Fungi occurring in the roots and basal parts of one‐ and two‐year‐old spruce and pine stumps

The occurrence of fungi in the underground parts of 1‐ and 2‐year‐old conifer stumps (Pimis sylveslris and Picea abies) was studied in two clear‐felled areas of eastern Uppland in Sweden, and most fungal isolates were identified to species.

Decay fungi (Basidiomycetes) were more frequent in spruce stumps than in pine stumps, and their incidence was highest in 2‐year‐old stumps. The occurrence of blue‐stain fungi and hyaline mycelia did not differ significantly between stumps of different species or age. Within stumps, decay fungi were most frequent in large roots close to the cut surface, whereas blue‐stain fungi and hyaline mycelia were most abundant in small roots. The occurrence of yeasts, bacteria and fast‐growing moulds was also recorded.     

Blue‐stain infections in roots,stems and branches of declining Pinus sylvestris trees in a dry inner alpine valley in Switzerland

Increased mortality rates in Scots pine (Pinus sylvestris) forests have recently been observed in the inner alpine Swiss Rhone valley. Drought, in combination with stand competition, mistletoe infections as well as nematode and insect infestations, appears to be the main factor for the decline. In focus of this study was the occurrence and role of fungal pathogens in the decline dynamics. Branches, stems and roots of 208 trees in five different crown transparency classes were collected and inspected for blue stain and fungal infections. Neither Armillaria species nor Heterobasidon annosum s. str. were detected, but blue stain was commonly observed. Visible blue stain increased with increasing crown transparency. Among the recently dead trees, 80% showed visible blue stain in the branches, 90% in the roots and 100% in the stems. In the crown transparency classes 2 and 3 (25–60% crown transparency), five of the 103 trees showed visible blue stain in the roots, one of 130 trees in the stem but none in the branches. Blue‐stain fungi were isolated from all parts of the trees and from all crown transparency classes. Overall incidence of blue stain was highest in the roots and lowest in the branches. In class 2, roots of 60% of the trees were visibly blue‐stained or developed blue stain in culture, but stems of only 24% and branches of 14% of the trees. In the roots Leptographium species, mostly L. serpens, dominated. From stems and branches, mainly Ophiostoma species were isolated. The positive relationship between the incidence of blue stain and crown transparency, in combination with the high infection levels of roots of fairly vigorous Scots pines, indicates the pathogenic potential of the blue‐stain fungi. Hence, these fungi together with their insect vectors may well act as an important contributing factor involved in pine decline.     

Ophiostomatoid fungi isolated from three different pine species in Argentinian Patagonia

Pine plantations in Argentinian Patagonia cover ca. 95,000 ha in Chubut, Río Negro and Neuquén provinces. Exotic bark beetles (Orthotomicus laricis, Hylastes ater and Hylurgus ligniperda) commonly occur in freshly cut logs, stumps and slash. These beetles are vectors of “ophiostomatoid” fungi which include primary tree pathogens as well as important agents of blue stain. The aim of this study was to identify these beetle‐associated fungi. Sawing mills and pine plantations were surveyed three consecutive years. Fungal isolates from stained logs, processed wood and insect galleries were identified based on morphological and DNA sequence comparisons of ITS and β‐tubulin gene regions. Two Grosmannia, one Graphilbum and three Ophiostoma species were identified. Ophiostoma piliferum and O. peregrinum sp. nov. were the most frequently isolated taxa. O. peregrinum occurred in all provinces, colonizing different conifer species and, interestingly, also the native broadleaved species Nothofagus dombeyi. Pine plantation forestry in southern South America includes Argentina, Brazil, Chile, Paraguay and Uruguay. Emerging data from Argentina, Chile and Uruguay revealed some coincidences between these countries, but also several differences, probably, as a result of multiple introduction events.     

