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.

First Report of Fusarium oxysporum f. sp. lactucae Race 1 Causing Fusarium Wilt of Lettuce in Norway.

Lettuce (Lactuca sativa L.) is produced in Norway both in field and greenhouses. In Norway, greenhouse lettuce is one of the most important vegetables grown year-round. In winter 2018, wilting symptoms were observed on soil-grown lettuce of the cultivar Frillice in a greenhouse in south east Norway (Buskerud county). Affected plants showed stunted growth, wilting of outer leaves, and brownish discoloration of vascular tissues of taproots and crowns. According to the producer, the disease led to an estimated 10% of yield losses. Fungal isolates were obtained from crowns and roots of diseased plants collected from the greenhouse in 2018 and 2019. Two single spore isolates, 231274 from 2018 and 231725 from 2019, were used in further studies. The isolates were incubated on synthetic nutrient-poor agar (SNA) at 18-20 °C, and a 12 hours dark, 12 hours UV light cycle. Isolate 231274 produced abundant macro- and microconidia characteristics of Fusarium oxysporum while macroconidia were never observed in isolate 231725. On potato dextrose agar (PDA), colonies of isolate 231274 were purple in color and colonies of isolate 231725 were pinkish with abundant aerial mycelium. For PCR-assay, DNA from mycelia was extracted using Easy-DNA kit (Invitrogen). A portion of the translation elongation factor 1-α (EF1-α) gene was amplified using primers F-728F (Carbone and Kohn. 1999) and EF2 (O'Donnell et al. 1998) as described by Aas et al. 2018. Blast analysis of both sequences (accession no. MW316853 for 231274 and MW316854 for 231275) obtained a 99% homology with the sequence of Fusarium oxysporum f.sp. lactucae (FOL) race 1 strain S1 (accession no. DQ837657)(Mbofung et al. 2007). Both isolates were identified as race 1 by using specific primers Hani3' and Hanilatt3rev (Pasquali et al. 2007) as described by Cabral et al. 2014. To complete Koch's postulate, lettuce plants of the cultivar Frillice were used. Race identity was confirmed using the differential lettuce cultivars Costa Rica No.4 (resistant to FOL race 1), Banchu Red Fire (resistant to FOL races 2 and 4) and Romana Romabella (resistant to FOL races 1 and 2) (Gilardi et al. 2017) provided by the breeding company Rijk Zwaan (De Lier, The Netherlands). For inoculation, roots of six 2-weeks old seedlings per cultivar were dipped in a spore suspension (1 x 106 CFU/ml) for 1 min, while controls were dipped in distilled water. Seedlings were planted in 250 ml pots containing fertilized potting substrate, and were placed in a greenhouse with temperature ranging from 15 to 35 °C and an average of 23 °C. After 10 days reduced growth was observed in cultivars Frillice and Banchu Red Fire for both fungal isolates. After 25 days wilting was observed in both cultivars. Affected plants presented discoloration of vascular tissue. No difference in growth was observed between cultivars Romana Romabella and Costa Rica No. 4 and their respective controls. FOL was re-isolated from all inoculated cultivars but not from controls. The colony patterns of the recovered isolates were the same than those of the isolates used for inoculation. These results confirm that the isolate belongs to race 1. Greenhouse lettuce in Norway is mainly produced in hydroponics. FOL is here reported to cause damages in soil- grown lettuce. Nevertheless FOL in hydroponic systems has been reported in Japan (Fujinaga et al. 2003) and Thailand (Thongkamngam and Jaenaksorn 2017). Thus, the possibility of infections in hydroponics remain a big concern for lettuce production in Norway.

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.

Localization of phenolics in phloem parenchyma cells of Norway spruce (Picea abies).

Norway spruce (Picea abies) bark contains specialized phloem parenchyma cells that swell and change their contents upon attack by the bark beetle Ips typographus and its microbial associate, the blue stain fungus Ceratocystis polonica. These cells exhibit bright autofluorescence after treatment with standard aldehyde fixatives, and so have been postulated to contain phenolic compounds. Laser microdissection of spruce bark sections combined with cryogenic NMR spectroscopy demonstrated significantly higher concentrations of the stilbene glucoside astringin in phloem parenchyma cells than in adjacent sieve cells. After infection by C. polonica, the flavonoid (+)-catechin also appeared in phloem parenchyma cells and there was a decrease in astringin content compared to cells from uninfected trees. Analysis of whole-bark extracts confirmed the results obtained from the cell extracts and revealed a significant increase in dimeric stilbene glucosides, both astringin and isorhapontin derivatives (piceasides A to H), in fungus-infected versus uninfected bark that might explain the reduction in stilbene monomers. Phloem parenchyma cells thus appear to be a principal site of phenolic accumulation in spruce bark.

