• The Plant Pathology Journal
• /
• v.18 no.1
• /
• pp.23-29
• /
• 2002

A total of 672 Cryphonectria parasitica was isolated from 2,536 blight lesions on chestnut twigs, which were collected from major chestnut plantations all over Korea. Isolation rates of each province ranged from 13.5% in Jeonbuk-ds to 37.4% in Gyeongnam-do, with an average rate of 25.6%. The isolates were classified into six groups according to color and shape of colony on PDA: smooth margin (S), irregular margin (I), yellow to brown (Y), white (W), and white with yellow center (C). Among these groups, IY was the most abundant with an isolation rate of 65%. On the other hand, SW, SC, IW, and SY were quite rare, with isolation rates ranging from 1.5% to 5.8%. When the 672 isolates were inoculated on the chestnut twigs,380 isolates (56.5%) caused lesions larger than the standard virulent isolate EPISS-2, while 158 isolates (23.4%) caused smaller lesions than the standard hypovirulent isolate UEP-1. However, 87.4% of the isolates belonged to the virulent group and only 12.6% belonged to the hypovirulent group based on Bavendamm test. In the provinces of Jeonnam-do, Jeonbuk-do, and Gyeongnamdo, which have high density of chestnut trees, the rates of hypovirulent-like isolates were over 20%.

• The Plant Pathology Journal
• /
• v.16 no.3
• /
• pp.142-146
• /
• 2000

Ordered differential display using RT-PCR (ODD-PCR) was conducted to have a profile of the differently expressed genes between a hypovirulent strain of Cryphonectria parasitica (UEP1) and its isogenic wild type strain (EP155/2). ODD-PCR has advantages of high sensitivity, reproducibility, proportional representation, and limited number of primer combinations comparing with other differential display methods. RNAs were prepared from 1 and 5 day liquid culture of both hypovirulent and wild type strains, and were further evaluated with the marker genes of C. parasitica such as cryparin and mating factor MF2-1, which were already proven to be specifically down-regulated by the presence of mycovirus CHV1-713. ODD-PCR was conducted using those RNAs and expressed genes were categorized to five groups according to their temporal and quantitative expression patterns. Those fives groups are CPC, CPE, CPL, CPD, and CPU which represent constitutively-expressed, early-expressed, late-expressed, down-regulated, and up-regulated, respectively. Ninety two primer combinations out of a total of 192 have been tested so far. Among the twenty to fifty distinct bands per each reaction, an average of four to ten genes was identified as viral-regulated fungal genes. Those viral-specifc genes were further analyzed by DNA sequencing followed by homology search. Characterization of 30 clones including all five groups were conducted as a preliminary data and more are under investigation.

• Korean Journal of Microbiology
• /
• v.40 no.1
• /
• pp.12-16
• /
• 2004

Acid proteinase has been discovered in Aspergillus niger (acid protease A) and Cryphonectria parasitica (acid proteinase EapC) and it plays major roles in cheese formation from milk. In this study, a partial gene encoding acid proteinase in Penicillium oxalicum HCLF-34 was cloned by using PCR with degenerate primers corresponding to highly conserved regions of the acid proteinase. The partial acid proteinase gene in P. oxalicum HCLF-34 contains an open reading frame of 438 base pairs and encodes an acid proteinase protein of 146 amino aicds. The predicted amino acid sequences showed 71 % homology with acid protease A and 67% homology with EapC.

• Molecules and Cells
• /
• v.26 no.5
• /
• pp.496-502
• /
• 2008

Cryparin, encoded as a single copy gene (Crp) of the chestnut blight fungus Cryphonectria parasitica, is the most abundant protein produced by this fungus. However, its accumulation is decreased remarkably in C. parastica strains containing the double-stranded (ds) RNA virus Cryphonectria hypovirus 1. To characterize the transcriptional regulatory element(s) for strong expression and viral regulation, promoter analysis was conducted. Serial deletion of the Crp promoter region resulted in a step-wise decrease in promoter activity, indicating a localized distribution of genetic elements in the cryparin promoter. Promoter analysis indicated two positive and a repressive cis-acting elements. Among them, the promoter region between nt -1,282 and -907 appeared to be necessary for hypoviral-mediated down-regulation. An electrophoretic mobility shift assay (EMSA) on the corresponding promoter region (-1,282/-907) indicated two regions at (-1,257/-1,158) and (-1,107/-1,008) with the characteristic AGGAGGA-N42-GAGAGGA and its inverted repeat TCCTCTC-N54-TCCTCCT, respectively, appeared to be specific binding sites for cellular factors.

