Fungal succession in decomposing ash leaves colonized by the ash dieback pathogen Hymenoscyphus fraxineus or its harmless relative Hymenoscyphus albidus.

Introduction: The ascomycete Hymenoscyphus fraxineus, originating from Asia, is currently threatening common ash (Fraxinus excelsior) in Europe, massive ascospore production from the saprotrophic phase being a key determinant of its invasiveness. Methods: To consider whether fungal diversity and succession in decomposing leaf litter are affected by this invader, we used ITS-1 metabarcoding to profile changes in fungal community composition during overwintering. The subjected ash leaf petioles, collected from a diseased forest and a healthy ash stand hosting the harmless ash endophyte Hymenoscyphus albidus, were incubated in the forest floor of the diseased stand between October 2017 and June 2018 and harvested at 2-3-month intervals. Results: Total fungal DNA level showed a 3-fold increase during overwintering as estimated by FungiQuant qPCR. Petioles from the healthy site showed pronounced changes during overwintering; ascomycetes of the class Dothideomycetes were predominant after leaf shed, but the basidiomycete genus Mycena (class Agaricomycetes) became predominant by April, whereas H. albidus showed low prevalence. Petioles from the diseased site showed little change during overwintering; H. fraxineus was predominant, while Mycena spp. showed increased read proportion by June. Discussion: The low species richness and evenness in petioles from the diseased site in comparison to petioles from the healthy site were obviously related to tremendous infection pressure of H. fraxineus in diseased forests. Changes in leaf litter quality, owing to accumulation of host defense phenolics in the pathogen challenged leaves, and strong saprophytic competence of H. fraxineus are other factors that probably influence fungal succession. For additional comparison, we examined fungal community structure in petioles collected in the healthy stand in August 2013 and showing H. albidus ascomata. This species was similarly predominant in these petioles as H. fraxineus was in petioles from the diseased site, suggesting that both fungi have similar suppressive effects on fungal richness in petiole/rachis segments they have secured for completion of their life cycle. However, the ability of H. fraxineus to secure the entire leaf nerve system in diseased forests, in opposite to H. albidus, impacts the general diversity and successional trajectory of fungi in decomposing ash petioles.

The Native Hymenoscyphus albidus and the Invasive Hymenoscyphus fraxineus Are Similar in Their Necrotrophic Growth Phase in Ash Leaves.

The populations of European ash and its harmless fungal associate Hymenoscyphus albidus are in decline owing to ash dieback caused by the invasive Hymenoscyphus fraxineus, a fungus that in its native range in Asia is a harmless leaf endophyte of local ash species. To clarify the behavior of H. albidus and its spatial and temporal niche overlap with the invasive relative, we used light microscopy, fungal species-specific qPCR assays, and PacBio long-read amplicon sequencing of the ITS1-5.8S-ITS2 region to examine fungal growth and species composition in attached leaves of European ash. The plant material was collected from a healthy stand in central Norway, where ash saplings in late autumn showed leaflet vein necrosis like that commonly related to H. fraxineus. For reference, leaflet samples were analyzed from stands with epidemic level of ash dieback in southeastern Norway and Estonia. While H. albidus was predominant in the necrotic veins in the healthy stand, H. fraxineus was predominant in the diseased stands. Otherwise, endophytes with pathogenic potential in the genera Venturia (anamorph Fusicladium), Mycosphaerella (anamorph Ramularia), and Phoma, and basidiomycetous yeasts formed the core leaflet mycobiome both in the healthy and diseased stands. In necrotic leaf areas with high levels of either H. albidus or H. fraxineus DNA, one common feature was the high colonization of sclerenchyma and phloem, a region from which the ascomata of both species arise. Our data suggest that H. albidus can induce necrosis in ash leaves, but that owing to low infection pressure, this first takes place in tissues weakened by autumn senescence, 1-2 months later in the season than what is characteristic of H. fraxineus at an epidemic phase of ash dieback. The most striking difference between these fungi would appear to be the high fecundity of H. fraxineus. The adaptation to a host that is phylogenetically closely related to European ash, a tree species with high occurrence frequency in Europe, and the presence of environmental conditions favorable to H. fraxineus life cycle completion in most years may enable the build-up of high infection pressure and challenge of leaf defense prior to autumn senescence.

