Fungi associated with Ips typographus on Picea abies in southern Poland and their succession into the phloem and sapwood of beetle‐infested trees and logs

This study dealt with the species distribution and frequency of fungi associated with the bark beetle Ips typographus (Scolytidae) on spruce trees of various states of health in southern Poland. The spruce trees were assessed by their degree of defoliation and damage of their crowns and trunks after attack by I. typographus. The state of health of trees from which samples were obtained was related to varying stages of brood development of I. typographus. Fungi were isolated from phloem taken from and around insect galleries and the sapwood underneath brood systems. Samples were taken from ‘healthy‐looking’, weakened, wind‐fallen and wind‐broken trees as well as from trap trees. The mycobiota associated with I. typographus was quite diverse in respect of the number of detected species, 65 fungal taxa were obtained from the phloem of trees infested by I. typographus, and 36 taxa occurred in the sapwood underneath insect galleries. The spectrum of fungi mainly consisted of ascomycetes and anamorphic fungi. The ophiostomatoid fungi were represented by 14 species and were the most numerously represented group in all niches examined. The most frequent ophiostomatoid species were Ceratocystis polonica, Ophiostoma ainoae, O. bicolor, O. penicillatum, O. piceae and O. piceaperdum. The frequency of occurrence of ophiostomatoid fungi differed significantly between the phloem and sapwood as well as in relation to the varying states of health of the spruce trees. These quantitative differences in the mycobiota of I. typographus between spruce trees belonging to different health categories can be explained by successional patterns of fungal colonization of host tissues following attack by I. typographus. The pathogenic species C. polonica was the primary invader, occurring most frequently in the sapwood of ‘healthy‐looking’ trees. Ophiostoma bicolor, O. penicillatum and O. piceaperdum also occurred during the early stages of brood development of I. typographus on ‘healthy‐looking’ trees, but they mainly colonized the phloem. In contrast, O. ainoae, O. minuta and O. piceae likely follow the aforementioned species as secondary and tertiary invaders into the phloem and the sapwood of spruce trees.     

Ophiostomatoid fungi associated with three spruce-infesting bark beetles in Slovenia

Context

Ophiostomatoid fungi can severely affect the health and economic value of Norway spruce trees (Picea abies). Although the diversity of ophiostomatoid species and their associations with insects have been well-investigated in central and northern Europe, little is known about the conditions in south-eastern Europe.

Aim

This study aims to study the assemblages of ophiostomatoid fungi associated with three bark beetle species (Ips typographus, Ips amitinus, and Pityogenes chalcographus) that infect Norway spruce in Slovenia.

Methods

Bark beetles were sampled in four phytogeographic regions in Slovenia. The fungi found on the bark beetles were identified based on morphology, DNA sequence comparisons of ITS regions and phylogenetic analysis. The species compositions of the fungal associates of the three insect species were compared and the pairwise associations of the occurrence of the fungal species were analysed.

Results

Thirteen different species were found. The most commonly encountered fungal associates of the beetles were Ophiostoma bicolor, Ophiostoma brunneo-ciliatum, Grosmannia piceiperda, Ophiostoma ainoae, Ceratocystiopsis minuta, and Grosmannia penicillata. The composition of the fungal associates differed among the bark beetle species, but not among the phytogeographic regions.

Conclusions

This study confirms that ophiostomatoid species are common associates of the investigated bark beetle species. Many ophiostomatoid species have strong host associations. I. typographus and P. chalcographus can act as effective vectors for O. bicolor, O. ainoae, G. piceiperda and O. brunneo-ciliatum, whereas I. amitinus often carries G. piceiperda and C. minuta in Slovenian forests.     

