Biopesticide를 위한 Verticillium lecanii CS-625 의 대량생산과 제제화

이진영, 김정준2, 윤철식3, 유재기2, 김승욱1*

고려대학교 화공생명공학과1, 농촌진흥청 농업과학기술원2, 고려대학교 공학기술연구소3,

kimsw@korea.ac.kr

The Mass Production and Formulation of Verticillium lecanii CS-625 for Biopesticide

Jinyoung Lee, Jungjun Kim2, Cheol-Sik Yoon3, Jai-Ki Yoo2, Seung Wook Kim1

Department of Chemical and Biological Engineering, Korea University1,

National Institute of Agriculture Science and Technology, Rural Development Administration2,

Institute of Engineering Technology, Korea University3

kimsw@korea.ac.kr

Introduction

Chemical pesticides have been harmful of environmental pollutants. They can threaten soil

organisms and ecosystem health via accumulation in food chains. Therefore, instead of such

pesticides, Verticillium lecanii CS-625 as an entomopathogenic fungus has been received great

attention in the biological control of insects and pests and could be useful with the mixture

of some insecticides and fungicides in integrated control programmes.

In this study, the mass production of the active spores of V. lecanii was examined on

liquid culture in a bioreactor and the formulation of these spores with various UV protectants

and wetting agents was investigated.

There are several important factors (pH, agitation speed, temperature, and supply of air etc.)

in order to increase productivity[1].

As the spores become dry, the humidity conditions at the plant surface are probably the

most important factor influencing the germination, growth and survival of V. lecanii[2]. UV

radiation effects on the colony germination. So it is necessary that the suitable wetting agent

and UV protectant should be selected for formualtion.

Experimental

Verticillium lecanii CS-625 was used. In the seed culture, the colony which was cultured

on PDA(Patato Dextrose Agar) substrate for 7 days was inoculated into PDB(Patato Dextrose

Broth) liquid medium and incubated in an incubator at 25℃ and 150rpm for 2 days. In the

main culture, the colony which was cultured in the seed culture was inoculated into 1.63% of

soybeen meal and 4.68% of sugar and was cultured in bioreactor at various experimental

conditions.

In the investigation of formulation of V. lecanii CS-625, five Montmorillonites(Cloisite-6A,

Cloisite-25A, Cloisite-30A, SCPX-1374, SCPX-1483, Southern Clay Products Inc.) as UV

protectants, wetting agents(Dongnam Chemical Ind, Ltd.) and spores were mixed in various

ratios. The colony germination was tested with a microscope and survival rate tested with UV

box (UVC).

Results and Discussion

The effect of the agitation speed(250, 300, 350, 400 rpm) was examined in bioreactor. the

result showed that operating conditions of 350 rpm in bioreactor gave significantly higher

spore production of 2.0 X 109 spores/ml at 120 hr (Fig. 1.). Batch type of culture without

pH control gave better productivity of spores than fedbatch type with pH control. The air

flow rate of 1.0 vvm showed better productivity of 1.7×109 spores/ml than  0.5 vvm

(0.78×109 spores/ml) at 120hr (Fig. 2.).

Fig. 1. The effect of agitation speed on the production of V. lecanii CS-625

Fig. 2. The effect of agitation speed on the production of V. lecanii CS-625

The influence of concentration of wetting agents and UV protectants to the colony

germination was tested. The presence of 1.0% of Cloisite 25A and EM-APW #2 gave the

highest results(96.2%), respectively. UV-radiation experiments with montmorillonites

(Cloisite-6A, Cloisite-25A, Cloisite-30A, SCPX-1374 and SCPX-1483) including 1.0% of

Fig. 3. The colony germination test            Fig. 4. the colony survival rate test

EM-APW#2 were performed. With SCPX-1374 and EM-APW #2, about 69.7% of the survival

rate of colony was obtained, which was significantly higher than any other montmorillonite

(Figs. 3, 4.).

Conclusion

In the investigation of mass production, operating conditions of 350 rpm, 1.0 vvm, and

batch type culture in bioreactor gave significantly higher spore production (2×109 spore/ml).

In formulation, the presence of 1.0% of SCPX-1374 (80%) and EM-APW #2 significantly

gave the highest results (96%) among others tested.

References

1. Feng, K.C, Liu, B.L. and Tzeng, Y.M. Verticillium lecanii spore production in solid-state

and liquid-state fermentations. Bioprocess Eng. 23, 25-29(2000)

2. Verhaar, M. A., Hijwegen T. and Zadoks J. C. Selection of Verticillium lecanii             isolates with high biocontrol capacity on cucumber powdery mildew by means of            components analysis at different humidity regimes Biocontrol Sci. Technol. 8, 465-477(1998)

Acknowledgement

The author is grateful to RDA(Rural Development Administration).