Host-Pathogen Interactions in Leaf Petioles of Common Ash and Manchurian Ash Infected with Hymenoscyphus fraxineus.

Some common ash trees (Fraxinus excelsior) show tolerance towards shoot dieback caused by the invasive ascomycete Hymenoscyphus fraxineus. Leaf petioles are considered to serve as a pathogen colonization route to the shoots. We compared four common ash clones with variation in disease tolerance, and included the native host, Manchurian ash (Fraxinus mandshurica), as a reference. Tissue colonization, following rachis inoculation by H. fraxineus, was monitored by histochemical observations and a quantitative polymerase chain reaction (qPCR) assay specific to H. fraxineus. Axial spread of the pathogen towards the petiole base occurred primarily within the phloem and parenchyma, tissues rich in starch in healthy petioles. In inoculated petioles, a high content of phenolics surrounded the hyphae, presumably a host defense response. There was a relationship between field performance and susceptibility to leaf infection in three of the four studied common ash clones, i.e., good field performance was associated with a low petiole colonization level and vice versa. Low susceptibility to leaf infection may counteract leaf-to-shoot spread of the pathogen in common ash, but the limited number of clones studied warrants caution and a larger study. The Manchurian ash clone had the highest petiole colonization level, which may suggest that this native host has evolved additional mechanisms to avoid shoot infection.

Patterns and roles of lignan and terpenoid accumulation in the reaction zone compartmentalizing pathogen-infected heartwood of Norway spruce.

MAIN CONCLUSION: Lignan impregnation of the reaction zone wood protects against oxidative degradation by fungi. Traumatic resin canals may play roles in the underlying signal transduction, synthesis, and translocation of defense compounds. Tree defense against xylem pathogens involves both constitutive and induced phenylpropanoids and terpenoids. The induced defenses include compartmentalization of compromised wood with a reaction zone (RZ) characterized by polyphenol deposition, whereas the role of terpenoids has remained poorly understood. To further elucidate the tree-pathogen interaction, we profiled spatial patterns in lignan (low-molecular-weight polyphenols) and terpenoid content in Norway spruce (Picea abies) trees showing heartwood colonization by the pathogenic white-rot fungus Heterobasidion parviporum. There was pronounced variation in the amount and composition of lignans between different xylem tissue zones of diseased and healthy trees. Intact RZ at basal stem regions, where colonization is the oldest, showed the highest level and diversity of these compounds. The antioxidant properties of lignans obviously hinder oxidative degradation of wood: RZ with lignans removed by extraction showed significantly higher mass loss than unextracted RZ when subjected to Fenton degradation. The reduced diversity and amount of lignans in pathogen-compromised RZ and decaying heartwood in comparison to intact RZ and healthy heartwood suggest that α-conindendrin isomer is an intermediate metabolite in lignan decomposition by H. parviporum. Diterpenes and diterpene alcohols constituted above 90% of the terpenes detected in sapwood of healthy and diseased trees. A significant finding was that traumatic resin canals, predominated by monoterpenes, were commonly associated with RZ. The findings clarify the roles and fate of lignan during wood decay and raise questions about the potential roles of terpenoids in signal transduction, synthesis, and translocation of defense compounds upon wood compartmentalization against decay fungi.

Biotic threats for 23 major non-native tree species in Europe.

For non-native tree species with an origin outside of Europe a detailed compilation of enemy species including the severity of their attack is lacking up to now. We collected information on native and non-native species attacking non-native trees, i.e. type, extent and time of first observation of damage for 23 important non-native trees in 27 European countries. Our database includes about 2300 synthesised attack records (synthesised per biotic threat, tree and country) from over 800 species. Insects (49%) and fungi (45%) are the main observed biotic threats, but also arachnids, bacteria including phytoplasmas, mammals, nematodes, plants and viruses have been recorded. This information will be valuable to identify patterns and drivers of attacks, and trees with a lower current health risk to be considered for planting. In addition, our database will provide a baseline to which future impacts on non-native tree species could be compared with and thus will allow to analyse temporal trends of impacts.

