SE513894C2 - Procedure for airtight storage of moist grain in the presence of a biocontrol agent - Google Patents

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

N) Oi 30 513 894 2 can be greatly reduced in airtight silos in which the yeast Pichia anomala (Hansen) Kurtzman was a dominant species (S. Lindgren, personal communication). This finding indicates that this yeast interacts competitively or antagonistically with molds. Pichia anomala (syn. Hansenula anomala) has also been frequently isolated in other studies regarding airtight stored cereals and from berries, gum, brewery and bakery yeast, finger millet, beer, soft drinks, fruit juices, maple syrup, condensed milk, sauerkraut, salty beans, molasses, miso, human and animal (Barnett mil. 1990; Lacey and Magan 1991).

Björnberg and Schnürer (1993) carried out a study in order to investigate the possibilities of using a yeast to suppress the growth of common grain pest molds. They evaluated the antagonistic effects of Pichía cmomala against the molds Penicillium roqueforti and Aspergillus candidus on agar plates. The organisms were co-cultured under controlled conditions in uitro, and the authors found that Pichia anomalous suppressed the plant in uitro of the two important harmful fungi, even during periods of unlimited air supply. Furthermore, the degree of inhibition of pests was related to the initial concentration of Pichia anomaZa cells. The inhibition was most pronounced at temperatures that were suboptimal for plant and spore formation of Penicillium roqueforti and Aspergillus candídus, i.e. below 15 ° C and above 30 ° C.

In a further study, Petersson and Schnürer (1995) determined the antagonistic activity of Pichia anomala, Pichia guilliermondii and Saccharomyces cereuisiae on agar against 17 mold species. It was found that Pichia anomala can be used in particular for biocontrol of moldy growth in moist wheat, which is stored under airtight conditions. This publication reveals that Pichia anomala reduces growth on agar plates of all tested fungal species in a dose-dependent manner, however, the different mold fungi show different sensitivity to the yeast species examined. Pichia anomala had the strongest antagonistic activity in wheat, with 105 and 106 CFU / g completely inhibiting the growth of Penicillium roqueforti. Also in this study, it was found that the best effect was obtained at temperatures that were suboptimal for the growth of the mold parts.

The use of antagonistic microorganisms in the biological control of fungal diseases on, for example, cereals, fruit and vegetables is also apparent from the patent literature, of which examples are given below. U.S. 5,670,368 and its division U.S. 5,413,783 (McLaughlin et al.) Relate to biological control of plant pathogens on agricultural products such as nuts, vegetables, cereals, etc. using an antagonistic microorganism selected from Pichia. guilliermondii and Hanseniasporum uuarum.

US 4,820,531 (Tomes) discloses a method for preserving the quality of stored hay with a high moisture content by treating fresh hay with the microorganism Bacillus pumílus or mutants thereof.

US 4,981,705 (Tomes) relates to a process for preserving silage by treating the silage with the microorganism Propionibacterium jensenii or genetic equivalents thereof.

A process for the preparation or preservation of an animal feed composition is disclosed in US 5,371,011, which comprises applying to a feed the antifungal bacterial strain Serratia rubidaea.

In US 4,842,871, Hill describes a process for preserving an agricultural product by treating it with the microorganism Lactobacillus plantarum. The agricultural product can be silage, baled hay or grain with a high moisture content.

The use of Pichia guilliermondii for biological control of rot in fruit after harvest is revealed by Wilson et al. iUS 5 041 384. Wilson m. fl. also discloses a method of using isolates of Candida oleophila to control diseases of fruit after harvest (US 5,425,941). A method of treating or prophylaxis of microorganism disease in fruits is also disclosed in US 5,525 132 (Shanmuganathan), the method comprising the use of one or more yeast strains.

However, none of the prior art references disclose or suggest the combination of features which is characteristic of the present invention.

The problem to be solved by the invention - In temperate climates, such as Sweden, cere alie grains are usually harvested at a water content of 20%, which corresponds to a water activity of 0.9 (N. Jonsson, Institute of Agricultural Engineering, personal communication). Mold growth in cereal cereals is often prevented by drying, but in temperate climates, drying procedures are expensive and energy-intensive. Feed grains can be stored at high moisture levels in airtight silos, where the growth of microorganisms is prevented by low O! 10 30 513 894 4 oxygen and high carbon dioxide levels, which are provided by respiration of the epiphytic micro och oran and the grain itself (Lacey and Magan 199 1). Difficulties in maintaining a low partial pressure of oxygen and a high carbon dioxide level make the storage system sensitive to air exchange between the silo and the external atmosphere. Incomplete sealing, daily temperature fluctuations and frequent removal of grain all contribute to air leakage factors into the silo atmosphere. The addition of a biocontroller, which is effective in preventing mold growth, such as the antagonistic yeast Pichia anomala, would improve the reliability of airtight grain storage systems with high moisture content.

The object of the invention is thus to improve the reliability of airtight systems for storing grain with a high moisture content and to counteract the effects of the above-mentioned air leakage problems. This object is achieved with the present invention.

Summary of the Invention The present invention is based on the surprising discovery that the growth of mold species belonging to the Penicillium roqueforti group, when storing high moisture grains and with limited air access, is dependent on the type of grain used, i.e. there are varieties that have an inherent resistance to pests under these conditions. Thus, the present invention provides a method for reliable airtight storage and preservation of moist cereals, the method being characterized by the combination of using as cereal a cereal variety which resists infection by storage fungi and carrying out the storage in the presence of an antifungal effective amount. of a biocontroller, which suppresses or inhibits storage damage fungi and enhances the effect of using said resistant cereal variety. The invention also provides a biocontrol composition for use in the method of the invention, the composition being characterized in that it comprises at least one biocontrol agent which suppresses or inhibits storage damage fungi, preferably a Pichia anomalous isolate.

Description of the drawings Fig. 1 The effect of increasing initial levels of Pichía anomala J 121 (Û) on the plant and the sporulation of Penicillium roqueforti Jö (Å) after 14 days of co-cultivation in 10 15 20 25 30 513 894 5 autumn wheat (Cossack variety) ( a), spring wheat (variety Dragon) (b), rye (variety Motto) (c), barley (variety Golf) (d) and oats (variety Svea) (e). Vaccination level of Penicillium roqueforti J 5 is indicated by (A). Grain grains (aw 0.95) were incubated with limited air supply at 25 ° C for 14 days. Data are given as log CFU / gram of grain (in SA, n = 3).

Fig. 2 The effect of increasing initial levels of Pichia anomala J 121 on the plant and the sporulation of the three Penicillium roqueforti isolates J 5 (Û), J 5A (I) and J 282 (Å), during co-cultivation on winter wheat (variety Cossack , aW 0.95) at 25 ° C after 14 days. The dashed line represents the mean of the grafted levels of Penicillium 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 The effect of increasing initial levels of the three Pichia anomala isolates J 121 (Û), J 281 (Å) and CBS5759 (I) on the plant of Penicillium roqueforti J 5, during co-cultivation on winter wheat (variety Cossack, aw 0 , 95) at 25 ° C after 14 days. The dashed line represents the mean of the inoculated levels of Penicillium roqueforti J 5.

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. 4 Plant and sporulation of the Penicillium roqueforti group in glass tubes with limited air supply, containing a) barley variety Golf, b) rye variety Motto, c) spring grazing variety Dragon (lot A), d) and autumn wheat variety Cossack (aw 0, 96), during incubation at 2500 for 14 days. Data are given as log mold CFU / gram cereal cereal on day 0 (n = 1, unfilled bars) and day 14 (filled bars).

Specified standard deviations were calculated on log-transformed values (n = 3).

Fig. 5 Growth of Penicillium roqueforti in wheat, which had been removed from the silo and exposed to air after 10 months of storage. Wheat was stored with full air supply at 2000. Non-inoculated (Û), inoculated with 103 CFU / g Penicillium roqueforti (I), 103 CFU / g Penicillium roqueforti and 104 CFU / g Pichia anomala (Å) or 10 20 30 513 894 6 103 CFU / g Penicillium roqueforti and 106 CFU / g Pichia cmomala (V).

