Species Delimitation and Host Specialization of Ceratocystis laricicola and C. polonica to Larch and Spruce

Bark beetles (Coleoptera: Scolytidae) are among the most important killers of trees in the family Pinaceae, especially on Pinus, Picea and Larix (15). Bark beetles are intimately associated with a variety of fungi, most notably, members of the genus Ophiostoma and their associated anamorphic species of Leptographium (5–7). Although species of Ceratocystis sensu stricto are generally associated with other insects, there are three species of Ceratocystis associated with tree-killing bark beetles (12,21). Two of these Ceratocystis species, C. laricicola Redfern & Minter and C. polonica Siemaszko, are associated with closely related species of Ips, I. cembrae and I. polonica, which attack Larix and Picea species, respectively, across Eurasia, from northern Europe to Japan (12–14,16–18,22,23,25,26). When inoculated into healthy trees, these Ceratocystis species induce extensive lesions (2,13,14, 16,18,25,26), and it is thought that the respective bark beetles kill their hosts through the actions of these plant pathogenic fungal species (2,7,13,14,16,18, 25,26). Of the fungi associated with tree killing bark beetles, these two species may be the most aggressive pathogens to trees (15), and thus, these species are important in the development of conceptual models of the interactions among fungi, bark beetles and their plant hosts. It is questionable if C. laricicola and C. polonica are distinct species. Siemaszko (17) mistakenly connected a Leptographium anamorph to C. polonica, and because of this confusion, Redfern et al. (16) did not compare their new species, C. laricicola, to the earlier described C. polonica (20). Harrington and Wingfield (12) recognized the two fungal species as distinct, though no morphological feature appeared to separate them. Analyses of the internal transcribed spacer region (ITS) of the rDNA operon demonstrated no difference between the species (24), though there appeared to be some differences in the DNA sequence of a portion of the MAT-2 mating type idiomorph of the two species (22). Also, isolates of C. polonica had a unique electromorph for the isozyme diaphorase (11). The two species also appear to have an interfertility barrier, but sexual compatibility is difficult to test in the homothallic Ceratocystis species (9). Most Ceratocystis species are capable of unidirectional mating type switching, in which one of the mating types (MAT-2) is capable of selfing, producing progeny that are self-sterile (MAT-1) or self-fertile (MAT-2) in a 1:1 segregation pattern (8). The MAT-1 progeny is always self-sterile but can cross with MAT-2 strains, whereas MAT-2 progeny is usually self-fertile but can also cross with MAT-1 strains. Selfing in MAT-2 strains of Ceratocystis species occurs via a deletion of the MAT-2 idiomorph, which transforms the strain to the MAT-1 mating type (23). Normal mating apparently occurs between the switched (MAT-1) and unswitched (MAT-2) nuclei (8). Testers of MAT-1 strains are easily identified as self-sterile progeny derived from selfings. The MAT-2 testers are more difficult to identify but can sometimes be found in self-sterile sectors of otherwise self-fertile MAT-2 strains. These MAT-2 testers are generally only able to form perithecia and ascospores when paired with MAT-1 testers, though sometimes they can be induced to self when paired with either MAT-1 or MAT-2 strains (9). Using such MAT-2 mutants, pairings between C. polonica and C. laricicola were only partially interfertile (9). Because of the importance of these respective bark beetle species and the fact that the Ceratocystis species are important plant-pathogenic symbionts, we made a detailed comparison of isolates of the two species from Europe, southwestern Siberia (south-central Russia), and Japan. We employed phylogenetic analysis of a portion of the MAT-2 idiomorph, conducted tests for sexual interfertility, and made morphological comparisons. Lastly, we inoculated mature larch and spruce trees to determine if the isolates were physiologically specialized to the respective tree hosts.