The invasive forest pathogen Hymenoscyphus fraxineus boosts mortality and triggers niche replacement of European ash (Fraxinus excelsior).

Determining the impacts of invasive pathogens on tree mortality and growth is a difficult task, in particular in the case of species occurring naturally at low frequencies in mixed stands. In this study, we quantify such effects by comparing national forest inventory data collected before and after pathogen invasion. In Norway, Fraxinus excelsior is a minor species representing less than 1% of the trees in the forests and being attacked by the invasive pathogen Hymenoscyphus fraxineus since 2006. By studying deviations between inventories, we estimated a 74% higher-than-expected average ash mortality and a 13% slower-than-expected growth of the surviving ash trees, indicating a lack of compensation by the remaining ash. We could confidently assign mortality and growth losses to ash dieback as no mortality or growth shifts were observed for co-occurring tree species in the same plots. The mortality comparisons also show regional patterns with higher mortality in areas with the longest disease history in Norway. Considering that ash is currently mostly growing in mixed forests and that no signs of compensation were observed by the surviving ash trees, a significant habitat loss and niche replacement could be anticipated in the mid-term.

Using the CODIT model to explain secondary metabolites of xylem in defence systems of temperate trees against decay fungi.

BACKGROUND: In trees, secondary metabolites (SMs) are essential for determining the effectiveness of defence systems against fungi and why defences are sometimes breached. Using the CODIT model (Compartmentalization of Damage/Dysfunction in Trees), we explain defence processes at the cellular level. CODIT is a highly compartmented defence system that relies on the signalling, synthesis and transport of defence compounds through a three-dimensional lattice of parenchyma against the spread of decay fungi in xylem. SCOPE: The model conceptualizes 'walls' that are pre-formed, formed during and formed after wounding events. For sapwood, SMs range in molecular size, which directly affects performance and the response times in which they can be produced. When triggered, high-molecular weight SMs such as suberin and lignin are synthesized slowly (phytoalexins), but can also be in place at the time of wounding (phytoanticipins). In contrast, low-molecular weight phenolic compounds such as flavonoids can be manufactured de novo (phytoalexins) rapidly in response to fungal colonization. De novo production of SMs can be regulated in response to fungal pathogenicity levels. The protective nature of heartwood is partly based on the level of accumulated antimicrobial SMs (phytoanticipins) during the transitionary stage into a normally dead substance. Effectiveness against fungal colonization in heartwood is largely determined by the genetics of the host. CONCLUSION: Here we review recent advances in our understanding of the role of SMs in trees in the context of CODIT, with emphasis on the relationship between defence, carbohydrate availability and the hydraulic system.We also raise the limitations of the CODIT model and suggest its modification, encompassing other defence theory concepts. We envisage the development of a new defence system that is modular based and incorporates all components (and organs) of the tree from micro- to macro-scales.

Invasive forest pathogens in Europe: Cross-country variation in public awareness but consistency in policy acceptability.

Political action can reduce introductions of diseases caused by invasive forest pathogens (IPs) and public support is important for effective prevention. The public's awareness of IP problems and the acceptability of policies aiming to combat these pathogens were surveyed in nine European countries (N = 3469). Although awareness of specific diseases (e.g., ash dieback) varied, problem awareness and policy acceptability were similar across countries. The public was positive towards policies for informational measures and stricter standards for plant production, but less positive towards restricting public access to protected areas. Multilevel models, including individual and country level variables, revealed that media exposure was positively associated with awareness of IP problems, and strengthened the link between problem awareness and policy acceptability. Results suggest that learning about IPs through the media and recognizing the associated problems increase policy acceptability. Overall, the study elaborates on the anthropogenic dimension of diseases caused by IPs.

Propagule Pressure Build-Up by the Invasive Hymenoscyphus fraxineus Following Its Introduction to an Ash Forest Inhabited by the Native Hymenoscyphus albidus.

