Dothistroma spp. in Western Ukraine and Georgia

Dothistroma needle blight (DNB) is among the most serious foliar diseases affecting Pinus spp. globally. Infected needles were collected from potential host species in four locations in western Ukraine and in four locations in eastern Georgia during spring–summer 2015 to update the knowledge on pathogen distribution in these countries. Dothistroma spp. were detected using isolation, sequencing and species‐specific priming (SSPP) PCR. Two new hosts for Dothistroma spp. were recorded in western Ukraine: D. septosporum on Pinus nigra var. australica and D. pini on P. nigra var. mollet. D. septosporum was found on 15‐year‐old P. strobus in western Ukraine. New hosts for D. septosporum were recorded in Georgia on 5‐ to 10‐year‐old naturally regenerated P. sylvestris var. hamata and on 40‐ to 50‐year‐old P. ponderosa trees. D. pini was found for the first time in Georgia on 30‐ to 40‐year‐old P. nigra trees. The work confirmed the presence of both D. septosporum and D. pini in western Ukraine and Georgia, and demonstrated new hosts for both Dothistroma species.     

Needle blight of pine caused by two species of Dothistroma in Hungary

Dothistroma septosporum and D. pini cause the serious pine needle disease, Dothistroma needle blight (DNB). Of these, D. septosporum has a global distribution, while D. pini is known only from the USA, Russia, Ukraine and France. During a study of D. septosporum isolates from Pinus nigra in Hungary, microsatellite markers revealed the presence of a second Dothistroma species. The aim of this study was to identify the DNB pathogens occurring in Hungary using four different molecular techniques. These included sequencing of the rDNA ITS region, a species‐specific ITS‐RFLP, mating type primers and a diagnostic microsatellite marker, Doth_A. Results showed that both D. septosporum and D. pini occur in Hungary and that both DNB pathogens were present on the same trees and in some cases, the same needles. Mating types MAT1 and MAT2 of D. septosporum were shown to be present in Hungary, but only the MAT2 of D. pini was found. In addition, examination of needles with DNB symptoms from Russia revealed the presence of D. pini on Pinus pallasiana and P. nigra as well as on a new host, Pinus mugo. The molecular markers applied in this study were sufficiently robust to identify and differentiate between the two DNB pathogens, both in culture and directly from needles. They will consequently be useful to document the geographical range and to monitor the spread of D. septosporum and D. pini in future studies.     

New host and country records of the Dothistroma needle blight pathogens from Europe and Asia

Dothistroma needle blight (DNB) is a serious disease of pines (Pinus spp.), with a worldwide distribution. It is caused by the ascomycete fungi Dothistroma septosporum (teleomorph: Mycosphaerella pini) and Dothistroma pini (teleomorph unknown). Recently, DNB was found on Pinus peuce in Austria, Pinus pallasiana in Ukraine and the European part of south-western Russia, as well as on Pinus radiata and Pinus wallichiana in Bhutan. Based on DNA sequence comparisons of the internal transcribed spacer and β-tubulin gene regions, isolates from Austria and Bhutan were identified as D. septosporum, while isolates from Ukraine and south-western Russia were identified as D. pini. Additional isolates studied from Pinus mugo in Hungary confirmed the presence of D. septosporum in this country. The record of D. septosporum on exotic P. peuce in Austria represents a new host report of this needle blight pathogen in Europe. Likewise, DNB and the associated pathogen, D. septosporum are reported from Bhutan, eastern Himalayas, for the first time. In addition, D. pini was found in two European countries and on a new host, P. pallasiana. These European records represent the only reports of D. pini from outside the north-central USA. Morphological examination of selected specimens from different hosts and countries showed that D. septosporum and D. pini overlap in the length of their conidia, while the width is slightly wider in D. pini than in D. septosporum. The differences in conidial width are so small, however, that identification of the two Dothistroma species solely based on morphology is virtually impossible. The new host and country records provided here are consistent with the continuing trend of reports of the DNB pathogens from new hosts and new geographical areas during the last two decades, particularly in the northern hemisphere.     

Dothistroma Needle Blight in Slovenia is caused by two cryptic species: Dothistroma pini and Dothistroma septosporum

Dothistroma Needle Blight (DNB) has been known in Slovenia since 1971, but the disease symptoms have intensified in recent years. With this study, the DNB symptoms in Slovenia are for the first time connected to both phytopathogenic Dothistroma species: D. pini and D. septosporum. Based on the ITS‐rDNA comparisons, the Slovenian isolates of D. pini group with the D. pini isolates from France, Hungary and the USA and not with the D. pini isolates from Russia and Ukraine. Both mating types MAT1 and MAT2 of D. septosporum are present in Slovenia, while for D. pini, only MAT2 was found. The current widespread occurrence of D. pini in native forest stands indicates the prolonged presence of this species in Slovenia.     

