Intraspecific variation in susceptibility to dothistroma needle blight within native Scottish Pinus sylvestris

Dothistroma needle blight (DNB), caused by Dothistroma septosporum, is the most important disease currently affecting pine plantations in Britain. Intraspecific variation in susceptibility to DNB has been observed in several pine species, but it is not clear if similar variation occurs in Pinus sylvestris (Scots pine), Britain's only native pine. In three separate experiments 2‐ and 3‐year‐old Scots pine saplings from six native Scottish populations were artificially inoculated with D. septosporum conidial suspensions and incubated under conditions optimal for disease development. Conidial suspensions were produced using a single isolate from northeast Scotland. In one experiment, plants were also treated with various spore suspension concentrations to assess the impact of inoculum load on disease severity. There were no significant interactions between host population, plant height, and experiment/inoculum load (anova , P > 0·05), but population, height and inoculum load all significantly affected disease severity (anova , P < 0·05). Among the 2‐year‐old trees, those from Amat were less susceptible than those from Glen Loyne and Glen Cannich (anova , P < 0·05). Among the 3‐year‐old trees, those from Beinn Eighe were less susceptible than those from Abernethy. Plant height and DNB susceptibility had a slightly negative relationship. The use of a spore suspension with a concentration of 1·6 × 10⁶ spores mL^?1 was optimum for disease development. In an in vitro experiment, production of conidia was greater when cultures were incubated in darkness. This paper is the first to report intraspecific variation in DNB susceptibility within Scots pine.     

Between‐site and ‐year variation in the relative susceptibility of native Scottish Pinus sylvestris populations to dothistroma needle blight

Dothistroma needle blight (DNB), caused by Dothistroma septosporum, is currently the disease causing most concern in British pine plantations. Previous artificial inoculation (AI) experiments showed that native Scottish Scots pine (Pinus sylvestris) populations vary in susceptibility to DNB. However, it is unclear if the relative susceptibility of Scots pine populations observed in these experiments can be replicated under natural conditions. It is also unknown whether relative susceptibility of Scots pine populations varies between sites or years. To answer these two questions, young Scots pine plants from six native Scottish populations (Abernethy, Allt Broighleachan, Amat, Beinn Eighe, Glen Cannich and Glen Loyne) were exposed to natural D. septosporum inoculum at two Scottish sites (Culbin and Torrs Warren) between 2012 and 2014. DNB disease incidence and severity was assessed each October. Relative susceptibilities of the Scots pine populations varied between sites and across years. In two of the three years at Torrs Warren (2012 and 2014), the relative susceptibilities of the populations were strongly positively correlated with those observed in previous AI experiments. In these years, trees from Glen Loyne and Glen Cannich were the most susceptible. Conversely, there was no correlation between the relative susceptibilities seen in any year at Culbin with those observed in AI experiments. At Culbin, Beinn Eighe was the most susceptible population. Across both sites, there was a strong positive relationship between total summer precipitation and DNB severity (R = 0·93, t = 8·2, P = 0·001).     

Dothistroma needle blight: an emerging epidemic caused by Dothistroma septosporum in Colombia

Plantation forestry in Colombia is based mainly on non‐native species of Pinus and Eucalyptus. Since 2008, a disease with symptoms similar to those of dothistroma needle blight (DNB) has been found affecting large areas planted to Pinus spp. The aim of this study was to identify the causal pathogen as well as to document the levels of disease incidence and severity. Isolates from each of three forestry zones, collected from different host species, were compared based on rDNA sequence of the ITS regions. These were conclusively identified as Dothistroma septosporum, one of two Dothistroma spp. known to cause DNB. Susceptibility was greatest on low elevation Pinus tecunumanii followed by Pinus kesiya and Pinus oocarpa. Pinus maximinoi and high elevation P. tecunumanii showed tolerance to D. septosporum. The disease incidence in the different zones varied significantly with the North zone being the most severely affected. This constitutes the first report of disease distribution and susceptibility of hosts, as well as the first consideration of the relative importance of D. septosporum in Colombia.     