Dieback of European ash, caused by the ascomycete Hymenoscyphus fraxineus originating from Asia, has rapidly spread across Europe, and is threatening this keystone tree at a continental scale. High propagule pressure is characteristic to invasive species. Consistently, the enormous production of windborne ascospores by H. fraxineus in an ash forest with epidemic level of disease obviously facilitates its invasiveness and long distance spread. To understand the rate of build-up of propagule pressure by this pathogen following its local introduction, during 2011-2017 we monitored its sporulation at a newly infested ash stand in south-western Norway characterized with mild winters and cool summers. We also monitored the propagule pressure by Hymenoscyphus albidus, a non-pathogenic native species that competes for the same sporulation niche with H. fraxineus. During the monitoring period, crown condition of ash trees had impaired, and 20% of the dominant trees were severely damaged in 2017. H. fraxineus showed an exponential increase in spore production between 2012 and 2015, followed by drastic decline in 2016 and 2017. During 2011-2013, the two Hymenoscyphus species showed similar sporulation level, but thereafter spores of H. albidus were no longer detected. The data suggest that following local introduction, the population of H. fraxineus reaches rapidly an exponential growth stage if the local weather conditions are favorable for ascomata maturation across years. In the North Atlantic climate, summer temperatures critically influence the pathogen infection pressure, warm summers allowing the population to grow according to its biotic potential, whereas cold summers can cause a drastic decline in propagule pressure.

The ash dieback invasion of Europe was founded by two genetically divergent individuals.

Accelerating international trade and climate change make pathogen spread an increasing concern. Hymenoscyphus fraxineus, the causal agent of ash dieback, is a fungal pathogen that has been moving across continents and hosts from Asian to European ash. Most European common ash trees (Fraxinus excelsior) are highly susceptible to H. fraxineus, although a minority (~5%) have partial resistance to dieback. Here, we assemble and annotate a H. fraxineus draft genome, which approaches chromosome scale. Pathogen genetic diversity across Europe and in Japan, reveals a strong bottleneck in Europe, though a signal of adaptive diversity remains in key host interaction genes. We find that the European population was founded by two divergent haploid individuals. Divergence between these haplotypes represents the ancestral polymorphism within a large source population. Subsequent introduction from this source would greatly increase adaptive potential of the pathogen. Thus, further introgression of H. fraxineus into Europe represents a potential threat and Europe-wide biological security measures are needed to manage this disease.

Fungal diversity and seasonal succession in ash leaves infected by the invasive ascomycete Hymenoscyphus fraxineus.

High biodiversity is regarded as a barrier against biological invasions. We hypothesized that the invasion success of the pathogenic ascomycete Hymenoscyphus fraxineus threatening common ash in Europe relates to differences in dispersal and colonization success between the invader and the diverse native competitors. Ash leaf mycobiome was monitored by high-throughput sequencing of the fungal internal transcribed spacer region (ITS) and quantitative PCR profiling of H. fraxineus DNA. Initiation of ascospore production by H. fraxineus after overwintering was followed by pathogen accumulation in asymptomatic leaves. The induction of necrotic leaf lesions coincided with escalation of H. fraxineus DNA levels and changes in proportion of biotrophs, followed by an increase of ubiquitous endophytes with pathogenic potential. H. fraxineus uses high propagule pressure to establish in leaves as quiescent thalli that switch to pathogenic mode once these thalli reach a certain threshold - the massive feedback from the saprophytic phase enables this fungus to challenge host defenses and the resident competitors in mid-season when their density in host tissues is still low. Despite the general correspondence between the ITS-1 and ITS-2 datasets, marker biases were observed, which suggests that multiple barcodes provide better overall representation of mycobiomes.

Identification of low temperature stress regulated transcript sequences and gene families in Italian cypress.

Cold acclimation is a complex transcriptionally controlled process regulated by many different genes and genic-interactions in plants. The northward spreading of woody species is mainly limited by winter harshness. To increase our knowledge about the biological processes underlying cold acclimation, plants evolved in warmer climates can serve as models. In this work, a Suppression Subtractive Hybridization approach using PCR-select was used to isolate Italian cypress (Cupressus sempervirens L.) transcript sequences putatively expressed under low temperature stress. After assessing the reliability of the subtractive step, a total of 388 clones were selected and sequenced. Following sequence assembly and removal of the redundant cDNAs, 156 unique transcripts were identified and annotated in order to assign them a putative functional class. Most of the identified transcripts were functionally classified pertaining to stress in cellular and chloroplast membranes, which are previously known to be severely damaged by cold treatment. Among the identified functional gene families, the extensively represented ones were dehydrins, early light-inducible proteins, senescence-associated genes and oleosins. The last three gene families were further selected for phylogenetic analysis, with the corresponding protein sequences across the complete genomes of the model plants Populus trichocarpa, Vitis vinifera, Physcomitrella patens, and Arabidopsis thaliana. The relationship with the ortholog sequences coming from these species and their further implications are discussed.

