• 한국균학회소식:학술대회논문집
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• 2015.05a
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• pp.28-28
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• 2015

Clubroot disease caused by Plasmodiophora brassicae induces severe losses of cruciferous vegetables worldwide. To control clubroot of Chinese cabbage, many CR (clubroot resistance) F1 hybrid cultivars have been bred and released in Korea, China and Japan. In this study, we determined the race of P. brassicae 12 field isolates, which collected from 10 regions in Korea, using Williams' differential varieties including two cabbage ('Jersey Queen', 'Badger Shipper') and two rutabaga ('Laurentian', 'Whilhelmsburger'). By Williams' differential varieties, 12 clubroot pathogens were assigned into one (GN2), two (HS and YC), two (HN1 and HN2), three (DJ, KS and SS) and four (GS, GN1, JS and PC) isolates for races 1, 2, 4, 5 and 9, respectively. In addition, the degree of resistance of 45 CR cultivars that were from Korea, China and Japan was tested with the 12 isolates. The 45 CR cultivars of Chinese cabbage were differentiated into three genotypes according to their resistance responses. Even though the 12 P. brassicae isolates were same race by Williams' differential varieties, three CR genotypes showed different resistance response to the isolates. These results indicate that races of P. brassicae by Williams' differentials were not related with resistance of CR cultivars, and three CR genotypes represented qualitative resistance to the P. brassicae isolates. CR genotype I including 'CR-Cheongrok' showed resistance to GN1, GN2, JS, GS, HS, DJ and KS isolates and susceptibility to YC, PC, HN1, HN2 and SS isolates. And CR genotype II such as 'Hangkunjongbyungdaebaekchae' was resistant to GN1, GN2, JS, GS, HS, YC, PC and HN1 and susceptible to DJ, KS, SS and HN2. CR genotype III including 'Chunhajangkun' and 'Akimeki' represented resistance to 10 isolates except for SS and HN2 isolates. Based on these results, we selected 'CR-Cheongrok', 'Hangkunjongbyungdaebaekchae', and 'Chunhajangkun' as a representative cultivar of three CR genotypes and 'Norangkimjang' as a susceptible cultivar. Furthermore, we investigated the resistance of 15 lines of Chinese cabbage, which were provided by seed companies, to 11 isolates except for HN1 of P. brassicae. The results showed that three lines were susceptible to all the tested isolates, whereas five, four, and three lines represented the similar responses corresponding to the CR genotypes I, II, and III, respectively; there is no line of Chinese cabbage showing different resistance patterns compared to three CR genotypes. In particular, line 'SS001' showing resistance responses of CR genotype II was a parent of 'Saerona' that have been commercialized as a CR $F_1$ cultivar of Chinese cabbage. Together, we divided 12 isolates of P. brassicae into 4 races, designated by wild type, mutant type 1, mutant type 2, and mutant type 3. Wild type including GN1, GN2, JS, GS, and HS isolates of P. brassicae was not able to infect all the cultivars of three CR genotypes, whereas, mutant type 3 such as SS and HN2 isolates developed severe clubroot disease on all the CR genotype cultivars. To mutant type 1 including DJ and KS isolates, CR genotypes I, II and III were resistant, susceptible and resistant, respectively. In contrast, to mutant type 2 including YC, PS, and HN1 isolates, CR genotypes I, II and III showed susceptibility, resistance and resistance, respectively. Taken together, our results provide the extended knowledge of classification of P. brassicae races, which is useful information for the breeding of resistant crops, with a suggestion that 'Norangkimjang', 'CR-Cheongrok', 'Saerona' and 'Chunhajangkun' cultivars of Chinese cabbage could be used as new race differentials of P. brassicae for clubroot disease assay.

• 한국균학회소식:학술대회논문집
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• 2014.05a
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• pp.27-28
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• 2014

