# Patent

## Title
Paecilomyces genus microorganism and microbial insecticide for controlling the soil pests containing the same

## Abstract

The present invention relates to a paecilomyces genus microorganism and microbial insecticide for controlling the soil pests using the same, more particularly to a paecilomyces genus microorganism having insecticidal activity to soil pests and a microbial insecticide for controlling the soil pests containing the same. Since the microbial insecticide of the present invention shows excellent insecticidal activity to harmful soil pests, it can effectively be used as a environment-compatible insecticide to prevent the soil pests from farmland crops.

## Background
This is the U.S. National Stage of International Application No. PCT/KR02/02040, filed Nov. 2. 2002 (published in English under PCT Article 21(2)). which in turn claims the benefit of Korean patent application no. 2001/68127. filed Nov. 2. 2001.
FIELD OF THE INVENTION
The present invention relates to a novel paecilomyces genus microorganism and a microbial insecticide for controlling the soil pests using the same, more particularly to a paecilomyces genus microorganism having insecticidal activity to soil pests, a medium for mass-production of the microorganism and a microbial insecticide for controlling the soil pests containing the same.
BACKGROUND ART OF THE INVENTION
Insects have excellent ability to adapt to environment and have the widest species on earth (Wilson, O. and F. N. Peter., Biodiversity., 1989, Washington, D.C. Natl. Acad. Press). Some of those insects harm to various major crops. Chemical insecticides have been used to control such pests. But such chemical insecticides have killed not only pests but also useful insects and parasites living in pests because of their wide spectrum. In addition, target pests cannot be controlled anymore by having resistance against chemical insecticides owing to the repeated exposure on them. Further, chemical insecticides are harmful for human body.
Various plants, trees and grasses are growing widely in golf courses, so that the appearance of pests is unique and the number of pests adapting to such ecosystem is increasing year after year. As a kind of soil pests, Mimela splendems (gold bug) is a euryphagous pest harming plants (42 families, 186 species), especially plants of Rossaceae, Salicaceae, Fagaceae, Betulaceae and Aceraceae. Some adults of gold bugs harm leaves of crops and plants and larvae do roots of various crops and grasses, making them major target pest of golf clubs. Indirectly, adults of the gold bugs steal onto greens and away with discharges during the night, causing interruption of a course of a golf ball. (Lee et al, Korean Journal of Applied Entomology, 1997, 36, 2, 156-165; Lee, PhD Thesis, 2000). Particularly, the larvae of soil pests including gold bugs harming major crops and grasses are troublesome since they damage the roots of plants and grasses. The larvae of gold bugs developing and living in golf courses directly harm grass roots to death and indirectly provide themselves as feed for birds, causing digging up the grass. Therefore, they have a bad effect on the preservation of grass quality.
To control such soil insects like gold bugs, fenitrothion emulsion, chlorpyrifos-methyl emulsion and ethoprophos granules have been used. But those chemical pesticides have effects on only just-hatched larvae (the first larva stage). Thus, catching the right time is essential for controlling those insects. By the way, those chemical pesticides weaken the grass and cause overdose (Korea Patent Application #1999-15472).
Great efforts have been made to control the soil pests like gold bugs, Encarcia formosa, Eretmocerus eremicus, Plutella xylostella, Spodoptera litura and Nilaparvata lugens. But using the conventional chemical pesticides causes not only high expense but also such problems that the destruction of ecosystem, the under water contamination, the residual toxicity in agricultural products and the appearance of insects having resistance. Thus, it is urgently required to develop an environment-friendly controlling method for controlling soil pests in order to minimize such problems.
As one way of environment-friendly controlling methods for pests, insect pathogenic microorganisms are now being used, which is characterized by working selectively for target pests only without harming human, animals and plants.
For the development of a microorganism for controlling soil pests, it is very important to determine the components and factors of medium for mass-production. Again, it is essential to prepare inexpensive and available medium in order to supply microbial insecticides that are less expensive than conventional chemical insecticides. Though, it is still hard to prepare such medium for mass-production since the growth speed and time of a target microorganism as well as biological, chemical and physical factors for the production ought to be considered together.
Thus, the present inventors have searched insect pathogenic microorganisms for controlling soil pests harming major crops and finally accomplished this invention by confirming the fact that a fungus of Paecilomyces genus killed soil pests including the larvae of gold bugs.
SUMMARY OF THE INVENTION
The present invention relates to a paecilomyces genus microorganism and microbial insecticide for controlling the soil pests using the same, more particularly to a paecilomyces genus microorganism having insecticidal activity to soil pests, a medium for mass-production of the microorganism and a microbial insecticide for controlling the soil pests containing the same. Since the microbial insecticide of the present invention shows excellent insecticidal activity to harmful soil pests, it can effectively be used as an environment-compatible insecticide to prevent the soil pests from farmland crops.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a diagram showing the manufacturing process of microbial insecticide wettable powders comprising the Paecilomyces lilacinus HY-4 of the present invention for controlling soil pests,
FIG. 2 is a diagram showing the manufacturing process of microbial insecticide granules comprising the Paecilomyces lilacinus HY-4 of the present invention for controlling soil pests,
FIG. 3 is a graph showing the insecticidal effect of the Paecilomyces lilacinus HY-4 of the present invention measured by dipping method,
FIG. 4 is a set of photographs showing the larva of an Adoretus tenuimaculatus killed by the infection with the Paecilomyces lilacinus HY-4 of the present invention,
FIG. 5 is a graph showing the insecticidal effect of the Paecilomyces lilacinus HY-4 of the present invention measured by contact toxicity test.



DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a novel fungus of Paecilomyces genus that is useful for controlling soil pests.
The present invention also provides a medium for mass-production of a fungus of Paecilomyces genus.
The present invention further provides a microbial insecticide comprising the above fungus of Paecilomyces genus for controlling soil pests and a preparation method thereof.
Further features of the present invention will appear hereinafter.
The present invention provides a Paecilomyces genus microorganism having insectisidal activity to soil pests.
The present inventors separated a microorganism having insecticidal effect on soil pests from dead insects infected with insect pathogenic microorganisms and soil samples where soil pests were inhabited. The microorganism of the present invention was identified as a kind of Paecilomyces lilacinus. The microorganism of the present invention was named as “Paecilomyces lilacinus HY-4” and deposited at Gene Bank of Korea Research Institute of Biosciene and Biotehnology on Oct. 27, 1997 (Accession No: KCTC 0395BP)
The present inventors investigated the insecticidal effect of the Paecilomyces genus microorganism of the present invention with the dipping method. As a result, the microorganism killed more than 50% larvae from the 1st instar through the 3rd instar and more larvae were proved to be infected with the Paecilomyces genus microorganism of the present invention as time went, suggesting the microorganism had excellent insecticidal effect on soil pests (see FIG. 3 and FIG. 4).
The present invention also provides a medium for mass-production of the above Paecilomyces genus microorganism.
In order to mass-produce the Paecilomyces genus microorganism economically, the present invention provides a standard medium prepared by mixing wheat bran and water. The above medium can contain one or more components selected from a group consisting of rice bran, glucose, yeast extract, cottonseed, KNO3, phosphate and zeolite.
The major factor influencing largely on the culture of microorganism, especially in the case of solid culture, is moisture content, that is, the productivity of bacteria increases, the consuming speed of medium becomes faster and the number of produced live bacteria also increase as moisture content is high. Among the above additives, rice bran is used as a supplementary material for the increase of ventilation and the improvement of property of solid medium, glucose as an early growth factor, yeast extract as a source of nitrogen and microelements, cottonseed as a nitrogen source, KNO3 as an inorganic nitrogen source, phosphate as a buffer for pH and zeolite is used as an absorbing inorganic additive having sustained releasing property of metallic elements and phosphate.
The present invention further provides a microbial insecticide comprising a Paecilomyces genus microorganism for controlling soil pests and a preparation method thereof.
Microbial insecticides of the present invention containing a Paecilomyces genus microorganism for controlling soil pests can be prepared in the form of either wettable powders, granules or capsules.
The wettable powders of the above microbial insecticides can be obtained by pulverization after drying solid media inoculated with Paecilomyces genus microorganism and mixing surfactant and diluents/nutrients with it.
Surfactants used for preparing wettable powders of the above microbial insecticides can be selected from a group consisting of polycarboxylate (Hannong chemicals Inc.), sodium lingosulfonate, calcium lignosulfonate, sodium dialkyl sulfosuccinate, sodium alkyl sulfonate, polyoxy ethylene alkyl phenyl ether, sodium tripolyphosphate (Hannong chemicals Inc.), sodium alkyl aryl sulfonate, polyoxyethylene alkyl phenyl ether, polyoxyethylene aryl phosphoric ester, polyoxyethylene alkyl aryl ether, polyoxyethylene alkyl aryl polymer, polyoxyethylene alkyl aryl polymer special, polyoxyalkylone alkyl phenyl ether, polyoxyethylene nonyl phenyl ether, sodium sulfonate naphthalene formaldehyde, triton 100 and tween 80. Diluents/nutrients can be selected from a group consisting of soybean flour, rice, wheat, yellow earth, diatomaceous earth, dextrin, glucose and starch.
For the preparation of the microbial insecticide granules for controlling soil pests containing the Paecilomyces genus microorganism, dried solid medium inoculated with the Paecilomyces genus microorganism, crushed thereof, and then added surfactants, diluents/nutrients and disintegrators.
The microbial insecticide granules of the present invention are preferably prepared with the following weight ratios: Paecilomyces genus microorganism spores 30-83 weight parts, surfactants 2-30 weight parts, diluents 5-20 weight parts, disintegrators 10-40 weight parts.
One or more surfactants can be selected from a group consisting of poly carboxylate, sodium lingosulfonate, calcium lignosulfonate, sodium dialkyl sulfosuccinate, sodium alkyl sulfonate, polyoxyethylene alkyl phenyl ether, sodium tripolyphosphate, sodium alkyl aryl sulfonate, polyoxyethylene alkyl phenyl ether, polyoxyethylene alkyl aryl phosphoric ester, polyoxyethylene alkyl aryl ether, polyoxyethylene alkyl aryl polymer, polyoxyethylene alkyl aryl polymer special, polyoxyalkylone alkyl phenyl ether, polyoxyethylene nonyl phenyl ether, sodium sulfonate naphthalene formaldehyde, triton 100 and tween 80, and used for the preparation of the above microbial insecticide granules. One or more diluents/nutrients can be selected from a group consisting of soybean flours, rice, wheat, diatomaceous earth, dextrin, glucose and starch. One or more disintegrators can be selected from a group consisting of bentonite, talc, dialite, kaoline and calcium carbonate.
In addition, the microbial insecticide granules of the present invention can additionally include one or more additives selected from a group consisting of surface active agents, inactive carriers, preservatives, wetting agents, supply accelerants, attractants, encapsulating agents, binders, emulsifiers, dyes, UV protectors, buffers and fluids.
The preparation method for microbial insecticide wettable powders of the present invention comprises the following steps:

    
    
        1) Pulverizing after drying solid media inoculated with Paecilomyces lilacinus HY-4; and
        2) Adding surfactants and diluents to the pulverized powders of the above step 1) and then pulverizing thereof again (see FIG. 1).
    
