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The infection process of Colletotrichum lagenarium, the causal agent of cucumber anthracnose disease, involves several key steps: germination; formation of melanized appressoria; appressorial penetration; and subsequent invasive growth in host plants. Here we report that the C. lagenarium CMK1 gene encoding a mitogen-activated protein (MAP) kinase plays a central role in these infection steps. CMK1 can complement appressorium formation of the Pmk1 MAP kinase mutant of Magnaporthe grisea. Deletion of CMK1 causes reduction of conidiation and complete lack of pathogenicity to the host plant. Surprisingly, in contrast to M. grisea pmk1 mutants, conidia of cmk1 mutants fail to germinate on both host plant and glass surfaces, demonstrating that the CMK1 MAP kinase regulates conidial germination. However, addition of yeast extract rescues germination, indicating the presence of a CMK1-independent pathway for regulation of conidial germination. Germinating conidia of cmk1 mutants fail to form appressoria and the mutants are unable to grow invasively in the host plant. This strongly suggests that MAP kinase signaling pathways have general significance for infection structure formation and pathogenic growth in phytopathogenic fungi. Furthermore, three melanin genes show no or slight expression in the cmk1 mutant when conidia fail to germinate, suggesting that CMK1 plays a role in gene expression required for appressorial melanization.

Striped jack nervous necrosis virus (SJNNV), a nodavirus, is the causative agent of viral nervous necrosis (VNN) in larval striped jack fish. In the present study, the SJNNV coat protein gene was sequenced and compared with that of four known insect nodaviruses and with four other fish nodaviruses causing VNN. The SJNNV coat protein gene was 1410 bases in length and contained a single ORF of 1023 bases encoding a protein of 340 amino acids. The sequence similarities between the coat protein gene of SJNNV and four insect nodaviruses were 28.6 % or less at the nucleotide level and 10.6% or less at the amino acid level. A portion of the coat protein gene from four additional fish VNN viruses was amplified by PCR using primers designed for SJNNV and the amplified fragments (870-876 bases) were sequenced. The sequence similarities among SJNNV and the four VNN viruses were 75-8 % or greater at the nucleotide level and 80.9 % or greater at the amino acid level. In the fish nodaviruses a highly conserved region of 134 amino acids with sequence similarity of 92.5 % or greater was detected. This conserved sequence was not found in the coat protein of insect nodaviruses. These results indicate that the fish nodaviruses that cause VNN are closely related to each other but are quite different from insect nodaviruses.

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Six cell clones were derived from the SSN-1 cell line, which is composed of a mixed cell population and persistently infected with a C-type retrovirus (SnRV). These clones were susceptible to 4 piscine nodavirus strains belonging to different genotypes (SJNNV, RGNNV, TPNNV and BFNNV [striped jack, redspotted grouper, tiger puffer and barfin flounder nervous necrosis viruses]). Three clones, designated A-6, E-9, and E-11, were highly permissive to nodavirus infection and production. The virus-induced cytopathic effects appeared as cytoplasmic vacuoles and intensive disintegration at 3 to 5 d post-incubation. These observations were highly reproducible and formed the basis for a successful virus titration system. Quantitative analysis using the cloned E-11 cell line clearly revealed differences in the optimal growth temperatures among the 4 genotypic variants: 25 to 30°C for strain SGWak97 (RGNNV), 20 to 25°C for strain SJNag93 (SJNNV), 20°C for strain TPKag93 (TPNNV), and 15 to 20°C for strain JFIwa98 (BFNNV). Electron microscopy demonstrated SnRV retrovirus particles only in A-6 and E-9 cells, but PCR amplification for the pol gene and LTR region of the proviral DNA indicated the presence of the retrovirus in the other clones, including E-11. The cell clones obtained in the present study will be more useful for qualitative and quantitative analyses of piscine nodaviruses than the SSN-1 cell line. KEY WORDS: Nodavirus · Viral nervous necrosis, VNN · SSN-1 cell line · Cell cloning · C-type retrovirus · Snakehead retrovirus Resale or republication not permitted without written consent of the publisherDis Aquat Org 43: [81][82][83][84][85][86][87][88][89] 2000 that a new cell line (GF-1) derived from grouper Epinephelus coioides was useful for the isolation and proliferation of a piscine nodavirus (GNNV, grouper nervous necrosis virus).Piscine nodaviruses can be divided into 4 genotypic groups based on partial sequences of the coat protein gene (Nishizawa et al. 1997): SJNNV (striped jack nervous necrosis virus), RGNNV (redspotted grouper nervous necrosis virus), TPNNV (tiger puffer nervous necrosis virus), and BFNNV (barfin flounder nervous necrosis virus). In a previous study, we demonstrated that the SSN-1 cell line was useful for propagating and differentiating 17 isolates of piscine nodavirus collected from 13 host fish species in 5 countries (Iwamoto et al. 1999). However, one problem with the practical use of the SSN-1 cell line was that this cell line was composed of a mixed population of cells, causing inconsistencies in the cytopathic effects (CPE) observed during virus infection. For this reason, FAT was used to titrate the virus instead of CPE as described in our previous study (Iwamoto et al. 1999). FAT was laborious and costly due to the requirement of special test chambers, and it has proved to be inadequate for the quantitative analysis of a large number of samples. In addition, the fact that the SSN-1 cell line is spontaneously infected by a C-type retrovirus designated as SnRV (Frerich...