European spruce (Picea abies) is one of the most important coniferous species on the European Continent. Its natural distribution ranges across the Pyrenees, Alps and Balkans, northwards to south Germany and Scandinavia, and eastwards through the Carpathian Mountains and Poland, to western Russia. So the family Pinaceae, which has existed since at least early Cretaceous, is the largest modern conifer family and forms the most widespread coniferous forests of the Northern Hemisphere (Nina. et al., 2000). However, Some bark-beetle-associated blue-stain fungi cause vascular stain diseases and can interrupt the water supply of living trees (Nelson 1934; Bramble et al., 1940; Basham, 1970; Horntvedt et al., 1983; Yamaoka et al., 2000; Lieutier et al., 2004).The blue-stain fungus Ceratocystis polonica, associated with the spruce bark beetle Ips typographus, is known to be pathogenic to European spruce (P. abies) in Europe (Christiansen et al., 1990; Solheim, 1992a; Kirisits, 1998; Krokene et al., 1998b). A very similar pathosystem exists in Japan (Hokkaido), where C. polonica occurs on Yezo spruce (P. jezoensis) and Sachalin spruce (P. glehnii) (Yoshida 1994; Yamaoka et al., 1997). At least two fungi, C. polonica and Ophiostoma penicillatum, have the ability to kill Yezo spruce trees whose trunks have been inoculated with high doses of each of these fungi (Yamaoka et al., 2000). In most inoculation trials with C. polonica, fungal virulence or levels of tree resistance were assessed based on needle symptoms and tree mortality. Only a few studies considered physiological effects of infection by C. polonica on the host trees (Horntvedt et al., 1983; Kirisits et al., 2002). In particular, a decrease of xylem sap flow after inoculation with C. polonica was reported in European spruce (Kirisits et al., 2002). A series of studies on other wilt diseases of trees, such as pine wilt caused by the nematode Bursaphelenchus xylophilus, a wilt disease of oak trees caused by Raffaelea quercivora, and a wilt disease of Yezo spruce caused by C. polonica in Japan have provided information on the mechanisms, which induce xylem dysfunction (Kuroda et al., 1988; 1996;Kuroda, 1991; 2001;2005). The xylem dysfunction covering a large portion of trunk cross-section is fatal to the host tree (Kuroda, 2001). In the wilt disease caused by C. polonica and other vascular diseases, the size of necrotic lesions in the phloem is not related to the virulence of the causal organisms (Solheim, 1988; Krokene et al., 1998b; Kuroda, 2001). The present study reports on investigations examining variation in growth ability of C. polonica, Ophiostoma piceaperdum and O. bicolor isolates, major fungal associates of I. typographus in southern Poland and France (Jankowiak, 2005; Viiri et al., 2004; Salle et al., 2005). Although the phytopathogenicity of C. polonica is well documented, the anatomical, physiological and biochemical analysis processes in the trunk of spruce trees following inoculation with C. polonica has not been investigated so far. The aims of the present study were to: (1) monitor changes of xylem dysfunction in spruce trees after inoculation with C. polonica, and (2) detect temporal changes of cellulase in xylem tissue of spruce trees after inoculation with C. polonica, the pathogenic fungi with I. typographus in European spruce in Austria where there have been no extensive surveys. This information will provide us with useful detail that will help us understand the effect of associated pathogenic fungi on host spruce in European.

The experiment was performed in an approximately 50-year-old stand of European spruce (48°05′N, 15°39′E, 480 m a.s.l), located near Kreisbach in Lower Austria. European spruce is the dominant species in the stand. Death of large number of trees in these stands was mainly caused by I. typographus. Isolation of fungi was made from phloem fragments taken from and around galleries of I. typographus, and also from the sapwood underneath insect galleries up to a depth of 30 mm into the sapwood. In the former case, fungi were isolated from pieces of phloem take from female and larval galleries, as well as from discoloured areas around these galleries. In the latter case, pieces of sapwood were taken along a radius. After drying of samples, the surface layer of phloem was removed with a sterile scalpel. Subsequently, fragments of phloem or sapwood, about 5 mm×5 mm, were cut down with a sterile scalpel, and placed on a culture medium. All isolation were made on 2% malt extract agar (2% MEA; 20 g malt extract, 20 g agar, 1 000 mL distilled water) supplemented with the antibiotic tetracycline(200 mg·L^-1 of culture medium) to inhibit bacterial growth. Pure culture of fungi were grown on 2% agar. The primary isolation plates were incubated at room temperature in the dark. Colonies of fungi growing from the phloem and sapwood fragments were compared on the basis of macro- and microscopic characteristics, and pure cultures were derived from representative colonies in order to identify the fungi. Culture typical of each C. polonica have been deposited in the culture collection of the Laboratory of Department of Forest and Soil Sciences, University of Natural Resources and Applied Life Sciences, Vienna, Austria.

