2. Background In biological control, antagonistic micro-organisms are used for the control of pathogenic micro-organisms (e. g. fungi and bacteria). Control is carried out for example by applying micro-organisms in a water suspension to the focus of control. Following application it is necessary to promote the growth of the antagonistic micro-organism so that it colonises the target growth site as quickly and completely as possible. The growth of pathogenic micro-organisms on the focus of control can be prevented in this way.

A problem occurs when applying spores in a water suspension during the spring and autumn.

When the temperature is below zero (0 °C), a suspension containing water will freeze in the application equipment. In the forest situation, this would happen at felling or respacing when applying the Phlebiopsis gigantea spore water suspension to tree stumps for control against Heterobasidion annosum or Heterobasidion parviporum. Control is diffcult if the water freezes at night or during the evening, and daytime temperatures require total and successful application.

A type of compound that prevents the fungal spore water suspension from freezing, maintains the spores (micro-organisms) viability and improves their growth, has not previously been presented or used. A compound which contains the qualities mentioned above should also be economically viable.

It is generally known that anti-freeze liquids e. g some alcohols, may damage small fauna and micro-organisms, and reduce their vitality.

Compounds known to affect the growth of micro-organisms have previously been presented in the following announcements and patent publications of The Board of Patents and Registration: * Utlaggningsskrift 449002 C 12 N 1/00 Satt att odla mikroorganismer i hydrofobt medium medelst mikroemulsion och flytande naringskomposition avsedd därför.

* International application PCT/US94/08548 AO1N 63/00, A61K37/00, C12N 1/20.

Biological control of post-harvest diseases by combining a sugar analog with an antagonistic yeast.

* International application PCT/GB93/00604 AO1N 63/00, A23B 7/155, C12N 1/20.

Biological control of post-harvest diseases.

3. Summary of the invention The invention focuses on the method presented in the characteristics part of claim number 1.

The objective of the invention is to provide a method for the use of chemical compounds in biological control that will increase and promote the growth of antagonistic hyphae and micro-organisms.

The objective of the invention is to provide a method for the use of chemical compounds which will prevent a micro-organism water suspension from freezing, while at the same time maintaining the micro-organisms (spores) viability. The antagonistic micro-organisms referred to include all spores, hyphae and bacteria of fungi, but in particular the spores and hyphae of the fungus Phlebiopsis gigantea that are used for control purposes in forests and gardens.

The freezing point of water with an alcohol content of 25-30 % is between-7°C and-12°C.

A fungal spore water suspension is generally not used in temperatures lower than this.

A further part of the invention is to provide chemical compounds (product) according to the method of the invention presented in the characteristics part of claim number 9.

Part 3.1 presents individual chemical compounds, and combinations of two or more compounds, which provide good biological control according to the methods of the invention.

These compounds and their combinations are particularly suited to biological control because for example they prevent the fungal spore water suspension of Phlebiopsis gigantea from freezing. They also maintain the viability of the spores and hyphae, and especially improve their growth.

3.1 The use of single compounds (reagents) as well as their derivatives and isomers in a fungal spore water suspension 1. G 1 y c e r o 1 (volume content 25 %) Glycerol prevents water from freezing. The spores remain viable over 3 days. Glycerol improves the growth of spores. The use of glycerol does not cause damage to the environment.

2. Normal salts (e. g. NaCI, NaI) and nutrients; phosphates, nitrates, ammoniums and sulphates (e. g. Na+Cl~, C1z+3lPO43-2, Na+N03-, NH4+, K+zS042-).

Normal salts prevent water from freezing. The spores remain viable from 3 to 5 days. Salts increase the growth of spores. If applied to soil, sodium chloride may increase the ion content of surface and groundwater. The nutrients will bind with vegetation.

3. Carbohydrates Carbohydrates prevent water from freezing to some extent. The spores remain viable from 3 to 5 days. Easily available carbohydrates (e. g. which dissolve or easily break up in suspension) improve the growth of spores.

4. Methanol (volume content 25 %) Methanol prevents water from freezing. The spores remain viable from 10 to 24 hrs. The growth of spores is satisfactory. The use of methanol does not cause damage to the environment.

