The purpose of this study was to breed new white strains of Agaricus bisporus with high yield and high quality by single spore crossbreeding program. In Agaricus bisporus the compatibility of strains is controlled by one mating type factor and different alleles are needed for fertility in . Fertile vegetative mycelium called heterokaryon consists of a network cells, each of which has a variable number of two genetically distinct nuclei with opposite mating type. In the basidia, specialized cells on the lamellae, a normal meiosis takes place after nuclear fusion and produces two spores, each containing two non-sister nuclei (n+n). Fruiting tests show that these single spore cultures can produce mushrooms, i.e. that they contain both mating types. Because of this secondarily homothallism, only a small percentage of the basidia produce 3 or 4 spores, which are mostly haploid (n) and do not fruit. Single spore cultures derived from these types of spores produce a vegetative mycelium that also contain a variable number of genetically identical nuclei per cell called homokaryon. Other characteristics of A. bisporus is the lack of clamp connections in heterokaryons. The typical life cycle and the lack of differences between homo- and heterokaryons are a serious drawback in breeding. Therefore, the availability of uninucleate self-sterile homokaryons is a pre-requisite for producing hybrids in crossbreeding program. Two homokaryons were isolated from two parental strains, SSU413 and CM0299021. They were crossed and cultivated on a small scale and on a commercial scale at a farm. For this, the spawn was made by a commercial spawn producer and the spawned compost by a commercial compost producer. The mature cap shape of new strain CM021002 is oblate spheroid and the immature cap shape is round to oblate spheroid. The cap diameter ranged from 30-43mm, with an average of 39mm. In comparison with white strain 505 Ho, the strain had a yield that was on average 9% higher. It produced fruiting bodies which had a higher weight on average per fruiting body and were 19% firmer with a good shelf life.

In the previous study it was demonstrated that most of sialolith contained bacterial colonies in their center of calcified laminated capsules1). Although it is still not determined whether the bacterial infection is a major pathogenic cause or not, many authors reported that the bacterial vegetation in sialolith can cause chronic suppurative inflammation in salivary gland. Actually a lot of different bacteria were found in oral cavity, most of them are non-pathogenic as normal flora, and only some of them are pathogenic for different mucosal infectious diseases. However, this study was aimed to identify a causative bacterial strain for the sialolith formation in human salivary gland. A strain of Bacillus subtilis (B. subtilis ) was found in the culture of sialolith, which was known to be a normal flora related to the fermentation of various Korean bean foods. In vitro culture experiment of B. subtilis it abundantly produced the thick biofilm aggregated around their cells and colony structure, which were subsequently attached on the surface of hydroxyapatite( HA) beads in scanning electronmicroscope(SEM) observation. The bacterial adherence on HA beads was strongest in the culture of B. subtilis, followed by Streptococcus mutans (Str. mutans ), oral mixed bacteria, and lactobacillus. Therefore, B. subtilis might be an important causative microorganism for the sialolithiasis in Korean people, and it is suggested that postmeal oral hygiene care should be required to reduce the number of normal flora, B. subtilis, after eating of fermented bean foods.

Plasmids from Bacillus thuringiensis have been implicated in pathogenicity as they carry the genes responsible for different types of diseases that in mammals and insects. A novel serogroup (H3a3b3d), B. thuringiensis strain K4 which showed mosquitocidal activity against Anopheles sinensis and Culex pipiens pallens, was isolated from fallen leaves in Mungyeong city, Republic of Korea. In contrast to the complicated plasmid profiles of B. thuringiensis H3 serotype strains, the strain K4 (designated as serovar mogi) had only one large plasmid (>200kb) on which the toxin genes were occasionally located. A 454 pyrosequencing was used for the complete sequencing of the large plasmid. The sequence analysis showed that k4 plasmid had at least seven putative cry genes, ending up to showing 84%, 75%, 73%, 58%, 84%, 39% and 75% homology with Cry27Aa, Cry19Ba, Cry20-like, Cry56Aa, Cry39ORF2, Cry8Ba and Cry40ORF2 toxins in amino acids, respectively. This novel 3a3b3d type strain, B. thuringiensis serovar mogi, can be used as a good resource for studying unknown mosquitocidal cry genes. The E. coli-B. thuringiensis shuttle vector, pHT1K was used to clone these cry genes for characterization. In each clone, the level of transcription and production of crystal proteins will be investigated in near the future.

