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Pitch canker is a serious disease of pines caused by the ascomycete fungus Gibberella circinata (anamorph = Fusarium circinatum). Three distinct mitovirus strains have been described in this fungus: Fusarium circinatum mitovirus 1 (FcMV1), FcMV2-1 and FcMV2-2. Here, we investigated the frequency and population variation of these viruses and closely related sequence variants in northern Spain using RT-PCR and sequencing. Each virus strain and similar sequence variants shared >95 % sequence identity and were collectively designated as virus types. All virus types were relatively common in Spain, with estimated prevalence of 18.5 %, 8.9 % and 16.3 % for FcMV1, FcMV2-1 and FcMV2-2, respectively.

Fusarium spp., as well as other endophytic or pathogenic fungi that form communities, have been reported to be phoretically associated with bark beetles (Coleoptera; Scolytinae) worldwide. This applies to Fusarium circinatum Nirenberg and O'Donnell, the causal agent of pine pitch canker (PPC), which threatens Pinus radiata D. Don plantations in northern Spain. The main objective of this study was to study the fungal communities associated with bark beetles and their galleries in stands affected by PPC, with special attention given to Fusarium species. Funnel traps and logs were placed in a P. radiata plot known to be affected by F. circinatum. The traps were baited with different attractants: four with (E)-pityol and six with ethanol and α-Pinene. In addition, fresh green shoots with Tomicus piniperda L. feeding galleries were collected from the ground in 25 P. radiata plots affected by PPC. Extracts of whole insects and gallery tissues were plated on agar medium to isolate and identify the associated fungi. A total of 24 different fungal species were isolated from the bark beetle galleries constructed in logs and shoots, while 18 were isolated from the insect exoskeletons. Ten different Fusarium species were isolated from tissue and insects. Fusarium circinatum was isolated from bark beetle exoskeletons (1.05% of the Pityophthorus pubescens Marsham specimens harboured F. circinatum) and from the galleries (3.5% of the T. piniperda feeding galleries harboured the pathogen). The findings provide information about the fungal communities associated with bark beetles in P. radiata stands in northern Spain.

Fusarium circinatum Nirenberg & O’Donnel is listed among the species recommended for regulation as quarantine pests in Europe. Over 60 Pinus species are susceptible to the pathogen and it also causes disease on Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) and species in genera such as Picea and Larix. The European Food Safety Authority considers the probability of new introductions—via contaminated seeds, wood material, soil and growing substrates, natural means and human activities—into the EU very likely. Due to early detection, constant surveillance and control measures, F. circinatum outbreaks have officially been eradicated in Italy and France. However, the global spread of F. circinatum suggests that the pathogen will continue to be encountered in new environments in the future. Therefore, continuous surveillance of reproductive material, nurseries and plantations, prompt control measures and realistic contingency plans will be important in Europe and elsewhere to limit disease spread and the “bridgehead effect”, where new introductions of a tree pathogen become increasingly likely as new environments are invaded, must be considered. Therefore, survey programs already implemented to limit the spread in Europe and that could be helpful for other EU countries are summarized in this review. These surveys include not only countries where pitch canker is present, such as Portugal and Spain, but also several other EU countries where F. circinatum is not present. Sampling protocols for seeds, seedlings, twigs, branches, shoots, soil samples, spore traps and insects from different studies are collated and compiled in this review. Likewise, methodology for morphological and molecular identification is herein presented. These include conventional PCR with a target-specific region located in the intergenic spacer region, as well as several real-time PCR protocols, with different levels of specificity and sensitivity. Finally, the global situation and future perspectives are addressed.