Needle blight caused by Dothistroma pini in Slovakia: distribution,host range and mating types

Dothistroma needle blight (DNB) has been observed in Slovakia during the last two decades. Up until 2017, Dothistroma septosporum has only been detected and molecularly confirmed to cause DNB in Slovakia. Here, we report the detection of Dothistroma pini at six localities around Slovakia, representing different plantation types. Four pine species (Pinus sylvestris, P. nigra, P. mugo and P. jeffreyi) were confirmed as hosts of D. pini in Slovakia, of which only P. mugo has been previously reported as host in Slovakia. Three gene regions (ITS, EF1 –α, and ß-tubulin) of each of the 13 isolates were sequenced and assigned as D. pini. Based on ITS sequences, the studied isolates represent the haplotypes Dp_HAP.1, Dp_HAP.2. Both mating types were detected but at different localities. Our results suggest that in addition to D. septosporum, D. pini may contribute to DNB also in Slovakia.     

Modelling of the spread of Dothistroma septosporum in Europe

Dothistroma needle blight (DNB), a disease affecting several pine species, is currently generating great concern in Europe. Caused by Dothistroma pini and Dothistroma septosporum, DNB affects pine needles and causes premature defoliation, which results in growth reduction and, in extreme cases, mortality. The disease has increased in importance in Europe over the last 20 years, with an increase in the number of observations of DNB in regions with large areas of Pinus sylvestris in northern Europe. This article presents a cell‐based spatiotemporal model for predicting the likelihood and intensity of the future spread of D. septosporum in Europe. Here, “spread” includes both invasion of new regions and infection of healthy stands within already‐colonized regions. Predicted spread depends on the availability of host species, climatic suitability of different regions to D. septosporum and dispersal of sexual and asexual spores from infected trees to surrounding forests via water splash, mist and wind. Long‐distance spread through transport of infected seedlings is also included in the model. Simulations of spread until 2007 and 2015 were used to validate the model. These simulations produced similar patterns of spread to those observed in Europe. Simulations for 2030 suggested that additional and new outbreaks are likely to occur in Scotland, southern Norway, southern and central Sweden, northern parts of Germany and Poland, Estonia, Latvia and south‐west Finland. Preventing the delivery of infected seedlings would be an effective method for reducing the spread of D. septosporum in the Nordic countries, Scotland and Ireland, the Baltic countries, and parts of Germany, Poland and Belarus. In these states, prevention of transport of infected seedlings can reduce the probability of additional spread by 15%–40%.     

Dothistromin biosynthesis genes allow inter‐ and intraspecific differentiation between Dothistroma pine needle blight fungi

Dothistroma septosporum and D. pini are the causal agents of Dothistroma needle blight (DNB) of Pinus spp. in natural forests and plantations. The main aim of this study was to develop molecular diagnostic procedures to distinguish between isolates within D. septosporum, for use in biosecurity and forest health surveillance programmes. This is of particular interest for New Zealand where the population is clonal and introduction of a new isolate of the opposite mating type could have serious consequences. Areas of diversity in the dothistromin toxin gene clusters were identified in D. septosporum (51 isolates) and D. pini (6 isolates) and used as the basis of two types of diagnostic tests. PCR‐restriction fragment length polymorphism (RFLP) of part of the dothistromin polyketide synthase gene (pksA) enabled distinction between two groups of D. septosporum isolates (A and B) as well as distinguishing D. septosporum and D. pini. The intergenic region between the epoA and avfA genes allowed further resolution between some of the A group isolates in RFLP assays. These regions were analysed further to develop a rapid real‐time PCR method for diagnosis by high‐resolution melting (HRM) curve analysis. The pksA gene enabled rapid discrimination between D. septosporum and D. pini, whilst the epoA–avfA region distinguished the New Zealand isolate from most other isolates in the collection, including some isolates from DNB epidemics in Canada and Europe. Although this study is focused on differences between the New Zealand isolate and other global isolates, this type of diagnostic system could be used more generally for high‐throughput screening of D. septosporum isolates.     