Single sequence repeat markers reflect diversity and geographic barriers in Eurasian populations of the conifer pathogen Ceratocystis polonica

The blue‐stain fungus and vascular stain pathogen Ceratocystis polonica and its associated bark beetle vectors, particularly Ips typographus and I. typographus japonicus, cause significant losses to several spruce species in Eurasia. Nothing is, however, known about the population genetics of this conifer pathogen. In this study, a set of single sequence repeat (SSR) markers were developed to determine the population structure and genetic diversity of C. polonica in Europe and Japan. ISSR‐PCR primers were used to target SSR‐rich regions and specific primers were designed flanking the SSR regions found in these amplicons. The SSR primers developed for C. polonica were found to be transferable to six other Ceratocystis species from conifers, residing in the Ceratocystis coerulescens complex. Ninety‐eight isolates representing four populations of C. polonica (Austria, Norway, Poland and Japan) were tested using 10 selected polymorphic SSR markers. A high level of gene diversity was found in C. polonica as a whole (H = 0.53). Analysis of G statistics showed a low degree of population structure in Europe and a high level of gene flow between populations (Gst = 0.05, Nm = 8.5). In contrast, the Japanese and the European populations of C. polonica displayed strong genetic separation, which is likely caused by geographic isolation. The low level of population structure of C. polonica in Europe and the differentiation between the European and the Japanese fungal populations mirror previous findings for I. typographus and I. typographus japonicus, the main insect vectors of this fungus. These results support the view that the fungus and the insect have closely co‐evolved together. This study also suggests that movement of C. polonica and its vectors between Europe and Asia pose a threat to forestry on both continents and this should clearly be avoided.     

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.     

Pathogenicity of ophiostomatoid fungi on Picea abies in Slovenia

Pathogenic fungi can survive and develop in living plants, often causing diseases in the host. Some theories speculate that pathogenic ophiostomatoid fungi provide benefits to its vectors – bark beetles – by overcoming the tree's defence mechanisms. This study reports the results of an experiment in south‐eastern Europe in which mature and seedling Norway spruce trees were artificially inoculated with various ophiostomatoid fungi. The aim of the experiment was to determine the relative virulence of ophiostomatoid fungi by assessing the ability of the fungi to stimulate host tree defence mechanisms through inoculation experiments. Experiments were performed by inoculation of Picea abies in seedling and mature trees. The following fungi were used in low‐density and seedling inoculations: Ophiostoma ainoae, O. brunneo‐ciliatum, Grosmannia cucullata and an unidentified Leptographium sp., O. bicolor, O. fuscum, O. piceae, G. penicillata and G. piceiperda. Endoconidiophora polonica was used in mass and seedling inoculations. Various characteristics such as host vitality, blue stain, lesion and resin outflow were measured before and after the trees were felled. E. polonica caused blue stain, induced large lesions and killed some of the mature trees and seedlings, confirming earlier reports that it is a strong wound pathogen. Only E. polonica, Leptographium sp. and O. ainoae caused blue stains in the sapwood of inoculated seedlings. In low‐density inoculations, G. piceiperda induced intense necrosis and had higher values for all the characteristics monitored. Some of the other ophiostomatoid fungi showed a moderate level of pathogenicity. Fungi with the capacity to stimulate a host defence mechanism could play a role in the establishment of bark beetle populations.     

Fungal succession in sapwood of Norway spruce infested by the bark beetle Ips typographus

Fungal invasion of Norway spruce sapwood was investigated during a period of 130 weeks fol-lowing attack by the spruce bark beetle Ips typographus. Heavy beetle attack tended to overwhelm the trees quickly and no secondary resinosis was produced. Nonetheless, fungal penetration in the sapwood was initiated only slowly, although it accelerated during the fourth week after attack. Typically the leading edge of fungal penetration was a few millimeters in advance of the visible blue-stain during the sapwood colonization. Fungi invaded the sapwood in a successional pattern with the most pathogenic species Ophiostoma polonicum first, followed by other beetle-transmitted Ophiostoma and Graphium species. The later succession was probably influenced by sapwood moisture. Strong decaying white-rotters invaded near the base of the trees, where sapwood moisture remained favourable, while cf. Trichoderma viride dominated in the drier parts or the trees.     

