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The anthracnose disease on pepper fruits in Korea was caused by Colletotrichum acutatum as well as C. gloeosporioides. Since C. acutatum showed less sensitivity to benomyl, it was analyzed whether the less sensitivity was given by the same mechanism for the fungicide resistance of C. gloeosporioides. The isolates of C. acutatum were less sensitive to the three benzimidazole fungicides tested, benomyl, carbendazim, and thiophanate-methyl. However, the of C. acutatum isolates were different from the resistant isolates of C. gloeosporioides in their response to diethofencarb, one of N-phenyl-carbamates; the former was still less sensitive to diethofencarb than the latter. The differences in the resistance mechanisms in two species were conspicuous in sequence analysis of the tub2 genes. The genes from C. acutatum did not show any non-synonymous base substitutions at the regions known to be correlated with the benzimidazole-resistance. All of these data may indicate that the less sensitivity of C. acutatum to benomyl is based on different mechanism(s) from that of C. gloeosporioides.

Rapid translation of genome sequences into meaningful biological information hinges on the integration of multiple experimental and informatics methods into a cohesive platform. Despite the explosion in the number of genome sequences available, such a platform does not exist for filamentous fungi. Here we present the development and application of a functional genomics and informatics platform for a model plant pathogenic fungus, Magnaporthe oryzae. In total, we produced 21,070 mutants through large-scale insertional mutagenesis using Agrobacterium tumefaciens-mediated transformation. We used a high-throughput phenotype screening pipeline to detect disruption of seven phenotypes encompassing the fungal life cycle and identified the mutated gene and the nature of mutation for each mutant. Comparative analysis of phenotypes and genotypes of the mutants uncovered 202 new pathogenicity loci. Our findings demonstrate the effectiveness of our platform and provide new insights on the molecular basis of fungal pathogenesis. Our approach promises comprehensive functional genomics in filamentous fungi and beyond.

Anthracnose severely occurred on begonia (Begonia semperflorens Link.) nurseries in Gyeongju, Gyeongbuk in July, 2004. More than of begonia seedlings were diseased in the greenhouse surveyed and diseased leaves per plant were in average. Yellowish spots occurred on the leaves of begonia as initial symptoms, and they coalesced irregularly to form large brown pleomorphic lesions. Severely infected leaves were defoliated, resulting in abnormal growth of the entire plant. Colletotrichum sp. was repeatedly isolated from the diseased plants and was identified as Colletotrichum acutatum on the basis of the mycological characteristics on potato dextrose agar and RAPD analysis. Pathogenicity of the fungus was also confirmed by artificial inoculation on healthy plants. The optimum temperature for mycelial growth of C. acutatum was around . The fungus was sensitive to azoxystrobin, bitertanol, diethofencarb-carbendazim, difenoconazole and tebuconazole. This is the first report on the anthracnose of begonia caused by C. acutatum in Korea.

Actinobacillus pleuropneumoniae is an important pig pathogen, which is responsible for swine pleuropneumonia, a highly contagious respiratory infection. To develop subunit vaccines forA. pleuropneumoniae infection, the Apx toxin genes,apxI andapxII, which are thought to be important for protective immunity, were expressed inSaccharomyces cerevisiae, and the induction of immune responses in mice was examined. TheapxI andapxII genes were placed under the control of a yeast hybridADH2-GPD promoter (AG), consisting of alcohol dehydrogenase II (ADH2) and theGPD promoter. Western blot analysis confirmed that both toxins were successfully expressed in the yeast. The ApxIA and ApxIIA-specific IgG antibody response assays showed dose dependent increases in the antigen-specific IgG antibody titers. The challenge test revealed that ninety percent of the mice immunized with ApxIIA or a mixture of ApxIA and ApxIIA, and sixty percent of mice immunized with ApxIA survived, while none of those in the control groups survived longer than 36 h. These results suggest that vaccination of the yeast expressing the ApxI and ApxII antigens is effective for the induction of protective immune responses againstA. pleuropneumoniae infections in mice.

