Occurrence and pathogenicity of fungi in necrotic and non-symptomatic shoots of declining common ash (<Emphasis Type="Italic">Fraxinus excelsior</Emphasis>) in Sweden

Currently, massive dieback of Fraxinus excelsior is observed in countries of eastern, northern and central Europe, and the reasons for it are unclear. The aims of the present work were (a) to study fungal communities in declining F. excelsior crowns; (b) to clarify role of fungi in the decline. Shoots from symptomatic crowns were collected in four localities in central Sweden, and distributed into the following categories: (a) visually healthy; (b) initial necroses; (c) advanced necroses; (c) dead tops. The most frequently isolated fungi were Gibberella avenacea, Alternaria alternata, Epicoccum nigrum, Botryosphaeria stevensii, Valsa sp., Lewia sp., Aureobasidium pullulans and Phomopsis sp., and these taxa were consistently found in shoots of all four symptomatic categories. Forty-eight taxa of other fungi were isolated, and fungal diversity was not exhausted by the sampling effort. The same taxa of fungi were dominant in F. excelsior shoots of different symptomatic categories, and moderate to high similarity of fungal communities was observed in shoots despite the symptoms. Forty-four isolates from 24 fungal taxa were used for artificial inoculations of 277 1-year-old F. excelsior seedlings in bare root nursery. After 2 years, only four fungi caused symptomatic necroses of bark and cambium: A. alternata, E. nigrum, Chalara fraxinea and Phomopsis sp. The most pathogenic was C. fraxinea, inducing symptoms on 50% of inoculated trees, while three other fungi caused necroses on 3–17% of inoculated trees. Infection biology of C. fraxinea and environmental factors determining susceptibility of F. excelsior to decline deserve future investigations.     

The culturable leaf mycobiome of Viscum album subsp. austriacum

In Poland's pine forests, mistletoe (Viscum album subsp. austriacum) abundance has increased over the past 20 years. This mistletoe infestation has decreased the growth and vigour of Scots pine trees. In this study, we surveyed the culturable fungi from healthy and diseased V. album subsp. austriacum leaves from two stands in Poland. In total, 63 distinct species were identified, 99.8% of which belonged to the phylum Ascomycota. The community compositions of fungi in mistletoe leaves were similar in healthy and diseased leaves as Alternaria alternata, Aureobasidium pullulans, Botryosphaeria visci, Fusarium paeoniae and Microsphaeropsis olivacea were consistently found in leaves of all symptom types. The most frequently isolated fungus from asymptomatic leaves was M. olivacea, followed by A. alternata, A. pullulans and Hypoxylon rubiginosum. In comparison, the most frequently isolated fungi from leaves with rusty-brown necrotic spots were (in decreasing order) M. olivacea, B. visci, F. paeoniae and A. alternata, while the most frequently isolated fungi from leaves with black or dark brown spots were M. olivacea, A. alternata, A. pullulans, Epicoccum layuense and F. paeoniae. This study was the first comprehensive report showing that certain fungal species may be pathogens of V. album subsp. austriacum in Poland. This study was also the first report of F. paeoniae, F. juglandicola, Diaporthe vacuae and Heterotruncatella spartii from V. album, and the first report of D. vacuae and H. spartii in Poland.     

Impregnated and covered birch plywood performance during external exposure

We evaluated the performance of covered and impregnated birch plywood (BP) in the outdoor environment. Fungal growth, crack formation, and surface hydrophobicity were regularly assessed over 36 months. After 2 months, uncovered BP was severely colonised by disfiguring fungi. During the test period, the surface of plywood with better coatings (solvent- or water-based) was not overgrown or was overgrown slightly with fungi, no cracks were formed, and the surface hydrophobicity was stable. Similar results were observed for laminates with phenol and melamine film. Plywood impregnated with copper and organic biocides, with retention conforming to Use Class 3 (EN 335:2013), did not protect the plywood from crack formation within several months and the severe growth of disfiguring fungi within a year. The blue stain fungus Aureobasidium pullulans was identified for all overgrown test variants. No rot fungi were detected after a 36-month exposure.     