    


And, the preparation method for microbial insecticide granules of the present invention comprises the following steps:

    
    
        1) Pulverizing after drying solid media inoculated with Paecilomyces lilacinus HY-4;
        2) Adding surfactants, adjuvant and diluents to the pulverized powders of the above step 1) and then kneading thereof with water; and
        3) Granulating the dough prepared in the above step 2) and then drying thereof (see FIG. 2).
    
    


The present inventors investigated the insecticidal effect of the microbial insecticides for controlling soil pests containing the Paecilomyces genus microorganism on the larvae of gold bugs. As a result, the microbial insecticides of the present invention could kill more than 50% of the larvae and the chance for the larvae to be infected with the Paecilomyces genus microorganism went higher as time passed (see FIG. 3-FIG. 5).
EXAMPLES
Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.
However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.
Example 1
Separation and Identification of Microorganisms
In order to separate microorganisms that are useful for controlling soil pests, the present inventors collected dead insects infected with insect pathogenic microorganisms and at the same time, picked insects from soil samples where soil pests are inhibiting as well. Particularly, the present inventors crushed soil pests dead by infection with insect pathogenic microorganisms and diluted with sterile water. Took 0.1 ml of suspension and smeared on microorganism test medium (Dermatophyte test medium; DTM medium) as presented in Table 1. Suspended 0.1 g of soil samples with 5 ml of sterile water and smeared on microorganism test medium as well. Culture thereof at 30° C. for 5 days and made a selection, which was inoculated on new DTM medium and further cultured under the same condition above.










TABLE 1







Ingredient
Amount (%)



















Glucose
2



Soytone
1



Phenol Red
0.02



Cycloheximide
0.05



Chloheximide
0.01



Gentamycine
0.01



Agar
1.5



Acetone (w/w)
2










As a result, the present inventors separated a microorganism showed excellent insecticidal effect on soil pests, and identified thereof as a kind of Paecilomyces lilacinus. The microorganism was named as “Paecilomyces lilacinus HY-4” and deposited at Gene Bank of Korea Research Institute of Biosciene and Biotehnology on Oct. 27, 1997 (Accession No: KCTC 0395BP).
Example 2
Preservation and Cultivation of the Separated Microorganism Paecilomyces lilacinus HY-4
In order to preserve and pre-culture the above separated Paecilomyces lilacinus HY-4 for the mass-culture thereof, the present inventors used ME medium represented in Table 2.










TABLE 2







Ingredient
Amount (%)



















Malt extract
2



Glucose
2



Peptone
0.1










Kept the microorganism in ME plate medium containing 2% agar. Cultured thereof at 26° C. for 15 days until spores were produced, after which added 3 ml of solution in which 0.05% tween 80 and distilled water were mixed to ME agar medium. Then collected spores using a scraper. Suspended the spores in 15% glycerol solution and distributed thereof to each container by 1 ml. Kept the containers at −70° C. refrigerator.
For the culture of the microorganism, autoclaved the ME medium at 121° C. for 20 minutes, inoculated the microorganism thereto and then shaking-cultured at 26° C. with 180 rpm for 4 days.
Example 3
Preparation of Solid Medium and Test of Culture Condition
The present inventors prepared solid medium for mass-production of Paecilomyces lilacinus HY-4 separated in the above Example 1 and investigated culture condition. Particularly, after mixing 330 g of wheat bran, 170 g of rice bran and 250 g of water together, put the mixture in a polypropylene bag (50×22×10 cm′) for autoclave to which 2 sheets of filter membrane (7×7 cm′, effective area diameter 3.5 cm) were attached for air-permeability. After sealing the bag, performed autoclave at 121° C for 30 minutes, resulting in the preparation of the solid medium for mass-production.
Meanwhile, inoculated 0.01-1% of pre-cultured liquid medium onto the above solid medium after the medium was completely cooled. Cultured thereof in a culture room for 3-4 weeks where the temperature was kept at 27° C., the radiation intensity was adjusted to 40 W×2×1.5 and the relative humidity was regulated to 40-70%.
Compared the spores collected from liquid medium cultured in a minimum medium with the spores collected from plate medium. As a result, it was proved that the production of inoculum in liquid medium was superior based on the investigation of culture speed, the generation of contaminated bacteria during culture and the maturing extent of spores after completing the culture. Cultured inoculum at 26° C. for 3 days using CYA medium represented in Table 3 and PDB medium shown in Table 4. Inoculated the above liquid-cultured inoculum solution into solid medium (500 g of dry weight) by 1 ml, 5 ml and 10 ml respectively.










