• Research in Plant Disease
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• v.18 no.2
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• pp.73-79
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• 2012

Phytophthora katsurae is a fungal pathogen responsible for chestnut ink disease. We designed two duplex primer sets (SOPC 1F/1R+KatI 3F/5R, SOPC 1-1F/1-1R+KatI 3F/5R) to detect P. katsurae. SOPC 1F/1R and SOPC 1-1F/1-1R primer pairs were designed for sequence characteristic amplification regions (SCAR) marker, and KatI 3F/5R primer pair was used for P. katsurae-specific primer designed from internal transcribed spacer (ITS) region. To assess the sensitivity of duplex PCR, genomic DNA was serially diluted 10-fold to make the final concentrations from 1 mg/ml to 1 ng/ml. The sensitivity for two primer sets were 1 ${\mu}g/ml$ and 100 ng/ml, respectively. To find detection limits for zoospores of P. katsurae, each zoospore suspension was serially diluted 10-fold to make the final concentrations from $1{\times}10^6$ to $1{\times}10^2$ cells/ml, and then DNA was extracted. The limits of detection for all of two primer sets were $1{\times}10^5$ cells/ml. All of two primer sets were specific to P. katsurae in PCR detection and did not produce any P. katsurae-specific PCR amplicons from other 16 Phytophthora species used as the control. This study shows that duplex PCR using two primer sets might be a useful tool for rapid and efficient detection of P. katsurae.

• The Plant Pathology Journal
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• v.32 no.1
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• pp.53-57
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• 2016

Major diseases in grafted cacti have been reported and Fusarium oxysporum, Bipolaris cactivora, Phytophthora spp. and Collectotrichum spp. are known as causal pathogens. These pathogens can lead to plant death after infection. Therefore, some European countries have quarantined imported cacti that are infected with specific fungal pathogens. Consequently, we developed PCR detection methods to identify four quarantined fungal pathogens and reduce export rejection rates of Korean grafted cacti. The pathogen specific primer sets F.oF-F.oR, B.CF-B.CR, P.nF-P.nR, and P.cF-P.CR were tested for F. oxysporum, B.cactivora, P. nicotinae, and P. cactorum, respectively. The F.oF-F.oR primer set was designed from the Fusarium ITS region; the B.CF-B.CR and P.nF-P.nR primers respectively from Bipolaris and Phytophthora ITS1; and the P.cF-P.CR primer set from the Ypt1protein gene region. The quarantine fungal pathogen primer pairs were amplified to the specific number of base pairs in each of the following fungal pathogens: 210-bp (F. oxysporum), 510-bp (B. cactivora), 313-bp (P. nicotinae), and 447-bp (P. cactorum). The detection limit for the mono- and multiplex PCR primer sets was 0.1 ng of template DNA under in vitro conditions. Therefore, each primer set successfully diagnosed contamination of quarantine pathogens in export grafted cacti. Consequently, our methodology is a viable tool to screen contamination of the fungal pathogen in exported grafted cacti.

• Journal of Industrial Technology
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• v.28 no.A
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• pp.141-147
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• 2008

Several contaminated bacteria such as Lactobacillus brevis and Pediococcus damnosus in beer production cause beer spoilage by producing off flavours and turbidity. Detection of these organisms is complicated by the strict anaerobic conditions and lengthy incubation times required for their cultivation, consequently there is a need for more rapid detection methods. Recently, two contaminated strains were isolated from vessel of beer production and identified as Lactobacillus species by API kit identificaton as well as 16S-23S ITS sequencing analyses. Two isolated strains were named as Lactobacillus sp. HLA1 and Lactobacillus HLB2, respectively. A polymerase chain reaction (PCR) method was developed for the rapid and specific detection of Lactobacillus sp.. Two sets of primer pairs (HLA1-F/HLA1-R and HLB2-F/HLB2-R) were designed for the amplification of a 1576 base pair (bp) fragment of the HLA1 16S-23S rRNA gene and 1888 bp fragement of the HLB2 16S-23S rRNA. Amplified PCR products were highly specific to detect corresponding bacteria when other contaminated strains were used as PCR templates. However, detection of both strains were limited when $100{\mu}{\ell}$ of cultured samples were mixed with $100m{\ell}$ of beer sample in arbitrary manner. The sensitivity of the assay still needs to be improved for direct detection of the small amounts of bacteria present in beer.

