Search

Colistin is an important antibiotic currently used to manage infections caused by multidrug-resistant pathogens in both humans and livestock animals. A new mobile colistin-resistance (mcr-9) gene w...

This study was conducted to characterize Escherichia coli strains and evaluate the spread of antimicrobial resistance among these strains from fresh produce and farm environments in Korea. We then conducted phenotypic and genetic studies on antimicrobial-resistant isolates. We determined the genetic epidemiological characteristics of isolates that produced extended-spectrum β-lactamase (ESBL) and confirmed plasmid transfer in isolates that carried blaCTX-M-type genes. E. coli strains were isolated from 8 samples of fresh produce and 152 samples from the farm environment collected from May 2014 to June 2016. Cephalosporin resistance was the most prevalent (61.8%) type of resistance among the isolates. Five ESBL-producing strains with high genetic homology with E. coli of human or livestock origin were identified. Lateral transfer of plasmids harboring blaCTX-M-type genes to transconjugants was successful. Two isolates from Chinese cabbage and from water samples collected from a nearby stream harbored the ISEcp1-blaCTX-M-55-orf477 operon and were confirmed as sequence type 1196 and the same type of plasmid replicon, suggesting that cross-contamination was highly likely. A high-risk clone of sequence type 69 (clonal complex 69) isolates was also recovered from the farm environment. This study provides genetic evidence that antimicrobial resistance factors in E. coli from farm environments originate in the clinic or in livestock, highlighting the fact that good agricultural practices in farming are important to inhibit the spread of antimicrobial resistance to bacteria on fresh produce. HIGHLIGHTS

Acidovorax citrulli, the gram-negative bacteria that causes bacterial fruit blotch (BFB), has been responsible for huge worldwide economic losses in watermelon and melon production since 1980. No commercial cultivar resistant to BFB has been reported. Of the two reported genotypes of A. citrulli, genotype I is the main causal agent of BFB in melon and genotype II causes disease in watermelon. After the isolation of the first bacteriophage against A. citrulli (ACP17), efforts have been made to isolate bacteriophages with wider host ranges by collecting samples from watermelon, pumpkin, and cucumber. The newly isolated phage ACPWH, belonging to the Siphoviridae family, has a head size of 60 ± 5 nm and tail size of 180 ± 5 nm, and can infect 39 out of 42 A. citrulli strains. ACPWH has genome size of 42,499 and GC content of 64.44%. Coating watermelon seeds with bacteriophage ACPWH before soil inoculation with A. citrulli resulted in 96% germination and survival, compared to 13% germination of uncoated control seeds. These results suggest that phage ACPWH may be an effective and low-cost biocontrol agent against BFB.

Fusarium infection rate of the paddy rice grain after harvest seemed to be influenced by the average temperature from late July (before heading) to the end of September (during ripening). In case of 2010 and 2013 in which average temperature of the same period was similar, Fusarium infection was related to cumulative precipitation, cumulative precipitation days, and precipitation durations over two days. The distribution ratio of Fusarium species complex isolated from paddy rice grains after harvest was 57% in 2010 and 45% in 2013 for Fusarium graminearum species complex (FGSC), 35% and 50% for Fusarium incarnatum-equiseti species complex, and 8% and 5% for Fusarium fujikuroi species complex (FFSC). The distribution ratios of FGSC and FFSC were higher in 2010 than 2013. Among the total 26 promoted rice varieties, the ‘Mihyang’ showed resistant response against the natural infection with Fusarium species belonging to FGSC and the varieties of ‘Nampyeong’, ‘Hi-ami’and ‘Younghojinmi’ showed resistant response against the natural infection with overall Fusarium pathogens. Majority of the promoted rice varieties could not be classified for resistance or susceptibility. These results are valuable as basic data to determine the resistance and susceptibility of rice variety against Fusarium spp. infection in the field.

Increased enterococcal infections in hospitals and multidrug-resistant and vancomycin-resistant enterococci (VRE) isolated from humans, animals, and food sources raised public health concern on the presence of VRE in multiple sources. We performed a comparative analysis of the antimicrobial resistance and genetics of VRE isolates derived from fresh produce and human fecal samples. Of 389 Enterococcus isolates, 8 fecal and 3 produce isolates were resistant to vancomycin and teicoplanin; all harbored vanA gene. The VRE isolates showed multidrug-resistant properties. The isolates from fresh produce in this study showed to have the common shared characteristics with the isolates from humans by the results of antimicrobial resistance, multilocus sequence typing, and Tn 1546 transposon analysis. Therefore, VRE isolates from fresh produce are likely related to VRE derived from humans. The results suggested that VRE may contaminate vegetables through the environment, and the contaminated vegetables could then act as a vehicle for human infections. Ongoing nationwide surveillance of antibiotic resistance and the promotion of the proper use of antibiotics are necessary.

Fusarium graminearum species complex (FGSC) causes Fusarium head blight in small grain cereals. To date, four species (F. graminearum, F. asiaticum, F. boothii, and F. meridionale ) belonging to FGSC frequently occur in Korean cereals. In addition, we first reported the occurrence of additional species (F. vorosii ) within FGSC, which was isolated from barley, corn, and rice in Korea. Phylogenetic analysis of the Fusarium isolates of this group using combined multi-gene sequences confirmed species identification. Moreover, the macroconidia produced by these isolates were morphologically similar to those of the F. vorosii holotype. Chemical analysis indicated that the F. vorosii isolates produced various trichothecenes such as nivalenol and deoxynivalenol with their acetyl derivatives along with zearalenone. Pathogenicity tests demonstrated that all of the F. vorosii isolates examined were pathogenic on barley, corn, and rice with variation in aggressiveness. This study is the first report of F. vorosii in Korean cereals, their pathogenicity towards barley and corn, and their ability to produce trichothecenes and zearalenone.