Beauveria bassiana (Cordycipitaceae, Hypocreales, Ascomycota) is an anamorphic fungus having a potential to be used as a biological control agent because it parasitizes a wide range of arthropod hosts including termites, aphids, beetles and many other insects. A number of bioactive secondary metabolites (SMs) have been isolated from B. bassiana and functionally verified. Among them, beauvericin and bassianolide are cyclic depsipeptides with antibiotic and insecticidal effects belonging to the enniatin family. Non-ribosomal peptide synthetases (NRPSs) play a crucial role in the synthesis of these secondary metabolites. NRPSs are modularly organized multienzyme complexes in which each module is responsible for the elongation of proteinogenic and non-protein amino acids, as well as carboxyl and hydroxyacids. A minimum of three domains are necessary for one NRPS elongation module: an adenylation (A) domain for substrate recognition and activation; a tholation (T) domain that tethers the growing peptide chain and the incoming aminoacyl unit; and a condensation (C) domain to catalyze peptide bond formation. Some of the optional domains include epimerization (E), heterocyclization (Cy) and oxidation (Ox) domains, which may modify the enzyme-bound precursors or intermediates. In the present study, we analyzed genomes of B. bassiana and its allied species in Hypocreales to verify the distribution of NRPS-encoding genes involving biosynthesis of beauvericin and bassianolide, and to unveil the evolutionary processes of the gene clusters. Initially, we retrieved completely or partially assembled genomic sequences of fungal species belonging to Hypocreales from public databases. SM biosynthesizing genes were predicted from the selected genomes using antiSMASH program. Adenylation (A) domains were extracted from the predicted NRPS, NRPS-like and NRPS-PKS hybrid genes, and used them to construct a phylogenetic tree. Based on the preliminary results of SM biosynthetic gene prediction in B. bassiana, we analyzed the conserved gene orders of beauvericin and bassianolide biosynthetic gene clusters among the hypocrealean fungi. Reciprocal best blast hit (RBH) approach was performed to identify the regions orthologous to the biosynthetic gene cluster in the selected fungal genomes. A clear recombination pattern was recognized in the inferred A-domain tree in which A-domains in the 1st and 2nd modules of beauvericin and bassianolide synthetases were grouped in CYCLO and EAS clades, respectively, suggesting that two modules of each synthetase have evolved independently. In addition, inferred topologies were congruent with the species phylogeny of Cordycipitaceae, indicating that the gene fusion event have occurred before the species divergence. Beauvericin and bassianolide synthetases turned out to possess identical domain organization as C-A-T-C-A-NM-T-T-C. We also predicted precursors of beauvericin and bassianolide synthetases based on the extracted signature residues in A-domain core motifs. The result showed that the A-domains in the 1st module of both synthetases select D-2-hydroxyisovalerate (D-Hiv), while A-domains in the 2nd modules specifically activate L-phenylalanine (Phe) in beauvericin synthetase and leucine (Leu) in bassianolide synthetase. antiSMASH ver. 2.0 predicted 15 genes in the beauvericin biosynthetic gene cluster of the B. bassiana genome dispersed across a total length of approximately 50kb. The beauvericin biosynthetic gene cluster contains beauvericin synthetase as well as kivr gene encoding NADPH-dependent ketoisovalerate reductase which is necessary to convert 2-ketoisovalarate to D-Hiv and a gene encoding a putative Gal4-like transcriptional regulator. Our syntenic comparison showed that species in Cordycipitaceae have almost conserved beauvericin biosynthetic gene cluster although the gene order and direction were sometimes variable. It is intriguing that there is no region orthologous to beauvericin synthetase gene in Cordyceps militaris genome. It is likely that beauvericin synthetase was present in common ancestor of Cordycipitaceae but selective gene loss has occurred in several species including C. militaris. Putative bassianolide biosynthetic gene cluster consisted of 16 genes including bassianolide synthetase, cytochrome P450 monooxygenase, and putative Gal4-like transcriptional regulator genes. Our synteny analysis found that only B. bassiana possessed a bassianolide synthetase gene among the studied fungi. This result is consistent with the groupings in A-domain tree in which bassianolide synthetase gene found in B. bassiana was not grouped with NRPS genes predicted in other species. We hypothesized that bassianolide biosynthesizing cluster genes in B. bassiana are possibly acquired by horizontal gene transfer (HGT) from distantly related fungi. The present study showed that B. bassiana is the only species capable of producing both beauvericin and bassianolide. This property led to B. bassiana infect multiple hosts and to be a potential biological control agent against agricultural pests.

• 한국균학회소식:학술대회논문집
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• 2015.11a
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• pp.16-16
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• 2015

Ectomycorrhizal (ECM) mushrooms play a major role in plant growth promotion through symbiotic association with roots of forest trees. They also provide an economically important food resource to us and therefore they have been studied for their artificial cultivation for decades in Korea. We have secured bio-resources of ECM mushrooms from Korean forests and performed their physiological studies. To investigate the cultural characteristics, the fungi were cultured under different conditions (medium, temperature, pH of the medium, inorganic nitrogen source). More than 90% of total 160 strains grew on three solid media (potato dextrose agar, PDA; sabouraud dextrose agar, SDA; modified Melin-Norkrans medium, MMN). The rate of mycelial growth on malt extract agar (MEA) was lower than those of three media (PDA, SDA, MMN). None of the Tricholomataceae strains grew on MEA. Many strains of ECM mushrooms were able to grow at the temperature range of $15{\sim}25^{\circ}C$ on PDA, while they showed poor growth at $10^{\circ}C$ or $30^{\circ}C$. In particular, the growth rates of both Gomphaceae and Tricholomataceae were significantly lower at $10^{\circ}C$ than at $30^{\circ}C$. The optimal pH of many strains was pH 5.0 when they cultured in potato dextrose broth (PDB). Fifty-seven percent of tested strains grew well on medium containing ammonium source than nitrate source. Many strains of Tricholomataceae showed a notable growth on ammonium medium than nitrate medium. Twenty-three percent of strains preferred nitrate source than ammonium source for their mycelial growth. The production and activity of two enzymes (cellulase and laccase) by ECM fungi were also assayed on the enzyme screening media containing CMC or ABTS. Each strains exhibited different levels of enzymatic activities as well as enzyme production. The number of laccase-producing strains was less than that of cellulase-producing strains. We found that 77% of tested strains produced both cellulase and laccase, whereas 2% of strains did not produce any enzymes. The morphological characteristics of mycelial colony were also examined on four different solid media. Yellow was a dominant color in mycelial colony and followed by white and brown on all culture media. ECM mushrooms formed mycelial colonies with a single or multiple colors within a culture medium depending on the strains and culture media. The most common shape of mycelial colony was a circular form on all media tested. Other families except for Amanitaceae formed an irregular colony on MMN than PDA. All strains of Tricholomataceae did not form a filamentous colony on all media. The pigmentation of culture media by mycelial colonies was observed in more than 50% of strains tested on both PDA and SDA. The degree of pigmentation on PDA or SDA was higher than MMN and brown color was dominant than yellow color. The production of exudates from mycelial colony was higher on PDA than MMN. Brown exudates were mainly produced by many strains on PDA or SDA, whereas transparent exudates were mainly produced by strains on MMN. We observed the mycelial colonies with a single or multiple textures in just one culture plate. Wrinkled or uneven colony surfaces were remarkably observed in many strains on PDA or SDA, while an even colony surface was observed in many strains on MMN. Sixty percent of Tricholomaceae strains formed wrinkled surface on PDA. However, they did not form any wrinkle on MMN plate. Cottony texture was observed in mycelia colonies of many strains. Velvety texture was often observed in the mycelial colonies on SDA than PDA and accounted for 60% of Suillaceae strains on SDA.

