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Summary Ophiostomatoid fungi are carried by various bark beetles. However, very little is known about the role of these fungi in conifer roots. We studied ophiostomatoid fungi in roots of dying and dead Pinus sylvestris trees and tested the potential phytotoxicity of some isolates using a sensitive bioassay with Lepidium sativum in Poland. Fungi were identified based on their morphology and DNA sequencing. Three ophiostomatoid fungi, Leptographium procerum, Sporothrix inflata and Ophiostoma pallidulum, were isolated from the roots. The most abundant soil-borne fungus, S. inflata, and relatively rare O. pallidulum were isolated for the first time from roots of dying and dead pine trees. The frequency of S. inflata and O. pallidulum correlated with tree decline. The fungi were isolated more frequently from roots of dead than dying trees. Sporothrix inflata and O. pallidulum slightly reduced the stem and root growth of L. sativum. Leptographium procerum reduced more significantly root than stem growth. This species reduced root elongation 32–54% after 10–17 days of incubation.

Two species of blue-stain fungi with similar morphologies, Ophiostoma brunneo-ciliatum and Ophiostoma clavatum, are associates of bark beetles infesting Pinus spp. in Europe. This has raised questions whether they represent distinct taxa. Absence of herbarium specimens and contaminated or mistakenly identified cultures of O. brunneo-ciliatum and O. clavatum have accentuated the uncertainty regarding their correct identification. The aim of this study was to reconsider the identity of European isolates reported as O. brunneo-ciliatum and O. clavatum by applying DNA-based identification methods, and to provide appropriate type specimens for them. Phylogenetic analyses of the ITS, βT, TEF-1α and CAL gene sequences revealed that the investigated isolates represent a complex of seven cryptic species. The study confirmed that ITS data is insufficient to delineate species in some Ophiostoma species clusters. Lectotypes and epitypes were designated for O. clavatum and O. brunneo-ciliatum, and three new species, Ophiostoma brunneolum, Ophiostoma macroclavatum and Ophiostoma pseudocatenulatum, are described in the newly defined O. clavatum-complex. The other two species included in the complex are Ophiostoma ainoae and Ophiostoma tapionis. The results suggest co-evolution of these fungi in association with specific bark beetles. The results also confirm the identity of the fungus associated with the pine bark beetle Ips acuminatus as O. clavatum, while O. brunneo-ciliatum appears to be mainly associated with another pine bark beetle, Ips sexdentatus.

Interakcje pomiędzy kalusem trzech genotypówAbies albaa grzybami o różnym statusie ekologicznym

Leptographium piriforme Greif, Gibas & Currah, described in 2006 from western Canada has recently been collected in Jaroszowiec in Poland. The ophiostomatoid fungus L. piriforme was isolated from fallen Pinus sylvestris L. shoots infested by Tomicus spp. Isolates were identified to species using morphological characteristics and nuclear ribosomal DNA sequences. Two-year-old P. sylvestris seedlings were wound-inoculated with one isolate of L. piriforme. Leptographium piriforme produced necrotic lesions in the phloem of the seedlings, but none of the plant inoculated with this fungus died. This is the first report of L. piriforme from Poland and entire Europe.