TABLE 3







Ingredient
Amount (%)




















Czapek-
Bacto saccharose
3



Dox
Sodium nitrate (NaNO3)
0.3



broth
Dipotassium phosphate (K2HPO4)
0.1




Mangnessium sulfate (MgSO4)
0.05




Potassium chloride (KCl)
0.001




Ferrious sulfate (FeSO4)
0.05




Yeast extract
0.5




















TABLE 4







Ingredient
Amount (%)




















PDB
Potato (Infusion from)
2



(Potato
Bacto Dextrose
0.2



Dextrose



Broth)










Yeast extract
0.5










After smearing the Paecilomyces lilacinus HY-4 cultured in the above Example 2 onto CYA and PDA plate medium, cultured thereof at 26° C. or 7-10 days until colonies were formed. After then, took 2-3 colonies from each solid plate medium and put the colonies into 1 ml Erlenmeyer flask containing 200 ml of CAY medium represented in Table 3 or PDB liquid medium represented in Table 4. Shaking-cultured thereof at 26° C. with 180 rpm for 3 days.
After measuring the weight of each solid medium prepared by mixing wheat bran, rice bran and water at the ratio of 4:2:1, put thereof into 125 ml Erlenmeyer flask. Inoculated with the inoculum cultured in liquid medium for 3 days and then cultured thereof at 26° C., with relative humidity (RH) 70% for 6 weeks, during which observed the growth of the inoculum. In order to investigate the growth of bacteria, measured the number of live bacteria (cfu/g) and condria using hemacytometer-counting (spore number/g) method. Took samples from solid medium and measured their weight. Suspended thereof in 20 ml of 0.05% tween 80 solution for successive dilution. Observed by a microscope to count the number of condria. Smeared the diluted solution by 0.1 ml onto plate medium and cultured thereof at 26° C. for 2-4 days, after which investigated all the bacteria. In order to measure the density of microorganism, took samples and counted the number of condria using hemacytometer. Measured specific growth rate by comparing the increased density of condria due to the cell growth with the earlier one, that is, specific growth rate was calculated by subtracting the early condria density from the later condria density after complete growth and then converting the value into log and finally dividing the obtained value by the time obtained by subtracting the early time from the later time.
As a result, the specific growth rate of Paecilomyces lilacinus HY-4 cultured in CAY medium was proved to be 0.04 hr−1 (inoculated amount was 1 ml), 0.034 hr−1 (inoculate amount was 5 ml) and 0.033 hr−1 (inoculate amount was 10 ml) on the first week after inoculation, suggesting that specific growth rate was not much affected by the amount of inoculum. Thus, the present inventors confirmed that inoculated amount did not affect the growth of Paecilomyces lilacinus HY-4 when CY medium was used as a medium for inoculum (Table 5).










TABLE 5










Number of conidia



Inoc-
(number/g−solid medium)














ulated 

1st
2nd
3rd
6th


Medium
amount
0
week
week
week
week





CY
1 ml
1.4 × 106
1.5 × 109
6.2 × 109
7.2 × 109
3.9 ×








109



5 ml
6.9 × 106
2.2 × 109
6.9 × 109
4.7 × 109
2.6 ×








109



10 ml 
1.4 × 107
3.7 × 109
2.9 × 109
4.2 × 109
3.9 ×








109


PDB
1 ml
2.2 × 105
1.3 × 109
5.2 × 109
4.9 × 109
7.2 ×








109



5 ml
1.1 × 106
2.0 × 109
4.3 × 109
5.7 × 109
4.4 ×








109



10 ml 
2.2 × 106
1.9 × 109
6.2 × 109
4.6 × 109
3.2 ×








109









There was not much difference in using PD medium as a medium for inoculum (0.051, 0.044, 0.040 hr−1, respectively). The number of condria was over 109/unit weight of medium and was constantly maintained during 6-week culturing.
Considering all the above results, the present inventors confirmed that more than 109 spores/unit weight of medium could be produced by the solid culture of Paecilomyces lilacinus HY-4 on wheat bran medium for 2 weeks.
Example 4
Measurement of the Number of Live Bacteria in Solid Culture
Since the number of live bacteria having insecticidal activity was more important for microbial insecticide, the present inventors measured the density of live bacteria after smearing samples onto CY agar plate medium. Particularly, diluted bacteria with sterilized 0.05% tween 80 solution (10−3-10−7) and inoculated 100 μl thereof onto antibiotic medium containing antibiotics or general medium. From the next day, counted the number of live bacteria, during which the temperature was maintained at 26° C. Antibiotic medium was prepared by adding oxygall (1.5% /l), a growth inhibitor, to Czapck-Dox agar medium (DIFCO) and additionally adding antibiotics and other growth inhibitors thereto as shown in Table 6.










TABLE 6







Ingredient
Amount (mg/ml)









Ampicilin
50



Nakidix acid
10



Rose Bengal
50










Confirmed contamination extent in the general medium and measured the numbers of contaminated bacteria and Paecilomyces lilacinus HY-4 in the microorganism test medium containing antibiotics.
As a result, it was confirmed 2-3 days later that Paecilomyces lilacinus HY-4 was growing slowly, compared with contaminated bacteria. On the second week after starting the culture, obtained the maximum cell density, which was 1012 colony forming units (CFU)/unit g of solid medium, that is, massive hyphae were formed, comparing to the result of Example 3 (Table 7).