Fusaric acid is a mycotoxin produced by species of the fungus Fusarium and can act synergistically with other Fusarium toxins. In order to develop a specific detection method for fusaric acid-producing fungus, PCR primers were designed to amplify FUB10, a transcription factor gene in fusaric acid biosynthetic gene cluster. When PCR with Fub10-f and Fub10-r was performed, a single band (~550 bp) was amplified from F. oxysporum, F. proliferatum, F. verticillioides, F. anthophilum, F. bulbicola, F. circinatum, F. fujikuroi, F. redolens, F. sacchari, F. subglutinans, and F. thapsinum, all of which were known for fusaric acid production. Whereas the FUB10 specific band was not amplified from Fusarium species known to be trichothecene producer. Because production of fusaric acid can co-occur in species that also produce fumonisin mycotoxins, we developed a multiplex PCR assay using the FUB10 primers as well as primers for the fumonisin biosynthetic gene FUM1. The assay yielded amplicons from fumonisin producers such as F. proliferatum and F. verticillioides, allowing for the simultaneous detection of species with the genetic potential to produce both types of mycotoxins.

Fascioliasis, a food-borne trematode zoonosis, is a disease primarily in cattle and sheep and occasionally in humans. Water dropwort (Oenanthe javanica), an aquatic perennial herb, is a common second intermediate host of Fasciola, and the fresh stems and leaves are widely used as a seasoning in the Korean diet. However, no information regarding Fasciola species contamination in water dropwort is available. Here, we collected 500 samples of water dropwort in 3 areas in Korea during February and March 2015, and the water dropwort contamination of Fasciola species was monitored by DNA sequencing analysis of the Fasciola hepatica and Fasciola gigantica specific mitochondrial cytochrome c oxidase subunit 1 (cox1) and nuclear ribosomal internal transcribed spacer 2 (ITS-2). Among the 500 samples assessed, the presence of F. hepatica cox1 and 1TS-2 markers were detected in 2 samples, and F. hepatica contamination was confirmed by sequencing analysis. The nucleotide sequences of cox1 PCR products from the 2 F. hepatica-contaminated samples were 96.5% identical to the F. hepatica cox1 sequences in GenBank, whereas F. gigantica cox1 sequences were 46.8% similar with the sequence detected from the cox1 positive samples. However, F. gigantica cox1 and ITS-2 markers were not detected by PCR in the 500 samples of water dropwort. Collectively, in this survey of the water dropwort contamination with Fasciola species, very low prevalence of F. hepatica contamination was detected in the samples.

The purposes of this study were to develop a small scale post-harvest facility, and consequently to evaluate the effects of applying the facility along with hygiene education on the level of microbial safety in Korean leeks production. A total of 135 samples were collected at three Korean leeks farms in Yangju, Gyeonggi province. Food safety indicators (Aerobic plate count (APC), coliform count, and Escherichia coli) and foodborne pathogens (E. coli O157:H7, Salmonella spp., Staphylococcus aureus, Listeria monocytogenes, and Bacillus cereus) on/in the samples were assessed. The microbial load measured as APC with harvesting tools such as comb, chopping board, and knife, at the farms where the small scale post-harvest facility had been operated (Farms A and B) was lower than that at another farm having no post-harvest facility (Farm C) by 1.44~2.33 log CFU / 100 cm. Moreover, the chopping board from Farm C was observed being contaminated with B. cereus at 6.03 log CFU / 100 cm. The coliform counts from the samples increased by 0.57~1.89 log CFU/g after leeks was submerged in ground water for washing. E. coli was recovered from leeks, soil, and the ground water used in the washing process, while no E. coli O157:H7, Salmonella spp., and L. monocytogenes was detected. Our results indicated that the small scale post-harvest facility developed in this study as well as the hygiene education played an important role in enhancing the level of microbial food safety in the leeks production environment. However, a disinfection technique could be needed during the washing step in order to prevent a potential contamination.

Antagonistic microbes were isolated from soils to control mycotoxin contamination of cereals by limiting the growth of mycotoxigenic Fusarium species. In total, 341 bacterial isolates were examined for antifungal activity against eight mycotoxigenic Fusarium species using dual culture assays. The screening identified 11 isolates that inhibited mycelial growth of all Fusarium species tested. The culture filtrates of 2 of the 11 isolates completely inhibited germination of conidia up to 21 days of incubation. These two isolates exhibited identical activity toward the fungi tested and were identified as Brevibacillus spp. based on 16S rRNA sequence homology. The most closely related species based on phylogenetic analysis was Brevibacillus reuszeri. Additional dual culturing using further fungal species showed that the antagonistic Brevibacillus inhibited the growth of most Fusarium species tested (39 of 46 species), two Epicoccum spp., one Alternaria sp., three Aspergillus spp. (3 of 11), and three Penicillium spp. (3 of 8). The in vivo assay was performed to test the efficacy of antagonistic Brevibacillus isolates on maize ears and revealed that the application of microbes suppressed ear rot (ANOVA, p = 0.0020). This Brevibacillus sp. may be an antagonist of the majority of Fusarium species, including mycotoxigenic species.

