Fusarium circinatum, the causal of pine pitch canker disease (PPC), is now considered among the most important pathogens of Pinaceae in the world. Although in Europe PPC is only established in the Iberian Peninsula, the potential endangered areas cover over 10 million hectares under the current host distribution and climatic conditions. It is therefore a priority to test the susceptibility of those species and their provenances, within Central and Northern Europe and find biological control agents (BCAs) against the disease. In this study, the susceptibility of Pinus sylvestris, P. mugo and Picea abies Romanian provenances to F. circinatum was tested using three inoculum doses. In parallel, the potential use of Trichoderma atroviride and Trichoderma viride as BCAs against F. circinatum was also tested. This study has demonstrated, for the first time, the susceptibility of P. mugo to F. circinatum. Likewise, the susceptibility of P. abies was also confirmed. The fact that the Romanian provenance of P. sylvestris has not been susceptible to F. circinatum suggests genetic resistance as a potential tool to manage the disease. This, together with the apparent effectiveness of Trichoderma species as BCAs, seems to indicate that an integrated management of the disease might be feasible.

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Laccase enzymes (benzenediol: oxygen oxidoreductase, EC 1.10.3.2) play a major role in the degradation of phenolic compounds such as lignin. They are common in fungi and have been suggested to participate in host colonization by pathogenic fungi. Putative mycoviruses have recently been isolated from the causal agent of pine pitch canker disease, Fusarium circinatum Nirenberg & O´Donell. In this study, the effects of single and double mycoviral infections on laccase activity, growth rate and pathogenicity were investigated in fourteen F. circinatum strains. Extracellular laccase activity was analyzed by the Bavendamm test, image processing and a spectrophotometric method. Mycelial growth, in vivo pathogenicity and seedling survival probability were also determined in Monterrey pine (Pinus radiata D. Don) seedlings. The findings showed that (i) mycelial growth of isolates from the same fungal population was homogeneous, (ii) the presence of mycovirus appears to increase the virulence of fungal isolates, (iii) co-infection (with two mycoviruses) caused cryptic effects in fungal isolates, and (iv) laccases embody a possible auxiliary tool in fungal infection. The prospects for biocontrol, the adaptive role of F. circinatum mycoviruses and the importance of laccase enzymes in host colonization are discussed.

The aim was to determine the inoculation density above which Scots pine (Pinus sylvestris) is overcome by the blue-stain fungus Ophiostoma ips that is associated with the bark beetle Ips sexdentatus. In north-western Spain, stems of 16 Scots pines were inoculated at various densities (0, 400, 800 or 1600 inoculi/m 2 ) along circumferential 100 or 150 cm wide inoculation belts. Each inoculum consisted of a 5 mm diameter cylinder of malt extract agar colonized by the fungus. Three months later, all trees were harvested and trunk resinosis and foliage colour were visually assessed. The percentage of healthy, desiccated, resin soaked, and blue-stained sapwood, as well as growth productivity indices, were calculated from stem disks cut from within the inoculated zone of each tree. Sapwood-specific hydraulic conductivity (Ks) of each tree was measured in the middle of the inoculated zone. All parameters of tree vigour changed dramatically to the worse when inoculation densities were above 400 inoculi/m 2 , and foliage changed from green to yellow-green or yellow when an inoculation density of 800 instead of 400 was used. The percentage loss of sapwood-specific conductivity (PLC) increased from 30 to 90% and the percentage of healthy, conductive sapwood dropped from 85 to 35% at 800 inoculi/m 2 . No effect of the width of the inoculation belt was observed, and there was no relationship between tree productivity indices and the level of resistance. A non-linear negative relationship was found between PLC and the percentage of healthy sapwood. It is concluded that tree resistance was overcome and that trees were going to die when the inoculation density was ‡800 inoculi/m 2 .