• 한국균학회소식:학술대회논문집
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• 2014.10a
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• pp.9-9
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• 2014

Null mutants generated by targeted gene replacement are frequently used to reveal function of the genes in fungi. However, targeted gene deletions may be difficult to obtain or it may not be applicable, such as in the case of redundant or lethal genes. Constitutive expression system could be an alternative to avoid these difficulties and to provide new platform in fungal functional genomics research. Here we developed a novel platform for functional analysis genes in Magnaporthe oryzae by constitutive expression under a strong promoter. Employing a binary vector (pGOF1), carrying $EF1{\beta}$ promoter, we generated a total of 4,432 transformants by Agrobacterium tumefaciens-mediated transformation. We have analyzed a subset of 54 transformants that have the vector inserted in the promoter region of individual genes, at distances ranging from 44 to 1,479 bp. These transformants showed increased transcript levels of the genes that are found immediately adjacent to the vector, compared to those of wild type. Ten transformants showed higher levels of expression relative to the wild type not only in mycelial stage but also during infection-related development. Two transformants that T-DNA was inserted in the promotor regions of putative lethal genes, MoRPT4 and MoDBP5, showed decreased conidiation and pathogenicity, respectively. We also characterized two transformants that T-DNA was inserted in functionally redundant genes encoding alpha-glucosidase and alpha-mannosidase. These transformants also showed decreased mycelial growth and pathogenicity, implying successful application of this platform in functional analysis of the genes. Our data also demonstrated that comparative phenotypic analysis under over-expression and suppression of gene expression could prove a highly efficient system for functional analysis of the genes. Our over-expressed transformants library would be a valuable resource for functional characterization of the redundant or lethal genes in M. oryzae and this system may be applicable in other fungi.

• 한국균학회소식:학술대회논문집
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• 2014.10a
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• pp.32-32
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• 2014

Meju is a brick of dried fermented soybeans and is the core material for Jang such as Doenjang and Ganjang. Jang is produced by addition of salty water to Meju and is considered the essential sauces of authentic Korean cuisine. Meju is fermented by diverse microorganisms such as bacteria, fungi and yeasts. It is known that fungi play an important role in the Meju fermentation and they degrade macromolecules of the soybeans into small nutrient molecules. In previous study, 26 genera and 0 species were reported as Meju fungi. However, it is not comprehensively examined where the fungi present on the Meju are originated. In order to elucidate the origin of the fungi present on the Meju, the mycobiota of 500 samples soybean kernels, 296 rice straw pieces and air samples of Jang factories was determined in 0, 2 and 7 Jang factories respectively. Forty-one genera covering 86 species were isolated from the soybeans and 33 species were identical with the species from Meju. From sodium hypochlorite untreated soybeans, Eurotium herbariorum, Eurotium repens, Cladosporium tenuissimum, Fusarium fujikuroi, Aspergillus oryzae/flavus and Penicillium steckii were the predominant species. In case of sodium hypochlorite-treated soybeans, Eurotium herbariorum, E. repens and Cladosporium tenuissimum were the predominant species. Of the 4 genera and 86 species isolated from soybeans, 3 genera and 33 species were also found in Meju. Thirty-nine genera and 92 species were isolated from the rice straws and 40 species were identical with the species from Meju. Fusarium asiaticum, Cladosporium cladosporioides, Aspergillus tubingensis, A. oryzae, E. repens and Eurotium chevalieri were frequently isolated from the rice straw obtained from many factories. Twelve genera and 40 species of fungi that were isolated in the rice straw in this study, were also isolated from Meju. Especially, A. oryzae, C. cladosporioides, E. chevalieri, E. repens, F. asiaticum and Penicillium polonicum that are abundant species in Meju, were also isolated frequently from rice straw. C. cladosporioides, F. asiaticum and P. polonicum that are abundant in low temperature fermentation process of Meju fermentation, were frequently isolated from rice straw incubated at $5^{\circ}C$ and $25^{\circ}C$, while A. oryzae, E. repens and E. chevalieri that are abundant in high temperature fermentation process of Meju fermentation, were frequently isolated from rice straw incubated at $25^{\circ}C$ and $35^{\circ}C$. This suggests that the mycobiota of rice straw have a large influence in mycobiota of Meju. Thirty-nine genera and 92 species were isolated from the air of Jang factories and 34 species were identical with the species from Meju. In outside air of the fermentation room, Cladosporium sp. and Cladosporium cladosporioides were the dominant species, followed by Cladosporium tenuissimum, Eurotium sp., Phoma sp. Sistotrema brinkmannii, Alternaria sp., Aspergillus fumigatus, Schizophyllum commune, and Penicillium glabrum. In inside air of the fermentation room, Cladosporium sp., Aspergillus oryzae, Penicillium chrysogenum, A. nidulans, Aspergillus sp., C. cladosporioides, Eurotium sp., Penicillium sp., C. tenuissimum, A. niger, E. herbariorum, A. sydowii, and E. repens were collected with high frequency. The concentrations of the genus Aspergillus, Eurotium and Penicillium were significantly higher in inside air than outside air. From this results, the origin of fungi present on Meju was inferred. Of the dominant fungal species present on Meju, Lichtheimia ramosa, Mucor circinelloides, Mucor racemosus, and Scopulariopsis brevicaulis are thought to be originated from outside air, because these species are not or are rarely isolated from rice straw and soybean; however, they were detected outside air of fermentation room and are species commonly found in indoor environments. However, A. oryzae, P. polonicum, E. repens, P. solitum, and E. chevalieri, which are frequently found on Meju, are common in rice straw and could be transferred from rice straw to Meju. The fungi grow and produce abundant spores during Meju fermentation, and after the spores accumulate in the air of fermentation room, they could influence mycobiota of Meju fermentation in the following year. This could explain why concentrations of the genus Aspergillus, Eurotium, and Penicillium are much higher inside than outside of the fermentation rooms.