Summary The aim of this study was to identify fungi associated with Cryphalus piceae on European silver fir(Abies alba) in Poland and to test the pathogenicity of selected isolates. Fungi were isolated from fivepopulations of overwintered adults and their galleries. A great diversity of taxa was associated withC. piceae. In total, 2487 isolates, including 58 species distributed in 25 genera, were obtained. The twomost frequently isolated fungi, an undescribed species of the genus Geosmithia and Ophiostomapiceae,appeared to be specifically associated with C. piceae, whereas Pesotum fragrans, Pesotum sp. andSporothrix sp. were sporadically associated. Two-year-old seedlings of silver fir were wound-inoculated with three species of fungi (Geosmithia sp., O. piceae and Pesotum sp.) recovered fromC. piceae. Only Pesotum sp. showed pathogenic ability, but we do not consider it to be an importantpathogen of A. alba. 1 Introduction The European silver fir (Abies alba Mill.) is a native species of the mountains of southernand central Europe. It is also found in Belarus, Asia and the Caucasus Mountains. InPoland, silver fir occurs in the Sudeten, the Carpathian and the S´wie˛tokrzyskie Mountains,but only forms pure stands in the eastern Carpathians (Boratyn´ski 1983). For many years,a gradual decline in its vigour has been observed in the Polish mountains, particularly in theSudeten Mountains (Bernadzki 1983). Recent studies have shown that, although abundantregeneration of the silver fir occurs in these areas (Jaworski et al. 1995; Filipiak andUfnalski 2004), significant tree damage is present. The fir stands, weakened by manyabiotic, biotic and anthropogenic factors, are attacked by phloem-feeding insects. Amongstthe bark beetles, which are secondary pests in these forests, considerable economic damageis caused by the silver fir bark beetle Cryphalus piceae (Ratz.) (Coleoptera: Scolytidae)(Michalski and Mazur 1999).Cryphalus piceae occurs in Europe and Asia, affecting several species of fir (Abies), buthas also been recorded from species of Pinus, Picea and Larix. It is usually found in themountainous regions of Poland where A. alba occurs. The fir bark beetle has a singlegeneration per year in mountain areas. Adults fly in May and June, and larvae develop inthe summer. Cryphalus piceae mainly attacks the branches and trunks of weakened, dyingand wind-fallen trees and speeds up the dieback process in weakened fir stands (Michalskiand Mazur 1999).Many bark beetle species carry ophiostomatoid fungi capable of causing blue-stain infreshly cut logs and, sometimes, damage to living trees. In some cases, the relationship isextremely close, as between Ips sexdentatus (Bo¨rn.) and Ophiostoma ips (Rumb.) Nannf.For. Path. 40 (2010) 133–144 doi: 10.1111/j.1439-0329.2009.00620.x 2009 Blackwell Verlag GmbH

Bark beetles (Coleoptera: Scolytidae) infesting Norway spruce trees are known to be associated with fungi, especially species of Ophiostoma sensu lato and Ceratocystis. However, very little is known about these fungi in Poland. In this study, we examined the ophiostomatoid species associated with seven species of bark beetles developing in Norway spruce (Picea abies). Fungi were isolated from the beetles and their galleries at ten sites in southern Poland. We identified a total of 2,769 fungal isolates that belong to 19 ophiostomatoid species. These 19 species included a total of 16 associations between fungi and bark beetles that had not been previously recorded. The isolated fungal species were similar to those previously reported. The species spectrum and relative abundance of fungal associates were similar in seven bark beetle species, despite some differences between species. Except for Pityophthorus pityographus, all bark beetles species were frequently associated with ophiostomatoid fungi. The most commonly encountered fungal associates of bark beetles were the following: Ophiostoma ainoae, O. bicolor, O. piceae sensu lato and Grosmannia piceiperda. The results support the hypothesis that pathogenic ophiostomatoid fungi are important for aggressive bark beetles to kill trees. The most virulent fungal associate of bark beetles, Ceratocystis polonica was most commonly associated with Ips typographus. The most frequent ophiostomatoid species isolated from the beetles and galleries of P. pityographus were O. ainoae and Graphium fimbriisporum.

Wstępne badania nad patogenicznością trzech grzybów siniznowych związanych zTetropiumspp. na świerku pospolitym w Polsce

The study identified and measured frequency of fungal species associated with Tomicus minor (Hart.) on Pinus sylvestris L. (Scots pine) in Poland. Additionally, fungal succession in P. sylvestris sapwood was investigated during a 12 week period following an attack by this insect. Fungi were isolated from five populations of overwintered adult beetles and their galleries with 59 species of fungi being represented among the 2880 cultures obtained. The most frequent species, Ophiostoma canum (Münch) Syd. & P. Syd., Hormonema dematioides Lagerb. & Melin, and Ambrosiella tingens (Lagerb. & Melin) L.R. Batra, appeared to be specifically associated with T. minor. The succesional changes in species composition during a 12 week period following an attack by T. minor were observed. The pattern of fungal succession in P. sylvestris sapwood essentially agreed with a general scheme of fungal succession in tree sapwood infested by bark beetles. Ambrosiella tingens was the first invader of sapwood and occurred most frequently in its deeper layers. Ophiostoma canum, H. dematioides, and other molds were also often isolated from the sapwood; however, they were most common at a depth of 5 mm during the initial phase of fungal colonization. Later, Ophiostoma canum followed A. tingens in the sapwood invasion.