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.

The Relationship between Fungal Diversity and Invasibility of a Foliar Niche-The Case of Ash Dieback.

European ash (Fraxinus excelsior) is threatened by the invasive ascomycete Hymenoscyphus fraxineus originating from Asia. Ash leaf tissues serve as a route for shoot infection but also as a sporulation substrate for this pathogen. Knowledge of the leaf niche partitioning by indigenous fungi and H. fraxineus is needed to understand the fungal community receptiveness to the invasion. We subjected DNA extracted from unwashed and washed leaflets of healthy and diseased European ash to PacBio sequencing of the fungal ITS1-5.8S-ITS2 rDNA region. Leaflets from co-inhabiting rowan trees (Sorbus aucuparia) served as a reference. The overlap in leaflet mycobiomes between ash and rowan was remarkably high, but unlike in rowan, in ash leaflets the sequence read proportion, and the qPCR-based DNA amount estimates of H. fraxineus increased vigorously towards autumn, concomitant with a significant decline in overall fungal richness. The niche of ash and rowan leaves was dominated by epiphytic propagules (Vishniacozyma yeasts, the dimorphic fungus Aureobasidion pullulans and the dematiaceous hyphomycete Cladosporium ramotenellum and H. fraxineus), and endophytic thalli of biotrophs (Phyllactinia and Taphrina species), the indigenous necrotroph Venturia fraxini and H. fraxineus. Mycobiome comparison between healthy and symptomatic European ash leaflets revealed no significant differences in relative abundance of H. fraxineus, but A. pullulans was more prevalent in symptomatic trees. The impacts of host specificity, spatiotemporal niche partitioning, species carbon utilization profiles and life cycle traits are discussed to understand the ecological success of H. fraxineus in Europe. Further, the inherent limitations of different experimental approaches in the profiling of foliicolous fungi are addressed.

Methyl jasmonate and oxalic acid treatment of Norway spruce: anatomically based defense responses and increased resistance against fungal infection.

To study the effect of chemical pretreatment on conifer resistance, 13-year-old Norway spruce (Picea abies (L.) Karst.) trees were treated with methyl jasmonate (MJ) or oxalic acid (OxA) on the outer bark and inoculated with the pathogenic blue-stain fungus Ceratocystis polonica (Siem.) C. Moreau 4 weeks later. Both chemicals significantly reduced symptoms of fungal infection, but MJ was more effective than OxA (51 versus 18% reduction in length of necrotic lesions in the phloem relative to untreated control trees). Anatomical examination of treated stem tissues showed that MJ induced extensive formation of traumatic resin ducts in the xylem and extra polyphenolic parenchyma (PP) cells in the secondary phloem between the cambium and the regular annual PP cell layer. No traumatic resin ducts were formed after treatment with OxA, and the coverage of extra PP cells in OxA-treated tissues was not significantly higher than in the controls. The anatomically based defense reactions induced by MJ were similar to the reactions observed after pathogen infection, mechanical wounding and bark beetle attack. Neither MJ nor OxA had apparent phytotoxic effects on Norway spruce at the concentrations used, with needle and stem tissues of all trees appearing normal without visible symptoms of toxicity. However, trees treated with MJ had 30% less radial sapwood growth than control trees. In conclusion, MJ treatment of Norway spruce appears to have practical potential as a tool for increasing plant resistance to fungal infection, but with a modest reduction in sapwood growth.

Anatomical-based defense responses of Scots pine (Pinus sylvestris) stems to two fungal pathogens.

We investigated the cellular responses of stem tissues of mature Scots pine (Pinus sylvestris L.) trees to inoculations with two fungal pathogens. The bark beetle vectored fungus, Leptographium wingfieldii Morelet, induced longer lesions in the bark, stronger swelling of polyphenolic parenchyma cells, more polyphenol accumulation and increased ray parenchyma activity compared with the root rot fungus, Heterobasidion annosum (Fr.) Bref., or mechanical wounding. Axial resin ducts in the xylem are a general feature of the preformed defenses of Scots pine, but there was no clear induction of additional traumatic axial resin ducts in response to wounding or fungal infection. The anatomical responses of Scots pine to pathogen infection were localized to the infection site and were attenuated away from bark lesions. The responses observed in Scots pine were compared with published studies on Norway spruce (Picea abies (L.) Karst.) for which anatomically based defense responses have been well characterized.