• Mycobiology
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• v.41 no.4
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• pp.252-255
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• 2013

Fungal pathogens have caused severe damage to the commercial production of Pleurotus eryngii, the king oyster mushroom, by reducing production yield, causing deterioration of commercial value, and shortening shelf-life. Four strains of pathogenic fungi, including Trichoderma koningiopsis DC3, Phomopsis sp. MP4, Mucor circinelloides MP5, and Cladosporium bruhnei MP6, were isolated from the bottle culture of diseased P. eryngii. A species-specific primer set was designed for each fungus from the ITS1-5.8S rDNA-ITS2 sequences. PCR using the ITS primer set yielded a unique DNA band for each fungus without any cross-reaction, proving the validity of our method in detection of mushroom fungal pathogens.

• Mycobiology
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• v.30 no.4
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• pp.197-201
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• 2002

Specific primer sets based on ribosomal DNA(rDNA) internal transcribed specer(ITS) sequences were designed for rapid detection of Phellinus linteus and P. baumii. Polymerase chain reaction(PCR) with these primers produced unique bands for each Phellinus species. The annealing temperature range is from $40^{\circ}C\;to\;55^{\circ}C$. The length of PCR products(P. linteus and P. baumii) using designed combinative primer sets of PL1F, PL2R, PB1F, PB2R, ITS5F and ITS4R, were from 520 by to 730 bp. Fifteen strains of Phellinus species including P. linteus, P. baumii, P. weirianus, P. johnsonianus, P. rhabarberinus, P. pini, P. gilvus, P. igniarius, P. nigricans and P. laevigatus were examined in this study. Five strains, including two isolated strains of P. linteus(MPNU 7001 and MPNU 7002), and two isolated strains of P. baumii(MPNU 7004 and MPNU 7005) were shown to have about 520 bp (PL1F-PL2R), 700 bp (TTS5F-PL2R) and 600 bp (PB1F-ITS4R) -sized PCR single bands respectively. This molecular genetic technique provided a useful method for rapid detection and identification of P. linteus and P. baumii.

• Mycobiology
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• v.29 no.1
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• pp.7-10
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• 2001

Based on the rDNA ITS sequences data, specific primer set for PCR detection of wood-decaying fungus Phellinus linteus was designed. The length of PCR products using designed primer set(SHF and SHR) was about 540 bp. Among 11 species, 17 isolates of Phellinus spp. including Phellinus linteus, P. pomaceus, P. spiculosus, P. baumi, P. pini, P. igniarius, P. gilvus, P. biscuspidatus, P. weirii, P. johnsonianus, P. robutus, and P. igniarius, seven isolates of Phellinus linteus showed about 540 bp-sized single band. This molecular technique could offer a useful tool for detecting and identifying Phellinus linteus.

• The Plant Pathology Journal
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• v.24 no.1
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• pp.17-23
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• 2008

A total of 33 isolates of Colletotrichum species obtained from pepper, apple, and strawberry in 2001 and 2002 were identified based on mycological characteristics, responses to fungicides carbendazim and the mixture of carbendazim and diethofencarb, and nucleotide sequence analysis of internal transcribed spacer (ITS) regionMost of the Colletotrichum isolates from pepper could be identified as C. acutatum. The pepper isolates produced grey white mycelia that gradually changed to dark gray. The conidia were variable in size, and almost cylindrical in shape with at least one rounded end. They could grow on PDA amended with carbendazim or with the mixture of carbendazim and diethofencarb at $10{\mu}g/ml$, to which the isolates from apple and strawberry were very sensitive. A part of the ITS regions from the Colletotrichum isolates was amplified with the specific primers designed for C. acutatum (Ca1-1) or C. gloeosporioides (Cg1-3). A primer pair of Ca1-1 and a universal primer (ITS4) amplified a 496-bp DNA fragment from all of the pepper isolates examined and one apple isolate. Taken together, it is conclusive that the Colletotrichum isolates causing the typical lesion of anthracnose on pepper fruits are C. acutatum.

