Lophodermium Piceae Research Articles

Palatability of Norway spruce needles infected with Lophodermium piceae to larvae of two sawfly species

Unequivocal evidence on the antagonistic effects of endophytic fungi associated with woody plants against insect herbivores has been documented in only a few cases so far. Experimental evidence of the significance of needle endophytes to coniferous trees has remained scant because it is difficult to obtain trees with needles free of endophytes that could be used as comparable controls for trees infected with endophytes. Previously we reported a new methodology to get Norway spruce (Picea abies (L.) Karst.) saplings without needle endophytes and to inoculate them with a needle endophyte Lophodermium piceae (Fuckel) Höhn. Here we describe the first trial where spruce saplings with and without needle endophytes were provided as substrate for insect larvae. We transferred larvae of two sawfly species, Neodiprion sertifer Geoffroy and Gilpina pallida Klug, to the seedlings. Even though the main host of these sawfly species is not Norway spruce, but Scots pine (Pinus sylvestris L.), they are also known to occasionally feed on spruce. In this experiment the larvae did not develop to pupae with the provided spruce substrate but consumed measurable amounts of needles. No significant difference was found between the extent of needles consumed by either of the two sawfly species in inoculated and uninoculated saplings.

Cloning and Characterization of Two Novel PR4 Genes from Picea asperata

Pathogenesis-related (PR) proteins are important in plant pathogenic resistance and comprise 17 families, including the PR4 family, with antifungal and anti-pathogenic functions. PR4 proteins contain a C-terminal Barwin domain and are divided into Classes I and II based on the presence of an N-terminal chitin-binding domain (CBD). This study is the first to isolate two PR4 genes, PaPR4-a and PaPR4-b, from Picea asperata, encoding PaPR4-a and PaPR4-b, respectively. Sequence analyses suggested that they were Class II proteins, owing to the presence of an N-terminal signal peptide and a C-terminal Barwin domain, but no CBD. Tertiary structure analyses using the Barwin-like protein of papaya as a template revealed structural similarity, and therefore, functional similarity between the proteins. Predictive results revealed an N-terminal transmembrane domain, and subcellular localization studies confirmed its location on cell membrane and nuclei. Real-time quantitative PCR (RT-qPCR) demonstrated that PaPR4-a and PaPR4-b expression levels were upregulated following infection with Lophodermium piceae. Additionally, PaPR4-a and PaPR4-b were induced in Escherichia coli, where the recombinant proteins existed in inclusion bodies. The renatured purified proteins showed antifungal activity. Furthermore, transgenic tobacco overexpressing PaPR4-a and PaPR4-b exhibited improved resistance to fungal infection. The study can provide a basis for further molecular mechanistic insights into PR4-induced defense responses.

The Large-Scale Investigation and Analysis of Lophodermium piceae in Subalpine Areas Based on Satellite Multi-Spectral Remote Sensing

Picea asperata, a common tree species in the subalpine areas of Li County, Sichuan Province, China, is susceptible to Lophodermium piceae. Remote sensing has the advantages of large-scale, fast information acquisition, and low cost, which can overcome the shortcomings of ground survey. Hence, we used Landsat 8 satellite multi-spectral images and forest resource distribution data to investigate and analyze this forest disease at a large scale. Firstly, we extracted the spatial distribution information of Picea asperata and chose a temporal sequence indicator to establish a regression model and obtained a significantly negative correlation between the damage degree of plants and the change rate of normalized difference vegetation index (NDVI). Accordingly, the investigation results of the disease have good consistency with the ground survey data in spatial distribution and damage degree. On this basis, a temporal regression analysis was performed by combining the remote sensing investigation results with climate variables, and canonical correspondence analysis (CCA) was utilized in the spatial comprehensive analysis of Lophodermium piceae with terrain, soil and forest stand factors. Conclusively, this study effectively coped with the difficulties in full investigation and analysis of Lophodermium piceae in ecologically fragile subalpine areas of Western Sichuan. It is of important reference value in the early warning and monitoring of this disease, and also provides objective and reliable information support for ecological restoration and management planning in the Wenchuan earthquake-stricken areas.

