Natural infection of Fraxinus excelsior seeds by Chalara fraxinea

The occurrence of Chalara fraxinea, the fungus responsible for dieback of European ash (Fraxinus excelsior), was investigated in the current and previous seed years collected from symptomatic trees in Latvia and Sweden using molecular techniques (DNA extraction, ITS‐PCR, Sanger sequencing). Molecular analysis of seeds revealed the presence of 30 different fungal taxa. Chalara fraxinea was detected in 8.3% of seeds tested from the current year originating from Latvia. The presence of C. fraxinea in seeds of F. excelsior is of great concern to phytosanitary protection authorities in countries outside the current zone of infestation.     

Fungal endophytes in woody roots of Douglas‐fir (Pseudotsuga menziesii) and ponderosa pine (Pinus ponderosa)

The fungal community inhabiting large woody roots of healthy conifers has not been well documented. To provide more information about such communities, a survey was conducted using increment cores from the woody roots of symptomless Douglas‐fir (Pseudotsuga menziesii) and ponderosa pine (Pinus ponderosa) growing in dry forests on the eastern slope of the Cascade Mountains in Washington state, USA. Fungal isolates were cultured on standard media, and then were identified using a combination of molecular and morphological methods. Fungal genera and species identified in this study will provide baseline data for future surveys of fungal endophytes. Examination of internal transcribed spacer (ITS1 and ITS2) and 5.8S rDNA sequences and morphology of cultured fungi identified 27 fungal genera. Two groups predominated: Byssochlamys nivea Westling (20.4% of isolations) and Umbelopsis species (10.4% of isolations). This is the first report of B. nivea within large woody roots of conifers. Both taxa have been previously identified as potential biological control agents. Although some trends were noted, this study found no significant evidence of host species or plant association effects on total recovery of fungal endophytes or recovery of specific fungal taxa.     

Fungi Isolated from Diseased Nursery Seedlings in Spain

Fungal endophytes in container-grown seedlings in forest nurseries located in the North Plateau of Spain have been studied. Influences of nursery, season (spring and autumn), host genus and plant part on those fungal assemblages have been also evaluated. Four nurseries were sampled in spring and three in autumn. Analysed material consisted of stems, roots and rhizosphere from five host species, Quercus ilex, Q. pyrenaica, Pinus pinea, P. nigra and P. sylvestris. A total of 581 isolates corresponding to 13 taxa were found in this study. Fusarium moniliforme, F. oxysporum and Trichoderma viride were the most frequently isolated fungi. Number of fungal species per observation differed significantly among the sampled parts. Multivariate Correspondence Analysis allowed the discrimination of samples in five groups based on nursery, season, host genus and plant part. Isolation frequencies of F. moniliforme and F. oxysporum were higher in the roots and in spring, whereas those of T. viride were higher in the rhizosphere and in autumn. A relationship was observed between presence of T. viride and absence of Fusarium spp. in forest nurseries. T. viride, could serve as a biological control agent against Fusarium spp. in forest nurseries.     

First report of Calonectria montana causing damping‐off disease on pine and spruce seedlings in Europe

In 2015–2016, damping‐off symptoms were observed on Pinus sylvestris and Picea abies seedlings in four forest nurseries in Poland. Some Calonectria isolates were obtained from symptomatic seedlings by culturing pieces of necrotic tissue on malt extract agar.Morphological and molecular analyses revealed that isolates represent Calonectria montana, a recently described species from natural forests in China. The isolates represent the first detection of this species outside of Asia. Pathogenicity tests demonstrated that C. montana is a severe pathogen of P. sylvestris seedlings.     

Identification of Lophodermium seditiosum and L. pinastri in Swedish forest nurseries using species‐specific PCR primers from the ribosomal ITS region

Lophodermium seditiosum is a serious needle pathogen on pine, particularly in nurseries, and there is a need to detect the pathogen during its latent phase. The internal transcribed spacer (ITS) regions of the rDNA of L. seditiosum and L. pinastri were amplified with universal primers and sequenced. Sequence comparisons of the two species allowed the design of species‐specific primers for the ITS regions. The primers were between 18 and 24 bp long with a minimum of 3 bp differences between the species. These primer pairs did not give any amplification of DNA from any other of the examined fungal species or from healthy Pinus sylvestris needles. It was also possible to identify either L. seditiosum or L. pinastri in infected needles with and without signs of infection using these primer pairs. The method was found to be very useful for detection of latent infections of L. seditiosum in P. sylvestris needles in nurseries.     

