• Korean journal of applied entomology
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• v.49 no.4
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• pp.409-416
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• 2010

An entomopathogenic bacterium, Photorhabdus temperata ssp. temperata (Ptt), suppresses insect immune responses and facilitates its symbiotic nematode development in target insects. The immunosuppressive activity of Ptt enhances pathogenicity of various microbial pesticides including Bacillus thuringiensis (Bt). This study was performed to select a cheap and efficient bacterial culture medium for large scale culturing of the bacteria. Relatively cheap industrial bacterial culture media (MY and M2) were compared to two research media, Luria-Bertani (LB) and tryptic soy broth (TSB). In all tested media, a constant initial population of Ptt multiplied and reached a stationary phase at 48 h. However, more bacterial colony densities were detected in LB and TSB at the stationary phase compared to two industrial media. All bacterial culture broth gave significant synergism to Bt pathogenicity against third instars of the diamondback moth, Plutella xylostella. Production of bacterial metabolites extracted by either hexane or ethyl acetate did not show any significant difference in total mass among four culture media. Reverse phase HPLC separated the four bacterial metabolites, which were not much different in quantities among four bacterial culture broths. This study suggests that two industrial bacterial culture media can be used to economically culture Ptt in a large scale.

• The Korean Journal of Pesticide Science
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• v.14 no.2
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• pp.123-132
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• 2010

Six populations of the diamondback moth, Plutella xylostella, were collected from the different national areas for resistance and reared in laboratory for two sensitive population. These populations of P. xylostella were examined the developed resistance against commercial products of Bacillus thuringiensis. There were 3 products with B. thuringiensis subsp. kurstaki including Tyuneup$^{(R)}$, Thuricide$^{(R)}$ and Geumulmang$^{(R)}$ and 2 products with B. thuringiensis subsp. aizawai including Tobagi$^{(R)}$ and Scorpion$^{(R)}$. The sensitive population of diamondback moths were provided from National Academy of Agricultural Science (NP) and Highland Agriculture Research Center (GR population) and field populations were caught from 6 different national areas. Resistance against Tyuneup$^{(R)}$ was developed 4.8 and 2.5 times in SP and HS compared with GR population of diamondback moth, respectively. In case of Geumulmang$^{(R)}$, it was developed 9.9 and 6.8 times in SP and NM population compared with NP population, respectively. Otherwise, Tobagi$^{(R)}$ was showed higher resistance in HS than any other population compared with GR population, however, Scorpion$^{(R)}$ that is a same strain with Tobagi$^{(R)}$, was showed only double resistance to SP population. It was supposed that the development of resistance to B. thuringiensis might be caused by the continuous application of the specific commercial product at the specific area. So, we need to use the commercial products of B. thuringiensis in rotation with different B. thuringiensis strains. In the other hand, when HS population with highest resistance were reared in laboratory, their resistance ratio was rapidly dropped to 1.1 times at second generation. We have to examined the resistance mechanism of the diamondback moth to B. thuringiensis strains.

• Journal of Microbiology and Biotechnology
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• v.22 no.6
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• pp.729-735
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• 2012

The PCR-RFLP method has been useful for detection of known genes and identification of novel genes. In the present study, degenerate primers were designed from five groups of cry1 genes for PCR-RFLP analysis. Bacillus thuringiensis (Bt) isolates from different regions were evaluated for PCR amplification of various cry1 genes using newly designed primers and cry2 genes using reported primers. PCR analysis showed an abundance of cry1A genes and especially cry1Ac genes in isolates from all regions. RFLP analysis revealed the presence of multiple cry1A genes in isolates from central and southern regions. Unique digestion patterns of cry1A genes were observed in isolates from each region. Few of the isolates represented a digestion pattern of cry1A genes that did match to any of the known cry1A genes. RFLP analysis suggested an abundance of cry2Ab along with a novel cry2 gene in Bt isolates from different regions of India. Sequence analysis of the novel cry2 gene revealed 95% sequence identity to cry2Ab and cry2Ah genes. Phylogenetic analysis revealed that the novel cry2 gene could have diverged earlier than the other cry2 genes. Our results encourage finding of more diverse cry2 genes in Bt isolates. Rarefaction analysis was used to compare cry1A gene diversity in isolates from different soil types. It showed a higher degree of cry1A gene diversity in isolates from central region. In the present study, we propose the use of novel degenerate primers for cry1 genes and the PCR-RFLP method using a single enzyme to distinguish multiple cry1A and cry2 genes as well as identify novel genes.

