Comparison of Diplodia tip blight pathogens in Spanish and North American pine ecosystems

microorganisms Article Comparison of Diplodia Tip Blight Pathogens in Spanish and North American Pine Ecosystems Ana Aragonés1, Tania Manzanos 1, Glen Stanosz 2, Isabel A. Munck 3, Rosa Raposo 4,5, Margarita Elvira-Recuenco 4, Mónica Berbegal 6, Nebai Mesanza 1, Denise R. Smith 2, Michael Simmons 7, Stephen Wyka 8and Eugenia Iturritxa 1,*   Citation: Aragonés, A.; Manzanos, T.; Stanosz, G.; Munck, I.A.; Raposo, R.; Elvira-Recuenco, M.; Berbegal, M.; Mesanza, N.; Smith, D.R.; Simmons, M.; et al. Comparison of Diplodia Tip Blight Pathogens in Spanish and North American Pine Ecosystems. Microorganisms 2021,9, 2565. https://doi.org/10.3390/ microorganisms9122565 Academic Editor: Lorenzo Brusetti Received: 15 November 2021 Accepted: 9 December 2021 Published: 11 December 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1Neiker-BRTA, Instituto Vasco de Investigación y Desarrollo Agrario, Granja Modelo s/n, Antigua Carretera Nacional 1, Km. 355, 01192 Arkaute, Spain; [email protected] (A.A.); [email protected] (T.M.); [email protected] (N.M.) 2Department of Forest and Wildlife Ecology, University of Wisconsin-Madison, Madison, WI 53706, USA; [email protected] (G.S.); [email protected] (D.R.S.) 3Northeastern Area State and Private Forestry, USA Department of Agriculture Forest Service, Durham, NH 03824, USA; [email protected] 4Intituto de Investigación Forestal_ Instituto Nacional de Investigación y Tecnología Agraria (CIFOR, INIA), Carretera La Coruña Km 7.5, 28040 Madrid, Spain; [email protected] (R.R.); [email protected] (M.E.-R.) 5Sustainable Forest Management Research Institute, University of Valladolid-INIA, Avenida Madrid 44, 34004 Palencia, Spain 6Instituto Agroforestal Mediterráneo, Universitat Politècnica de València, Camino de Vera S/N, 46022 Valencia, Spain; [email protected].es 7Department of Natural Resources and the Environment, University of New Hampshire, Durham, NH 03824, USA; [email protected] 8LifeMine Therapeutics, Cambridge, MA 02140, USA; [email protected] *Correspondence: [email protected]; Tel.: +34-637436343 Abstract: Diplodia tip blight is the most ubiquitous and abundant disease in Spanish Pinus radiata plantations. The economic losses in forest stands can be very severe because of its abundance in cones and seeds together with the low genetic diversity of the host. Pinus resinosa is not genetically diverse in North America either, and Diplodia shoot blight is a common disease. Disease control may require management designs to be adapted for each region. The genetic diversity of the pathogen could be an indicator of its virulence and spreading capacity. Our objective was to understand the diversity of Diplodia spp. in Spanish plantations and to compare it with the structure of American populations to collaborate in future management guidelines. Genotypic diversity was investigated using microsatellite markers. Eight loci (SS9–SS16) were polymorphic for the 322 isolates genotyped. The results indicate that Diplodia sapinea is the most frequent Diplodia species present in plantations of the north of Spain and has high genetic diversity. The higher genetic diversity recorded in Spain in comparison to previous studies could be influenced by the intensity of the sampling and the evidence about the remarkable influence of the sample type. Keywords: Sphaeropsis sapinea; Diplodia tip blight; dieback; SSRs; Diplodia scrobiculata 1. Introduction Pinus radiata D. Don. is the most widespread exotic forest species susceptible to the fungal pathogen Diplodia sapinea (Fr.) Fuckel (syn. Diplodia pinea (Desm.) Kickx., Sphaeropsis sapinea (Fr.:Fr./Dyko and Sutton)) in Spain. The first record of the introduction of P. radiata D. Don in Spain is its presence in a garden in Lekeitio (Bizkaia) in the mid-19th century [ 1 ]. It was considered an appropriate candidate for forestry in Spain based on previous acclimatization studies [ 2 ]. The first plantations of this tree species in Spain were established at the end of the 19th century in the Basque Country. A policy of reforestation of public lands [ 3 ] led to an increase in the area covered by plantations, which reached Microorganisms 2021,9, 2565. https://doi.org/10.3390/microorganisms9122565 https://www.mdpi.com/journal/microorganisms Microorganisms 2021,9, 2565 2 of 17 over 160,000 ha by the 1970s [ 3 – 5 ]. Due to the environmental requirements of P. radiata, cold sensitivity and high humidity, its distribution within the northern Iberian Peninsula is limited mainly to the Cantabrian coast, which has an Atlantic climate, where this species is an important feature of the landscape [ 4 , 6 ]. It is difficult to determine the origin of P. radiata germplasm at any particular location in Spain [ 3 ], P. radiata seeds were obtained from collections performed during the first thinnings of the Basque Country pine forests and from providers located in New Zealand, the USA, Chile, France and Denmark. However, a study of the population of P. radiata growing in Spain showed low genetic diversity [ 7 ]. The local landrace and the three Californian natural provenances (Año Nuevo, Monterey and Cambria) have been compared using genetic diversity analysis by molecular markers (RAPDs), growth and characteristics morphological and survival. The local population was found to be most similar to the Año Nuevo provenance. The Año Nuevo and the local landrace showed the lowest mortality (2.1 and 8.1%, respectively). Mortality was greater for Monterey provenance (29.3%) and particularly high for the Cambrian provenance (52.6%). The differential genotype adaptation to local conditions of northern Spain and survival there may explain in part the detected low genetic diversity. Diplodia