Ceratocysts polonica inoculated in Norway spruce: Blue-staining in relation to inoculum density,resinosis and tree growth

When Norway spruce trees were inoculated with Ceratocystis polonica, a dose-dependent response was recorded. Local resinosis near the inoculation sites decreased with increasing inoculum density; parallelled by an increasing degree of sapwood staining, and tree mortality. Suppressed trees, and trees showing growth decline appeared more susceptible than trees suffering less competition.     

Fungi associated with the spruce bark beetle Ips typographus in an endemic area in Norway

To investigate the fungal flora associated with the spruce bark beetle Ips typographus in an endemic area, beetles were collected during the flight period in six locations in Tr?ndelag, Norway. The beetles were inoculated into fresh Norway spruce logs, and the frequency of different species was monitored by fungal isolation from bark and wood. The most common species were Ophiostoma bicolor, O. penicillatum, O. polonicum and a previously undescribed Graphium species, the same species which were the most important associates of I. Typographus in an epidemic area in southeastern Norway. The frequency of the primary invader, O. polonicum, was, however, lower in Tr?ndelag, which may indicate that this species is less frequent in endemic areas than in epidemic areas.     

Virulence of ophiostomatoid fungi associated with the spruce bark beetleIps typographus f.japonicus in Yezo spruce

Yezo spruce trees (Picea jezoensis), approximately 40-year-old were inoculated with eight ophiostomatoid fungi associated withIps typographus f.japonicus to compare relative virulence of the fungi. Among them,Ophistoma penicillatum formed the longest necrotic lesion on inner bark around inoculation points, followed byO. aenigmaticum, Ceratocystis polonica, andO. bicolor, whileC. polonica formed a larger dry zone in sapwood than the other fungi. Yezo spruce trees were also mass inoculated withC. polonica, O. penicillatum, O. piceae singly or mixed to demonstrate the ability of the fungi to kill Yezo spruce trees. The trees inoculated withC. polonica, O. penicillatum or the mixed inoculum showed discoloration of needles in the early summer of the next year and died by autumn. However, the trees inoculated withO. piceae or the control inocula did not die, except for one tree. These results indicated thatC. polonica andO. penicillatum were more virulent thanO. piceae and suggested that they might be at least partially responsible for the mortality of the beetle-infested Yezo spruce trees. Part of this study was supported by the Sumitomo Foundation, Japan to Y. Yamaoka and I. Takahashi. Part of this study was presented at the 107th meeting of the Japanese Forestry Society, April 1–4, 1996, Tsukuba, Ibaraki, at the 42nd annual meeting of the Mycological Society of Japan, May 16–17, 1998, Kyoto, and at the 110th meeting of the Japanese Forestry Society, April 2–5, 1999, Matsuyama, Ehime. Contribution No. 143, Laboratories of Plant Pathology and Mycology, Institute of Agriculture and Forestry, University of Tsukuba.     

Induced resistance of Norway spruce,variation of phenolic compounds and their effects on fungal pathogens

Three clones of Norway spruce (Picea abies) were studied for their response to mass‐inoculation with the blue‐stain fungus Ceratocystis polonica. The effect of different pretreatments (fungal inoculation and wounding) before mass‐inoculation was investigated for their possible role in an acquired resistance reaction. Pretreated trees showed enhanced resistance to the subsequent mass‐inoculation relative to control trees that received no pretreatment. Furthermore, the fungal colonization of inoculated trees was less than that of wounded trees. The phenolic content of the bark, analysed by RP‐HPLC, was compared in trees receiving different treatments. Trees inoculated with C. polonica had higher average concentration of (+)‐catechin, taxifolin and trans‐resveratrol than wounded trees. Both inoculated and wounded trees had higher average concentrations of these compounds than control trees. The effect of the phenolic extract of Norway spruce bark on the growth of the root rot fungus Heterobasidion annosum and the blue‐stain fungi C. polonica and Ophiostoma penicillatum were investigated in vitro. Heterobasidion annosum was not negatively affected, and the extracts had fungistatic effects on the blue‐stain fungi. The growth of O. penicillatum was more inhibited than the growth of the more aggressive C. polonica.     