The green peach aphid (Myzus persicae) is a serious pest of agricultural and horticultural crops all over the world. M. persica has rapidly developed resistance to a wide variety of insecticides, including carbamates. The E4/FE4 carboxylesterase is known to be involved in carbamate resistance. To compare the E4/FE4 carboxylesterase gene copy number, as a genetic resistance marker, between seven field strains, quantitative real-time PCR (qPCR) was performed. In addition, quantitative sequencing (QS) was employed to predict the frequencies of acetylcholinesterase (AChE) mutations (A301S and S431F) that are associated with target site insensitivity. All M. persica strains examined possessed the S431F mutation in the heterozygous state except for a susceptible strain, implying the possibility of AChE duplication. In contrast, no A301S mutation was found. Frequency prediction equation was generated from the plots of signal ratios and amplification critical time, which showed a high correlation (r2>0.996). QS analysis of M. persicae populations revealed that the allele frequency of S431F ranged 4% to 63%. Taken together, the AChE resistance allele frequencies determined by QS and the E4/FE4 gene copy number by qPCR should facilitate the detection and monitoring of carbamate resistance in M. persicae in the field.

An entomopathogenic filamentous fungus, Paecilomyces lilacinus strain HY-4, has a great potential as a promising bio-pesticide due to its superior pathogenicity against Adoretus tenuimaculatus and Tetranychus urticae. When the fungal strain infects host cuticle, it secrets a combination of hydrolytic enzymes including chitinase to solubilize the cuticle. Thus, we investigated effects of different carbon and nitrogen sources on the production of a chitinase from P. lilacinus strain HY-4. The organism produced an extracellular chitinase at a relatively high level (45.4 mU/ml) when cultivated for 5 days on a medium supplemented with insect pupa (0.5%) and colloidal chitin (1%), which was prepared by treating chitin from crab shells (Sigma-Aldrich Co. Ltd.) with 12 N HCl solution. However, extracellular secretion of chitinase by strain HY-4 was found to be significantly repressed in the presence of glucose (1%).

The extracellular GH11 β-1,4-xylanase (XylY) gene (633-bp) of Paenibacillus sp. strain KYJ-16 was molecularly cloned by repeated DNA walking and nested PCR method. The xylY gene was predicted to encode an extracellular protein consisting of 611 amino acids with a nesuced molecular mass of 23 kDa and a calculated pI of 9.55. Protein blast search revealed that the enzyme consisted of a putative catalytic domain, which is homologous to a catalytic GH11 domain. The highest sequence identity (92%) was obtained as the catalytic GH11 domain of XylY was compared to that of Paenibacillus sp. strain HGF5 (GenBank accession number: EGG35584) that has not yet been characterized. Enzymatic properties of the recombinant His-tagged enzyme (rXylY) overexpressed in E. coli BL21 harboring pET-28a(+)/xylY will be also presented.

The gene (2,304-bp) encoding a novel xylanolytic enzyme (XylD) with a catalytic domain, which is 70% identical to that of Cellulomonas flavigena DSM 20109 GH6 β-1,4-cellobiohydrolase, was identified from an earthworm (Eisenia fetida)-symbiotic bacterium, Cellulosimicrobium sp. strain HY-13. The enzyme consisted of an N-terminal catalytic GH6-like domain, a fibronectin type 3 (Fn3) domain, and a C-terminal carbohydrate-binding module 2 (CBM 2). XylDΔFn3-CBM 2 displayed high transferase activity (788.3 IU mg-1) toward p-nitrophenyl (PNP) cellobioside, but did not degrade xylobiose, glucose-based materials, or other PNP-sugar derivatives. Birchwood xylan was degraded by XylDΔFn3-CBM 2 to xylobiose (59.2%) and xylotriose (40.8%). The transglycosylation activity of the enzyme, which enabled the formation of xylobiose (33.6%) and xylotriose (66.4%) from the hydrolysis of xylotriose, indicates that it is not an inverting enzyme but a retaining enzyme. The endo-β-1,4-xylanase activity of XylDΔFn3-CBM 2 increased significantly by approximately 2.0-fold in the presence of 50 mM xylobiose.