Aleppo pine provenances vary in susceptibility and secondary chemical response to Gremmeniella abietina infection

Aleppo pine is a thermophilic species that has become a popular tree species in plantings even beyond its natural habitat. In 1999 the pathogenic fungus Gremmeniella abietina was isolated for the first time in Aleppo pine plantations in Spain. The main objective of this study was to analyse the variation in susceptibility to G. abietina (European race, biotype A) infections among trees from five Aleppo pine provenances. Artificial inoculations were performed at two different times in January 2012. The susceptibility of the trees was evaluated by recording disease severity and the length of internal stem necrosis produced by the pathogen. In addition, the concentrations of two flavanone compounds were quantified as putative indicators of resistance. Provenances and G. abietina isolates both significantly affected levels of necrosis although the results were dependent on the time of inoculation. There was a relationship between provenance and resistance, with trees of higher elevation provenances being more susceptible to disease than trees of lower elevation provenances. The amount of naringenin flavanone from trees was significantly different between provenances, suggesting that it is a possible indicator value for resistance. Conversely, no differences were found in the concentration of eriodictyol in trees between provenances. The results show that the provenance‐dependent variation in the susceptibility of Aleppo pine to G. abietina should be considered in selection of seed sources for restoration.     

Dothistromin toxin is a virulence factor in dothistroma needle blight of pines

Dothistromin is a broad‐spectrum mycotoxin produced by the Dothideomycete pine needle pathogen Dothistroma septosporum. It accumulates in lesions, causing characteristic red bands on needles infected with this fungus. Dothistromin is similar in structure to the aflatoxin precursor versicolorin B and the biosynthetic pathways of these toxins share many common gene products. Although dothistromin is not essential for pathogenicity in dothistroma needle blight, its presence in infected needles suggests it might have a role in the disease process. The hypothesis that dothistromin is a virulence factor was tested by studying Pinus radiata infected with dothistromin‐deficient mutants of D. septosporum. The mutants were able to infect pine needles and complete their life cycle as previously shown, and were unaffected in spore germination, epiphytic growth or needle penetration. However, colonization of the mesophyll by the mutants was restricted compared to the wild type. Correspondingly, lesions produced by the mutants were smaller and produced significantly fewer spores than lesions produced by wildtype strains. Interestingly, ‘green islands’, in which chlorophyll was maintained at a higher level than in adjacent chlorotic and necrotic regions, surrounded early‐appearing lesions caused by both wildtype and mutant strains. At a later stage of disease development green islands were still present in the mutant but appeared to be masked by the extended dothistromin‐containing lesions in the wild type. Overall the results support a role for dothistromin in virulence in dothistroma needle blight.     

Alternative species to replace Monterey pine plantations affected by pitch canker caused by Fusarium circinatum in northern Spain

The pitch canker pathogen Fusarium circinatum was first found to cause damage in nurseries and pine plantations in northern Spain in 2004. Since then, establishment of pine plantations in the region has decreased as a result of the prohibitions placed on planting Pinus spp. and Pseudotsuga menziesii in areas affected by the disease. However, although most pine species have been found to be susceptible to the pathogen under nursery conditions, little is known about how the fungus affects the trees in the field. Furthermore, it is not known whether some of the native or exotic species commonly planted in the area are also susceptible to F. circinatum. The aim of this study was to evaluate the susceptibility of several conifer species commonly planted in northern Spain to the pitch canker pathogen. For this purpose, two different trials were carried out, one under controlled laboratory conditions and the other in the field. Although most of the conifers were affected by the pathogen in the laboratory tests, only Pinus radiata, Pinus nigra, Pinus pinaster and Pinus uncinata were susceptible to the pathogen in the field.     

Distribution and characterization of Dothistroma needle blight pathogens on <Emphasis Type="Italic">Pinus mugo</Emphasis> in Slovakia

The occurrence and distribution of Dothistroma needle blight (DNB) on Pinus mugo was studied in 2014–2015 around the Slovakia. In total, 42 localities were investigated both native and planted ones. Symptoms of DNB were observed on 35 localities only on planted shrubs. All these 35 localities are new P. mugo DNB stands. No DNB symptoms were observed in natural and naturally regenerated plantations. DNA was extracted from a total of 236 isolates and eight needle samples. Based on the ITS-rDNA comparisons and using species specific primers, both pathogenic Dothistroma species were detected: D. septosporum and D. pini. Isolates of D. septosporum had ITS sequences identical to D. septosporum from Europe and both mating types were identified with slight predominance of MAT2. The ratio of D. septosporum mating types varies significantly between sites, ranging from an equal proportion of each mating type to single mating type populations. D. pini ITS sequence grouped with D. pini from Ukraine, Russia and Switzerland and only MAT2 was found.     