• Food Science and Biotechnology
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• v.17 no.2
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• pp.357-361
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• 2008

One of the major allergenic proteins in common buckwheat (Fagopyrum elculentum) was found to be a BW10KD. In this work, allergenic BW10KD genomic DNAs from the native common buckwheat 'Pyeongchang' and Tartarian buckwheat 'Clfa47' were cloned by polymerase chain reaction (PCR), and their nucleotide sequences were determined. In addition, a novel PCR assay targeting the allergenic BW10KD gene was developed to detect and differentiate both buckwheat species in food. The nucleotide sequences of the BW10KD genomic DNA from 'Pyeongchang' and 'Clfa47' were 94% identical. Base differences in the nucleotide sequences of the BW10KD genes are probably useful as a molecular marker for species-specific identification. The 'Pyeongchang'-specific primer set 154PF/400PR and the 'Clfa47'-specific primer set 154DF/253DR generated 247 and 100 bp fragments in singleplex PCR, respectively. A duplex PCR assay with 2 species-specific primer sets simultaneously differentiated the 'Pyeongchang' and 'Clfa47' in a single reaction. The PCR assay also successfully allowed for the rapid detection of buckwheat ingredients in foods.

• The Korean Journal of Mycology
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• v.31 no.3
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• pp.121-128
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• 2003

This study was carried out to identify the phylogenetic relationship of Phellinus species and to know its distribution by comparing the DNA sequences of internal transcribed spacer regions(ITS1 and IST2) and 5.8S ribosomal DNA (rDNA) repeat unit. The Phellinus species had their specific sequences in IST1 and 2 regions depending on suedes. The comparison of the ITS sequences of standard strains indicated that the sequences of ITS1 were more variable than those of ITS2. Nine strains of the commercial products of Phellinus species used in this study were identified as P. lintues, P. baumii, P. igniarius, and P. pini. Most of commercial species were P. pini and P. baumii, and P. gilvus was not found. Also, P. linteus was only found in form of mycelial culture rather than fruiting body. Moreover, the species-specific primers were designed based on ITS sequence data. Each species-specific primers were bound in P. lintues(ITSF-PL2R), P. baumii(PB1F-ITS4R), P. igniarius(IF1-IR3), P. pini(PF1-PR3), and P. gilvus(GF2-GR4), respectively. These primer sets would be useful fer the detection of specific-species among unidentified Phellinus species rapidly.

• Mycobiology
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• v.33 no.2
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• pp.104-108
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• 2005

Genus Phellinus taxonomically belongs to Aphyllophorales and some species of this genus have been used as a medicinal ingredients and Indian folk medicines. Especially, P. linteus and morphological-related species are well-known medicinal fungi that have various biological activities such as humoral and cell-mediated, anti-mutagenic, and anti-cancer activities. However, little is known about the rapid detection for complex Phellinus species. Therefore, this study was carried out to develop specific primers for the rapid detection of P. linteus and other related species. Designing the species-specific primers was done based on internal transcribed spacer sequence data. Each primer set detected specifically P. linteus (PL2/PL5R) and P. baumii (PB1/PB4R). These primer sets could be useful for the rapid detection of specific-species among unidentified Phellinus species. Moreover, restriction fragment length polymorphism analysis of the ITS region with HaeIII was also useful for clarifying the relationship between each 5 Phellinus species.