NISK, Hogskolevn. 12, 1432 Ås, Norway

Background and objectives
The blue-stain fungus Ceratocystis polonica is a pathogenic fungus associated associated with the spruce bark beetle Ips typographus, a serious threat to Norway spruce (Picea abies). The root rot fungus Heterobasidion annosum is the most serious pathogen in Norway spruce. Previous research has established that there is considerable genetic variation among Norway spruce clones in resistance against both fungi. Correlation between in vivo and in vitro resistance against fungal pathogens has been demonstrated in several plant species. We have tested several clones for in vivo resistance against both fungi. We wanted to study the growth of C. polonica and H. annosum when coinoculated with callus tissue of a susceptible and a much less susceptible clone of Norway spruce under different culture conditions.

Materials and methods
In the field, 25 years old trees of two Norway spruce clones, nos 409 and 589, were inoculated with C. polonica and H. annosum by using the cork borer method. After 15 weeks the lesion lengths in the bark were measured as a parameter of tree resistance.

Callus from the same clones was established and cultured on BMI-S1 medium with auxin (2,4-D), and cytokinins (kinin and BA). For the cocultivation studies, one piece of callus (ca 100 mg) was placed close to the edge of a 90 mm Petri dish filled with fresh medium with or without auxin. One and 14 days later mycelium of freshly growing fungus was inoculated near the centre of the dish (30 mm from the callus tissue). Fungal growth was recorded every 12th hours, staring after 48 hours, on the back of the dish along a line directly towards and away from callus. Every 12th hours after the mycelium had reached the callus we visually estimated how much of the callus area which were covered by mycelium. As control fungal growth was measured in dishes without callus tissue.

Results and conclusions
In the field test the two clones differed much in their susceptibility to both fungi. Based on lesion length, clone 409 was consistently more susceptible than clone 589.

When the fungi were inoculated one day after the callus tissue, only minor effects of clone and auxin on fungal growth could be observed. In all experiments the most clear differences was found when the callus tissue was cultured on medium without auxin for 14 days before the fungus inoculation. These data are therefor presented. The growth of both fungi were strongly stimulated by the presence of callus tissue. After 48 hours the growth of C. polonica in dishes with callus tissue was almost twice of the growth in dished without callus tissue.

Callus of the two clones differed in their ability to inhibit growth of both fungi. Fungal growth was inhibited after 48 and 60 hours for C. polonica and H. annosum, respectively, when coinoculated with clone 589. Clone 409 did not inhibit growth of C. polonica, but inhibition was evident for H. annosum after 108 hours when the mycelium was near the callus tissue. With H. annosum the growth stimulatory effect exceeded the inhibitory effect for both clones. The strong stimulation caused by clone 589 on growth of C. polonica was exceeded by an even stronger inhibition. For both fungi callus tissue of clone 409 was earlier overgrown by mycelium than did clone 589.

Clone 409 produced longer lesions than clone 589 when both C. polonica and H. annosum was inoculated in living trees and the inhibition was weaker when callus tissue of clone 409 was coinoculated with both fungi. These results are thus promising with respect to developing reliable in vitro tests for studying resistance of Norway spruce clones.

1. Ostry ME, Skilling DD, 1992. Pp 405-423 in Blanchette RA, Biggs AR (Eds). Springer Verlag, Berlin.