Distribution and genetic diversity of Dothistroma septosporum in Pinus brutia forests of south-western Turkey

Dothistroma needle blight (DNB) is a serious disease of the Pinaceae, mainly Pinus species, caused by the fungi Dothistroma septosporum and D. pini. Both species are regarded as invasive forest pathogens worldwide, with rising incidence in central and northern Europe over the last three decades. In this work, 29 sites were investigated between 2013 and 2015 in south-western Turkey. Morphological examination of needles confirmed DNB infection (i.e., Dothistroma conidiospores observed) at 18 sites, and a total of 108 Dothistroma sp. isolates were obtained from 11 of the sites. Host age seemed to be an important factor in both occurrence and severity of DNB in Pinus brutia forests. Continuous rainy days, especially in December, may increase severity of disease; however, extreme rain events may reduce available conidiospores on plant tissues or in the air. Species-specific mating type primers showed that all isolates were D. septosporum; D. pini was not detected. The mating type ratio was close to 1:1, indicating sexual recombination was occurring. Eleven microsatellite markers revealed 59 unique multilocus haplotypes (MLHs) among the 73 isolates originating from different conidiomata. The majority of MLHs were represented by a single isolate (n = 52) and only one MLH was shared between two localities. Analyses showed high genetic diversity, isolation-by-distance, and clear population clusters. These findings suggest that D. septosporum is well established in south-western Turkey and is probably not a recent introduction.     

The gene flow and mode of reproduction of Dothistroma septosporum in the Czech Republic

Since 1911, dothistroma needle blight, caused by Dothistroma septosporum, has been recorded in most European countries. In the Czech Republic, the fungus has become an important disease of pines since 2000, especially Austrian pines in plantations of Christmas and ornamental trees. The aim of this study was to analyse the population structure, gene flow and mode of reproduction of this pathogen. Microsatellite and mating‐type markers were analysed in a Dothistroma population in the southeastern part of the country using reference isolates from other European countries. The haplotypic diversity was high, with 87 unique and 13 shared haplotypes (probable clones) identified in 121 samples. Based on structure analysis, the isolates were divided into two populations, with an uneven distribution over the sampling sites. The grouping of the sites to the populations did not follow a geographical pattern because certain isolates that were sympatrically co‐occurring at the same site were placed in different populations. Tests for random mating (the index of association and a parsimony tree‐length permutation test) showed a significant clonal mode of reproduction in most cases, but the intrapopulation haplotypic diversity is unexpectedly high. Although a teleomorphic stage of D. septosporum has not been previously observed in the Czech Republic, the high intrapopulation haplotypic diversity can be explained by infrequent sexual reproduction consistent with the occurrence of both mating types.     

A reconsideration of natural dispersal distances of the pine pathogen Dothistroma septosporum

The natural spread of Dothistroma septosporum, the causal agent of a foliar disease of pines, was investigated at three sites in the south of England using trap plants. The pathogen is considered to be primarily rain‐splash dispersed, but this study shows that it can be spread many hundreds of metres from an inoculum source, demonstrating that dispersal is not solely via rain splash. The maximum distance the pathogen was recorded from any infection source was in excess of 1400 m, over five times the distance defined in the only previous work of this kind. Consequently, a reassessment of forest and production nursery management practices is called for, as these assume that the pathogen only spreads naturally over limited distances. Detection of the pathogen on trap plants over 100 m from the inoculum source was, in most cases, only possible using quantitative real‐time PCR diagnosis. The entire diagnostic procedure, from DNA extraction to amplification, was able to detect a minimum of approximately 17 D. septosporum cells in a pine needle sample, assuming only a moderate DNA extraction efficiency of 30%.     