In fungi, genes involved in biosynthesis of a secondary metabolite (SM) are often located adjacent to one another in the genome and are coordinately regulated. These SM biosynthetic gene clusters typically encode enzymes, one or more transcription factors, and a transport protein. Fusaric acid is a polyketide-derived SM produced by multiple species of the fungal genus Fusarium. This SM is of concern because it is toxic to animals and, therefore, is considered a mycotoxin and may contribute to plant pathogenesis. Preliminary descriptions of the fusaric acid (FA) biosynthetic gene (FUB) cluster have been reported in two Fusarium species, the maize pathogen F. verticillioides and the rice pathogen F. fujikuroi. The cluster consisted of five genes and did not include a transcription factor or transporter gene. Here, analysis of the FUB region in F. verticillioides, F. fujikuroi, and F. oxysporum, a plant pathogen with multiple hosts, indicates the FUB cluster consists of at least 12 genes (FUB1 to FUB12). Deletion analysis confirmed that nine FUB genes, including two Zn(II)2Cys6 transcription factor genes, are required for production of wild-type levels of FA. Comparisons of FUB cluster homologs across multiple Fusarium isolates and species revealed insertion of non-FUB genes at one or two locations in some homologs. Although the ability to produce FA contributed to the phytotoxicity of F. oxysporum culture extracts, lack of production did not affect virulence of F. oxysporum on cactus or F. verticillioides on maize seedlings. These findings provide new insights into the genetic and biochemical processes required for FA production.

To evaluate microbiological and aflatoxin safety on traditional dried persimmon, a total of 315 samples were collected from 105 farms. The collected samples were assessed on aflatoxin and microorganisms (Aer- obic plate count, coliform count, Escherichia coli, Escherichia coli O157:H7, Salmonella spp., Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus). The the APC of sliced dried persimmon, dried persimmon, and semi dried persimmon were 3.93 ± 0.96, 2.12 ± 0.93, and 1.50 ± 1.08 log CFU/g, respectively. S. aureus was detected in 40.0% of sliced dried persimmon, 29.5% of dried persimmon, and 23.5% of semi dried persimmon. E. coli recovered from dried persimmon and semi dried persimmon was 6.6%, and 2.9%, respectively. However, E. coli O157:H7, Sal- monella spp., and L. monocytogenes were not detected. According to the result of aflatoxin by ELISA and UPLC, aflatoxin was not detected in any sample. These data suggested that safety management system should be introduce to the farms producing traditional dried persimmon to enhance the safety of traditional dried persimmon.

Rice samples including paddy, husk, brown rice, blue-tinged rice, broken rice, discolored rice and polished rice were collected from rice processing complexes(RPC) nationwide to determine the contamination of fungi and mycotoxins on rice during 2010-2013. Among the samples, paddy rice had the highest frequencies of fungal and Fusarium occurrence, and the frequencies decreased along with milling as husk was the next. Blue-tinged rice or discolored rice was similar with brown rice for fungal occurrence, and polished rice showed the lowest frequency. Among Fusarium species, F. graminearum species complex occupied 87% in 2012 but did 35-39% in 2011 and 2013. Aspergillus and Penicillium species appeared at low frequency in most samples but occurred at higher frequency in certain RPC samples. Alternaria, Nigrospora, and Epicoccum species occurred similarly to the pattern of total fungi. The rice samples from 2010-2012 were analyzed for the occurrence of Fusarium mycotoxins including deoxynivalenol, nivalenol, zearalenone, and other trichothecenes. The most highly con- taminated sample was discolored rice in terms of frequency, level, ratio of simultaneous contamination with multiple toxins, followed by blue-tinged rice.

This study was conducted to investigate survival of Bacillus cereus (B. cereus) on stainless steel and polyvinyl chloride (PVC) and its transfer from two material to lettuce. The stainless steel and PVC were innoculated with B. cereus and stored at 6 combination conditions (temperature : 20oC and 30oC, relative humidity (RH) : 43%, 69%, and 100%). Although the total numbers of B. cereus at RH 43% and RH 69% were reduced by 3.53-4.00 log CFU/coupon within 24 h regardless of material type, the spore numbers of B. cereus was lasted at 3.0 log CFU/ coupon. When two materials were stored at 30oC, RH 100%, the spore numbers of B. cereus was rapidly increased by 3.0 log CFU/coupon. In addition, the reduction rate of B. cereus was decreased in the presence of organic matter. Transfer rate of B. cereus from surface of stainless steel and PVC to lettuce was increased by 10 times in the presence of water on the lettuce surface. As a result of this study, the presence of B. cereus on produce contact surfaces can increase the risk of cross-contamination. Thus, it is important that the packing table and conveyer belt in post harvest facility should be properly washed and sanitized after working to prevent cross-contamination.

Foodborne illness due to the consumption of contaminated raw strawberries is a continuing food safety concern. This study investigated and evaluated contamination levels of bacteria on strawberries at farms stage to evaluate potential hazards associated with fresh strawberries. A total of 315 samples, 105 samples from 5 sampling sites (A to E) of 21 farms and 210 samples from 1 sampling site of 6 farms, was collected every month for four months and analyzed to enumerate aerobic bacterial counts, Coliforms/E. coli, Bacillus cereus and Staphylococcus aureus. In addition, the prevalence study of five pathogens (S. aureus, E. coli, E. coli O157:H7, Salmonella spp. and Listeria monocytogenes) was performed on each sample. Aerobic bacterial counts ranged from 0.48 to 6.36 Log CFU/g, with the highest bacterial cell counts recorded for D and E sites. Coliforms were detected in 71 samples (22.5%) with a minimum of 0.48 cfu/g and a maximum of more than 4 Log CFU/g. B. cereus was detected in 98 samples (31.1%) among total samples analyzed. S. aureus was detected in 2 samples with a minimum of 0.48 Log CFU/g and a maximum of 1.38 Log CFU/g. E. coli, E. coli O157:H7, Salmonella spp. and L. monocytogenes were not isolated from any of the samples. The microbial contamination levels of strawberries determined in this study may be used as the fundamental data for microbiological risk assessment.