Blue stain fungi associated withIps cembrae (Coleoptera: Scolytidae) in Japanese larch in connection with their brood development

The association between blue stain fungi andIps cembrae Heer (Coleoptera: Scolytidae) was investigated in the Japanese larch,Larix leptolepis Gordon, in the Nagoya University Forest, Aichi Prefecture, central Japan.I. cembrae had one or two generation(s) in a year in this study area. Two blue stain fungi,Ophiostoma piceae andLeptographium sp., were isolated from the body surface of both male adults in mating chambers and female adults in parent galleries, suggesting that this beetle species was a vector of these fungi. Although no blue stain fungi were isolated from non-stained wood, both fungi were isolated from the mating chambers, the center and the uppermost end of the galleries throughout the season. The fact thatO. piceae was consistently isolated with high frequency from adults and from their galleries strongly suggested that this species would be the principal blue stain fungus infecting the beetle-attacked larch trees.     

Association of Diplodia mutila with blue stain of Caribbean pine in Venezuela

Diplodia mutila was isolated from blue‐stained Caribbean pine timber (Pinus caribaea var. hondurensis) in eastern Venezuela. The ability of D. mutila to cause blue stain was demonstrated in vitro.     

Chestnut Red Stain: Identification of the fungi associated with the costly discolouration of Castanea sativa

In Europe, fungal pathogens have reduced the overall productivity of sweet chestnut (Castanea sativa) stands and continue to threaten the economic viability of forestry operations. Chestnut Red Stain (CRS) in north‐eastern Spain, locally referred to as Roig, is capable of decreasing the market value of chestnut timber to the point of rendering chestnut coppices uneconomical. Despite its economic importance, the specific cause of this red discolouration is unknown. With the objective of verifying the presence of fungi within the symptomatic wood, and identifying the fungus or suite of fungi associated with the red stain, wood samples were collected and cultured from 37 stumps found in eight recently harvested stands in the Montseny and Montnegre‐Corredor Natural Parks. To separate the fungi associated with CRS from other species inhabiting the chestnut wood, the origin of each fungal culture was mapped in every stump. The fungi were isolated from cultures and identified by sequencing the ITS region. The results provide insight into the fungal community inhabiting chestnut wood and the potential cause of CRS; nine species were identified including two species known to cause decay in chestnut. One of them, Fistulina hepatica, appears to be a likely candidate for the causal agent of CRS. This is the first study reporting the fungi associated with CRS and opens the door to new epidemiological studies focused on F. hepatica.     

Factors affecting the fungal colonization of pine lumber

This study investigated whether the physiological condition of wood influenced patterns of colonization by mould and sapstain fungi. The extent of fungal defacement on sawn pine lumber Pinus nigra var. maritima that had been killed by gamma‐irradiation was compared with the defacement on untreated, still living timber that had been aged for up to 16 weeks prior to being sawn. All the sawn lumber was exposed to the natural inoculum of sapstain and mould fungi in a working sawmill environment over a 4‐week period. The results indicated that the pattern of fungal defacement differed markedly in dead or aged wood compared with untreated wood. Mould fungi were most prevalent on the dead irradiated wood, whereas sapstain fungi dominated the freshly sawn lumber which was still living. The differences appear to be independent of wood moisture content and may be related to the production of inhibitory compounds by living cells in wood as it becomes senescent and dies.     

Fungi isolated from Picea abies infested by the bark beetle Ips typographus in the Białowieża forest in north‐eastern Poland

The assemblage of fungi occurring in the sapwood of Norway spruce (Picea abies) and in bark beetle galleries following attack by the Eurasian spruce bark beetle Ips typographus was investigated in the Bia?owie?a forest in north‐eastern Poland. Fungi were isolated from blue‐stained sapwood of beetle‐infested spruce trees in June 2002, and a few isolates were also obtained from ascospores and conidia taken from perithecia and asexual structures occurring in the gallery systems of the insects. The mycobiota of I. typographus in the Bia?owie?a forest was dominated by ophiostomatoid fungi, which were represented by seven species. Four species, including Ceratocystis polonica, Grosmannia penicillata, Ophiostoma ainoae and Ophiostoma bicolor were isolated at high frequencies, whereas three other taxa, Ceratocystiopsis minuta, Ceratocystiopsis alba and a Pesotum sp. were rare. The anamorphic fungus Graphium fimbriisporum and yeasts also occurred occasionally. In addition, the basidiomycete Gloeocystidium ipidophilum was relatively common. The pathogenic blue‐stain fungus C. polonica was the dominant fungal associate of I. typographus in the Bia?owie?a forest, which is consistent with a previous study at this area in the 1930s. Ceratocystis polonica was the most frequently isolated species at the leading edge of fungal colonization in the sapwood and had on an average penetrated deeper into the wood than other fungal associates. This suggests that it acts as a primary invader into the sapwood after attack by I. typographus in the Bia?owie?a forest, followed by O. bicolor, O. ainoae, G. ipidophilum and G. penicillata. Thus far, the Bia?owie?a forest is one of the few areas in Europe, where C. polonica has been reported as a dominate fungal associate of I. typographus.     