tip blight caused by D. sapinea is the most ubiquitous and abundant disease in Spanish P. radiata plantations, which are monocultures and susceptible to various diseases. In a field survey performed in 2009 in the Basque Country [ 8 ], the incidence of Diplodia tip blight in surveyed plots was 100%, compared to 17% for pitch canker disease caused by Fusarium subglutinans f. sp. pini. The severe economic losses in forest stands of P. radiata can be attributed to both the low genetic diversity of the host [ 7 , 9 ] and abundance of the pathogen in cones and seeds [ 8 , 10 – 12 ] which are the major sources of inoculum in the area [13]. Similar to P. radiata in Spain, P. resinosa Ait. is one of the least genetically diverse conifer species in North America [ 14 ]. The native range of P. resinosa is a narrow latitudinal band running east–west across southeastern Canada and the northeastern USA. This range extends north to Maine (USA), southern Quebec (Canada), New Brunswick (Canada), and Nova Scotia (Canada), west to central Ontario (Canada), and south to Minnesota (USA), Wisconsin (USA), Michigan (USA), northern Pennsylvania (USA), northern New Jersey (USA), Connecticut (USA), and western Massachusetts (USA) [ 15 ]. In addition, separated patches of endemic P. resinosa also occur in Newfoundland (Canada), northern Illinois (USA), and eastern West Virginia (USA) [ 15 ]. Red pine is naturally found in pure stands or more commonly in mixtures with Pinus strobus (eastern white pine) or Pinus banksiana (jack pine) on well-drained sandy soils [15]. Red pine was widely planted in the 1930s to 1960s to stabilize abandoned agricultural lands [ 15 ]. Today, red pine is one of the most commonly planted trees in the northern USA and Canada [ 15 ]. Diplodia shoot blight, collar rot and canker are common diseases in these red pine plantations [ 16 – 22 ]. The persistence of D. sapinea in seed orchards and forest nurseries may have contributed to the widespread dissemination of this pathogen [13,23–25] . Diplodia sapinea was considered a variable organism, both in morphological and virulence of different strains but a recent study confirms that the European D. sapinea population is homogeneous and little differentiated except for subpopulations from Italy and Georgia [ 26 ]. Historically, three morphotypes were differentiated [ 27 – 29 ]. In 2003, de Wet et al. [ 30 ], however, proposed the separation of morphotypes into species. Based on comparison of multiple genes and microsatellite markers, they concluded that strains of morphotypes A and C corresponded to D. sapinea. For the morphotype B strains, they proposed a new taxon called Diplodia scrobiculata J. de Wet, Slippers & M. J. Wingf. Both species have been detected in Spain on P. radiata [31] and North America on P. resinosa [32,33]. Control of D. sapinea is complicated because it is capable of surviving on needles, branches, shoots, wood and pine cones for long periods [ 20 , 23 , 34 – 37 ]. It is commonly isolated from seedlings, needles, cones, branches, seed scales, seeds and pits of cones and mature wood [ 25 , 38 – 43 ]. Furthermore, D. sapinea can persist asymptomatically as a latent pathogen [ 10 , 11 ]. Only after trees experience a stress event such as drought, physical Microorganisms 2021,9, 2565 3 of 17 damage or hail may the characteristic Diplodia tip blight symptoms develop [ 44 – 47 ]. In addition, its genetic diversity could be an indicator of its potential virulence and capacity to spread. The frequent importation of P. radiata seeds in Spain from different suppliers of uncertain origin and the negative impact of these diseases on the productivity of our forest stands led us to carry out this study. Our objective was to understand the diversity of Diplodia spp. in Spanish P. radiata plantations and to compare the structure of American D. sapinea populations with populations obtained from P. radiata in northern Spain. Most of the North American D. sapinea isolates used in this study came from P. resinosa plantations, which are similar to P. radiata plantations in the low genetic variation of the hosts and potential spread of the pathogen via nursery stock. Improved knowledge about the genetic diversity and mode of reproduction in the Basque Country D. sapinea populations might help to design specific management options at a local scale. In addition, the potential influence of the sampling strategy (density and sample type) will be discussed. 2. Materials and Methods 2.1. Fungal Collection and Isolation Symptomatic (dieback) P. radiata trees were sampled in the major pine-growing regions of the Basque Country during spring and summer of 2016–2020. Plantations located in Laukiniz (P1), Sollano (P2), Hernani (P3), Luyando (P4) and Oiartzun (P5) were intensively sampled. Otherwise, only one strain of Diplodia spp. per plot was isolated. Fragments of cone scales bearing pycnidia were soaked in 30% commercial bleach (1.6% sodium hypochlorite) for 1 min and rinsed with sterile water. A single pycnidium from the cone surface was transferred to water agar medium (Panreac, Barcelona, Spain), and a single conidium was selected to initiate a monosporic culture. Single conidial isolates were grown on potato dextrose agar (Panreac, Barcelona, Spain) in petri plates in darkness at 20 ±3◦C for 4 to 6 days. Fungal species were initially identified by colony and conidium morphology [ 30 , 48 ]. Diplodia species were confirmed by molecular methods. All isolates were maintained in the Culture Collection of the Forestry Department, Neiker, BRTA Granja Modelo Arkaute, Vitoria-Gasteiz, Spain. Roots were carefully washed under tap water to remove any adhered soil particles. For surface disinfestation, roots were dipped into 70% EtOH for 1 min, submerged in a 