• 한국균학회소식:학술대회논문집
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• 2014.10a
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• pp.25-25
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• 2014

Three species of Fusarium, F. fujikuroi, F. verticillioides and F. proliferatum, are known to be associated with bakanae disease of rice [1, 2]. F. fujikuroi infects rice flowers and survive in endosperm and embryo of the seeds. Infected seed is an important source of primary inoculum of pathogens [3]. Seeds of rice (Oryza sativa cv. Boramchan) collected from bakanae-infected field were found to be 96% infected with Fusarium sp., 52% with F. fujikuroi, 42% with F. verticillioides, and 12% with F. proliferatum as determined by incubation method and species-specific PCR assays. F. fujikuroi was detected at lemma/palea, endosperm and embryo whereas F. verticillioides and F. proliferatum were recovered only from lemma/palea by means of component plating test. Seed disinfection methods have been developed to control bakanae disease and prochloraz has been most widely used for rice seeds. Two chemicals formulated with prochloraz (PC 1) and prochloraz + hexaconazole (PC 2) that inhibit biosynthesis of ergosterol strongly reduced the incidence of Fusarium spp. on selective media to 4.7% and 2.0%, respectively. Disease symptoms of rice seedlings in nursery soil were alleviated by chemical treatment; seedlings with elongated leaves or wide angle between leaf and stem were strikingly reduced from 15.6 to 3.2% (PC 1) and 0 (PC 2), stem rots were reduced from 56.9 to 26.2% (PC 1) and 32.1% (PC 2), and normal seedling increased from 0.4 to 13.3% (PC 2). Prochloraz has some disadvantages and risks such as the occurrence of tolerant pathogens [4] and effects on the sterol synthesis in animals and humans [5]. For these reasons, it is necessary to develop new disinfection method that do not induce fungal tolerance and are safe to humans and animals. Chlorine dioxide ($ClO_2$), that is less toxic, produces no harmful byproducts, and has high oxidizing power, has been reported to be effective at disinfection of several phytopathogenic fungi including Colletotrichum spp. and Alternaria spp. [6]. Gaseous $ClO_2$ applied to rice seeds at a concentration of 20 ppm strongly suppressed mycelial growth of Fusarium fujikuroi, F. verticillioides and F. proliferatum. The incidence of Fusarium spp. in dry seed with 8.7% seed moisture content (SMC) tended to decrease as the concentration of $ClO_2$ increased from 20 to 40 ppm. Applying 40 ppm $ClO_2$ at 90% relative humidity, incidence was reduced to 5.3% and resulted in significant reduction of disease symptoms on MS media. In nursery soil, stem rot was reduced from 56.9 to 15.4% and the number of normal seedlings increased from 0.4 to 25.5%. With water-soaked seeds (33.1% SMC) holding moisture in the endosperm and embryo, the effectiveness of disinfection using $ClO_2$ increased, even when treated with only 20 ppm for four hours. This suggests that moisture was a key element for action of $ClO_2$. Removal of the palea and lemma from seeds significantly decreased the incidence of Fusarium spp. to 3.0%. Seed germination appeared to decrease slightly by water-soaking at $30^{\circ}C$ because of increased SMC and by physical damage of embryos from hulling. These results indicate that the use of gaseous $ClO_2$ was effective as a means to disinfect rice seeds infected with Fusarium spp. and that moisture around the pathogens in the seed was an important factor for the action of $ClO_2$. Further investigations should be conducted to ascertain the best conditions for complete disinfection of Fusarium spp. that infect deep site of rice seeds.