The aim of this study was to develop Prevotella intermedia ATCC 49046-specific PCR primers designed based on the nucleotide sequence of a DNA probe Pig28. The strainspecificity of the PCR primers, Pig28-F1/Pig28-R1, was confirmed with 9 strains of P. intermedia and 25 strains (15 species) of Prevotella species. The detection limit of the PCR primers was 2 pg of the purified genomic DNA of P. intermedia ATCC 49046. These PCR primers were found to be useful for identifying P. intermedia ATCC 49046, particularly for determining the authenticity of the strain.

Canine parvovirus (CPV) type 2a (CPV-2a) has recently been identified as the main genotype circulating in the dog population in South Korea. Although CPV vaccines protect domestic dogs from CPV-2 infection, the efficacy of commercial live or inactivated CPV vaccines against CPV-2a has not been reevaluated. In this study, dogs were immunized with one of 7 commercial CPV vaccines (4 modified live and 3 inactivated vaccines) followed by challenge with CPV-2a strain, KV0901 that had been isolated from naturally infected dog in 2009. All dogs vaccinated twice with 4 commercial modified live CPV vaccines were seroconverted (geometric mean HI titer > 190.2) and most of dogs were completely protected against virulent CPV-2a strain infection. The dogs inoculated with 3 commercial inactivated CPV vaccines were also seroconverted and showed a slight loss of appetite and light diarrhea for 4 days after challenge and returned to normal at 5 days post challenge. However, the non-vaccinated dogs revealed the typical clinical signs of CPV infection including haemorrhgic diarrhea. In conclusion, the 4 live CPV vaccines licensed in Korea cross-protected dogs against virulent challenge with CPV-2a and are applicalble to pet dogs for the prevention of CPV infection.

Single basidiospores were isolated from a Pleurotus ostreatus strain collected in Boryeong, Korea showing gray and semispherical in color and shape of pileus. Mycelial growth and several other characters were good to be a breeding material. Mating experiments were performed using 23 monokaryons to provide mating compatibility data. Pairings were performed in 90 mm petri dishes on PDA. They were allowed to grow at 25℃ until two fronts of the advancing mycelia met and developed a conspicuous contact zone. The contact zone and the outer edges of paired colonies on each plate were examined for clamp connections and the crosses were considered positive when clamp connections were found at the growing margin of either side of the interacting monokaryons. The collected strain resulted in tetrapolar incompatibility system in intrastrain crosses. Compatible matings could be visibly distinguished from incompatible ones by the rapid growth and gross morphology. The dikaryotic colony was also confirmed microscopically by the presence of clamp connections from incompatible pairings. The four classes of tester strains representing the four incompatibility types were chosen from intrastrain mating tests as expected. The mating tests with the tester strain were performed for the determination of the rest monokaryons. As a result of these tests it was found that the strain share the same A and B incompatibility factors showing tetrapolar incompatibility system.

Agaricus bisporus is a nutritious edible fungus that is cultivated worldwide. Because of its secondarily homothallic life cycle, 90% or more of the basidia lining the gills at fruit bodies are predominantly two-spored, with each spore receiving two of the four meiotic nuclei. Such homothallic system results in scarcity of monokaryotic basidiospores in its progeny but the availability of uninucleate self-sterile homokaryons is a pre-requisite for producing hybrids in breeding program. This experiment screened 64 single spore isolates collected from CN0210-021, which is white button mushroom cultivated in Chungnam area. They were germinated on PAD meduium at 25℃. After 30 days of incubation colony diameter and morphology type of each isolates was determined. Based on growth rate, 5 strains showing very slow growing morphology than that of parental heterokaryotic strain were selected. Out of 5 slow growing isolates, 2 of them fruited and the rest 3 isolates did not fruit, which were considered as putative homokaryons. These data will provide the outcrossing sources that could be used in commercial breeding programs later. However, in the identification of homokaryotic strains, there is no direct morphological distinguishing features such as a clamp connection that would permit the reliable sorting between homokaryons and heterokaryons. Traditional method to verify a homokaryon thorough the fruiting trial is also time-consuming and not perfect. Therefore, to accelerate the breeding of Agaricus bisporus, quick and reliable methods such as molecular markers to identify the infrequent homokaryons from heterokaryons are needed to be carried out.

