First Record of Dothistroma pini on Pinus nigra in Switzerland.

The mitosporic ascomycetes Dothistroma septosporum s.s. (Dorog.) Morelet and D. pini Hulbary are closely related species (1) causing red band needle blight on Pinus spp. D. septosporum (teleomorph Mycosphaerella pini Rostr.) is considered as a cosmopolitan species, whereas D. pini (no teleomorph known) seems to have a more restricted distribution area. Detected in the United States on Pinus nigra for the first time, it was later found in Russia, Ukraine, Hungary, and France on different pine species (P. radiata, P. mugo, P. pallasiana) (3). In Switzerland, Dothistroma sp. (species not further determined) was recorded in 1989 for the first time and since then only damages on planted pines (mainly P. mugo and P. nigra) in urban areas were reported (R. Engesser, personal communication). In September 2012 and in April 2013, several planted mature trees and naturally regenerated young trees of P. nigra with Dothistroma needle blight were detected on a climatically mild forest site on limestone at the shore of Lake Walensee (47°07'48.0″ N, 9°13'54.4″ E, 420 m asl). In 2012, symptomatic needles were collected from the litter under one planted mature P. nigra tree and in 2013, symptomatic needles were collected from green twigs from a 2 m tall naturally regenerated P. nigra specimen. Conidiomata were frequently observed in the red bands but no conidia were detected. For fungal isolation, the surface of infected needles was shortly disinfected with 95% ethanol. The epi- and hypo-dermis covering the still closed conidiomata was removed and small tissue samples from the mesophyll (less than 0.5 mm length) were placed on malt extract agar (15 g/liter agar, 20 g/liter malt extract) amended by 50 mg/liter oxytetracycline. Conidia were observed after one year at 4°C in the resulting pure colonies (3 to 4 cm diameter on malt extract agar medium). The conidia formed by strain OH_120923_2_1_1 (KJ878557 = D. pini) were hyaline, smooth, thin-walled, 2- to 4-celled, and 31.6 (22 to 37) × 2.8 (2 to 3.5) µm. While conidial morphology of both Dothistroma species overlap, DNA was extracted and the internal transcribed spacer (ITS) region (primers ITS 1 and ITS 4) sequenced (KJ878557 to 81). From the 25 obtained ITS sequences, seven were identical with AY808275 (D. septosporum from P. radiata, South Africa, CMW 684), three were identical with AY808302 (D. pini from P. nigra, Michigan, CMW 10951), and 15 were identical with DQ926964 (D. pini from P. pallasiana, Ukraine, CMW 23767). The North American and Ukrainian D. pini sequences (AY808302 and DQ926964) showed only 1 bp difference. In addition, mating type genes were amplified using the method described by Groenewald et al. (2) for D. pini and scored using gel electrophoresis. Analyses showed that both D. pini ITS-sequence variants (e.g., KJ878557 and KJ878558) and both mating types were sometimes present in the same needle. In two cases, both mating types and ITS-sequence variants were also present within the same lesion. Interestingly, D. pini and D. septosporum were found on the same tree but not on the same needles. This is the first report of D. pini in Switzerland. Although symptoms of red band needle blight (species not determined) were repeatedly observed on this site during the last 20 years, the disease level always remained low and no tree mortality was noted. However, due to the presence of two ITS-sequence variants and both mating types, the incidence of D. pini in Switzerland deserves attention. References: (1) I. Barnes et al. Stud. Mycol. 50:551, 2004. (2) M. Groenewald et al. Phytopathology 97:825, 2007. (3) D. Piou and R. Ioos. Plant Dis. 98:841, 2014.

Biogeographical patterns and determinants of invasion by forest pathogens in Europe.

A large database of invasive forest pathogens (IFPs) was developed to investigate the patterns and determinants of invasion in Europe. Detailed taxonomic and biological information on the invasive species was combined with country-specific data on land use, climate, and the time since invasion to identify the determinants of invasiveness, and to differentiate the class of environments which share territorial and climate features associated with a susceptibility to invasion. IFPs increased exponentially in the last four decades. Until 1919, IFPs already present moved across Europe. Then, new IFPs were introduced mainly from North America, and recently from Asia. Hybrid pathogens also appeared. Countries with a wider range of environments, higher human impact or international trade hosted more IFPs. Rainfall influenced the diffusion rates. Environmental conditions of the new and original ranges and systematic and ecological attributes affected invasiveness. Further spread of established IFPs is expected in countries that have experienced commercial isolation in the recent past. Densely populated countries with high environmental diversity may be the weakest links in attempts to prevent new arrivals. Tight coordination of actions against new arrivals is needed. Eradication seems impossible, and prevention seems the only reliable measure, although this will be difficult in the face of global mobility.

Stereum sanguinolentum: Is it an amphithallic basidiomycete?