After‐effects of drought did not predispose young Picea abies to infection by the bark beetle‐transmitted blue‐stain fungus Ophiostoma polonicum

The defence of Norway spruce against a combined attack of the bark beetle Ips typographus and its associated blue‐stain fungi is based upon a) constitutive resin stored in ducts of the bark and sapwood, and b) induced resinosis in reaction zones surrounding the point of infection. Empirically, beetle epidemics are associated with external stresses, drought being a particularly prominent factor. An attempt was made to mechanistically explain the apparent link between drought and infestations, through studying after‐effects of drought on the induced defence in stressed trees. In the field, 3–5.5 m tall trees were exposed to artificial drought over three growth seasons to investigate whether this treatment would predispose them to fungal infection in a fourth season when drought was absent. Pre‐dawn xylem water potentials down to ‐1.85 MPa and a considerable foliage depletion were recorded. In the fourth season, the trees were inoculated with Ophiostoma polonicum, a pathogenic associate of Ips typographus. No difference in susceptibility could be seen between drought stressed trees and unstressed controls. Carbohydrate concentrations of foliage and branch bark were slightly enhanced and mineral nutrient concentrations strongly reduced in stressed trees.     

Types of bark beetle (Coleoptera: Scolytidae) infestation in spruce forest stands affected by air pollution, bark beetle outbreak and honey fungus (Armillaria mellea)

583 spruce stands in an area affected by air pollution and bark beetle outbreak in Eastern Slovakia were studied in 1996. According to bark beetle infestation of dominant and codominant trees, stands were classified into following types of spruce stand decline:Ips typographus-A,Ips typographus-B,Polygraphus poligraphus, I. typographus/P. poligraphus—A,I. typographus/P. poligraphus—B. The presence of attacked trees in forest edges, bark beetle spots and forest interior was the key important factor for the classification. Data from forest inventory and forest management evidence together with data on types of spruce stands decline were used in further analyses. Results shows that the distribution of forest stands classified into different types or uninfested stands is related mainly to host size and site quality. The percentage of spruce, exposition of stands and stand density showed significant effects. The mechanisms of spreading of studied bark beetle outbreak could be explained by direct effects of stress of trees caused by an abrupt increase of level of solar irradiation and by weakening of trees by the honey fungus.     

The threshold of successful attack by Ips typographus on Picea abies: A field experiment

The spruce bark beetle, Ips typographus, was induced to attack Norway spruce by means of pheromone dispensers. The degree of attack on each tree was recorded and the trees were later categorized as surviving or dying, according to the degree of sapwood blue-staining caused by the attacks. A threshold of successful attack was observed; i.e. above a certain number of attacks the trees were successfully invaded by the beetles and their mutualistic blue-stain fungi. The height of this threshold increased with increasing tree vigour, measured as the relative increment of the sapwood cross-sectional area.     

Ophiostomatoid fungi associated with bark beetles onAbies veitchii in wave-regenerated forests

Relationships between tree mortality and bark beetle infestation onAbies veitchii at the wave-regenerated forest in Mt. Asahi, Okuchichibu area were investigated. Most of the firs with green needles and newly developed current year’s shoots in the dieback zone were heavily infested by bark beetles before the death of the trees. After heavy infestation of beetles, about half of the infested firs died within the year, and the other half died in the next year. When the species composition of bark beetles and associated ophiostomatoid fungi were investigated in Mt. Asahi and also at a typical wave-regenerated forest in Mt. Shimagare, Yatsugatake area,Cryphalus montanus andC. piceae were dominant beetle species for Mt. Asahi and Mt. Shimagare, respectively.Ophiostoma subalpinum andO. europhioides were dominant fungal species at both wave-generated forests. BecauseO. subalpinum was detected more frequently from deeper areas of sapwood thanO. europhioides, it was suggested that the fungal species may accelerate the death of stressed firs in wave-regenerated forests. Contribution No. 169, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba.     