Detailed Description of the Invention The present invention relates to a process for reliable airtight storage and preservation of moist cereals, the process being characterized by the combination of using as cereal a cereal variety which resists infection by storage damage fungi, and carrying out the storage in the presence of a antifungal effective amount of a biocontroller, which suppresses or inhibits storage damage fungi and enhances the effect of using said resistant cereal variety.

The invention is based on the surprising discovery that the growth of mold parts, when storing cereals with a high moisture content under conditions involving low oxygen and high carbon dioxide concentrations, is dependent on the type of grain used, ie. there are varieties with an inherent resistance to pests under these conditions.

The cereal used is preferably a feed cereal which includes cereal cereals. Any variety of grain that resists pest infestation can be used. In order to determine whether a cereal variety is resistant to pest infestation, in particular Penicillium roqueforti, those skilled in the art can readily determine this using the procedures proposed in this specification under the heading "IL Cereal Cereals with Intrinsic Resistance to Pest Infection", "Materials and Methods ".

By "cereal cereals" is meant, throughout this specification and claims, wheat, rye, barley, oats, triticale, maize, sorghum, millet, rice, etc.

The most preferred cereal cereal varieties are rye variety Motto, spring wheat variety Dragon and the barley varieties Maud and Mentor.

At present, yeast species appear to be the most promising biocontrol agents as an alternative to chemical fungicides for post-harvest storage. This is due to the fact that yeast species do not produce mycotoxins or allergenic spores, that the majority are not pathogenic to humans or animals, that many yeast strains can grow at low water availability and on very different substrates at low oxygen arterial pressures.

As a biocontrol agent, any yeast strain capable of suppressing the growth of cereal pest fungi and enhancing the effect of using a resistant cereal variety can be used in the process of the invention, provided that it meets the following three selection criteria: (1) it should be naturally present in cereals; (2) it should show good growth at low water activity; and (3) it should grow well at low temperature. All these criteria are met by Pichia anomala, the suitability of which is further substantiated by observations indicating that it is predominant in mold-free, airtight stored cereals. The ability of a yeast strain to suppress the growth of common cereal pests can be determined by the inhibition experiments described by Björnberg and Schnürer (1993), p. 624, or by Petersson and Schnürer (1995), p. 1028

The use of a Pichia anomala strain as a biocontrol agent is a preferred embodiment of the invention. The use of Pichia anomala as a biocontroller in cereals can be considered as an additional addition by a regular member of the micro-organ in cereals. This is an advantage in gaining a general recognition of this biotechnological approach to feed storage. In general, Pichia has anomalous variable growth at both 350 ° C and body temperature (Barnett et al. 1990).

Human infections, caused by Pichia anomala, are extremely rare.

Particularly useful strains are Pichia anomala J 121, Pichia anomala J 281 and Pichia anomala CBS5759.

The strain Pichia anomala J121 is described by Björnberg and Schnürer (1993) and Petersson and Schnürer (199 5). The said strain was deposited with the Centraalbureau voor Schimmelcultures (CBS), Baarn, the Netherlands on 27 February 1998 under deposit number CBS 100487.

A characterization of Pichia anomala J 121 is given below.

Ch 10 30 513 894 Plant temperature 25OC.

Morphology: Lemon-shaped cells - + Pink colonies ~ Budding cells Dividing cells - Filament + Pseudohyphae + Arthroconidia - Ballistoconidia - Ascospores: +, hat-shaped Buds on stems Septathyroid - Symmetrical ballistoconidia - Fermentation: D-glucose Saltose - Lactose-Galactose + Lactose + Plant: D-glucose + Maltose + Glycerol + D-galactose OAOL-trehalose + Erythritol L-sorbos - Methyl-oc-D-glucoside + D-glucitol D-glucosamine - Cellobiosis - D-mannitol + Dmibos - Melibios - Myo- inositol - D-xylose + Lactose - Z-keto-D-gluconate - L-arabinose - Raffinose + D-gluconate - L-ramnose - Melezitose + D-glucuronate - Sucrose + DL-lactate Nitrate + Ethylamine + L-lysine Cadaverine + 0.01% cycloheximide - Acetic acid production - at; ss ° o s7 ° c + 4o ° c Pichía anomala Jl2l has the ability to grow at pH values from about 2 to about l 1 and has the ability to grow at temperatures from about 6OC to about 37OC. A special feature of Pichia anomala J 121 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 according to the invention, the presence of the biocontroller may be a consequence of its natural presence in the grain or a consequence of the biocontroller being applied to the cereal, for example in the form of a biocontroller composition.

The antifungal yeast strains of the present invention can be prepared in the required quantity by fermenting a sample of said strain under suitable conditions and in a suitable medium. Such conditions and media are well known in the art. An example is given below under the heading "I. Pichia anomala as a biocontroller of Penicillium roqueforti in cereals with high moisture content under airtight conditions", "Materials and methods", "Inoculum".

The yeast strain can be obtained from the fermentation broth by concentration or ultra-filtration, optionally followed by the addition of a preparation agent, such as surfactants, solid or surface carriers or diluents, dispersants, etc.

Thus, according to a further aspect of the present invention, there is provided a biocontrol composition comprising at least one biocontroller which suppresses or inhibits storage fungi and enhances the effect of using a cereal variety which resists infestation of pests, and which preferably comprises a yeast strain and in particular any of the above preferred Pichia anomala isolates.

When performing the procedure according to the invention, the biocontrol composition can be applied as a liquid or a particulate solid. When applied as a liquid, the carrier may be water while, when applied as a solid, the carrier is a solid. Examples of solid carriers include cereals such as ground barley and ground wheat, ground corn cobs and other materials such as clay, chalk, magnesite, limestone and talc.

Any additional materials added to the biocontrol composition of the invention include nutrients, growth factors, antioxidants and materials intended to reduce the dusting propensity of the additives, improve adhesion to the grain for storage or selectively enhance the growth of the yeast strain used as a biocontroller.

The relative proportions of carrier and yeast strain incorporated into the biocontrol composition for use in the storage of cereals depend on the particular yeast strain in question and its activity and viability. The concentration of living cells, as defined by the number of colony forming units (CFU), can be used as a guide for determining appropriate dosages. As a guide, the yeast cells can be applied as a surface suspension or solid additive in concentrations ranging from about 103 CFU to about 106 CFU per gram of stored grain, depending on the nature and condition of the grain. Preferably, the concentration is in the range of about 104 to about 105 CFU per gram of grain.

In preparing the biocontrol composition of the invention, the yeast strain is usually dried by suitable methods, for example freeze-dried or spray-dried, or a live culture is resuspended in water and then mixed with other components by conventional mixing methods.

According to a further aspect of the invention there is provided a method of reliable airtight storage and preservation of moist cereals, the method being characterized in that a biocontrol composition is applied to the cereal, which comprises a biocontrol agent which suppresses or inhibits the growth of storage fungi, the cereal used being a cereal variety that resists infection by storage fungi. The biocontroller is preferably a Pichia anomala isolate as defined above. The biocontrol composition may consist of the yeast cells as such or be a preparation which contains, in addition to the yeast cells, preparation agents as defined above.

The application of the biocontrol composition according to the invention can be effected by mixing, powdering, injection, rubbing, spraying, brushing, dipping or rolling.

The airtight storage can be achieved in a silo with limited air access or in airtight bags of plastic, rubber or similar materials. In the pilot scale experiments described below, Pichia anomala cells were applied by spraying on the grain in a feed mixer to obtain an even distribution. In full scale, the yeast can be added by arranging a spray nozzle in the transport screw used for feeding into the silo. The subsequent mixing distributes the yeast cells evenly over the span. The inoculum was added to the growth medium, comprising a small amount of the nutrients. It is also possible to add the cells of Píchia cmomala washed in tap water.

Commercial use and benefits The results presented in the experimental section that follows indicate the suitability of large-scale application of Pichia anomala as a biocontroller against damage to airtight stored feed grain of high moisture content after harvest. The yeast inhibits the growth of the Penicillium roqueforti group, the most significant harmful fungi in airtight storage, in provocation inoculation to a very high level during silo storage and also delays mold growth when cereals, which are removed from the silo, are exposed to air.