This study assessed microbiological hazards at postharvest stage of dropwort farms (A, B, C, D, E, F, G, H, I) located in 4 different areas in Korea. The samples were assessed for sanitary indication bacteria (total aerobic bacteria, coliform, and Escherichia coli) and pathogenic bacteria (Escherichia coli O157:H7, Listeria monocytogenes, Staphylococcus aureus and Bacillus cereus). Total aerobic bacteria and coliform in 9 dropwort farms were detected at the levels of 0~7.00 and 0~4.25 log CFU/g, mL, of 100 cm. In particular, microbial contamination in worker’s hand showed higher than cultivation environment factors. Escherichia coli was detected in several farms of soil, irrigation water, washing water and worker’s hand and also, dropwort in these farms was contaminated with E. coli (positive reaction). In case of pathogenic bacteria, B. cereus was detected at the highest levels in soil. S. aureus was detected qualitatively from only one sample of dropwort washed by water. E. coli O157:H7 and L. monocytogenes were not detected. Although dropwort pass through 2 process (trimming and washing), the microbial contamination was not differ significantly before and after which indicates that current washing system was not effect on reduction of microorganism. From these results, the postharvest environment and workers have been considered as cross-contamination factors. Thus, processing equipments and personal hygiene should be managed to reduce the microbial contamination of dropwort. Accordingly management system such as good agricultural practices (GAP) criteria is needed for the safety of dropwort

While searching for healthier diets, people became more attentive to agricultural organic products. However, organic foods may be more susceptible to microbiological contamination because of the use of livestock manure compost and liquid manure, potential sources of pathogenic bacteria. This study was undertaken to investigate the persistence of Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes in soil, liquid manure amended soil, and liquid manure. Loamy soil, liquid manure amended soil, and liquid manure were inoculated with S. enterica, E. coli O157:H7, and L. monocytogenes. Samples were incubated in consistent moisture content at 25°C. Samples had been periodically collected during 120 days depending on the given conditions. S. enterica and E. coli O157:H7 survived over 120 days in loamy soil and over 60 days in liquid manure amended soil, respectively. L. monocytogenes decreased faster than other pathogens in soil. S. enterica, E. coli O157:H7, and L. monocytogenes survived for up to 5 days in liquid manure. S. enterica and E. coli O157:H7 in soil decreased by 2 to 2.5 log CFU g -1 for 120 days. S. enterica and E. coli O157:H7 in liquid manure amended soil decreased slowly for 21 days. However, S. enterica, E. coli O157:H7, and L. monocytogenes sharply decreased after 21 days. S. enterica, E. coli O157:H7, and L. monocytogenes in soil increased by 0.5 to 1.0 log CFU g -1 for 7 days. Foodborne pathogens in soil and liquid manure amended soil gradually decreased over time.

This study investigated the effect of adaptation of Staphylococcus aureus strains to the acidic condition of tomato in response to environmental stresses, such as heat and acid. S. aureus ATCC 13565, ATCC 14458, ATCC 23235, ATCC 27664, and NCCP10826 habituated in tomato extract at 35°C for 24 h were inoculated in tryptic soy broth. The culture suspensions were then subjected to heat challenge or acid challenge at 60°C and pH 3.0, respectively, for 60 min. In addition, transcriptional analysis using quantitative real-time PCR was performed to evaluate the expression level of acid-shock genes, such as clpB, zwf, nuoF, and gnd, from five S. aureus strains after the acid habituation of strains in tomato at 35°C for 15 min and 60 min in comparison with that of the nonhabituated strains. In comparison with the nonhabituated strains, the five tomato-habituated S. aureus strains did not show cross protection to heat, but tomato-habituated S. aureus ATCC 23235 showed acid resistance. In quantitative real-time-PCR analysis, the relative expression levels of acid-shock genes (clpB, zwf, nuoF, and gnd) were increased the most in S. aureus ATCC 23235 after 60 min of tomato habituation, but there was little difference in the expression levels among the five S. aureus strains after 15 min of tomato habituation. These results indicate that the variation of acid resistance of S. aureus is related to the expression of acid-shock genes during acid habituation.

The objective of this study was to analyze the microbiological hazards of Astragalus membranaceus Bunge on the post-harvest processing. Samples from processing equipments (cleaner, water, cart, table, tray and pack- aging machine), personal hygiene (hand) and harvested crops (before washing, after washing, after sorting, and after drying) were collected from four farms (A, B, C, and D) located in Chungchengbuk-do, Korea. The samples were analyzed for sanitary indication bacteria and pathogenic bacteria. First, total aerobic bacteria and coliform in process- ing facilities were detected at the levels of 0.93~4.86 and 0.33~2.28 log CFU/100 cm 2 and /mL respectively. In partic- ular, microbial contamination in hand (5.43~6.11 and 2.52~4.12 log CFU/Hand) showed higher than processing equipments. Among the pathogenic bacteria, Bacillus cereus was detected at the levels of 0.33~2.41 log CFU/100 cm 2 , 1.48~3.27 log CFU/Hand and 0.67~3.65 log CFU/g in equipments, hands, and plants and Staphylococcus aureus were detected in cleaner, table, hand and harvested crops (before washing and after sorting) by qualitative test. Escher- ichia coli O157:H7, Listeria monocytogenes, and Salmonella spp. were not detected. These results indicated that per- sonal hygiene and processing equipments should be managed to reduce the microbial contamination of A. membranaceus Bunge. Therefore, management system such as good agricultural practices (GAP) criteria is needed for hygienic agricultural products.

BACKGROUND: Azadirachta Indica extract(AIE) and Sophorae radix extract(SRE) are widely used as environment-friendly organic materials of plant origin in South Korea. METHODS AND RESULTS: In this study, the in vitro micronucleus(vitMN) tests of two samples of AIE and SRE were conducted to evaluate their genotoxicity using the Chinese hamster lung(CHL) cell. This study was composed of two parts; cytochalasin B(cyto B) test and non-cyto B test. Mitomycin C and colchicine were used as positive controls. As a result, the incidence of micronucleus(MN) in all AIE and SRE treated groups increased in dose- dependent manner, but were less than 2.2% in 1,000 binucleated cells. In addition, there were no significant increases of MN incidence in all AIE and SRE treated groups, compared with the negative control group. CONCLUSION: Therefore, we suggest that AIE samples and SRE samples used in this study may have no genotoxicity in the in vitro micronucleus test using the CHL cells. In our previous study, we reported that AIE and SRE did not cause genotoxicity in Ames test. According to the genotoxicity battery system, we concluded that AIE and SRE used in this study have no genotoxic effects to humans.