Breeding performance of the second generation in some bivoltine populations of <Emphasis Type="Italic">Ips typographus</Emphasis> (Coleoptera Curculionidae) in the south-eastern Alps

The spruce bark beetle, Ips typographus, is one of the most extensively studied European forest pests. Gaps exist in the knowledge about second generation breeding performance in bivoltine populations. In this study, the breeding performance of the second generation was evaluated in three bivoltine populations of I. typographus in the SE Alps. Length of the maternal galleries (from 40.5 to 44.8 mm), population growth rate (PGR; from 0.7 to 3.6), and emerged adults per m² (from 669 to 1,570 insects/m²) varied among populations and were negatively correlated with bark colonisation density. Pheromone traps set up in the three investigated forests differed in the number of trapped beetles, with mean captures ranging between 5,310 and 19,850 insects per trap. The populations giving the highest captures in the traps showed the lowest bark colonisation density (248 vs. 489 maternal galleries per m²) and the best breeding performance. The populations of parasitoids and predators corresponded to just 1–9 and 2–10% of the emerging I. typographus adults, respectively, and phloem temperature never reached thresholds lethal to I. typographus. Interspecific competition was negligible, whereas intraspecific competition was found to be the main factor affecting the breeding performance of the second generation, although with different intensity according to the colonisation density. It is hypothesised that competition with the first generation and spring precipitation influence the number of suitable hosts available to the second generation.     

Biocontrol of decay in seasoning utility poles. I. Growth rate and colonizing ability of bluestain and decay fungi in vivo and in vitro

Seasoning (air drying) of utility poles for 6–12 months is essential before preservative treatment can be achieved. However, during seasoning, pine sapwood is often colonized by decay fungi, thereby compromising the performance and service life of the poles. This study investigated the potential of bluestain fungi to act as short‐term biocontrol agents against decay during seasoning. An important attribute for biocontrol is rapid growth, so growth rates of common bluestain (Ceratocystis coerulescens, Ophiostoma minus, Ophiostoma piceae, Ophiostoma piliferum, Sphaeropsis sapinea) and decay fungi (Heterobasidion annosum, Phlebiopsis gigantea, Stereum sanguinolentum) were compared on agar medium and pine in logs at various temperatures. On agar, the growth temperature optimum of most bluestain fungi and all the decay fungi was ～25°C, with little growth at ≤5°C or above 32.5°C. Overall, the fastest growing were S. sapinea and O. minus. In logs, the most effective colonizers were S. sapinea and O. minus with pathogenic abilities that made them well fitted to colonize the sapwood of freshly felled pine. Within these species, certain isolates produced much larger lesions in phloem and the sapwood tangential plane than all the decay fungi. Notably, there was significant variation in colonizing ability between different isolates within a species, emphasizing the need for testing a range of isolates when selecting a potential biocontrol agent.     

Insect natural enemies of <Emphasis Type="Italic">Ips typographus</Emphasis> (L.) (Coleoptera,Scolytinae) in managed and unmanaged stands of mixed lowland forest in Poland

We tested the effects of forest management type (managed vs. unmanaged) on the abundances of Ips typographus (L.) (Col., Curculionidae, Scolytinae) and related parasitoids and predators reared from spruce bolts exposed in Białowieża, Poland. Habitat availability for these species differed between forest types mainly as a result of regular sanitary fellings occurring in managed forests. Populations of the common polyphagous insect predators Paromalus paralellepipedus (Herbst) and Plegaderus vulneratus (Panz.) (Col., Histeridae) or parasitoids Rhopalicus tutela (Walk.), Dinotiscus eupterus (Walk.) and Roptrocerus xylophagorum Ratz. (Hym., Pteromalidae) related to I. typographus did not differ between managed and unmanaged forests. Only Thanasimus spp. (Col., Cleridae) was significantly more abundant in managed forest, possibly because it was favored by the more open, sunny stands. However, the experiment was performed in stands where spruce is only a minor component and during the non-epidemic phase of the bark beetle population, so further experiments may be necessary to determine if the effects of sanitary fellings are greater in different forest types and at different stages of the population cycle.     