Advantages obtained with the various aspects of the present invention are: the invention improves the preservation of the grain; - the invention improves the biological value of the stored grain; - the invention makes it possible to store grain at a higher moisture content, which means energy savings; - the invention provides grain with better durability properties even after the end of the airtight storage; - the invention exercises biocontrol mainly through competition for nutrients and oxygen; - Pichia anomala grows strongly on cheap growth media using simple technology, which are important factors for commercial use of this yeast on a large scale.

The invention is further illustrated by the experimental section which follows.

Experimental sections To substantiate the recovery experimentally, we evaluated the use of Pichia anomala as a biocontroller during storage of wheat (autumn and spring forms), rye, barley and oats, which are the most commonly grown cereals in Sweden.

These cereals can all be used as feed grain and can be stored under airtight conditions without prior drying. To imitate a malfunctioning airtight system for storing grain of high moisture content, glass tubes were filled with grafted grain (water activity 0.95) and fitted 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 wheat, which were subjected to limited air supply. Furthermore, the sensitivity of three Penicillium roqueforti isolates against Pichia anomala in autumn wheat was evaluated.

I. Pichia anomala as a biocontrol agent of Penicillium roqueforti in cereals with high moisture content under airtight conditions Materials and methods Sponge isolate Pichia anomala (Hansen) Kurtzman J 121 with previously shown antagonistic effect (Petersson and Schnürer 1995) was originally isolated from airtight stored 10 N) O ! 30,515,894 12 wheat, as well as our latest isolate Pichia anomala J281. For comparison purposes, we used the Pichia anomaZa strain CBS57ö9 from the "Centraalbureau voor Schimmelkultures" (CBS), Baarn, the Netherlands. Penicillium roqueforti Thom J5 and Penicillium roqueforti J 5A, originally isolated from airtight stored grain, and Penicillium roqueforti J282, recently isolated from silage, were gifts from Dr. P. Häggblom, Statens Veterinärmedícinska Anstalt, Uppsala.

All used fungi were available from the fungus collection at the Department of Microbiology, Swedish University of Agricultural Sciences, SLU, Uppsala. The yeast strains were identified by the ID 32 C test (Biomerieux, Marcy l'Etoile, France) and by morphological studies (Kreger-van Rij 1984; Kurtzman 1984). The mold isolates were identified as Penicillium roqueforti through morphological studies (Pitt 1991; Samson mil. 1995). Svarnp cultures were held on malt extract days (MEA, Oxoid, Basingstoke, England) at ZZOC.

Cereal cereals Autumn wheat (variety Cossack), spring wheat (variety Dragon), rye (variety Motto), barley (variety Golf) and oats (variety Svea), all of seed quality, were purchased from the Swedish Farmers' Association, Uppsala.

Inoculum 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, England), 1.0 g sucrose). (BDH, Poole, England) in 1.0 liter of distilled water) with a loop of cells from a culture stored at MEA at ÉZOC. After incubation in a rotary shaker (100 rpm) at 25 ° C for 48 hours, the number of yeast cells was determined using a hemocytometer. The spore suspension of mold was prepared by harvesting spores from 7 day old colonies (grown on MEA at 25 ° C) in peptone water (2 g "PhytoneTM" peptone (BBL, Becton Dickson and Co., Cockeysville, USA) per liter of distilled water. ) with 0, 0 15% "TWeen 80" added to facilitate the dispersion of conidia. The trace concentration was determined with a hemocytometer.

UI 10 15 20 25 30 35 513 894 13 Inhibition of Penicillium roqueforti J5, grafted on wheat, rye, barley and oats, by the yeast Pichia anomala J 121 Non-sterile cereal cereals, which had been stored at a water content of 10% by 2000, were moistened with tap water to obtain a water activity of 0.95 (about 22% water content). In order for the moisture content to reach equilibrium, the grain was stored for 3-5 days at 200 with frequent mixing. The water activity (aw) was measured at 25 ° C with a "Novasina TH-2 / RTD-33 / BS", equipped with a temperature constant holder (Defensor AG, formerly Novasína AG, Pfäf fi kon, Switzerland).

Grain with aw 0.95 was inoculated with the mold spore suspension to about 3 x 103 mold spores per gram. The spores were applied by adding the suspension dripping onto the grain and mixing to obtain an even spore distribution. The yeast Pichia anomala was similarly inoculated to obtain 0, 101, 102, 103, 104, 105 and 106 cells per gram of grain in separate experiments.

The inoculum, <1 ml of fungal suspension per 100 grams of cereal cereals, did not measurably affect the water activity of the cereals. Thick-walled glass tubes (27 ml) were filled with approximately 17 g portions of grafted grain and sealed with a butyl rubber stopper.

To simulate air leakage, a needle (diameter 0.6 mm) was inserted through each butyl rubber stopper. The tubes were incubated at 25 ° C. According to previous observations, these conditions (aW 0.95, 2500, 3 x 103 mold spores per gram) represent an environment conducive to the growth of Penicillium roqueforti and were chosen in order to subject the antagonistic activity of Pichia anomala to a very rigorous test (Petersson and Schnürer 1995).

On day 14, three tubes from each treatment were opened, and the contents were diluted ten-fold with peptone water, soaked for 0.5 hours and homogenized for 2 minutes at normal speed in a "Stomacher 400" (Colworth, England).

Yeast plant was quantified on MEA, supplemented with 100 ppm chloramphenicol (C-0378 Sigma Chemical Co., St. Louis ,; Mo. USA) (MEAC plates). To avoid plant inhibition of antagonistic yeast on agar plates, intended for counting fungal colony forming units (CFU), MEACC (MEAC supplemented with 10 ppm cycloheximide C-7698 Sigma Chemical Co., St. Louis; Mo. USA) was used for surface smear of 0.1 ml aliquots. Chloramphenicol is a bacteriostatic broad-spectrum antibiotic and cycloheximide inhibits the growth of Pichia anomala (Björnberg and Schnürer 1993). Lactic acid bacteria and the total number of aerobic bacteria were counted on "LBS" (BBI, Meyland, France) and plate counting agar (Oxoid, Basingstoke, England), respectively, both supplemented with 0.1% "Delvocid®" 10 15 20 30 513 894 14 ( Gist-Brocades, Delft, The Netherlands). "Delvocide" is a broad-spectrum fungicide with natamycin as the active ingredient. Only the control tubes during the experiments, ie. tubes that had not been inoculated with yeast were analyzed for bacteria.

Inhibition of three isolates of Penicillium roqueforti using Píchia anomala J12l To evaluate isolate-related variation in susceptibility of the antagonistic yeast Pichia anomala J 121 in airtight stored high wheat wheat with high moisture content, three isolates of Penicillium Jquef, roqueforti were used. J 5A and J 282) in three separate experiments, which were performed as described above.

Inhibition of Penicillium roqueforti Jö using three isolates of Pichia anomala Three isolates of Píchia anomala were compared for their antagonistic activity against Penícíllium roquefortí Jö in airtight, high-moisture stored winter wheat. The Píchía anomaZa isolates J121, J281 and CBS5759 were used in three separate experiments, which were performed as described above.

All inhibition experiments were performed in triplicate and repeated twice.

RESULTS Inhibition of Penicillium roquefortí Jö, grafted on wheat, rye, barley and oats, by the yeast Pichia anomala J12l In autumn wheat (Cossack variety), Pichia reduced the anomalous plant of Penícíllium roqueforti in a dose-dependent manner (Fig. 1a). Penicillium roqueforti was also reduced in spring wheat (variety Dragon) in a dose-dependent manner, although its growth was not as strong as in autumn wheat (Fig. 1b). On rye (variety Motto), Penicillium roqueforti did not grow, regardless of whether the grain had been inoculated with Pichia anomala or not (Fig. 1c). In fact, even a slight decrease in Penicillium roquefortí-CF U (compared to inoculated amount) was observed on rye without inoculated Pichia anomala.

Penicillium roqueforti grew well in barley (variety Svea) and increased from 5 x 103 to 106 CFU / g, which was comparable to its growth on winter wheat (Figs. 1a and d). Pichia anomala reduced the growth of Penicillium roqueforti in barley to 104 CFU / g at 103 CF U / g of inoculated Pichia anomala and totally inhibited the growth at 105 CF U / g (Fig. Ld). The growth of Penicillium roqueforti on oats was comparable to that of winter wheat and barley (Fig. Le). Pichia anomala totally inhibited the growth of Penicillíum roqueforti on oats at 104 CFU / g.