Genetic diversity of Dothistroma septosporum in Estonia, Finland and Czech Republic

Dothistroma needle blight is one of the most damaging foliage diseases in pine plantations worldwide. Recently it has become more aggressive in native pine stands in northern America and has been found frequently on Scots pine stands in northern Europe. In Estonia and Finland it was noticed for the first time in 2006 and 2008, respectively and in Central Europe in the late 1990s. We show considerable diversity in allele patterns of several microsatellite loci in populations of these countries which does not support the hypothesis of a recent introduction. We investigated 104 isolates by using eight microsatellite loci. Estonian and Finnish isolates originated from P. sylvestris and those from the Czech Republic from six species of Pinus spp. and Pseudotsuga menziesii. The genetic diversity was considerable in all three populations, and did not differ significantly between populations. The results suggest slight migration from south to north, even if no similar haplotypes were found between any of the populations. Both, the pairwise genetic differentiation and Nei’s genetic distance reflected geographic distances between the populations. Differentiation between the studied populations of D. septosporum was low but statistically significant. Only 6 % of the genetic variation was due to differences between populations. The high haplotypic diversity, low number of identical haplotypes, and low degree of genetic disequilibrium in all investigated populations suggested occurrence of sexual proliferation in this area, although the sexual state of the fungus has not been recorded in Estonia and Finland. The high diversity may suggest a long presence of D. septosporum in northern Europe, or alternatively, its recent introduction as a massive inoculum from an unknown direction.     

Screening Corymbia populations for resistance to Puccinia psidii

The exotic rust pathogen Puccinia psidii is now widespread along the east coast of Australia from temperate Victoria to tropical far north Queensland, with a current host range exceeding 200 species from 37 myrtaceous genera. To determine the threat P. psidii poses to plantation and native eucalypts, artificial inoculation was used to screen germplasm of spotted gum (Corymbia spp.) for resistance to the biotype of P. psidii that has become established in Australia. The objective was to characterize resistance to P. psidii within the Corymbia species complex so that management strategies for the deployment of germplasm from existing breeding programmes of these spotted gum species could be developed. Symptom development initiated 7 days after inoculation, with resistant and susceptible seedlings identified within all species, provenances and families. Inter‐ and intraspecific variability in rust resistance was observed among spotted gum species. There was no apparent relationship between climatic conditions at the provenance origin and disease resistance. The heritability estimates for all assessments are moderate to high and indicate a significant level of additive genetic variance for rust resistance within the populations. The results of this study clearly identify potential to select for resistance at the family level within the tested populations. While the potential for P. psidii to detrimentally impact upon Corymbia in the nursery and in young plantations was demonstrated, estimations of the heritability of resistance suggest that efforts to enhance this trait through breeding have reasonable prospects for success.     

Risk assessment for Puccinia psidii becoming established in South Africa

Puccinia psidii, the causal agent of myrtle rust, was first recorded from Latin America more than 100 years ago. It occurs on many native species of Myrtaceae in Latin America and also infects non‐native plantation‐grown Eucalyptus species in the region. The pathogen has gradually spread to new areas including Australia and most recently South Africa. The aim of this study was to consider the susceptibility of selected Eucalyptus genotypes, particularly those of interest to South African forestry, to infection by P. psidii. In addition, risk maps were compiled based on suitable climatic conditions and the occurrence of potential susceptible tree species. This made it possible to identify the season when P. psidii would be most likely to infect and to define the geographic areas where the rust disease would be most likely to establish in South Africa. As expected, variation in susceptibility was observed between eucalypt genotypes tested. Importantly, species commonly planted in South Africa show good potential for yielding disease‐tolerant material for future planting. Myrtle rust is predicted to be more common in spring and summer. Coastal areas, as well as areas in South Africa with subtropical climates, are more conducive to outbreaks of the pathogen.     