Fungi in foliage and shoots of Fraxinus excelsior in eastern Ukraine: a first report on Hymenoscyphus pseudoalbidus

In eastern Ukraine, the first symptoms of dieback on common ash (Fraxinus excelsior) were observed in 2010, as sparse flushing of leaves, bark necrosis and wood discoloration of shoots. The aim of this study was to assess possible causal agents of the damage by studying fungal communities in both symptomatic and healthy‐looking shoots, and leaf petioles. Field sampling was carried out in 2010 in Kharkiv and Sumy regions in eastern Ukraine and included 68 segments of symptomatic shoots, 68 segments of healthy‐looking shoots and 240 segments of petioles. DNA isolation from individual segments and direct sequencing of fungal ITS rRNA resulted in 430 fungal sequences representing 29 distinct taxa. Results showed that Hymenoscyphus pseudoalbidus – the primary causal agent of ash dieback in Europe – was present at low proportion (5.6%) in symptomatic shoots. Other more frequently detected fungi were Epicoccum nigrum, Venturia fraxini, Colletotrichum truncatum, Aureobasidium pullulans, Alternaria alternata, Alternaria sp. and Lophiostoma corticola. In conclusion, the study reports on the first incidence of ash decline in the Ukraine and other possible causal agents of damage which may help to evaluate and forecast the future situation with F. excelsior stands in the region.     

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.     

Susceptibility of different provenances of Pinus sylvestris,Pinus Contorta and Picea abies to Gremmeniella abietina

In a randomized block factorial experiment, 1200 seedlings from four provenances, each of Pinus sylvestris, Pinus contorta and Picea abies were inoculated with conidia (2 × 10⁴ and 1 × 10⁶ conidia/seedling) of Gremmeniella abietina (Brunchorstia pinea), isolated from P. contorta plantations in northern Sweden. A further 600 seedlings were left as controls. The occurrence of symptoms and the extension of dead tissues on the annual shoots were recorded 13 months after inoculation. Only the higher spore dose resulted in significant infection. P. sylvestris and P. contorta seedlings were equally susceptible (53% infected), and significantly more infected than P. abies seedlings (39%). The annual shoots of P. sylvestris and P. abies were affected to 43% and 37% of their length, which was significantly more than 15% of the length of P. Contorta shoots. The frequency of affected seedlings differed between the most southern and northern provenances of P. sylvestris and P. abies, and between the northwestern and the south-eastern provenance of P. contorta Twenty-six months after inoculation, a higher proportion of P. contorta seedlings than P. sylvestris and P. abies seedlings had recovered, and a lower proportion of P. contorta than P. sylvestris and P. abies seedlings had died. This paper discusses why P. sylvestris was found to be more susceptible to G. abietina than P. contorta in this experiment, while the reverse is found in plantations in northern Sweden.     

Diversity of ectomycorrhizal fungus assemblages on nursery grown European beech seedlings

? Context

An important feature of forest nursery management is the production of first-rate planting stock, which is closely connected with an adequate development of ectomycorrhizas on seedling roots.

? Aims

The aim of this study was to provide a detailed assessment of the ectomycorrhizal (ECM) fungal diversity of European beech grown in bare-root forest nursery conditions.

? Methods

Morphological and molecular analyzes were used to identify ECM fungi, quantify fungal richness, and record differences in the relative abundance of individual taxa.

? Results

Twenty-seven different mycorrhizal fungal taxa were found in four tested nurseries. Of these 27, only 6 were present in more than one nursery. Taxa richness in individual nurseries ranged from 6 to 13, indicating that ECM colonization potential is nursery-dependent. The most frequent taxa were: Tuber sp. 1, Cadophora sp., Cenococcum geophilum agg., Hebeloma sacchariolens, and Laccaria tortilis. According to the analysis of similarity, ECM fungal communities were significantly different between nurseries (R?=?0.7741; p?<?0.0001).

? Conclusion

Beech seedlings grown in bare-root nurseries are colonized by a highly diverse suite of ECM fungi; thus, artificial inoculation of seedlings is generally not necessary in nursery practice.     

Microbial communities in roots of Pinus sylvestris seedlings with damping‐off symptoms in two forest nurseries as determined by ITS1/2 rDNA sequencing

A methodological molecular procedure, which included extraction and cloning of the ITS1/2 rDNA of root‐associated organisms with subsequent transformation and sequencing of representative clones, was effective for detection, discrimination and determination of the frequency of the main damping‐off pathogens in roots of Pinus sylvestris seedlings growing in different forest‐tree nursery soils and exhibiting different rates of disease progress. Roots exhibiting slower damping‐off progression were colonized by Fusarium oxysporum, Neonectria radicicola (Ascomycota) and Pythium spp. (Oomycota), which comprised 50% of the microbial community. Roots exhibiting faster damping‐off progression were dominated by Thanatephorus cucumeris (Basidiomycota), which comprised 80% of the microbial community. The microbial community was more diverse in roots with slower damping‐off progression (14 species) than in roots with faster disease progression (seven species).     