Summary The association between Pityogenes bidentatus and fungi was studied in young, managed Pinus sylvestris stands in Poland. Fungi were isolated from emerged adults and their galleries collected from four populations. In total, 2089 fungal isolates including 42 species, were obtained. Penicillium sp. 1 and Geosmithia sp. 1 were the most commonly isolated fungi from beetles (49% and 41% of beetles respectively). Geosmithia sp. 1 species was the dominant species in P. bidentatus galleries with a frequency of occurrence of 57.9%. Hormonema dematioides was the second most abundant fungus in gallery systems (17.1% of wood samples). Two of the isolated Geosmithia species were previously undescribed. Pityogenes bidentatus also vectored three ophiostomatoid species: Ophiostoma minus, O. piceae and Graphium sp. ‘W’. These species were occasionally isolated from beetles and their galleries, suggesting a non-specific relationship.

The species composition of fungi associated with Ips cembrae was studied in the Raciborskie forests, Poland. The fungi were isolated from overwintered adults, larvae, new adults and from galleries at various stages of development. The results showed that there was a great diversity of fungi associated with this insect. We isolated 2877 cultures, including 61 species. The most important group of fungi were ophiostomatoid species. From these, Ceratocystis laricicola, Ophiostoma brunneo-ciliatum and Graphium laricis were commonly detected, whereas eight other species occurred less frequently or sporadically. Ophiostoma ips is documented here as a new associate of I. cembrae. Ceratocystis laricicola was shown to be the primary invader occurring most frequently at early stages of brood development, particularly in the sapwood. Ophiostoma brunneo-ciliatum and G. laricis were secondary invaders following C. laricicola. In the later stages of brood development other ophiostomatoid fungi appeared.

The association between Tomicus piniperda L. (Col., Scolytinae) and fungi was studied in a Pinus sylvestris L. forest in Mielec-Mościska. Fungi were isolated from overwintered adult beetles taken from two stands situated in different distance from timber yard. Two media were used for isolation. The results showed great diversity of fungi associated with T. piniperda: 1895 cultures, representing 64 species, were isolated. Penicillia and Hormonema dematioides were the dominant species, found in 20.2% and 17.8% of all beetles, respectively. A frequently isolated ophiostomatoid fungi was Ophiostoma minus. Qualitative and quantitative differences in the mycobiota composition of this insect between two stands were detected. The highest richness and diversity of fungal species appeared in the samples taken from the location where the trees were heavily damaged by shoot-feeding of T. piniperda. Differences were most clear for the pathogenic O. minus, which was a common fungal associate of the insects in this stand.

The species composition and virulence of fungi associated with Tomicus piniperda were studied at eight locations in Poland. The fungi were isolated from phloem and sapwood samples taken from insect galleries and then identified. The virulence of the most common ophiostomatoid species was evaluated through inoculations using two-year-old Scots pine seedlings. A great diversity of fungi were found associated with T. piniperda, including 4 837 cultures and 67 species. The most important groups of fungi were the ophiostomatoids and moulds, including mainly Penicillium, Trichoderma and Mucor species. Among ophiostomatoid fungi, Ophiostoma minus and O. piceae dominated, with a frequency of occurrence of 32.4 and 11.5% of inspected galleries, respectively. Occasionally isolated species included Leptographium lundbergii, L. procerum, L. wingfieldii, Graphium pycnocephalum and Graphium sp. 'W'. In general, the frequency of the ophiostomatoid species was highly variable among locations. Leptographium wingfieldii and O. minus, were the only species capable of killing whole plants and penetrated deeper into the sapwood than other species (87-100% mortality during the 11 week incubation period). Other fungi, including O. piliferum, O. piceae and L. procerum, were considerably less virulent. associated fungi / Leptographium / Ophiostoma / Pinus sylvestris / Tomicus piniperda / virulence

