Blue-stain Fungus Ceratocystis Polonica Research Articles

Antioxidant defences of Norway spruce bark against bark beetles and its associated blue-stain fungus

Abstract Bark beetles and their fungal associates are integral parts of forest ecosystems, the European spruce bark beetle (Ips typographus Linnaeus, 1758) and the associated pathogenic blue stain fungus Ceratocystis polonica (SIEM.) C. MOREAU, are the most devastating pests regarding Norway spruce [Picea abies (L.) H. KARST.]. Bark beetles commonly inhabit weakened and felled trees as well as vital trees. They cause physiological disorders in trees by destroying a phloem and cambium or interrupt the transpiration -ow in the xylem. Conifers have a wide range of effective defence mechanisms that are based on the inner bark anatomy and physiological state of the tree. The basic function of bark defences is to protect the nutrient-and energy-rich phloem, the vital meristematic region of the vascular cambium, and the transpiration -ow in the sapwood. The main area of defence mechanisms is secondary phloem, which is physically and chemically protected by polyphenolic parenchyma (PP) cells, sclerenchyma, calcium oxalate crystals and resin ducts. Conifer trunk pest resistance includes constitutive, inducible defences and acquired resistance. Both constitutive and inducible defences may deter beetle invasion, impede fungal growth and close entrance wounds. During a successful attack, systemic acquired resistance (SAR) becomes effective and represents a third defence strategy. It gradually develops throughout the plant and provides a systemic change within the whole tree’s metabolism, which is maintained over a longer period of time. The broad range of defence mechanisms that contribute to the activation and utilisation of SAR, includes antioxidants and antioxidant enzymes, which are generally linked to the actions of reactive oxygen species (ROS). The presented review discusses the current knowledge on the antioxidant defence strategies of spruce inner bark against the bark beetle (Ips typographus) and associated blue stain fungus (Ceratocystis polonica).

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.

Erratum to: Low-density Ceratocystis polonica inoculation of Norway spruce (Picea abies) triggers accumulation of monoterpenes with antifungal properties

Among the most devastating pests of Norway spruce (Picea abies) are the European spruce bark beetle (Ips typographus) and the associated pathogenic blue-stain fungus Ceratocystis polonica. Following attack and colonization, the beetle and the fungus must cope with induced host chemical defenses, such as monoterpenes that are generally thought to be toxic to both symbionts. The goal of this study was to better understand the response of Norway spruce following C. polonica inoculation at low density that does not overwhelm the tree and to identify monoterpenes mobilized toward the fungus. We inoculated healthy mature trees and monitored monoterpene profiles 2, 3, and 5 months post-inoculation. We also exposed three different C. polonica strains to the most abundant or significantly up-regulated monoterpenes to determine differences in monoterpene toxicity and resistance among strains. Total monoterpene levels, including limonene, were increased at 2 and 3 months after inoculation and had dropped after 5 months. In in vitro assays, all monoterpenes were inhibitory to C. polonica. Limonene and β-pinene were the most potent inhibitors of fungal growth. The extent of inhibition varied between the three strains tested. These results showed a defense response of Norway spruce to C. polonica, in which limonene may play a critical role in inhibiting the spread of the fungus. We also showed that differences between strains of C. polonica must be taken into account when assessing the role of the fungus in this bark beetle–symbiont system.

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.

The influence of Ceratocystis polonica inoculation and methyl jasmonate application on terpene chemistry of Norway spruce, Picea abies

Constitutive and inducible terpene production is involved in conifer resistance against bark beetles and their associated fungi. In this study 72 Norway spruce ( Picea abies) were randomly assigned to methyl jasmonate (MJ) application, inoculation with the bluestain fungus Ceratocystis polonica, or no-treatment control. We investigated terpene levels in the stem bark of the trees before treatment, 30 days and one year after treatment using GC–MS and two-dimensional GC (2D-GC) with a chiral column, and monitored landing and attack rates of the spruce bark beetle, Ips typographus, on the trees by sticky traps and visual inspection. Thirty days after fungal inoculation the absolute amount and relative proportion of (+)-3-carene, sabinene, and terpinolene increased and (+)-α-pinene decreased. Spraying the stems with MJ tended to generally increase the concentration of most major terpenes with minor alteration to their relative proportions, but significant increases were only observed for (−)-β-pinene and (−)-limonene. Fungal inoculation significantly increased the enantiomeric ratio of (−)-α-pinene and (−)-limonene 1 month after treatment, whereas MJ only increased that of (−)-limonene. One year after treatment, both MJ and fungal inoculation increased the concentration of most terpenes relative to undisturbed control trees, with significant changes in (−)-β-pinene, (−)-β-phellandrene and some other compounds. Terpene levels did not change in untreated stem sections after treatment, and chemical induction by MJ and C. polonica thus seemed to be restricted to the treated stem section. The enantiomeric ratio of (−)-α-pinene was significantly higher and the relative proportions of (−)-limonene were significantly lower in trees that were attractive to bark beetles compared to unattractive trees. One month after fungal inoculation, the total amount of diterpenes was significantly higher in putative resistant trees with shorter lesion lengths than in putative susceptible trees with longer lesions. Thus, terpene composition in the stem bark may be related to resistance of Norway spruce against I. typographus and C. polonica.

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.

