Genetic evidence for sexual reproduction and multiple infections of Norway spruce cones by the rust fungus Thekopsora areolata.

Rust fungi are obligate parasites, of plants, with complex and in many cases poorly known life cycles which may include host alteration and up to five spore types with haploid, diploid, and dikaryotic nuclear stages. This study supports that Thekopasora areolata, the causal agent of cherry-spruce rust in Norway spruce, is a macrocyclic heteroecious fungus with all five spore stages which uses two host plants Prunus padus and Picea abies to complete its life cycle. High genotypic diversity without population structure was found, which suggests predominantly sexual reproduction, random mating and a high gene flow within and between the populations in Fennoscandia. There was no evidence for an autoecious life cycle resulting from aeciospore infection of pistillate cones that would explain the previously reported rust epidemics without the alternate host. However, within cones and scales identical multilocus genotypes were repeatedly sampled which can be explained by vegetative growth of the fertilized mycelia or repeated mating of mycelium by spermatia of the same genotype. The high genotypic diversity within cones and haplotype inference show that each pistillate cone is infected by several basidiospores. This study provides genetic evidence for high gene flow, sexual reproduction, and multiple infections of Norway spruce cone by the rust fungus T. areolata which expands the general understanding of the biology of rust fungi.

New genetic markers for identifying Cronartium flaccidum and Peridermium pini and examining genetic variation within and between lesions of Scots pine blister rust in Sweden.

Microsatellite markers were developed as an identification tool and for analysis of the genetic variation in the pathogens Cronartium flaccidum and Peridermium pini, causing Scots pine blister rust in Pinus spp. Six reference aeciospore samples from Finland were used to examine genetic differences between the two pathogens. Genetic variation within and between 27 lesions on Scots pines from seven locations in Sweden was also investigated. Aeciospores were collected from single aecia within the lesions. Reference samples from P. pini were homozygous for all seven microsatellite loci investigated, while the three C. flaccidum samples contained heterozygous loci. These results confirm previous studies, where homozygous aeciospores were indicated to be characteristic for P. pini. The majority of aeciospores had two nuclei in both heterozygotic and homozygotic samples. Five of the Swedish lesions contained only homozygotic aecia, while the aecia in the remaining 22 lesions were heterozygotic. All lesions with homozygotic aecia contained only one single multilocus genotype, while many of the lesions with heterozygotic aecia contained several genotypes. The latter indicates the occurrence of multiple matings within a lesion between the resident spermogonia and alien fertilizing spermatia.

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.

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.