A method based on real-time polymerase chain reaction (PCR) and the use of rotating-arm spore traps was developed for quantifying airborne Hymenoscyphus pseudoalbidus ascospores. The method was sensitive and reproducible, and the collection efficiency was 10% of the spores present in the air. The temporal ascospore dispersal pattern was studied over 3 years by collecting spores every 15 days for a 24 h air-sampling period during the ash-growing season. The highest production was detected from the end of June to the beginning of September. The overall ascospore production did not differ significantly among stands within a specific year but there were differences from year to year. There was a positive correlation between air temperature and the number of ascospores trapped, with most of the positive samples being observed at temperatures above 12°C. The vertical profile of ascospore dispersal showed a strong decrease in ascospore density within a height of 3 m, regardless of date of collection. An analysis of the spore traps installed at increasing distances from an infected stand showed that most of the ascospores were deposited downwind within 50 m of the stand. These data are discussed in context of the epidemiology of the disease.

Fusarium circinatum, the causal agent of pine pitch canker (PPC), is currently one of the most important threats of Pinus spp. globally. This pathogen is known in many pine-growing regions, including natural and planted forests, and can affect all life stages of trees, from emerging seedlings to mature trees. Despite the importance of PPC, the global distribution of F. circinatum is poorly documented, and this problem is also true of the hosts within countries that are affected. The aim of this study was to review the global distribution of F. circinatum, with a particular focus on Europe. We considered (1) the current and historical pathogen records, both positive and negative, based on confirmed reports from Europe and globally; (2) the genetic diversity and population structure of the pathogen; (3) the current distribution of PPC in Europe, comparing published models of predicted disease distribution; and (4) host susceptibility by reviewing literature and generating a comprehensive list of known hosts for the fungus. These data were collated from 41 countries and used to compile a specially constructed geo-database. A review of 6297 observation records showed that F. circinatum and the symptoms it causes on conifers occurred in 14 countries, including four in Europe, and is absent in 28 countries. Field observations and experimental data from 138 host species revealed 106 susceptible host species including 85 Pinus species, 6 non-pine tree species and 15 grass and herb species. Our data confirm that susceptibility to F. circinatum varies between different host species, tree ages and environmental characteristics. Knowledge on the geographic distribution, host range and the relative susceptibility of different hosts is essential for disease management, mitigation and containment strategies. The findings reported in this review will support countries that are currently free of F. circinatum in implementing effective procedures and restrictions and prevent further spread of the pathogen.

Abstract:Hymenoscyphus fraxineus is an invasive fungal species causing the most serious disease of ashes (Fraxinus spp.) in Europe-ash dieback. The biology of this fungus is not totally elucidated, neither its relation to the saprophytic species Hymenoscyphus albidus, native in Europe. Our study is focused on the description of seasonal spore dispersal of both fungi and its relation to meteorological conditions, which is needed for more precise and effective control of the disease. For this experiment one long time infected mixed forest in the SE Czech Republic was chosen. A seven-day automatic volumetric spore trap and a weather station were installed to continuously sample the aerospora from April to October 2014. In seven periods a rotating arm spore trap was also used to obtain 48-h air samples to compare the efficiency of these two types of air samplers. Air samples were evaluated solely by qPCR with a very low detection limit. Results show co-occurrence of inoculum of both fungi throughout the entire sampling period with peak levels in August. The origin of the inoculum sampled in the periods without apothecia is discussed. Air-inoculum occurrence of both fungi is significantly correlated with each other, suggesting their coexistence in this forest.

Abstract:The airborne inoculum of Fusarium circinatum Nirenberg & O'Donnell, the fungal pathogen causing Pine Pitch Canker (PPC), is one of the main means of spread of the disease in forest stands and forest nurseries. Since this world-wide known pathogen was introduced in Europe, its biology in this newly infested area still remains scarcely known. To shed more light on this topic, we set up an experiment on a naturally PPC infested forest of Monterey pine in Galicia (NW Spain) with the following two goals: (i) to describe the seasonal spore dispersal pattern during one year of regular sampling and (ii) to assess the spatial spore dispersal pattern around the infested plot. Portable rotating arm spore traps were used and complemented with meteorological measurements. The abundance of F. circinatum spores in the samples was assessed by quantitative PCR (qPCR) with a hydrolysis probe. The results showed almost permanent occurrence of the air inoculum throughout the whole year, being detected in 27 of the 30 samplings. No clear temporal trends were observed, but a higher air inoculum was favoured by previous lower air temperatures and lower leaf wetness. Conversely, neither rainfall nor air humidity seemed to have any significant importance. The spatial spread of the inoculum was noted to be successful up to a distance of 1000 m in the wind direction, even with winds of just 5 m·s −1 . Our study shows that rotating arm spore traps combined with qPCR may be an efficient tool for F. circinatum detection.

Pine pitch canker (PPC), caused by Fusarium circinatum, is considered among the most important diseases affecting pines in many locations throughout the world. In Europe, F. circinatum is currently present in the Iberian Peninsula, posing a high risk of its spread into currently disease-free countries in Europe. In the present study, the susceptibility of Pinus sylvestris, Picea abies, and Larix decidua originating in the Czech Republic to F. circinatum was tested. Furthermore, the presence of asymptomatic yet infected seedlings was also checked. This study demonstrated the pathogenicity of F. circinatum to the Czech provenance of P. sylvestris, whereas Picea abies and Larix decidua proved to be tolerant. The reisolation of F. circinatum beyond the inoculation point demonstrated that this quarantine pathogen is able to infect the three conifers tested, giving rise to asymptomatic seedlings for at least eight and a half months. To our knowledge, this is the first study in which the presence of symptomless seedlings has been recorded in the genera Picea and Larix. This finding points out that the European legislation would fail to avoid the risk of new introductions via symptomless seedlings, since this legislation is only restricted to plants of the genus Pinus and the species Pseudotsuga menziesii.

Fusarium circinatum is a harmful pathogenic fungus mostly attacking Pinus species and also Pseudotsuga menziesii , causing cankers in trees of all ages, damping-off in seedlings, and mortality in cuttings and mother plants for clonal production. This fungus is listed as a quarantine pest in several parts of the world and the trade of potentially contaminated pine material such as cuttings, seedlings or seeds is restricted in order to prevent its spread to disease-free areas. Inspection of plant material often relies on DNA testing and several conventional or real-time PCR based tests targeting F . circinatum are available in the literature. In this work, an international collaborative study joined 23 partners to assess the transferability and the performance of nine molecular protocols, using a wide panel of DNA from 71 representative strains of F . circinatum and related Fusarium species. Diagnostic sensitivity, specificity and accuracy of the nine protocols all reached values >80%, and the diagnostic specificity was the only parameter differing significantly between protocols. The rates of false positives and of false negatives were computed and only the false positive rates differed significantly, ranging from 3.0% to 17.3%. The difference between protocols for some of the performance values were mainly due to cross-reactions with DNA from non-target species, which were either not tested or documented in the original articles. Considering that participating laboratories were free to use their own reagents and equipment, this study demonstrated that the diagnostic protocols for F . circinatum were not easily transferable to end-users. More generally, our results suggest that the use of protocols using conventional or real-time PCR outside their initial development and validation conditions should require careful characterization of the performance data prior to use under modified conditions (i.e. reagents and equipment). Suggestions to improve the transfer are proposed.

This chapter focuses on foliar diseases of conifers, pines, spruces, firs, larches, yews and Cupressaceae. Information is given on their detection, infection biology, epidemiology, and management strategies and tactics.

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