Prevalence and Toxin Characteristics of Bacillus thuringiensis Isolated from Organic Vegetables.

The prevalence and toxin characteristics of Bacillus thuringiensis isolated from 39 organic vegetables were investigated. B. thuringiensis was detected in 30 out of the 39 organic vegetables (76.9%) with a mean value of 2.60 log CFU/g. Twenty-five out of the 30 B. thuringiensis isolates (83.3%) showed insecticidal toxicity against Spodoptera exigua. The hblCDA, nheABC, and entFM genes were found to be the major toxin genes, but the ces gene was not detected in any of the tested B. thuringiensis isolates. The hemolysin BL enterotoxin was detected in all 30 B. thuringiensis isolates (100%). The non-hemolytic enterotoxin complex was found in 27 out of 30 B. thuringiensis isolates (90.0%). The B. thuringiensis tested in this study had similar toxin gene characteristics to B. cereus, which possessed more than one toxin gene. B. thuringiensis could have the potential risk of foodborne illness based on the toxin genes and toxin-producing ability.

Effects of some pesticides on development of Ascaris suum eggs.

To evaluate the effects of pesticides to parasite eggs, Ascaris suum eggs were incubated with 5 different pesticides (1:1,500-1:2,000 dilutions of 2% emamectin benzoate, 5% spinetoram, 5% indoxacarb, 1% deltamethrin, and 5% flufenoxuron; all v/v) at 20℃ for 6 weeks, and microscopically evaluated the egg survival and development on a weekly basis. The survival rate of A. suum eggs incubated in normal saline (control eggs) was 90±3% at 6 weeks. However, the survival rates of eggs treated with pesticides were 75-85% at this time, thus significantly lower than the control value. Larval development in control eggs commenced at 3 weeks, and 73±3% of eggs had internal larvae at 6 weeks. Larvae were evident in pesticide-treated eggs at 3-4 weeks, and the proportions of eggs carrying larvae at 6 weeks (36±3%-54±3%) were significantly lower than that of the control group. Thus, pesticides tested at levels similar to those used in agricultural practices exhibited low-level ovicidal activity and delayed embryogenesis of A. suum eggs, although some differences were evident among the tested pesticides.

Actin Cytoskeleton and Golgi Involvement in Barley stripe mosaic virus Movement and Cell Wall Localization of Triple Gene Block Proteins.

Barley stripe mosaic virus (BSMV) induces massive actin filament thickening at the infection front of infected Nicotiana benthamiana leaves. To determine the mechanisms leading to actin remodeling, fluorescent protein fusions of the BSMV triple gene block (TGB) proteins were coexpressed in cells with the actin marker DsRed: Talin. TGB ectopic expression experiments revealed that TGB3 is a major elicitor of filament thickening, that TGB2 resulted in formation of intermediate DsRed:Talin filaments, and that TGB1 alone had no obvious effects on actin filament structure. Latrunculin B (LatB) treatments retarded BSMV cell-to-cell movement, disrupted actin filament organization, and dramatically decreased the proportion of paired TGB3 foci appearing at the cell wall (CW). BSMV infection of transgenic plants tagged with GFP-KDEL exhibited membrane proliferation and vesicle formation that were especially evident around the nucleus. Similar membrane proliferation occurred in plants expressing TGB2 and/or TGB3, and DsRed: Talin fluorescence in these plants colocalized with the ER vesicles. TGB3 also associated with the Golgi apparatus and overlapped with cortical vesicles appearing at the cell periphery. Brefeldin A treatments disrupted Golgi and also altered vesicles at the CW, but failed to interfere with TGB CW localization. Our results indicate that actin cytoskeleton interactions are important in BSMV cell-to-cell movement and for CW localization of TGB3.

Quantitative evaluation of viability- and apoptosis-related genes in Ascaris suum eggs under different culture-temperature conditions.