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.     

Carbon and nitrogen stocks in soil,trees and field vegetation in conifer plantations 10 years after deep soil cultivation and patch scarification

Abstract

Biomass, total nitrogen (N) and total carbon (C) stocks were determined in trees, roots, field vegetation and soil in plots given two different site preparation treatments, deep soil cultivation (DSC) approximately 50?cm deep and patch scarification (PS), at three locations in Sweden 10?years after treatment. One location was planted with Pinus contorta, one with Picea abies and one with a mixture of P. abies and Pinus sylvestris. No differences were found in total ecosystem (trees, roots, field vegetation and soil) C and N stocks between the DSC and PS plots. In the DSC plots the tree biomass, tree N and C contents and total biomass were higher than in the PS plots, but the opposite was found for stocks in field vegetation. Biomass and C stocks in the total vegetation (trees, roots and field vegetation) were higher in the DSC plots. However, vegetation N stocks did not differ between the soil treatments, probably because the combined amount of leaf tissue in the trees and field vegetation did not differ between them. The proportions of biomass allocated to roots, stems and needles did not differ between the two treatments. However, the rooting was deeper in DSC plots, possibly because nutrient availability was higher, and subsoil density lower, following DSC than following PS.     

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.     

Fungal root endophytes of Quercus robur subjected to flooding

In August 2011, fungal endophytes were isolated from fine (0.1–0.5 cm diameter) and structural (0.6–2.0 cm) roots from eighteen Q. robur trees at six sites in a Q. robur‐dominated forest displaying symptoms of oak decline. Five sites were subjected to periodic flooding for 3 months in 1997 and 1 month in 2010. Fungal colonization was detected in 94% of 1296 root fragments examined. Fungi representing 126 taxa from Zygomycota (four species), Ascomycota (117 species) and Basidiomycota (five species) were isolated using culture‐based methods and identified by morphotyping and sequencing of the ITS 1 and 2 rDNA. The most frequent eudominants (with colonization frequencies ≥10%) were Aspergillus spp., Cosmospora sp., Ilyonectria radicicola, Pezicula radicicola, Pyrenochaeta cava and six species of Trichoderma. Species composition did not vary greatly between sites, but only 3% of species occurred in all sites. Number of species was higher in roots subjected to floods. Eudominants and dominants present only on flooded sites included Aspergillus spp., Chaetomium globosum, Cosmospora sp., I. radicicola, Sporothrix inflata, Trichoderma harzianum, T. pubescens and T. virens. Structural roots were usually colonized by a higher number of species than fine roots. The majority of species detected belong to Class 2 NC‐endophytes. Pezicula radicicola and Phialocephala fortinii belong to Class 4 NC‐endophytes.     

Two studies to assess the risk to Pinus sylvestris from Heterobasidion spp. in southern Sweden

Abstract

Pine stumps are not being treated against Heterobasidion spp. in Sweden. To determine whether they should be, the frequency of stump infections and the species of Heterobasidion involved were investigated in nine newly thinned pure Scots pine (Pinus sylvestris L.) stands in southern Sweden. The incidence of Heterobasidion was measured in roots of standing Scots pine in another 15 stands. Infections by both H. annosum (Fr.) Bref. s.s. and H. parviporum Niemelä & Korhonen were numerous in stumps six months after a summer thinning. The pathogen, mostly H. annosum, was found in 44 of 60 sampled root systems, from 14 of the 15 stands. Twenty of the infected pines were assessed as healthy on the basis of crown symptoms while 24 trees had defoliated crowns. Infected root systems were most frequent among trees with thin crowns in stands on former agricultural land, where previous thinnings had been carried out during the growing season when airborne spores are plentiful. The study suggests that stump treatment may be a profitable way to reduce disease development in Scots pine monocultures on sandy soils as well as in mixtures with Norway spruce [Picea abies (L.) Karst.] on any soil.     