• 한국균학회소식:학술대회논문집
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• 2018.05a
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• pp.27-27
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• 2018

The production scale of mushroom cultivation in Korea is approximately 600 billion won, which is 1.6% of the Korean gross agricultural output. Annually, ca. 190,000 tons of mushrooms are harvested in Korea. Although the numbers of mushroom farms and cultivators are constantly decreasing, the total mushroom yields are increasing due to the large-scale cultivation facilities and automation. The recent expansion of the well-being trend causes increase in mushroom consumption in Korea: annual per capita consumption of mushroom was 3.9kg ('13) that is a little higher than European's average. Thus the exports of mushrooms, mainly Flammulina velutipes and Pleurotus ostreatus, have been increased since the middle of 2000s. Recently, however, it is slightly reduced. However, Vietnam, Hong Kong, the United States, the Netherlands and continued to export, and the country has increased recently been exported to Australia, Canada, Southeast Asia and so on. Canned foods of Agaricus bisporus was the first exports of the Korean mushroom industry. This business has reached the peak of the sale in 1977-1978. As Korea initiated trade with China in 1980, the international prices of mushrooms were sharply fall that led to shrink the domestic markets. According to the high demand to develop new items to substitute for A. bisporus, oyster mushroom (Pleurotus ostreatus) was received the attention since it seems to suit the taste of Korean consumers. Although log cultivation technique was developed in the early 1970s for oyster mushroom, this method requires a great deal of labor. Thus we developed shelf cultivation technique which is easier to manage and allows the mass production. In this technique, the growing shelf is manly made from fermented rice straw, that is the unique P. ostreatus medium in the world, was used only in South Korea. After then, the use of cotton wastes as an additional material of medium, the productivity. Currently it is developing a standard cultivation techniques and environmental control system that can stably produce mushrooms throughout the year. The increase of oyster mushroom production may activate the domestic market and contribute to the industrial development. In addition, oyster mushroom production technology has a role in forming the basis of the development of bottle cultivation. Developed mushroom cultivation technology using bottles made possible the mass production. In particular, bottle cultivation method using a liquid spawn can be an opportunity to export the F.velutipes and P.eryngii. In addition, the white varieties of F.velutipes were second developed in the world after Japan. We also developed the new A.bisporus cultivar "Sae-ah" that is easy to grown in Korea. To lead the mushroom industry, we will continue to develop the cultivars with an international competitive power and to improve the cultivation techniques. Mushroom research in Korea nowadays focuses on analysis of mushroom genetics in combination with development of new mushroom varieties, mushroom physiology and cultivation. Further studied are environmental factors for cultivation, disease control, development and utilization of mushroom substrate resources, post-harvest management and improvement of marketable traits. Finally, the RDA manages the collection, classification, identification and preservation of mushroom resources. To keep up with the increasing application of biotechnology in agricultural research the genome project of various mushrooms and the draft of the genetic map has just been completed. A broad range of future studies based on this project is anticipated. The mushroom industry in Korea continually grows and its productivity rapidly increases through the development of new mushrooms cultivars and automated plastic bottle cultivation. Consumption of medicinal mushrooms like Ganoderma lucidum and Phellinus linteus is also increasing strongly. Recently, business of edible and medicinal mushrooms was suffering under over-production and problems in distribution. Fortunately, expansion of the mushroom export helped ease the negative effects for the mushroom industry.

• The Korean Journal of Mycology
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• v.7 no.1
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• pp.13-73
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• 1979