Molecular Identification and Antifungal Properties of Four Thaumatin-like Proteins in Spruce (Picea likiangensis)

Thaumatin-like proteins (TLPs) are involved in the plant defense response against pathogens, and most of them exhibit antifungal activity. However, the role of TLPs in pathogen-induced defense responses in spruce is not fully understood. In this study, four TLP genes encoding thaumatin-like protein, designated as PlTLP1–4, were isolated and identified from Picea likiangensis needles. Sequence analysis showed that PlTLP1, PlTLP3, and PlTLP4 contained 16 conserved cysteine residues, while PlTLP2 had only 10 conserved cysteine residues. qPCR analysis showed that PlTLPs were expressed in all tissues tested, PlTLP1, PlTLP3, and PlTLP4 had the highest expression levels in young fruits, while PlTLP2 had the highest expression levels in roots. In addition, the expression levels of four PlTLPs were significantly upregulated during infection by Lophodermium piceae. Four recombinant PlTLPs expressed in Escherichia coli exhibited obvious β-1,3-glucanase activity. The antifungal activity assay showed that four recombinant PlTLPs had significant inhibitory effects on the mycelial growth of L. piceae, Fusarium proliferatum, Botrytis cinerea, and Roussoella doimaesalongensis. Microscopic observation revealed that the recombinant PlTLP1–4 induced the morphological changes of the mycelia of L. piceae, and the recombinant PlTLP2 and PlTLP3 induced the morphological changes of the mycelia of F. proliferatum and R. doimaesalongensis, while all the recombinant PlTLPs had no obvious negative effect on the morphology of B. cinerea mycelium. These results suggest that PlTLP genes may play an important role in the defense response of P. likiangensis against L. piceae invasion.

Eradication of endophytes from needles, their inoculation with Lophodermium piceae and persistence of this fungus in needles of Norway spruce

Lophodermium piceae is the most common endophyte of Norway spruce (Picea abies) needles, and it probably occurs in the total distribution area of Norway spruce. Its significance to the host tree is still largely unknown mainly because no methodology is known for generating endophyte-free saplings nor for inoculation of intact needles with this fungus. We report here how a daily heat treatment at 40 °C for 8 h eradicates endophytes of Norway spruce needles within a few days without causing visible harm to the plants. We compared various methods for inoculation of endophyte-free saplings. In order to obtain effective inocula, we investigated what factors activate dormant L. piceae infections in the needle tissue resulting in ascomata formation. Best inoculation success was achieved by suspending naturally shed, highly infected needles above the saplings. Infection frequencies of up to 70% of needles were achieved with repeated inoculations in the course of 3 years. Once established in needles, individual infections by L. piceae can persist for at least 5 years. Individual infections remain small in young needles, but at a needle age of 6 years, they start to grow and spread resulting in presence all over the needle from base to tip at a needle age of 7 years, but still without causing visible symptoms on the needles. The presented methodology for generating both endophyte-free and endophyte-infected saplings of Norway spruce provides a new approach for testing the significance of infections by L. piceae to insect herbivory and diseases of Norway spruce needles.

Molecular Identification and Antifungal Activity of a Defensin (PaDef) from Spruce

Defensins, as part of the PR protein family, not only have antimicrobial activity in vitro, but also play an important role in plant disease resistance. To explore the molecular function of defensin in spruce, a defensin gene PaDef was isolated and characterized from Picea asperata. The full-length cDNA sequence of the PaDef was 264 bp and contained an ORF of 252 bp, encoding a total of 83 amino acids. The deduced PaDef protein belongs to the gamma-thionin family and contains the Knot1 domain and eight conserved cysteine residues. Blast and phylogenetic analysis showed that the PaDef protein shared high homology with other plant defensin proteins. The tertiary structure of PaDef was obtained by homologous modelling using the structure of Pinus sylvestris defensin as a template and exhibited a βαββ structure. SDS-PAGE analysis showed that the molecular mass of recombinant PaDef expressed in Escherichia coli was 23.8 kDa. Plate assay showed that purified PaDef exhibited strong antifungal activity inhibiting the growth of Pestalotiopsis neolitseae, but not Colletotrichum gloeosporioides and Botrytis cinerea. Microscopic observation revealed that purified PaDef had a negative effect on the hyphal morphology of all three phytopathogens. qRT-PCR analysis indicated that the PaDef gene had the highest expression level in needles of P. asperata compared with roots, phloem, twigs, and could be significantly upregulated by the pathogenic fungus Lophodermium piceae, which causes needle cast disease. These results provide a molecular basis for determining the molecular function of defensin PaDef in P. asperata and its potential as an antifungal agent.