5. Ethanol (volume content 25 %)

Ethanol prevents water from freezing. The spores remain viable from 10 to 24 hours. The growth of spores is satisfactory. The use of ethanol does not cause damage to the environment.

6. Monoethylene glycol (volume content 25 %) Monoethylene glycol prevents water from freezing. The spores remain viable for approximately 24 hours. The growth of spores is satisfactory. Potential damage to the environment must be researched.

7. Sugar alcohols (e. g. mannitol, sorbitol, xylitol) As derivatives of sugar they are suitable for the purpose mentioned above. The use of sugar alcohols does not cause damage to the environment.

For comparison: 8.2-Propanol (volume content 25 %) 2-Propanol prevents water from freezing. The spores remain viable from 1 to 6 hrs. The growth of spores is satisfactory. The use of 2-propanol does not cause any damage to the environment.

3.2 In accordance with the invention method, the use of combinations of two or more chemical compounds (reagents) and their derivatives in a fungal spore water suspension 1. Carbohydrates (e. g. saccharose) and methanol; The spores remain viable over 24 hours.

The growth of spores improves.

2. Carbohydrates (e. g. saccharose) and ethanol; The spores remain viable over 24 hours. The growth of the spores improves.

3. Carbohydrates (e. g. saccharose) and monoethylene glycol + additives (anti-freezers); The spores remain viable over 24 hours. The growth of spores improves.

4. Polyvinylpyrrolidone and ethanol; The spores remain viable over 24 hours. The growth of spores is normal.

5. Polyvinylpyrrolidone and methanol; The spores remain viable over 24 hours. The growth of spores is normal.

For comparison: 6. Carbohydrates (e. g. saccharose) and 2-propanol; The spores remain viable over 10 hours.

The growth of spores is poor.

7. Polyvinylpyrrolidone and 2-propanol; The spores remain viable over 10 hours. The growth of spores is poor.

The combined use of two or more chemical compounds (which prevent water from freezing, protect the spores, and promote their growth) at the same time has a positive effect on the viability and growth of spores. The 1 or 2-OH alcohols normally have a negative reaction on the viability of the spores after a long (over 10 hours) reaction time, but using additional compounds (one or more) with these alcohols preserves the spores'viablity for over 24 hours.

For example, these compounds protect the reaction surfaces of the spores from the effect of the hydroxyl groups of alchohols.

Carbohydrates as protection for spores: Carbohydrates (as well as polyvinylpyrrolidone) provide protection to fungal spores and other micro-organisms e. g. against the reactions of hydroxyl groups of alcohols. For example, where the compound that prevents water from freezing has a negative effect on the fungal spores, adding saccharose can provide protection both to the spores and the hyphae.

Carbohydrates improve the growth of micro-organisms significantly. Fungal spores utilize carbohydrates for growth. Saccharose clearly improves the speed of germination and the volume growth of hyphae in both pure water suspensions and other liquid suspensions which prevent water freezing. Carbohydrates must be quickly and easily available for use by micro-organisms.

By adding carbohydrates and alcohols (one, two or more-OH groups) to the fungal spore water suspension, the suspension will be prevented from freezing and the viability of the fungal spores will remain normal for 24 hours. This method also improves fungal spore germination and volume of growth. Carbohydrates do not cause damage to the environment when applied to soil. The majority of carbohydrates, their different forms, additives and isomers are suitable for this purpose (e. g. monosaccharides, disaccharides and polysaccharides; cellulosa, hemicellulosa, starch, aldoses and ketoses; trioses, tetroses, pentoses and hexoses; all stereoisomers, D-and L- (+ or-) forms as well as alfa-and beta-forms.

Polyvinylpyrrolidone as protection for the spores: Polyvinylpyrrolidone (PVP) provides protection for fungal spores and other micro-organisms for example against reactions with the hydroxyl groups of alcohols. Where the compound that prevents water freezing has a negative effect on the spores, protection for both spores and hyphae can be provided by PVP.

By adding PVP (Da 5 000-15 000) with alcohol (1-, 2-or more-OH groups) to the fungal spore water suspension, freezing of the suspension will be prevented and the fungal spores viability will remain normal for 24 hours. PVP does not particularly enhance the growth of spores.

