Standardized tools are needed to identify and prioritize the most harmful non-native species (NNS). A plethora of assessment protocols have been developed to evaluate the current and potential impacts of non-native species, but consistency among them has received limited attention. To estimate the consistency across impact assessment protocols, 89 specialists in biological invasions used 11 protocols to screen 57 NNS (2614 assessments). We tested if the consistency in the impact scoring across assessors, quantified as the coefficient of variation (CV), was dependent on the characteristics of the protocol, the taxonomic group and the expertise of the assessor. Mean CV across assessors was 40%, with a maximum of 223%. CV was lower for protocols with a low number of score levels, which demanded high levels of expertise, and when the assessors had greater expertise on the assessed species. The similarity among protocols with respect to the final scores was higher when the protocols considered the same impact types. We conclude that all protocols led to considerable inconsistency among assessors. In order to improve consistency, we highlight the importance of selecting assessors with high expertise, providing clear guidelines and adequate training but also deriving final decisions collaboratively by consensus.

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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.

Mitoviruses have been found in several forest pathogens (i.e. Cryphonectria parasitica, Gremmeniella abietina), and because they have been shown to reduce the virulence of host fungi there is a growing interest in studying their use as a biocontrol. This study was carried out to test the effect of temperature (5°C, 15°C, 25°C and 35°C), pH (4, 5, 7 and 9) and osmotic potential (-0.6, -1.2, -1.8 and -2.4 MPa) on the mycelial growth of seven G. abietina isolates under controlled laboratory conditions. Four of the isolates hosted mitoviruses and three of them did not. During the experiment, mycelial growth was recorded every week for a period of 8 weeks. Results showed no differences in growth behavior between mitovirus infected and non-infected isolates when placed under different pH modifications. However, the mitovirus-infected isolates presented larger mycelial growth than the mitovirus-free ones when at the fungi's optimal growing temperature of 15°C. When growing at certain osmotic potentials (-0.6 and -1.8 MPa) a reduction in growth of the mitovirus-infected isolates was observed. The results of this experiment suggest that mycelial growth among non-infected isolates and isolates naturally infected by mitovirus vary under different culture conditions, thus providing further insight into the effects of mitovirus on Gremmeniella abietina isolates.Key words: mitoviruses; Scleroderris canker; in vitro; biological control; Gremmeniella abietina; dsRNA. Resumen Efecto de posibles mitovirus en el crecimiento in vitro de aislados de Gremmeniella abietina bajo diferentes condiciones de laboratorioLos mitovirus son virus exclusivamente fúngicos que han sido aislados de algunos patógenos forestales (i.e. Cryphonectria parasitica, Gremmeniella abietina) y ya que pueden reducir la virulencia del hongo existe un creciente interés por su posible papel como agentes de control biológico. Se ha llevado a cabo un estudio para evaluar el efecto de la temperatura (5°C, 15°C, 25°C y 35°C), el pH (4, 5, 7 y 9) y el potencial osmótico (-0.6, -1.2, -1.8, -2.4 MPa) en el crecimiento micelial de siete aislados de G. abietina bajo condiciones controladas de laboratorio. Cuatro de los aislados albergaban mitovirus y tres de ellos no. Durante el experimento, el crecimiento micelial fue registrado semanalmente hasta completar 8 mediciones. Los aislados infectados con mitovirus presentaron mayor crecimiento micelial que los no infectados a la temperatura de crecimiento óptimo del hongo de 15°C. No se observaron efectos de la presencia de mitovirus entre los aislados infectados y los no infectados en los tratamientos de modificación del pH. Cuando se modificaron los potenciales osmóticos se observó una reducción del crecimiento micelial de los aislados infectados con mitovirus en los potenciales osmóticos de -0.6 y -1.8 MPa. Los resultados de este experimento sugieren que la presencia de los mitovirus afecta al crecimiento micelial del hongo bajo distintas condiciones de laboratorio. Este estudio proporciona un conocimiento más prof...

Abiotic and biotic factors may shape the mycobiome communities in plants directly but also indirectly by modifying the quality of host plants as a substrate. We hypothesized that nitrogen fertilization (N) would determine the quality of aspen (Populus tremula) leaves as a substrate for the endophytic fungi, and that by subjecting the plants to N, we could manipulate the concentrations of positive (nutritious) and negative (antifungal) chemicals in leaves, thus changing the internal “chemical landscape” for the fungi. We expected that this would lead to changes in the fungal community composition, in line with the predictions of heterogeneity–diversity relationship and resource availability hypotheses. To test this, we conducted a greenhouse study where aspen plants were subjected to N treatment. The chemical status of the leaves was confirmed using GC/MS (114 metabolites, including amino acids and sugars), LC/MS (11 phenolics), and UV-spectrometry (antifungal condensed tannins, CTs), and the endophytic communities were characterized using culture-dependent sequencing. We found that N treatment reduced foliar concentrations of CT precursor catechin but not that of CTs. Nitrogen treatment also increased the concentrations of the amino acids and reduced the concentration of some sugars. We introduced beetle herbivores (H) as a second treatment but found no rapid changes in chemical traits nor strong effect on the diversity of endophytes induced by herbivores. A few rare fungi were associated with and potentially vectored by the beetle herbivores. Our findings indicate that in a controlled environment, the externally induced changes did not strongly alter endophyte diversity in aspen leaves.

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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.