Characterization of Cylindrocarpon‐like species associated with litter in the old‐growth beech forests of Central Europe

Cylindrocarpon‐like fungi are globally distributed plant pathogens and have a wide range of host species. However, very little is known about the species that live in the topsoil of forests and their potential role in reducing the natural regeneration of tree species, particularly of forest trees that produce abundant fruit only once every few years. To enhance our understanding of the species that inhabit the topsoil, we studied the diversity and pathogenicity of Cylindrocarpon‐like fungi inhabiting the litter in old‐growth mixed‐beech forests in the Carpathians (Poland) and in the Alps (Austria), and in a managed beech stand in the Krakowsko‐Cz?stochowska Highland (Poland). The fungi inhabiting the beech litter were investigated using beechnuts and pine seedlings as bait. Isolates were identified based on morphology and DNA sequencing. The pathogenicity of the most common species was investigated by inoculating beech germinants. A wide range of Cylindrocarpon‐like fungi were associated with the beech litter: 718 cultures representing 12 species were isolated. Five taxa were identified down to species level: namely Ilyonectria crassa, I. pseudodestructans, I. rufa, Neonectria candida and N. obtusispora, and seven species were identified to genus level (Neonectria or Ilyonectria species). Ilyonectria destructans, which is considered to be the sexual morph of ‘Cylindrocarpon destructans’, was not found. There were qualitative and quantitative differences between the different forest sites in terms of Neonectria and Ilyonectria species composition in beech litter. The isolation frequency and species richness of Cylindrocarpon‐like fungi were greatest in beech litter taken from old‐growth mixed‐beech forests. Neonectria and Ilyonectria species were capable of killing beech germinants, suggesting that they may play a negative role in natural beech regeneration.     

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.     

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.     

Pathogenicity of Ceratocystis resinifera to Norway spruce

The blue‐stain fungus Ceratocystis resinifera colonizes wounds on living Picea spp. and other conifers in Europe and North America. Little is known regarding the pathogenicity of this fungus and consequently, four Norwegian C. resinifera isolates were inoculated on to Norway spruce (Picea abies) using two different techniques. These included single‐point inoculations on young trees (two inoculations per tree on 14‐year‐old trees) and mass‐inoculations on older trees (～200 inoculations per tree on 34‐year‐old trees). In both experiments, C. resinifera induced minor symptoms that in most cases did not differ significantly from inoculation with sterile agar. The virulent blue‐stain fungus C. polonica, which was inoculated for comparative purposes, induced extensive symptoms, causing 83% dead cambium circumference and 82% blue‐stained sapwood, and long necrotic lesions in the phloem. The results suggest that C. resinifera is non‐pathogenic or only mildly pathogenic to Norway spruce and does not present a threat to these trees.     

Botryosphaeriaceae associated with die‐back of Schizolobium parahyba trees in South Africa and Ecuador