Ascaris suum eggs are inactivated by composting conditions; however, it is difficult to find functional changes in heat-treated A. suum eggs. Here, unembryonated A. suum eggs were incubated at 20°C, 50°C, and 70°C in vitro, and the gene expression levels related to viability, such as eukaryotic translation initiation factor 4E (IF4E), phosphofructokinase 1 (PFK1), and thioredoxin 1 (TRX1), and to apoptosis, such as apoptosis-inducing factor 1 (AIF1) and cell death protein 6 (CDP6), were evaluated by real-time quantitative RT-PCR. No prominent morphological alterations were noted in the eggs at 20°C until day 10. In contrast, the eggs developed rapidly, and embryonated eggs and hatched larvae began to die, starting on day 2 at 50°C and day 1 at 70°C. At 20°C, IF4E, PFK1, and TRX1 mRNA expression was significantly increased from days 2-4; however, AIF1 and CDP6 mRNA expression was not changed significantly. IF4E, PFK1, and TRX1 mRNA expression was markedly decreased from day 2 at 50° and 70°C, whereas AIF1 and CDP6 mRNA expression was significantly increased. The expressions of HSP70 and HSP90 were detected for 9-10 days at 20°C, for 3-5 days at 50°C, and for 2 days at 70°C. Taken together, incremental heat increases were associated with the rapid development of A. suum eggs, decreased expression of genes related to viability, and earlier expression of apoptosis-related genes, and finally these changes of viability- and apoptosis-related genes of A. suum eggs were associated with survival of the eggs under temperature stress.

Mass production of aphicidal Beauveria bassiana SFB-205 supernatant with the parameter of chitinase.

Beauveria bassiana SFB-205 supernatant can effectively control cotton aphid populations, which is closely associated with its chitinase activity. The present work extends to optimizing a culture medium to produce more efficacious supernatant in flask conditions, followed by scale-up in 7 L, 300 L and 1.2 KL fermentors with the parameter of chitinase. In flask conditions, a combination of soluble starch and yeast extract produced the greatest amount of chitinase (5.1 units/ml) and its supernatant had the highest aphicidal activity. An optimal quantitative combination of the two substrates, estimated by a response surface method, enabled the supernatant to have 15.7 units/ml of chitinase activity and 3.7 ml/l of median lethal concentration (LC50) of toxicity against cotton aphid adults in laboratory conditions. In the scale-up conditions, overall supernatant had 25-28 units/ml of chitinase activity. Decrease in pH and limitation of dissolved oxygen (DO) during cultures were significantly related to the yield of chitinase. These results suggest that the substrate-dependent chitinase production can be background information for optimizing a culture medium, and pH and DO are critical factors in maximizing the production in scale-up conditions.

Intestinal parasite infections in pigs and beef cattle in rural areas of Chungcheongnam-do, Korea.

The present study was performed to investigate the infection status of intestinal parasites in pigs and beef cattle in rural areas of Chungcheongnam-do, Korea. From November 2009 to April 2010, a total of 241 fecal samples of pigs and beef cattle (136 and 105, respectively) were examined by direct smear and centrifugal sedimentation methods. The overall positive rates of intestinal parasites among pigs and beef cattle were 73.5% and 4.8%, respectively, and the double-infection rate was 10.3% in pigs. Of 136 specimens from pigs, Balantidium coli, Ascaris suum, and Entamoeba spp. infections were found in 88 (64.7%), 24 (17.6%), and 5 cases (3.7%), respectively. Of 105 beef cattle, Entamoeba spp. infections were detected in 5 cases (4.8%). From these results, it is shown that pigs raised on rural farms in Chungcheongnam-do had a high B. coli infection rate and a moderate A. suum infection rate. These results demonstrate that environmentally resistant cysts or eggs could be widespread on the farms examined, and thus an effective hygienic management system is needed to prevent them from serving as the source of infection for human beings.

Acinetobacter antiviralis sp. nov., from Tobacco plant roots.

Acinetobacter strain KNF2022T was isolated from tobacco plant roots during the screening of antiviral substances having inhibitory effects on Tobacco mosaic virus (TMV) and examined by phenotypic, chemotaxonomic, and genetic characterization. It was a nonmotile, Gram-negative bacterium. This strain contained Q-9 as the main respiratory quinone. The major cellular fatty acids of the isolate were 16:0, 18:1 w9c, and 16:1 w7c/15 iso 2OH. The DNA base composition was 44 mol%. Phylogenetic analysis based on the 16S rRNA sequence revealed that the isolate formed an evolutionary lineage distinct from other Acinetobacter species. Based on the evaluation of morphologic, physiologic, and chemotaxonomic characteristics, DNA-DNA hybridization values, and 16S rRNA sequence comparison, we propose the new species Acinetobacter antiviralis sp. nov., the type strain of which is KNF2022T(=KCTC0699BPT).

