Finnish Forest Research Institute, P0 Box 18, 01301 Vantaa, Finland

Background and objectives
Based on previous studies the most common endophyte in Norway spruce (Picea abies) needles is Lophodermium piceae, which in Fennoscandia is found more often than other species together. In this work we looked answer to two questions related to the diversity of fungal community in Norway spruce needles: How many ITS-RFLP-groups can be found within single needles? - How many haplotypes can be found within single needles based on random amplified microsatellite (RAMS) markers?

Materials and Methods
We examined thirty-six healthy looking green needles by cutting them after surface sterilization into approximately 0.2 mm thick slices (46-63 slices/needle) which then were incubated on water agar. Single fungal hypha were removed from the water agar after a two month incubation and cultivated on modified orange serum agar. Isolates from four needles with at least four infected slices were used for the analysis of geneuc variation within the needles.
Total DNA was isolated from 47 fungal isolates and their ITS regions were PCR-amplified using fungus specific primers ITS1-F and 1T54. The resulting amplification products were digested with restriction enzymes Mspl and CfoI. In addition, RAMS markers were amplified from total DNAs of the same 47 isolates using primers DHB(CGA)5 and DDB(CCA)5. Finally, in order to identify the mycelial cultures to species level 18 control isolates belonging to five species known to be spruce needle endophytes were analysed for ITS-RFLP. All DNA fragments were separated in agarose gels.

Results and conclusions
Seventy-five % of the analysed needles were infected by endophytic fungi. From those we selected four needles for further analysis of genetic diversity. The number of ITS-types within single Norway spruce needles was either three or four. Most isolates had ITS-types similar to those obtained from our control cultures originating from L. piceae sporocarps. This supports the idea that L. piceae would be the most common endophytic fungus in Norway spruce. However, among the twelve L. piceae controls we observed altogether six ITS alleles. This large number of ITS alleles suggests that the species is either extremely variable or that what is known as L. piceae is actually a species complex composed of two or more biological species.
In RAMS analysis all isolates within single needles differed from each other indicating that the endophytic community within each needle was composed of many genotypes. Thus, the spruce needles are occupied by a high number of separate fungal mycelia, and also the space dominated by single endophytes (individuals) is small.