Adaptation of subpopulations of the Norway spruce needle endophyte Lophodermium piceae to the temperature regime

Lophodermium piceae represents the most common Norway spruce needle endophyte. The aim of this study was to find out whether subpopulations of L. piceae in climatically different environments (in which Norway spruce occurs natively) are adapted to local thermal conditions. L. piceae’s ability for thermal adaptation was investigated by determining growth rates of 163 isolates in vitro at four different temperatures: 2, 6, 20 and 25 °C. Isolates were obtained between 1995 and 2010 from apparently healthy needles sampled in Finland, Poland, Switzerland, Italy and southeastern Siberia. The sampling sites represent seven climatically distinct locations. Results were evaluated in relation to the age and geographic origin of the isolate, in addition to the highest and lowest average monthly temperature of the sampling location. We found a significant correlation between the growth rate and the age of the isolate at 25 °C. Variation in growth rates between subpopulations was low compared to within subpopulations. Only at 2 °C did statistically significant differences between the average growth rates of subpopulations emerge. These results suggest that L. piceae covers the whole distribution area of Norway spruce but that generally the thermal reaction norm of its subpopulations has not changed according to local temperature ranges, despite high contrast in thermal conditions across this vast area. Therefore, it would appear that the thermal environment is not a crucial factor in assessing the fitness of this fungal species within the native range of Norway spruce.

Cloning, Characterization and Expression of the Phenylalanine Ammonia-Lyase Gene (PaPAL) from Spruce Picea asperata

Phenylalanine ammonia-lyase (PAL) is the crucial enzyme of the phenylpropanoid pathway, which plays an important role in plant disease resistance. To understand the function of PAL in Picea asperata, in this study, the full-length cDNA sequence of the PAL gene from this species was isolated and named PaPAL. The gene contains a 2160-bp open reading frame (ORF) encoding 720 amino acids with a calculated molecular weight of 78.7 kDa and a theoretical isoelectric point of 5.88. The deduced PaPAL protein possesses the specific signature motif (GTITASGDLVPLSYIA) of phenylalanine ammonia-lyases. Multiple alignment analysis revealed that PaPAL has high identity with other plant PALs. The tertiary structure of PaPAL was predicted using PcPAL from Petroselinum crispum as a template, and the results suggested that PaPAL may have a similar function to that of PcPAL. Furthermore, phylogenetic analysis indicated that PaPAL has a close relationship with other PALs from the Pinaceae species. The optimal expression condition of recombinant PaPAL in Escherichia coli BL21 (DE3) was 0.2 mM IPTG (isopropyl β-D-thiogalactoside) at 16 °C for 4 h, and the molecular weight of recombinant PaPAL was found to be approximately 82 kDa. Recombinant PaPAL was purified and exhibited high PAL activity at optimal conditions of pH 8.6 and 60 °C. Quantitative real-time PCR (qRT-PCR) showed the PaPAL gene to be expressed in all tissues of P. asperata tested, with the highest expression level in the needles. The PaPAL gene was induced by the pathogen (Lophodermium piceae), which caused needle cast disease, indicating that it might be involved in defense against needle cast disease. These results provide a basis for understanding the molecular mechanisms of the PAL gene in the process of P. asperata disease resistance.

Gemmamyces bud blight of Picea pungens: a sudden disease outbreak in Central Europe

Due to its high tolerance to acid rain, the North American Colorado blue spruce (Picea pungens) has been among the most frequent substitute tree species used in reforestation programmes in spruce mountain forests in the Ore and Sudeten Mountains in the Czech Republic after destruction by sulphur dioxide pollution in the second half of the last century. This species was planted on more than 8800 ha of destroyed mountain forest stands in the Ore Mountains alone. New plantations prospered until a massive outbreak of the gemmamyces bud blight, first identified in 2009. The causal agent, Gemmamyces piceae, induced massive bud blight in infected trees – bud loss progressively increased to 70–80% or more, and because the trees were not able to replace the natural loss of older needles, they quickly withered and died. Many plantations disrupted by the pathogen had to be cut down. Although several resistant genotypes have been identified, the cultivation of this species in forest stands has no future in this area. Because this disease was also identified in several ornamental plantations in the area, the bud blight epidemic haunts public green space administrators and nursery and Christmas tree plantation owners. Other pathogens and pests, such as Lophodermium piceae, Sirococcus conigenus and Elatobium abietinum, have also been identified as involved in the damage to P. pungens.