Pichia anomala grew to the same level on four of the five cereals tested, i.e. to about 5 x 107 CFU / g, when inoculated to densities in excess of 103 cells per gram, the exception being spring wheat (variety Dragon), where it reached a level of about 1 x 109 CFU / g (Fig. 1b). At inoculum levels below 103 CFU / g, the growth of Pichia anomala was most pronounced in spring wheat and rye (Figs. 1b and c).

After 14 days of incubation, lactic acid bacteria at levels between 102 and 103 CFU per gram were found in all cereals (data not shown). The total number of aerobic bacteria was 105 CFU per gram in rye and 107 CFU per gram in other cereals (data not shown).

Inhibition of three isolates of Penicillium roqueforti using Píchia anomala J 121 Without the addition of yeast, Penicillium roqueforti J 5, J 5A and J 282 all grew to the same degree on winter wheat, ie. from 5 x 103 to 106 CFU / g (Fig. 2). Penicillium roqueforti J 5, J 5A and J282 were very similar in their sensitivity to the antagonistic yeast Pichia anomala J 121. The plant was significantly affected at an initial level of 104 CFU / g and was totally inhibited at 105 CFU / g (Fig. 2). ).

Inhibition of Penicillium roqueforti J5 using three isolates of Pichia anomala Pichia anomala J121, J 281 and CBS57 59 all reduced the growth of Penicillium roqueforti J 5 on winter wheat (Cossack variety) (Fig. 3). The growth of Penicillium roquefonrti was inhibited in total by an initial level of 105 CFU of Pichia anomala per gram. In the experiment with Pichia anomala J 121, higher mold numbers were found at both 0 and low yeast inoculations (<108 CFU / g) than in the other two experiments (Fig. 3). in: 'kívkirk ************** inhfrkakicärškie-Ic * irkie fi kulriewkak * From the results above, the following conclusions can be drawn.

Pichia anomala inhibits plant and spore formation (measured as CFU) O! 10 30 513 894 16 of Penicillium roqueforzfi on winter wheat, barley and oats in a dose-dependent manner. The antagonistic activity of Pichia anomala in cereal cereals, stored in a restricted air supply tube, was not dependent on the cereal variety. Pichia anomala grew strongly on all five cereals tested.

For some reason, the cereal infection with Penicíllium roqueforti was clearly lower on rye and to some extent also on spring wheat. Differences in the microenvironment in the cereals due to the presence of shells, competing microorganisms, nutrient availability or antifungal compounds can all be expected to affect the growth of both the grafted yeast and the mold. The availability of nutrients in cereal cereals probably differs between varieties and is also dependent on the micro-organism and the previous handling of the cereal. We have observed bacterial levels between 103 and 5 x 107 CFU per gram and yeast levels melan 102 CFU (detection level) and ö x 105 CFU per gram ivete stored at 10% water content (unpublished data). If these differences are caused by microbial growth on the grain, and not only by temporary contamination, significant differences in consumed nutrients in the grain can be expected. However, the bacterial level in the cereals does not explain the presence of Penicillium roqueforti in rye and the weak growth in spring wheat, compared with autumn wheat, oats and barley. The number 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, at which Penicillium roqueforti did not grow, was at least as high as that reached on the other cereals. However, antagonistic microorganisms other than Pichia cmomala could have been present in the rye, but no bacteria or yeast were identified or characterized.

Long-term storage and cultivation of microorganisms can change their properties. Accordingly, a strain of Pichia anomala, recently isolated from winter wheat, was evaluated. Furthermore, one of the three Penicillium roqueforti used from silage was recently isolated. The antagonistic activity of Pichia anomala against Penicillium roquefortí in our model system was not isolation specific. Furthermore, the three Penicillium roqueforti isolates were equally suppressed by Pichia anomalous J121 on high-moisture winter wheat, stored with limited air supply. The ability of Pichia anomala J 121 to effectively reduce the growth of Penicillíum roqueforti reflects the stability of this antagonistic activity, even after years of storage under laboratory conditions. 10 15 30 513 894 17 In summary, Pichia reduced the anomalous growth of Penicillium roqueforti in winter wheat with high moisture content, oats and barley in a container with limited air supply. This finding indicates that Pichia anomala could be used to improve the reliability of airtight storage of feed grains.

Because Penicillium roqueforti did not grow on rye, it was not possible to evaluate the fungal inhibitory activity of Pichia anomala on rye. However, the surprising discovery that Penicillium roqueforti did not grow on rye initiated us to investigate the growth of Penicillium roqueforti and a number of different mold species on different cereals. The surprising results obtained are discussed in more detail below.

II. Cereal cereals with inherent resistance to fungal infection The species Penicillium roqueforti includes fl era taxa, and it has recently been suggested that it should be divided into three different species called Penicillium roqueforti, Pencillium carneum and Penicillium panum, based on i.a. different secondary metabolite profiles and nucleic acid sequence differences (Boysen et al., 1996). The three species, Penicíllium roqueforti, Penicillium carneum and Penicillium panum, are morphologically very similar to each other and can, for practical purposes, 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 probably have the potential to destroy food as well as feed, especially in silage and airtight stored grains with high moisture content.

Below is an account of our investigations regarding the infectivity of Penicillium carneum, Pencillium paneum and Penicillium roqueforti during airtight storage of winter wheat, spring wheat, rye and barley with a high moisture content. The pathogenicity after harvest of Penicillium roqueforti, ie. Penicillium roqueforti, Penicillium carneum and Penicillium paneum, against different varieties and batches of spring wheat and varieties of barley are also compared.

Materials and Methods Fungi studied Penicillium roqueforti Thom J 5 and Penicillium roqueforti J 5A were gifts from Dr. P. Häggblom (Statens Veterinärmedicinska Anstalt, Uppsala). Penicillium UI 10 20 N.) CJ! 515 894 18 carneum lBTl4042, Penicillium carneum lBT6889, Penicillium paneum IBT13929, Penicillium paneum lBT13321, Penicillium roqueforti IBT18689 and Penicíllium roqueforti IBT14088 were gifts from Dr. J. Frisvad, Biotechnology Institute, Lyngby, Denmark. Species identities were confirmed based on morphology and RAPD profiles (Boysen mil., 1996). The fungal cultures were held on malt extract days (LNIEA; Oxoid, Basingstoke, England) at 200. All used fungi have been deposited in the fungal collection at the Department of Microbiology, Swedish University of Agricultural Sciences, SLU, Uppsala.

Studied cereals Autumn wheat (lat. Tríticum aestíuum) variety Cossack, spring wheat (lat. Triticum aestiuum) variety Dragon (designated as A), rye (lat. Secale cereale) variety Motto and barley (lat. Hordeum vulgare, spring form) variety Golf were all purchased from Svenska Lantmännens Riksförbund, Uppsala. Barley, the varieties Baronesse, Kinnan, Maud and Mentor, were gifts from Jörgen Löhde (Svalöf Weibull AB, Svalöv). Baronesse and Kinnan are used as feed grains while Maud and Mentor are barley malt varieties.

Spring wheat, the varieties Alve, Curry, Dragon (referred to as B), Thasos and Tjalve, were gifts from Jan Persson (Svalöf Weibull AB, Svalöv). Four extra batches of spring wheat variety Dragon from Svalöf Weibull AB were used to investigate variations in plant resistance of Penicillium roqueforti (Jö) among batches of the same variety.

The lots are designated as C (P151703 -95), D (M. Elite 88621), E (> 96) and F (-97).

All lots represent the same genotype of the spring wheat variety Dragon; however, each was allowed to grow under different conditions.

Inoculum preparation Conidia suspensions of the fungi were prepared by harvesting spores from 7 day old colonies (grown on malt extract agar (MEA) [Oxoid, Basingstoke, England] at 25 ° C) in peptone water (2 g "Phytone TM" peptone [BBL, Becton Dickinson and Company, Cockeysville] per liter of distilled water) with 0,05% "Tween 80", which was added to facilitate dispersion of the conidia. The spore concentration was determined using a hemocytometer. 15 15 25 25 30 513 894 19 Infectivity of Penicillium Roqueforti, Pencillium carneum and Penicillium paneum in barley, rye and wheat Non-sterile cereal cereals, previously stored at a water content of 10% at 20 DEG C., were moistened with tap water to obtain a water activity (aw) of 0.96 (about 25% water) To equalize the moisture content, the cereals were stored for 2-4 days at 200 with frequent mixing.The water activity was measured at 22-2400 using a "CX-Z AquaLab" equipment (Decagon Devices, Inc., Washington, USA).