Aureobasidium pullulans (de Bary) Arnaud: An associate of Alnus rubra and Lactarius obscuratus mycorrhizae1

Aureobasidium pullulans was isolated from Alnus rubra+Lactarius obscuratus mycorrhizae in 50% of cases. Antibiotic effects against the fine root pathogen, Fusarium oxysporum, were found.     

Fungal colonisation of outside weathered modified wood

Specimens of Scots pine sapwood (Pinus sylvestris L.) and beech wood (Fagus sylvatica L.) were treated with an amino-alkyl-functional oligomeric siloxane, a sodium water glass solution and 1,3-dimethylol-4,5-dihydroxyethylene urea (DMDHEU). Treated and untreated wood specimens were exposed outdoors without ground contact. After 9?months of outside exposure, all specimens showed discolouration caused by infestations of mould and staining fungi on the exposed wood surface. Fungi grown on the sample surface were isolated and identified by microscopic technique and sequencing of PCR-amplified DNA from the ITS region. Primarily, an infestation by ascomycetes and related deuteromycetes was found. The most dominant fungi were Trichoderma sp. and Epicoccum sp.. An infestation of Aureobasidium pullulans was only detected on untreated and DMDHEU-treated samples. There were only marginal differences of fungal infestation between the two wood species.     

Fungi in leaves,twigs and stem bark of Populus tremula from northern Spain

Fungal species were recorded from Populus tremula leaves, twigs and bark (trunk) at eight sites in the northern region of Spain, with the aim of evaluating the effect of sampling site, sampling tissue and isolation method on the frequency and species distribution of the fungi recovered from necrotic and healthy tissues. Two different isolation methods were used: the first consisted of finding fruitbodies on plant tissues after incubating them in wet and warm conditions, and the second included growing mycelia from plant fragments plated on potato dextrose agar (PDA). A total of 48 fungal species were isolated from 960 plant fragments. Cytospora chrysosperma, previously reported to be a pathogen of poplar, was recovered very frequently and was recorded as much in healthy as in dead or dying tissues. Cladosporium maculicola, Elsinoe veneta, Alternaria alternata, Aureobasidium pullulans and Pollaccia radiosa were also frequently isolated. The isolation frequencies from necrotic and healthy plant tissues were significantly different between sites and isolation methods. The incubation in moist chambers revealed significantly more fungal isolates and a higher species richness (38 species) than isolations on PDA. However, 16% of the fungal species were exclusively recorded from cultures. Therefore, a combination of several isolation methods is recommended for surveys of fungal communities associated with trees.     

Occurrence of common phyllosphere fungi of horse-chestnut (Aesculus hippocastanum) is unrelated to degree of damage by leafminer (Cameraria ohridella)

The horse chestnut leaf-miner (Cameraria ohridella Deschka & Dimi?, Lepidoptera, Gracillariidae) is an invasive pest causing extensive damage to leaves of the horse-chestnut (Aesculus hippocastanum L.) in Europe. In Lithuania, C. ohridella invaded in 2002 causing wilting, browning and premature fall of A. hippocastanum leaves. The aim was to get a better understanding of possible linkages between foliar fungal communities and leaf-miner damage in A. hippocastanum. Leaves of A. hippocastanum, differentially damaged by C. ohridella, were collected in 10 sites in Lithuania. The fungal communities were described through DNA isolation and amplification using an ITS rRNA marker and Ion Torrent-sequencing. Clustering of 214,897 high-quality sequences resulted in 1017 non-singleton fungal taxa, among which Aureobasidium pullulans (28.2% of all fungal sequences), Endoconidioma populi (27.7%), Phoma fungicola (11.3%), Cladosporium ramotenellum (7.6%) and Cryptococcus sp. 2185_4 (5.0%) were most common. Correspondence analysis showed that fungal communities from heavily and slightly damaged leaves were largely intermingled, showing that in both types of samples fungal communities were similar. In conclusion, the study demonstrated that the phyllosphere of A. hippocastanum is inhabited by a high diversity of fungal species, the majority of which constitute generalist endophytes, epiphytes and saprotrophic fungi. The occurrence of common phyllosphere fungi was unrelated to the degree of damage by C. ohridella.     