Differences in constitutive and inducible defences in pine species determining susceptibility to pinewood nematode

The pinewood nematode, Bursaphelenchus xylophilus, originating from North America (NA), is a major invasive pine pest in Eurasia. It was first detected in Portugal in 1999 associated with maritime pine, Pinus pinaster, and has been differently affecting the main local pine species, P. pinaster and P. pinea. Field studies and direct inoculation experiments in Pinus spp. seedlings, under controlled conditions, were performed to assess whether the differences in constitutive and inducible defences are determining the different susceptibility of pine host species to B. xylophilus. Host co‐evolution with the pathogen was also assessed, including the NA P. radiata, widely used in forestry in the northeast of the Iberian peninsula. Pine mortality in the field was positively related with the abundance of B. xylophilus, and concentration of phenolics and condensed tannins in pines. In the greenhouse assay, seedling tissues were analysed for constitutive investment in defences, as well as the potential inducibility of those defences as driven by B. xylophilus inoculation. Slower growing P. pinea presented higher levels of constitutive defences than faster growing P. pinaster, with only P. pinaster being affected by B. xylophilus. Furthermore, co‐evolution with the pathogen is important, with the fast‐growing NA P. radiata presenting an inducible and effective response to B. xylophilus. Results point to the importance of integrating data on pine life history traits, including growth rate, and production of constitutive and inducible defences, into predictive models for this invasive forest pest.     

Occurrence and ITS diversity of wood‐associated Bursaphelenchus nematodes in Scots pine forests in Switzerland

The quarantine pathogen Bursaphelenchus xylophilus (pine wood nematode, PWN) represents a serious threat for Pinus species in Europe. To exclude its presence in Switzerland, in 2010 and 2011 a countrywide survey was conducted in 102 Pinus sylvestris stands, chosen according to whether they contained dying or dead trees or were located in areas at risk of PWN introduction. In total, 285 trees (1–5 per site) were sampled. Nematodes were extracted from wood chips using a standard procedure, and identified to species by internal transcribed spacer (ITS) sequencing. Bursaphelenchus species were present in 34% of the trees, but no B. xylophilus was identified, i.e. PWN is still not present in Switzerland. The nematodes found belonged to seven different species, with B. vallesianus the most frequent species, followed by B. sexdentati, B. mucronatus kolymensis and B. eggersi. Three other species (B. borealis, B. pinophilus, B. poligraphi) were each only present in one or two trees. Three groups of sequences could not be assigned to a species because of the lack of matching reference sequences. The species composition found in Switzerland suggests co‐existence of southern and central European Bursaphelenchus species. Intraspecific ITS variability differed considerably among the four most common species. Bursaphelenchus eggersi, B. mucronatus kolymensis and B. sexdentati had several variable sites in the ITS region, resulting in multiple ITS genotypes in each species. In contrast, all 99 B. vallesianus isolates had an identical ITS region. This could indicate a founder effect, and possibly that B. vallesianus is not native to the Alpine region.     

Population structure of Dothistroma septosporum in Poland: revealing the genetic signature of a recently established pathogen

The incidence and geographic range of Dothistroma septosporum, a serious foliar pathogen of pine, has increased dramatically in central Europe in the last three decades. In Poland, it was first detected in 1990, and by the mid-2010s became established throughout the country, meaning the population of D. septosporum in Poland is relatively young. In this study we analysed site populations of the pathogen in Poland using highly variable microsatellite markers in order to identify the genetic signatures of a recent migration and to track the mode of its introduction. As a result, we found that in Poland the population of D. septosporum is highly structured, the strongest division occurring between a portion of isolates from south-central Poland, including the original outbreak site, and the isolates from the rest of the country. Further genetic structure allowed identification of population clusters specific to particular regions of Poland. Random mating tests show that some population groups have a much lower tendency to reproduce sexually than others, a factor that helps maintain the strong population structure observed. Such a pattern of diversity indicates multiple independent introduction events/migration waves: a single introduction, probably human-mediated, at the original outbreak site, and at least two waves representing a general shift in the distribution of D. septosporum as a consequence of natural spread, resulting in most of the local populations having similar diversity parameters.     

Fungal Infection and Mechanical Wounding Induce Disease Resistance in Scots Pine

Scots pine trees (Pinus sylvestris) recovering from a 90–100% defoliation 2–3 years previously were pretreated with small mechanical wounds or inoculations with the blue-stain fungi Leptographium wingfieldii and Ophiostoma canum. Pretreated trees were less susceptible to a subsequent massive inoculation with L. wingfieldii than untreated control trees, which were extensively colonised by the mass-inoculation. A low pretreatment dosage of L. wingfieldii was somewhat more effective in inducing disease resistance than a higher dosage. Pretreatment with L. wingfieldii, O. canum, and mechanical wounding were about equally effective inducers of resistance in Scots pine, even though L. wingfieldii is known to produce much more extensive phloem necrosis than the other pretreatments. Thus, the strength of the induced resistance response did not depend on the amount of host tissues that was destroyed by the pretreatment. Previously, induced disease resistance has been demonstrated in Norway spruce (Picea abies), and the present study shows that similar responses can be activated in Scots pine.     