30% commercial bleach with Tween 20 (1 drop/100 mL) solution for 15 min and rinsed twice in sterile distilled water. The thinnest roots (less than 1 mm diameter) were immersed in the same commercial bleach solution but for 10 min instead of 15 min. These thin surface disinfested roots were aseptically transferred to sterilized filter paper and when dried, transversally cut into 5 mm long segments and placed on PDA petri dishes. The thickest roots were first longitudinally divided into two pieces and then cut into 5 mm long pieces. In addition, branches, needles, pieces of wood from cankers and cores were sampled. Cores were collected from tree trunks with a Pressler’s 5-mm-diameter increment borer at 130 cm height [ 49 ]. Needles, fragments of branches and wood were separately collected in paper bags, and cores were introduced into sterilized tubes. All the tools in contact with the samples were disinfected before and after sampling with 70% EtOH. All the samples were labelled and stored at 4 ◦ C. In the laboratory, the samples were immersed for 2 min in a sodium hypochlorite solution (1% active chlorine) and rinsed with sterile water. Thin disks cut from whole cross sections of the cores and branches were placed on potato dextrose agar (Panreac, Bareclona, Spain) and cultivated under the same conditions as the conidial cultures. Three to six petri dishes per sample were used. Dishes were incubated in darkness at 25 ◦ C and evaluated every 3 days. Putative colonies of D. sapinea were transferred to potato dextrose agar (PDA) dishes, which were incubated for 7 days at 25 ◦ C, and mycelial growth characteristics were observed. Isolates were then grown on 2% water agar with sterilized pine needles at 25 ◦C under near-ultraviolet light (near-UV light) to induce sporulation. Microorganisms 2021,9, 2565 4 of 17 North American isolates were obtained from an extensive collection at the University of Wisconsin-Madison and P. resinosa cones collected in New England, USA. Isolates U1– U50 were obtained as part of a study evaluating the factors and effects associated with widespread red pine mortality. Pinus resinosa branch and cone samples were obtained from asymptomatic trees and trees expressing crown dieback symptoms associated with Matsucoccus matumurae Kuwana (pine bast scale) infestation within both plantations and natural stands, the latter of which were located in Hancock County, Maine.. Pinus resinosa cones from New England were sampled using methods described previously [ 50 ]. Cones were bagged, placed on ice in a cooler for transportation, and stored in a freezer until processed in the laboratory. Conidia were extracted from each cone, and Diplodia species were identified by morphological and molecular methods described below. Isolates were sent to Neiker’s lab to implement the molecular work. 2.2. Species Identification Morphological and molecular methods were used to identify isolates. Conidial shape, color, presence of septa, width and length were observed, as well as mycelial growth. Mycelium grown on medium in petri dishes was scraped off and collected in a 2 mL tube with five sterile tungsten carbide beads (300 µ M diameter). The fungal material was disrupted using a Qiagen-Retsch MM300 Tissuelyser (Qiagen, Hilden, Germany) at a speed of 30 m/s for 3 min at room temperature. In all cases, fungal DNA was extracted from 200 mg pure monosporic cultures using a DNA Plant Mini Kit (Analytik Jena AG, Life Science). Extractions were performed following the manufacturer’s instructions. DC-PCR with species-specific primers was used to differentiate D. sapinea DpF (5 0 -CTTATATATCAAACTATGCTTTG-TA-3 0 ) and D. scrobiculata DsF (5 0 - CTTATATATCAAACTAATGTTTG-CA-3 0 ); a Botryosphaeria-specific primer was used as the reverse primer BotR (5 0 -GCTTACACTTTCATTTATAGACC-3 0 ) [ 18 ] and was used for the identification of species. PCR amplification was performed in a total volume of 25 µ L containing 1 x reaction buffer, 2 mM MgCl 2 , 0.25 mM dNTPs, 0,8 µ M of each specific primers, 40 ng of DNA and 1.25 U Platinum Taq polymerase (Roche Diagnostic GmbH, Mannheim, Germany). The cycling profile was as follows: denaturation at 94 ◦ C for 60 s, followed by 35 cycles at 94 ◦ C for 30 s, 67 ◦ C for 30 s, and 72 ◦ C for 30 s, and a final extension at 70 ◦ C for 5 min. Fragment sizes were verified on 0.7% agarose gels in Tris-boric acid-EDTA buffer (TBE) with DNA loading buffer, 5 × DNA (Bioline Merdidian Bioscience, London, UK). 2.3. PCR Amplification of SSR Loci and Data Analysis Ten microsatellite loci, SS1-5-9-10-11, previously described by Burgess et al. [ 51 ], and SS12-14-15-16, described by Bihon et al. [ 52 ], were amplified for 322 D. sapinea isolates (Table 1). Positive controls with known DNA and negative controls without DNA were included. All SSR-PCR products were multiplexed and run in a single lane. SSR-PCR was conducted with a PCR mixture containing 1 × QIAGEN ® Multiplex PCR kit, 0.15 µ M (each) primer, 15 ng of DNA template and water to a final volume of 13 µ L. The reactions were carried out in a thermocycler (Eppendorf, Hamburg, Germany) programmed for an initial denaturation of 1 min at 95 ◦ C, followed by 2 min at 94 ◦ C, 15 cycles of 30 s at 58 ◦ C, 45 s at 60 ◦ C and 1 min at 72 ◦ C, and 20 cycles of 55 ◦ C. Dilution of 1:50 for the thermocycler products was conducted before multiplex analysis to avoid detection error. The forward primers were labelled with a phosphoramidite fluorescent dye indicated as FAM, NED, PET and VIC. One µ L of these multiplexed PCR products was separated on an ABI Prism 3130 Genetic Analyser (Applied Biosystems, Foster, CA, USA). The amplicon peaks were determined based on the four fluorescent dyes used and the sizes of the DNA fragments. The mobility of SSR products