Degradation of Norway spruce (Picea abies) heartwood and sapwood during 5.5 years’ above-ground exposure

Abstract

Differences in durability between heartwood and sapwood of Norway spruce [Picea abies (L.) Karst.] were investigated to determine wood qualities most favourable for use in outdoor constructions above ground. Trees grown on sites with either good or poor access to water were used. Seventy-eight specimens measuring 20 × 50 × 300?mm³ separated into heartwood and sapwood, half untreated, half painted, were exposed horizontally outdoors above ground for 5.5?years with the pith side up and the bark side down. Crack length and crack number were measured. Fungus growth and surface changes were visually estimated. Fungus type was determined by microscopic analysis. The main finding was that spruce heartwood had fewer and shorter cracks and less surface-discolouring fungus growth than sapwood. This was valid for both painted and untreated wood. After 2?years’ exposure, the cracks in sapwood (upper surface) were more than three times longer and about five times more numerous than in heartwood for both painted and untreated boards. Microscopic study showed that surface discoloration was due mainly to Aureobasidium pullulans, together with a few other discolouring fungi. After 5.5?years, initial decay was established on the surface and in the end grain of four untreated test objects.     

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Abstract

The effect of soaking on germination and occurrence of fungal infections on Norway spruce (Picea abies (L.) Karst.) seeds and germinants was studied in three commercial seed lots. Treatments in which seeds were soaked in water for 24 h or in which the water was changed during the soak did not have an effect on the species diversity or on the abundance of the fungi isolated from the seeds. Different fungi were found in different seed lots. Most of those isolated are saprophytic or weakly pathogenic, but pathogens such as Sirococcus conigenus (D.C.) P.F. Cannon &; Minter and Trichothecium roseum (Pers.) Link were also isolated. Soaking increased germination energy but had no effect on final germination percentage. The number of mouldy seeds and germinants with disease symptoms was different among seed lots when seeds were germinated on water agar. When germinated in low humified Sphagnum peat, no differences in the emergence of disease symptoms were observed among seed lots. The frequency of disease was lower in peat than in water agar. Soaking had no effect on disease emergence in germination trials on either water agar or in Sphagnum peat.     

Newly recognized bacteria involved in trunk scab and canker of white and hybrid poplars

In a 4‐year‐old collection of native Italian Populus alba, growing in the eastern Po valley, many trunks showed severe symptoms characterized by brown spots, similar to trunk scab and/or by cankers. Canker‐like growths, often with gall‐like‐formations in the earlier developmental phases were also found on some clones of P. trichocarpa and their hybrids, as well as on P. deltoides growing in several Northern Italian nurseries. Bacteria and fungi present in the affected tissues and potentially involved in the disease were examined. No fungi were detected, however, several species of bacteria were isolated and identified: Pseudomonas mendocina and Erwinia herbicola group, from the brown spots; Erwinia carotovora sub sp. carotovora and, occasionally, E. herbicola group, from cankers. Only E. carotovora sub sp. carotovora produced clear symptoms of canker when artificially inoculated on young plants of different poplar species. This is the first time this symptomology on poplar species has been clearly related to E. carotovora sub sp. carotovora.     

Mycobiota in needles of Abies alba with and without symptoms of Herpotrichia needle browning

The ascomycete Nematostoma parasiticum (syn.: Herpotrichia parasitica) is commonly perceived as the causal agent of the so‐called Herpotrichia needle browning in silver fir (Abies alba). However, its fruitbodies are rarely present on symptomatic needles, which are also colonized by many presumably saprotrophic fungi. We compared the internal colonization of healthy and symptomatic needles on two sites in Poland. In addition, the endophytic mycobiota in needles of various age was recorded on two other sites without disease symptoms. Fungi were isolated from 95.6% of the dead needles and from 62.9% of the living needles on symptomatic trees, whereas on healthy trees, only 45.0% of the needles were colonized internally. Colonization frequency increased with needle age. From a total of 2017 isolates, 116 fungal taxa were identified. Frequency of many species was influenced by needle type. Anthostomella formosa, Gloeosporidiella sp., Hypoxylon fragiforme, Xylaria hypoxylon and X. polymorpha were the most common fungi isolated from living needles. In symptomless living needles, fungi occurred significantly more often in the basal than in the apical parts. In dead needles, the most common fungi were Alternaria alternata, Paraconiothyrium sporulosum, Fusarium sp., Mollisia cinerea, Rhizoctonia sp., Rhizosphaera oudemansii, Thysanophora penicillioides, Xylaria hypoxylon and X. polymorpha. Rhizoctonia sp. was the most frequently isolated fungus in dead needles (23.4%) but occurred rarely also in living needles (0.3–1.1%). The supposed pathogen N. parasiticum was detected only sporadically (at most in 0.6% of the needles). Our findings demonstrate the need for understanding the role of Rhizoctonia sp. in Herpotrichia needle browning disease aetiology.     