A novel rice (Oryza sativa L.) gene, homologous to Arabidopsis pathogenesis-related NDR1 gene, was cloned from cDNA library prepared from 30 min Magnaporthe grisea -treated rice seedling leaves, and named as OsNDR1. OsNDR1 encoded a 220-aminoacid polypeptide and was highly similar to the Arabidopsis AtNDR1 protein. OsNDR1 is a plasma membrane (PM)-localized protein, and presumes through sequence analysis and protein localization experiment. Overexpression of OsNDR1 promotes the expression of PBZ1 that is essential for the activation of defense/stressrelated gene. The OsNDR1 promoter did not respond significantly to treatments with either SA, PBZ, or ETP. Exogenously applied BTH induces the same set of SAR genes as biological induction, providing further evidence for BTH as a signal. Presumably, BTH is bound by a receptor and the binding triggers a signal transduction cascade that has an ultimate effect on transcription factors that regulate SAR gene expression. Thus OsNDR1 may act as a transducer of pathogen signals and/or interact with the pathogen and is indeed another important step in clarifying the component participating in the defense response pathways in rice.

Severe wilt disease epidemic was found on winter daphnes (Daphne odora Thunb.) cultivated in farmers, nurseries in Suncheon, Jeonnam in 2003. Typical symptoms appeared on the leaves of winter daphne as yellowish wilts and turned brown from the lower leaves on the same plant. Severely infected leaves were defoliated, resulting in blight of stems and eventual death of the entire plant. Black decayed vascular tissues were distinctly observed in a wilted plant. Fusarium sp. was isolated from the diseased plants repeatedly and its pathogenicity was confirmed by artificial inoculation on healthy plants. The fungus was identified as Fusarium oxysporum on the basis of the morphological and cultural characteristics on potato dextrose agar and carnation leaf agar. The optimum temperature for fungal growth was around and the fungal growth was inhibited by metconazole, triflumizole and trifloxystrobin on potato dextrose agar. This is the first report on the wilt disease of winter daphnes caused by F.oxysporum in Korea.

Sensitivity of Phytophthora infestans isolates to fungicides metalaxyl and ethaboxam in Korea was examined with 260 isolates for 3 years (9 isolates in 2000,93 isolates in 2001, and 158 isolates in 2002). Both Al and A2 mating types were found from the isolates collected for 3 years. Al mating type was dominant in the population with 8 isolates (88.9%) in 2000, 84 isolates (89.4%) in 2001, and 138 isolates (87.3%) in 2002. Only some isolates from diseased tomatoes in Buyergun and diseased potatoes in Pyeongchanggun were of the A2 mating type. As for metalaxyl sensitivity, 77.0% of the isolates were moderately resistant with 8 isolates (88.9%) in 2000, 73 isolates (77.7%) in 2001, and 120 isolates (75.9%) in 2002. Meanwhile, those found resistant were 1 isolate (11.1%) in 2000, 16 isolates (17.0%) in 2001, and 33 isolates (20.9%) in 2002. Only 5 isolates (3.2%) were sensitive to metalaxyl in 2002. There was no significant difference in the sensitivity among years. As for ethaboxam, no isolate was able to grow at 5.0 /ml, and only four isolates (1.5 %) grew at 1.0 /ml with heavy retardation compared with the untreated control. Based on these 3-year results, the minimum inhibitory concentration (MIC) of ethaboxam to p. infestans was determined to be 0.2-1.0 /ml. Results indicate that resistance development by p. infestans to ethaboxam is not likely to occur in the natural condition. furthermore, there was no indication of cross resistance between metalaxyl and ethaboxam because all the isolates, regardless of classification for their sensitivity to metalaxyl, were not able to grow at 5.0 /ml of ethaboxam.

The ascomycete Magnaporthe grisea is a pathogen of rice blast and is known to form specialized infection structures called appressoria for successful infection into host cells. To understand the molecular mechanism underlying infection process, appressorium-related genes were identified through global approaches including EST sequencing, differential hybridization, and suppression subtractive hybridization. EST database was generated on >2,000 cDNA clones randomly selected from appressorium stage cDNA library. Large number of ESTs showed homology to known proteins possibly involved in infection-related cellular development (attachment, germination, appressorium formation, and colonization) of rice blast fungus. The 1051 ESTs showing significant homology to known genes were assigned to 11 functional categories. Differential hybridization and suppression subtractive hybridization were applied to identify genes showing an appressorium stage specific expression pattern. A number of genes were selected as up-regulated during appressorium formation compared with the vegetative growing stage. Clones from various cDNA libraries constructed in different developmental stages were arrayed on slide glass for further expression profiling study. Functional characterization of genes identified from these global approaches may lead to a better understanding of the infection process of this devastating plant disease, and the development of novel ways to protect host plant.