The hosts and potential geographic range of Dothistroma needle blight

Dothistroma needle blight, one of the most important foliar diseases of Pinus spp., is caused primarily by the fungus Dothistroma septosporum (Dorog.) Morelet, and to a lesser extent by Dothistroma pini Hulbary. The potential distribution and abundance of Dothistroma spp. was determined by (i) developing a process-oriented model of potential distribution of Dothistroma spp. from known locations, (ii) compiling a comprehensive list of susceptible host species from existing scientific literature and (iii) determining the distribution of susceptible hosts in areas predicted to be suitable for range expansion of Dothistroma spp. Using these three sources of information regions at risk were identified as those that were predicted to be suitable for range expansion by Dothistroma spp. and included significant areas of susceptible host species.     

Fungicide sensitivity of Dothistroma septosporum isolates in the UK

Dothistroma septosporum, a causal agent of Dothistroma needle blight (DNB), is a damaging fungal pathogen of pines that has recently started to affect native Scots pine woodlands in the UK. In addition to silvicultural methods, fungicide spraying of forest nursery stock can help prevent the spread of DNB. However, the effectiveness of modern single‐site fungicides against D. septosporum and the risk of fungicide resistance evolution remain largely unknown. In this project, we aimed to establish sensitivity profiles of D. septosporum to some widely used single‐site fungicide classes in vitro, and to determine whether fungicide resistance is already present, as this could increase the spread of D. septosporum genotypes on planting stock in native woodlands. For this purpose, we compared isolates of D. septosporum, originating from pine stands unexposed to fungicides, with isolates from nursery outbreaks, for sensitivity to a range of commonly applied fungicides. Most of the fungicides we tested were effective in vitro and we observed no significant shifts in sensitivity in forest nurseries. Although further tests in planta are required to confirm effectiveness of single‐site fungicides against D. septosporum, our results suggest that they can be successfully used in DNB control, although appropriate measures to prevent the evolution of fungicide resistance are strongly recommended.     

The hemibiotrophic lifestyle of the fungal pine pathogen Dothistroma septosporum

Dothistroma needle blight is a forest disease of increasing international importance due to its ability to kill as well as to retard the growth of pines. It is caused by fungi in the genus Dothistroma that produce dothistromin, a non‐host selective toxin and virulence factor that is involved in necrosis of pine tissue. Recent studies of the genome of one of the main pathogenic species, Dothistroma septosporum, showed that it contains many similarities to that of the biotroph Cladosporium fulvum, including the presence of candidate biotrophic effector genes, which supported the hypothesis that D. septosporum has a hemi‐biotrophic lifestyle. Using Pinus radiata as a host, we used a combination of microscopy, histological and molecular tools to further test this hypothesis and to determine the stage of the disease cycle in which dothistromin toxin is produced. The results showed a biotrophic‐type phase in which the fungus grew over the needle surface, penetrated through stomatal pores and colonized epistomatal chambers. The subsequent necrotrophic phase was characterized by colonization of the mesophyll and production of dothistromin, with a >100‐fold increase in dothistromin levels from early necrotic lesion to sporulating lesion stages. This is consistent with the role of dothistromin as a virulence factor that is involved in lesion expansion.     

Dothistroma septosporum on firs (Abies spp.) in the northern Baltics

In the spring of 2008, Dothistroma septosporum, a damaging quarantine pathogen of pine foliage, was documented in Järvselja (southeastern Estonia), apparently for the first time, on white fir (Abies concolor). Thus, a new host species and genus for the fungus were recorded. Infection of white fir by D. septosporum, and also of some other fir species, was monitored annually on a transect proceeding from southeastern Estonia to northern Latvia. As a result, D. septosporum was detected also on silver fir (Abies alba). It is not clear why this fungus with a worldwide distribution appeared on this new host genus definitely in northern Europe.     

Artificial inoculation and susceptibility of Pinus armandii to Dothistroma septosporum

Dothistroma needle blight (DNB) is a serious needle disease of conifers that primarily affects pine species (Pinus spp.). Dothistroma septosporum is one of the DNB pathogens that has a diverse range of host species excluding Pinus armandii. In 15 inoculated P. armandii seedlings, D. septosporum acervuli were observed in 43 infected needles of ten seedlings with a mean disease severity of 1.11% at 25 weeks after inoculations, demonstrating the potential of D. septosporum to cause symptoms on the needles of P. armandii via artificial inoculation. The disease severity of P. armandii was similar to the positive control, Pinus nigra (median 0.75 for P. armandii to 0.70 for P. nigra), thus, P. armandii acts under artificial conditions as a susceptible host species.     