Pines are major components of native forests and plantations in Europe, where they have both economic significance and an important ecological role. Diseases of pines are mainly caused by fungal and oomycete pathogens, and can significantly reduce the survival, vigor, and yield of both individual trees and entire stands or plantations. Pine pitch canker (PPC), caused by Fusarium circinatum (Nirenberg and O’Donnell), is among the most devastating pine diseases in the world, and is an example of an emergent invasive disease in Europe. The effects of microbial interactions on plant health, as well as the possible roles plant microbiomes may have in disease expression, have been the focus of several recent studies. Here, we describe the possible effects of co-infection with pathogenic fungi and oomycetes with F. circinatum on the health of pine seedlings and mature plants, in an attempt to expand our understanding of the role that biotic interactions may play in the future of PPC disease in European nurseries and forests. The available information on pine pathogens that are able to co-occur with F. circinatum in Europe is here reviewed and interpreted to theoretically predict the effects of such co-occurrences on pine survival, growth, and yield. Beside the awareness that F. circinatum may co-occurr on pines with other pathogens, an additional outcome from this review is an updating of the literature, including the so-called grey literature, to document the geographical distribution of the relevant pathogens and to facilitate differential diagnoses, particularly in nurseries, where some of them may cause symptoms similar to those induced by F. circinatum. An early and accurate diagnosis of F. circinatum, a pathogen that has been recently introduced and that is currently regulated in Europe, is essential to prevent its introduction and spread in plantings and forests.

Ips sexdentatus (Coleoptera; Scolytidae) is one of the main vectors of ophiostomatoid blue stain fungi that can cause mortality of healthy conifers. For this reason, our objective was to identify the fungal species carried by this bark beetle in Maritime pine (Pinus pinaster) in northwestern Spain. We collected insects from naturally infected pines placed them on malt extract agar (MEA) and left to walk freely on culture plates. Plant tissues (phloem and xylem) from adult pines were cultivated in moist chambers and also on MEA. At the same time, we inoculated pine logs with living insects in the laboratory. Four ophiostomatoid fungi appeared: Ophiostoma ips, Ophiostoma brunneo-ciliatum, Ceratocystiopsis minuta and Ophiostoma sp., as well as Graphium and Sporothrix imperfect stages. Moreover there were seven saprophytic species: Penicillium sp., Trichoderma sp., Verticillium sp., Mucor sp., Aspergillus niger, Gliocladium viride and Scopulariopsis brevicaulis, and the pathogenic Ophiostoma ips. The fructification percentage of the ophiostomatoid species was low, however; its imperfect stage Sporothrix/Hyalorhinocladiella produced high quantity of conidiophores.

Bark beetles (Coleoptera, Scolytinae) carry a diverse filamentous fungal community sometimes acting as vectors or carriers of phytopathogens. In this study, mycobiota carried by two Tomicus species (Tomicus piniperda and Tomicus destruens) were investigated through (i) morphological and molecular identification of taxa; (ii) taxonomic richness, diversity, evenness, dominance and phoresy indices; (iii) ecological network analysis and (iv) statistical co-occurrence analysis. The studied mycobiota were formed by eleven taxa and showed a moderate fungal diversity with low evenness. The fungus Sydowia polyspora was significantly abundant and dominated the community. All the fungal taxa were randomly associated. Both insect species (T. piniperda and T. destruens) were collected from plantations of Pinus radiata infected by Fusarium circinatum. The ecological factors that could drive community ecology and phoretic links between fungi and bark beetles are discussed.

Deep sequencing of small RNAs has proved effective in the diagnosis of mycovirus infections. In this study, the presence of mycoviruses in ten isolates of the phytopathogenic fungus Fusarium circinatum was investigated by high-throughput sequencing (HTS) of small RNAs. The contigs resulting from de novo assembly of the reads were aligned to viral genome sequences. The presence of each mycovirus detected in the isolates was confirmed by RT-PCR analysis with four previously described primer pairs and seven new pairs designed on the basis of sequencing data. The findings demonstrate the potential use of HTS for reconstructing previously identified mitoviruses infecting F. circinatum.