was compared to those of internal size standards (LIZ-500), and allele sizes were estimated by GeneMapper 4.0 computer software (Applied Biosystems, Foster, CA, USA). A reference sample was run on every gel to ensure reproducibility. Microorganisms 2021,9, 2565 5 of 17 For each population defined by country of origin (the Basque Country in Spain and USA), nursery location within the Basque Country and sample type, the total number of alleles at each SSR locus was estimated. A multilocus genotype (MLG) was constructed for each isolate by combining data for single SSR alleles, and the expected multilocus genotype (eMLG) based on rarefaction was calculated using the R package poppr V.2.3.0 [ 53 , 54 ]. Given the clonality observed analyses were conducted for the clone-corrected dataset, with only one isolate of each MLG considered. Stoddart and Taylor’s diversity index (G) [ 55 ] and evenness index E5 [56] were calculated using the same R package. The standardized index of association (rbarD) as an estimate of linkage disequilibrium was calculated to investigate the mode of reproduction [ 54 , 57 ]. The expectation of rbarD for a randomly mating population is zero, and significant deviation from this value would suggest clonal reproduction. Significance was tested based on 1000 permutations and conducted in the R package poppr using the clone-corrected data [54]. The standardized measure of genetic differentiation G ´ st described by Hedrick [ 58 ] was calculated to estimate subdivision among populations. This index ranges from 0 to 1, independent of the extent of population genetic variations and locus mutation rates [ 58 ]. Pairwise G ´ st values within the clone-corrected data were calculated using the R packages strata G V.1.0.5 [ 59 ] and mmod V.1.3.3 [ 60 ]. Statistical significance was calculated based on 1000 permutations. Discriminant analysis of principal components (DAPC) was performed to infer clusters of populations without considering previous geographic/nursery location/isolation tissuebased assignment criteria [ 61 ]. DAPC was conducted with the R package adegenet V. 2.0.1 [ 62 ] using the Bayesian information criterion (BIC) to infer the optimal number of groups. Important advantages of DAPC are that it maximizes variation between the groups, minimizes the within-group genetic variability and does not require assumptions regarding evolutionary models [61]. To assess the relationships among MLGs, minimum spanning networks (MSNs) were constructed from the clone-corrected dataset. Bruvos ´ s genetic distance matrix and MSNs were generated using the R package poppr V.2.3.0 [ 53 , 54 ]. The genetic distance described by Bruvo et al. [ 63 ] takes the SSR repeat number into account, with a distance of 0.1 equivalent to one mutational step (one repeat). 2.4. DNA Sequencing and Phylogenetic Analysis Based on different MLGs, 47 D. sapinea isolates were selected. The internal transcribed spacer (ITS) region was amplified using the primers ITS1 and ITS4 [ 64 ], and translation elongation Factor 1α (TEF1α ) was amplified using the primers EF1-728F and EF1-986R [ 65 ]. PCRs for each region contained 20ng DNA, 3 µ L 10 × PCR Complete KCl reaction buffer (IBIAN Technologies) containing 15 mM MgCl2, 200 nM of each primer, 200 µ M of each dNTP and 1U IBIAN-Taq DNA polymerase (IBIAN Technologies, Zaragoza, Spain). The PCR profile for the ITS region was as follows: 94 ◦ C for 10 min, 35 cycles at 94 ◦ C for 30 s, 58 ◦ C for 45 s, 72 ◦ C for 60 s, and 72 ◦ C for 10 min. For the TEF1α region, the same PCR conditions were used, but the annealing temperature was set at 52 ◦ C. PCR products were sequenced by Macrogen (Seoul, South Korea). Sequence data were edited using FinchTV software version 1.4.0 (https://finchtv. software.informer.com/1.4/, accessed on 8 January 2021) and aligned, and a phylogenetic tree was constructed from the aligned sequences with MEGA X software version 10.0.4 (https://www.megasoftware.net/, accessed on 3 October 2021). 3. Results 3.1. Species Identification The presence of Diplodia scrobiculata was detected only in a single tree, from a wood core sample, of all the 253 analyzed trees in the Basque Country. Diplodia scrobiculata detection is considered something exceptional in this region where both Diplodia species Microorganisms 2021,9, 2565 6 of 17 co-occurred in the same tree. Only D. sapinea was isolated from red pine cones collected in New England. Table 1shows the strains identified as D.sapinea. Table 1. Diplodia sapinea isolates used in this study: origin, host species, sample type and strain identification code. Origin Host Species Sample Type Sample ID Origin Host Species Sample Type Sample ID Bajo Deba, Spain Pinus pinaster Canker BCI2 P1 (Laukiniz), Spain Pinus radiata Canker BCM198 Plentzia-Munguia, Spain Pinus radiata Root BCI5 P1 (Laukiniz), Spain Pinus radiata Canker BCM199 Alto Deba, Spain Pinus radiata Cone BC14 P1 (Laukiniz), Spain Pinus radiata Canker BCM200 Estribaciones del Gorbea, Spain Pinus radiata Cone BC15 P1 (Laukiniz), Spain Pinus radiata Canker BCM201 Encartaciones, Spain Pinus radiata Cone BC17 P1 (Laukiniz), Spain Pinus radiata Canker BCM202 Encartaciones, Spain Pinus radiata Cone BC18 P2 (Sollano), Spain Pinus radiata Cone BC22 Encartaciones, Spain Pinus radiata Cone BC19 P2 (Sollano), Spain Pinus radiata Cone BC23 Alto Deba, Spain Pinus radiata Cone BC39 P2 (Sollano), Spain Pinus radiata Cone BC24 Alto Deba, Spain Pinus radiata Cone BC40 P2 (Sollano), Spain Pinus radiata Cone BC25 Goierri, Spain Pinus nigra Cone BC41 P2 (Sollano), Spain Pinus radiata Cone BC26 Goierri, Spain Pinus nigra Cone BC42 P2 (Sollano), Spain Pinus radiata Cone BC27 Montaña Alavesa, Spain Pinus nigra Cone BC44 P2 (Sollano), Spain Pinus radiata Cone BC29 Llanada Alavesa, Spain