Endophyte communities vary in the needles of Norway spruce clones

Endophytic fungi show no symptoms of their presence but can influence the performance and vitality of host trees. The potential use of endophytes to indicate vitality has been previously realized, but a standard protocol has yet to be developed due to an incomplete understanding of the factors that regulate endophyte communities. Using a culture-free molecular approach, we examined the extent to which host genotype influences the abundance, species richness, and community composition of endophytic fungi in Norway spruce needles. Briefly, total DNA was extracted from the surface-sterilized needles of 30 clones grown in a nursery field and the copy number of the fungal internal transcribed spacer (ITS) region of ribosomal DNA was estimated by quantitative PCR. Fungal species richness and community composition were determined by denaturing gradient gel electrophoresis and DNA sequencing. We found that community structure and ITS copy number varied among spruce clones, whereas species richness did not. Host traits interacting with endophyte communities included needle surface area and the location of cuttings in the experimental area. Although Lophodermium piceae is considered the dominant needle endophyte of Norway spruce, we detected this species in only 33 % of samples. The most frequently observed fungus (66 %) was the potentially pathogenic Phoma herbarum. Interestingly, ITS copy number of endophytic fungi correlated negatively with the richness of ectomycorrhizal fungi and thus potential interactions between fungal communities and their influence on the host tree are discussed. Our results suggest that in addition to environmental factors, endophyte communities of spruce needles are determined by host tree identity and needle surface area.

Diversity of foliar endophytes in wind-fallen Picea abies trees

The unmanaged Norway spruce montane forests in the Bohemian Forest National Park (Czech Republic) suffered from repeated large-scale bark beetle outbreaks in last decade. In this study, the diversity of culturable foliar endophyte microfungi in needles originating from eleven recently wind-fallen trees in this area was surveyed. Our aims were to describe their diversity and to determine the relative host and organ specificities of isolated endophyte species to estimate the species pool and abundance of foliar endophytes before the forest degradation. Microfungi were isolated from surface-sterilized needles, and the outgrowing strains were identified based on morphological and molecular characteristics (analyses of ITS1, ITS2 and partial 28S rDNA). Fungal communities in the needles were diverse, with ascomycetes (mostly anamorphs of Helotiales) dominating basidiomycetes. The most frequent species (Phacidiopycnis sp., Cistella acuum, Sirococcus sp. and two species of Chalara) did not correspond with those recorded in previous studies of Picea spp. For example, the widely distributed Lophodermium piceae was rarely recorded in this study. This pattern may be caused by different methods of sterilization and cultivation or by physiological characteristics of the needles, or it may reflect the species distribution in the studied area. Members of the Helotiales, along with sequences from GenBank, showed substantial overlap in host affinities, most prominently between Pinus and Picea, and also among species from distant plant lineages.

Two species of Lirula on Abies from Yunnan, Southwest China

Based on a recent investigation of species of the Rhytismatales in the eastern Himalayas in China, two species of Lirula on Abies were found, one of which is described as a new species, Lirula yunnanensis, which differs from Lirula japonica mainly by ascomatal structure and conidiomatal features, while the other is Lirula exigua, a new record for China. An analysis of the combined sequences of ITS + LSU r DNA showed that Lirula macrospora and Lophodermium piceae were closely related and that Lirula yunnanensis and Lophodermium autumnale were grouped together. The genus of Lirula should be redefined based on more molecular data. Lirula exigua was distantly related to Lirula species and its systematic position has to be confirmed by further molecular data. A key for known species of Lirula worldwide is provided.

Lophodermium piceae and Rhizosphaera kalkhoffii in Norway spruce: correlations with host age and climatic features.

The study was carried out in 4 similar Norway spruce stands and it demonstrated that the spreading structures produced by Lophodermium piceae and Rhizosphaera kalkhoffii are not correlated. The two fungi were always detected with opposite growth trends, probably due to different needle colonization and spreading strategies, and this was most likely also why they were able to co-exist, colonizing different parts of the needle. Independently of the year, site, sampling period and amount of precipitation, the two fungi were significantly less common in saplings and more common in mature trees, with frequencies also depending on both the minimum and the maximum temperatures.