• Proceedings of the Korean Society of Sericultural Science Conference
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• 2003.10a
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• pp.114-115
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• 2003

A Bacillus thuringiensis isolate, Bt 2385-1, which showed toxicity to lepidopteran, was isolated from Korean soil sample and characterized. PCR-RFLP showed that this isolate contains two novel cryl-type crystal protein genes. In this study, we designed cryl-type specific primer set (ATG1-F and N400-R) to clone the toxic domain of the all cryl-type genes. The two novel rlyl-type toxin genes in addition to crylJal gene were cloned and sequenced. (omitted)

• Journal of Microbiology and Biotechnology
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• v.19 no.8
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• pp.749-759
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• 2009

A survey of Bacillus thuringiensis (Berliner) strains isolated from Spanish citrus orchards has been performed, and the strains were tested for insecticidal activity against the Mediterranean fruit fly Ceratitis capitata (Wiedemann), a key citrus pest in Spain. From a total of 150 environmental samples, 376 isolates were selected, recording a total B. thuringiensis index of 0.52. The collection was characterized by means of phase-contrast microscopy, SDS-PAGE, and PCR analysis with primer pairs detecting toxin genes cry1, cry2, cry3, cry4, cry5, cry7, cry8, cry9, cry10, cry11, cry12, cry14, cry17, cry19, cry21, cry27, cry39, cry44, cyt1, and cyt2. Diverse crystal inclusion morphologies were identified: bipyramidal (45%), round (40%), adhered to the spore (7%), small (5%), and irregular (3%). SDS-PAGE of spore-crystal preparations revealed 39 different electrophoresis patterns. All primer pairs used in PCR tests gave positive amplifications in strains of our collection, except for primers for detection of cry3, cry19, cry39, or cry44 genes. Strains containing cry1, cry2, cry4, and cry27 genes were the most abundant (48.7%, 46%, 11.2%, and 8.2% of the strains, respectively). Ten different genetic profiles were found, although a total of 109 strains did not amplify with the set of primers used. Screening for toxicity against C. capitata adults was performed using both spore-crystal and soluble fractions. Mortality levels were less than 30%. We have developed a large and diverse B. thuringiensis strain collection with huge potential to control several agricultural pests; however, further research is needed to find out Bt strains active against C. capitata.

• Korean Journal of Environmental Biology
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• v.37 no.4
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• pp.585-591
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• 2019

Most insect-resistant LMOs have been produced by applying Cry and Vip3Aa proteins. Vip3Aa protein is activated during the vegetative stage of Bacillus thuringensis (Bt) and the inhibitory activity of the Vip3Aa protein against pathogenic attacks from lepidopteran insect species is well known. However, a risk assessment of the Vip3Aa protein compared to the Cry protein has not been conducted in South Korea. This study demonstrates a possible risk assessment method for Vip3Aa protein against honeybees (Apis mellifera). For the risk assessment of the protein, we purified the recombinant Vip3Aa protein in Escherichia coli. The survival rate and symptoms of general intoxication of 4 months honeybees were measured after Vip3Aa exposure. These results indicated that there was no significant difference in the survival rate and the symptom between Vip3Aa and the control buffer. In this study, we established standard methods of Vip3Aa protein purification and oral adult toxicity test using A. mellifera as an LMO risk assessment technique for preserving the natural ecosystem of South Korea.

• Food Science and Biotechnology
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• v.15 no.3
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• pp.385-391
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• 2006

In Korea, different kinds of genetically modified (GM) crops are under development, including GM-rice expressing insecticidal crystal (Cry) proteins of Bacillus thuringiensis (Bt) modified to change a single amino acid. In this study, amino acid (aa) sequences of modified Cry proteins were compared to that of known allergens, and Cry proteins expressed in GM-rice were identified by using Cry protein specific polyclonal antibody. The antigen-antibody reactions were compared between GM and commercial rice to assess the allergic risk of Cry proteins. This analysis showed no known allergen to have more than 35% aa sequence homology with modified Cry proteins in Bt rice over an 80 aa window or to have more than 8 consecutive identical aa. Sera from allergic patients showed some IgE reactivity via immunoblotting and enzyme-linked immunosorbent assay (ELISA), although no differences were seen between GM and commercial rice. Based on these results we conclude that GM rice with modified Cry proteins has no differences in its protein composition or allergenicity relative to commercial rice.