Pinus radiata Cone BC45 P2 (Sollano), Spain Pinus radiata Cone BC30 Llanada Alavesa, Spain Pinus radiata Cone BC46 P2 (Sollano), Spain Pinus radiata Cone BC31 Llanada Alavesa, Spain Pinus radiata Cone BC47 P2 (Sollano), Spain Pinus radiata Cone BC33 Gernika Bermeo, Spain Pinus radiata Cone BC48 P2 (Sollano), Spain Pinus radiata Cone BC34 Gernika Bermeo, Spain Pinus radiata Cone BC49 P2 (Sollano), Spain Pinus radiata Cone BC35 Gernika Bermeo, Spain Pinus radiata Cone BC50 P2 (Sollano), Spain Pinus radiata Cone BC36 Gernika Bermeo, Spain Pinus radiata Cone BC51 P2 (Sollano), Spain Pinus radiata Cone BC38 Gernika Bermeo, Spain Pinus radiata Cone BC52 P3 (Hernani), Spain Pinus radiata Cone BC60 Duranguesado, Spain Pinus radiata Cone BC53 P3 (Hernani), Spain Pinus radiata Cone BC61 Markina-Ondarroa, Spain Pinus radiata Cone BC54 P3 (Hernani), Spain Pinus radiata Cone BC62 Donostia San Sebastian, Spain Pinus radiata Cone BC55 P3 (Hernani), Spain Pinus radiata Cone BC63 Valles Alaveses, Spain Pinus attenuata Cone BC56 P3 (Hernani), Spain Pinus radiata Cone BC64 Gernika Bermeo, Spain Pinus radiata Cone BC57 P3 (Hernani), Spain Pinus radiata Cone BC65 Goierri, Spain Pinus radiata Cone BC76 P3 (Hernani), Spain Pinus radiata Cone BC66 Goierri, Spain Pinus radiata Cone BC77 P3 (Hernani), Spain Pinus radiata Cone BC67 Goierri, Spain Pinus radiata Cone BC78 P3 (Hernani), Spain Pinus radiata Cone BC68 Tolosa, Spain Pinus radiata Cone BC79 P3 (Hernani), Spain Pinus radiata Cone BC69 Tolosa, Spain Pinus radiata Cone BC80 P3 (Hernani), Spain Pinus radiata Cone BC70 Tolosa, Spain Pinus radiata Cone BC81 P3 (Hernani), Spain Pinus radiata Cone BC71 Tolosa, Spain Pinus nigra Cone BC82 P3 (Hernani), Spain Pinus radiata Cone BC72 Tolosa, Spain Pinus nigra Cone BC83 P3 (Hernani), Spain Pinus radiata Cone BC73 Tolosa, Spain Pinus nigra Cone BC84 P3 (Hernani), Spain Pinus radiata Cone BC74 Tolosa, Spain Pinus radiata Cone BC86 P3 (Hernani), Spain Pinus radiata Cone BC75 Donostia San Sebastian, Spain Pinus radiata Cone BC87 P4 (Luyando), Spain Pinus radiata Root BCM157 Donostia San Sebastian, Spain Pinus radiata Cone BC89 P4 (Luyando), Spain Pinus radiata Root BCM175 Donostia San Sebastian, Spain Pinus radiata Cone BC90 P4 (Luyando), Spain Pinus radiata Root BCM176 Donostia San Sebastian, Spain Pinus radiata Cone BC91 P4 (Luyando), Spain Pinus radiata Root BCM177 Donostia San Sebastian, Spain Pinus radiata Cone BC92 P4 (Luyando), Spain Pinus radiata Root BCM178 Donostia San Sebastian, Spain Pinus radiata Cone BC93 P4 (Luyando), Spain Pinus radiata Core BCM179 Urola Costa, Spain Pinus radiata Cone BC94 P4 (Luyando), Spain Pinus radiata Root BCM181 Urola Costa, Spain Pinus radiata Cone BC95 P4 (Luyando), Spain Pinus radiata Root BCM183 Urola Costa, Spain Pinus radiata Cone BC96 P4 (Luyando), Spain Pinus radiata Core BCM187 Urola Costa, Spain Pinus radiata Cone BC97 P5 (Oiarztun), Spain Pinus radiata Core BCM158 Bajo Deba, Spain Pinus radiata Cone BC98 P5 (Oiarztun), Spain Pinus radiata Core BCM159 Bajo Deba, Spain Pinus radiata Cone BC99 P5 (Oiarztun), Spain Pinus radiata Core BCM160 Encartaciones, Spain Pinus radiata Cone BC100 P5 (Oiarztun), Spain Pinus radiata Core BCM162 Encartaciones, Spain Pinus radiata Cone BC101 P5 (Oiarztun), Spain Pinus radiata Core BCM163 Encartaciones, Spain Pinus nigra Cone BC104 P5 (Oiarztun), Spain Pinus radiata Core BCM164 Encartaciones, Spain Pinus radiata Cone BC105 P5 (Oiarztun), Spain Pinus radiata Core BCM173 Encartaciones, Spain Pinus radiata Cone BC106 P5 (Oiarztun), Spain Pinus radiata Core BCM180 Encartaciones, Spain Pinus radiata Cone BC108 P5 (Oiarztun), Spain Pinus radiata Root BCM209 Valles Alaveses, Spain Pinus pinaster Cone BC109 P5 (Oiarztun), Spain Pinus radiata Root BCM211 Estribaciones del Gorbea, Spain Pinus radiata Cone BC112 P5 (Oiarztun), Spain Pinus radiata Root BCM212 Arratia Nervión, Spain Pinus nigra Cone BC113 P5 (Oiarztun), Spain Pinus radiata Root BCM213 Arratia Nervión, Spain Pinus radiata Cone BC114 Barbour County, West Virginia USA Pinus sylvestris Needle W171 Cantábrica Alavesa, Spain Pinus radiata Cone BC115 Black Hills, South Dakota USA Pinus ponderosa Stem W172 Cantábrica Alavesa, Spain Pinus radiata Cone BC116 Idaho USA Pinus ponderosa Unknown W174 Valles Alaveses, Spain Pinus halepensis Cone BC120 Grant County, Wisconsin USA Pinus resinosa Unknown W175 Valles Alaveses, Spain Pinus sylvestris Cone BC121 Oktibbeha County, Mississippi USA Pinus palustris Unknown W177 Valles Alaveses, Spain Pinus contorta Cone BC123 Pennsylvania USA Pinus sylvestris Unknown W180 Estribaciones del Gorbea, Spain Pinus radiata Cone BC124 Riverside County, California USA Pinus jeffreyi Unknown W185 Cantábrica Alavesa, Spain Pinus radiata Cone BC125 Florida USA Pinus elliottii Cone-seed W186 Arratia Nervión, Spain Pinus radiata Cone BC127 Bennington County, Vermont USA Pinus resinosa Cone W190 Llanada Alavesa, Spain Pinus radiata Cone BC128 Maui, Hawaii USA Pinus radiata Unknown W191 Microorganisms 2021,9, 2565 7 of 17 Table 1. Cont. Origin Host Species Sample Type Sample ID Origin Host Species Sample Type Sample ID Llanada Alavesa, Spain Pinus radiata Cone BC129 Itasca St. Park, Minnesota USA Pinus resinosa Unknown W192 Spain Pinus nigra Cone BC131 Dallas County, Texas USA Pinus eldarica Cone W194 Llanada Alavesa, Spain Pinus nigra Cone BC132 Georgia USA Pinus taeda Needle W206 Llanada Alavesa, Spain Pinus radiata Cone BC133 Wisconsin USA Pinus banksiana Needle W210 Llanada Alavesa, Spain Pinus radiata Cone BC134 Wisconsin USA Pinus banksiana Needle W211 Llanada Alavesa, Spain Pinus radiata Cone BC135 Waushara County, Wisconsin USA Pinus resinosa Needle W212 Llanada Alavesa, Spain Pinus radiata Cone BC137 Portage County, Wisconsin USA Pinus banksiana Needle W213 Llanada