To analyze the hazards associated with cucumber and hot pepper cultivation areas, a total of 72 samples were obtained and tested to detect the presence of biological (sanitary indicative, pathogenic bacteria and fungi) and chemical hazards (heavy metals and pesticide residues). The levels of sanitary indicative bacteria (aerobic plate counts and coliforms) and fungi were ND-7.2 and ND-4.8 log CFU/(g, mL, hand, or 100 cm 2 ) in cucumber cultivation areas, and ND- 6.8 and 0.4-5.3 log CFU/(g, mL, hand, or 100 cm 2 ) in hot pepper cultivation areas. More specifically, the soil of hot pepper cultivation areas was contaminated with coliforms at a maximum level of 5.6 log CFU/g. Staphylococcus aureus was detected only in glove samples at a level of 1.4 log CFU/100 cm 2 and Bacillus cereus was detected in the majority of samples at a level of ND-4.8 log CFU/(g, mL, hand, or 100 cm 2 ). Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella spp. were not detected. Heavy metal (Zn, Cu, Ni, Pb, and Hg) chemical hazards were detected at levels lower than the regulation limit. Residual insecticides were not detected in cucumbers; however, hexaconazole was detected at a level of 0.016 mg/kg (maximum residue limit: 0.3 mg/kg) in hot peppers.

Livestock manure is a valuable source of nutrients and organic matter for plant. Thus, livestock manure compost is commonly used fertilizer in organic vegetable and fruit production in many countries. However, contaminated or inadequate manure compost can give negative effect to soil microorganisms. This study was conducted to investigate the survival difference of Salmonella enterica and Listeria monocytogenes in chicken and pig manure compost under the selected environmental conditions. Commercially available manure compost (pig, chicken) was inoculated with S. enterica and L. monocytogenes. Manure compost was incubated at 25℃ and consistent moisture content. Samples had been collected during 200 days depending on the given conditions. S. enterica survived for 130 days in pig manure compost and over 200 days in chicken manure compost, respectively. L. monocytogenes persisted for 120 days in pig manure compost and over 200 days in chicken manure compost, respectively. It is noted that the number of S. enterica and L. monocytogenes gradually decreased over time. The results indicate that S. enterica survived longer than L. monocytogenes in manure compost at 25℃. S. enterica and L. monocytogenes survived longer in chicken manure compost than in pig manure compost. Increased knowledge of pathogen behavior in agricultural environments is a valuable part of future work on improving risk evaluations and, in a longer perspective, in providing data for guidelines regarding safe handling of pathogen-contaminated manure compost and soil.

Microbial Safety Division, National Academy of Agricultural Science-Rural Development Administration,Suwon 441-707, Korea(Received on November 4, 2013; Revised on November 28, 2013; Accepted on November 28, 2013)To investigate Fusarium mycotoxin contamination in rice samples from rice processing complexes (RPCs),paddy rice and rice-milling products such as husks, brown rice, blue-tinged rice, broken rice, rice bran,discolored rice, and polished rice were collected from nationwide in 2012. Three hundred seventy one samplesof rice and its by-products were analyzed for three trichothethenes including nivalenol (NIV), deoxynivalenol(DON), and zearalenone (ZEA) by LC/MS. Discolored rice samples were found to have the highestcontamination of DON, NIV or ZEA, followed by broken rice. Polished rice samples were largely free frommycotoxins, except three samples which were contaminated with NIV or DON at safety level. The rice by-product samples were contaminated at higher level and frequencies than polished rice samples.Keywords : Fusarium, Mycotoxins, Rice, RPCs

This study investigated and evaluated contamination levels of bacteria on tomatoes at farms stage to evaluate potential hazards associated with fresh tomatoes. A total of 170 samples, 90 samples from 5 sampling sites from 18 farms and 80 samples from 1 sampling site from 4 farms every month for four months, were analyzed to enu- merate aerobic bacterial counts, coliforms, E. coli, Bacillus cereus, Staphylococcus aureus, Escherichia coli, E. coli O157:H7, Salmonella spp., and Listeria monocytogenes. Aerobic bacterial counts ranged from 0.48 to 6.15 Log CFU/ g, with the lowest and the highest bacterial cell counts recorded for A site and E site, respectively. Thirty five percent of the samples from the E site contained more than 2 Log CFU/g. Six samples (6.6%) of 90 samples contained B. cereus less than 1 Log CFU/g. In addition, the contamination level of indicator bacteria and B. cereus in tomatoes were higher on March than on April, May and June (P < 0.05). S. aureus, E. coli, E. coli O157:H7, Salmonella spp. and L. monocytogenes were not detected in the tomato samples. The microbial contamination levels of tomatoes deter- mined in this study may be used as the data for microbiological risk assessment.

