid or solid form. 12. The process of claim 10 or claim 11, characterized in that the biocontrol composition comprises, in addition to the biocontrol agent, one or more additives selected from liquid or solid carriers and diluents, surfactants, dispersing agents, nutrients, growth factors and antioxidants and mixtures thereof. 13. The process of any of claims 10-12, characterized in that the application of the biocontrol composition is effected by mixing, dusting, injecting, rubbing, spraying or brushing, dripping or rolling. 14. A biocontrol composition for use in the process of any of claims 1-13, characterized by comprising at least one biocontrol agent suppressing or inhibiting storage spoilage fungi. 15. The composition of claim 14, characterized by being in liquid, semiliquid or solid form. 16. The composition according to claim 14 or 15, characterized by additionally comprising an additive selected from liquid or solid carriers and diluents, surfactants, dispersing agents, nutrients, growth factors and antioxidants and mixtures thereof. 17. The composition according to any of claims 14-16, characterized by con- taining at least one yeast strain as biocontrol agent being antagonistic to storage spoilage fungi. 18. The composition according to claim 17, characterized in that the yeast 36 strain is a Pichia anomala isolate having the ability to suppress or inhibit storage spoilage fungi while reinforcing the effect of using a grain cultivar resisting infection by such fungi. 19. The composition according to claim 18, characterized in that the Pichia anomala isolate is selected from the group consisting of Pichia anomala strain J121 deposited with Centraalbureau voor Schimmelcultures (CBS), Baarn, The Netherlands under accession number CBS 100487, Pichia anomala strain J281 and Pichia anomala strain CBS5759. 37 AMENDED CLAIMS [received by the International Bureau on 17August 1999 (17.08.99); original claims 1-19 replaced by amended claims 1-19 ( 3 pages)] 1. A process for reliable airtight storage and preservation of moist grain, characterized by the combination of using, as grain, a grain cultivar resisting infection by storage spoilage fungi but not resisting infection by a biocontrol agent, and effecting the storage in the presence of an antifungally effective amount of a biocontrol agent having the ability to suppress or inhibit storage spoilage fungi while reinforcing the effect of using a grain cultivar resisting infection by such fungi. 2. The process of claim 1, characterized in that the grain cultivar used is intended for use as animal feed. 3. The process of claim 2, characterized in that the grain cultivar used is a cereal grain cultivar. 4. The process of claim 3, characterized in that the cereal grain cultivar is selected from wheat, rye, barley, oats, rye wheat, corn, sorghum, millet and rice. 5. The process of claim 4, characterized in that the cereal grain cultivar used is rye cv. Motto, spring wheat cv. Dragon or barley cv. Maud or cv. Mentor. 6. The process of any of claims 1-5, characterized in that the biocontrol agent is a Pichia anomala isolate having the ability to suppress or inhibit storage spoilage fungi while reinforcing the effect of using a grain cultivar resisting infection by such fungi. 7. The process of claim 6, characterized in that the Pichia anomala isolate is Pichia anomala J121, deposited with the Centraalbureau voor Schimmelcultures (CBS) under accession number CBS 100487, Pichia anomala J281 or Pichia anomala CBS 5759. 38 8. The process of any of claims 1-7, characterized in that the storage spoilage fungi belong to the Penicillium species group. 9. The process of claim 8, characterized in that the Penicillium species are Penicillium roqueforti, Penicillium carneum, Penicillium panum and Penicillium verrucosum. 10. The process of any of claims 1-9, characterized in that the presence of the biocontrol agent is due to its natural occurence in the grain and/or due to application to the grain of a biocontrol composition containing the biocontrol agent. 11. The process of claim 10, characterized in that the biocontrol composition is in liquid, semiliquid or solid form. 12. The process of claim 10 or claim 11, characterized in that the biocontrol composition comprises, in addition to the biocontrol agent, one or more additives selected from liquid or solid carriers and diluents, surfactants, dispersing agents, nutrients, growth factors and antioxidants and mixtures thereof. 