TABLE 7









Inoc-
Number of condria (number/g−solid medium)














ulated

1st
2nd
3rd
6th


Medium
amount
0
week
week
week
week





CY
1 ml
2.8 × 106
2.0 × 1010
8.1 × 1012
1.0 × 1010
3.9 ×








105



5 ml
1.4 × 107
1.7 × 1010
4.8 × 1012
8.7 × 109
8.2 ×








105



10 ml 
2.8 × 107
4.4 × 1010
7.0 × 1012
6.5 × 109
2.7 ×








106


PDB
1 ml
1.3 × 105
1.1 × 1010
6.0 × 1012
1.3 × 1011
1.8 ×








106



5 ml
6.7 × 105
1.4 × 1010
1.7 × 1012
4.3 × 1011
5.5 ×








105



10 ml 
1.3 × 106
1.4 × 1010
6.1 × 1011
4.4 × 1010
9.6 ×








105









The number of live bacteria was decreased three weeks later and down to the beginning level 6 weeks later. Thus, it was confirmed that the optimum time was on the second week after starting solid culture. Especially two weeks later, most bacteria turned into spores showing high maturity, so that they were expected to have high activity as an insect pathogenic microorganism.
The specific growth rate of the first week was 0.052 hr−1 (inoculated amount was 1 ml), 0.043 hr−1 (inoculate amount was 5 ml ) and 0.042 hr−1 (inoculate amount was 10 ml) respectively as Paecilomyces lilacinus HY-4 was inoculated in CAY medium for inoculum, and 0.067 hr−1 (inoculated amount was 1 ml), 0.059 hr−1 (inoculate amount was 5 ml) and 0.055 hr−1 (inoculate amount was 10 ml) respectively as Paecilomyces lilacinus HY-4 was inoculated in PD medium for inoculum. Resultingly, such conditions as medium for inoculum and the amount of inoculation could hardly affect on solid culture of Paecilomyces lilacinus HY-4, by which basic conditions of solid culture for the mass-culture of Paecilomyces lilacinus HY-4 were determined.
Example 5
Investigation of Conditions for Mass-Solid Culture of Paecilomyces lilacinus HY-4 by Analysis of Culturing Factors
The present inventors investigated the culture conditions for mass-solid culture of Paecilomyces lilacinus HY-4. Precisely, prepared basic medium by mixing wheat bran and water at the ratio of 2:1 and regarded each medium condition as a culture factor to search out advantageous factors for yield of mass-solid culture. Used rice bran as a supplementary element for the improvement of ventilation and property of solid medium. Used glucose as an early growth factor, yeast extract as a nitrogen source and a mineral source, cottonseed as a nitrogen source, KNO3 as an inorganic nitrogen source, phosphate as a buffer for pH and used zeolite as an absorbing inorganic additive having sustained releasing property of metallic elements and phosphate. Investigated moisture content as another culture factor. Marked the investigated conditions by t-value and % according to Plackett-Burman method, one of factorial methods. The effect of each variable on bacteria production was determined by calculating the difference between test results done with a variable of higher value and a variable of lower value. Experimental errors were calculated with dummy variables, which were represented by the variation extent presumed in the range of an experimental section. Effective variation (Jeff) and standard deviation (S.E.) of a variable were calculated as Jeff=(Ed)2/n=(S.E.)2. Ed meant each dummy effect and n represented the number of dummy variables. Ex of each variable effect was calculated by Student's t test (t=Ex/S.E.). The basic plan of the test is shown in Table 8.


















TABLE 8





Fermenting
Rice





Yeast

Contton




factor
bran
Dummy
Zeolite
Moisture
Glucose
Dummy
extract
Dummy
seed
KNO3
K2HPO4


























Higher
2 g

1 g
45.8
1%

1%

1%
0.5%
0.1%


value


Lower
0

0
38.9
0  

0  

0  
0
0


value


Test
+
+
−
+
+
+
−
−
−
+
−


group 1


Test
+
−
+
+
+
−
−
−
+
−
+


group 2


Test
−
+
+
+
−
−
−
+
−
+
+


group 3


Test
+
+
+
−
−
−
+
−
+
+
−


group 4


Test
+
+
−
−
−
+
−
+
+
−
+


group 5


Test
+
−
−
−
+
−
+
+
−
+
+


group 6


Test
−
−
−
+
−
+
+
−
+
+
+


group 7


Test
−
−
+
−
+
+
−
+
+
+
−


group 8


Test
−
+
−
+
+
−
+
+
+
−
−


group 9


Test
+
−
+
+
−
+
+
+
−
−
−


group


10


Test
−
+
+
−
+
+
+
−
−
−
+


group


11


Test
−
−
−
−
−
−
−
−
−
−
−


group


12









How much the each factor had meaning influence on the production of Paecilomyces lilacinus HY-4 was summarized in Table 9.









TABLE 9









Response, t-value/valency(%)











Additives
Cell
Medium
Number















Max-
Min-
amount
con-
of live
Medium


Factors
imum
imum
(NAcGa)
sumption
bacteria
pH





Rice
2.0 g
0
−0.78
−1.29
−0.35
−1.71  


bran





(−90%) 


Zeolite
1.0 g
0
  4.15
−0.23
−2.89
5.96





(97.5%)   

(−97.5%)
(99.5%)


Moisture
38.9%
5.8%
42.47
1.52
1.12
4.90





(99.95%)  


(99%)  


Glucose
1.0%
0
−4.70
−1.58
−0.01
1.38





(−99.5%) 


Yeast
1.0%
0
0.51
0.50
−0.50
0.52


extract


Cotton-
1.0%
0
−5.65
−0.78
  1.27
0.52


seed


(−99%)


KNO3
0.5%
0
−14.63
−1.63
−1.55
3.13





(−99.95%)


(95%)  


K2HPO4
0.1%
0
−6.76
−0.29
−0.51
−1.86  





(−99.5%) 


(−90%) 