Methyl jasmonate treatment of mature Norway spruce (Picea abies) trees increases the accumulation of terpenoid resin components and protects against infection by Ceratocystis polonica, a bark beetle-associated fungus

When conifers such as Picea abies Karst. (Norway spruce) are attacked by insects or pathogens, they often respond by producing increased quantities of terpenoid oleoresin. This response can be mimicked in young P. abies seedlings by treatment with methyl jasmonate (MJ). In this study, we determined the effects of MJ on terpenoids and other chemical defenses of mature P. abies, and investigated whether this treatment protected trees against attack by the blue-stain fungus Ceratocystis polonica (Siem.) C. Moreau, the most important fungal associate of the spruce bark beetle Ips typographus L. Methyl jasmonate treatment induced the formation of traumatic resin ducts in the developing xylem, enhanced resin flow and stimulated increased accumulation of monoterpenes, sesquiterpenes and diterpene resin acids. However, only minor changes were detected in terpene composition in response to MJ treatment and no changes in soluble phenolic concentration were measured. There was much variability in the timing and degree of response to MJ among clones. The observed chemical and anatomical changes in response to MJ treatment were correlated with increased resistance to C. polonica, suggesting that terpenoid oleoresin may function in defense against this pathogen.

Inducible anatomical defense responses in Norway spruce stems and their possible role in induced resistance.

Norway spruce (Picea abies (L.) Karst.) trees were preinoculated with a sublethal dose of the blue-stain fungus Ceratocystis polonica Siem. (C. Moreau) 1 to 52 weeks before they were mass inoculated with the same fungus. Trees pretreated 1 week before mass inoculation had similar, severe symptoms of fungal infection as the control trees. Pretreatment 3, 6 or 9 weeks before mass inoculation resulted in effective protection of the trees, reducing pathogenic symptoms by 63-90% relative to the control trees, whereas pretreatment 52 weeks before mass inoculation gave intermediate protection (44-71% reduction in symptoms). Thus, pretreatment induced resistance to the blue-stain fungus in Norway spruce by a process that requires more than 1 week to become activated and protects trees for at least one year after pretreatment. Pretreatment induced formation of traumatic resin ducts (TDs) in the sapwood and swelling and proliferation of polyphenolic parenchyma cells (PP cells) in the phloem. Trees pretreated 3-9 weeks before mass inoculation had more TDs and showed greater swelling of existing PP cells than control trees or trees pretreated 1 week before mass inoculation. We conclude that induced disease resistance in Norway spruce is probably associated with PP cell activation and TD induction, because resistance was enhanced within the same time frame as the induction of these defense responses.

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.

Variation in Tree Size and Resistance to Ceratocystis polonica in a Monoclonal Stand of Picea abies

Tree resistance to the pathogenic blue-stain fungus Ceratocystis polonica was studied in a monoclonal stand of Norway spruce [ Picea abies (L.) Karst.] in relation to tree social status and diameter at breast height (DBH). The DBH distribution of the 33-yr-old stand ranged from 5 to 35 cm. There were clear differences in tree height between the suppressed (DBH 7.4-10.3 cm), codominant (DBH 11.8-17.4 cm) and dominant (DBH 18.6-23.9 cm) tree classes. The resistance was tested by mass inoculating trees with a low (400 inoculations m -2 , 60 cm inoculation belt) or high (400 inoculations m -2 , 120 cm inoculation belt) dosage. The small, suppressed trees were more susceptible to inoculation than the codominant and dominant trees, based on the amount of blue-stained and occluded sapwood, lesion length and dead cambium/phloem. A threshold in tree social status or tree size may be important in the overall resistance to fungal infection.

Induced resistance to pathogenic fungi in norway spruce

Norway spruce (Picea abies) trees (approximately 16 m high) of a single clone were used to study the effects of fungal infection and wounding on induction of resistance to the bark beetle-associated bluestain fungus Ceratocystis polonica. A dose-response experiment was designed involving three different dosages of fungal (fungus and wound) and sterile agar (wound) pretreatment inoculations (10, 50, or 100 inoculations/m(2) on the stem between 0.8 and 2.0 m high). Three weeks after pretreatment, trees were challenged with a massive C. polonica inoculation (400 inoculations/m(2)). Control trees that received no pretreatment were heavily colonized and killed by the challenge inoculation. The high and medium fungal pretreatments reduced subsequent fungal colonization success by 76% to 97% relative to the control, and fungal pretreatments protected the trees much more efficiently than sterile agar pretreatments. The protection was demonstrated to be local and not systemic in a subsequent experiment, where trees were pretreated with the medium fungal dosage on the lower bole and challenge inoculated further up the stem. Protection was also demonstrated to be pathogen nonspecific, as trees that had been pretreated with a medium dosage of the root rot fungus Heterobasidion annosum showed enhanced resistance to challenge inoculation with C. polonica.

Mechanical injury and fungal infection induce acquired resistance in Norway spruce.

Norway spruce trees (Picea abies (L.) Karst.) pretreated by wounding and fungal infection showed highly enhanced resistance to a subsequent challenge inoculation with the pathogenic bluestain fungus Ceratocystis polonica (Siem.) C. Moreau. This is the first time the effectiveness of the constitutive and inducible defenses has been shown to depend on prior wounding and infection in conifers, although such acquired resistance has previously been found in several angiosperms. Trees that were pretreated with a combination of 12 bark wounds (1.6 x 10 cm), four fungal inoculations and four sterile inoculations 1-15 days before mass inoculation with C. polonica at 400 inoculations per square meter over a 0.8 m stem section had significantly shorter necroses in the phloem, less bluestained sapwood, and less dead cambium than untreated control trees. Pretreatment with four fungal or sterile inoculations alone did not lead to enhanced resistance. Pretreatment by bark wounding alone seemed to provide an intermediate degree of resistance compared to bark wounding, fungal inoculations and sterile inoculations combined. All trees had a marked increase in the number of resin ducts in the year of inoculation compared with previous years, suggesting that formation of traumatic resin ducts play an important role in the development and maintainance of enhanced resistance.