Animal manure compost is a commonly used fertilizer in organic vegetable and fruit production in Korea. However, livestock manure compost produced from animal feces can contain a lot of the non-pathogenic and pathogenic bacteria. Of particular concern are bacteria causing human food-borne illness such as Escherichia coli O157:H7 and Listeria monocytogenes. The objective of this study was to investigate effect of temperature on survival of E. coli O157:H7 and L. monocytogenes in livestock manure compost. Commercial livestock manure compost (manure 60%, sawdust 40%) was inoculated with E. coli O157:H7 and L. monocytogenes. Compost was incubated at four different temperatures (10, 25, 35, and 55℃) for 20 weeks. Samples were taken every week during incubation depending on the given conditions. E. coli O157:H7 persisted for up to 1 day in livestock manure compost at 55℃, over 140 days at 10℃, 140 days at 25℃, and 120 days at 35℃, respectively. L. monocytogenes persisted for up to 1 day in livestock manure compost at 55℃ and 140 days at 10℃, 70 days at 25℃, and 40 days at 35℃, respectively. The results indicated that E. coli O157:H7 and L. monocytogenes persisted longer under low temperature condition. E. coli O157:H7 survived longer than L. monocytogenes at three different temperatures (10, 25, and 35℃). The results are being used to develop guidelines on the management of manure to reduce the risks of E. coli O157:H7 and L. monocytogenes transmission to foods produced in the presence of animal waste.

Fusaric acid (FA) is a mycotoxin produced by Fusarium species. Its toxicity is relatively low but often associated with other mycotoxins, thus enhancing total toxicity. To date, biosynthetic genes or enzymes for FA have not been identified in F. oxysporum. In order to explore the genetic element(s) for FA biosynthesis, restriction enzyme mediated integration (REMI) procedure as an insertional mutagenesis was employed using FA producing-F. oxysporum strains. Genetic transformation of two F. oxysporum strains by REMI yielded more than 7,100 transformants with efficiency of average 3.2 transformants/μg DNA. To develop a screening system using phytotoxicity of FA, eleven various grains and vegetable seeds were tested for germination in cultures containing FA: Kimchi cabbage seed was selected as the most sensitive host. Screening for FA non-producer of F. oxysporum was done by growing each fungal REMI transformant in Czapek-Dox broth for 3 weeks at 25oC then observing if the Kimchi cabbage seeds germinated in the culture filtrate. Of more than 5,000 REMI transformants screened, fifty-three made the seeds germinated, indicating that they produced little or fewer FA. Among them, twenty-six were analyzed for FA production by HPLC and two turned out to produce less than 1% of FA produced by a wild type strain. Sequencing of genomic DNA regions (252 bp) flanking the vector insertion site revealed an uncharacterized genomic region homologous (93%) to the F. fujikuroi genome. Further study is necessary to determine if the vector insertion sites in FA-deficient mutants are associated with FA production.

To investigate contamination of ground red pepper with fungi and mycotoxin, we obtained 30 ground red pepper samples from 15 manufacturers in the main chili-pepper-producing areas in Korea. Fungal contamination was evaluated by spreading diluted samples on potato dextrose agar plates. The total fungi counts ranged from 0 to 7.3 × 103 CFU/g. In the samples, the genus Aspergillus had the highest incidence, while Paecilomyces was isolated most frequently. The next most frequent genera were Rhizopus, Penicillium, Cladosporium, and Alternaria. Within Aspergillus, A. ruber was predominant, followed by A. niger, A. amstelodami, A. ochraceus, A. terreus, A. versicolor, A. flavus, and A. fumigatus. The samples were analyzed for aflatoxins, ochratoxin A, and citrinin by ultra-perfomance liquid chromatography (UPLC) with a fluorescence detector. Ochratoxin A was detected from three samples at 1.03‒2.08 μg/kg, whereas no aflatoxins or citrinin were detected. To test the potential of fungal isolates to produce aflatoxin, we performed a PCR assay that screened for the norB-cypA gene for 64 Aspergillus isolates. As a result, a single 800-bp band was amplified from 10 A. flavus isolates, and one Aspergillus sp. isolate. UPLC analyses confirmed aflatoxin production by nine A. flavus isolates and one Aspergillus sp. isolate, which produced total aflatoxins at 146.88‒909.53 μg/kg. This indicates that continuous monitoring of ground red pepper for toxigenic fungi is necessary to minimize mycotoxin contamination.

Staphylococcus haemolyticus S167 has the ability to produce biofilms in large quantities. Genomic analyses revealed information on the biofilm-related genes of S. haemolyticus S167. Detailed studies of biofilm formation at the molecular level could provide a foundation for biofilm control research.

Microbial Safety Division, National Academy of Agricultural Science, Rural Development Administration,Suwon 441-707, Korea(Received on November 14, 2011; Revised on November 25, 2011; Accepted on November 29, 2011)Fusarium species are worldwide causal agents of Fusarium head blight (FHB) in cereals such as barley andwheat. Their toxigenic potential is a health risk for both humans and animals. To survey the naturaloccurrence of FHB and mycotoxins produced by Fusarium, total 126 barley or wheat grains grown in 2009-2010 season in Korea were collected. The incidence of FHB was 30.7% in silage barley, 26.9% in wheat,20.7% in naked barley, 19.4% in malting barley, 16.4% in unhulled barley. Overall FHB incidence of barleyand wheat in 2010 was 23.0% and 10% higher than that of 2009. The incidences and level of deoxynivalenol(DON), nivalenol (NIV), and zearalenone (ZEA) were 34%, 0.89 mg/kg, 84.9%, 1.86 mg/kg, and 10.3%, 0.06mg/kg respectively. The both levels and incidences of NIV were found to be highest in barley, whereas thelevel of DON was found to be highest in wheat. Incidences of DON and NIV and the level of NIV in thesamples from southern regions of Korea were higher than those from central region, whereas the level ofDON from central region was higher than that from southern regions. This is the first paper demonstratingregional difference in natural occurrence of DON and NIV in wheat and barley. Keywords : Deoxynivalenol, Fusarium head blight (FHB), Natural occurrence, Nivalenol, Zearalenone

Fusarium verticillioides (teleomorph: Gibberella moniliformis), a member of the Gibberellea fujikuroi species complex, causes rots of corn stalks and ears, and produces a group of mycotoxins known as fumonisins that are harmful to animals and humans. Here, we focus on the development of a species-specific PCR primer set for differentiating F. verticillioides from other fumonisin-producing Fusarium species belonging to the species complex, such as F. proliferatum, F. fujikuroi, and F. subglutinans that are frequently associated with corn. The specific primers (RVERT1 and RVERT2) derived from the nucleotide sequences of RNA polymerase II beta subunit (RPB2) gene amplified a 208 bp-DNA fragment from only F. verticillioides isolates among the potential fumonisin-producing species examined; all of these isolates were shown to carry FUM1 required for fumonisin biosynthesis. The PCR detection limit using this specific primer set was approximately 0.125 pg/ genomic DNA of F. verticillioides. In addition, the F. verticillioides-specfic fragment was successfully amplified from genomic DNAs of corn samples contaminated with Fusarium spp. This primer set would provide a useful tool for the detection and differentiation of potential fumonisin-producing F. verticillioides strains in cereal samples.