Effects of prolonged drought on the anatomy of sun and shade needles in young Norway spruce trees.

Predicted increases in the frequency and duration of drought are expected to negatively affect tree vitality, but we know little about how water shortage will influence needle anatomy and thereby the trees' photosynthetic and hydraulic capacity. In this study, we evaluated anatomical changes in sun and shade needles of 20-year-old Norway spruce trees exposed to artificial drought stress. Canopy position was found to be important for needle structure, as sun needles had significantly higher values than shade needles for all anatomical traits (i.e., cross-sectional needle area, number of tracheids in needle, needle hydraulic conductivity, and tracheid lumen area), except proportion of xylem area per cross-sectional needle area. In sun needles, drought reduced all trait values by 10-40%, whereas in shade needles, only tracheid maximum diameter was reduced by drought. Due to the relatively weaker response of shade needles than sun needles in drought-stressed trees, the difference between the two needle types was reduced by 25% in the drought-stressed trees compared to the control trees. The observed changes in needle anatomy provide new understanding of how Norway spruce adapts to drought stress and may improve predictions of how forests will respond to global climate change.

Cytological and enzymatic responses to aluminium stress in root tips of Norway spruce seedlings.

• Aluminium (Al) stress reduces plant growth. However, some species such as Norway spruce (Picea abies) seem to tolerate high Al concentrations. The aim of this study was to investigate characteristics possibly involved in Al tolerance in Norway spruce seedlings. • Seedlings (10-d-old) were exposed to Al3+ concentrations of 0.5 and 5 mm for up to 168 h. The effect of Al stress on root growth, cell morphology and Al distribution, callose production, and peroxidase and chitinase activity was analysed. • Root growth decreased after 1 d and 2 d with 5 and 0.5 mm Al, respectively. Callose concentration increased strongly after 6 h treatment with 5 mm Al. The activity of many peroxidase and chitinase isoforms decreased after 1-24 h exposure of both treatments. Several isoforms increased after 48-168 h exposure to 5 mm Al. • We postulate that, with external Al concentrations 0.5 mm or lower, an increased production above constitutive levels of peroxidase or chitinase is not required for Al tolerance in young Norway spruce seedlings. High constitutive levels of peroxidase and chitinase in this species may be part of this Al tolerance.

Effects of Rhizoctonia infection and drought on peroxidase and chitinase activity in Norway spruce (Picea abies).

Seedlings of Norway spruce were exposed to fungal infection and drought in order to investigate differences in their stress responses on the enzymatic level. Six-week-old seedlings were infected with the root rot fungus Rhizoctonia, or subjected to drought, respectively. Changes at the enzymatic level were more rapid and significantly higher in infected plants in comparison with drought-stressed spruce plants. Rhizoctonia infection resulted in early local and systemic increase in peroxidase and chitinase activity. The most prominent isoforms responding were highly basic peroxidases and chitinases (pI 9-9.5) and several acidic chitinases (pI3-4). An increased intensity of similar peroxidase isoforms was found in drought-affected plants. Two peroxidase isoforms (with pI < 9) accumulated exclusively in response to drought. These results suggest that at an early stage of infection and drought stress, the two stresses can be distinguished by the temporal appearance and isoform profile of peroxidases and chitinases. Changes in enzyme activity appeared before changes in physiological parameters, thus these isoform profiles could be used as early markers of stress conditions in spruce.

Induced responses to pathogen infection in Norway spruce phloem: changes in polyphenolic parenchyma cells, chalcone synthase transcript levels and peroxidase activity.