High diversity of fungi associated with living parts of boreal forest bryophytes

Bryophytes are a dominant and functionally important component of the forest floor vegetation in boreal forests, yet little is known about the fungal diversity associated with these abundant plants. Using molecular identification, we document an ecologically and phylogenetically diverse array of fungi associated with the living parts of three widespread and abundant boreal forest bryophytes, i.e., Hylocomium splendens (Hedw.) Schimp. in B.S.G., Pleurozium schreberi (Brid.) Mitt., and Polytrichum commune Hedw. From 376 cloned ITS sequences, 158 operational taxonomic units (OTUs), roughly corresponding to the number of species, were defined based on sequence similarity searches (Fasta) and phylogenetic analyses. A main portion (62.8%) of the OTUs belonged to Ascomycota, 32.0% to Basidiomycota, 3.9% to Chytridiomycota, and 1.3% to Glomeromycota. The most common orders were Helotiales (18.6%), Agaricales (11.5%), Chaetothyriales (9.6%), and Tremellales (9.0%). Frequently detected OTUs of potentially high ecologic importance included two with high sequence similarity (>99%) to the agaric Entoloma conferendum (Britzelm.) Noordel and the endophyte Lophodermium piceae (Fckl.) Hoehn., and various OTUs with affinity to the Helotian genus Hyphodiscus. Several ectomycorrhiza-forming basidiomycetes were also detected. Most OTUs (77.2%) were only detected once and a very small overlap in composition of OTUs was observed among the three bryophytes, indicating the occurrence of an immense fungal diversity associated with boreal forest bryophytes that deserves further study.

Fungal Diversity of Norway Spruce Litter: Effects of Site Conditions and Premature Leaf Fall Caused By Bark Beetle Outbreak

Fungi play an important role in leaf litter decomposition due to their ability to break down the lignocellulose matrix, which other organisms are unable to digest. However, little is known regarding the factors affecting components of fungal diversity. Here, we quantified richness of internal fungi in relation to litter nutrient and phenolic concentrations, sampling season (spring or fall), and premature leaf shedding due to low precipitation and infestation of bark beetles (mainly Ips typographus and Ips duplicatus). The study was conducted in 37-year-old Norway spruce [Picea abies (L.) Karst.] stands, with three plots each in mixed forest (MF) and coniferous forest (CF) site conditions in south-central Poland. Fifty-four species of sporulating fungi were identified in 2,330 freshly fallen needles sampled during 2003-2005, including 45 species in MF and 31 in CF. The significantly higher number of species in MF was likely related to moister conditions at that site. Among isolated fungi, 22% (12 species) were identified as endophytes of Norway spruce in prior studies. During spring of 2005, we found less than half the number of isolates and fungal species at each forest site as compared to fall for the two prior years. This pattern was observed in typical soil fungi (e.g., Penicillium daleae, Penicillium purpurogenum) and endophytes/epiphytes (e.g., Aureobasidium pullulans, Alternaria alternata, Cladosporium spp., and Lophodermium piceae). Premature shedding of needles was the most likely cause of this decline because it shortened the time period for fungi to infect green needles while on the tree. For all sites and sampling periods, richness of internal fungi was strongly and positively related to the age of freshly fallen litter (assessed using needle Ca concentration as a needle age tracer) and was also negatively related to litter phenolic concentration. Richness of internal fungi in freshly fallen litter may be adversely affected by low soil moisture status, natural inhibitors slowing fungal colonization (e.g., phenolics) and biotic (e.g., insect infestation) and abiotic (e.g., drought) factors that shorten leaf life span.

Decomposition and Fungi of Needle Litter from Slow- and Fast-growing Norway Spruce (Picea abies) Clones

The fungal species involved in the decomposition of needle litter and their response to intraspecific genetic variation of trees are poorly known. First, we compared the needle decomposition and fungal decomposers underneath eight different Norway spruce clones in situ. This experiment revealed 60-70% loss of needle mass in two years. Although spruce clones differed considerably in growth (twofold height difference) and their needles differed in chemical composition, no significant difference was found for loss of needle mass under the spruce clones. Furthermore, the spruce clones did not affect the community structure of the fungal decomposers. Fungi inhabiting needle litter were identified by extracting ribosomal RNA (rRNA) and sequencing complementary DNA (cDNA) of internal trascribed spacer 1 (ITS1) region. The most frequent identifications were Lophodermium, Pezizales, Mycena, and Marasmius, suggesting that endophytic fungi were involved in the decomposition process. Second, we evaluated the potential of endophytes to decompose needle material in a microcosm experiment in which all other fungi than endophytes were excluded. Within 2 years, the endophytes had decomposed 35-45% of the needle mass. Sequences of Mollisia, Lophodermium, Lachnum, and Phialocephala were most frequently found in rRNA and rDNA extracted from the needles at the end of the microcosm experiment. The dominant needle endophyte in fresh, green needles was Lophodermium piceae, and this species was also found frequently in the needle material after 2 years of decay both in the field and laboratory experiments. Moreover, the relative abundance of Lophodermium-derived denaturing gradient gel electrophoresis (DGGE) bands correlated positively with the decomposition in the microcosm experiment. Hence, our results suggest a significant role of endophytic fungi, and particularly L. piceae, in the process of needle decomposition in boreal forests.