The moisture content of a medium was proved to be the most important factor in the event. Precisely, the factor got all “+” in every test fields, suggesting that high moisture content prompted the production of bacteria, accelerated the consuming speed of medium and produced more amount of live bacteria. At that time, the statistical valency was 99.95%
The typical (−) factor was proved to be phosphate. Adding phosphate caused rather interruption in the growth of bacteria. Although rice bran and glucose were confirmed to be (+) factors, starch particles contained in rice bran or glucose was not preferable for Paecilomyces lilacinus HY-4 medium. Especially, rice bran accelerated the dryness of medium owing to its too good ventilating property, resulting in the fast drop of moisture content of medium. Thus adding rice bran was beneficial for the maturation of spores but not for the production of bacteria in early stage.
Zeolite was proved to be a culture factor promoting the production of bacteria. Zeolite was observed to moderate the growth inhibition effect caused by phosphate by absorbing and releasing out phosphate. Therefore, zeolite, an absorbent keeping the density of medium low by absorbing culture factors, was proved to be superior to glucose or nitrate that were the factors that bacteria could use easily and fast.
Example 6
Preparation of Microbial Insecticide Containing Paecilomyces lilacinus HY-4 for Controlling Soil Pest
<6-1> Preparation of Microbial Insecticide Wettable Powders
In order to provide the raw powders of Paecilomyces lilacinus HY-4 stably, the present inventors prepared wettable powders (WP) that were hydrated when being diluted with water. Particularly, heated the solid medium and then dried thereof. Added surfactants and diluents thereto, and then pulverized thereof. More precisely, dried raw powders in a drier at 70° C. for about 2 hours and then pulverized thereof. Next, mixed the raw powders, surfactants and diluents together well. Pulverized the mixture with a grinder (Jet-O-mill, Aljet) and then tested hydrating capacity for the preparation of microbial insecticide wettable powders (FIG. 1).
In order to confirm the stability of the microbial insecticides for controlling soil pests prepared above, performed preservation test on the wettable powders of the present invention. As a result, the microbial insecticides showed about 38% recovery rate (2.10×107 CFU/g).
<6-2> Preparation of Microbial Insecticide Granules 1-13
The present inventors prepared granules (GR) that could be used as they were in order to provide Paecilomyces lilacinus HY-4 raw-powders stably. Particularly, dried the raw-powders in a dry oven at 70° C. for 2 hours and then pulverized thereof with a grinder (Jet-O-mill, Aljet). Mixed the crushed powders, surfactants, diluents/nutrients and disintegrators together according to the ratios represented in Table 10. Added water (35% of the total weight of the mixture) to the mixture and kneaded thereof. Upon finishing kneading, granulated thereof (diameter: 1 mm, length: 1-5 mm) using Basket type extruder (Fuji powder Co) and then dried in a dry oven at 70° C. Removed dusts by sieving the dried materials with a 16-30 mesh sieve, resulting in the preparation of microbial insecticide granules (FIG. 2).