The aim of this study was to determine the immediate effects of single treatment of strain-counter strain (SCS) on pressure pain threshold (PPT) and muscle activity during scapular plane abduction with 3% body weight load. Fifteen asymptomatic male adults with upper trapezius latent trigger point (LTrP) (PPT<2.9 ) participated in this study. Pressure algometer was used to measure PPT and surface electromyography was used to record upper, middle arid lower trapezius, serratus anterior, infraspinatus and middle deltoid muscle activity and relative ratio during scapular plane abduction between pre- and post-intervention. There was a significant increase in upper trapezius PPT after a 90-second SCS (p<.05). The activity of the upper trapezius and middle deltoid was significantly decreased (p=.014, p=.001), coupled with a decreased muscle activity ratio between the upper and lower trapezius (p<.05). These results indicate that the SCS may effectively deactivate upper trapezius activity, thereby alleviating muscle balance and reducing pain sensitivity.

xylanolytic gut bacterium isolated from Eisenia fetida, Cellulosimicrobium sp. strain HY-13, produced an extracellular glycoside hydrolase capable of efficiently degrading mannose-based substrates such as locust bean gum (LBG), guar gum, mannotetraose, and mannopentaose. The purified mannan-degrading enzyme (ManS, 34,926 Da) from strain HY-13 was found to have an N-terminal amino acid sequence of DEATTDGLHVVDD, which has not yet been identified. Under the optimized reaction conditions of 50℃ and pH 7.0, ManK exhibited extraordinary high specific activities of 7,109 IU/mg and 5,158 IU/mg toward LBG and guar gum, respectively, while the enzyme showed no effect on sugars substituted with p-nitrophenol and various non-mannose carbohydrates. ManK strongly attached to Avicel, lignin, β-cyclodextrin, and poly(3-hydroxybutyrate) granules, but not bound to chitin, chitosan, curdlan, or insoluble oat spelt xylan. The aforementioned characteristics of ManS suggest that it is a unique endo-β -1,4-mannanase with out additional carbohydrolase activities, which differentiates it from other well-known carbohydrolases.

Lentinula edodes is an important cultivated mushroom in China. The development of Lentinula edodes production promotes more studies on it. In our previous work, degenerate PCR and chromosome walking technologies were used to obtain one pheromone receptor gene and one pheromone precursor gene from Lentinula edodes. In this study, four pairs of specific primers were designed according to the whole genome sequencing of the protoplast monokaryon of Lentinula edodes strain 135, to amplify STE3-like pheromone receptor gene and its flanking conserved genes in the protoplast monokaryon strain SUP2 derived from Lentinula edodes strain Suxiang and 33655bp DNA sequence was obtained. By BlastX search, seven putative genes were identified, and three of them are pheromone receptor encoded genes. Furthermore, near to two pheromone receptor genes, four genes encoding proteins with conserved motifs of pheromone precursors were found. This study firstly reveals the molecular organization of the B mating type locus of Lentinula edodes.

[Aims] Lentinula edodes (Berk.) Pegler has many commercial strains both morphologically and physiologically similar to each other. At present, detection of polymorphism in rDNA-IGS region (Babasaki, 2006) and/or RAPD marker (Zhang and Molina, 1995) is generally used for strain typing of L. edodes. However, it is rather time-and-cost consuming. Inter-retrotransposon amplified polymorphism (IRAP)-PCR method mainly used for horticultural crops takes less time and lower in cost in strain typing (Kalendar et al, 1999). In this study, we designed IRAP primers for L. edodes and verified their strain typing efficiency. [Method] Thirty three strains were provided for this study. Either fungal cultures on PDA or fungal tissues of fruit bodies were cut into approximately 4 x 4 x 4 mm. Total DNA of each samples were extracted by DNeasy Plant Mini Kit (QIAGEN). For PCR, IRAP primer set and Pfu-X polymerase (greiner) were performed. Based on LTR (Long Terminal Repeat) sequence in L. edodes, we designed one set of primers amplifying the regions between retrotransposons. Ampricons were electrophored for 50 min at 100 V on 1.7 % agarose gel with GelRed (Biotium) and evaluated under UV irradiation. [Results] The products obtained by IRAP-PCR were determined using mini-gel electrophoresis system. The band patterns of IRAP-PCR products differ among strains except the ones having the same parental cultivar. The detected bands were bright and clear without smearing. The IRAP-PCR products of fungal cultures on PDA and correlating fungal tissues of fruit bodies showed the same band pattern, suggesting that the procedure is highly reproducible. Thus, it is considered that IRAP-PCR with short ranged (ca. 1 kb) electrophoresis is a time-efficient and practical strain typing method of L. edodes.