Description of Streptomyces neopeptinius sp. nov., an actinobacterium with broad spectrum antifungal activities.

A streptomycete strain producing broad-spectrum antifungal substances was taxonomically characterized. The strain, designated KNF 2047(T) (= SH-09(T) = KCTC 10586BP(T)), was found to form extensively branching aerial and substrate mycelia, and produce spiny-ornamented spores with loose spiral chains. The whole cell hydrolyzates contained major amount of LL-diaminopimelic acid. The major fatty acids of the phospholipids were saturated and branched fatty acids containing 14~17 carbons, and the major isoprenoid quinones were hexa-and octa-hydrogenated menaquinones with 9 isoprene units. The phylogenetic analysis using the 16S rRNA gene indicated that the strain belongs to the genus Streptomyces but forms an independent phyletic line. These results clearly demonstrate that strain KNF2047(T) forms a new center of taxonomic variation within Streptomyces, for which the name Streptomyces neopeptinius sp. nov. is proposed.

Occurrence of parasporin-producing Bacillus thuringiensis in Vietnam.

A total of 63 Bacillus thuringiensis isolates were recovered from urban soils of Hanoi, Vietnam. Of these, 34 were identified to 12 H serogroups. None of the isolates showed larvicidal activities against three lepidopterous insects. Three isolates belonging to the two serovars, colmeri (H21) and konkukian (H34), were highly toxic to larvae of the mosquito Aedes aegypti. Parasporal inclusion proteins of four isolates exhibited cytocidal activities against HeLa cells. Immunologically, proteins of four isolates were closely related to parasporin-1 (Cry31Aa), a parasporal protein that preferentially kills human cancer cells. Haemolytic activities were associated with parasporal proteins of the three mosquitocidal isolates but not with those of the four cancer-cell-killing isolates. PCR experiments and nucleotide sequence analysis revealed that the genes of four anti-cancer isolates are closely related to the gene parasporin-1 (cry31Aa) but are dissimilar to those of the three other existing parasporins. Our results suggest that the soil of northern Vietnam is a good reservoir of parasporin-producing B. thuringiensis.

Streptomyces luridiscabiei sp. nov., Streptomyces puniciscabiei sp. nov. and Streptomyces niveiscabiei sp. nov., which cause potato common scab disease in Korea.

Three plant-pathogenic isolates of Streptomyces spp., isolated from potatoes with common scab disease lesions in Korea, are described as novel species. Morphological and physiological properties of these isolates were distinct from those of previously described Streptomyces species. Strain S63(T) has yellow-white, smooth, cylindrical spores that are borne in monoverticillus flexuous spore-chains. Strain S77(T) has purple-red, spiny spores that are borne in simple rectus flexuous spore-chains. Strain S78(T) has white, smooth, cylindrical spores that are borne in simple rectus flexuous spore-chains. These three isolates differed from known pathogenic strains by analysis of 16S rRNA gene sequences in a previous study. Furthermore, genetic uniqueness of our isolates was confirmed by sequencing of the 16S-23S internal transcribed spacer (ITS) region, which indicated that isolates S63(T) and S78(T) belong to the genus Streptomyces and have low homology to other Streptomyces species (less than 71.2 and 75.7 %, respectively). The 16S-23S ITS region of strain S77(T) was not amplified by these primer sets. DNA-DNA hybridization results for all three isolates show distant relationships to previously described Streptomyces species; therefore, on the basis of polyphasic evidence, the names Streptomyces luridiscabiei sp. nov. for strain S63(T) (=LMG 21390(T)=KACC 20252(T)), Streptomyces puniciscabiei sp. nov. for strain S77(T) (=LMG 21391(T)=KACC 20253(T)) and Streptomyces niveiscabiei sp. nov. for strain S78(T) (=LMG 21392(T)=KACC 20254(T)) are proposed.