Cereals (aw 0.96) were inoculated with mold spore suspension to reach a level of about 104 mold spores per gram. The spores were applied by adding the suspension dripping onto the grain, followed by careful mixing to obtain an even spore distribution in the grain. Thick-walled glass tubes (27 ml) were filled with approximately 17 g portions of inoculated grain and sealed with butyl rubber stoppers. To simulate air leakage, a needle (diameter 0.6 mm) was inserted through each butyl rubber stopper. The tubes were incubated at 2500. On day 14, three tubes from each treatment were opened and the contents were diluted ten-fold with peptone water, soaked for 0.5 h and homogenized for 2 min at normal speed in a "Stomacher 400" (Colworth, England). To avoid plant inhibition of bacteria on agar plates, intended for fungal CFU counting, 0.1 ml aliquots were smeared on the surface of malt extract agar, supplemented with 100 ppm chloramphenicol (C-0378 Sigma Chemical Co. St. Louis; Mo. USA) (MEAC) . Chloramphenicol is a bacteriostatic broad-spectrum substance. Lactic acid bacteria and the total number of aerobic bacteria were counted on M.R.S. (de Man, Rougosa, Sharpe; Oxoid, Unipath Ltd., Basingstoke, Hampshire, England) and flat-counting glucose yeasts (Oxoid, Unipath Ltd., Basingstoke, Hampshire, England), respectively, both supplemented by 0.1% Delvocid ® "(Gist-Brocades, Delft, The Netherlands).

"Delvocide" is a broad-spectrum fungicide that contains natamycin as an active ingredient.

RESULTS Infectivity in Penicillium roqueforti, Penicillium carneum and Penicillium paneum in barley, rye and wheat In barley (variety Golf) all eight mold isolates increased in iP. roqueforti group from the inoculation level 5 x 103 to 4 x 105 CFU / g (Fig. 4a). The total number of molds other than those belonging to the added P. Roqueforti group decreased during the incubation period from 1 x 103 to below the detection limit.

The amount of yeast was below the detection limit on day 0 as well as on day 14. The amount of aerobic bacteria in barley was 1 x 107 CFU / g both at the start and after 14 days of incubation, regardless of the nature of the added mold. The lactic acid bacteria increased in number from below the detection limit, ie. 102 CFU / g, to 8 x 103 CFU / g for all treatments. In rye (variety Motto) none of the eight mold isolates belonging to the P. roqueforti group grew (Fig. 4b). Instead, the mean number of mold CFUs decreased from δ x 103 CFU / g to 3 x 102 CFU / g between day 0 and day 14.

The total number of molds other than those belonging to the added P. roqueforti group decreased from 1 x 104 to below the detection limit. The amount of yeast in the samples was below the detection limit on day 0 as well as on day 14. The aerobic bacteria in rye decreased from 1 x 107 to 2 x 105 regardless of the type of added fungus. The lactic acid strains grew from below the teaking level to 5 x 103 CFU / g. Spring wheat variety Dragon (A) showed all isolates bad plant. The mean value of CFU increased from 4 x 104 to 7 x 104 per gram (Fig. 40). The number of P. paneum and P. carneum increased slightly but significantly more compared to isolates of P. roqueforti (P <0.01). The total number of molds other than those belonging to the added P. roqueforti group increased from 2 x 102 to 3 x 108 CFU / g. The concentration of yeast increased during the incubation period from 2 x 105 to 3 x 107 CFU / g. The aerobic bacteria in spring wheat increased in number from 1 x 107 to 4 x 107 during the 14-day incubation. Lactic acid bacteria increased from below the detection limit to 1 x 103 CPU / g. In autumn wheat (Cossack variety), all three species belonging to the P. roqueforti group increased in number to about 5 x 105 CFU / g (Fig. 4d), fi from a detected inoculum level ranging from 2 x 103 to 9 x 104 CFU / g . The total number of other molds increased from below the detection limit to 4 x 105 CFU / g. The yeast in autumn wheat remained at the O-day level 8 x 104 CFU / g during the entire incubation. The aerobic bacteria increased in number from 6 x 106 to 6 x 107 CFU / g during the incubation, regardless of the nature of the added fungus. The lactic acid bacteria increased in number from below the detection limit to 3 x 103 CFU / g. 10 15 20 'w 513 894 21 Growth of Penicillium roqueforti, Penicillium carneum and Penicillium paneum in different varieties of barley and spring wheat Barley For all strains of P. roqueforti, paneum and P. carneum, CFU levels increased significantly (P <0,0Û1 ) in the feed varieties Baronesse and Kinnan, but not in the malt varieties Maud and Mentor (Table 1). In the Maud and Mentor varieties, P. paneum and P. carneum increased to slightly higher CFU values than those obtained with P. roqueforti (Table 1).

The amount of aerobic bacteria on day 0 was 1.8 x 106 CFU / g in the Baronesse variety, 4.6 x 105 CFU / g in Kinnan, 1.8 x 107 CFU / g in Maud and 3.7 x 106 CFU / g in Mentor.

After 14 days of incubation, the aerobic bacteria had grown to 3.9 x 107 CFU / g in the Baronesse variety and to 4.7 x 106 CFU / g in the Kinnan variety. In the malt varieties Maud and Mentor, there was no detectable growth of aerobic bacteria. In the Baronesse and Kinnan varieties, the aerobic bacteria grew significantly more in cereals inoculated with P. roqueforti than in cereals inoculated with P. paneum or P. carneum (P <0.001, data not shown). The number of lactic acid bacteria increased to 103 CFU / g in the grain variety Kinnan in combination with P. roqueforti but was not detectable with the other inoculated Penicillium species or in the other varieties.

The initial amount of yeast was 1.5 x 104 CFU / g in barley, Baronesse, 1.2 x 105 CFU / g in Maud, 2.6 x 104 CFU / g in Mentor and 5.5 x 103 CFU / g in Kinnan. . The number of yeast colony forming units increased during the experiment to 9 x 103 CFU / g in the Baronesse variety, 1.5 x 103 CFU / g in the Mentor variety and to less than 103 CFU / g in the Maud variety and in the Mentor variety.

Spring wheat In the spring wheat varieties Alve, Curry, Tjalve and Thasos, all strains of P. roqueforti, P. paneum and P. carneum increased from an inoculated level of about 104 CFU / g to 106 CFU / g over 14 days (P <0.001). However, in the two batches of the Dragon variety (A and B), there was no significant increase in the number of CFUs for any of the eight fungal isolates tested (Table 2).

Aerobic bacteria increased in the spring wheat varieties from about 104 to 107 CFU / g in the sorrows Alve and Tjalve and from 107 to 108 CFU / g in the varieties Curry, Dragon (batch A) and Thasos during the 14-day incubation, regardless of the species of grafted mold. The lactic acid bacteria grew during the incubation in the spring wheat variety Curry to 104 CFU / g but could not be detected in the other varieties. Yeast 10 30 515 894 22 grew in the spring wheat variety Dragon (batch A) to 6 x 103 CFU / g during the incubation but could not be detected in the other varieties.

Six different batches of the spring wheat variety Dragon were inoculated to about log 4.6 CFU / g with P. roqueforti J 5 to evaluate the variety's resistance to the fungus under conditions where the air supply is limited. In lots A and F, P. roqueforti J 5 did not increase significantly in CFU, while a weakly significant (P <0.05) growth was observed in B and significant growth (P <0.01) in lot C. In contrast, a sharply significant (P <0.001) increase in P. roqueforti Jö in lots D and E and in the control cereal autumn wheat variety Cossack (Table 3). In general, the species belonging to the Penicillium roqueforti group differed, i.e. Penicillium roqueforti, Penicillium carneum and Penicillium paneum, did not affect their ability to grow in the tested cereal cereal. Among the cereals tested, ie. barley (four varieties), rye, winter wheat and spring wheat (five varieties), only the rye variety Motto and two batches of the spring wheat variety Dragon were resisted by the Penicillium roquefortt group.

