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Fungal virulence may be studied using tissues cultures of host plants in dual cultures in vitro, enabling analyses of interactions with undifferentiated cells of their host plants. Three genotypes of Pinus sylvestris callus, initiated by somatic embryogenesis, were used for establishing dual cultures with fungi pathogenic, endophytic or saprotrophic on pine needles or shoots. Fungal growth towards the plant callus tissue differed, depending on the life strategy of the fungus. The pathogen Gremmeniella abietina proved the slowest colonizer of callus whereas the saprotrophic Phacidium lacerum was the fastest. Gremmeniella abietina partially overgrew the callus, causing extensive necrosis and death within 10 days after inoculation. Anthostomella formosa, an endophyte of pines, did not cause evident symptoms of callus degradation: after 10 days of dual culture, the callus cells remained greenish and at least 50% of cells were alive. In dual cultures Ph. lacerum, callus remained alive until the end of the experiment, maintaining a white-creamy colour with a loose cell structure. Electrophoresis of protein extracts from the callus showed the presence of additional bands of 25-35 kDa only in host tissues challenged with the pathogen G. abietina, possibly indicating the production of pathogenesis-related proteins. This work has shown that pine callus does not respond equally to challenge with different fungal isolates. In general, one-third of the isolates of each fungus examined showed greater virulence compared to other isolates.

Dothistroma septosporum is one of the most damaging fungi-attacking pines wherever they are grown. Specific symptoms on pine include the formation of red bands on needles and the appearance of which is attributed to dothistromin, a toxin produced by this pathogen. Many reports have suggested that such red bands do not appear in all infections, although the cause of this phenomenon is unclear. When grown in vitro, some strains of D. septosporum cause an intense blue, rather than red, coloration of the medium. This study presents results of experiments designed to determine the effects of culture conditions on growth of D. septosporum in vitro and the accompanying coloration of the medium. Growth of D. septosporum is characterized by slow mycelial extension and high morphological diversity in all culture conditions. The most rapid growth occurred at 20°C and on a medium at pH 6. Colour of the secreted pigment was not a consistent feature of a given fungal isolate, but depended on temperature and pH of the medium, along with a significant interaction among these factors. Lower temperature and lower pH favoured the production of the blue pigment. Moreover, pigmentation around the D. septosporum colonies was affected by other fungi inhabiting the same ecological niche. These results are discussed in terms of the possible reasons for the failure to produce the characteristic red banding symptoms on needles infected by D. septosporum in some in vivo situations.

Interactions between three genotypes of a silver fir (Abies alba Mill.) embryogenic callus and Heterobasidion abietinum, H. parviporum, and H. annosum were examined in dual cultures. The aim of this study was to determine whether dual cultures can be used to evaluate the degree of fungal virulence at an embryogenic level, and whether different genotypes of a callus show different susceptibility. The dual cultures were performed on Schenk and Hildebrandt medium. Mycelial growth of H. parviporum and H. annosum was significantly stimulated in the presence of the callus but was not directional in nature. The embryogenic callus died between six and nine days after being colonized by H. parviporum suggesting that this was the most virulent species. By contrast, the callus remained healthy for up to 50 d after colonization with H. abietinum suggesting that this was the least virulent species. The callus of the A. alba genotype which originated in the mountain region of Poland remained healthy and alive for a significantly longer period than the other two genotypes in the dual cultures with all three Heterobasidion species even though overgrown by mycelium suggesting that the mountain genotype had the strongest defence response to Heterobasidion infection.

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