Prevalence, distribution, enterotoxin profiles, antimicrobial resistance, and genetic diversity of Bacillus cereus group isolates from lettuce farms in Korea.

Lettuce wraps are popular in Korean cuisine for their high nutritional value and versatility as healthy additions to multiple dishes. Microbial contamination of lettuce is a major concern, as lettuce is consumed fresh without cooking. Among foodborne pathogens, the spore-forming, facultative anaerobic bacterium, Bacillus cereus is one of the frequently detected pathogen in lettuce in Korea. In this study, we investigated the prevalence and distribution of Bacillus cereus strains in lettuce production farms and further evaluated the enterotoxin gene profiles, antibiotic susceptibility, multidrug resistance pattern, and genetic differences among the B. cereus group isolates. Of the 140 samples isolated from 10 lettuce production farms, 30 samples (21.42%) were positive for B. cereus in which 19 (31.6%) and 10 (23.25%) were from soil and lettuce, respectively. The enterotoxin patterns A (hblCDA, nheABC, entFM, and cytK genes) and B (hblCDA, nheABC, and entFM genes) accounted for 50% and 20% of all the isolates, whereas the emetic gene cesB was not detected in any of the B. cereus group isolates. Antibiotic susceptibility testing of the B. cereus group isolates revealed that all the strains were predominantly resistant to ß-lactam antibiotics except imipenem and generally susceptible to most of the non ß-lactam antibiotics, including gentamycin, streptomycin, chloramphenicol, and tetracycline. ERIC-PCR and MLST analysis revealed high genetic diversity among the 30 B. cereus group isolates, which belonged to 26 different sequence types (STs) and seven new STs. Moreover, isolates with identical STs exhibited similar patterns of antibiotic resistance and enterotoxin profiles. Results of this study indicate a high prevalence of B. cereus group isolates in lettuce production farms in the Republic of Korea.

Effect on Colony Growth Inhibition of Soil-Borne Fungal Pathogens by Available Chlorine Content in Sodium Hypochlorite.

Our study investigated the available chlorine content, contact time and difference among strains of each pathogen for sodium hypochlorite (NaOCl) to control chemically against soil-borne fungal pathogens, such as Phytophthora rot by Phytophthora cactorum, violet root rot by Helicobasidium mompa, and white root rot by Rosellinia necatrix, causing die-back symptom on apple trees. As a result, the colony growth of Phytophthora cactorum was inhibited completely by soaking over 5 s in 31.25 ml/l available chlorine content of NaOCl. Those of H. mompa and R. necatrix were inhibited entirely by soaking over 160 s in 62.5 and 125 ml/l available chlorine content in NaOCl, respectively. Also, inhibition effect on available chlorine in NaOCl among strains of each soil-borne pathogen showed no significant difference and was similar to or better than that of fungicides.

Re-exploration of U's Triangle Brassica Species Based on Chloroplast Genomes and 45S nrDNA Sequences.

The concept of U's triangle, which revealed the importance of polyploidization in plant genome evolution, described natural allopolyploidization events in Brassica using three diploids [B. rapa (A genome), B. nigra (B), and B. oleracea (C)] and derived allotetraploids [B. juncea (AB genome), B. napus (AC), and B. carinata (BC)]. However, comprehensive understanding of Brassica genome evolution has not been fully achieved. Here, we performed low-coverage (2-6×) whole-genome sequencing of 28 accessions of Brassica as well as of Raphanus sativus [R genome] to explore the evolution of six Brassica species based on chloroplast genome and ribosomal DNA variations. Our phylogenomic analyses led to two main conclusions. (1) Intra-species-level chloroplast genome variations are low in the three allotetraploids (2~7 SNPs), but rich and variable in each diploid species (7~193 SNPs). (2) Three allotetraploids maintain two 45SnrDNA types derived from both ancestral species with maternal dominance. Furthermore, this study sheds light on the maternal origin of the AC chloroplast genome. Overall, this study clarifies the genetic relationships of U's triangle species based on a comprehensive genomics approach and provides important genomic resources for correlative and evolutionary studies.

Evaluation of Resistance to Ralstonia solanacearum in Tomato Genetic Resources at Seedling Stage.

Bacterial wilt of tomatoes caused by Ralstonia solanacearum is a devastating disease that limits the production of tomato in Korea. The best way to control this disease is using genetically resistant tomato plant. The resistance degree to R. solanacearum was evaluated for 285 tomato accessions conserved in the National Agrobiodiversity Center of Rural Development Administration. These accessions of tomato were originated from 23 countries. Disease severity of tomato accessions was investigated from 7 days to 14 days at an interval of 7 days after inoculation of R. solanacearum under greenhouse conditions. A total of 279 accessions of tomato germplasm were susceptible to R. solanacearum, resulting in wilt and death in 70 to 90% of these plants. Two tomato accessions were moderately resistant to R. solanacearum. Only four accessions showed high resistance against R. solanacearum. No distinct symptom of bacterial wilt appeared on the resistant tomato germplasms for up to 14 days after inoculation of R. solanacearum. Microscopy of resistant tomato stems infected with R. solanacearum revealed limited bacterial spread with thickening of pit membrane and gum production. Therefore, these four resistant tomato germplasms could be used in tomato breeding program against bacterial wilt.

Comparison of sample preparation methods for the recovery of foodborne pathogens from fresh produce.