The absence of general differences in the infectivity of Penicillium roqueforti, Penicillium carneum and Penicillium paneum in the cereals tested does not contradict or substantiate the division of Penicillium roqueforti into three species. Nor is it possible to predict, on the basis of the results presented, whether one of the species is the main damage fungus in front of the Others during airtight storage of cereals with a high moisture content.

The micro. In the grain can protect against invading fungi. Fungi can reduce the growth of other fungi through parasitism, antibiosis or nutritional competition. The yeast in all tested cereals was reduced in number during the incubation period. In spring wheat, however, the number of yeasts, after the incubation period, was higher in the Dragon variety than in other varieties. The yeast may have contributed to the resistance to fungal growth in the spring wheat variety Dragon. The bacterial content can also inhibit fungal invasion by consuming nutrients or secreting antibiotic substances. In the feed grain varieties, both aerobic bacteria and molds grew well, while in the malt varieties there was less growth of both aerobic bacteria and molds.

Thus, the bacterial growth did not inhibit the mold growth.

In summary, the Maud and Mentor barley varieties, the Motto rye variety and two batches of spring wheat, Dragon variety, were resistant to the Penicillium roquefortí group. The resistance is assumed to be dependent on treatments both before and after harvest. All three species in the Penicillium roqueforti group, ie. Penicillium carneum, 10 20 30 513 894 23 Penicillium paneum and Penicillium roqueforti, infected the tested cereals evenly. Consequently, all three species can destroy airtight stored grains with high moisture content. The choice of less sensitive varieties with inherent resistance to fungal infection becomes of great importance in ensuring better microbial quality of the final feed or food while minimizing the use of agrochemicals.

III. Experiments on a pilot scale Petersson and Schnürer (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, although this is close to the optimal temperature for this mold (Björnberg and Schnürer 1993). However, antagonists, which appear promising under laboratory conditions, do not necessarily perform as well in a large-scale experiment. For example, interactions may vary depending on the type of substrate or other environmental conditions (Magan and Lacey 1985). As a biocontrol agent, a microorganism must compete with other fungi to establish an active population in the new environment. The ability to quickly build up a population is assumed to be a prerequisite for successfully competing with harmful microorganisms or pathogens.

In Sweden, cereal cereals are usually harvested at a water content of 20-22%, corresponding to a water activity of 0.9. Mold growth in cereal cereals is often prevented by high-temperature drying, although it is expensive and energy-intensive in a temperate climate. On farms, where cereals are allowed to grow using conventional cultivation methods, about 60% of the total amount of fuel used in crop cultivation operations is consumed in connection with the high-temperature drying of cereals (Pick et al., 1989). Airtight storage, which is an alternative method of storing feed grain, presupposes perfect sealing in combination with the anaerobic conditions caused by respiration of the grain itself and the adhering microorganisms. Airtight storage requires only about 2% of the energy consumed during high-temperature drying (Pick et al., 1989). However, air leakage during airtight storage can easily lead to a deterioration of the conditions required for preservation. Changes in the internal temperature of the silo (mostly due to solar radiation) and atmospheric pressure, together with an increasing gas volume as the silo is emptied, lead to air movements through the reduction valves and perforations in the casing or through the outlet system. 10 20 æl 515 894 24 In practice, grain is not taken from the silo every day. Instead, larger batches are stored with full access to air for several days. Measures to prevent mold damage and mycotoxin production would be beneficial even during this short-term storage.

To evaluate the practical potential of Pichia anomala as a fungal biocontroller, we designed a 0.2 m3 "pilot scale silo" for airtight storage of moist grain. Wheat with a high moisture content and with different graft concentrations of Pichia anomala and Penicillium roqueforti was stored in silos and samples were taken twice a week. Concentrations of oxygen and carbon dioxide were monitored and the microbial quality of the cereals was monitored during 18 months of storage. The mold-suppressing ability of Pichía anomala during aerobic storage of wheat, which had been removed from the silo, was evaluated after 10 and 12 months of airtight storage. The survival of Pichia anomala in wheat with high moisture content was also evaluated at different temperatures during 15 months of airtight storage.

Silon Silona each contained 0.21 m3 of grain, corresponding to 160 kg. To equalize the differences between internal and external air pressures, each silo was equipped with a "lung" and a Z-way reduction valve made of a U-tube, filled with a liquid that did not freeze. Except when the grain was removed through the outlet pipes, no leakage could occur through the reduction valve until the pressure exceeded # 60 mm static water pressure. This pressure difference could not be built up until the "lung" had either been filled with 14 liters of air or 7 liters of air had been deducted from it.

Gas analysis equipment The concentrations of CO2 and O2 in the silos were measured with an infrared monitor (accuracy in (15%) and a galvanic cell (accuracy in 1%), respectively (GA 90, Geotechnical Instruments Limited, Leamington Spa, Warwickshire, England).

Pressure variations were recorded continuously with a differential pressure gauge (model 600D, Innovex, Hopkins, MN, USA) in one of the silos. The temperature of the wheat was measured at each sampling time with an infrared thermometer (model C-600, Linear Laboratories, Mountain View, Ca, USA). Inhibition Attempts Yeast and mold spore suspensions were prepared as described above.

Conventionally grown winter wheat (Cossack variety) was harvested at a water content of 24% corresponding to a water activity of 0.94. Water activity was measured at 22-2400 with an Aqua Lab CX-2 (Decagon Devices Inc., Pullman, Washington, USA).

The water activity in the wheat was measured on samples after 0, 8, 10 and 57 weeks.

Grafting Freshly harvested wheat grain was grafted with the Penicillium roqueforti spore suspension to about 3 x 108 mold spores per gram. The spores were applied by adding the suspension dripping onto the grain while mixing in a feed mixer to obtain an even spore distribution in the grain. The yeast was similarly inoculated to obtain 0, 104 and 106 cells per gram of wheat. During the control treatment, only tap water was added. The same volume of inoculum was used in all experiments. The silo was filled with about 160 kg of grafted wheat and each experiment was run in triplicate. After sealing, the silo was placed in an outdoor shelter, protected from rain and direct sunlight, for 13 months.

Intentional air intake After 54 weeks of storage, the silo was opened to achieve an increased oxygen concentration in the silo. First, the lids for the outlet pipes (diameter 44 mm) were removed and after 3 days also the connections to the "lungs" (diameter 8 mm) until the oxygen concentration was about 18% before closing, ie. 5 days. The concentrations of CO 2 and O 2 were monitored continuously, first in a silo with wheat inoculated with 106 CFU of Pichia anomala per gram and then in one of the silos with ungrafted cereals. The microorganism was analyzed one week after the silo was closed.

Sampling and quantification of microbial plant Sampling from silos took place initially after one month of storage and then twice a week. The wheat samples, 1 kg each, were removed through the withdrawal tubes.

Samples were taken after 5, 5.5, 6 and 8 weeks and then monthly. After careful mixing, aliquots of about 20 grams were diluted ten-fold with peptone water, soaked for 0.5 hour and homogenized for 2 minutes at normal speed in a "Stomacher 400" (Colworth, England). The yeast plant was quantified on MEA, supplemented with 100 ppm chloramphenicol (C-0378 Sigma Chemical Co. St. Louis; 10 NJ O! 5 515 894 26 Mo, USA) (LN1EAC plates). To avoid plant inhibition of 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 coating of 0.1 ml. -aliquots. Cycloheximide at 10 ppm inhibits the growth of Pichia anomala (Björnberg and Schnürer, 1993). Lactic acid bacteria and the total number of aerobic bacteria were counted on MRS (Oxoid, Basingstoke, England) and flat count agar (Oxoid, Basingstoke, England), respectively, both supplemented with 0.1% "Delvocid®" (Gist-Brocades, Delft, The Netherlands).

"Delvocide" is a broad-spectrum fungicide that contains natamycin as an active ingredient.