New hosts for Lecanosticta acicola and Dothistroma septosporum in newly established arboreta in Spain

A historical outbreak of needle blight disease was recorded during 2018 to 2019 in plantations of Pinus radiata and Pinus nigra in the North of Spain. The main pathogens involved in this historical outbreak were identified as Lecanosticta acicola and Dothistroma septosporum. Recently, a variety of tree species in three arboreta planted between 2011 and 2013 in the Basque Country as part of the European project REINFFORCE were showing symptoms of needle blight and defoliation. The aim of this study was to determine which pine species were affected with these pathogens. Tree species sampled included several provenances of P. brutia, P. elliottii, P. nigra, P. pinaster, P. pinea, P. ponderosa, P. sylvestris and P. taeda. Using molecular identification methods, Lecanosticta acicola was confirmed infecting Pinus brutia (Provenance: Alexandropolis, Greece and var. eldarica, Crimea) and represents a new host species for this pathogen. Pinus elliottii (Provenance: Georgia, USA) and P. ponderosa (Provenance: Central California, USA) are new host reports of L. acicola for Spain. Dothistroma septosporum was found for the first time on P. brutia (Provenance: Marmaris, Turkey) and P. ponderosa (Provenance: Oregon, USA) in Spain and was also detected infecting P. nigra (Provenance: Sologne Vayrières, France).     

Swiss stone pine trees and spruce stumps represent an important habitat for Heterobasidion spp. in subalpine forests

In the Western Italian Alps (WIA), the three European species of the forest pathogen Heterobasidion spp. can coexist in the same area. Heterobasidion parviporum Niemelä & Korhonen and Heterobasidion abietinum Niemelä & Korhonen are normally found in areas with a significant presence of their respective primary hosts, spruce (Picea spp.) and fir (Abies spp.). The host/niche occupied by Heterobasidion annosum (Fr.) Bref. in the region still remains unclear. Although Scots pine (Pinus sylvestris), a major host for this fungal species in other parts of Europe, is abundant in the region, little or no evidence of disease caused by H. annosum is visible in this tree species. Two different, but not mutually exclusive, hypotheses can explain the presence of H. annosum: (1) Scots pines are infected but largely asymptomatic and (2) H. annosum has adapted to different hosts. An analysis of Heterobasidion species was performed in two natural, mixed‐conifer forests using traditional isolation techniques and novel direct molecular diagnosis from wood. In a subalpine stand of mixed spruce (Picea abies), larch (Larix spp.), and Swiss stone pine (Pinus cembra), 18 naturally infected spruces and larches only yielded H. parviporum. A Swiss stone pine in the same stand was extensively colonized by both H. parviporum and H. annosum. In a second subalpine stand, an analysis of 18 spruce stumps and nine Swiss stone pine stumps yielded both H. parviporum and H. annosum isolates. Pine stumps had been mostly colonized by H. parviporum prior to tree felling, suggesting that this species may be secondarily infected by the locally predominant Heterobasidion species (i.e. H. parviporum). Results of our analysis also indicated that primary colonization of spruce stumps (e.g. through basidiospores) was caused by both H. parviporum and H. annosum, while secondary infection of such stumps was mostly because of H. parviporum.     

New and unexpected host associations for Diplodia sapinea in the Western Balkans

Diplodia sapinea is an important pathogen of pine trees in plantations and urban areas in many parts of the world. This pathogen has recently also been isolated from diseased Cedrus atlantica, C. deodara and Picea omorika planted as ornamentals across the Western Balkans. The aim of this study was to consider the host range of D. sapinea in Serbia and Montenegro. Diplodia sapinea was identified from a broader collection of Botryosphaeriaceae from the Western Balkans region, based on the DNA sequence data for the internal transcribed spacer (ITS) rDNA and the translation elongation factor 1α (TEF 1‐ α). The D. sapinea isolates were obtained from sixteen tree species in the genera Abies, Cedrus, Chamaecyparis, Juniperus, Picea, Pinus, Pseudotsuga and Fagus. Four species represented new hosts in the Balkans, and this is the first report of D. sapinea from F. sylvatica anywhere in the world. Pathogenicity tests were conducted on the tree hosts from which D. sapinea was isolated, as well as on P. abies, Thuja occidentalis, Prunus laurocerasus, Eucalyptus grandis and P. patula. Inoculations were made on seedlings in the field, in the greenhouse or on freshly detached branches. Inoculations on P. pungens, P. omorika, P. abies, P. menziesii, A. concolor, P. nigra and P. sylvestris resulted in death of the seedlings 5–16 weeks after inoculation. Diplodia sapinea produced lesions on J. horizontalis and P. patula seedlings and F. sylvatica cut branches. Reciprocal inoculations showed that D. sapinea is not a pine‐specific pathogen, causing disease on tree species, including those from which it had not been isolated. Not surprisingly, the pathogen was most aggressive on some species of Pinaceae.     