This investigation was undertaken to survey toxigenic fungal contamination of various rice samples in 93 rice processing complexes (RPC) in Korea. Rice was grown in 2009 and the samples were collected in 2010. Seven types of rice samples such as unhusked, brown, blue-tinged, discolored, polished, half-crushed, and rice husks were obtained from each RPC. One-hundred and five grains of each sample were placed on PDA plates after surface disinfection. The incidence of fungal contaminants was 26.8%. Aspergillus spp. was the most dominant fungal contaminants and Fusarium spp. was the most frequently occurred in samples. The heaviest Fusarium contamination was found in unhusked grain, rice husks, and bare blue-tinged rice and followed by colored rice whereas broken rice was the least contaminated. Regional difference of fungal contamination was distinctive. Fusarium incidence in the rice samples from southern region of Korea including Jeolla and Gyeongsang Provinces was higher than those from central region including Chungcheong, Gyeonggi, and Gangwon Provinces. In contrast to Fusarium spp., Aspergillus spp. and Penicillium spp. were dominated in brown and polished rice samples and their incidences were more severe in central region than southern region. The major contaminants shown more than 1% of kernels infected were Aspergillus (5.0%), Fusarium (2.0%), Alternaria (1.4%), Dreschlera (1.3%), Penicillium spp. (1.3%), and Nigrospora spp. (1.0%). Collectotrichum, Pyricularia, Myrothecium, Epicoccum, Cladosporium, Moniliella, Gloeocercospora, Chaeto- mium, Curvularia, Phialopora, Acremonium, Gliomastix, Trichoderma, Rhizopus, Phomopsis, Paecilomyces, Genicularia, Geotrichum, Acremoniella, Rhizoctonia, Phoma, Oidiodendran, and Candida spp. were among the rest observed at low incidence. The major contaminants of rice samples were well-known as toxigenic fungal genera so toxin producibility of these fungal isolates is necessary to be examined in future. It is also needed to study Myrothecium spp. on species level as it was detected for the first time in rice.

A total of 201 samples of brown rice, polished rice, and two types of by-products, blue-tinged rice and discolored rice, were collected from rice stores maintained at 51 rice processing complexes in Korea. These samples were analyzed for the presence of Fusarium mycotoxins such as deoxynivalenol (DON), nivalenol (NIV), and zearalenone (ZEA). Contaminants (and their ranges) found in discolored rice samples were DON (59 to 1,355 ng g(-1)), NIV (66 to 4,180 ng g(-1)), and ZEA (25 to 3,305 ng g(-1)); those found in blue-tinged (less-ripe) rice were DON (86 to 630 ng g(-1)), NIV (50 to 3,607 ng g(-1)), and ZEA (26 to 3,156 ng g(-1)). Brown rice samples were contaminated mostly with NIV and ZEA (52 to 569 ng g(-1) and 47 to 235 ng g(-1), respectively). Polished rice samples were largely free from mycotoxins, although one sample was contaminated with NIV (77 ng g(-1)). When the fungal flora associated with each rice sample was investigated, blue-tinged rice was the most often contaminated with Fusarium graminearum (3.8%), followed by the discolored rice (2.4%) and brown rice (1.6%) samples. Using PCR, toxin genotyping of 266 isolates of F. graminearum revealed that most isolates (96%) were NIV producers. In conclusion, this survey is the first report of the cocontamination of Korean rice and its by-products with trichothecenes and ZEA. Importantly, it also provides new information on the natural contamination of rice by Fusarium mycotoxins.

To detect Fusarium mycotoxins, grain samples were collected from 32 rice fields all over the country and from 19 maize fields in eastern and midland provinces in Korea in 2009. Maize contamination with Fusarium species (54.9%) was higher than in rice (8.2%). Using Fusarium species specific PCR primer sets (Fg16 and VERT), 58 and 354 of total 506 isolates from maize samples were putatively identified as F. graminearum (11.5%) and F. verticillioides (70.0%), respectively. From rice samples, 276 of 315 isolates (87.8%) were putatively identified as F. graminearum but F. verticillioides was not identified. LC or LC-MS analysis of the samples revealed that fumonisin was the most commonly detected mycotoxin in maize samples but its level was below the regulation limit. Only two maize samples were contaminated with deoxynivalenol and zearalenone at the levels above the regulation limit. In rice samples, contamination with zearalenone was common but the levels were below the regulation limit. This study showed that most of the Korean maize and rice samples collected in 2009 were contaminated with Fusarium mycotoxins but the levels were below the Korean regulations for deoxynivalenol, fumonisin and zearalenone.