Aerobic stability after airtight long-term storage of wheat After 10 and 12 months of storage, 700 g of cereals from each silo were stored for 3 weeks at 20 ° C in Erlenmeyer flasks, sealed with cotton. Before sampling, the cereals were mixed well. Microbial analysis was performed as described above. The growth of Penicillium roqueforti, Píchia anomala, aerobic bacteria and lactic acid bacteria was frequently determined for 3 weeks, as described above.

RESULTS Environmental conditions The temperature of the wheat intended for testing followed closely the changes in the average daily temperature of the outdoor air during the experiment. Differences in air pressure between the gas in the silo and the external atmosphere were close to zero most of the time. The pressure inside the silo was low enough to cause the air to move into the silo through the reduction valve on only seven occasions during the first two months and on one occasion during weeks 28, 29, 44 and 46 respectively.

The water activity of the wheat husk with high moisture content remained between 0.93 and 0.94 during the storage period (data not shown).

Gas analysis Already 5 hours after sealing, no O 2 could be detected in the air inside the silo, and the concentration of CO 2 increased rapidly during the first experimental week.

After two months, the concentration of CO2 had increased to its maximum, about 70%, in all silos. When the temperature dropped below zero, the concentration of CO 2 in the triplicates decreased and stabilized at a level between 50 and 60%. During the same time, oxygen concentrations could time only shortly after sampling.

Microbial plant The inoculation with Pichia anomala, intended to reach 104 and 106 CFU / g, resulted in 4 x 104 CFU / g and 5 x 105 CFU / g, respectively. Furthermore, the cereal had 1 x 104 CFU / g of Pichia anomala as a natural fl ora. After the first five weeks of storage, in closed silos, the yeast had grown to 6 x 104 CFU / g in the ungrafted cereals, to 2 x 104 CFU / g in grains inoculated with Penicillium roqueforti, to 4 x 105 CFU / g when inoculated with 1 x 104 CFU of Pichia anomala / g and to 9 x 105 CFU / g when inoculated with 5 x 105 CFU of Pichia anomala / g. After eight weeks of storage, the amount of Pichia anomalous in the two inoculation treatments was 4 x 10 6 CFU / g, while the treatments in which yeast was not inoculated showed 4 x 10 5 CFU / g cereals. After 10 weeks of storage, the inoculated Pichia anomala had grown to 7 x 10 6 CFU / g in both treatments. Naturally occurring Pichia anomala had grown from 1 x 104 CFU / g to about 6 x 105 CFU / g in the ungrafted wheat. This difference persisted during weeks 20 to 35, ie. January to the end of April. During the month of May, the yeast in the ungrafted grain increased to the same level as in the grafted grains, ie. to close to 107 CFU / g.

Penicillium roqueforti was inoculated at 3 x 103 CFU / g in order to represent an extremely high level of contamination. Despite this, the inoculated level of Penicillium roqueforti in wheat was detected only at one time during the year. The amount inoculated was reduced to the detection limit, i.e. 102 CFU / g, during the first five weeks in the closed silos. Furthermore, at the end of the storage period, about 102 CFU / g of Penicillium roqueforti were found. In cereals which had not been inoculated with either Pichia anomala or Penicillium roqueforti, endogenous Penicillium roqueforti was found in a sampling case, ie. after 36 weeks of storage.

At the start of the experiment, approximately 102 CFU / g of lactic acid shares were found in all treatments, with the exception of unvaccinated ones. After up to week 39 of storage, <103 CFU / g was found in one of the silos during this treatment. On rare occasions, less than 103 CFU / g lactic acid bacteria were found during the other treatments in concentrations.

Initially, the wheat contained 5 x 105 CFU / g of aerobic bacteria. Under the five O! 10 20 NJ O! During the weeks of closed storage, the amount decreased but it was detected to 105 CFU / g in the inoculated treatment, to 106 CFU / g in the treatment with Penicillium roqueforti alone and to 104 CFU / g in both treatments with inoculated Pichia anomala. During weeks 20 to 35 (January to late April), the amount of aerobic bacteria remained stable, followed by a decrease in all treatments around week 40 (early June).

Microbial stability after a simulated major air leakage During the simulated air leakage after 54 weeks of storage, the oxygen concentration in all silos increased to about 18% in five days (data not shown). Once the silo with wheat, inoculated with 106 CFU Pichía anomala per gram, all oxygen was consumed within 20 hours, while it took 46 hours in the ungrafted grain. One week after the planned air leakage, no increase in Penicillium roqueforti- CFU could be 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, grafted Penicillium roqueforti reached only 105 CFU / g after 6 days, while it took 12 days to reach the same level in wheat, inoculated with 104 CFU / g, and 14 days in wheat, inoculated with 106 CFU / g of Pichia anomala (Fig. ö). Similar results were obtained with wheat after 12 months of airtight storage. * ivävlcvkirícvkücicärkvë fl cvkvkäckir ** icieslrickakvkwëürírkkicvirvkvkvš-íricic * Growth of Penicillium roqueforti, measured as colony-forming units (CFU), in wheat with high moisture content was totally inhibited during the aeration.

When the stored grains were removed from the silo and exposed to air, the growth of Penicillium roqueforti of grafted Pichia anomala was delayed in a dose-dependent manner. Since Penicíllíum roqueforzti grew when the cereals were exposed to air, it can be concluded that at least some of the grafted fungal spores were viable. Thus, the growth of Penicillium roqueforti in silona was inhibited by adverse conditions. Probably both the atmosphere of the silona and the high levels of Píchía anomala in the grain contributed to the suppression of the plant by Penicillium roqueforti. 10 20 30 513 894 29 Our previous tests using Pichia anomala as a biocontroller in cereals have been performed with 1-2 year old moistened cereals. There are probably physiological differences between freshly harvested and moistened cereals.

However, in these pilot-scale experiments, the antagonistic effect of Pichia anomala was evident even with the use of freshly harvested cereals.

Upon exposure to air, grains that had been stored airtight for 10-12 months were destroyed more rapidly when not previously inoculated with Píchia anomala, despite the high level of endogenous Pichia anomala in the grain prior to inoculation. In cereals taken from the silo after 10 months, inoculated Pichia delayed the anomalous growth of inoculated Penicillium roqueforti to 5 x 104 CFU / g by 6 days.

During the same period, Pichia anornala did not increase in number. Thus, the antagonistic activity of Pichia anomala is not strictly associated with detectable growth. Inhibition of the growth of Aspergillus auus by Pichia guilliermondii in soybeans depends on the active plant of the antagonistic yeast. Aspergillus fl auus, which was added when the extensive yeast growth had decreased during the first 2-3 days, could compete with the yeast (Paster et al., 1993). Thus, an active plant of Pichia guilliermondii, in contrast to Pichia anomala, affects the fungal inhibition.

In test tube experiments with 18 g of wheat with a high moisture content, the plant is limited by Penicillium roqueforti of Pichia anomala, grafted to 103 CFU / g, and is practically inhibited by a total of 104 CFU / g during 14 days of air leakage (Petersson and Schnürer, 1995). In the present study with silo, containing 160 kg of wheat, 104 CFU / gram and 105 CFU / gram were used to determine the amount of Pichia anomala required to inhibit the growth of Penicillium roqueforti during the 13 month storage period. By way of comparison, other antagonistic yeasts are often used in very high concentrations in experiments, for example 106 CFU / g or even higher concentrations.

The designed silos mimicked the function of airtight silos at full scale and could be easily handled. However, the impermeability to air may have been too great in the silo, compared to a full scale silo. When cereals with a moisture content of 23% are stored in a completely closed container, levels of carbon dioxide up to 95% have been measured.

In practice, the concentration of carbon dioxide, due to incomplete sealing, etc., often increases to only 15-25%. In this experiment, the carbon dioxide level in the silos was stabilized at 50-60%. There were not as many air leaks through the air regulator as expected, probably due to the protection from direct solar radiation in the silos and the capacity of the rubber lungs. Nevertheless, the silo was opened twice a week for about 1 minute each time. These opening events probably did not give rise to any significant amounts of gas exchange but equalized the pressure in the silos to the outside pressure. However, not even the incoming oxygen at the end of storage gave rise to any plant of Penicillium roqueforti. The system appeared to be robust in its mold-inhibiting ability, even in the event of minor air leakage.