• Mycobiology
• /
• v.45 no.4
• /
• pp.362-369
• /
• 2017

We assessed the regulation of cryparin, a class II hydrophobin, using three representative mitogen-activated protein kinase (MAPK) pathways in Cryphonectria parasitica. Mutation of the CpSlt2 gene, an ortholog of yeast SLT2 in the cell wall integrity (CWI) pathway, resulted in a dramatic decrease in cryparin production. Similarly, a mutant of the CpBck1 gene, a MAP kinase kinase kinase gene in the CWI pathway, showed decreased cryparin production. Additionally, mutation of the cpmk1 gene, an ortholog of yeast HOG1, showed decreased cryparin production. However, mutation of the cpmk2 gene, an ortholog of yeast Kss1/Fus3, showed increased cryparin production. The easy-wet phenotype and accumulation of the cryparin transcript in corresponding mutants were consistent with the cryparin production results. In silico analysis of the promoter region of the cryparin gene revealed the presence of binding motifs related to downstream transcription factors of CWI, HOG1, and pheromone responsive pathways including MADS-box- and Ste12-binding domains. Real-time reverse transcriptase PCR analyses indicated that both CpRlm1, an ortholog of yeast RLM1 in the CWI pathway, and cpst12, an ortholog of yeast STE12 in the mating pathway, showed significantly reduced transcription levels in the mutant strains showing lower cryparin production in C. prasitica. However, the transcription of CpMcm1, an ortholog of yeast MCM1, did not correlate with that of the mutant strains showing downregulation of cryparin. These results indicate that three representative MAPK pathways played a role in regulating cryparin production. However, regulation varied depending on the MAPK pathways: the CWI and HOG1 pathways were stimulatory, whereas the pheromone-responsive MAPK was repressive.

• The Korean Journal of Mycology
• /
• v.26 no.4 s.87
• /
• pp.450-457
• /
• 1998

The cryparin of Cryphonectria parasitica belongs to a cell wall associated fungal hydrophobin. The cryparin, though it is encoded by a single copy gene, is known for the high expression during the liquid culture of C. parasitica, and it turns out that 22% of total mRNA was transcribed for cryparin at 48hr after the liquid culture. In addition, it is also known as one of down-regulated fungal proteins by the presence of double stranded RNA virus, Cryphonectria hypovirus 1. In previous studies (Kim et al., 1999), we have constructed a cryparin-null mutant by replacing the cryparin gene with hygromycin B resistance gene due to site directed homologous recombination. In order for the promoter analysis of cryparin which seems to be very strong as well as mycoviral specific, it is preferable to have a strain with only a target promoter replaced and a discernable target site for incoming vectors. However, the cryparin-null mutant revealed the presence of an additional copy of transforming vector except the one which replaced the cryparin gene. In addition, the cryparin-null mutant did not contain any markers for targeted integration of incoming vectors. This prompts us to design an experiment to obtain a strain for promoter analysis of cryparin gene. A different mating type strain EP6(Mata, $met^-$) was mated with the cryparin-null mutant ${\triangle}$Crp194-7(MatA, Crp${\triangle}$::hph) to make the progenies with only a single replacement vector and $met^-$ characteristic remained. Nutritional assay as well as Southern blot analysis revealed that the progeny, ${\triangle}$Crp194-a6, was the methionine auxotroph with a single replacing vector in genome. Northern blot analysis and PAGE showed that there was no cryparin produced in this bred strain either.

• Korean journal of applied entomology
• /
• v.21 no.4 s.53
• /
• pp.211-215
• /
• 1982

Incidences of Phytophthora blight in plant of sesame (Sesamum indicum L.) were observed in southern sesame production areas, Gochang of Jeonbug, Yeonggwang of Jeonnam, Jinyang of Gyeongnam and Dalseong of Gyeongbug province where disease survy was conducted from July 29 to August 1, 1981. The rate of disease incidence ranged from none to $61\%$ depending upon the field observed. The causal species of the Phytophthora was identified as P. nicotianae var. parasitica (Dastur) Waterhouse based on specific pathogenicity to sesame and morphological characteristics of sporangia. Diseased plants of sesame generally showed dark discoloration on the stem leading to plant death.

• Proceedings of the Korean Society of Plant Pathology Conference
• /
• 2003.10a
• /
• pp.23-23
• /
• 2003