The conidial germ tube of the fungus Magnaporthe grisea differentiates an infection-specific structure, an appressorium, for penetration into the host plant. Formation of the appressorium is also observed on synthetic solid substrata such as polycarbonate. We found that a plant lectin, concanavalin A, specifically suppressed the appressorium formation without affecting the germling adhesion if it was applied within 2-3 hours after germination. Standing on the result, we constructed a cDNA library that represents the early stage of germ tube development and/or appressorium formation from the 2.5-hour-old germ tubes using a cDNA subtraction strategy by the combination of the biotin labeled driver method and adapter-primed PCR method. Out of 686 colonies of the library, 158 distinct clones' nucleotide sequences were partially analyzed. Some clones' expression patterns were detected by RT-PCR and from those results, our library seemed to well represent the objective developmental stage of M. grisea.

Magnaporthe grisea, the causal agent of rice blast disease, differentiates an appressorium to penetrate through the host cuticle with an infection peg elaborated from the appressorium. Similar reaction is observed on various synthetic substrata. By using a hardness-adjustable material, Hycel A-342, the correlation between appressorium formation and substratum hardness was evaluated. The results suggested that substratum hardness played an important role in triggering appressorium formation of M. grisea. Furthermore, growth orientation of germ tubes was coerced by the hydrophobicity of the contact surfaces. When conidia germinated at the interface of two differently hydrophobic phases, the germ tubes grew preferentially towards the more hydrophobic phase. Mutagenesis studies suggested that loss of this anti-hydrotactic behavior impaired appressorium formation. Since water is a pre-requisite for germination, we propose that the anti-hydrotactic response initiates attempted penetration into the plants by ger...

Magnaporthe grisea, the causal agent of rice blast, forms a dome-shaped melanized infection structure, an appressorium, to infect its host. Environmental cues that induce appressorium formation in this fungus include the hydrophobicity and hardness of the contact surface and chemicals produced by the host. An elevated concentration of intracellular cyclic AMP (cAMP) has been implicated in appressorium differentiation in M. grisea. Polyamines (putrescine, spermidine, and sper-mine) are involved in cell growth and differentiation in a wide range of organisms. To understand the role of polyamines in appressorium differentiation in M. grisea, intracellular polyamines were quantified, and the effects of polyamines and polyamine biosynthesis inhibitors on conidial germination and appressorium formation were tested. High levels of polyamines were detected in freshly collected spores, but the levels decreased during conidial germination. Spermidine was found to be the major component. Polyamines and polyamine biosynthesis inhibitors did not affect conidial germination, but polyamines specifically impaired appressorium formation. Furthermore, exogenous addition of cAMP restored appressorium formation inhibited by poly-amines. These results suggest that polyamines may reduce intracellular cAMP levels in M. grisea, leading to the inhibition of appressorium formation.

Colletotrichum gloeosporioides forms a specialized infection structure, an appressorium, to infect its host, red pepper. Polyamines (putrescine, spermidine, and spermine) as well as S-adenosyl methionine inhibitor, methylglyoxal-bis-guanyl hydrazone (MGBG), impaired conidial germination and appressorium formation of C. gloeosporioides. Curtailment of cell differentiation by polyamines and MGBG was more evident in conidial germination than in appressorium development. Exogenous addition of calcium restored conidial germination and appressorium formation and expression of calmodulin-encoding gene (CgCaM) inhibited by polyamines. Taken together, proper regulation of intracellular polyamine concentration is indispensable for conidial germination and appressorium formation, and involved in Ca(2+)/calmodulin-dependent signaling pathways of C. gloeosporioides infecting red pepper.