Growth of Phlebiopsis gigantea in wood of seven conifer species

Heterobasidion parviporum and Heterobasidion annosum are widely distributed root‐rot fungi that infect conifers throughout Europe. Infection of conifer stumps by spores of these pathogens can be controlled by treating fresh stumps with a competing non‐pathogenic fungus, Phlebiopsis gigantea. In this study, growth of three Latvian strains of P. gigantea and the biological control agent ‘Rotstop’ strain was evaluated in stem pieces of Norway spruce, Scots pine, lodgepole pine, Douglas‐fir, Weymouth pine, Siberian larch and Sitka spruce. The growth rates of one H. parviporum and one H. annosum isolate were also measured in the same stem pieces. The growth rate of P. gigantea varied greatly in wood of different conifer species. It was higher in the three pine species, lower in Norway spruce and lowest in Sitka spruce and Siberian larch, and in Douglas‐fir, this fungus did not grow. The largest area of wood occupied by P. gigantea was in lodgepole pine. Growth of Latvian isolates of P. gigantea in the wood of Pinus and Picea species was comparable to that of the Rotstop isolate. Consequently, stump treatment with local P. gigantea isolates should be recommended. However, our results suggest that Douglas‐fir stump treatment against Heterobasidion by P. gigantea may be ineffective and other stump treatment methods should be considered.     

Larval performances and life cycle completion of the Siberian moth, <Emphasis Type="Italic">Dendrolimus sibiricus</Emphasis> (Lepidoptera: Lasiocampidae), on potential host plants in Europe: a laboratory study on potted trees

The Siberian moth, Dendrolimus sibiricus, Tschtv. is the most harmful defoliator of coniferous forests in North Asia. The pest has already spread over the Urals and continues moving westwards. Recently, it has been recommended for quarantine in member countries by European and Mediterranean Plant Protection Organization (EPPO). The performances of the pest on coniferous species planted in Europe were assessed on a range of potted trees corresponding to the spectrum of economically important conifers in the EU: European larch Larix decidua, Norway spruce Picea abies, Scots pine Pinus sylvestris, European black pine Pinus nigra, and the North American species: Douglas fir Pseudotsuga menziesii and grand fir Abies grandis. Larvae showed a potential to survive and complete the development on all these host tree species. Favorable hosts were grand fir, European larch, and Douglas fir that allowed higher survival, better larval development, and as a result, yielded heavier pupae and adult moths with higher longevity. Black pine was a poor host but, however, could still support larval and pupal development. Norway spruce and Scots pine had an intermediate behavior. If accidentally introduced to Europe, the Siberian moth may become especially damaging in forest stands predominated by European larch and by the North American firs. Norway spruce and especially the two-needle pines will be less prone to intensive defoliation by this species. The fact that the pest may damage the range of economically important coniferous species should be taken into account in the pest risk assessment for Europe and also for North America where the Siberian moth occurrence is considered likely.     

First record of Ektaphelenchoides pini associated with Ips sexdentatus on Pinus nigra laricio in Italy

In February 2015, an unexpected windstorm downed five hectares of a European black pine Pinus nigra subsp. laricio forest formation located close to Vallombrosa, Florence (Central Italy). In the following spring, an extensive survey was conducted in the area. Felled trees, stumps and all the suitable plant material were screened for the presence of the pinewood nematode (PWN), Bursaphelenchus xylophilus, by sampling wood and bark. Bark beetles were then collected from the gallery systems on the inner side of bark samples and observed in the laboratory. The following bark beetles were morphologically identified: Ips sexdentatus, Orthotomicus erosus, O. laricis and Pityogenes bidentatusa. The dissection of Ips sexdentatus allowed the extraction of numerous nematodes that were morphologically and molecularly identified as Ektaphelenchoides pini. Conversely, only few nematode specimens were isolated from either pine bark or wood. These individuals could be only molecularly identified and belonged to an undescribed nematode taxon. Even though no PWN was recorded in the investigated sites, our survey allowed the detection of a new association between E. pini and I. sexdentatus on P. nigra.     

Beobachtungen über Wechselwirkungen dreier Nadelpilze der Kiefer in vitro

Interactions in vitro among three pine needle fungi . Interactions in vitro among Lophodermium pinastri, Naemacyclus niveus and Dothistroma pini, all fungi on conifers, were related to their various host spectra. The results were remarkably conformable, with those species which were mutually tolerant in vitro being joint colonisers of many host species and vice versa.