TABLE 10







Preparation
Compositions











of
Pulverized

Diluents/



granules
powders
Surfactants
Nutrients
Disintegrators


















<6-2-1>
HY-4
50%
Sodiumtripolyphosphate
15%


Talc
20%


Preparation 1
spores

Polycarboxylate
15%


<6-2-2>
HY-4
50%
Polycarboxylate
3%
Glucose
10%
Bentonite
20%


Preparation 2
spores

Sodiumlignosulfonate
3%


Calciumcarbonate
13%





Sodium dialkyl
1%





sulfosuccinate


<6-2-3>
HY-4
50%
Sodium alkyl aryl
5%
Starch
10%
Talc
30%


Preparation 3
spores

sulfonate



Dialite
3%





Polyoxyethylene
1%





alkyl phenyl ether





Sodium dialkyl
1%





sulfosuccinate


<6-2-4>
HY-4
50%
Polycarboxylate
3%
Starch
10%
Bentonite
20%


Preparation 4
spores

Polyoxyethylene
1%


Talc
13%





alkyl phenyl ether





Calcium
3%





lignosulfonate


<6-2-5>
HY-4
50%
Polyoxyethylene
5%
Glucose
10%
Bentonite
20%


Preparation 5
spores

alkyl aryl



Talc
12%





phosphoricester





Polyoxyethylene
3%





alkyl aryl ether





and polyoxyethylene





alkyl aryl polymer


<6-2-6>
HY-4
50%
Polyoxyethylene
1%
Starch
10%
Bentonite
20%


Preparation 6
spores

alkyl phenyl ether



Talc
16%





Sodiumtripolyphosphate
3%


<6-2-7>
HY-4
50%
Polycarboxylate
3%
Starch
10%
Bentonite
20%


Preparation 7
spores

Polyoxyethylene
5%


Kaolin
12%





alkyl aryl ether





and polyoxyethylene





alkyl aryl polymer


<6-2-8>
HY-4
50%
Polycarboxylate
3%
Starch
10%
Dialite
20%


Preparation 8
spores

Sodium alkyl aryl
5%


Calciumcarbonate
11%





sulfonate





Sodium dialkyl
1%





sulfosuccinate


<6-2-9>
HY-4
50%
Sodiumtripolyphosphate
3%
Starch
10%
Bentonite
10%


Preparation 9
spores

Polyoxyethylene
5%
Dextrin
10%
Dialite
12%





alkyl aryl





phosphoricester


<6-2-10>
HY-4
50%
Polyoxyethylene
5%
Dextrin
5%
Bentonite
20%


Preparation
spores

alkyl aryl polymer



Talc
12%


10


special





Calciumlignosulfonate
5%





Polycarboxylate
3%


<6-2-11>
HY-4
50%
Polyoxyalkylone
3%
Dextrin
5%
Calciumcarbonate
28%


Preparation
spores

alkyl phenylether

Starch
10%


11


Sodiumtripolyphosphate
3%





Sodium dialkyl
1%





sulfosuccinate


<6-2-12>
HY-4
50%
Sodium
3%
Glucose
10%
Kaolin
20%


Preparation
spores

lignosulfonate



Talc
14%


12


Polyoxyethylene
3%





alkyl aryl ether





polymer


<6-2-13>
HY-4
50%
Polyoxyethylene
3%
Dextrin
5%
Kaolin
20%


Preparation
spores

nonyl phenyl ether



Dialite
19%


13


Sodium sulfonate
2%





naphthalene





formaldehyde





Sodium dialkyl
1%





sulfosuccinate









In order to confirm the stability of the microbial insecticides for controlling soil pests prepared above, performed preservation test on the granules of the present invention. As a result, the microbial insecticides showed about 52% recovery rate (3.40×107 CFU/g).
<6-3> Preparation of Formulations for Capsulation of Microbial Insecticide
The present inventors prepared formulations for capsulation of microbial insecticide having the same effect as the microbial insecticide granules for controlling soil pests prepared in the above Example 6. Particularly, mixed 11% soybean flour, 6% mung beans, 4% rice flour, 4% potato starch, 4% rye flour and 3% yellow earth together and autoclaved thereof at 121° C. for 20 minutes. Before completely cooling down, mixed thereof well again and then cool it down in cold water. Mixed thoroughly over 1 minute using a stirrer (d=5 cm, over 200 rpm). While keeping on stirring, added additives and Paecilomyces lilacinus HY-4 (Accession No: KCTC 0395BP) spores and mixed well again, resulting in the preparation of formulations for capsulation of microbial insecticide for controlling soil pests.
Example 7
Security of Test Insect
The present inventors secured test insects to confirm the insecticidal effect of Paecilomyces lilacinus HY-4 on soil pests. Particularly used Adoretus tenuinaculatus among gold bugs that caused trouble in golf courses and parks, a kind of soil pests, as a test insect. The imagoes (adult insects) were collected in Yusung country club, Taejeon, Korea and bred through generations using a chestnut tree as a host plant. Tests were done within 2-4 generations. The larvae were bred with artificial feeds (sawdust:bed soil:water=5:3:2). Both imagoes and larvae of the test insects were bred in cages (30 cm×30 cm×50 cm) in which temperature was maintained at 25±2° C., and relative humidity was kept 60-70%.
Example 8
Confirmation of the Insecticidal Effect by the Dipping Method
The present inventors confirmed the insecticidal effect of Paecilomyces lilacinus HY-4 of the present invention on gold bugs by dipping method. Particularly, shaking cultured Paecilomyces lilacinus HY-4 on ME medium for 3 days (26° C., 180 rpm). Measured the density of spores with hemacytometer and adjusted the density to 1×108 spores/ml using distilled water containing 0.05% tween-80. After dipping 10 larvae each from 1st instar, 2nd instar and 3rd instar of Adoretus tenuinaculatus in spore suspension for 10 seconds, transferred them to artificial feeds (sawdust:bed soil:water=5:3:2) and raised them at 26-28° C. in dark condition. While keeping required humidity for 15 days, investigated pathogenesis. At that time, regarded insects on which hyphae of fungus were generated as dead insects and insects grown to next instar as live insects. Counted both numbers and presented them with percentage. All experiments were performed 5 times. Used distilled water containing 0.05% tween-80 for a control group.
As a result, Paecilomyces lilacinus HY-4 of the present invention showed excellent insecticidal effect (52-56%) while control group showed just 13-26% insecticidal effect (FIG. 3). In addition, more larvae of Adoretus tenuinaculatus were confirmed to be infected with Paecilomyces lilacinus HY-4 as time went (FIG. 4).
Example 9
Confirmation of Insecticidal Effect by the Contact Toxicity Test
The present inventors confirmed the insecticidal effect of Paecilomyces lilacinus HY-4 of the present invention on gold bugs by contact toxicity test. Particularly, shaking cultured Paecilomyces lilacinus HY-4 on ME medium for 3 days (26° C., 180 rpm). Put 750 g of solid medium (wheat bran:rice bran:water=4:2:3) into a polypropylen bag (50×22×10 cm′) having 2 sheets of filter membranes (7×7 cm′, effective area diameter 3.5 cm) in it for air permeability and then sealed, which was autoclaved at 121° C. for 30 minutes. Inoculated 9-10 ml of the culture fluid on the solid medium prepared above and then cultured thereof in a culture room for 3-4 weeks in which temperature was maintained at 27° C., the radiation intensity was kept 40 W×2×1.5 and the relative humidity was kept 40-70%. Measured the density of spores with hemacytometer. After crushing the solid medium, adjusted the density to 1×108 spores/ml and then distributed thereof over humidified artificial feeds (sawdust:bed soil:water=5:3:2). Transferred 10 larvae each from 1st instar, 2nd instar and 3rd instar of Adoretus tenuinaculatus into the above artificial feeds and raised them at 26-28° C. in dark condition. While keeping required humidity for 15 days, investigated pathogenesis. At that time, regarded insects on which hyphae of fungus were generated as dead insects and insects grown to next instar as live insects. Counted those numbers and presented them with percentage. All experiments were performed 5 times. Used distilled water containing 0.05% tween-80 for a control group.
As a result, Paecilomyces lilacinus HY-4 of the present invention showed excellent insecticidal effect (53-60%) while control group showed just 20-27% insecticidal effect (FIG. 5).
INDUSTRIAL APPLICABILITY
As described hereinbefore, the microbial insecticide containing a Paecilomyces genus microorganism of the present invention having insecticidal activity to soil pests was proved to have excellent insecticidal effect on soil pests including the larvae of gold bugs, a kind of soil pests harming agricultural products. Therefore, it can be effectively used as an environment-compatible insecticide to prevent the soil pests from farmland crops.