Polyphenolic parenchyma cells (PP cells) in Norway spruce (Picea abies (L.) Karst.) stem phloem play important roles in constitutive and inducible defenses. To determine whether anatomical and molecular changes in PP cells are correlated with tree resistance, we infected two Norway spruce clones with the pathogenic fungus Ceratocystis polonica (Siem.) C. Moreau. The fungus induced significantly different lesion lengths in the two clones, indicating that one clone was more resistant to the fungus (short lesions) than the other (long lesions). After infection, the cross-sectional area of PP cells and their vacuolar polyphenol bodies increased in the three most recent annual rings of PP cells in both clones. The more resistant clone had larger PP cells with denser polyphenol bodies than the less resistant clone, whereas the less resistant clone accumulated relatively more polyphenols after infection. Compared with the less resistant clone, the more resistant clone contained higher starch concentrations before infection that were reduced more quickly after infection before returning to original values. Low transcript levels of chalcone synthase were detected in uninfected tissues of both clones, but the levels increased dramatically after infection. Transcript levels were higher and peaked 6 days earlier in the more resistant clone than in the less resistant clone. The activity of at least one highly basic peroxidase isoform was greatly enhanced after infection, and this increase occurred earlier in the more resistant clone.

Indications of heightened constitutive or primed host response affecting the lignin pathway transcripts and phenolics in mature Norway spruce clones.

Two mature clones of Norway spruce (Picea abies (L.) Karst.) that have previously been shown to have differential degrees of resistance towards the necrotrophic pathogen Heterobasidion parviporum (Niemelä & Korhonen) were compared with respect to the primed defense expression of transcripts related to biosynthesis of lignin, stilbenes and other phenolic compounds from one year to the next. The host's response to physical wounding and pathogen inoculation was examined in the initial year, whereas indications of heightened basal defense level or primed response, and responses to re-wounding, were examined the following year. The responses of the two clones to wounding and pathogen inoculation, examined in the initial year, differed; the increases in lignin and phenolics were more distinct in response to the pathogen than to wounding alone. The more resistant clone 589 had higher initial lignin concentrations in the cell walls when compared with clone 409, and these remained higher in clone 589 over both years and increased after the treatments. Both clones responded at the transcriptional and chemical levels to wounding; changes were evident both in the initial wounds and when re-wounded the following year. There were distinct differences in the basal transcript levels of the lignin pathway-related genes, phenolics and total lignin levels in healthy tissue from the initial year to the following year indicative of a primed host response or at least altered constitutive level of defense expression.

Effect of thinning on anatomical adaptations of Norway spruce needles.

Conifers and other trees are constantly adapting to changes in light conditions, water/nutrient supply and temperatures by physiological and morphological modifications of their foliage. However, the relationship between physiological processes and anatomical characteristics of foliage has been little explored in trees. In this study we evaluated needle structure and function in Norway spruce families exposed to different light conditions and transpiration regimes. We compared needle characteristics of sun-exposed and shaded current-year needles in a control plot and a thinned plot with 50% reduction in stand density. Whole-tree transpiration rates remained similar across plots, but increased transpiration of lower branches after thinning implies that sun-exposed needles in the thinned plot were subjected to higher water stress than sun-exposed needles in the control plot. In general, morphological and anatomical needle parameters increased with increasing tree height and light intensity. Needle width, needle cross-section area, needle stele area and needle flatness (the ratio of needle thickness to needle width) differed most between the upper and lower canopy. The parameters that were most sensitive to the altered needle water status of the upper canopy after thinning were needle thickness, needle flatness and percentage of stele area in needle area. These results show that studies comparing needle structure or function between tree species should consider not only tree height and light gradients, but also needle water status. Unaccounted for differences in needle water status may have contributed to the variable relationship between needle structure and irradiance that has been observed among conifers.

The putative gymnosperm plant defensin polypeptide (SPI1) accumulates after seed germination, is not readily released, and the SPI1 levels are reduced in Pythium dimorphum-infected spruce roots.

The putative plant defensin SPI1 cDNA from the conifer Norway spruce (Picea abies) is the only known plant defensin-like sequence from a gymnosperm. The predicted translational product SPI1 was not detected in the embryo or other parts of the seed by means of antibodies, but it accumulated in the root cortex after germination. In roots of seedlings infected with the root pathogenic oomycete Pythium dimorphum and the blue stain fungus Ceratocystis polonica, variable levels of SPI1 was detected during the first day as a response to the infection, however a significant increase was seen as an initial response to the root-rot fungus Heterobasidion annosum. After the first day of infection, the amount of SPI1 polypeptide was dramatically reduced in response to either of the pathogens, but not in response to the ectomycorrhizal fungus Laccaria bicolor. During the same time of infection, extensive damage to cortical root cells resulted from the infecting pathogens, but not from the mycorrhiza. These results indicate that pathogens may reduce the level of SPI1 by suppressing its expression, but may also reduce the SPI1 level by invading and disrupting the root cortical cells or by a combination of these mechanisms.