Description

METHOD FOR ENHANCING THE THERMAL TOLERANCE OF ENTOMOPATHOGENIC FUNGAL SPORES, BLASTOSPORES AND ENZYMES

Technical Field

[1] The present invention relates to a method for enhancing the thermal tolerance of spores and enzymes produced by entomopathogenic fungi, which can be advantageously used as a material for preparing biopesticides. Background Art

[2] Recently, many problems have been created by an excessive use of chemical pesticides, which causes a high level of resistance for harmful insects in agriculture, a disturbance in ecological system, an adverse effect on human health, etc., and many concerns related thereto have been continuously raised. As an alternative to such chemical pesticides, more attention is being paid to the development of biopesticides which utilize entomopathogenic fungi that are present in nature.

[3] Biological pesticides comprising microbes can be understood as a biological formulation which is prepared from microbe itself or from other formulations comprising microbes and is used for an efficient control of various kinds of weeds, phytopathogenic fungi, or pests such as insects, mites and nematodes, etc. that are harmful to many crops.

[4] Meanwhile, since the biological function of chitinase has been recently determined for fungi, microbes and plants, its important role as a mediator for an ecological interaction among them is being noticed. In this connection, biological control using chitinase is also getting more attention.

[5] Biological pesticides comprising such entomopathogenic fungi have many advantages that they are less toxic and therefore are less harmful to crops compared to synthetic organic pesticides, and they have less impact on ecological system and do not cause a tolerance or resistance to pesticides, etc. However, because activity of enzymes that are produced by fungi themselves cannot be maintained for a long period of time during manufacturing process, the short shelf life has been remained as a problem. Disclosure of Invention Technical Problem

[6] The present invention is a result of works to solve the problems as described above.

Specifically, purpose of the present invention is to provide a method for enhancing the thermal tolerance of spores and enzymes produced by entomopathogenic fungi, which can be advantageously used as a material for preparing biopesticides.

[7] The present invention is accomplished through a series of studies for increasing the stability of entomopathogenic fungal spores by using an oil as a preservation medium so as to prevent exposure of the spores to moisture as much as possible, after confirming the fact that the activity of the spores is significantly lowered by the presence of moisture in general, and for the enzyme (e.g., chitinase), considering a tendency that the enzyme activity is lowered when the enzyme is dissolved in water, the enzyme dissolved in water is first pulverized to reduce its contact with water and then kept in oils to increase their thermal tolerance. Technical Solution

[8] In order to accomplish the above-described purpose, the present invention provides a method for enhancing the thermal tolerance of spores and enzymes that are produced by entomopathogenic fungi. More specifically, the present invention provides a method for enhancing the thermal tolerance of spores and enzymes produced by entomopathogenic fungi, characterized in that spore powder of entomopathogenic fungi or powder to which enzymes originating from entomopathogenic fungi are adsorbed is added to vegetable oils, and then the resulting mixtures are stored until further use.

[9] The present invention also relates to a novel entomopathogenic fungal species of

Beauveria sp. DBB2507 (Deposit No.: KCCM 10892P) having a property of efficiently controlling the harmful insects in agriculture by treatment of spores or enzymes that are produced by entomopathogenic fungi.

[10] The above-described vegetable oils of the present invention can be selected from soybean oil, olive oil, cotton seed oil, or corn oil. Preferably, olive oil can be selected.

[11] Meanwhile, the powder to which enzymes originating from entomopathogenic fungi are adsorbed can be obtained by freeze-drying of the pellets that are formed by adding enzyme adsorbents to a supernatant resulting from centrifuge of culture media comprising entomopathogenic fungi. The above-described drying step is not limited to freeze-drying, and it is evident that a skilled person can freely select any of known drying methods depending on his or her needs. In addition, for the above-described enzyme adsorbent of the present invention, kaoline can be preferably used.

[12] The above-described enzymes originating from entomopathogenic fungi can be chitinase, but are not limited thereto. It can include several kinds of enzymes relating to virulence.

[13] Meanwhile, according to the present invention the novel entαnopathogenic fungal species of Beauveria sp. DBB2507 (Deposit No.: KCCM 10892P) was used to determine the claimed method for enhancing the thermal tolerance of the spores and the enzymes produced by entomopathogenic fungi.

Advantageous Effects

[14] As it has been described above, the method for enhancing the thermal stability of entomopathogenic fungal spores, blastospores and enzymes according to the present invention is found to be a useful method to maintain and enhance the activities of the spores, blastospores and the enzymes produced by entomopathogenic fungi, which can be used as a material for producing biopesticides.