Impacts of exotic forest pathogens on Mediterranean ecosystems: four case studies

Mediterranean ecosystems are hotspots of biodiversity. Because of a coincidence of high species richness and human presence, Mediterranean biodiversity is particularly threatened by processes such as habitat degradation, fragmentation and loss, pollution, climate change and introduction of invasive species. Invasive tree pathogens are among the problematic exotic species of California, Chile, the Mediterranean, South Africa and Australia. In this review, we provide an update on a selection of non-native tree pathogens currently posing a threat in Mediterranean ecosystems. The impact of exotic forest pathogens range from large-scale tree and shrub mortality in native ecosystems (Phytophthora ramorum on the West Coast of the USA) to disruption of plantations of exotic (e.g., Seiridium cardinale on planted Monterey cypress in California, Fusarium circinatum on Monterey pine worldwide) and native trees (introduction of the North American Heterobasidion irregulare in stone pine woodland in Italy). Genetic analyses are instrumental in improving our understanding and management of these outbreaks. There is a need for more empirical data on how novel pathosystems are likely to develop under novel climates, as well as interdisciplinary collaborations among forest pathologists, theoretical modellers and climatologists. The magnitude of the observed effects of some exotic tree diseases makes it important to try and minimize the risk of the inadvertent movement of plant pathogens when planning assisted migration activities to enable plant species to cope with rapid climate change.     

Pinewood nematode population growth in relation to pine phloem chemical composition

The pinewood nematode, Bursaphelenchus xylophilus, native to North America (NA), is now a major invasive pine pest in Eurasia. Pine species differ greatly in their susceptibility to B. xylophilus, and differences in susceptibility might be related to differences in phytochemistry. This study addressed two major questions: (i) which plant chemical profiles (including compounds that act as plant defences and those important for nutrition) favour pinewood nematode development; and (ii) are NA and Portuguese pinewood nematodes different in their capacity to subsist on plant tissues? For this, B. xylophilus isolates from NA and Portugal were tested on pine phloem and xylem of different pine species. It was found that unaltered pine phloem can sustain the population growth of B. xylophilus, but only in a few cases, and this was clearly related to distinct chemical profiles in some pine species. No evidence was found of evolutionary divergence between B. xylophilus populations in Portugal and NA. The pine species with phloem on which pinewood nematodes could grow tended to have low levels of total phenols, condensed tannins, flavonoids and lignin. Evidence was also found that nutrients such as N may be important.     

Quantitative trait loci associated with resistance to a potato isolate of Verticillium albo‐atrum in Medicago truncatula

Verticillium albo‐atrum is responsible for considerable yield losses in many economically important crops, among them alfalfa (Medicago sativa). Using Medicago truncatula as a model for studying resistance and susceptibility to V. albo‐atrum, previous work has identified genetic variability and major resistance quantitative trait loci (QTLs) to Verticillium. In order to study the genetic control of resistance to a non‐legume isolate of this pathogen, a population of recombinant inbred lines (RILs) from a cross between resistant line F83005.5 and susceptible line A17 was inoculated with a potato isolate of V. albo‐atrum, LPP0323. High genetic variability and transgressive segregation for resistance to LPP0323 were observed among RILs. Heritabilites were found to be 0·63 for area under the disease progress curve (AUDPC) and 0·93 for maximum symptom score (MSS). A set of four QTLs associated with resistance towards LPP0323 was detected for the parameters MSS and AUDPC. The phenotypic variance explained by each QTL (R²) was moderate, ranging from 4 to 21%. Additive gene effects showed that favourable alleles for resistance all came from the resistant parent. The four QTLs are distinct from those described for an alfalfa V. albo‐atrum isolate, confirming the existence of several resistance mechanisms in this species. None of the QTLs co‐localized with regions involved in resistance against other pathogens in M. truncatula.