From 6-7 June 2006, four dominant, apparently vigorous European spruce trees of similar diameter, height, crown length and general appearance were mass-inoculated with C. polonica, while two similar trees were inoculated with sterile malt agar to serve as controls (Tab. 1). Both fungal and control inoculations were carried out at a density of four inoculations per dm² within a 120-cm-wide band around the trunk circumference at a height of about 0.5~1.7 m above ground. Trees were inoculated by removing a bark plug with a 7 mm cork borer, inserting inoculum into the wound and replacing the plug. Inoculum consisted of plugs of malt agar (20 g·L^-1 malt extract, 16 g·L^-1 agar) bearing mycelium from 17 to 18 day-old cultures of C. polonica or sterile malt agar (control treatment). C. polonica from the culture collection of the Institute of Forest Entomology, Forest Pathology and Forest Protection, Vienna, isolated in May 2006 from stained sapwood of a spruce trap tree infested by I. typographus, was used.

External symptoms of fungal infection, i.e. sapwood blue-stain on the European spruce, was recorded at 10 days intervals for 9 weeks. Some of the inoculated specimens were harvested 8 weeks after inoculation in order to examine them for internal symptoms of infection by C. polonica. For long-term observation of external symptoms, two control trees inoculated with sterile agar and four trees inoculated with C. polonica were left in the nursery and were checked at irregular intervals until Aug. 2006 (Tab. 1). To visualize symptoms of fungal infection in the main stems, an anatomical test was conducted on five specimens at harvest (Tab. 1). Immediately after being cut at the base, the cut ends of the main stems of the spruce were soaked in 1% aqueous acid fuchsin for 6 h in the open air (Kuroda et al., 1988; Hillis, 1987; Ruzin, 1999). Thereafter, the main stems were cut into 20 cm pieces and then divided into shorter sections. On the cut surfaces, the changes of xylem area and changes of the blue-stains in the host tissue were observed macroscopically (PlateⅠ 1\-7).

Tab.1 Characteristics of the test trees and experiments on European spruce inoculated on 6-7 June 2006 with C. polonica or with sterile agar^①

Tab.1 Characteristics of the test trees and experiments on European spruce inoculated on 6-7 June 2006 with C. polonica or with sterile agar①

Plate Ⅰ   1.C. polonica caused blue-stains and induced xylem dysfunction on European spruce in Austria; 2. Mass-inoculated with C.polonica and sterile malt agar on European spruce in the Alps; 3. Inoculated trunks deposited in the culture collection were incubated at room temperature; 4.C. polonica caused blue-stains and induced phloem and cambial region dysfunction on European spruce; 5. The maximum length of blue sapwood area caused by C. polonica can be extend to 39.6cm; 6. Mass infection of the pathogen associated with the vector beetle's mass attack; 7. Ips typographus occurring long after the trees had been severely weakened by inoculation with C. polonica.

At the time of the inoculations on 6-7 June of 2006, a sample of fresh bark and sapwood was taken from each tree after 3, 6, and 9 weeks later, one ring per tree was sampled, starting with the uppermost ring. Samples with bark and sapwood (Φ:2 cm) centered around the inoculation point were excised with a hammer and chisel and immediately immersed in a fixative of 3% glutaraldehyde in 100 mmol·L^-1 Hepes buffer, pH 7.0. About 15 mg of fresh xylem fraction were transferred into the tip of tubes (2.5 mL) and crushed carefully after addition of 200 mL buffer solution (2 mL 1 mol·L^-1 Tris-HCI, 63 mL 0.5 mol·L^-1 EDTA, 13.2 mL H₂O, 10 mL 10% SDS, pH 8.0) with a macerator at room temperature. The crushed samples were incubated at 75 ℃ for 30 min and spun at 8 000 r·min^-1 for 10 min at 4 ℃. As much as possible of the aqueous phase was transferred into new 1.5 mL tubes. To clean the extracts, the same quantity of cold is opropanol was added and kept for 20 min at -18 ℃. After centrifugation for 15 min at 12 000 r·min^-1 the aqueous phases were carefully poured away. To clean the protein cellulose 100 μL 70% ethanol was added and carefully shaken. After pouring the ethanol the pellets were dried for about 1~2 h. The dried pellets were suspended in about 100 μL TE-buffer and kept in the fridge at 7 ℃ for 1~2 h. An isoelectric-focusing electrophoresis of the extracted protein cellulose method was used to assess changes and distribution of cellulase in the xylem tissue which inoculated with sterile agar and four trees inoculated with C. polonica. All chemicals used for this procedure were drawn from University of Natural Resources and Applied Life Sciences, Vienna, Austria.