Using laser micro-dissection and qRT-PCR to analyze cell type-specific gene expression in Norway spruce phloem.

The tangentially oriented polyphenolic parenchyma (PP) and radially organized ray parenchyma in the phloem are central in the defense of conifer stems against insects and pathogens. Laser micro-dissection enables examination of cell-specific defense responses. To examine induced defense responses in Norway spruce stems inoculated with the necrotrophic blue-stain fungus Ceratocystis polonica, RNA extracted from laser micro-dissected phloem parenchyma and vascular cambium was analyzed using real-time RT-PCR (qRT-PCR) to profile transcript levels of selected resistance marker genes. The monitored transcripts included three pathogenesis-related proteins (class IV chitinase (CHI4), defensin (SPI1), peroxidase (PX3), two terpene synthesis related proteins (DXPS and LAS), one ethylene biosynthesis related protein (ACS), and a phenylalanine ammonia-lyase (PAL). Three days following inoculation, four genes (CHI4, PAL, PX3, SPI1) were differentially induced in individual cell and tissue types, both close to the inoculation site (5 mm above) and, to a lesser degree, further away (10 mm above). These resistance marker genes were all highly induced in ray parenchyma, supporting the important role of the rays in spruce defense propagation. CHI4 and PAL were also induced in PP cells and in conducting secondary phloem tissues. Our data suggests that different cell types in the secondary phloem of Norway spruce have overlapping but not fully redundant roles in active host defense. Furthermore, the study demonstrates the usefulness of laser micro-dissection coupled with qRT-PCR to characterize gene expression in different cell types of conifer bark.

Genes associated with lignin degradation in the polyphagous white-rot pathogen Heterobasidion irregulare show substrate-specific regulation.

The pathogenic white-rot basidiomycete Heterobasidion irregulare is able to remove lignin and hemicellulose prior to cellulose during the colonization of root and stem xylem of conifer and broadleaf trees. We identified and followed the regulation of expression of genes belonging to families encoding ligninolytic enzymes. In comparison with typical white-rot fungi, the H. irregulare genome has exclusively the short-manganese peroxidase type encoding genes (6 short-MnPs) and thereby a slight contraction in the pool of class II heme-containing peroxidases, but an expansion of the MCO laccases with 17 gene models. Furthermore, the genome shows a versatile set of other oxidoreductase genes putatively involved in lignin oxidation and conversion, including 5 glyoxal oxidases, 19 quinone-oxidoreductases and 12 aryl-alcohol oxidases. Their genetic multiplicity and gene-specific regulation patterns on cultures based on defined lignin, cellulose or Norway spruce lignocellulose substrates suggest divergent specificities and physiological roles for these enzymes. While the short-MnP encoding genes showed similar transcript levels upon fungal growth on heartwood and reaction zone (RZ), a xylem defense tissue rich in phenolic compounds unique to trees, a subset of laccases showed higher gene expression in the RZ cultures. In contrast, other oxidoreductases depending on initial MnP activity showed generally lower transcript levels on RZ than on heartwood. These data suggest that the rate of fungal oxidative conversion of xylem lignin differs between spruce RZ and heartwood. It is conceivable that in RZ part of the oxidoreductase activities of laccases are related to the detoxification of phenolic compounds involved in host-defense. Expression of the several short-MnP enzymes indicated an important role for these enzymes in effective delignification of wood by H. irregulare.

The pathogenic white-rot fungus Heterobasidion parviporum responds to spruce xylem defense by enhanced production of oxalic acid.