콩은 식물성 단백질 및 지방의 주요 공급원이고 콩 종실에는 기능성 성분이 많이 함유되어져 있어 소비 가 점차 증가하고 있지만 Lipoxygenase, Kunitz Trypsin inhibitor, 7S α′-subunit 단백질과 같은 성 분들이 존재하는데 이는 품질과 영양가치를 떨어뜨리고 섭취시 알러지를 일으키기도 한다. 콩에서 유전적 으로 이러한 성분이 결핍되어져 있는 유전자형의 선발은 품질이 우수한 콩 육종의 기초단계이다. “개척2 호”와 PI506876의 교배로부터 434개의 F2 종자를 얻어 F2 종자의 일부를 사용하여 SDS-PAGE로 각각 의 종자를 분석한 결과 Lipoxygenase와 Kunitz Trypsin inhibitor 및 7S의 α′-subunit 단백질이 모두 결핍되어져 있는 lx1lx1lx2lx2lx3lx3titicgy1cgy1 유전자형을 가진 종자를 선발하여 F2 식물체로 길러 성 숙 후 F3 종자를 수확하였다. F3 종자로부터 Lipoxygenase, Kunitz Trypsin inhibitor, 7S α′-subunit 단백질이 모두 결핍되어져 있음을 재확인하였으며 선발된 종자는 고품질 콩 품종 육성에 유용하게 활용될 것으로 기대된다.

A new commercial strain "Dan Bi" of Pleurotus eryngii was developed by intra-specific crossing between monokaryotic spores derived from KNR2312 and KNR2596, respectively. The optimum temperature of mycelial growth was 25℃ and that of fruiting body development was 15 -16℃. The primordia formation of "Dan Bi" was one day later than that of control strain KNR2312. The color of pileus and stipe surface was neutral-brown and pure white, respectively. The shape of pileus was dome and has a scale like as cobweb. The yield was 93±4.8g/850cc plastic bottle. Analysis of the genetic characteristics of the new commercial strain "Dan Bi" showed a different profile as that of the control strain KNR2312 when RAPD primer 8001, 8002, 8003, 8004, and 8005 were used. This new variety of King Oyster mushroom is characterized by a few primordia formation after scratching. It would be of much help to mushroom cultivators in a process of culling of P. eryngii.

Pleurotus ostreatus 'Miso' is a mutant strain showing white color in pileus from the known parent strain 'Wonhyeong No. 1'. Shape and several other characters also vary with culture conditions. Mating experiments were performed to understand interstrain mating relationship using monokaryons of the parent and the mutant strains. All monokaryons were grown from single spores isolated from freshly collected fruit bodies. Pairings were performed in 90 mm petri dishes on PDA. They were allowed to grow at 25℃ until two fronts of the advancing mycelia met and developed a conspicuous contact zone. The contact zone and the outer edges of paired colonies on each plate were examined for clamp connections. The crosses were considered positive when clamp connections were at the growing margin of either side of the interacting monokaryons. The parent strain 'Wonhyeong No. 1' and the mutant strain 'Miso' resulted in tetrapolar incompatibility system in intrastrain crosses. Normal compatible matings could be visibly distinguished from incompatible ones by the rapid growth and gross morphology. The resultant dikaryotic colony was also confirmed microscopically by the presence of clamp connections from incompatible pairings. In interstrain crosses, each monokaryotic tester strains of the parent strain was out-crossed to monokaryotic tester strains of the mutant. The four classes of tester strains representing the four incompatibility types were chosen from each mutant and parent strains. As a result of these crosses it was found that both strains share the same A and B incompatibility factors yielding 25% compatibility.