Sample preparation methods (pummeling, pulsifying, sonication, and shaking by hand) were compared for achieving maximum recovery of foodborne pathogens from iceberg lettuce, perilla leaves, cucumber, green pepper, and cherry tomato. Antimicrobial and dehydration effects also were examined to investigate causes of poor recovery of pathogens. Each produce type was inoculated with Escherichia coli O157:H7, Salmonella Typhimurium, Listeria monocytogenes, Staphylococcus aureus, and Bacillus cereus at 6.0 log CFU/cm(2), and samples were prepared using the four methods. Bacterial populations recovered from the five types of produce were significantly different (P < 0.05) according to sample preparation methods and produce type. The bacterial populations recovered from pummeled and pulsified samples were higher (P < 0.05) than those recovered from sonicated and hand-shaken samples, except for cherry tomato. The number of bacteria recovered from produce was reduced (P < 0.05) from that of the inoculum by 0.16 to 2.69 log CFU/cm(2). Although extracts of iceberg lettuce, perilla leaves, cucumber, and green pepper had no antimicrobial activity, the populations of E. coli O157:H7, Salmonella Typhimurium, B. cereus, and L. monocytogenes in cherry tomato extract were slightly reduced after these treatments (P < 0.05). The pathogen populations on perilla leaves and cherry tomatoes decreased by >2 log CFU/cm(2) after exposure to 40% relative humidity for 1 h. No reduction was observed when the five pathogens were exposed to 90% relative humidity. These data suggest that pummeling and pulsifying are optimal sample preparation methods for detection of microorganisms. Acidic produce such as cherry tomato should be treated with a method that does not cause sample breakdown so that acid stress on the bacteria can be minimized. Dehydration stress also affects recovery of pathogens from produce.

Genetic diversity of cultivable plant growth-promoting rhizobacteria in Korea.

To elucidate the biodiversity of plant growth-promoting rhizobacteria (PGPR) in Korea, 7,638 bacteria isolated from the rhizosphere of plant species growing in many different regions were screened. A large number of PGPR were identified by testing the ability of each isolate to promote the growth of cucumber seedlings. After redundant rhizobacteria were removed via amplified rDNA restriction analysis, 90 strains were finally selected as PGPR. On the basis of 16S ribosomal RNA sequences, 68 Gram-positive (76%) and 22 Gram-negative (24%) isolates were assigned to 21 genera and 47 species. Of these genera, Bacillus (32 species) made up the largest complement, followed by Paenibacillus (19) and Pseudomonas (11). Phylogenetic analysis showed that most of the Grampositive PGPR fell into two categories: low- and high- G+C (Actinobacteria) strains. The Gram-negative PGPR were distributed in three categories: alpha-proteobacteria, beta- proteobacteria, and gamma-proteobacteria. To our knowledge, this is the largest screening study designed to isolate diverse PGPR. The enlarged understanding of PGPR genetic diversity provided herein will expand the knowledge base regarding beneficial plant-microbe interactions. The outcome of this research may have a practical effect on crop production methodologies.

Thiosulfate Oxidation and mixotrophic growth of Methylobacterium goesingense and Methylobacterium fujisawaense.

The mixotrophic growth with methanol plus thiosulfate was examined in nutrient-limited mixotrophic condition for Methylobacterium goesingense CBMB5 and Methylobacterium fujisawaense CBMB37. Thiosulfate oxidation increased the growth and protein yield in mixotrophic medium that contained 150 mM methanol and 20 mM sodium thiosulfate, at 144 h. Respirometric study revealed that thiosulfate was the most preferable reduced inorganic sulfur source, followed by sulfite and sulfur. M. goesingense CBMB5 and M. fujisawaense CBMB37 oxidized thiosulfate directly to sulfate, and intermediate products of thiosulfate oxidation such as polythionates, sulfite, and sulfur were not detected in spent medium and they did not yield positive amplification for tested soxB primers. Enzymes of thiosulfate oxidation such as rhodanese and sulfite oxidase activities were detected in cell-free extracts of M. goesingense CBMB5, and M. fujisawaense CBMB37, and thiosulfate oxidase (tetrathionate synthase) activity was not observed. It indicated that both the organisms use the "non-S4 intermediate" sulfur oxidation pathway for thiosulfate oxidation. It is concluded from this study that M. goesingense CBMB5, and M. fujisawaense CBMB37 exhibited mixotrophic metabolism in medium containing methanol plus thiosulfate and that thiosulfate oxidation and the presence of a "Paracoccus sulfur oxidation" (PSO) pathway in methylotrophic bacteria are species dependant.

Chemolithoautotrophic oxidation of thiosulfate and phylogenetic distribution of sulfur oxidation gene (soxB) in rhizobacteria isolated from crop plants.

Twenty-one thiosulfate-oxidizing bacteria were isolated from rhizosphere soils and 16S rRNA analysis revealed that the isolates were affiliated with seven different phylogenetic groups within the Beta and Gamma subclasses of Proteobacteria and Actinobacteria. Among these, five genera, including Dyella, Burkholderia, Alcaligenes, Microbacterium and Leifsonia sp., represented new sulfur oxidizers in rhizosphere soils. The thiosulfate-oxidizing Dyella, Burkholderia, Alcaligenes, Microbacterium, Leifsonia and Pandoraea were able to grow chemolithotrophically with a medium containing thiosulfate and exhibited growth coupled with thiosulfate oxidation. They accumulated intermediate products such as sulfur, sulfite and trithionate in the spent medium during the time course of thiosulfate oxidation, and these products were finally oxidized into sulfate. Furthermore, they possessed thiosulfate-metabolizing enzymes such as rhodanese, thiosulfate oxidase, sulfite oxidase and trithionate hydrolase, suggesting that these bacteria use the 'S4 intermediate' (S4I) pathway for thiosulfate oxidation. Phylogenetic analysis of the soxB gene revealed that Pandoraea sp. and Pandoraea pnomenusa strains formed a separate lineage within Betaproteobacteria.