Alavesa, Spain Pinus radiata Cone BC138 Portage County, Wisconsin USA Pinus resinosa Needle W214 Montaña Alavesa, Spain Pinus radiata Cone BC141 Wood County, Wisconsin USA Pinus resinosa Needle W215 Alto Deba, Spain Pinus radiata Cone BC142 Adams County, Wisconsin USA Pinus resinosa Needle W216 Bajo Deba, Spain Pinus radiata Cone BC143 Marathon County, Wisconsin USA Pinus resinosa Needle W217 Bajo Deba, Spain Pinus radiata Cone BC144 Wallowa County, Oregon USA Pinus ponderosa Cone W218 Bajo Deba, Spain Pinus radiata Cone BC145 Wallowa County, Oregon, USA Pinus ponderosa Needle W219 Markina Ondarroa, Spain Pinus radiata Cone BC146 Bennington County, Vermont USA Pinus resinosa Stem tip W220 Estribaciones del Gorbea, Spain Pinus radiata Cone BC147 Sawyer County, Wisconsin USA Pinus banksiana Stem W221 Arratia Nervión, Spain Pinus radiata Cone BC148 Wood County, Wisconsin USA Pinus banksiana Stem W222 Arratia Nervión, Spain Pinus radiata Cone BC149 Vilas County, Wisconsin USA Pinus resinosa Unknown W223 Encartaciones, Spain Pinus radiata Cone BC150 South Dakota Pinus ponderosa Unknown W224 Encartaciones, Spain Pinus radiata Cone BC151 Dallas County, Texas USA Pinus nigra Cone W225 Encartaciones, Spain Pinus radiata Cone BC152 Bayfield County, Wisconsin USA Pinus resinosa Needle W226 Duranguesado, Spain Pinus radiata Cone BC154 Sumter County, Alabama USA Pinus taeda Cone W227 Gernika Bermeo, Spain Pinus radiata Cone BC155 Adams County, Wisconsin USA Pinus sylvestris Bark W228 Gernika Bermeo, Spain Pinus radiata Cone BC156 Vilas County, Wisconsin USA Pinus ponderosa Unknown W229 Markina-Ondarroa, Spain Pinus radiata Cone BC157 Vilas County, Wisconsin USA Pinus resinosa Unknown W231 Markina-Ondarroa, Spain Pinus radiata Cone BC158 Pine County, Minnesota USA Pinus resinosa Unknown W232 Duranguesado, Spain Pinus radiata Cone BC159 Morgantown, WV Pinus nigra Needle W233 Gernika Bermeo, Spain Pinus radiata Cone BC160 Jackson County, Wisconsin USA Pinus resinosa Needle W234 Spain Pinus radiata Cone BC161 Dane County, Wisconsin USA Pinus nigra Needle W235 Plentzia-Munguia, Spain Pinus radiata Cone BC162 Northern Highland American Legion State Forest, Wisconsin USA Pinus banksiana Twig W236 Goierri, Spain Pinus radiata Cone BC163 Marquette County, Wisconsin USA Pinus resinosa Needle W238 Tolosa, Spain Pinus radiata Cone BC164 Trempealeau County, Wisconsin USA Pinus resinosa Needle W239 Tolosa, Spain Pinus radiata Cone BC165 Fairfield County, Connecticut USA Pinus sylvestris Needle W240 Urola Costa, Spain Pinus radiata Cone BC166 Adair County, Iowa USA Pinus nigra Needle W241 Markina-Ondarroa, Spain Pinus radiata Cone BC169 Codington County, South Dakota USA Pinus sylvestris Needle W242 Cantábrica Alavesa, Spain Pinus radiata Cone BC196 Polk County, Iowa USA Pinus nigra Needle W243 Arratia Nervión, Spain Pinus radiata Cone BC197 Lacrosse County, Wisconsin USA Pinus resinosa Needle W244 Arratia Nervión, Spain Pinus radiata Cone BC198 Wood County, Wisconsin USA Pinus resinosa Stem W245 Arratia Nervión, Spain Pinus radiata Cone BC199 Centre County, Pennsylvania USA Pinus nigra Needle W246 Arratia Nervión, Spain Pinus radiata Cone BC200 Upshur County, West Virginia USA Pinus sylvestris Needle W247 Duranguesado, Spain Pinus radiata Cone BC201 Lafayette County, Wisconsin USA Pinus resinosa Needle W248 Gran Bilbao, Spain Pinus radiata Cone BC202 Monroe County, Wisconsin USA Pinus banksiana Needle W250 Microorganisms 2021,9, 2565 8 of 17 Table 1. Cont. Origin Host Species Sample Type Sample ID Origin Host Species Sample Type Sample ID Plentzia-Munguia, Spain Pinus radiata Cone BC203 Cheboygan County, Michigan USA Pinus banksiana Needle W251 Plentzia-Munguia, Spain Pinus radiata Stem BC208 Manistee National Forest, Michigan USA Pinus resinosa Unknown W252 Plentzia-Munguia, Spain Pinus radiata Stem BC209 Jefferson County, West Virginia USA Pinus nigra Needle W253 P1 (Laukiniz), Spain Pinus radiata Cone BC2 Stanislaus National Forest, California USA Pinus ponderosa Unknown W254 P1 (Laukiniz), Spain Pinus radiata Cone BC3 Morgan County, West Virginia USA Pinus sylvestris Needle W255 P1 (Laukiniz), Spain Pinus radiata Cone BC4 Marion County, Indiana USA Pinus nigra Cone W256 P1 (Laukiniz), Spain Pinus radiata Cone BC5 Foxborough, Massachusetts, USA Pinus resinosa Cone U1 P1 (Laukiniz), Spain Pinus radiata Cone BC6 Washington, Vermont, USA Pinus resinosa Cone U2 P1 (Laukiniz), Spain Pinus radiata Cone BC7 Hancock County, Maine, USA Pinus resinosa (natural)Cone U3 P1 (Laukiniz), Spain Pinus radiata Cone BC8 Hancock County, Maine, USA Pinus resinosa (natural)Cone U4 P1 (Laukiniz), Spain Pinus radiata Cone BC9 Andover, Massachusetts, USA Pinus resinosa Cone U5 P1 (Laukiniz), Spain Pinus radiata Cone BC10 Hancock County, Maine, USA Pinus resinosa (natural)Cone U6 P1 (Laukiniz), Spain Pinus radiata Cone BC11 Washington, Vermont, USA Pinus resinosa Cone U7 P1 (Laukiniz), Spain Pinus radiata Core BCI1 Washington, Vermont, USA Pinus resinosa Cone U8 P1 (Laukiniz), Spain Pinus radiata Core BCI3 Andover, Massachusetts, USA Pinus resinosa Cone U9 P1 (Laukiniz), Spain Pinus radiata Core BCI4 Foxborough, Massachusetts, USA Pinus resinosa Cone U10 P1 (Laukiniz), Spain Pinus radiata Core BCI6 Shrewsbury, Vermont, USA Pinus resinosa Cone U11 P1 (Laukiniz), Spain Pinus radiata Core BCI7 Hancock County, Maine, USA Pinus resinosa (natural) Cone U12 P1 (Laukiniz), Spain Pinus radiata Core BCI8 Shrewsbury, Vermont, USA Pinus resinosa Cone U13 P1 (Laukiniz), Spain Pinus radiata Core BCI9 Hancock County, Maine, USA Pinus resinosa (natural) Cone U14 P1 (Laukiniz), Spain Pinus radiata Core BCI10 Hudson, Massachusetts, USA Pinus resinosa Cone U15 P1 (Laukiniz), Spain Pinus radiata Core BCI11 Shrewsbury, Vermont, USA Pinus resinosa Cone U16 P1 (Laukiniz), Spain