These studies were conducted to investigate nutrient sources and supplementary materials of synthetic compost media for Agaricus bisporus culture. Investigation were carried out to establish the optimum composition for compost of Agaricus bisporus methods of out-door fermentation and peakheating with rice straw as the main substrate of the media. The incidence and flora of harmful organisms in rice straw compost and their control were also studied. 1. When rice straw was used as the main substrate in synthetic compost as a carbon source. yields were remarkably high. Fermentation was more rapid than that of barley straw or wheat straw, and the total nitrogen content was high in rice straw compost. 2. Since the morphological and physico-chemical nature of Japonica and Indica types of rice straw are greatly dissimilar. there were apparent differences in the process of compost fermentation. Fermentation of Indica type straw proceeded more rapidly with a shortening the compost period, reducing the water supply, and required adding of supplementary materials for producing stable physical conditions. 3. Use of barley straw compost resulted in a smaller crop compared with rice straw. but when a 50%, barley straw and 50% rice straw mixture was used, the yield was almost the same as that using only rice straw. 4. There were extremely high positive correlations between yield of Agaricus bisporus and the total nitrogen, organic nitrogen, amino acids, amides and amino sugar nitrogen content of compost. The mycerial growth and fruit body formation were severely inhibited by ammonium nitrogen. 5. When rice straw was used as the main substrate for compost media, urea was the most suitable source of nitrogen. Poor results were obtained with calcium cyanamide and ammonium sulfate. When urea was applied three separate times, nitrogen loss during composting was decreased and the total nitrogen content of compost was increased. 6. The supplementation of organic nutrient activated compost fermentation and increased yield of Agaricus bisporus. The best sources of organic nutrients were: perilla meal, sesame meal, wheat bran and poultry manure, etc. 7. Soybean meal, tobacco powder and glutamic acid fermentation by-products which were industrial wastes, could be substituted for perilla meal, sesame meal and wheat bran as organic nutrient sources for compost media. B. When gypsum and zeolite were added to rice straw. physical deterioration of compost due to excess moisture and caramelization was observed. The Indica type of straw was more remarkable in increase of yield of Agricus bisporus by addition of supplementing materials than Japonica straw. 9. For preparing rice straw compost, the best mixture was prepared by 10% poultry manure, 5% perilla meal, 1. 2 to 1. 5% urea and 1% gypsum. At spring cropping, it was good to add rice bran to accelerate heat generation of the compost heap. 10. There was significantly high positive correlation (r=0.97) between accumulated temperature and the decomposition degree of compost during outdoor composting. The yield was highest at accumulated temperatures between 900 and $1,000^{\circ}C$. 11. Prolonging the composting period brought about an increase in decomposition degree and total nitrogen content, but a decrease in ammonium nitrogen. In the spring the suitable period of composting was 20 to 25 days. and about 15 days in autumn. For those periods, the degree of decomposition was 19 to 24%. 12. Compactness of wet compost at filling caused an increase in the residual ammonium nitrogen. methane and organic acid during peak heating. There was negative correlation between methane content and yield (r=0.76)and the same was true between volatile organic acid and yield (r=0.73). 13. In compost with a moisture content range between 69 to 80% at filling. the higher the moisture content, the lower the yield (r=0.78). This result was attributed to a reduction in the porosity of compost at filling the beds. The optimum porosity for good fermentation was between 41 and 53%. 14. Peak heating of the compost was essential for the prevention of harmful microorganisms and insect pests. and for the removal of excess ammonia. It was necessary to continue fer mentatiion for four days after peak heating. 15. Ten species of fungi which are harmful or competitive to Agaricus bisporus were identified from the rice compost, including Diehliomyces microsporus, Trichoderma sp. and Stysanus stemoites. The frequency of occurrance was notably high with serious damage to Agaricus bisporus. 16. Diehliomyces microsporus could be controlled by temperature adjustment of the growing room and by fumigating the compost and the house with Basamid and Vapam. Trichoderma was prevented by the use of Bavistin and Benomyl. 17. Four species of nematodes and five species of mites occured in compost during out-door composting. These orgnanisms could be controlled through peakheating compost for 6 hours at $60^{\circ}C$.

• 한국균학회소식:학술대회논문집
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• 2015.05a
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• pp.45-46
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• 2015

