Juha Kaitera

A novel putative virus of Gremmeniella abietina type B (Ascomycota: Helotiaceae) has a composite genome with endornavirus affinities

Ascospore and mycelial isolates of Gremmeniella abietina type B were found to contain three different dsRNA molecules with approximate lengths of 11, 5 and 3 kb. The 11 kb dsRNA encoded the genome of a putative virus and is named Gremmeniella abietina type B RNA virus XL (GaBRV-XL). GaBRV-XL probably exists in an unencapsulated state. We identified two distinct dsRNAs (10 374 and 10 375 bp) of GaBRV-XL, both of which coded for the same putative polyprotein (3249 amino acids) and contained four regions similar to putative viral methyltransferases, DExH box helicases, viral RNA helicase 1 and RNA-dependent RNA polymerases. While a cysteine-rich region with several CxCC motifs in GaBRV-XL was similar to that of putative endornaviruses, cluster analyses of conserved regions revealed GaBRV-XL to be distinct from a broad range of viral taxa but most closely related to Discula destructiva virus 3. Collectively, these findings suggest that GaBRV-XL represents a novel virus group related to endornaviruses.

Occurrence of Gremmeniella abietina var. abietina large- and small-tree types in separate Scots pine stands in northern Finland and in the Kola Peninsula, Russia

Variation in Gremmeniella abietina var. abietina was studied in three stands of Scots pine in northern Finland and in the Kola Peninsula. Eighty-four isolates of large- and small-tree types of G. abietina var. abietina (LTT and STT, respectively) were identified on the basis of tentative characteristics (spore morphology, disease type and host size), fatty acid and sterol profiles (FAST), and random amplified microsatellite technique (RAMS). Both LTT and STT occurred in all three stands. In general the classifications obtained using the three methods agreed with one another, although a few contradicting results were observed. Variation in fatty acids and sterols in G. abietina var. abietina was rather low, although the amounts of some individual extractives showed statistically significant differences between the stands. All pathogenic and asymptotic G. abietina var. abietina isolates originating from branches located at heights above the annual snow cover were identified as LTT based on RAMS, but some were grouped to STT according to their FAST profiles. Both STT and LTT were detected among the isolates obtained from seedlings according to both FAST and RAMS. In addition, in two cases RAMS markers thought to be STT- or LTT-specific were found in the same isolate. The results presented here suggest that LTT of G. abietina var. abietina caused the devastating epidemics on pines in the first-thinning stage or middle age similar to pines in this study reported in northern Finland and in the Kola Peninsula during the 1980s.

Inoculation of known and potential alternate hosts with Peridermium pini and Cronartium flaccidum aeciospores

The ability of Peridermium pini and Cronartium flaccidum aeciospores and mycelium to infect known (Vincetoxicum spp., Pedicularis spp., Paeonia spp.) and potential (Melampyrum spp., Pyrola sp., Dactylorhiza sp., Solidago sp., Salix sp., Geranium sp. and Maianthemum sp.) alternate hosts was tested. None of the mycelial cultures and only 10% of the aeciospore samples produced uredinia or telia on the tested species suggesting that most aeciospores in Finland belong to the autoecious P. pini. Aeciospores from three locations in northern Finland, however, produced uredinia or telia on Vincetoxicum mongolicum, V. nigrum, V. fuscatum, Paeonia anomala, three P. officinalis cultivars, Melampyrum sylvaticum and Pedicularis palustris either in vitro or in vivo, indicating that these aeciospores belong to the heteroecious C. flaccidum, which occurs sporadically in Finland. Interestingly, the host-specificity of C. flaccidum encountered in Finland was very low (e.g. one sample produced uredinia or telia on eight species). This, added to the wide distribution of Melampyrum spp. over northern Fennoscandia, suggests that the main alternate hosts for C. flaccidum in Finland may be in Melampyrum rather than Pedicularis.

Melampyrum sylvaticum, a new alternate host for pine stem rust Cronartium flaccidum.

Cronartiumflaccidum was observed for the first time on Melampyrum sylvaticum. Uredinia and telia were found on M. sylvaticum growing in Scots pine (Pinus sylvestris) stands representing both moist and subdry forest site types. The finding suggests that the occurrence of Cronartium flaccidum is much wid- er in northern Fennoscandia than has been reported over the past century.

Two types of the European race of Gremmeniella abietina can be identified with immunoblotting

Gremmeniella abietina, the causative agent of Scleroderris canker in conifers, has been separated into three races: European, North American and Asian. Recent studies have suggested that the European race in Fennoscandinavia may be divided into two types. We show that these types cannot be identified with their protein patterns obtained under denaturing conditions, but the two types can be separated based upon presence or absence of a 26-28 kD immunoreactive double band in western blotting. Polyclonal antibodies, either crude or purified with preparative western blotting, detected this double band in all tested 25 isolates classified as type B or short tree type but not in any of 27 isolates of type A or large tree type. This immunoassay was used to type 10 unclassified isolates. The results indicated that short tree type of the Swedish classification is immunologically identical to the Finnish type B and large tree type is identical to the type A. Thus, in Fennoscandinavia the European race can be divided into two serovars as previously suggested by random amplified polymorphic DNA markers.