Pinus radiata Core BCI12 Hancock County, Maine, USA Pinus resinosa (natural) Cone U17 P1 (Laukiniz), Spain Pinus radiata Core BCI13 Hancock County, Maine, USA Pinus resinosa (natural) Cone U18 P1 (Laukiniz), Spain Pinus radiata Core BCI14 Hancock County, Maine, USA Pinus resinosa (natural) Cone U19 P1 (Laukiniz), Spain Pinus radiata Core BCI15 Shrewsbury, Vermont, USA Pinus resinosa Cone U20 P1 (Laukiniz), Spain Pinus radiata Core BCI16 Hudson, Massachusetts, USA Pinus resinosa Cone U21 P1 (Laukiniz), Spain Pinus radiata Core BCI17 Washington, Vermont, USA Pinus resinosa Cone U22 P1 (Laukiniz), Spain Pinus radiata Core BCI18 Foxborough, Massachusetts, USA Pinus resinosa Cone U23 P1 (Laukiniz), Spain Pinus radiata Core BCI19 Hudson, Massachusetts, USA Pinus resinosa Cone U24 P1 (Laukiniz), Spain Pinus radiata Core BCI20 Hudson, Massachusetts, USA Pinus resinosa Cone U25 P1 (Laukiniz), Spain Pinus radiata Core BCI21 Washington, Vermont, USA Pinus resinosa Cone U26 P1 (Laukiniz), Spain Pinus radiata Core BCI22 Andover, Massachusetts, USA Pinus resinosa Cone U27 P1 (Laukiniz), Spain Pinus radiata Canker BCM161 Washington, Vermont, USA Pinus resinosa Cone U28 P1 (Laukiniz), Spain Pinus radiata Canker BCM165 Foxborough, Massachusetts, USA Pinus resinosa Cone U29 P1 (Laukiniz), Spain Pinus radiata Canker BCM167 Washington, Vermont, USA Pinus resinosa Cone U30 P1 (Laukiniz), Spain Pinus radiata Canker BCM168 Hancock County, Maine, USA Pinus resinosa (natural) Cone U31 P1 (Laukiniz), Spain Pinus radiata Canker BCM169 Hudson, Massachusetts, USA Pinus resinosa Cone U32 P1 (Laukiniz), Spain Pinus radiata Canker BCM170 Hancock County, Maine, USA Pinus resinosa (natural) Cone U33 P1 (Laukiniz), Spain Pinus radiata Canker BCM171 Andover, Massachusetts, USA Pinus resinosa Cone U34 Microorganisms 2021,9, 2565 9 of 17 Table 1. Cont. Origin Host Species Sample Type Sample ID Origin Host Species Sample Type Sample ID P1 (Laukiniz), Spain Pinus radiata Canker BCM172 Hancock County, Maine, USA Pinus resinosa (natural) Cone U35 P1 (Laukiniz), Spain Pinus radiata Canker BCM174 Andover, Massachusetts, USA Pinus resinosa Cone U36 P1 (Laukiniz), Spain Pinus radiata Root BCM182 Hancock County, Maine, USA Pinus resinosa (natural) Cone U37 P1 (Laukiniz), Spain Pinus radiata Canker BCM184 Hancock County, Maine, USA Pinus resinosa (natural) Cone U38 P1 (Laukiniz), Spain Pinus radiata Canker BCM185 Foxborough, Massachusetts, USA Pinus resinosa Cone U39 P1 (Laukiniz), Spain Pinus radiata Canker BCM186 Andover, Massachusetts, USA Pinus resinosa Cone U40 P1 (Laukiniz), Spain Pinus radiata Canker BCM188 Washington, Vermont, USA Pinus resinosa Cone U41 P1 (Laukiniz), Spain Pinus radiata Canker BCM189 Washington, Vermont, USA Pinus resinosa Cone U42 P1 (Laukiniz), Spain Pinus radiata Canker BCM190 Hancock County, Maine, USA Pinus resinosa (natural) Cone U43 P1 (Laukiniz), Spain Pinus radiata Canker BCM191 Hancock County, Maine, USA Pinus resinosa (natural) Cone U44 P1 (Laukiniz), Spain Pinus radiata Canker BCM192 Washington, Vermont, USA Pinus resinosa Cone U45 P1 (Laukiniz), Spain Pinus radiata Canker BCM193 Hudson, Massachusetts, USA Pinus resinosa Cone U46 P1 (Laukiniz), Spain Pinus radiata Canker BCM194 Washington, Vermont, USA Pinus resinosa Cone U47 P1 (Laukiniz), Spain Pinus radiata Canker BCM195 Washington, Vermont, USA Pinus resinosa Cone U48 P1 (Laukiniz), Spain Pinus radiata Canker BCM196 Shrewsbury, Vermont, USA Pinus resinosa Cone U49 P1 (Laukiniz), Spain Pinus radiata Canker BCM197 Shrewsbury, Vermont, USA Pinus resinosa Cone U50 3.2. PCR Amplification of SSR Loci and Data Analysis All primer pairs evaluated successfully amplified SSR loci for D. sapinea from the Basque Country and the USA. Eight loci (SS9, SS10, SS11, SS12, SS13, SS14, SS15 and SS16) were polymorphic for the 322 isolates genotyped. The number of observed alleles per locus ranged from two to nine (Table 1), resulting in a total of 48 MLGs (Table 2). The Basque Country population exhibited 19 MLGs, the USA population exhibited 34 MLGs, and both populations shared five MLGs (Figure 1). A clone correction of the dataset was performed to remove the bias of resampled MLG in the analysis, resulting in a total of 53 representative isolates. Table 2. Genetic diversity and linkage disequilibrium among loci based on the standardized index of association (rbarD) of Diplodia sapinea populations defined by country of origin, by nursery location in the Basque Country and by sample type a . Parametersb Country Nursery Location in the Basque Country Sample Type Spain (Basque Country) USA Laukiniz Sollano Hernani Luyando Oiarztun Canker Root Cone Core Sample size (N)c216 106 58 14 16 9 12 28 13 145 30 MLG/Diversity (G) 19 34 6 4 8 4 4 5 5 19 4 eMLG 19 19 6 4 8 4 4 5 5 10 4 Evenness (E5) 1 1 0.427 0.3 0.407 0.333 0.3 1 1 1 1 Diversity (H) 2.94 3.53 1.79 1.39 2.08 1.39 1.39 1.61 1.61 2.94 1.39 rbarD −0.074 0.087 − 0.080 − 0.316 − 0.066 − 0.115 − 0.333 − 0.056 − 0.194 − 0.074 − 0.115 p-value 0.999 0.001 a The set of nonredundant indices of genotypic diversity recommended by Arnaud-Haond et al. [ 66 ] was calculated for each population clone-corrected dataset. b MLG, number of multilocus genotypes observed; G, Stoddart and Taylor’s diversity [ 55 ] genotypic diversity; eMLG, expected multilocus genotypes based on rarefaction; E 5 , evenness index adapted from Simpson diversity; H, Shannon-Weiner diversity index [67]; rbarD, standardized index of association; p-value for rbarD. cSample size before clone correction. Microorganisms 2021,9, 2565 16 of 17 53. R Core Team. R: A Language and Environment for Statistical Computing; R Foundation for Statistical Computing: Vienna, Austria, 2017. 54. Kamvar, Z.N.; Brooks, J.C.; Grünwald, N.J. Novel R tools for analysis of genome-wide population genetic data with emphasis on clonality. Front. Genet. 