Susceptibility of germinating seedlings of European and Eurasian populations of Pinus sylvestris to damping‐off caused by Fusarium circinatum

Abstract The effect of inoculation with Fusarium circinatum on survival of seed and seedlings of 19 populations of Pinus sylvestris was examined under environmentally controlled conditions, with four treatments (0, 50, 103, 106 spores ml−1). A single seed source of P. radiata was included as a positive control. Germination (emergence of the plumule above the compost) and health of seedlings was assessed daily, for 85 days. Spore density had a significant effect on germination: at 50 spores ml−1, only germination of a Northeast Scotland population was reduced. Treatment with 1000 spores ml−1, however, reduced germination of six populations of P. sylvestris and of P. radiata. Survival of emerged seedlings also varied with inoculum dose. Approximately 75% of seedlings survived 85 days after germination after inoculation with 50 spores ml−1. Seedlings of all populations were killed within 12–16 days of germination by the 103 and 106 spores ml−1 treatments. Emerged seedlings of the Austrian populations showed the highest susceptibility to F. circinatum following treatment with 50 spores ml−1, although 15% of seedlings of one Austrian population (AU3) survived to the end of the experiment (85 days after germination). There was no clear pattern in survival rates of the P. sylvestris seedlings from other populations treated with 1000 or 1 million spores ml−1 due to death of all emerged seedlings within a short period. Variations in susceptibility of different populations of P. sylvestris to F. circinatum may be used in future selection and breeding programmes to reduce the impact of the pathogen as it spreads over wider areas in Europe and Eurasia.


Fusarium circinatum was first detected in 1945 infecting Pinus
virginiana Mill. in the southeastern USA (Hepting and Roth, 1946).

The European and Mediterranean Plant Protection Organization
(EPPO) currently includes F. circinatum in the A2 list (present in the EPPO region but not widely distributed) of organisms recommended for regulation as quarantine pathogens.
There are at least 60 species of Pinus, along with Pseudotsuga menziesii, known to be susceptible to F. circinatum (Bezos et  However, susceptibility within the genus Pinus varies significantly, with Pinus radiata considered the most susceptible species (Wingfield et al., 2008).
Pinus sylvestris (Scots pine) is the most widespread pine species in nature, covering over 28 million hectares in Europe (Stanners et al., 1995); it is also one of the most commercially important pines and delivers a range of ecosystem services (Houston Durrant et al., 2016). Many provenances of Scots pine have been delineated and within these provenances, further sub-populations (hereafter 'populations') have been distinguished, based on a range of phenotypic characters (Salmella et al. 2013). Scots pine is affected by many different pests and diseases (e.g. Ips spp., Heterobasidion spp.); however, potential introductions of alien pathogens with which the host has not coevolved are major concerns (Stenlid and Oliva, 2016).
Variations in susceptibility to pitch canker have also been found at an intraspecific level among provenances of Pinus and Pseudotsuga in work conducted in Brazil, Colombia, El Salvador, Guatemala, Mexico, South Africa, USA Dvorak, 2000, 2007;Gordon et al., 2006;Dvorak et al., 2009;Mitchell et al., 2012;Steenkamp et al., 2012). In Europe, most work to date has been on screening variations in susceptibility of P. pinaster (Vivas et al., 2012(Vivas et al., , 2013Elvira-Recuenco et al., 2014).
Climatic conditions are considered as limiting for spread of F. circinatum in Northern and Central/Eastern Europe (Möykkynen et al., 2015). However, the potential distribution of pitch canker in Europe was expected to include the Netherlands and Denmark, based on climatic change scenarios used in modelling (Watt et al., 2011;Möykkynen et al., 2015); climate change could, in addition, render pines in parts of the British Isles, susceptible to the disease. Climatic limitations are less of a constraint to F. circinatum causing damping-off in forest nurseries, however, particularly where plants are raised under protection, and any international trade in live plant material further amplifies the high risk of the pathogen spreading to disease-free regions of Europe where Scots pine plantations and native forests occur.
In the present work, it was hypothesized that high genetic variability in European and Eurasian Scot pine populations (Belletti et al., 2012;Donnelly et al., 2016;Wójkiewicz et al., 2016) will result in differences in susceptibility of these populations to F. circinatum.
The aim of the work reported here was to determine variations in susceptibility to pitch canker of germinating seed and young plants of nineteen Scots pine populations from several European and Eurasian provenances.

| Fungal material
Isolate FcCa6 of F. circinatum was selected for this work as it was Flasks were incubated on an orbital shaker at 180 rpm for 24 h.
Spores were recovered by passing cultures through two layers of cheesecloth to remove mycelial fragments. Spore density was adjusted to 50, 10 3 or 10 6 spores ml −1 using replicate haemocytometer counts.