• Korean journal of applied entomology
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• v.53 no.3
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• pp.271-280
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• 2014

Some bacterial metabolites of Xenorhabdus nematophila (Xn) inhibit phospholipase $A_2$ ($PLA_2$) activity to shutdown eicosanoid biosynthesis in target insects. However, little has been known about the target insect $PLA_2$ of these bacterial metabolites. Eight bacterial metabolites identified in Xn culture broth exhibited significant insecticidal activities against larvae of both lepidopteran species of Plutella xylostella and Spodoptera exigua. Moreover, these bacterial metabolites significantly enhanced insecticidal activities of Bacillus thuringiensis (Bt). To determine target $PLA_2$, we cloned and over-expressed cellular $PLA_2$ ($SecPLA_2$) of S. exigua. Purified $SecPLA_2$ catalyzed phospholipids derived from the fat body and released several polyunsaturated fatty acids. Most Xn metabolites significantly inhibited $SecPLA_2$ activity, but were different in their inhibitory activities. There was a positive correlation between the inhibition of $SecPLA_2$ and the enhancement of Bt insecticidal activity. These results indicate that $SecPLA_2$ is a molecular target inhibited by Xn metabolite.

• Korean journal of applied entomology
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• v.52 no.2
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• pp.115-123
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• 2013

A microbial insecticide "Bt-Plus" has been developed to enhance an insecticidal efficacy of an entomopathogenic bacterium, Bacillus thuringiensis (Bt). However, its wettable powder formulation is not preferred by farmers and industry producers due to relatively high cost. This study aimed to develop a soluble concentrate formulation of Bt-Plus. To this end, an optimal mixture ratio of two bacterial culture broths was determined to be 5:4 (v/v) of Bt and Xenorhabdus nematophila (Xn) along with 10% ethanol preservative. In addition, Bt broth was concentrated by 10 times to apply the mixture at 1,000 times fold dilution. The resulting liquid formulation was sprayed on cabbage crop field infested by late instar larvae of the diamondback moth, Plutella xylostella. The field assay showed about 77% control efficacy at 7 days after treatment, which was comparable to those of current commercial biopesticides targeting P. xylostella. For storage test in both low and room temperatures, the liquid formation showed a relatively stable control efficacy at least for a month. To develop a quality control technique to exhibit a stable control efficacy of Bt-Plus, Bt spore density ($5{\times}10^{11}$ spores/mL) and eight active component concentrations of Xn bacterial metabolites in the formulation products have been proposed in this study.

• Korean Journal of Agricultural Science
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• v.45 no.2
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• pp.185-196
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• 2018

The fungus gnat, Bradysia difformis, damages various crops in greenhouses and is recognized as an important pest around the world. Additionally, in the future, many other greenhouse crops will be added to the list of crops damaged by the fungus gnat. In this study, to find effective control methods for the fungus gnat, the insecticidal effect of 20 chemical synthetic insecticides was tested with the potato disc and pot treatment methods; additionally, the control effect of 16 strains of B. thuringiensis was examined with the potato disk method. The fungus gnat larvae were treated for 2 days with each of the synthetic insecticides to determine insecticidal effect using the potato disc method. The results were as follows. Among the highly insecticidal active pesticides, chlorfenapyr exhibited a 100% insecticidal activity, and fenazaquin, acetamiprid, dinotefuran, fenthion and thiamethoxam exhibited more than a 90% insecticidal activity. For the pot treatment method, chlorfenapyr exhibited a 3.3% insecticidal effect, and thiamethoxam, acetamiprid, dinotefuran, fenthion, etc. exhibited an insecticidal effect of less than 10% of the emergence rate to adult fungus gnat after 14 days of treatment. To select the B. thuringiensis strains that have an insecticidal effect on the fungus gnat, 16 strains were biologically assayed using the potato disc method. Among the 16 strains, Bt-3, Bt-8 and Bt-13 had more than a 70% insecticidal effect. The $LC_{50}$ and $LC_{95}$ values of Bt-3, Bt-8 and Bt-13 were $3.7{\times}10^5$ and $4.7{\times}10^8cfu/ml$, $1.4{\times}10^5$ and $1.1{\times}10^7cfu/ml$, and $1.4{\times}10^5$ and $1.3{\times}10^7cfu/ml$, respectively.