Xylem dysfunction in Yezo spruce (Picea jezoensis) after inoculation with the blue‐stain fungus Ceratocystis polonica

The blue‐stain fungus Ceratocystis polonica is pathogenic to Norway spruce (Picea abies) in Europe, as well as to Yezo spruce (Picea jezoensis) and Sachalin spruce (Picea glehnii) in Japan. The wilting mechanism in P. jezoensis saplings after inoculation with C. polonica was examined based on anatomical studies of the phloem and xylem of periodically harvested trees. In addition, the course of sap ascent in the trunks was traced by injection of acid fuchsin solution at harvest. As an initial external symptom, needle discolouration was observed. In dye conduction tests, xylem dysfunction in the xylem of inoculated trees became obvious. The dehydrated xylem area (dry zone) had extended more than 20 cm above the inoculation wounds, within 1 month after inoculation. When the sap flow to the branches had nearly stopped, the leaves began to discolour. Hyphae of C. polonica colonized the ray tissue around the inoculation wounds, but were absent at the front of the dry zones. Defence reactions occurred in ray parenchyma cells adjacent to the penetrating hyphae. It is suggested that secondary metabolites, which are formed by the ray cells and epithelial cells of resin canals, are involved in the obstruction of sap flow. Limited necrotic lesions of the phloem and cambium were not associated with foliar symptoms. It is proposed that the dry zone formation caused by C. polonica is the main mechanism leading to tree death.     

Bia?owie?a Primeval Forest as a remnant of culturally modified ancient forest

The present and past population status of oak (Quercus robur) in the Bia?owie?a National Park preserve (BNP) was analyzed with respect to the historic use of the ecosystem. I assessed average parameters (density, dbh distribution, differences between habitat categories) of the oak population in the whole BNP preserve, and performed detailed analyses of local populations on three 26.5-ha and one 3.5-ha plots, representing eutrophic sites of deciduous forest (3) and a mesotrophic site of mixed forest (1). Based on the tree ring data, I reconstructed the historic dbh distribution back to 1750–1825. The results of the reconstruction confirm the early 1800s expert account and the 1889 forestry survey report. The high oak concentration in eutrophic habitats are legacies of either active game management (e.g., bison habitat improvement measures, supplementary feeding), forest recovery after the ban on forest industries (baking potash, tar, charcoal), or abandonment of inner small farms. The emergence of oak on poorer sites in the mid 1800s coincides with the ban on use of fire—a common practice that earlier had perpetuated the dominating position of fire-resistant pine. The present oak regeneration in declining spruce stands is a further step of the forest natural adaptation to environmental changes. The study supports the view that BNP is a remnant of an ecosystem substantially shaped by human uses. Modern forestry, as practiced in the managed part of the Bia?owie?a forest, does not mimic either natural processes (as observed in BNP) or historic forest uses.     

Occurrence of pathogens in associated living bark beetles (Col., Scolytidae) from different spruce stands in Austria

Pathogen occurrence was studied in 16?099 adult specimens of 10 different bark beetle species, which live associated on Norway spruce (Picea abies (L.) Karst.). Beetles (mainly Ips typographus L. and Pityogenes chalcographus L.) were collected from 6 different localities in Austria (4 secondary spruce stands and 2 natural forest type stands, 9 sampling plots in total) in elevations between 400?m and 1600?m. Various viral, protozoan, and fungal pathogens could be diagnosed with a light microscope in the examined beetles. Numerous pathogen species were known from former studies, some pathogens were totally new or could be found in a new host species beside their type host. The most dominant pathogen species were Protozoa, Gregarina cf. typographi, Malamoeba cf. scolyti, and Chytridiopsis cf. typographi. Over the whole investigation period, the highest pathogen diversity with eight pathogen species was found in I. typographus. Differences were observed in the pathogen complex of each beetle species from the different collection sites and in different years of investigation. Several species showed an overlapping in their host range and infected various bark beetle species. Furthermore, pathogen occurrence and prevalence differed in bark beetles from 4 different sampling plots in an area (one locality) within a distance of a few kilometres.