| Inoculations of plant materials
A total of 19 populations of Pinus sylvestris and one of Pinus radiata (used as a positive control) were tested against F. circinatum in the inoculation assay (Table 1). Seventy six seeds per population were sown in cell trays (cell volume 96 ml), with one seed per cell, in a substrate of peat -vermiculite 1:1 (v/v), previously autoclaved twice at 120°C for 20 min. Four different treatments were tested for each population: three spore concentrations (50, 10 3 , 10 6 spore ml −1 ) plus the control treatment. After sowing the seeds, each cell was inoculated with 100 μl of the respective spore suspension or with sterile distilled water. Trays were maintained at 25°C with a photoperiod of 16/8 h of light/darkness and watered periodically with sterile distilled water.
Seed germination was evaluated daily, recording the number of living and dead seedlings. At the end of the experiment, F. circinatum was re-isolated from the seedlings, processing needles, stems and roots separately. Each plant part was immersed in water for 3 min, followed by 3% sodium hypochlorite for 2 min, and finally 70% alcohol for 2 min. Subsequently, tissues were rinsed in sterile distilled water for 5 min before plating on PDA amended with streptomycin sulphate (0.5 g/L) in 90 mm Petri dishes and incubating at 25°C in dark for 7 days. Fungal isolates were identified morphologically (Leslie and Summerell, 2006).
Yates' correction for continuity was applied in those cases where the expected frequencies were below 5. Survival analysis based on the Kaplan-Meier nonparametric estimator (Kaplan and Meier, 1958) was carried out with the 'Survival' package (Therneau, 2017) to test post-emergence mortality to the end of the experiment. Survival curves were created with the 'Survfit' function and differences between the curves tested with the 'Survdiff' function. All analyses were performed using R software environment (R Foundation for Statistical Computing,). TA B L E 1 Populations of Pinus sylvestris and Pinus radiata inoculated with Fusarium circinatum, coded and sorted by country of origin, giving geographic coordinates. Germination rate given is based on negative control treatments
Differences among populations were found at each inoculum dose. At the lowest inoculum dose (50 spores ml −1 ), only Austrian provenances showed high susceptibility to F. circinatum (Figures 2   and 3). Mortality of P. sylvestris in the Austrian provenances was greater at 35 days after germination than in P. radiata seedlings at 80 days after germination. All seedlings of provenances AU1, AU2 and AU4 died within 35 days of germination, but ca. 15% of AU3 seedlings survived to the end of the experiment (85 days after germination). However, no significant differences in time to mortality were found among these Austrian provenances (χ2 = 5.1, p = 0.17).
Although survival analyses on seedlings treated with 1000 or 1 million spores ml −1 showed significant differences among populations (χ2 = 42.8, p < 0.001 and χ2 = 60.2, p < 0.001, respectively), no clear pattern in survival was found due to death of all emerged seedlings within a short period (Figures 4 and 5).

| DISCUSS ION
The   (Dwinell, 1999;Landeras et al., 2005), the disease is causing substantial damage to the highly susceptible P. radiata. Other pine species are at risk, however, since many species in the genus and native to or grown in Europe and Eurasia are known to be susceptible to F. circinatum (Iturritxa et al., 2012, Martínez-Alvarez et al., 2014; moreover, changing climate is likely to lead to an expansion in the area suitable for establishment of the pathogen (Watt et al., 2011;Möykkynen et al., 2015). Among the pine species at risk to this disease in Europe, P. sylvestris is arguably the most important because of its wide distribution from western  Table 1 for population codes F I G U R E 4 Plot of survival probability determined using the Kaplan-Meier estimate of the survival function for each population of Pinus sylvestris inoculated with 1000 spores ml −1 Fusarium circinatum. See Table 1  Other authors reported lesions developing on seedlings of several pine species, after inoculating 25 or 50 spores (Gordon et al., 1998;Iturritxa et al., 2012Iturritxa et al., , 2013, but to our knowledge symptoms have never been reported previously when using such a low number of spores.
The unsuberized tissues of newly germinated seedlings used in this work could have favoured colonization by the pathogen, ultimately contributing to the high rates of mortality. In contrast, the two higher doses of inoculum applied in the work, 10 3 and 10 6 spores ml −1 resulted in rapid death of all seedlings, with no clear discernible pattern in susceptibility among populations/provenances.
In other experiments performed with 10 6 spores ml −1 , a similar rapid death of pine seedlings also occurred (Martínez-Alvarez et al., 2014): fewer than 10% of P. sylvestris seedlings survived after inoculation. This work confirmed that the selection of a provenance is an important consideration when planning forest plantations in the presence of pathogen threats. It is generally recommended that, when feasible, the provenance native to the region in which the plantations will be established is the preferred option, as it is adapted to local conditions. In the altered conditions presented by the establishment of an alien invasive pathogen in a given region; however, exploring potential variations in resistance between provenances of a tree species should be considered. It is important, therefore, to conduct screening for relative resistance/susceptibility between F I G U R E 5 Plot of survival probability determined using the Kaplan-Meier estimate of the survival function for each population of Pinus sylvestris inoculated with 10 6 spores ml −1 Fusarium circinatum. See Table 1 for population codes provenances of pines so that appropriate forest management decisions can be made when F. circinatum spreads to and establishes in other regions of Europe. Foundation.

DATA AVA I L A B I L I T Y S TAT E M E N T
Raw data are available to legitimate users on request from the corresponding author.