Monobasidiospore isolates were prepared from basidiocarps of Stereum sanguinolentum. Five isolates per basidiome were paired with each other and with isolates from the trama. Interbasidiome pairings of the trama isolates and of a selection of single-spore isolates also were performed. Thin sections of the hymenium were stained with DAPI and examined by fluorescence microscopy to study the nuclei in the basidia. Spore prints were stained with DAPI to count the number of nuclei per spore. SEM was used to determine the number of basidiospores per basidium. All intrabasidiome pairings were compatible. In contrast, interbasidiome pairings, except one, were incompatible, independent of whether single-spore or trama isolates were paired. Fertile basidiomes were formed in single-basidiospore cultures. Basidia were regularly four-spored. On average, 5% of the basidiospores possessed one nucleus, 82% two, 2% three and 1% four nuclei. Ten percent of the spores appeared to be empty. Karyogamy, meiosis and postmeiotic mitosis were observed in the basidia. Nuclei resulting directly from meiosis, i.e., without having undergone postmeiotic mitosis, sometimes were observed in the sterigmata or spore primordia. The high number of vegetative compatibility groups (VCG) of S. sanguinolentum observed in this study and earlier studies is difficult to explain without sexual or parasexual recombination. We suppose that the majority of spores with ≥2 nuclei are amphithallic, possessing at least one nucleus of each mating type. Recombination could occur by exchange of nuclei among VCGs via anastomoses between homothallic compartments. Transfer of nuclei from heterothallic to homothallic mycelia or matings between homothallic mycelia, which originate from monokaryotic spores, might be other paths for gene exchange.

DNA changes in tissues entrapped in plant resins (the precursors of amber).

There have been many reports characterizing DNA from amber, which is a fossil version of plant resin. Here we report an investigation of the effects of plant resin (from Pseudotsuga menziesii) and drying conditions on the preservation of DNA in biological tissues. We examined the degree of degradation of the DNA by agarose gel electrophoresis of extracted DNA, by polymerase chain reaction, and by DNA sequencing. The plant resin alone appeared to cause little or no damage to DNA. Tissue immersed in plant resin that dried rapidly (exposed to sunlight) contained DNA with little apparent damage. Tissue immersed in the resin that was dried slowly (in shade without sunlight) contained DNA with some degradation (3.5% nucleotide changes). The tissue that was immersed in the resin that was constantly hydrated (by immersion in water) yielded DNA that was severely damaged (50-62% nucleotide changes). Transversions outnumbered transitions in these samples by a ratio of 1.4 : 1. A piece of Baltic amber immersed in water for 5 days appeared to be impervious to the water. Thus amber inclusions that initially dried rapidly have the potential to yield undamaged DNA. Those that dried slowly may contain damaged DNA and may be unsuitable for phylogenetic and other studies.

Population structure of the wood decay fungus Fomitopsis pinicola.

Three populations of the wood decay fungus Fomitopsis pinicola, one from each of three countries (Sweden, Russia and Lithuania), were studied by means of arbitrary primed PCR. The genetic structure of the populations was assessed by inferring the genotype of the genets by studying the haplotypes of several single-spore isolates from one sporocarp for each individual. Heterozygotes could therefore be detected with a dominant genetic marker. The amplified band and the null allele of all loci segregated in a way that was in agreement with a 50:50 ratio. Genetic analysis showed that the total population as well as the subpopulations had heterozygote frequencies in agreement with Hardy-Weinberg expectations. No population differentiation was detected in spite of large geographical distances among the populations studied. We also compared the methods of somatic incompatibility and AP-PCR in terms of their value in detecting fungal genets. This was tested for a sample of dikaryotic mycelia from Switzerland. For the tested material the two methods gave congruent results.

Population structure and responses to disturbance of the basidiomycete Resinicium bicolor.

Resinicium bicolor (Alb. & Schw. ex Fr.) Parm. [=Odontia bicolor (Alb. & Schw. ex Fr.) Bres.] is an outcrossing resupinate basidiomycete associated with root and butt rots of trees, but is itself only very weakly pathogenic. The distribution of genets among every spruce stump in a 70-year-old 1250 m2 spruce stand was analysed using somatic incompatibility testing. R. bicolor was present on 40% of 8-to 10-year-old stumps. Nineteen genets were found occupying 32 stumps; yielding probabilities of colonisation following establishment by basidiospores of 0.20-0.24 and by mycelial extension or dispersal of 0.16-0.20. The probability of colonisation decreased with increasing distance from a point of establishment. R. bicolor responded to both enrichment and destructive disturbances by the formation of an extensive cord system which enabled it to colonise discontinuously distributed resources and to overgrow fungi adjacent to it in a single resource unit, including Heterobasidion annosum.

Secondary fungal metabolites and their biological activities, I. Isolation of antibiotic compounds from cultures of Heterobasidion annosum synthesized in the presence of antagonistic fungi or host plant cells.

Heterobasidion annosum (Syn. Fomes annosus), one of the most pathogenic basidiomycetes in conifer forests, produces a series of new metabolites specifically in the presence of antagonistic fungi or some plant cells. These "ecological metabolites" have been isolated and chemically characterized. The time course of production was measured with regard to their biosynthetic pathway. In bio-assays with other fungi, E. coli and cell cultures of Picea abies and Nicotiana tabacum, it could be shown that some of the compounds have antibiotic and growth-inhibiting properties.