Screening And Identification Of Entomogenous Bacteria For Biocontrol Of Colorado Potato Beetle(Leptinotarsa Decemlineata)

Colorado potato beetle （CPB）, Leptinotarsa decemlineata （Say）, is one of the most destructive pests in the world. It is also an important invasive and quarantine species in China, which has distributed widely in northern of Xinjiang Autonomous Region of China. Several biopesticide products using Bacillus thuringiensis and Saccharopolyspora spinosa had been developed for application in biocontrol of the pest, but there were very few related reports in China. The objectives of the present work were to isolate entomogenous bacteria from diseased samples of CPB and to screen and identify the highly pathogenic isolates which were biological control agents （BCAs） and potentially available in CPB biocontrol in potato production.1. Isolation of pathogenic bacteria from diseased CPB and determination of the degrading enzyme activity:A total of126entomogenous bacteria isolates were obtained from diseased CPB samples collected from Qitai, Yiling and Fukang in Xinjiang using conventional tissue culture methods in lab. Skim milk and chitin were used to detect the activities of degrading enzyme of isolates. There were20isolates which had chitinase activity,26isolates had protease activity and10isolates had both of activities, respectively. Isolates with whichever degrading enzyme activity were potential BCAs which were Bacillus through preliminary morphological identification.2. Pathogenicity tests on silkworms:2nd-instar larvae of silkworm （Bombyx mori） were inoculated with cell suspensions （2.0x10cfu/mL） of the36active isolates. Results showed different pathogenicities among21isolates to silkworm:8of21isolates exhibited higher pathogenicities than the others and the death of insects was first observed only in1-2d. The cumulative mortality rate of silkworms after7d （LR7d） was over50%and the half-lethal time （LT5o） was less than7.4d. Isolates of CPB008, CPB012and CPB016had the strongest pathogenicities with LR7d of over70%and LT50of3.2d,3.3d and3.6d, respectively. The results are useful in determining the pathogenicity to CPB of these isolates in CPB epidemic area.3. Pathogenicity tests on CPB:CPBs （1st,2nd,3rd,4th-instar larvae and adults） were inoculated with cell suspensions of21pathogenic isolates at a concentration of2.0×109cfu/mL. All of these isolates had no pathogenicity to3rd-intar,4th-instar CPB larvae and adults.11isolates showed different pathogenicity to1st-and2nd-instar CPB larvae.6of11isolates exhibited higher pathogenicity to lst-instar larvae and the death of insects was first observed only in1-2d, with LR7d of over70%, LT50of3.8-6.8d and the half-lethal concentration （LC50） of0.088-2.555×109cfu/mL. CPB008, CPB012and CPB016showed the strongest pathogenicity, with LR7d of over70%, LT50of3.8d,4.1d,4.3d and LC50of0.088,0.383,0.316×109cfu/mL, respectively. The pathogenicity of isolates to2nd-instar CPB larvae was lower than that to1st-instar larvae, with LR7d of21.0-62.6%and LT50of4.1-10.3d. But LR7d of CPB008, CPB012and CPB016were all over50%. Dead CPB larvae caused by entomogenous bacteria became black, rot and some of them secreted brown liquid. All of these isolates were entomopathogenic bacteria of CPB confirmed by Koch’s postulates. The results indicated that the isolates CPB008, CPB012, CPB016, CPB072, CPB108and CPB111were important pathogens of CPB with strong virulence to CPB and they would have great applications in biocontrol of CPB.4. Field tests of6highly pathogenic isolates and screening BCAs:1st-instar CPB larvae were inoculated with the6highly pathogenic isolates at a concentration of2.0×109cfu/mL under field conditions. Three isolates （CPB008, CPB012and CPB016） had obviously significant pathogenicity to lst-instar CPB larvae in the field, with LR1Od of49.9%,43.1%and41.0%, respectively. The other three isolates showed lower pathogenicity, with LR10d of37.5%（CPB108）,28.4%（CPB072） and21.1%（CPB111）, respectively. Control efficacies of all isolates except CPB111were significantly higher than that of regular Bt WP preparations （control, LR10d of24.9%）. It can be concluded that CPB008, CPB012and CPB016may be thought of as BCAs and be used as candidates to develop bacterial biopesticides against CPB.5. Identification of6highly pathogenic isolates:1). Morphological characteristics. Colonies of these6isolates were smooth, wet, cycloid and white on Luria-Bertani （LB） plates. Single cell rod-shaped, forming blastic spores at one end, gram-positive. Colonies of CPB008, CPB012, CPB016and CPB111were cycloid and white with irregular edge. The two ends of their cells were smooth, sized1.17-1.94x6.12-7.24μm. Colonies of CPB072and CPB108were similar to CPB008. The two ends of them were spinous, sized1.79-2.40x6.32-7.29μm.2). Physiological and biochemical characteristics. All isolates utilized effectively D-glucose, D-fructose, mannitol, NH4H2PO4and KNO3but not D-xylose. Tests including catalase, amylolysis, V-P and gelatin liquefaction were positive while benzpyrole test was negative. All of these6isolates grew well at temperature of30℃,37℃and at2%concentration of NaCl.3).16S rDNA sequencing. The16S rDNA sequences of these6isolates were obtained from their genomic DNA with bacterial16S rDNA universal primer through PCR. These sequences were comparatively analyzed with Blast tool and registered in GenBank with accession numbers of JF795483_CPBO08, JN896990（CPB012）, JN896992_CPB016, JN896991（CPB072）. JF505384（CPB108） and JN896993（CPB111）.4). Identification. Using the combination of16S rDNA sequences, morphological, physiological and biochemical characteristics, CPB008, CPB012, CPB016and CPB111were identified as Bacillus thuringiensis; CPB072and CPB108were nomenclatured as Bacillus atrophaeus.6. Cultural characteristics of BCAs:Among the8cultural media tested, LB was the best medium for CPB008and nutrient agar （NA） was the best for CPB012and CPB016. In LB liquid medium, the optimum temperature, acidity and saltility for growth of CPB008, CPB012and CPB016were37℃, pH7.0and2%NaCl, respectively. The best carbon and nitrogen sources for the bacteria were glucose and peptone. The growth dynamics of the three BCAs were essentially the same; the growth curves showed a lag phase within the first2hours, a logarithmic increase from2to16hours, and a stationary phase appeared after16hours following inoculation.7. Synergistic insecticidal effect of CPB008and Beauveria bassiana MJ-07:Control efficiency of MJ-07+CPB008was enhanced significantly to CPB under greenhouse （LR14d of over95%for1st and2nd-instar larvae, over60%for3rd and4th-instar larvae） and field （LR14d of45.2%for1st-instar larvae and42.2%for2nd-instar larvae） conditions. They were significantly higher than that of common pesticide based on Bacillus thuringiensis （control, LR14d of24.9%for1st-instar larvae and23.2%for2nd-instar larvae）. CPB larvae inoculated with MJ-07+CPB008grew slower and longer time was needed for development.As a valuable biological resource, entomogenous bacteria are distributed widely in natural environment. It will play a more important role in crop pest management with the rapid development of science and technology, In the present work,6isolates with high pathogenicities to CPB larvae were investigated and selected as potential BCAs and identified as Bacillus thuringiensis and Bacillus atrophaeus..Three B. thuringiensis isolates were thought of as BCAs against CPB. The combined treatment of B. thuringiensis （one of BCAs） and entomogenous fungus Beauveria bassiana produced a significant higher larval motality than that of using the two agents separately. It is possible that some biopesticides may be developed using these bacterial biocontrol agents.