Jae-Gee Ryu

Identification of a 12-Gene Fusaric Acid Biosynthetic Gene Cluster in Fusarium Species Through Comparative and Functional Genomics

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.

Occurrence of Fusarium mycotoxins in rice and its milling by-products in Korea.

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.

Toxigenic Fungal Contaminants in the 2009-harvested Rice and Its Milling-by products Samples Collected from Rice Processing Complexes in Korea

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.

Natural Occurrence of Fusarium Head Blight and Its Mycotoxins in 2010-harvested Barley and Wheat Grains in Korea

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

Incidence and Distribution of Virus Diseases on Paprika (Capsicum annuum var. grossum) in Jeonnam Province of Korea

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.

Natural Occurrence of Fusarium Mycotoxins in Field-collected Maize and Rice in Korea in 2009

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.

Detection of Fusarium verticillioides Contaminated in Corn Using a New Species-specific Primer

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.

Mycobiota of ground red pepper and their aflatoxigenic potential

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.

Occurrence of Fungi and Fusarium Mycotoxins in the Rice Samples from Rice Processing Complexes

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.

Effects of Small Scale Post-Harvest Facility and Hygiene Education on the Level of Microbial Safety in Korean Leeks Production

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.