Clubroot disease of crucifers has occurred since 1957. It has spread to the whole China, especially in the southwest and nourtheast where it causes 30-80% loss in some fields. The disease has being expanded in the recent years as seeds are imported and the floating seedling system practices. For its effective control, the Ministry of Agriculture of China set up a program in 2010 and a research team led by Dr. Yueqiu HE, Yunnan Agricultural University. The team includes 20 main reseachers of 11 universities and 5 institutions. After 5 years, the team has made a lot of progresses in disease occurrence regulation, resources collection, resistance identification and breeding, biological agent exploration, formulation, chemicals evaluation, and control strategy. About 1200 collections of local and commercial crucifers were identified in the field and by artificiall inoculation in the laboratories, 10 resistant cultivars were breeded including 7 Chinese cabbages and 3 cabbages. More than 800 antagostic strains were isolated including bacteria, stretomyces and fungi. Around 100 chemicals were evaluated in the field and greenhouse based on its control effect, among them, 6 showed high control effect, especially fluazinam and cyazofamid could control about 80% the disease. However, fluzinam has negative effect on soil microbes. Clubroot disease could not be controlled by bioagents and chemicals once when the pathogen Plasmodiophora brassicae infected its hosts and set up the parasitic relationship. We found the earlier the pathogent infected its host, the severer the disease was. Therefore, early control was the most effective. For Chinese cabbage, all controlling measures should be taken in the early 30 days because the new infection could not cause severe symptom after 30 days of seeding. For example, a biocontrol agent, Bacillus subtilis Strain XF-1 could control the disease 70%-85% averagely when it mixed with seedling substrate and was drenching 3 times after transplanting, i.e. immediately, 7 days, 14 days. XF-1 has been deeply researched in control mechanisms, its genome, and development and application of biocontrol formulate. It could produce antagonistic protein, enzyme, antibiotics and IAA, which promoted rhizogenesis and growth. Its The genome was sequenced by Illumina/Solexa Genome Analyzer to assembled into 20 scaffolds then the gaps between scaffolds were filled by long fragment PCR amplification to obtain complet genmone with 4,061,186 bp in size. The whole genome was found to have 43.8% GC, 108 tandem repeats with an average of 2.65 copies and 84 transposons. The CDSs were predicted as 3,853 in which 112 CDSs were predicted to secondary metabolite biosynthesis, transport and catabolism. Among those, five NRPS/PKS giant gene clusters being responsible for the biosynthesis of polyketide (pksABCDEFHJLMNRS in size 72.9 kb), surfactin(srfABCD, 26.148 kb, bacilysin(bacABCDE 5.903 kb), bacillibactin(dhbABCEF, 11.774 kb) and fengycin(ppsABCDE, 37.799 kb) have high homolgous to fuction confirmed biosynthesis gene in other strain. Moreover, there are many of key regulatory genes for secondary metabolites from XF-1, such as comABPQKX Z, degQ, sfp, yczE, degU, ycxABCD and ywfG. were also predicted. Therefore, XF-1 has potential of biosynthesis for secondary metabolites surfactin, fengycin, bacillibactin, bacilysin and Bacillaene. Thirty two compounds were detected from cell extracts of XF-1 by MALDI-TOF-MS, including one Macrolactin (m/z 441.06), two fusaricidin (m/z 850.493 and 968.515), one circulocin (m/z 852.509), nine surfactin (m/z 1044.656~1102.652), five iturin (m/z 1096.631~1150.57) and forty fengycin (m/z 1449.79~1543.805). The top three compositions types (contening 56.67% of total extract) are surfactin, iturin and fengycin, in which the most abundant is the surfactin type composition 30.37% of total extract and in second place is the fengycin with 23.28% content with rich diversity of chemical structure, and the smallest one is the iturin with 3.02% content. Moreover, the same main compositions were detected in Bacillus sp.355 which is also a good effects biocontol bacterial for controlling the clubroot of crucifer. Wherefore those compounds surfactin, iturin and fengycin maybe the main active compositions of XF-1 against P. brassicae. Twenty one fengycin type compounds were evaluate by LC-ESI-MS/MS with antifungal activities, including fengycin A $C_{16{\sim}C19}$, fengycin B $C_{14{\sim}C17}$, fengycin C $C_{15{\sim}C18}$, fengycin D $C_{15{\sim}C18}$ and fengycin S $C_{15{\sim}C18}$. Furthermore, one novel compound was identified as Dehydroxyfengycin $C_{17}$ according its MS, 1D and 2D NMR spectral data, which molecular weight is 1488.8480 Da and formula $C_{75}H_{116}N_{12}O_{19}$. The fengycin type compounds (FTCPs $250{\mu}g/mL$) were used to treat the resting spores of P. brassicae ($10^7/mL$) by detecting leakage of the cytoplasm components and cell destruction. After 12 h treatment, the absorbencies at 260 nm (A260) and at 280 nm (A280) increased gradually to approaching the maximum of absorbance, accompanying the collapse of P. brassicae resting spores, and nearly no complete cells were observed at 24 h treatment. The results suggested that the cells could be lyzed by the FTCPs of XF-1, and the diversity of FTCPs was mainly attributed to a mechanism of clubroot disease biocontrol. In the five selected medium MOLP, PSA, LB, Landy and LD, the most suitable for growth of strain medium is MOLP, and the least for strains longevity is the Landy sucrose medium. However, the lipopeptide highest yield is in Landy sucrose medium. The lipopeptides in five medium were analyzed with HPLC, and the results showed that lipopeptides component were same, while their contents from B. subtilis XF-1 fermented in five medium were different. We found that it is the lipopeptides content but ingredients of XF-1 could be impacted by medium and lacking of nutrition seems promoting lipopeptides secretion from XF-1. The volatile components with inhibition fungal Cylindrocarpon spp. activity which were collect in sealed vesel were detected with metheds of HS-SPME-GC-MS in eight biocontrol Bacillus species and four positive mutant strains of XF-1 mutagenized with chemical mutagens, respectively. They have same main volatile components including pyrazine, aldehydes, oxazolidinone and sulfide which are composed of 91.62% in XF-1, in which, the most abundant is the pyrazine type composition with 47.03%, and in second place is the aldehydes with 23.84%, and the third place is oxazolidinone with 15.68%, and the smallest ones is the sulfide with 5.07%.

• 한국균학회소식:학술대회논문집
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• 2016.05a
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• pp.19-20
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• 2016