The forrn of polyvinylpyrrolidone: (QH, n In addition to polyvinylpyrrolidone, spores can also be protected with other similar macromolecyles.

Salts as protection for spores: Ions formed by salts (e. g. normal salts) in a water suspension will protect spores. Salt prevents water from freezing. Preventing water from freezing can be further enhanced by the combination effects of salt and alcohol. Salts improve the growth of spores.

3.3 Other observations The compounds, their derivatives and isomers, as well as other similar compounds mentioned above, can be used several at a same time. The co-use of alcohols, carbohydrates, and normal salts, as well as their derivatives and isomers, in the fungal spore water suspension prevents the suspension freezing, improves the viability of the fungal spores and improves their growth.

Lignin has a positive effect on the growth of spores. Lignin can be used in conjunction with carbohydrates, alcohols or different salts in the fungal spore water suspension.

The alcohol used in a fungal spore water suspension vaporizes soon after application.

Therefore the potential reaction caused by alcohol is reduced or stops completely. Adding easily available carbohydrates (e. g. saccarose, syrup, molasses, starch) to the micro-organsim water suspension maintains the viability of the fungal spores and growth can begin already in the water suspension. This increases the volume of the micro-organism in the suspension to be applied and improves its effectivness for biological control. Therefore, it is possible to carry out control with less suspension and spores because the amount of the growing units (volume) has increased.

Normal salts (and fertilizers like garden"Y-fertilizer") can increase the growth of the fungal spores and hyphae. The use of carbohydrates and salts together, or separately, is also suitable for this purpose. Micro-organisms use salt nutrient ions for growth. Glycerol quickens fungal spore germination and significantly increases the volume growth of hyphae.

A product for biological control can be manufactured from chemical compounds according to the methods described above. This product, when added to the water suspension creates a positive effect for the micro-organism. The use of water in the application suspension can be minimized or completely replaced by another substance.

The viability of the spores in water is maintained for many days when the temperature is between 0-28 ° C. When a small amount of saccarose (1 %) is added to the water, the spores will remain viable even longer (5-7 days). If the growth of spores has begun already in this nutritious liquid and the mass of hyphae has increased, the result achieved in stump treatment is probably better than if using spores which have not started to grow.

By using the chemical compounds, their derivatives and other similar compounds described above, freezing of the micro-organism water suspension is prevented, the viability of the micro-organism (fungal spores) is maintained and their growth is improved.

The invention is described according to the results of the following growth tests: Fig 1. Growth test: Phlebiopsis gigantea spore water suspension without a compound to prevent freezing (standard).

Water has not damaged the spores. Growth is good after all the reaction times.

Fig 2. Growth test: Phlebiopsis gigantea spore water suspension and glycerol.

Glycerol does not damage the spores or hyphae of the fungus during reaction times less than 24 hours. Growth after all the reaction times is very good. Glycerol improves the germination of spores and increases the volume growth of hyphae.

Fig 3. Growth test: Phlebiopsis gigantea spore water suspension and sodium chloride.

Normal salts do not damage the fungal spores or hyphae. Growth after all the reaction times is very good. The salts improve the germination of spores and increases the volume growth of hyphae.

Fig. 4 Growth test: Phlebiopsis gigantea spore water suspension and ethanol.

Ethanol has a moderately harmful effect on the fungal spores and hyphae. Growth after all the reaction times is however fairly good.

Fig. 5 Growth test: Phlebiopsis gigantea spore water suspension, saccharose and ethanol.

The saccharose (carbohydrate) protects the fungal spores and hyphae from the negative effects of the ethanol (alcohols). Growth is very good after all the reaction times. Saccharose improves the germination of spores and increases the volume growth of hyphae.

Fig. 6 Growth test: Phlebiopsis gigantea spore water suspension, polyvinylpyrroledone and ethanol.

Polyvinylpyrrolidone protects the fungal spores and hyphae from the negative effects of the ethanol (alcohols). Growth after all the reaction times is good.

Test reaction times: Growth points on the same horizontal level in the picture have the same reaction time. 24hours------> 10hours------> 1 hour------> 6 hours------> Growth point The growing time of the spores: left 5 days, right 8 days. 5 days 8 days