Accumulation of a porin-like mRNA and a metallothionein-like mRNA in various clones of Norway spruce upon long-term treatment with ozone

In a long-term study six Norway spruce (Picea abies) clones from two different provenance regions were exposed to 150/μg/m³ and 300μg/m³ ozone in open top chambers. Differential screening of subtractive cDNA libraries made from mRNA of ozone-treated trees identified several cDNA clones of ozone-responsive genes, two of which were studied in more detail. One cDNA clone corresponded to a porin-like mRNA, the other to a metallothionein-like mRNA. Under moderate ozone exposure the accumulation of these two mRNAs was already detectable before damage symptoms became visible. The spruce clones varied in the level of accumulation for the mRNAs. This is discussed under the aspect of ozone-induced early senescence rather than a defense reaction suppressing symptoms, because the two phenotypically most ozone-‘tolerant’ spruce clones showed the lowest levels of accumulation for both mRNAs.     

Effect of ozone treatment of wood on its liquefaction

The effects of ozone treatment were investigated to improve the process of liquefaction of wood with polyhydric alcohol solvents. The liquefied wood having a high wood to polyhydric alcohol ratio (W/P ratio) could be prepared by using the wood treated with ozone in the liquid phase. The liquefied wood with a W/P ratio of 2 : 1 had enough fluidity to act as a raw material for chemical products. To get some information about the effects of ozone treatment toward the wood components, cellulose powder and steamed lignin were treated with ozone and liquefied. In particular, ozone treatment in the liquid phase was found to be effective for wood and cellulose powder. On the other hand, steamed lignin self-condensed during liquefaction after treatment with ozone in the liquid phase. Thus, ozone treatment provided lignin with reactive functional groups, and caused the subsequent condensation reaction. Although lignin was converted to a more condensable structure by ozone treatment, the condensation reaction was found to be suppressed for wood during its liquefaction. The wood liquefied products displayed good solubilities in N,N-dimethyl formamide (DMF) even after treatments of long duration. It was suggested that one of the main effects of ozone treatment toward wood was the decomposition of cellulose.Part of this report was presented at the 53rd Annual Meeting of the Japan Wood Research Society, Fukuoka, April 2003     

Etiology of oak decline in Spain

In different areas of Extremadura, Western Spain, soil samples were taken at the bottom of holm oak (Quercus ilex) trees that were showing decline symptoms. Half of each sample was sterilized, and acorns were sown in both sterilized and nonsterilized soil samples. The resulting seedlings were used as baits for the isolation of fungi. Seedlings growing on the natural, nonsterilized substrate were characterized by having a lower vegetative growth than the ones growing on the sterilized soil samples, and most of them died. Phytophthora dnnamomi was consistently isolated from their roots. Fusarium oxysporum was also isolated as well as different species of Pythium, although to a lesser extent. Pathogenicity tests were performed on holm oak seedlings with five different isolates of P. chinnamomi, with F. oxysporum, Pythium and with a mixture of the three fungi. All the inoculated seedlings with P. chinnamomi developed root rot and grew slowly, and 35.7% of them died up to the end of the experiments. P. chinnamomi was consistently isolated from their roots, indicating that this fungus is the causal agent of holm oak decline. However, F. oxysporum caused similar symptoms on oak seedlings as P. chinnamomi, and was isolated also from the roots, although its frequency was lower than that of. P. cinnamomi.     

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.     