Experiment of four trees (No.1-4) inoculated with C. polonica in 2006 showed that C. polonica was the most important pathogenic fungi which caused blue-stain and induced xylem dysfunction on European spruce in Austria. And these four fungi inoculated trees generally induced significantly more extensive symptoms than the other two trees which inoculated with sterile agar. None of the agar inoculated spruce trees in 2006 showed any sapwood blue-stain. 3~4 weeks after inoculation with C. polonica, European spruce showed many blue-stains of some sapwood. No sapwood blue-stain was observed on the two control trees which had been inoculated with sterile agar. By using of a anatomical test, xylem dysfunction in the xylem of inoculated trees became obvious. The dysfunctional xylem area was observed (PlateⅠ-1—7). In two control trees (No.5, 6), most of the xylem was normal except in narrow areas around the inoculation wounds. Such dysfunctional xylem areas infected with C. polonica were dehydrated and desiccated. Furthermore, compared with healthy-looking trees which inoculated with sterile agar and weakened trees which inoculated with fungi C. polonica, C. polonica were more numerously and at higher frequencies isolated from the sapwood underneath galleries of I. typographus. At 18th and 36th d after inoculation, heavy fungal infection led to massive destruction of the cambial zone in areas close to the wound (PlateⅠ-4). The zone, extending from secondary phloem, through the cambium and into the secondary xylem, and the maximum length of blue sapwood area can be extend to 39.6 cm (PlateⅠ-5). The control trees were characterized by intensive and sustained exudation of resin within the inoculated area. In contrast, the four seriously affected trees showed very little resin exudation around the inoculation points, which supports the view that the defence mechanisms of these trees were rapidly overcome by the fungus (Basham, 1970). When stems of four trees were dissected after inoculation for 3 months, inspections of the boles of the killed trees revealed that they had been attacked by bark and cerambycid beetles in 2006 (PlateⅠ-6). However, the infestations of the insects were secondary, occurring long after the trees had been severely weakened by inoculation with C. polonica (PlateⅠ-7). The sapwood of the trees that had been killed by C. polonica showed intensive blue-staining within at least part of the inoculated trunk section.

Biochemical analysis and histochemical localization techniques were used to assess changes and distribution of cellulase in the xylem fraction. It was shown that, in maturing xylem tissue that inoculated with sterile agar, a small amount of the cellulase activity signal was found. However, high cellulase activity was detected in the xylem tissue of four trees that had been inoculated with C. polonica. An isoelectric-focusing electrophoresis of the extracted protein displayed two obvious isozyme bands of cellulase. (Fig. 1) This proved that C. polonica can secrete cellulase to utilize cellulose of xylem in host trees. The results confirm the ability of C. polonica to kill mature European spruce trees in the Alps.

Fig.1 Comparison of changes and distribution of isozyme bands of cellulase in the xylem tissue of European spruce trees that had been inoculated with C. polonica and agar M: Standard protein marker; 1, 2, 3, 4:Four European spruce trees that had been inoculated with C. polonica; 5, 6:Two European spruce trees that had been inoculated with agar