Fusarium circinatum, the causal agent of pitch canker disease (PCD), poses a threat to Pinus radiata plantations due to the presence of bleeding cankers on the trunk that can cause the tree to die. This pathogen has been reported to be phoretically associated with bark beetle species, specifically, with Pityophthorus species in California. Pityophthorus pubescens is a secondary pest, attacking weak trees or broken branches in healthy trees. The aim of this study was to know the association between P. pubescens and F. circinatum in PCD affected plantations in northern Spain. Specific aims were determined: i) to assess the phoretic association between P. pubescens and F. circinatum, ii) to study the presence of F. circinatum in P. pubescens infested twigs and iii) to evaluate whether PCD damages were enhanced in (E)-pityol baited P. radiata trees. Funnel traps baited with (E)-pityol were established and twigs from infested trees were sampled to collect insects and plant tissues in PCD affected plots, with the aim of testing the presence of F. circinatum. Moreover, an experiment was carried out in nature in which P. radiata trees were baited with (E)-pityol and PCD symptoms were evaluated 4 times during one year. A total of 263 specimens were collected from funnel traps between June and

Fusarium circinatum is the causal agent of pine pitch canker disease (PPC), affecting Pinus species and other conifers (i.e., Pseudotsuga menziesii (Mirb.) Franco.), forming resinous cankers on the main stem and branches and causing dieback in the terminal guide. This pathogen is spreading worldwide, causing economic losses by converting plantations into standing timber without any potential for future production. The disease was recently detected in Northern Spain in plantations of Pinus radiata and forest nurseries. The aim of the work reported here was to study the role of climatic and topographic variables, soil properties, and stand characteristics on PPC. For this purpose, we surveyed 50 pine stands in Cantabria and quantified the percentage of trees showing three symptoms in each stand: canker, defoliation, and dieback. We investigated the predictive power of 30 variables using generalized linear models and hierarchical partitioning. Both approaches yielded similar results. We found that the three symptoms correlated with different explanatory variables. In addition, more trees exhibited cankers in the proximity of the coast and the Basque Country. Additionally, our results showed that low canopy cover is related to a high level of the dieback symptom. Overall, this study highlights the important variables affecting the distribution of PPC in Cantabria.

The basidiomycete Heterobasidion annosum (Fr.) Bref. (=Fomes annosus (Fr.) Cooke), one of the most important pathogens in coniferous forests in Europe, Asia, and North America, causes root and butt rot. H. annosum was first recorded on Pinus pinaster Ait. (commonly known as Maritime pine) in France and Great Britain in 1961 (4) and Portugal in 1986 (2). P. pinaster is the most widespread conifer in Spain, with more than 700,000 and 600,000 ha in pure and mixed stands, respectively. Over the last few years, P. pinaster decline was observed in several stands in the center of the Iberian Peninsula. Unusual crown transparency, small needles, foliage discoloration, and early tree death are characteristic decline symptoms associated with the high mortality rate on this species. In June of 2010, 11 trees (40 to 60 years old) with a different degree of decline were felled in two zones (42°2′41″N, 3°18′14″W, elevation 1,096 m and 41°55′40″N, 3°12′3″W, elevation 1,128 m) and cut into sections (stump height, breast height, and near the top). Wood slices were removed from each section and taken to the laboratory. Samples were placed in moist chambers with optimal conditions of humidity and temperature to enhance pathogen growth. After 20 days of incubation in darkness at 25°C, H. annosum (anamorph Spiniger meineckellum [A. Olson] Stalpers) occurred on most of these slices. Conidiophores with subglobose to pyriform conidia (5.8 × 4.2 μm) were observed with a compound microscope. The fungus was isolated to extract DNA by disruption of the mycelium followed by washes with phenol/chloroform/isoamyl alcohol solution (25:24:1). DNA was precipitated with 20% polyethylene glycol solution. PCR was carried out according to the instructions of the manufacturer of Dynazyme II DNA polymerase (Finnzymes Ltd, Espoo, Finland) with ITS primers, 1F (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and 4 (5′-TCCTCCGCTTATTGATATGC-3′). After DNA purification, samples were sequenced (SECUGEN, Madrid, Spain) and aligned and corrected with Geneious Pro 5.3 to obtain the consensus sequences. Resulting DNA sequences of two isolates were deposited in GenBank (Nos. FR850494 and FR850495), and compared with a Blastn search at GenBank showing 100% identity and 100% coverage with H. annosum sensu stricto, former ISG-P (intersterility group of pines). For pathogenicity tests, 10 seedlings (2 year old) were inoculated with autoclaved P. pinaster wood chips colonized by H. annosum, and 10 control seedlings were inoculated with noncolonized wood chips. Inoculums were prepared by growing H. annosum on 4-mm-diameter wood chips placed on potato dextrose agar media for 3 weeks. The wood chips were put inside an oblique incision made at 6 cm above the soil line and wrapped with Parafilm. After 8 weeks in a growth chamber at 22.5°C with a 14-h photoperiod, the inoculated seedlings showed typical symptoms and 3 seedlings of 10 were dead. H. annosum was previously recorded on P. sylvestris in central Spain (1), causing needle drop, swelling at the stump height, and presence of dead trees by circular areas. This pathogen was also reported on P. nigra in northeastern Spain, associated with defoliation and mortality (3). To our knowledge, this is the first record of H. annosum on P. pinaster in Spain. References: (1) J. Benito-Martínez. An. Jardín Bot. Madrid 3:23, 1943. (2) N. Neves et al. EPPO Bull. 16:505, 1986. (3) J. Oliva et al. Bol. Sanidad Vegetal. Plagas. 34:415, 2008. (4) P. Spaulding. US Dep. Agric. Agric. Handb. 197:100, 1961.