The incidence and occurrence of virus infecting paprika (Capsicum annuum var. grossum) in Jeonnam province, the main areas of cultivation in Korea is undetermined. In this study, a total of 1,020 samples with virus-like symptoms were collected in Jeonnam province during summer season for 3 consecutive years (2002-2005) and were tested using enzyme linked immunosorbent assay (ELISA). Results showed that Pepper mottle virus (PepMoV), Broad bean wilt virus (BBWV), and Cucumber mosaic virus (CMV) were found to be the most prevalent viruses with a 3-year average percent incidence of 41.3, 19.8, and 4.4 respectively. Mixed infection with more than two viruses was also found with 3.5%, 17.0%, and 8.3%, respectively. Symptoms of these virus diseases were not evident at the seedling stage but slowly appeared at the transplanting stage and increased to the middle stage (4-5 months after transplanting) during the 3-year cultivation periods. Symptom appearance of infected plants however varied largely with transplanting time. Those plants transplanted from November to January were found to be infected with viruses in June, whereas symptoms appeared with in a month those plants transplanted from June to August. There were differences in the virus incidence from primary factor such as district, type of green house and variety, but these were not statistically significant (data not shown). Recommended control measures of paprika against these viruses is also discussed in this paper.

Powdery mildew of lettuce that is a newly reported disease became a threat to organic cultivation of lettuce in Korea since the disease caused by Podosphaera fusca resulted in a half of yield loss in heavily infected fields. To improve micro-environmental conditions around lettuce, ACF (air-circulation fan) was installed on inside roof of plastic house at 6 m intervals. The ACF increased 57% of lettuce yield and reduced 71.4% of lettuce seedling death. COY (cooking oil and yolk mixture) consisted of cooking oil 0.3% and egg yolk 0.08% reduced lettuce seedling death from 89.3% to 92.9% under the greenhouse. Seven-day interval spray of COY resulted in high control values of powdery mildew of lettuce ranging from 89.6% to 96.3%, which was comparable to a fungicide, Azoxystrobin. Lettuce yield was increased about two times compared to a non-treated conventional cultivation. Qualities of lettuce such as hardness and chlorophyll content were also improved by COY and ACF combination. Effect of COY on control of the disease was improved when or 1,000 ppm was supplemented. Results indicated that the COY made of cooking oil such as canola emulsified with yolk was highly effective on control of powdery mildew of lettuce and suitable for organic agriculture, especially when combined with ACF.

The 1080 epiphytic bacteria obtained from 370 samples of pome and stone fruits including apple, pear, peach, grape, apricot and Chinese quince were screened for antagonistic activity against postharvest pathogens, Penicillium expansum, Alternaria alternata and Botrytis cinerea. Among tested antagonistic bacteria, eight bacterial isolates inhibited mycelial growth of the postharvest pathogens and were identified as Bacillus amyloliquefaciens (three strains), B. megaterium, B. subtilis var. gladioli, B. licheniformis, B. pumilus and Serratia marcescens based on biochemical characteristics and utility of carbon and nitrogen compounds (Biolog system). Eight carbohydrates were evaluated for their effect on mycelial growth and germination of the postharvest pathogen, P. expansum to select nutrients for enhancing bio-control efficacy. The growth of four selected antagonists, B. amyloliquefaciens P43-2, B. amyloliquefaciens A71-2, B. licheniformis P94-1, and S. marcescens P76-9 were also tested. As a result, 1% glucose (w/v) strongly stimulated growth of the antagonists, suppressed mycelial growth of the postharvest pathogen, and had a little comparatively stimulatory effect on germination of the the postharvest pathogen. It was confirmed that the addition of 1% glucose (w/v) greatly enhanced biocontrol effect of B. amyloliquefaciens P43-2, B. licheniformis P94-1, and S. marcescens P76-9. Application of B. amyloliquefaciens P43-2, B. licheniformis P94-1, and S. marcescens P76-9 with the addition of 1% glucose (w/v) increased the control efficacy up to 48%, 46%, 14% compared with those of the antagonists without glucose, respectively. When the antagonists were applied to control postharvest disease caused by P. expansum in apple wounds, the population of B. amyloliquefaciens P43-2 and B. licheniformis P94-1 increased until 4 days after inoculation (DAI) of the antagonists and then decreased from 10 DAI. Meanwhile the population of S. marcescens P76-9 decreased at early stage (4 DAI), but increased from 7 DAI, and finally maintained constantly until 10 DAI in apple wounds.

A total of 509 rice panicle samples were collected at harvest time from fields in 8 provinces from 2010 to 2014. One hundred five grains per sample were plated on potato dextrose agar and 6,658 Fusarium isolates were obtained; among them, 67 were identified as Fusarium armeniacum by sequencing the translation elongation factor () and confirmed by their morphological and cultural characteristics. Considerable variation in conidial size, colony color and sequences was observed among the fungal isolates. The ability of 24 F. armeniacum isolates to produce T-2 and HT-2 toxin in potato sucrose agar was determined using liquid chromatography-mass spectrometry. Twenty one isolates produced T-2 and HT-2 toxin, resulting in varying toxin levels among the isolates. The results show that Korean isolates of F. armeniacum have diversity with respect to morphological, cultural, genetic, and toxigenic properties.

This study conducted microbiological assessment in tunnel style strawberry greenhouses and packaging centers and suggested recommendations to establish a good agricultural practice for strawberry production. The samples from irrigation water, workers' gloves, harvest bins, soil, strawberry leaves and strawberries in greenhouses, packers' gloves, conveyor belts, packaging tables, and door knobs of entrances in packaging centers were collected. Bacterial cell counts of aerobic plate counts, coliforms, Escherichia coli, E. coli O157:H7, Salmonella, Staphylococcus aureus, and Bacillus cereus were then enumerated on appropriate selective media. In general, bacterial populations were similar (p ≥ 0.05) among strawberry greenhouses but not among packaging houses. E. coli and E. coli O157:H7 were negative in all samples, and low levels of Salmonella and B. cereus were detected. However, high bacterial cell counts of aerobic plate counts, coliforms, and S. aureus were found in most samples. These results suggest that food safety practice in strawberry greenhouses and packaging centers should be improved, and the results may be useful in the establishment of a good agricultural practice system for strawberry production.