[15] Furthermore, it is expected that the method for enhancing the thermal stability of entomopathogenic fungal spores, blastospores and enzymes according to the present invention can be advantageously used for the formulation and mass production of biopesticides having a high level of controlling activity. Brief Description of the Drawings

[16] Figure 1 shows the thermal stability of Beauveria sp.DBB2507 spores and blastospores in water, as an embodiment of the present invention (left: spores, right: blastospores).

[17] Figure 2 shows the stability of Beauveria sp.DBB2507 spores in several kinds of oils between room temperature and heat-treatment condition, as an embodiment of the present invention (left: at room temperature, right: under heat treatment condition).

[18] Figure 3 shows the thermal stability of Beauveria sp.DBB2507 spores and blastospores in oil at different time of thermal-stress, as an embodiment of the present invention (left: spores, right: blastospores).

[19] Figure 4 shows the thermal stability of Beauveria sp.DBB2507 chitinase existed as liquid form at different time of thermal-stress, as an embodiment of the present invention.

[20] Figure 5 shows the amount of chitinase harvested during the process of centrifuge, wherein said chitinase has been produced by Beauveria sp.DBB2507 in liquid culture, as an embodiment of the present invention.

[21] Figure 6 shows the absorption capacity of enzyme adsorbents for chitinase that has been produced by Beauveria sp.DBB2507 in liquid culture, as an embodiment of the present invention.

[22] Figure 7 shows the thermal stability of Beauveria sp.DBB2507 chitinase adsorbed

onto enzyme adsorbent at several conditions wherein said chitinase has been produced by liquid culture of Beauveria sp.DBB2507, as an embodiment of the present invention.

Mode for the Invention

[23] The present invention will now be described in greater detail with reference to the following preferred examples. However, it is only to specifically exemplify the present invention and in no case the scope of the present invention is limited by these examples. It is construed that the scope of the present invention is limited only by the attached claims of the invention.

[24]

[25] <Example 1> Method of enhancing the thermal stability of the spores and blastospores that are produced by entomopathogenic fungi

[26] 1-1. Test for determining a thermal stability of entomopathogenic fungal spores and blastospores

[27] Beauveria sp.DBB2507 strain (Deposit No.; KCCM 10892P) was subjected to a plate culture using SDA+Y agar (Saubraud dextrose culture media + 0.5 wt% of yeast extract) and also to a liquid culture using SDA+Y broth to obtain spores and blastospores, respectively, which were then subjected to a freeze-drying for thermal stability test. Specifically, suspensions comprising the spores or blastospores were kept in an incubator at 5O ⁰ C for 60 min, or 120 min. \QμJl of the resulting suspensions were then added dropwise to SDA+Y agar plate. After culturing at 28 ⁰ C for 12 hours, number of the germinated spores per one hundred spores was counted to obtain the spore germination ratio.

[28] It was found that the suspension of the spores and the blastospores that had been produced by entomopathogenic fungi had a very low level of germination ratio (i.e., only about 10% under the condition of 5O ⁰ C heat treatment for an hour), indicating that the spores and the blastospores have a very low thermal stability (see Figure 1).

[29] 1-2. Gbmparative test to determine the ambient temperature stability and the thermal stability of the entomopathogenic fungal spores prepared in different kinds of oils

[30] Beauveria sp.DBB2507strain was subjected to a solid culture for three weeks using

SDA+Y agar plate. Spores were harvested with a brush and then freeze-dried to produce spore powder that is used for a next test. Specifically, thus-obtained spore powder was added to several kinds of oils including soybean oil, olive oil, cotton seed oil, corn oil, mineral oil, and methyl oleate, etc. After mixing the spore powder with oils, the mixture was kept at ambient temperature (25 ⁰ C) or heat-treated (5O ⁰ C

incubator) for 120 min. Thereafter, lOβfl of the spore suspension was added dropwise to SDA+Y agar plate. After culturing at 28 ⁰ C for 12 hours, number of the germinated spores per one hundred spores was counted to obtain the spore germination ratio.

[31] As a result, it was found that the ambient temperature stability of the spores present in oils such as soybean oil, olive oil, cotton seed oil and corn oil is similar to that of the non-treated spores (i.e., almost 90% of the spores have germinated; see Figure 2). In addition, under 5O ⁰ C heat treatment condition, the spores present in olive oil showed spore germination ratio similar to that of the non-treated spores (see Figure X). Therefore, it was confirmed that when spores are stored in an olive oil, their thermal tolerance is improved compared to those stored in water.

