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This work describes a simple protocol for long-term preservation of strains of Rosellinia necatrix based on sclerotia production combined with storage at 4°C in liquid substrate, without affecting the growth and pathogenic characteristics of the fungal isolates recovered. The sclerotization process was set up in both liquid and solid media, and the sclerotia-like structures (pseudosclerotia) obtained were preserved in liquid media or water at 4°C. R. necatrix pseudosclerotia viability after 6 years of preservation at 4°C was confirmed by growth and microscopic characteristics, with no differences when compared with the fungal strains routinely preserved by periodic transfers. Additionally, pathogenicity on avocado plants by the preserved R. necatrix strains showed no difference from those preserved by periodic transfers. The albino strain used in this study should continue to be preserved by periodic subculturing.

Hygromycin B resistance was conferred to the mycelium of the white root rot fungus Rosellinia necatrix by transformation with the hygromycin B phosphotransferase gene (hph) of Escherichia coli under the control of the heterologous fungal Aspergillus nidulans P-gpd (glyceraldehyde 3-phosphate dehydrogenase) promoter and the trpC terminator. In all three transformants, the presence of hph and single-copy integrations of the marker gene were demonstrated by Southern analysis. This is the first report describing A. tumefaciens-mediated transformation of R. necatrix

To prepare homologous DNA fragments as restriction fragment length polymorphism (RFLP) markers, the genes encoding phenol oxidase, chitinase, and xylanase were amplified from genomic DNA of Rosellinia necatrix strains. RFLP analysis using the amplified DNA fragments as probe was carried out, with segregation of the markers among two sets of F1 progenies isolated from an independent perithecium. RFLP was frequently found using rpo1 as the RFLP marker among strains of R. necatrix, which was isolated from single ascospores and the circumference of the perithecium. In each set, RFLPs of some F1 progenies were different from that of the parent strain. Random amplified polymorphic DNA (RAPD) also revealed that several strains, which were of different genotypes from the parent strain, were contained in the single ascospore culture isolated from the same perithecium. From these results, it is suggested that another strain, which was genetically different, was required for mating and development of the ascus in R. necatrix. Therefore, the life cycle in R. necatrix was presumed to be heterothallism. This is the first report about a heterothallic life cycle in R. necatrix.

Conidiomata of the white root rot fungus were produced in axenic culture under near-ultraviolet light radiation. Pieces of sterilized Japanese pear twigs were placed on 7-day-old oatmeal agar culture in plates. The plates were further incubated for 5 days and then illuminated by near-ultraviolet light. Synnemata developed on the twigs within 5 weeks in 19 of 20 isolates tested, and conidia were observed in 12 of the 19 isolates. The synnemata and conidia produced were morphologically identical to those of Dematophora necatrix.

Double-stranded (ds)RNA of various types was detected in 65 (21.8%) of 298 isolates from vegetative hyphae of Rosellinia necatrix by electrophoresis, but dsRNA was not detected from 39 ascosporic isolates. There were 45 distinct dsRNA profiles in the 65 isolates: they varied in the number of electrophoretic bands from 1 to 12 and in size from less than 1000 bp to more than 10 kbp. Each dsRNA profile was unique to each locality. dsRNAs having the same profiles were restricted to isolates of the same mycelial compatibility groups (MCG) from the same trees, with an exception where different profiles were detected in different isolates of the same MCGs.

The process of teleomorph development in the white root rot fungusRosellinia necatrix is described on diseased roots of Japanese pear. Stromata were also found on dead plants in nonagricultural lands such as yards and forests. The stroma ofR. aquila is also described.