Table 1 Plant and spore formation, measured as colony forming units (CFU) per gram of the Penicillium roquefortí complex, on different varieties of barley (off, 0.96) incubated with limited air supply in glass tubes at 25 ° C for 14 days. The values are given as logarithms and represent the mean values for three tubes. The smallest 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 (* **).

Kornsort P. ro ue orti P. Qaneum P. carneum daa Jö 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 kk * 72k * * ii 'käšk * kit * kk 72k * kk * 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 6,1 716% * irš * * vkvï fit * * kärr * írvë kk * 'kvšiv lvlaud 0 4,5 4,4 4,5 4,4 4,5 4,4 4,4 4,4 14 4,4 4,4 4,4 4,2 5,0 5, 0 4.9 4.7 sing. es? es es es ** *** ** es Mentod 0 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 sing. *** es es es *** es es ** Note: 1. "sing." indicates significance level for the increase in CFU between values on day 14 and day 0 2. "no" does not indicate significant. 10 15 20 25 30 513 894 31 Table 2 Plant and spore formation, measured as colony forming units (CFU) per gram of the Penicillium roqueforti complex, on different varieties of spring wheat (aw 0,96) incubated with limited air supply in glass tubes at 25 ° C for 14 days. "Dragon A" and "Dragon B" represent independent samples of the same variety. The values are given as logarithms and represent the mean values for three tubes. The smallest 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 <0.00l (***).

Vårvete- P. ro ue orti P. Qaneum P. carneum sort dag J5 J 5A 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 6.8 * ink kit * * kk ink * * ink kit * vl- * k * ate 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 * cock kit * kit * * i fl š ink * * kick ink * 1696 * 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. esz es es es es es es es es es Dragon B 0 4.3 4.3 3.8 4.6 4.0 4.8 4.0 4.3 14 3.8 4.2 3.8 4.2 3, 6 4.0 3.7 3.9 sign. es es es es es es es es es Thasos 0 3.8 4.0 8.9 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. ** ~ k ~ k * 'k kß fi * kit * ink * ivk * 12k * * ink Tjalve' o 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 ätit * 1216 * * å-i 'ätit * 121% * ink * * Skit kit * Anm .: 1. "singï "indicates the significance level for the increase in CFU between values on day 14 and day 0 2." no "does not indicate a significance. 10 20 513 894 32 Table 3 'Vl'ax, measured as colony forming units (CFU) per gram, of Penicillium roquefortí J 5 on different lots of spring wheat (aw 0,96) variety Dragon and autumn wheat variety Cossack as a control, in glass tubes at 2500 for 14 days. All lots represent independent samples of the same variety. The values are given as logarithms and represent the mean value for three tubes. The statistical significance of differences in the CFU value between days 0 and 14 is stated (n = 8).

Dragon Detected P. roqueforti J 5 batch (mean value for log CFU / g) day 0 day 14 A 4.6 4.7 ”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 * Control: Cossack 4.6 6.6 *** Anzn: l. "Es" does not indicate significant, * (P <0 , 05), ** (P <0.01l) and *** (P <0.00l). 10 15 20 25 513 894 33 REFERENCES Barnett, J.A., Payne, R.W. and Yarrow, D., 1990. Yeasts: characteristics and identification. Cambridge University Press, Cambridge.

Bj Örnberg, A. and Schnürer J., 1993. Inhibition of the growth of grain-storage molds in uitro by the yeast Pichia anomala (Hansen) Kurtzman. Can. J. Microbiol. 39: 623-628.

Boysen, M., Skouboe, P., Frisvad, J., och Rossen, L., 1996. Reclassification of the Penicillium roqueforti group into three species on the basis of molecular genetic and biochemical proñles. Microbiology, 142: 541-549.

Chalutz, E. 1990. Postharvest biocontrol of green and blue mold and sour rot of citrus fruit by Debaryomyces hansenii. Flat. Dís. 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 fi uits and vegetables. Posthar- vest News and Information. Vol. 2, p. 169-173.

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Kreger-van Rij, N.J.W. (Ed.), 1984. The yeasts. A Taxonomic study. North- Holland Publishing Co., Amsterdam.

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Lacey, J., and Magan, N. 1991. Fungi in cereal grains; their occurrence and water 10 20 513 894 34 and temperature relationships. In Cereal grain. Mycotoxins, fungi and quality in drying and storage. Edited by J. Chelkowski, Elsevier, Amsterdam. p. 77-118.

Magan, N., and Lacey, J. 1984. Effects of gas composition and Water activity on growth ofñeld 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 Schnürer, J. 1995. Biocontrol of mold in high-moisture Wheat stored under airtight conditions by Pichia anomala, Pichia guillerimondii and Saccharomyces cereuisiae. Applied and Environmental Nlicrobiology, 6 1: 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, Nederlän- derna.

VVilson, C.L. and Chalutz, E. 1989. Postharvest biological control of Peniczlllium rock of citrus With antagonistic yeasts and bacteria. Sci. Hortic. (Amsterdam), 40: 105-112.

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Claims (9)

A method for reliable airtight storage and preservation of moist cereals, characterized by the combination of using as cereals a cereal variety which resists infection by storage fungi, and of carrying out the storage in the presence of an antifungal effective amount. of a biocontrol agent capable of suppressing or inhibiting storage damage fungi while enhancing the effect of using a cereal variety that resists infection by such fungi. The method according to claim 1, characterized in that the cereal used is intended for use as animal feed. The method according to claim 2, characterized in that the cereal used is a cereal cereal. The method according to claim 3, characterized in that the cereal seed variety is selected from wheat, rye, barley, oats, rye wheat, maize, sorghum, millet and rice. The method according to claim 4, characterized in that the cereal grain variety used is rye variety Motto, spring wheat variety Dragon or barley variety Maud or variety Mentor. The method according to any one of claims 1-5, characterized in that the biocontroller is a Pichia anomala isolate capable of suppressing or inhibiting storage damage fungi while enhancing the effect of using a cereal variety that resists infection by such fungi. The method according to claim 6, characterized in that the Pichia anomala isolate is Pichia anomala J 121, which has been deposited with Centraalbureau voor Schimmelcultures (CBS) under deposit number CBS 100487, Pichia anomala J 281 or Pichia anomala CBS 5759. 8. The method according to any of claims 1-7, characterized in that the stock damage fungi belong to the group of Penicillium species. The method according to claim 8, characterized in that the PenicilZium species are Penicillium roqueforti, Penicillium carneum, Penicilliumpanum and Penicillium UCFTLLCOSLLITI. The method according to any one of claims 1-9, characterized in that the presence of the biocontroller is a consequence of its natural occurrence in the grain and / or is a consequence of application to the grain of a biocontroller composition containing the biocontroller. The method according to claim 10, characterized in that the biocontrol composition is in liquid, semi-liquid or solid form. The method of claim 10 or claim 11, characterized in that the biocontrol composition comprises, in addition to the biocontroller, one or more additives selected from liquid or solid carriers and diluents, surfactants, dispersants, nutrients, growth factors and antioxidants and mixtures thereof. 1 The method according to any one of claims 10-12, characterized in that the application of the biocontrol composition is effected by mixing, powdering, injection, rubbing, spraying or brushing, dripping or rolling. 1 Biocontrol composition for use in the method according to any one of claims 1 to 13, characterized in that it comprises at least one biocontrol agent which suppresses or inhibits storage damage fungi. 1 The composition according to claim 14, characterized in that it is in liquid, semi-liquid or solid form. 1 The composition according to claim 14 or 15, characterized in that it further comprises an additive selected from liquid or solid carriers and diluents, surfactants, dispersants, nutrients, growth factors and antioxidants and mixtures thereof. 1 The composition according to any one of claims 14-16, characterized in that it contains at least one yeast strain as a biocontrol agent which is antagonistic against stock damage fungi. 1 The composition according to claim 17, characterized in that the yeast strain is a Pichia anomala isolate capable of suppressing or inhibiting storage damage fungi while enhancing the effect of using a grain variety which resists infection by such fungi. 1 The composition according to claim 18, characterized in that the Pichia anomala isolate is selected from the group consisting of Pichia anomala strain J 121, deposited with the Centraalbureau voor Schimmelcultures (CBS), Baarn, the Netherlands, under deposit number CBS 100487, Pichia anomala strain J 281 and Pichia anomala strain CBS 5759.