Die‐back of Schizolobium parahyba var. amazonicum is a serious problem in plantations of these trees in Ecuador. Similar symptoms have also been observed on trees of this species in various parts of South Africa. The most common fungi isolated from disease symptoms on S. parahyba var. amazonicum in both locations were species of the Botryosphaeriaceae. The aim of this study was to identify these fungi from both Ecuador and South Africa, and to test their pathogenicity in greenhouse and field trials. Isolates obtained were grouped based on culture morphology and identified using comparisons of DNA sequence data for the internal transcribed spacer (ITS) and translation elongation factor 1α (TEF‐1α) gene regions. The β‐tubulin‐2 (BT2) locus was also sequenced for some isolates where identification was difficult. Three greenhouse trials were conducted in South Africa along with a field trial in Ecuador. Neofusicoccum parvum was obtained from trees in both areas and was the dominant taxon in South Africa. Lasiodiplodia theobromae was the dominant taxon in Ecuador, probably due to the subtropical climate in the area. Isolates of Neofusicoccum vitifusiforme (from South Africa only), Neofusicoccum umdonicola and Lasiodiplodia pseudotheobromae (from Ecuador only) were also obtained. All isolates used in the pathogenicity trials produced lesions on inoculated plants, suggesting that the Botryosphaeriaceae contribute to the die‐back of S. parahyba trees. While the disease is clearly not caused by a single species of the Botryosphaeriaceae in either region, N. parvum has been introduced into at least one of the regions. This species has a broad host range and could have been introduced on other hosts.     

First report of botryosphaeriaceous fungi causing canker on Cedrela fissilis and leaf spots on Cariniana estrellensis in forest nursery in Brazil

Cariniana estrellensis and Cedrela fissilis are native forest species from Brazil that are widely used for arborization of parks and recuperation of degraded areas. The production of seedlings from these two forest trees in nurseries is severely affected by fungal diseases. Morphological identification, sequencing analysis of three gene regions (TEF+ACT+ITS or TEF+TUB2 + ITS) and pathogenicity tests confirmed that fungi belonging to Botryosphaeriales are the aetiological agent of the diseases. This is the first report of Phyllosticta capitalensis causing leaf spots on C. estrellensis and Botryosphaeria dothidea causing canker on C. fissilis in forest nurseries in Brazil.     

Dieback and decline of wild almond (Amygdalus scoparia Spach) in the Harat protected forest of Yazd Province,Iran

Wild almond (Amygdalus scoparia) is a dominant shrub species in mountain forests of the Irano‐Turanian region. Dieback and decline symptoms of wild almond shrubs were first observed in Harat protected forest (Yazd, Iran) in the autumn of 2014. Since then, the incidence and severity of the disease have increased. To study the aetiology and estimate losses from the disease, field studies were conducted in 2016 and 2017. For this purpose, eight stands were selected, in which 24%–99% of the trees were wild almond in their species composition. In total, 50 cankers and 50 infected twigs of wild almond shrubs were collected. Fungal isolates were identified based on morphological and cultural characteristics, as well as sequence data of ITS1 + 5.8S + ITS2 rDNA. Pathogenicity of fungi was checked on detached shoots and evaluated after 40 days by measuring the discoloured lesion length at the inoculation site. Among 681 wild almond shrubs checked, 84.44% showed decline symptoms with different severities; only 15.56% did not show observable disease symptoms. The most frequent colonizers of infected tissues taken from cankers were Wilsonomyces carpophilus (56%), Thyrostroma cornicola (40%) and Collophorina paarla (30%). Infected twigs were extensively colonized by W. carpophilus (36%), Th. cornicola (24%) and Ulocladium consortiale (24%). Other species, such as Endoconidioma populi, Microsphaeropsis olivacea, Trichoderma asperellum, Paecilomyces formosus, Saccothecium rubi, Preussia sp. and Chaetomium globosum, had lower isolation frequencies. W. carpophilus, Th. cornicola, C. paarla and U. consortiale were found as pathogens on detached wild almond shoots. Based on the frequency of the isolates and the pathogenicity tests, four fungi, such as W. carpophilus, Th. cornicola, C. paarla and U. consortiale, are considered serious contributing agents playing a significant role in the dieback and decline of wild almond. All isolated species are reported for the first time on the wild almond shrubs of the world.     

Botryosphaeriaceae species causing canker and dieback of English walnut (Juglans regia) in Italy

English walnut (Juglans regia) is an important nut crop worldwide and is currently considered emerging in Italy. Botryosphaeriaceae fungi cause symptoms including cankers, discoloration and dieback, and several species are reported across the world on walnut. In this study, symptomatic trees from an orchard in Southern Italy showing branch dieback, cankers, wood discoloration and gummosis were surveyed. Three different fungi from the Botryosphaeriaceae were consistently isolated from symptomatic tissues. Representative isolates were characterized using morphological and molecular approaches based on conidial morphology, optimum growth temperature and the comparison of DNA sequence data from the ITS, tef1‐α and tub2 loci. Three species were identified: Botryosphaeria dothidea, Neofusicoccum mediterraneum and Neofusicoccum parvum. Pathogenicity tests on detached fruits and potted plants showed that all three species were pathogenic. To our knowledge, this is the first report of these Botryosphaeriaceae species causing canker and dieback on English walnut in Italy.     