Genetic diversity of Lophodermium piceae in South Finland

Summary Lophodermium piceae, the dominant needle endophyte of Norway spruce (Picea abies), is known to be extremely diverse. This work aimed to test the possible occurrence of cryptic species within the morphological species L. piceae. Genetic variation in 36 South Finnish L. piceae isolates originating from six localities was investigated by comparing DNA sequences of three genetic markers. One of the markers was the internal transcribed sequence (ITS) of the ribosomal DNA and the other two (LP1 and LP2) were based on sequence characterized amplified regions designed for L. piceae. The LP2 marker could be detected only from isolates of L. piceae but not from 20 other ascomycete species tested. This sequence, therefore, is considered as a species‐specific marker for L. piceae. For comparison, ITS sequences of isolates representing two other Lophodermium species, L. pinastri and L. seditiosum, were also investigated. In a neighbor‐joining analysis of ITS sequences all L. piceae isolates fell into one cluster, which was clearly separate from those of L. pinastri and L. seditiosum. Dendrograms of the three markers were incongruent indicating that the L. piceae population examined consisted of a single phylogenetic species. No geographical differentiation was observed. Our results confirm that L. piceae is a genetically highly diverse endophytic species.

Black spruce (Picea mariana) foliage hosts numerous and potentially endemic fungal endophytes

Fungal foliar endophytes (hereinafter endophytes) were isolated on malt agar from black spruce ( Picea mariana (Mill.) BSP) needles from the Quebec boreal forest during 2 successive years to study their distribution along a latitudinal transect. Twenty sites were sampled in 2002, 8 of which were sampled again in 2003, in an area located between 47°N and 54°N in the province of Quebec. The endophytes were named whenever possible, using homologies of ITS rDNA sequences from GenBank. In 2002, 232 morphotypes were classified in 40 operational taxonomic units (OTUs), and in 2003, 93 morphotypes were classified in 16 OTUs, for a total of 44 OTUs. Isolation frequencies of the most common endophyte species were 28% for Lophodermium piceae (Fuchel) Höhn., 17% for Darkera parca Whitney, Reid & Piroz, and 9% for Dwayaangam colodena Sokolski & Bérubé. Of the 44 OTUs identified, 18 were unique to P. mariana and found on one site only, possibly constituting rare endemic species. There was some evidence that needles colonized with rare endophyte species were more abundant in the western and southern regions than in the northern region. This supports our hypothesis that as the diversity of tree species surrounding the P. mariana decrease from the southern region to the northern region, the diversity of endophytes also decreases.

Genetic Variation among European Lophodermium piceae Populations - Preliminary Results

Lophodermium piceae is a common needle endophyte of Norway spruce (Picea abies). The aim of the present study was to examine the degree of differentiation within and among European populations separated by various distances and geographical obstacles. For this purpose, populations (including > 10 isolates/subpopulation) were collected along a north-south transect stretching from the northern timberline in Finnish Lapland to the southern border of the distribution area of Norway spruce in northern Italy. Differentiation between L. piceae populations was determined from DNA sequences of three genetic markers. One of the markers was the internal transcribed spacer (ITS) of the ribosomal DNA and the other two (LP1 and LP2) were based on sequence characterized amplified regions (SCAR) designed for L. piceae. Preliminary results including sequences of Finnish, Swiss and Italian isolates show low differentiation among populations. According to analysis of molecular variance the among population variation was 1%, 5% and 0% in ITS, LP1 and LP2 markers, respectively.

Holocene occurrence of Lophodermium piceae, a black spruce needle endophyte and possible paleoindicator of boreal forest health

Holocene occurrences of conifer needle endophytes have not previously been reported. We report the fossil remains of Lophodermium piceae (Fckl.) Hoehn., a fungal endophyte of black spruce ( Picea mariana (Mill.) B.S.P.) needles, in macrofossils dating back to 8000 cal yr BP. Spruce budworm head capsules and L. piceae remains were found preceding charcoal layers delineating the transformation of four spruce−moss forest sites to spruce−lichen woodland. As L. piceae is found solely on senescent needles, its increased presence during these transformation periods likely indicates that the forests were in decline due to the spruce budworm ( Choristoneura fumiferana (Clem.)) when they burned. Future paleoecological studies incorporating needle fungi observations could be used to investigate the historical occurrence of tree disease and the role of fungi in forest health and decline.