Characterization of Streptomycetes Causing Potato Common Scab in Korea.

Six representative Korean strains of streptomycetes (S33, S27, S71, S63, S77, and S78) that were pathogenic to potato were characterized based on phenotypic properties, analysis of 16S rRNA genes, production of thaxtomin A, and presence of nec1 and ORFtnp gene homologs. Strains S33 and S27 had typical characteristics of Streptomyces scabies and S. turgidiscabies, respectively, producing thaxtomin A and hybridizing to genes of nec1 and ORFtnp. Strain S71 produced thaxtomin A and had phenotypic and phylogenetic properties similar to those of S. acidiscabies, except having a greater minimum growth pH (4.5), production of a melanoid pigment on tyrosine agar, and failure to hybridize with nec1 and ORFtnp gene probes. In contrast, strains S63, S77, and S78 were phenotypically different from described scab pathogens. Spore colors of strains S63 and S77 were yellow-white or pale orange, respectively, with rectiflexuous chains. Strain S78 had thin and compact spores unlike typical S. acidiscabies (ATCC 49003). Phylogenetic analysis of strains S63, S77, and S78 based on 16S rRNA gene sequences showed low homology to that of described scab pathogens (less than 97.3, 96.0, and 96.3%, respectively). Strain S78 produced thaxtomin A, but did not have homologous sequences to nec1 and ORFtnp genes. Production of thaxtomin A and gene homologs of nec1 and ORFtnp were not detected in strains S63 and S77. All three strains grow at low pH, with minimal growth at pH 3.5 (S77 and S78) or 4.5 (S63). Streptomyces strains S63, S77, and S78 are novel pathogenic streptomycetes adapted to acidic soil conditions in Korea.

Effect of gamma-irradiation on degradation of alginate.

The aqueous solution of alginate was irradiated by 60Co gamma-rays in the dose range of 10-500 kGy. To assess the effect of irradiation on the degradation of alginate, the irradiation-induced changes in the viscosity, molecular weight, color, monomer composition, and sequence were measured. The molecular weight of raw alginate was reduced from 300000 to 25000 when irradiated at 100 kGy. The degradation rate decreased and the chain breaks per molecule increased with increasing irradiation dose. The viscosity of irradiated alginate solution reached a near minimum as low as at 10 kGy. No appreciable color changes were observed in the samples irradiated at up to 100 kGy, but intense browning occurred beyond 200 kGy. The 13C NMR spectra showed that homopolymeric blocks, MM and GG, increased and the M/G ratio decreased with irradiation. Considering both the level of degradation and the color change of alginate, the optimum irradiation dose was found to be 100 kGy.

Seasonal distribution and characterization of Bacillus thuringiensis isolated from sericultural environments in Korea.

To isolate naturally occurring novel Bacillus thuringiensis strains, we investigated the distribution and characteristics of B. thuringiensis from samples of sericultural farms in various regions of Korea in the spring and fall. Fifty-four B. thuringiensis strains out of 164 samples and 34 B. thuringiensis strains out of 135 samples were isolated in the spring and fall, respectively. Seventy percent of the isolates in the spring and 15% in the fall were toxic to lepidopteran larvae. Dipteran-active isolates were rare (7% in spring and 3% in fall isolation). Particularly, B. thuringiensis isolates, which are toxic to both Lepidoptera and Diptera, were widely distributed (19% in spring and 62% in fall isolation). Non-toxic isolates were also found (4% in spring and 20% in fall isolation). B. thuringiensis isolates in the sericultural farms represented 11 H serotypes; they were principally B. thuringiensis subsp. aizawai in the spring and kurstaki in the fall. B. thuringiensis isolates of serotypes 1, 3a, 3a3b, 4a4c, 6, 7 and 12 were toxic to Lepidoptera. Seventy isolates produced typical rhomboidal inclusions, and the remainder produced parasporal inclusions with various morphologies. PCR analysis using cryI gene type-specific primers showed that cryIAa and cryIC genes are frequently found in the spring and cryIAa gene is a predominant type in the fall. Toxicity, H serotype and the cryI gene contents of B. thuringiensis isolated from sericultural farms showed that distribution varied depending on the season.