First report of Sphaeropsis sapinea on Scots pine (Pinus sylvestris) and Austrian pine (P. nigra) in Sweden

Abstract

In January 2013, an infected Pinus sylvestris cone was detected in Fjällnora (central Sweden: 59° 50' 0.05“ N, 17° 54' 46.19” E). The cone displayed abundant pycnidia on the scales (Fig. 1). Conidia, 30-37 üm x 12-15 üm, were dark-brown, subglobose, mostly non-septate, although septate and even triseptate conidia were observed. These characteristics matched well with those reported for Sphaeropsis sapinea (syn. Diploidia pinea) (Punithalingam & Waterston, 4). Single spore isolates were grown on 2% malt extract agar (MEA) with chloramphenicol (200 mg/l). Cultures on MEA were initially white with profuse aerial mycelia that after one week turned grey to blackish. DNA was extracted with a regular CTAB protocol (3%) from cultures and scales, and amplified by PCR using specific primers of both D. pinea and D. scrobiculata (Smith & Stanosz, 5). Only the D. pinea primers gave positive results amplifying a product of 700 bp in all cases. S. sapinea was later also found causing tip blight (Figs. 2, 3) on three Austrian pines (P. nigra) in Alnarp (southern Sweden: 55° 39' N, 13° 5' E) as well as infection on cone scales. Isolations showed the same morphological characters while PCR revealed the same diagnostic 700 bp band as the samples retrieved from P. sylvestris. A total of 60 isolates were deposited in the Department of Forest Mycology and Plant Pathology collection under the codes DP1-60. Pathogenicity tests were run and Koch's postulates fulfilled by inoculating six two-year-old P. sylvestris seedlings with a 1 cm agar plug from a two-week-old culture of DP1 growing on MEA at an incision made in the seedling tip. After three weeks, shoot necrosis was observed on average 3 cm beyond the infection point of all inoculated seedlings (Fig. 4b) from where S. sapinea was re-isolated. Control plants, inoculated only with MEA plugs, remained healthy (Fig. 4a). This is the first report of S. sapinea affecting forest pines in Sweden. The only previous report in Sweden mentioned that the fungus was isolated from nursery stock (Molin et al., 3). However, no data on the species identification procedure or isolates were available from this study. Interestingly, S. sapinea has never been definitely identified as causing damage in nurseries in the last 40 years (E. Stenström, P. Barklund, pers. comm.). While widespread in Central and Southern Europe, S. sapinea has not been reported in neighbouring countries such as Norway or Finland, denoting its observation in Fjällnora as the northernmost report of this pathogen in Europe to date. S. sapinea can cause severe damage to stressed pines, especially during drought (Stanosz et al., 6). The prevalence of S. sapinea on cones has been linked to high winter temperatures and rainy summers (Fabre et al., 1), conditions that may become more prevalent in Sweden due to the changing climatic conditions. Our observations, together with the recent discovery in Estonia (Hanso & Drenkhan, 2) support the suggested northward range expansion of this pathogen from southern Europe. This research has been partly funded by the FORMAS project 215-2012-1255 and by ‘Future Forests’, financed by the MISTRA Foundation for Strategic Research.