Chestnut blight, caused by Cryphonectria parasitica, is the most destructive disease of American and European chestnut trees. A total of 672 C prasitica was isolated from blight lesion on chestnut twigs, which were collected from major chestnut plantations all over Korea in 1999. Isolation rates were over 30% in Kyunggj-, Kyongnam-, and Chonnam-do. The highest isolation rate was 37.4% and recorded in Kyongnam-do. On the other hand, Chonbuk-do had the lowest isolation rate as 13.5%. In grouping of C parasitica by colony shape and color, yellow colony with irregular margin were the most dominant colony type with a frequency of 65.2%. When the 672 isolates were inoculated on the chestnut twigs, 380 isolates (56.5%) caused lesions larger than the standard virulent isolate EP155-2, while 158 isolates (23.4%) caused smaller lesions than the standard hypovirulent isolate UEP-1. In Bavendamm test that determines phenol oxidase activity, 97.1% of all the isolates resulted the same or darker discoloration than EP155-2, and only 12.2% resulted the same or lighter discoloration than UEP-1. In the vegetative compatibility (VC) tests, total 670 isolates were divided into 121 VC groups (VCGs). Kyongnam-, Chonnam-, and Chungnam-do, the three principal chestnut plantation area, had 49, 33, and 27 VCGs, respectively. Among the VCGs, the biggest VCG, KR-VC104, was composed of 164 isolates and the second biggest VCG had 62 isolates. But, 64 of 121 VCGs consisted of sole member. More than 65.8% of KR-VC104, was isolated from the three provinces, Kyongnam-, Kangwon-, and Chungbuk-do. In KR-VC104, 62.8%, 59.1%, and 85.9% of the isolates looked like virulent in colony type, pathogenicity test, and Bavendamm test. In ds-RNA detection tests using cellulose chromatography, 77 of total 650 isolates were ds-RNA positive and detected ds-RNA segments were approximately 12kb, 3kb, 2.7kb, 2kb, and 1.8kb in size. Among the 77 isolates, 46 isolates had 12kb and 25 isolates had 12kb and 2.7kb. Other 6 Isolates had small ds-RNA segments. Kyongnam-, Chonnam-, and Chungnam-do had 43, 16, and 5 ds-RNA positive isolates, respectively. Among the 121 VCGs, only 29 VCGs had ds-RNA positive isolates.(중략)

• Proceedings of the Microbiological Society of Korea Conference
• /
• 2005.05a
• /
• pp.159-161
• /
• 2005

Hypovirus infection of the chestnut blight fungus Cryphonectria parasitica is a useful model system to study the hypoviral regulation of fungal gene expression. The hypovirus is known to downregulate the fungal laccase1 (lac 1), the modulation of which is tightly governed by the inositol triphosphate ($IP_3$) and calcium second messenger system in a virus-free strain. We cloned the gene cplc1 encoding a phosphatidyl inositol-specific phospholipase C (PLC), in order to better characterize the fungal gene regulation by hypovirus. Sequence analysis of the cplc1 gene indicated that the protein product contained both the X and Y domains, which are the two conserved regions found in all known PLCs, with a 133 amino acid extension between the 2nd ${\beta}$-strand and the ${\alpha}$-helix in the X domain. In addition, the gene organization appeared to be highly similar to that of a ${\delta}$ type PLC. Disruption of the cplc1 gene resulted in slow growth and produced colonies characterized by little aerial mycelia and deep orange in color. In addition, down regulation of lac1 expression was observed. However, temperature sensitivity, osmosensitivity, virulence, and other hypovirulence-associated characteristics did not differ from the wild-type strain. Functional complementation of the cplc1-null mutant with the PLC1 gene from Saccharomyces cerevisiae restored lac1 expression, which suggests that the cloned gene encodes PLC activity. The present study indicates that the cplc1 gene is required for appropriate mycelial growth, and that it regulates the lac1 expression, which is also modulated by the hypovirus. Although several PLC genes have been identified in various simple eukaryotic organisms, the deletion analysis of the cplc1 gene in this study appears to be the first report on the functional analysis of PLC in filamentous fungi.

• Journal of the Korean Wood Science and Technology
• /
• v.31 no.1
• /
• pp.1-9
• /
• 2003

Antimicrobial activities of plant extractives were investigated to develop a natural fungicide. Two stilbenoids and five flavonoids were isolated from wood extractives of Hovenia dulcis (Rhamnaceae) which had been selected due to its high antifungal activity among the tested species. The chemical structures of isolated compounds were determinded as : 5-hydroxy-7-methoxyflavone, 5,7-dihydroxyflavone (chrysin), 5,7-dihydroxyflavanone (pinocembrin), 3,5,7-trihydroxyflavanone (pinobanksin), 3,4',5,7-tetrahydroxyflavanone (aromadendrin), 3-hydroxy-5-methoxystilbene and 3,5-dihydroxystilbene (pinosylvin) on the basis of Mass and NMR spectroscopic data. According to the results of antifungal test, 3-hydroxy-5-methoxystilbene was evaluated as the strongest antifungal compound among the tested compounds and next were pinocembrin and pinosylvin, but those also had high hyphal growth inhibition activities against C. parasitica, T. versicolor, T. palustris and T. viride. However, pinobanksin, 5-hydroxy-7-methoxyflavone, chrysin and aromadendrin showed very low antifungal activity. In this regard, it could inferred that high antifungal activity of wood extractives of H. dulcis were derived from 3-hydroxy-5-methoxystilbene, pinocembrin and pinosylvin, respectively.