Sesquiterpenoids are defined as $C_{15}$ compounds derived from farnesyl pyrophosphate (FPP), and their complex structures are found in the tissue of many diverse plants (Degenhardt et al. 2009). FPP's long chain length and additional double bond enables its conversion to a huge range of mono-, di-, and tri-cyclic structures. A number of cyclic sesquiterpenes with alcohol, aldehyde, and ketone derivatives have key biological and medicinal properties (Fraga 1999). Fungi, such as the wood-rotting Polyporus brumalis, are excellent sources of pharmaceutically interesting natural products such as sesquiterpenoids. In this study, we investigated the biosynthesis of P. brumalis sesquiterpenoids on modified medium. Fungal suspensions of 11 white rot species were inoculated in modified medium containing $C_6H_{12}O_6$, $C_4H_{12}N_2O_6$, $KH_2PO_4$, $MgSO_4$, and $CaCl_2$ for 20 days. Cultivation was stopped by solvent extraction via separation of the mycelium. The metabolites were identified as follows: propionic acid (1), mevalonic acid lactone (2), ${\beta}$-eudesmane (3), and ${\beta}$-eudesmol (4), respectively (Figure 1). The main peaks of ${\beta}$-eudesmane and ${\beta}$-eudesmol, which were indicative of sesquiterpene structures, were consistently detected for 5, 7, 12, and 15 days These results demonstrated the existence of terpene metabolism in the mycelium of P. brumalis. Polyporus spp. are known to generate flavor components such as methyl 2,4-dihydroxy-3,6-dimethyl benzoate; 2-hydroxy-4-methoxy-6-methyl benzoic acid; 3-hydroxy-5-methyl phenol; and 3-methoxy-2,5-dimethyl phenol in submerged cultures (Hoffmann and Esser 1978). Drimanes of sesquiterpenes were reported as metabolites from P. arcularius and shown to exhibit antimicrobial activity against Gram-positive bacteria such as Staphylococcus aureus (Fleck et al. 1996). The main metabolites of P. brumalis, ${\beta}$-Eudesmol and ${\beta}$-eudesmane, were categorized as eudesmane-type sesquiterpene structures. The eudesmane skeleton could be biosynthesized from FPP-derived IPP, and approximately 1,000 structures have been identified in plants as essential oils. The biosynthesis of eudesmol from P. brumalis may thus be an important tool for the production of useful natural compounds as presumed from its identified potent bioactivity in plants. Essential oils comprising eudesmane-type sesquiterpenoids have been previously and extensively researched (Wu et al. 2006). ${\beta}$-Eudesmol is a well-known and important eudesmane alcohol with an anticholinergic effect in the vascular endothelium (Tsuneki et al. 2005). Additionally, recent studies demonstrated that ${\beta}$-eudesmol acts as a channel blocker for nicotinic acetylcholine receptors at the neuromuscular junction, and it can inhibit angiogenesis in vitro and in vivo by blocking the mitogen-activated protein kinase (MAPK) signaling pathway (Seo et al. 2011). Variation of nutrients was conducted to determine an optimum condition for the biosynthesis of sesquiterpenes by P. brumalis. Genes encoding terpene synthases, which are crucial to the terpene synthesis pathway, generally respond to environmental factors such as pH, temperature, and available nutrients (Hoffmeister and Keller 2007, Yu and Keller 2005). Calvo et al. described the effect of major nutrients, carbon and nitrogen, on the synthesis of secondary metabolites (Calvo et al. 2002). P. brumalis did not prefer to synthesize sesquiterpenes under all growth conditions. Results of differences in metabolites observed in P. brumalis grown in PDB and modified medium highlighted the potential effect inorganic sources such as $C_4H_{12}N_2O_6$, $KH_2PO_4$, $MgSO_4$, and $CaCl_2$ on sesquiterpene synthesis. ${\beta}$-eudesmol was apparent during cultivation except for when P. brumalis was grown on $MgSO_4$-free medium. These results demonstrated that $MgSO_4$ can specifically control the biosynthesis of ${\beta}$-eudesmol. Magnesium has been reported as a cofactor that binds to sesquiterpene synthase (Agger et al. 2008). Specifically, the $Mg^{2+}$ ions bind to two conserved metal-binding motifs. These metal ions complex to the substrate pyrophosphate, thereby promoting the ionization of the leaving groups of FPP and resulting in the generation of a highly reactive allylic cation. Effect of magnesium source on the sesquiterpene biosynthesis was also identified via analysis of the concentration of total carbohydrates. Our current study offered further insight that fungal sesquiterpene biosynthesis can be controlled by nutrients. To profile the metabolites of P. brumalis, the cultures were extracted based on the growth curve. Despite metabolites produced during mycelia growth, there was difficulty in detecting significant changes in metabolite production, especially those at low concentrations. These compounds may be of interest in understanding their synthetic mechanisms in P. brumalis. The synthesis of terpene compounds began during the growth phase at day 9. Sesquiterpene synthesis occurred after growth was complete. At day 9, drimenol, farnesol, and mevalonic lactone (or mevalonic acid lactone) were identified. Mevalonic acid lactone is the precursor of the mevalonic pathway, and particularly, it is a precursor for a number of biologically important lipids, including cholesterol hormones (Buckley et al. 2002). Farnesol is the precursor of sesquiterpenoids. Drimenol compounds, bi-cyclic-sesquiterpene alcohols, can be synthesized from trans-trans farnesol via cyclization and rearrangement (Polovinka et al. 1994). They have also been identified in the basidiomycota Lentinus lepideus as secondary metabolites. After 12 days in the growth phase, ${\beta}$-elemene caryophyllene, ${\delta}$-cadiene, and eudesmane were detected with ${\beta}$-eudesmol. The data showed the synthesis of sesquiterpene hydrocarbons with bi-cyclic structures. These compounds can be synthesized from FPP by cyclization. Cyclic terpenoids are synthesized through the formation of a carbon skeleton from linear precursors by terpene cyclase, which is followed by chemical modification by oxidation, reduction, methylation, etc. Sesquiterpene cyclase is a key branch-point enzyme that catalyzes the complex intermolecular cyclization of the linear prenyl diphosphate into cyclic hydrocarbons (Toyomasu et al. 2007). After 20 days in stationary phase, the oxygenated structures eudesmol, elemol, and caryophyllene oxide were detected. Thus, after growth, sesquiterpenes were identified. Per these results, we showed that terpene metabolism in wood-rotting fungi occurs in the stationary phase. We also showed that such metabolism can be controlled by magnesium supplementation in the growth medium. In conclusion, we identified P. brumalis as a wood-rotting fungus that can produce sesquiterpenes. To mechanistically understand eudesmane-type sesquiterpene biosynthesis in P. brumalis, further research into the genes regulating the dynamics of such biosynthesis is warranted.