Mountain pine beetle‐associated blue‐stain fungi in lodgepole × jack pine hybrids near Grande Prairie,Alberta (Canada)

The mountain pine beetle (MPB), the most serious pest of lodgepole pine in mountainous western Canada, spread northeastward into lodgepole × jack pine hybrids in the boreal forest of Alberta in 2006. The MPB vectors three species of blue‐stain fungi, which contribute to the success of the beetles. These fungi were isolated from MPB larvae and galleries in several lodgepole × jack pine stands in the Grande Prairie region of northwestern Alberta in autumn 2006 and winter and spring 2007. Fungi were recovered from more than 95% of gallery systems. The three fungi were similarly prevalent but Ophiostoma montium was the most frequently isolated fungus at each sampling point, isolated from 72% to 90% of gallery systems compared with 63% to 78% for Grosmannia clavigera, and 61% to 86% for Leptographium longiclavatum. Ophiostoma montium and G. clavigera were isolated from more larvae than gallery samples, with the opposite true for L. longiclavatum. Most gallery systems contained multiple fungi with three fungi per gallery system being more common in autumn and winter and two fungi more common in the spring. The combination of G. clavigera and L. longiclavatum was less common among gallery systems with two fungi than either of the pairwise combinations containing O. montium. Fungal prevalence was the same above and below snow level. The prevalence of the three fungi did not differ significantly among stands sampled in the spring but stands with more G. clavigera tended to have less L. longiclavatum. The winter of 2006–2007 was colder than average throughout Alberta with temperatures below ?30°C in November, January and February, and all three fungi were present after the cold winter while most larvae had died, suggesting that overwintering mortality of the fungi will not limit persistence and spread of MPB in the boreal forest.     

Antifungal effect of bark extracts from some European tree species

The inhibitory effect of methanol bark extracts from six deciduous and three coniferous European tree species were bioassayed against eight fungi from the different damage categories, brown rot, white rot, canker and blue-stain. This is the first report providing data on the antifungal activity of several Europaen tree species against fungi within these damage categories. Generally the decay fungi were more inhibited by the bark extracts than the blue-stain fungi, while the lowest inhibition was found among the cancer fungi. The main pattern found between the fungal groups in relation to the bark extracts in this study is believed to be caused by the route of ingress. Acer platanoides bark extract proved to be the most efficient bark extract tested, significantly reducing the growth rate of all tested fungi. Betula pubescens bark extract generally gave the weakest reduction in growth rate. In this study, the conifer bark extracts were in general more active against the canker and blue stain ascomycete fungi than the deciduous trees extracts.     

A comparison of heat pulse velocity and lesion lengths for assessing the relative virulence of mountain pine beetle‐associated fungi on jack pine

The mountain pine beetle (MPB) vectors three blue‐stain fungi, Grosmannia clavigera, Ophiostoma montium and Leptographium longiclavatum, which contribute to the success of the beetles and the death of the trees. The utility of two methods, heat pulse velocity (HPV) and lesion length, for assessing the relative virulence of these fungi were compared on jack pine in central Alberta. The HPV monitoring apparatus failed to detect xylem sap flow in any of the trees and, thus, could not be used to assess fungal virulence. In contrast, measurement of lesion lengths was more sensitive and provided further evidence that G. clavigera and L. longiclavatum are more virulent than O. montium. The failure of the HPV apparatus to detect sap flow suggests that the study trees were moisture stressed, a factor likely to increase their susceptibility to MPB. Thus, this method is not appropriate for assessing the response of the most susceptible (i.e. drought stressed) trees to MPB and its associated fungi.     

Ophiostomatoid fungi associated with root‐feeding bark beetles on Scots pine in Poland

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.