Summary 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. Resume La frequence et la distribution des especes fongiques associees avec le scolyte I. typographus (Scolytidae) a eteetudiee sur des epiceas presentant differents etats sanitaires dans le sud de la Pologne. Les epiceas ont ete caracterises par leur degre de defoliation et de degât sur tronc et houppier apres attaque par I. typographus. L’etat de sante des arbres dont sont issus les echantillons a ete reliea differents stades de developpement des generations-filles de I. typographus. Les champignons ont ete isoles a partir de phloeme dans et autour des galeries d'insectes et dans l'aubier sous-jacent. Les echantillons ont ete preleves a partir d'arbres apparemment sains ou affaiblis, de volis, chablis et d'arbres pieges. Les champignons associes aI. typographus se sont averes tres diversifies en terme de nombre d'especes. 65 taxons ont ete obtenus a partir du phloeme des arbres infestes par I. typographus et 36 taxons a partir de l'aubier situe sous les galeries. Ces champignons sont majoritairement des ascomycetes ou representent des anamorphes. Les Ophiostomatoides, representes par 14 especes, sont le groupe le plus represente, dans toutes les niches etudiees. Les especes ophiostomatoides les plus frequentes sont C. polonica, O. ainoae, O. bicolor, O. penicillatum, O. piceae et O. piceaperdum. La frequence des differents champignons ophiostomatoides a montre des variations significatives entre le phloeme et l'aubier et en relation avec l’etat sanitaire des arbres. Ces differences quantitatives relatives aux champignons associes aI. typographus entre epiceas appartenant a differentes classes d’etat sanitaire peuvent s'expliquer par des processus de succession dans la colonisation fongique des tissus apres l'attaque de I. typographus. L'espece pathogene C. polonica est l'envahisseur primaire, tres souvent rencontree dans l'aubier d'arbres apparemment sains. O. bicolor, O. penicillatum et O. piceaperdum sont aussi frequemment trouves en relation avec des stades precoces des generations-filles de I. typographus sur des arbres apparemment sains, mais ils colonisent preferentiellement le phloeme. Au contraire, O. ainoae, O. minuta et O. piceae suivent probablement les especes precedentes, comme envahisseurs secondaires ou tertiaires dans le phloeme ou l'aubier des epiceas. Zusammenfassung In Sudpolen wurde das Artenspektrum und die Haufigkeit von Pilzen, die mit dem Borkenkafer I. typographus assoziiert sind, an Fichten mit unterschiedlichem Gesundheitszustand untersucht. Die Baume wurden nach dem Kaferbefall aufgrund des Zustandes von Krone und Stamm klassifiziert. Der Gesundheitszustand der beprobten Baume hing von den unterschiedlichen Stadien der Brutentwicklung von I. typographus ab. Die Pilze wurden aus dem Phloem und dem Splint aus den Brutgangen und ihrer Umgebung entnommen. Das Probenmaterial stammte von ‘gesund’ aussehenden, geschwachten, vom Wind geworfenen und gebrochenen Baumen sowie von Fangbaumen. Die mit I. typographus assoziierte Pilzflora war bezuglich iher Artenzahl sehr divers; aus dem Phloem kaferbefallener Baume wurden insgesamt 65 Taxa erhalten und 36 Taxa kamen im Splint unter den Brutgangen vor. Uberwiegend waren dies Ascomyceten und imperfekte Pilze. Die Gruppe der ophiostomatoiden Pilze war mit 14 Arten in allen untersuchten Nischen am haufigsten vertreten. Die dominierenden Arten waren hier C. polonica, O. ainoae, O. bicolor, O. piceae und O. piceaperdum. Die Haufigkeit des Vorkommens von ophiostomatoiden Pilzen war sowohl zwischen Phloem und Splint als auch zwischen Baumen mit unterschiedlichem Gesundheitszustand signifikant verschieden. Diese quantitativen Unterschiede in der Pilzgemeinschaft von I. typographus zwischen Baumen mit unterschiedlichem Zustand kann durch das Suckzessionsmuster der Pilze im Wirt nach einem Kaferbefall erklart werden. Als Primarbesiedler trat die pathogene Art C. polonica auf, sie kam am haufigsten im Splint von noch ‘gesund’ aussehenden Baumen vor. Opiostoma bicolor, O. penicillatum und O. piceaperdum kamen ebenfalls in fruhen Stadien der Brutgangentwicklung von I. typographus auf noch ‘gesund’ aussehenden Baumen vor, sie besiedelten aber vorwiegend das Phloem. Im Gegensatz dazu treten O. ainoae, O. minuta und O. piceae wahrscheinlich nach den genannten Arten als sekundare und tertiare Besiedler des Phloems und Splints auf.