2015,208, 6. [CrossRef] [PubMed] 55. Stoddart, J.A.; Taylor, J.F. Genotypic diversity: Estimation and prediction in samples. Genetics 1988 ,118, 705–711. [CrossRef] [PubMed] 56. Grünwald, N.J.; Goodwin, S.B.; Milgroom, M.G.; Fry, W.E. Analysis of genotypic diversity data for populations of microorganisms. Phytopathology 2003,93, 738–746. [CrossRef] 57. Agapow, P.M.; Burt, A. Indices of multilocus linkage disequilibrium. Mol. Ecol. Notes 2001,1, 101–102. [CrossRef] 58. Hedrick, P.W. A standardized genetic differentiation measure. Evolution 2005,59, 1633–1638. [CrossRef] 59. Archer, F.I.; Adams, P.E.; Schneiders, B.B. STRATAG: An R package for manipulating, summarizing and analysing population genetic data. Mol. Ecol. Resour. 2017,17, 5–11. [CrossRef] 60. Winter, D.J. MMOD: An R library for the calculation of population differentiation statistics. Mol. Ecol. Resour. 2012 ,12, 1158–1160. [CrossRef] 61. Jombart, T.; Devillard, S.; Balloux, F. Discriminant analysis of principal components: A new method for the analysis of genetically structured populations. BMC Genet. 2010,11, 94. [CrossRef] [PubMed] 62. Jombart, T. Adegenet: A R package for the multivariate analysis of genetic markers. Bioinformatics 2008 ,24, 1403–1405. [CrossRef] [PubMed] 63. Bruvo, R.; Michiels, N.K.; D’Souza, T.G.; Schulenburg, H. A simple method for the calculation of microsatellite genotype distances irrespective of ploidy level. Mol. Ecol. 2004,13, 2101–2106. [CrossRef] [PubMed] 64. White, T.J.; Bruns, T.; Lee, S.; Taylor, J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR Protocols: A Guide to Methods and Applications; Academic Press: San Diego, CA, USA, 1990; pp. 315–322. 65. Carbone, I.; Kohn, L. A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia 1999 ,91, 553–556. [CrossRef] 66. Arnaud-Haond, S.; Duarte, C.M.; Alberto, F.; Serrão, E.A. Standardizing methods to address clonality in population studies. Mol. Ecol. 2007,16, 5115–5139. [CrossRef] 67. Shannon, C.E. A mathematical theory of communication. Bell Syst. Tech. J. 1948,27, 379–423. [CrossRef] 68. Batista, E.; Lopes, A.; Alves, A. Botryosphaeriaceae species on forest trees in Portugal: Diversity, distribution and pathogenicity. Eur. J. Plant Pathol. 2020,158, 693–720. [CrossRef] 69. Burgess, T.I.; Gordon, T.R.; Wingfield, M.J.; Wingfield, B.D. Geographic isolation of Diplodia scrobiculata and its association with native P. radiata.Mycol. Res. 2004,108 Pt 12, 1399–1406. [CrossRef] [PubMed] 70. Morelet, M.; Chandelier, P. A case of variability in Sphaeropsis sapinea.Eur. J. Plant Pathol. 1993,23, 317–320. 71. Blodgett, J.T.; Stanosz, G.R. Differences in aggressiveness of Sphaeropsis sapinea RAPD marker group isolates on several conifers. Plant Dis. 1999,83, 853–856. [CrossRef] [PubMed] 72. Munck, I.A.; Stanosz, G. Quantification of Conidia of Diplodia spp. Extracted from Red and Jack Pine Cones. Plant Dis. 2009 ,93, 81–86. [CrossRef] [PubMed] 73. Munck, I.A.; Stanosz, G.R. Longevity of inoculum production by Diplodia pinea on red pine cones. For. Pathol. 2010 ,40, 58–63. [CrossRef] 74. Hanso, M.; Drenkhan, R. Diplodia pinea is a new pathogen on Austrian pine (P. nigra) in Estonia. Plant Pathol. 2009 ,58, 797. [CrossRef] 75. Oliva, J.; Boberg, J.; Stenlid, J. First report of Sphaeropsis sapinea on Scots pine (P. sylvestris) and Austrian pine (P. nigra) in Sweden. New Dis. Rep. 2013,27, 23. [CrossRef] 76. Müller, M.; Hantula, J.; Wingfield, M.; Drenkhan, R. Diplodia sapinea found on Scots pine in Finland. For. Pathol. 2018 ,49, e12483. [CrossRef] 77. Adamson, K.; Klavina, D.; Drenkhan, R.; Gaitnieks, T.; Hanso, M. Diplodia sapinea is colonizing the native scots pine (P. sylvestris) in the northern baltics. Eur. J. Plant Pathol. 2015,143, 343–350. [CrossRef] 78. Swart, W.J.; Wingfield, M.J. Biology and control of Sphaeropsis sapinea on P. species in South Africa. Plant Dis. 1991 ,75, 761–766. [CrossRef] 79. Santini, A.; Pepori, A.; Ghelardini, L.; Capretti, P. Persistence of some pine pathogens in coarse woody debris in a forest. For. Ecol. Manag. 2008,256, 502–506. [CrossRef] 80. Manzanos, T.; Stanosz GSmith DMuenchow, J.; Schratz, P.; Brenning, A.; Aragones, A.; Iturritxa, E. Mating type ratios and pathogenicity in Diplodia shoot blight fungi populations: Comparative analysis. Eur. J. For. Pathol. 2018,49, e12475. [CrossRef] 81. Taherdoost, H. Sampling Methods in Research Methodology, How to Choose a Sampling Technique for Research. Int. J. Acad. Res. Manag. 2016,5, 18–27. [CrossRef] 82. Brodde, L.; Adamson, K.; Julio Camarero, J.; Castaño, C.; Drenkhan, R.; Lehtijärvi, A.; Luchi, N.; Migliorini, D.; Sánchez-Miranda, Á.; Stenlid, J.; et al. Diplodia tip blight on its way to the North: Drivers of disease emergence in Northern Europe. Front. Plant Sci. 2019,9, 1818. [CrossRef] 83. Burgess, T.I.; Wingfield, M.J.; Wingfield, B.D. Global distribution of Diplodia pinea genotypes revealed using simple sequence repeat (SSR) markers. Australas. Plant Pathol. 2004,33, 513–519. [CrossRef] Microorganisms 2021,9, 2565 17 of 17 84. Do˘gmu¸s-Lehtijärvi, T.; Aday, G.; Lehtijärvi, A.; Oskay, F.; Kaya, Ö.D. Occurrence and Genetic Similarity of Diplodia pinea on Shoots and Cones in Seed Orchards of P. spp. in North-Western Turkey. Plant Prot. Sci. 2014,50, 217–220. [CrossRef] 85. Monat, J.P. The benefits of global scaling in multi-criteria decision analysis. Judgm. Decis. Mak. 2009,4, 492–508. 86. Ramos, P.; Petisco, E.; Martín, J.M.; Rodríguez, E. Downscaled climate change projections over Spain: Application to water resources. Int. J. Water Resour. Dev. 2012,29, 201–218. [CrossRef] 87. Bachi, P.R.; Peterson, J.L. Emhancement of Sphaeropsis sapinea stem invasion of pines by water deficits. Plant Dis. 1985 ,69, 798–799.