Effect of fertilization on growth and ectomycorrhizal development of container-grown and bare-root nursery conifer seedlings

The effect of three levels of fertilizer on thegrowth of three species of containerized-grownconifer seedlings (Pinus contorta, Picea glauca, and Picea mariana) and twospecies of bare-root conifer seedlings (Pinus sylvestris and Larix sibirica),and on the colonization of these seedlings bysix species of ectomycorrhizal fungi (Hebeloma longicaudum, Laccaria bicolor,Paxillus involutus, Pisolithustinctorius, Rhizopogon vinicolor andSuillus tomentosus), was studied. Thegrowth of the seedlings in both container-grownand bare-root nurseries increased as the levelsof fertilizer increased. For better seedlinggrowth and environmental quality it may be possible to reduce the level of fertilizers in commercial nurseries upto 33% by using selected mycorrhizal fungi.Ectomycorrhizal colonization in all seedlingswas not affected by fertilizer levels. Hebeloma longicaudum, L. bicolor, P.involutus, and P. tinctorius formedwell-developed ectomycorrhizae, whereasectomycorrhizal development by R.vinicolor and S. tomentosus was poor.Native mycorrhizal fungi colonizednon-inoculated control seedlings; however,their colonization was always lower than withinoculated fungi.     

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.     

Statistical interferometric investigation of nano-scale root growth: effects of short-term ozone exposure on ectomycorrhizal pine (<Emphasis Type="Italic">Pinus densiflora</Emphasis>) seedlings

This study presents the effects of short-term ozone exposure on the nano-scale growth behavior of the fine roots of Pinus densiflora (Japanese red pine) seedlings. Root elongation measurements were obtained in nanometers for very short (sub-second) time intervals by using the optical interference method called statistical interferometry, developed by the authors. Three categories of P. densiflora seedlings were investigated; two categories were infected with ectomycorrhiza of Pisolithus sp. (Ps) and Cenococcum geophilum (Cg), while the third was without any fungal infection. In experiments, two points on a root with a separation of 3 mm were illuminated by laser beams and the elongation was measured continuously by analyzing speckle patterns successively taken by a CCD camera. The ectomycorrhizal fungi-infected and uninfected seedlings were exposed to ozone at concentrations of 120 and 240 ppb for periods of 1, 3, or 5 h in separate treatments. The root elongations of P. densiflora seedlings were measured before and immediately after the each ozone treatment and then the root elongation rates (RER) were determined for growth-measurement periods of 5.5 s and 9.5 min. From the measurements obtained for 9.5 min, we found that the RERs of uninfected and Cg-infected seedlings were reduced by 42 and 18%, respectively, after 5 h of exposure to 120 ppb ozone compared with that before exposure, while the reduction in RER of Ps-infected seedlings was not significant. When the concentration of ozone was increased to 240 ppb, the RERs of Ps-infected and Cg-infected seedlings were reduced by 32 and 44%, respectively, after exposure for 5 h, while the reduction in RER of uninfected seedlings was 59%. These observations prove that the non-mycorrhizal seedling roots are more sensitive to ozone stress. From this study, we found that the RERs of both mycorrhizal and non-mycorrhizal seedlings apparently fluctuated throughout the measurements, even within a few minutes.     

Interactions between Tricholomopsis rutilans and ectomycorrhizal fungi in paired culture and in association with seedlings of lodgepole pine and Sitka‐spruce

Interactions between two isolates (Avondhu and CBS) of Tricholomopsis rutilans and ectomycorrhizal fungi (Hebeloma crustuliniforme, Laccaria laccata and Paxillus involutus) were examined on agar medium in the presence or absence of woodchips. The CBS isolate showed more competitiveness than the Avondhu isolate, when paired with ectomycorrhizal fungi. There was an inhibition of the growth of mycelium of the ectomycorrhizal fungi ranging from overgrowth to avoidance. The ectomycorrhizal fungi exhibited hyphal abnormalities such as discoloration, excessive vacuolation and curling. Formation of mycorrhizas by H. crustuliniforme in Pinus contorta and Picea abies was unaffected by the presence of the CBS isolate, whereas a reduction occurred in the presence of the Avondhu isolate. In non‐mycorrhizal seedlings of both conifers, the Avondhu isolate reduced root and shoot dry mass and number of root laterals and caused a lower number of short roots in lodgepole pine. The significance of these interactions between T. rutilans and ectomycorrhizal fungi in paired culture and during symbiosis is discussed.