Phytophthora plurivora and other Phytophthora species are known to be serious pathogens of forest trees. Little is known, however, about the presence of P. plurivora in Polish oak forests and their role in oak decline. The aims of this study were to identify P. plurivora in healthy and declining Quercus robur stands in southern Poland and to demonstrate the relationship between different site factors and the occurrence of P. plurivora. In addition, the virulence of P. plurivora and other Phytophthora species was evaluated through inoculations using 2-year-old oak seedlings. Rhizosphere soil was investigated from 39 oak stands representing different healthy tree statuses. The morphology and DNA sequences of the internal transcribed spacer regions (ITS) of the ribosomal DNA and the mitochondrial cox1 gene were used for identifications. P. plurivora, an oak fine root pathogen, was isolated from rhizosphere soil samples in 6 out of 39 stands. Additionally, Phytophthora cambivora, Phytophthora polonica and Phytophthora rosacearum-like were also obtained from several stands. The results showed a significant association between the presence of P. plurivora and the health status of oak trees. Similar relationships were also observed for all identified Phytophthora species. In addition, there was evidence for a connection between the presence of all identified Phytophthora species and some site conditions. Phytophthora spp. occurred more frequently in declining stands and in silt loam and sandy loam soils with pH ≥ 3.66. P. plurivora and P. cambivora were the only species capable of killing whole plants, producing extensive necrosis on seedling stems.

Phytophthora cambivora (Straminipila, Oomycota) causes root rot and stem canker on several deciduous tree species in Europe. However, very little is known about the variation in susceptibility to P. cambivora colonisation among provenances and families of pedunculate oak (Quercus robur L.). We studied variation in susceptibility of one French and 16 Polish provenances, representing 62 families. Samples were taken from three test plots located in the Brzesko Forest District. Oak susceptibility to P. cambivora was assessed by measuring lesion length following inoculation of excised shoots with two isolates of P. cambivora. There was significant variability in susceptibility among the 17 provenances tested. The highest susceptibility to P. cambivora was apparent in several provenances including Tronçais, Zaporowo, Runowo, Opole and Krotoszyn; while the most resistant provenances originated from Chojnów, Siedlce, Płock, Krotoszyn-90 and Wioska. There was also significant within-provenances variation in susceptibility to P. cambivora.

Summary Ophiostomatoid fungi are known to be associated with various species of bark beetles. However, information about fungal associates of root-feeding bark beetles in Europe is still fragmentary. For this reason, the fungal associates of Hylastes ater, H. opacus and Hylurgus ligniperda on Pinus sylvestris were isolated and identified. A total of 743 fungal isolates were collected and separated into 10 morphological groups. Analyses of ITS rDNA and partial β-tubulin gene sequences confirmed that these groups represented distinct species. The 10 species included a total of 13 associations between fungi and bark beetles that had not been recorded previously. All of the bark beetles examined were frequently associated with ophiostomatoid fungi. The fungal diversity and relative abundance of species were very similar in the three species of root-feeding bark beetles. The most commonly encountered associates of these beetles were Grosmannia radiaticola, Leptographium lundbergii, L. procerum and L. truncatum. Insect infestation data furthermore suggest that Hylastes spp. and Hg. ligniperda are also important vectors of the fungal pathogen Sphaeropsis sapinea.

The ophiostomatoid fungi associated with cerambycid beetles Tetropium spp. (their symbiotic vectors) colonizing Norway spruce in Poland (six species collected) were isolated. The virulence of representative isolates was evaluated through inoculations using 2-year-old Norway spruce seedlings. A total of 1325 isolates (Ophiostoma piceae, O. tetropii, O. minus, Grosmannia piceiperda, G. cucullata, and five other less frequent taxa) were obtained. Tetropium castaneum and T. fuscum were vectors of similar spectra of ophiostomatoid fungi although some differences in fungal frequency between these Tetropium spp. were found. Among the fungal associates of the Tetropium spp. collected only G. piceiperda was pathogenic, which suggests that it can play a role in the death of spruce trees following attack by Tetropium spp.