Spanish population of Gremmeniella abietina is genetically unique but related to type A in Europe.

Genetic structure of the European Gremmeniella abietina var. abietina was analyzed in this study. Ninety-two Spanish isolates, six Swiss isolates of Alpine biotype, 76 Finnish isolates of biotype A and 54 Finnish and seven Russian isolates of biotype B were collected. Genetic variation of different populations was analyzed using sequence analysis of specifically amplified markers GAAA1000, GAAA800 and ACA900. Variation in the GAAA1000 marker was significant, and composed of 33 alleles divided into the following four studied populations: five alleles in the Alpine type, 12 in biotype B, 16 in biotype A and two in the Spanish population. Based on variation in GAAA1000 marker, a subset of isolates were further analyzed using GAAA800 and ACA900 sequences, which showed lower overall genetic variability, and no variation among the Spanish population. Genetic differentiation analysis revealed a high genetic differentiation among populations. Finally, clustering analysis of GAAA1000 sequences showed that the Spanish isolates clearly separated from the rest of the biotypes, whereas the Alpine type was closely related to the B type. However, one of the A-type isolates had an identical GAAA1000 allele with the prevailing allele among Spanish isolates. Altogether, our data suggest that the Spanish population is genetically highly differentiated from any other G. abietina population in Europe with a probable A-type origin.

Morphological and ecological variation of Gremmeniella abietina var. abietina in Pinus sylvestris, Pinus contorta and Picea abies sapling stands in northern Finland and the Kola Peninsula.

The morphological and ecological variation of two types of Gremmeniella abietina var. abietina causing scleroderris canker on conifers was investigated in Pinus spp. and Picea sp. sapling stands in northern Finland and the Kola Peninsula. Small - tree type (STT or B type) of G. abietina was detected alone in 13 Scots pine, three lodgepole pine and two Norway spruce sapling stands out of 26 stands investigated, both STT and large - tree type (LTT or A type) were observed in six Scots pine stands, and LTT was detected alone in two Scots pine stands. For the first time, G. abietina was found to injure Norway spruce saplings in a respective plantation in northern Fennoscandia. STT isolates produced statistically significantly more conidia in vitro than LTT isolates. Morphological variation in conidia septation revealed that STT produced conidia with more than five septa more frequently than did LTT. There was a greater range in variation in septation in STT than in LTT, with overlapping between the types. Isolates of both types were equally associated with cankers, coloured wood, pycnidia or apothecia in the infected saplings.

Effect of resin‐top disease caused by Peridermium pini on the volume and value of Pinus sylvestris saw timber and pulpwood

The role of the resin‐top disease caused by Peridermium pini in volume and value losses to Scots pine (Pinus sylvestris L.) was surveyed in two heavily infected stands in northern Finland. Of the Scots pines, 26% were infected by the disease. Peridermium pini caused 2% volume losses to saw timber trees and 3% volume losses to pulpwood trees in the stem‐lesion class and 10% and 14% in the dead‐top class, respectively. The disease caused saw timber volume losses to saw timber trees of 34% and 22% in the stem‐lesion and dead‐top class, respectively. However, saw timber volume losses increased the pulpwood volume in both disease classes. The disease reduced the marketing value of saw timber trees and pulpwood trees by 18% and 3% in the stem‐lesion class and by 15% and 14% in the dead‐top class.

Analyses of genetic variation suggest that pine rusts Cronartium flaccidum and Peridermium pini belong to the same species.

Cronartium flaccidum and Peridermium pini are rust fungi occurring on two-needle hard pines. According to previous molecular and morphological analyses, they are very closely related despite differences in their life-cycles. In this study we showed that although a low level of genetic differentiation occurs among populations of both P. pini and C. flaccidum, there is no overall differentiation between the two rusts, and in this respect they resemble a single taxon. We also observed evidence for linkage disequilibrium occurring between different alleles of separate loci of Peridermium pini suggesting that its population structure would be clonal. This suggests that strains of P. pini would have originated as asexual or self-fertilizing host range mutants of C. flaccidum.

Relative Susceptibility of Four Melampyrum Species to Cronartium flaccidum

The relative susceptibility of Melampyrum arvense, M. pratense, M. sylvaticum and M. nemorosum to Cronartium flaccidum was investigated by artificial inoculations. Uredinia and telia were artificially produced on plants from sown seed or transplanted from the wild in the greenhouse. This is the first report in which C. flaccidum telia were artificially produced on leaves of whole plants of M. arvense and M. pratense, the first report of successful artificial disease establishment on M. arvense and the first report of telia production on the upper surface of Melampyrum leaves. As an earlier unreported observation, telia were occasionally formed on both upper and lower leaf surfaces of M. arvense, M. sylvaticum and M. pratense. Host species, spore source and their interaction each affected some aspects of disease development. Melampyrum arvense and M. sylvaticum were the most susceptible hosts, while M. nemorosum was least susceptible. Melampyrum arvense should be considered a potentially important alternate host of C. flaccidum with its limited area of distribution.