European spruce forests suffer regularly from extensive outbreaks of the Eurasian spruce bark beetle I. typographus (Coleoptera: Scolytidae). During recent years, European spruce bark beetles together with associated pathogenic fungi have killed millions of cubic meters of spruce in western and central Europe(Viiri et al., 2004). Among these most harmful fungi species, C. polonica is based its ability to grow rapidly through the tracheids of moist wood and to disrupt water transport in the host trees, finally leading to high levels of mortality (Christiansen, 1985; Horntvedt et al., 1983; Kirisits, 1998; Krokene et al., 1996; 1998;2001). In the present study, the pathogenicity of C. polonica to Yezo spruce, which has previously been shown by Yamaoka et al. (2000), was demonstrated by the development of needle discolouration on trees after inoculation with this fungus. The symptom development was less intensive compared with the results by Yamaoka et al. (2000). The dye injection tests and anatomical studies indicated that sap ascent of the spruce that had been inoculated with C. polonica was considerably reduced. The rapid enlargement of the desiccated xylem in horizontal and longitudinal directions within 4 weeks after inoculation confirms the observations of Krokene et al.(1997) on European spruce. The extension of the dysfunctional xylem observed in the present experiment was similar to that reported by Yamaoka et al.(2000) after the inoculation of mature Yezo spruce trees with C. polonica for 5 or 8 weeks. Indeed, a sharp decrease of the water supply occurred as a result of the expansion of the dysfunctional area in the desiccated xylem. This view is supported by sap flow measurements on European spruce trees that have been mass inoculated with C. polonica, in which sap flow in the outer sapwood decreased abruptly within the first weeks after inoculation until no sap flow was measurable 4~6 weeks after inoculation (Kirisits et al., 2002). Visible external symptoms and anatomical dysfunctional xylem observed in the present experiment on mature trees of European spruce occurred in various periods from 6 weeks to 16 weeks after the inoculation were similar to former studying (Solheim, 1992a; Yamaoka et al., 2000; Kirisits et al., 2002). The expression of different external symptoms and anatomical symptoms seems to be influenced by the dimensions of trees, inoculation densities and the environmental conditions as suggested by Krokene et al. (1998b). According to the former research, during the early stages of infection prior to the appearance of visible symptoms, a sharp decrease of the water supply occurred in the saplings which inoculated with C. polonica (Yamaoka et al., 2000; Kirisits et al., 2002). However, visible external symptoms occurred in various periods from 6 weeks to 12 weeks after the inoculation of C. polonica on mature European spruce. In the mature trees, it is reasonable that the foliar symptoms are delayed because needles can use water kept in the branches and a part of the thick trunk for a while after the sap flow had stopped (Kuroda, 2005). In dye conduction tests, xylem dysfunction in the xylem of inoculated trees became obvious. However, the finding that fungal hyphae are not present in the margin of the dry zones at the initiation of needle discolouration is in agreement with Hobson et al. (1994) and Krokene et al. (1997). The present results clearly indicate that dry (desiccated) zones in the sapwood of Yezo spruce develop in advance of extensive colonization of the xylem tissues by blue-stain fungi. This lag of fungal colonization by blue-stain fungi behind the enlargement of desiccated area is detectable only by the observation of specimens during the initial stage of infection(Kuroda, 2005). This suggests that necrotic lesions in the phloem around inoculation wounds are clearly not the direct cause of needle discolouration and shoot dieback on spruce trees after infection by C. polonica (Krokene et al., 1997). The natural infection with C. polonica originates from numerous wounds initiated in the course of mass attacks of spruce trees by I. typographus (Christiansen et al., 1990; Solheim, 1992b). At high inoculation densities, blockage of the sap flow is accelerated by the merging of dehydrated xylem areas (Horntvedt et al., 1983; Christiansen et al., 1990; Krokene et al., 1998a; 1998b; Yamaoka et al., 2000; Kirisits et al., 2002). Also in other wilt diseases, the complete blockage of sap flow occurs by the mass infection of the pathogen associated with the vector beetle's mass attack (Kuroda et al., 1996). After the approximately 50-year-old European spruce was inoculated with C. polonica and agar, the changes and distribution of cellulase in the xylem fraction showed that in maturing xylem tissue that inoculated with sterile agar and served as controls, a small amount of the cellulase activity signal was found. However, high cellulase activity was detected in the xylem tissue of four trees that had been inoculated with C. polonica. This suggest that some blue-stain fungi can secrete cellulase to utilize cellulose of xylem in host trees (Chen et al., 2004). The results confirm the ability of C. polonica to kill mature European spruce trees in the Alps.

We wish to thank institute of Forest Entomology, Forest Pathology and Forest Protection of University of Natural Resources and Applied Life Sciences in Vienna for providing isolates of C. polonica and sharing information on the characteristics of the fungus with us. This research was conducted as part of the Special Research Program "Forest Ecosystem Restoration (SF008)", funded by the Austrian Science Foundation and the Ministry of Agriculture and Forestry. The forest enterprise of Lilienfeld convent kindly supplied experimental trees. We thank Thomas Kirisits and Hans Peter for their help with the mass inoculation of the trees. Thanks are also due to Peter Baier, Roman and other members (Institute of Forest Entomology, Forest Pathology and Forest Protection, University of Natural Resources and Applied Life Sciences in Vienna) for their assistance with the fieldwork and helpfulness and technical assistance during the course of the study and to Prof. Kirisits (Department of Forest and Soil Sciences, University of Natural Resources and Applied Life Sciences in Vienna, Austria), who gave valuable comments on an early draft of the manuscript.