[32] 1-3. Test for determining a thermal stability of entomopathogenic fungal spores with different period of heat treatment time

[33] Beauveria sp.DBB2507strain was subjected to a solid culture for three weeks using

SDA+Y agar plate. Spores were harvested with a brush and then freeze-dried to prepare spore powder that is used for a next test. Specifically, thus-obtained spore powder was added to olive oil. After mixing the powder with the oil, the mixture was subjected to a heat treatment (5O ⁰ C incubator) for 60 min and 120 min. Thereafter, \QμJl of the spore suspension was added dropwise to SDA+Y agar plate. After culturing at 28 ⁰ C for 12 hours, number of the germinated spores per one hundred spores was counted to obtain the spore germination ratio.

[34] As a result, as it is shown in Figure 3, it was found that not only the spores but also the blastospores of the entomopathogenic fungi are heat stable when they were kept in olive oil (see Figure 3).

[35]

[36] <Example 2> Method for enhancing the thermal stability of enzymes produced by entomopathogenic fungi.

[37] 2-1. Test for determining the thermal stability of an enzyme

[38] Beauveria sp. DBB2507 strain was subjected to a liquid culture for three days using

SDA+Y broth. After centrifuging the culture broth at 15,000 rpm for 5 min, the supernatant was prepared and kept in 5O ⁰ C incubator for 60 min and 120 min. Enzyme activity of chitinase was then measured. Specifically, the enzyme solution (100/i2), p- nitrophenyl β-D-N-acetylglucosaminide (PNG; 100 //#) as a substrate, and 0.1M citrate-phosphate buffer, pH 6.2 (3OO/i6) were mixed and then the enzyme reaction was carried out at 37 ⁰ C for one hour. Then the adsorbance of p-nitrophenol, the reaction product, was measured at 405 nm.

[39] As a result, it was found that the activity of chitinase existed as solution that has been produced by entomopathogenic fungi ( Beauveria sp.DBB2507) was reduced from 4.2 unit/hr at the beginning to 0.34 unit/hr after two hours of heat treatment at 5O ⁰ C (see Figure 4).

[40] 2-2. Test for determining the amount of enzyme harvested during centrifuge process

[41] Beauveria sp.DBB2507 strain was subjected to a liquid culture for three days in

SDA+ Y broth. After centrifuge at 15,000 rpm for 5 min, supernatant and pellet were separated. Resulting pellet comprising blastospores and mycelium was adjusted to the concentration before the centrifuge by using 0.1M citrate-phosphate buffer, pH 6.2. The chitinase activity was measured for the supernatant and the pellet suspension obtained above, respectively.

[42] It was also found that the harvest efficiency for chitinase by general centrifugal precipitation of the enzyme which is present in an aqueous solution is rather low (4.5%; 0.3/6.7, see Figure 5) although above process is an essential step.

[43] 2-3. Test for selecting adsorbents that can be used for improving the harvesting efficiency of enzyme

[44] Beauveria sp.DBB2507 strain was subjected to a liquid culture for three days in

SDA+ Y broth. After centrifuge at 15,000 rpm for 5 min, supernatant was obtained. To the resulting supernatant, several kinds of enzyme adsorbents including silica gel, cellulose, pyrophillite, skim milk, bentonite, celite, kaoline, and polyvinyl alcohol, etc. were added to the concentration of 0.5% (w/v). The mixture was ketp at ambient temperature for 30 min with shaking, and then centrifuged at 15,000 rpm for 5 min to obtain the pellet to which the enzyme was adsorbed. The chitinase activity was determined for the supernatant obtained above and the pellet to which the enzyme was adsorbed to find out the absorption ratio for each of the above-described adsorbents.

[45] In order to improve the harvesting efficiency of chitinase, mineral substances and polysaccharides were further used and the absorption ratio was determined therefor. As a result, it was found that among various candidate substances, the pellet treated with kaoline exhibited the highest chitinase activity of 11.1 unit/hr. At the same time, the lowest chitinase activity of 0.2 unit/hr was observed for the remaining solution. The absorption ratio for kaoline was about 90.2% (see Figure 6).

[46] 2-4. Test for determining thermal stability of the enzyme-adsorbed powder in oil

[47] The above-described pellet to which the enzyme has been adsorbed was subjected to a freeze-drying. The resulting enzyme- adsorbed powder was added to a vegetable oil, i.e., olive oil, and mixed. The mixture was then heat-treated in 5O ⁰ C incubator for 60

min and 120 min. By comparing with a sample which was prepared by adding said enzyme-adsorbed powder into water, the thermal stability of chitinase in oil was determined. [48] As a result, it was found that when chitinase was adsorbed first to kaoline and the resulting powder was dispersed in an olive oil, thermal stability thereof was about 8 to 10 times higher than the enzyme present in liquid form. In addition, compared to the bare enzyme present in powder form, it had a thermal stability that is about 1.25 times higher (see Figure 7).