Fusarium circinatum Nirenberg and O'Donnell (1998) is the causal agent of Pitch Canker Disease (PCD) in Pinus species, producing damage to the main trunk and lateral branches as well as causing branch dieback. The disease has been detected recently in northern Spain in Pinus spp. seedlings at nurseries and in Pinus radiata D. Don adult trees in plantations. Fusarium circinatum seems to require a wound to enter the tree, not only that as caused by insects but also that resulting from damage by humans, i.e. mechanical wounds. However, the effects of pruning on the infection process have yet to be studied. The aim of the present study was to know how the presence of mechanical damage caused by pruning affects PCD occurrence and severity in P. radiata plantations. Fifty P. radiata plots (pruned and unpruned) distributed throughout 16 sites affected by F. circinatum in the Cantabria region (northern Spain) were studied. Symptoms of PCD presence, such as dieback, oozing cankers and trunk deformation were evaluated in 25 trees per plot and related to pruning effect. A significant relationship between pruning and the number of cankers per tree was observed, concluding that wounds caused by pruning increase the chance of pathogen infection. Other trunk symptoms, such as the presence of resin outside the cankers, were also higher in pruned plots. These results should be taken into account for future management of Monterrey Pine plantations.Key words: pitch canker; Cantabria; Spain; wound; Pinus radiata. Resumen Efecto de la poda en plantaciones de pino radiata afectadas por Fusarium circinatumFusarium circinatum Nirenberg and O'Donnell (1998) es el agente causante de la enfermedad del chancro resinoso del pino, que afecta a especies del género Pinus y provoca la aparición de chancros resinosos en el tronco y en ramas gruesas, además de puntisecado en la guía terminal. Esta enfermedad fue detectada recientemente en el norte de España asociada a plántulas de coníferas en vivero y a plantaciones de Pinus radiata D. Don. Fusarium circinatum suele requerir una herida en el árbol para poder infectarlo. Estas heridas pueden estar causadas por insectos o ser de origen antrópico, como las heridas mecánicas. Con la finalidad de conocer cómo las heridas producidas durante la poda afectan a la severidad de la enfermedad del chancro resinoso del pino, se estudiaron 50 parcelas de P. radiata (podadas y no podadas) distribuidas a lo largo de la provincia de Cantabria. En cada una de las parcelas fueron evaluados 25 árboles, en los que se estudiaron los síntomas más característicos de la enfermedad, como son puntisecado, presencia de chancros resinosos y deformación del tronco, relacionándolos con la presencia de poda. Se observó una relación significativa entre la poda y el número de chancros presentes en el árbol, lo que indica que la herida producida en este tratamiento selvícola es susceptible de infección por parte del patógeno. Otros síntomas también presentes en el tronco, como los exudados de resina fuera del chancro, apa...