Involvement of Cylindrocarpon destructans in root death of Pinus sylvestris seedlings: Pathogenic behaviour and predisposing factors

The common rhizospheric fungus Cylindrocarpon destructans was investigated in relation to its role in root death of Pinus sylvestris in Nordic nurseries and plantations. Laboratory methods were developed for studying similar root problems as well as the early effects of phytotoxicity and fungal infection. Seedlings grown under standardized optimal conditions were exposed to controlled stress (known to occur in nurseries), with or without C. destructans in the rhizosphere. Low light conditions, anaerobic root environment, and fungicide treatment were each found to predispose pine seedlings to invasion by the pathogen. The pathogen was very sensitive to competition as well as antagonism on the root, and fungicide‐induced inhibition of antagonists such as Trichoderma spp. also increased the severity of attack by the pathogen. To compete successfully, the pathogen would have to invade and dominate weakened roots prior to the arrival of saprophytes. Toxic metabolites produced by the pathogen weakened or killed nearby root tissues, and pathogen metabolites seemed to prevent saprophytes from taking hold in the infected roots. Such heavily infected, dead roots may act as inoculum sources allowing the pathogen to invade adjacent living roots, even healthy ones. Dead roots left in nursery soil after earlier harvests may act as reservoirs of inoculum for long periods and pose a threat to new plants.     

Analysis of Norway spruce dieback phenomenon in Latvia – a belowground perspective

In 2010, dieback of Norway spruce (Picea abies) was observed in Latvia. As mostly stands on seasonally or permanently water-saturated soils were damaged, we hypothesized that the recorded foliar damage might be associated with belowground factors. We established 48 sample plots in stands with damage symptoms for soil parameter evaluation and fine root sampling to determine the number of fine roots in different growth stages and to describe the associated fungal community. Among sample plots on organic soils, there were fewer viable older fine roots and greater number of dead fine roots in more damaged sites than in less damaged. These root parameters were significantly correlated with higher groundwater level and soil pH. The fungal community was dominated by ectomycorrhizal (ECM) species (the most common was Tylospora asterophora), the endophyte Oidiodendron maius and saprotrophic species Cryptococcus magnus. Saprotrophic species had higher abundance in more damaged sites, suggesting a shift in fungal communities from ECM fungi. In conclusion, the results of this work suggest Norway spruce root system response to short-term climatic stress, which should be considered when planning spruce forest management, especially on organic soils with a high groundwater level.     

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.     

Incidence of Gremmeniella abietina in planted seedlings of Picea abies and Pinus sylvestris in northern Finland

The ascomycete Gremmeniella abietina causes a disease (scleroderris canker) on conifers. The pathogen kills terminal shoots and branches of Pinus sylvestris and Picea abies, sporulates on host shoots and causes leader changes (i.e. dieback). Damage caused by G. abietina was investigated in young P. abies and P. sylvestris plantations in northern Finland. Side branches from seedlings of both species and the main stems of P. sylvestris were collected from three sites in two locations (Poksa 1 and 2, Kivalo) in northern Finland. The number of cankers and leader changes was counted in branches of each age (i.e. year) in a total of ca. 6300 shoots of P. abies and 1200 shoots of P. sylvestris. Cankers were common on both P. abies and P. sylvestris in Poksa 1 (2002–2008) and on P. abies in Poksa 2 (1998–2001). In Kivalo, cankers occurred only sporadically on P. sylvestris. Leader changes were most frequent in 1999–2009 in Poksa 1, in 2001–2009 in Poksa 2 and in 2003 in Kivalo. Cankers and other symptoms of infection were more obscure on P. abies than on P. sylvestris. According to both conidial morphology and molecular analysis, the strain of G. abietina infecting both P. abies and P. sylvestris was small‐tree type (STT). This is the first report of G. abietina, STT or B type, injuring P. abies in plantations.     

Potential for Biological Control of Botrytis cinerea in Pinus sylvestris Seedlings

Grey mould (Botrytis cinerea Pers.: Fr.) is the most common economically important fungal disease in Swedish forest nurseries. In tests in a growth room, foliage of predisposed (preinoculation incubation at 35°C for 4 days) Scots pine (Pinus sylvestris L.) seedlings was sprayed with suspensions containing Mycostop®, Binab® TF.WP or GlioMix® at concentrations of 0.5, 1 or 0.5 g l^?1, respectively, and/or conidia of B. cinerea (10⁶ spores ml^?1). Binab and GlioMix reduced grey mould in needles by 94 and 92%, respectively, and were as effective as the fungicide Euparen® M 50 WG, while Mycostop reduced disease by 51%. In one trial in a forest nursery, Mycostop, Binab and GlioMix, each applied two and four times during the growing season, suppressed spontaneous B. cinerea infections in needles of first year container-grown P. sylvestris seedlings by 16–57%, and were as effective as recommended fungicidal sprays. It was concluded that biological control has potential to effectively suppress grey mould in seedlings in forest nurseries.