## Claims

1. An isolated Paecilomyces lilacinus HY-4 fungus having insecticidal activity to soil pests, deposited at Gene Bank of Korea Research Institute of Bioscience and Biotechnology under Accession No: KCTC 0395BP.

2. A microbial insecticide for controlling soil pests containing the isolated Paecilomyces lilacinus HY-4 fungus of claim 1.

3. The microbial insecticide of claim 2, wherein the microbial insecticide is prepared in the form of wettable powders, granules or capsules.

4. The microbial insecticide of claim 3, wherein the wettable powders are prepared by:
pulverizing dry solid media inoculated with a Paecilomyces lilacinus fungus designated HY-4 deposited at Gene Bank of Korea Research Institute of Bioscience and Biotechnology under Accession No: KCTC 0395BP, thereby generating a pulverized powder; and
adding surfactants and diluents/nutrients to the pulverized powder.

5. The microbial insecticide of claim 3, wherein the granules are prepared by pulverizing dry solid media inoculated with a Paecilomyces lilacinus fungus designated HY-4 deposited at Gene Bank of Korea Research Institute of Bioscience and Biotechnology under Accession No: KCTC 0395BP, thereby generating a pulverized powder; and
adding surfactants, diluents/nutrients and disintegrators to the pulverized powder.

6. The insecticide as set forth in claim 5, wherein the granules comprise by weight, 30-83 parts pulverized powders of the microorganism spores, 2-30 parts surfactants, 5-20 parts diluents and 10-40 parts disintegrators.

7. The insecticide as set forth in claim 6, wherein the granules additionally contain one or more components selected from a group consisting of surface active agents, inactive carriers, preservatives, wetting agents, attractants, encapsulating agents, binders, emulsifiers, dyes, UV protectors, buffers and fluids.

8. The microbial insecticide of claim 4, wherein the insecticide contains one or more surfactants selected from a group consisting of polycarboxylate, sodium lignosulfonate, calcium lignosulfonate, sodium dialkyl sulfosuccinate, sodium alkyl sulfonate, polyoxyethylene alkyl phenyl ether, sodium tripolyphosphate, sodium alkyl aryl sulfonate, polyoxyethylene alkyl phenyl ether, polyoxyethylene alkyl aryl phosphoric ester, polyoxyethylene alkyl aryl ether, polyoxyethylene alkyl aryl polymer, polyoxyethylene alkyl aryl polymer special, polyoxyalkylene alkyl phenyl ether, polyoxyethylene nonyl phenyl ether, sodium sulfonate naphthalene formaldehyde, triton 100 and tween 80.

9. The microbial insecticide of claim 4, wherein the insecticide contains one or more diluents/nutrients selected from a group consisting of soybean flour, rice, wheat, yellow earth, diatomaceous earth, dextrin, glucose and starch.

10. The insecticide as set forth in claim 5, wherein the insecticide contains one or more disintegrators selected from a group consisting of bentonite, talc, dialite, kaolin and calcium carbonate.

11. The microbial insecticide of claim 5, wherein the insecticide contains one or more surfactants selected from a group consisting of polycarboxylate, sodium lignosulfonate, calcium lignosulfonate, sodium dialkyl sulfosuccinate, sodium alkyl sulfonate, polyoxyethylene alkyl phenyl ether, sodium tripolyphosphate, sodium alkyl aryl sulfonate, polyoxyethylene alkyl phenyl ether, polyoxyethylene alkyl aryl phosphoric ester, polyoxyethylene alkyl aryl ether, polyoxyethylene alkyl aryl polymer, polyoxyethylene alkyl aryl polymer special, polyoxyalkylene alkyl phenyl ether, polyoxyethylene nonyl phenyl ether, sodium sulfonate naphthalene formaldehyde, triton 100 and tween 80.

12. The microbial insecticide of claim 6, wherein the insecticide contains one or more surfactants selected from a group consisting of polycarboxylate, sodium lignosulfonate, calcium lignosulfonate, sodium dialkyl sulfosuccinate, sodium alkyl sulfonate, polyoxyethylene alkyl phenyl ether, sodium tripolyphosphate, sodium alkyl aryl sulfonate, polyoxyethylene alkyl phenyl ether, polyoxyethylene alkyl aryl phosphoric ester, polyoxyethylene alkyl aryl ether, polyoxyethylene alkyl aryl polymer, polyoxyethylene alkyl aryl polymer special, polyoxyalkylene alkyl phenyl ether, polyoxyethylene nonyl phenyl ether, sodium sulfonate naphthalene formaldehyde, triton 100 and tween 80.

13. The insecticide of claim 5, wherein the insecticide contains one or more diluents/nutrients selected from a group consisting of soybean flour, rice, wheat, yellow earth, diatomaceous earth, dextrin, glucose and starch.