Pathogen challenge of tree sapwood induces the formation of reaction zones with antimicrobial properties such as elevated pH and cation content. Many fungi lower substrate pH by secreting oxalic acid, its conjugate base oxalate being a reductant as well as a chelating agent for cations. To examine the role of oxalic acid in pathogenicity of white-rot fungi, we conducted spatial quantification of oxalate, transcript levels of related fungal genes, and element concentrations in heartwood of Norway spruce challenged naturally by Heterobasidion parviporum. In the pathogen-compromised reaction zone, upregulation of an oxaloacetase gene generating oxalic acid coincided with oxalate and cation accumulation and presence of calcium oxalate crystals. The colonized inner heartwood showed trace amounts of oxalate. Moreover, fungal exposure to the reaction zone under laboratory conditions induced oxaloacetase and oxalate accumulation, whereas heartwood induced a decarboxylase gene involved in degradation of oxalate. The excess level of cations in defense xylem inactivates pathogen-secreted oxalate through precipitation and, presumably, only after cation neutralization can oxalic acid participate in lignocellulose degradation. This necessitates enhanced production of oxalic acid by H. parviporum. This study is the first to determine the true influence of white-rot fungi on oxalate crystal formation in tree xylem.

Substrate-specific transcription of the enigmatic GH61 family of the pathogenic white-rot fungus Heterobasidion irregulare during growth on lignocellulose.

The GH61 represents the most enigmatic Glycoside Hydrolase family (GH) regarding enzymatic activity and importance in cellulose degradation. Heterobasidion irregulare is a necrotizing pathogen and white-rot fungus that causes enormous damages in conifer forests. The genome of H. irregulare allowed identification of ten HiGH61 genes. qRT-PCR analysis separate the HiGH61 members into two groups; one that show up regulation on lignocellulosic substrates (HiGH61A, HiGH61B, HiGH61D, HiGH61G, HiGH61H, and HiGH61I) and a second showing either down-regulation or constitutive expression (HiGH61C, HiGH61E, HiGH61F, and HiGH61J). HiGH61H showed up to 17,000-fold increase on spruce heartwood suggesting a pivotal role in cellulose decomposition during saprotrophic growth. Sequence analysis of these genes reveals that all GH61s except HiGH61G possess the conserved metal-binding motif essential for activity. The sequences also divide into groups having either an insert near the N terminus or an insert near the second catalytic histidine, which may represent extensions of the substrate-binding surface. Three of the HiGH61s encode cellulose-binding modules (CBM1). Interestingly, HiGH61H and HiGH61I having CBM1s are up-regulated on pure cellulose. There was a common substrate-specific induction patterns of the HiGH61s with several reference cellulolytic and hemicellulolytic GHs, this taken together with their low transcript levels on media lacking lignocellulose, reflect the concerted nature of cell wall polymer degradation.

Xylem defense wood of Norway spruce compromised by the pathogenic white-rot fungus Heterobasidion parviporum shows a prolonged period of selective decay.

Heterobasidion parviporum, a common pathogenic white-rot fungus in managed Norway spruce forests in northern and central Europe, causes extensive decay columns within stem heartwood of the host tree. Infected trees combat the lateral spread of decay by bordering the heartwood with a fungistatic reaction zone characterized by elevated pH and phenol content. To examine the mode of fungal feeding in the reaction zone of mature Norway spruce trees naturally infected by H. parviporum, we conducted spatial profiling of pectin and hemicellulose composition, and established transcript levels of candidate fungal genes encoding enzymes involved in degradation of the different cell wall components of wood. Colonized inner heartwood showed pectin and hemicellulose concentrations similar to those of healthy heartwood, whereas the carbohydrate profiles of compromised reaction zone, irrespective of the age of fungal activity in the tissue, indicated selective fungal utilization of galacturonic acid, arabinose, xylose and mannose. These data show that the rate of wood decay in the reaction zone is slow. While the up-regulation of genes encoding pectinases and hemicellulases preceded that of the endoglucanase gene during an early phase of fungal interaction with xylem defense, the manganese peroxidase gene showed similar transcript levels during different phases of wood colonization. It seems plausible that the reaction zone components of Norway spruce interfere with both lignin degradation and the associated co-hydrolysis of hemicelluloses and pectin, resulting in a prolonged phase of selective decay.