13. The process of any of claims 10-12, characterized in that the application of the biocontrol composition is effected by mixing, dusting, injecting, rubbing, spraying or brushing, dripping or rolling. 14. A biocontrol composition for use in the process of any of claims 1-13, characterized by comprising at least one biocontrol agent suppressing or inhibiting storage spoilage fungi. 15. The composition of claim 14, characterized by being in liquid, semiliquid or solid form. 16. The composition according to claim 14 or 15, characterized by additionally comprising an additive selected from liquid or solid carriers and diluents, surfac- tants, dispersing agents, nutrients, growth factors and antioxidants and mixtures thereof. 39 17. The composition according to any of claims 14-16, characterized by containing at least one yeast strain as biocontrol agent being antagonistic to storage spoilage fungi. 18. The composition according to claim 17, characterized in that the yeast strain is a Pichia anomala isolate having the ability to suppress or inhibit storage spoilage fungi while reinforcing the effect of using a grain cultivar resisting infection by such fungi. 19. The composition according to claim 18, characterized in that the Pichia anomala isolate is selected from the group consisting of Pichia anomala strain J121 deposited with Centraalbureau voor Schimmelcultures (CBS), Baarn, The Netherlands under accession number CBS 100487, Pichia anomala strain J281 and Pichia anomala strain CBS5759. 40 STATEMENT UNDER ARTICLE 19 In the enclosed "Amended claims under Article 19 PCT", claim 1 has been amended by the addition of "but not resisting infection by a biocontrol agent" after "resisting infection by storage spoilage fungi". No other amendments to the claims have been effected. An implicit counterpart to the added statement is to be found in Fig. lc compared to Fig. lb, as will be further explained below. The basic problem underlying the present invention is the well-known fact that moist cereal grain is subjected to mold growth when stored. One solution of the problem is to dry the grains. However, the drying of grains in temperate climates is expensive. For moist grains used as animal feed, airtight storage provides a less costly alternative by maintaining an atmosphere low in oxygen and high in carbon dioxide. In reality, however, air leakage cannot be prevented and considerable mold infestations can develop. Thus, additional measures to control mold growth in grain storage systems are necessary. The growth of spoilage molds can be further suppressed in airtight storage by using competetive or antagonistic biocontrol agents such as the fungus Pichia anomala. However, not even this mode of action provides a 100% reliable effect. The present invention is based upon the novel concept of combining, in airtight storage systems, the use of a grain cultivar resisting infection by storage spoilage fungi and the use of a biocontrol agent having the ability to suppress or inhibit storage spoilage fungi while reinforcing the effect of using such a resistant grain cultivar. 41 The inventive feature of the invention is based on the following two surprising and non-obvious facts: the finding that there are grain cultivars with in-born resistance to spoilage fungi when stored with restricted air access; the finding that such a resistant grain cultivar, in spite of the fact that it inhibits the growth of spoilage fungi, does not inhibit the growth of the antagonistic biocontrol agent, such as Pichia anomala. The last-mentioned finding is quite evident when comparing Fig. la (non-resistant wheat cultivar Kosack) with Fig. lc (resistant rye cultivar Motto) which, in the Applicant's opinion, justifies the addition of the statement "but not resisting infection by a biocontrol agent" to claim 1. In view of the above, it is the Applicant's firm belief that the present invention meets the patentability criteria over the relevant prior art.

Inventors: JONSSON ANDERS, PETERSSON STINA, SCHNUERER JOHAN, JONSSON, ANDERS, PETERSSON, STINA, SCHNUERER, JOHAN
Applicants: BIOAGRI AB, JONSSON ANDERS, PETERSSON STINA, SCHNUERER JOHAN, JONSSON, ANDERS, PETERSSON, STINA, SCHNUERER, JOHAN
Application year: 1999
Main classification: A23B9
IPC codes: A01N63, A23B9, C12N1