Spatial patterns in hyphal growth and substrate exploitation within norway spruce stems colonized by the pathogenic white-rot fungus Heterobasidion parviporum.

In Norway spruce, a fungistatic reaction zone with a high pH and enrichment of phenolics is formed in the sapwood facing heartwood colonized by the white-rot fungus Heterobasidion parviporum. Fungal penetration of the reaction zone eventually results in expansion of this xylem defense. To obtain information about mechanisms operating upon heartwood and reaction zone colonization by the pathogen, hyphal growth and wood degradation were investigated using real-time PCR, microscopy, and comparative wood density analysis of naturally colonized trees with extensive stem decay. The hyphae associated with delignified wood at stump level were devoid of any extracellular matrix, whereas incipient decay at the top of decay columns was characterized by a carbohydrate-rich hyphal sheath attaching hyphae to tracheid walls. The amount of pathogen DNA peaked in aniline wood, a narrow darkened tissue at the colony border apparently representing a compromised region of the reaction zone. Vigorous production of pathogen conidiophores occurred in this region. Colonization of aniline wood was characterized by hyphal growth within polyphenolic lumen deposits in tracheids and rays, and the hyphae were fully encased in a carbohydrate-rich extracellular matrix. Together, these data indicate that the interaction of the fungus with the reaction zone involves a local concentration of fungal biomass that forms an efficient translocation channel for nutrients. Finally, the enhanced production of the hyphal sheath may be instrumental in lateral expansion of the decay column beyond the reaction zone boundary.

Genotype-specific regulation of cold-responsive genes in cypress (Cupressus sempervirens L.).

Cold acclimation in plants involves a very complex molecular response, with the regulation of many different genes and metabolic pathways. In this work fifteen cypress (Cupressus sempervirens) genes putatively regulated during cold exposure were isolated and their expression was studied in five cypress genotypes, along 15 days of treatment at 3 degrees C. Treated samples of shoots were collected from four year old cypress seedlings and a subtractive hybridization approach (PCR-Select) was performed after mRNA extraction. Fifteen genes were selected according to sequence similarities after a GenBank search and their expression was studied using Real-time PCR. Among these genes, five (ELIP, aquaporin, dehydrin and two cold-induced proteins) and four (oleosin, chlorophyll a/b-binding protein, oxidoreductase and rubisco activase) resulted respectively up- and down-regulated by the treatment in all tested genotypes. Finally, three genes (metal-binding protein, nodulin-like protein and beta-amylase) showed remarkable different pattern among genotypes. A consistent relationship was found between the cold regulation of the genes studied and their putative function, suggesting the existence of different cold response pathways in cypress. The possible roles of the low temperature-regulated sequences and of the individual expression differences during cypress cold acclimation are proposed and discussed.

Real-time PCR-based monitoring of DNA pools in the tri-trophic interaction between Norway spruce, the rust Thekopsora areolata, and an opportunistic ascomycetous Phomopsis sp.

The difficulty in subculturing biotrophic fungi complicates etiological studies related to the associated plant diseases. By employing internal transcribed spacer rDNA-targeted quantitative real-time polymerase chain reaction, we now show that the heteroecious rust Thekopsora areolata, commonly associated in natural conditions to sapling shoots and cones of Norway spruce and leaves of wild bird cherry, frequently infects nursery-grown seedlings of the conifer. A spatial sampling scheme was used to investigate seedlings and saplings of Norway spruce showing phloem necrosis: the highest concentration of DNA of T. areolata was recorded in the area with necrotic phloem. The separate analysis of bark and wood tissues suggested that the initial spread of the rust to healthy tissues neighboring the infection site takes place in the bark. A Phomopsis species found to coexist with T. areolata in several seedlings showed very high DNA levels in the upper part of the lesion, and even in the visually healthy proximal tissues above the lesions, which indicates that the ascomycete, most probably a secondary invader following primary infection by T. areolata, has a latent stage during early host colonization. We hypothesize that this hemibiotrophic mode of infection contributes to the successful coexistence of Phomopsis with a biotrophic rust.