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.     

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.     

Temporal variations in endophytic fungal assemblages of Ginkgo biloba L.

We isolated endophytic fungi from living healthy leaves, petioles, and current-year twigs of Ginkgo biloba L. from April to November 2004 with the objective of identifying the dominant endophytic fungal taxa, and monitoring their occurrence and frequency. A total of 9 fungal taxa were identified to the genus level. Diversity measures inferred from the Shannon–Wiener, Morisita–Horn, and S?rensen indices indicated that leaves and petioles harbored more diverse endophytic fungal assemblages than twigs, and that fungal taxa involved in twigs shared less with those in leaves and petioles. Among the organs, the occurrence pattern of overall endophytic fungi differed significantly, and two taxa, Phomopsis sp. and Phyllosticta sp., were the most frequently isolated and thus regarded as the dominant endophytic fungi. Phomopsis sp. was isolated frequently from twigs (84.8%) but rather few from leaves (16.1%) and petioles (24.3%). Phyllosticta sp. was isolated frequently from leaves (72.9%) and petioles (65.7%) but was never isolated from twigs. Temporal changes in relative frequency of total endophytic fungi tended to differ among sampling dates for all three organs. The occurrence of Phyllosticta sp. in both leaves and petioles was first detected in August and peaked in October. Phomopsis sp. was detected in twigs throughout the growing season. These results suggest that the distribution of the two dominant endophytic fungi was organ-specific and differed within seasons.     

Detection of Chalara fraxinea from tissue of Fraxinus excelsior using species‐specific ITS primers

Chalara fraxinea (teleomorph: Hymenoscyphus albidus) is known as a serious pathogen of Fraxinus excelsior, causing massive dieback of trees in Europe. The fungus is able to cause latent infections, and has been previously detected as an endophyte in asymptomatic tissues. Chalara fraxinea is a slow grower in culture, and is thus likely to be overgrown by faster growing fungi whenever pure culture isolations are being attempted. This study reports species‐specific ITS primers allowing fast and reliable detection of the pathogen directly from infected tissues of F. excelsior.     

Phytophthora root and collar rot of mature Fraxinus excelsior in forest stands in Poland and Denmark

In recent years, Common ash (Fraxinus excelsior) throughout Europe has been severely impacted by a leaf and twig dieback caused by the hyphomycete Chalara fraxinea. The reasons for its current devastating outbreak, however, still remain unclear. Here, we report the presence of four Phytophthora taxa in declining ash stands in Poland and Denmark. Phytophthora cactorum, Phytophthora plurivora, Phytophthora taxon salixsoil and Phytophthora gonapodyides were isolated from rhizosphere soil samples and necrotic bark lesions on stems and roots of mature declining ash trees in four stands. The first three species proved to be aggressive to abscised roots, twigs and leaves of F. excelsior in inoculation experiments. Soil infestation tests also confirmed their pathogenicity towards fine and feeder roots of ash seedlings. Our results provide first evidence for an involvement of Phytophthora species as a contributing factor in current decline phenomena of F. excelsior across Europe. Specifically, they may act as a predisposing factor for trees subsequently infected by C. fraxinea. Phytophthora species from ash stands also proved to be aggressive towards a wide range of tree and shrub species commonly associated with F. excelsior in mixed stands. Although damage varied considerably depending on the Phytophthora species/isolate–host plant combination, these results show that many woody species may be a potential source for survival and inoculum build‐up of soilborne Phytophthora spp. in ash stands and forest ecosystems in general.     

Pathogenicity of fungi isolated from Quercus suber in Catalonia (NE Spain)

Summary Thirty‐four fungal species isolated from cork oak (Quercus suber) in Catalonia (NE Spain) during 1992–95 were tested for pathogenicity either in stem, leaf or root inoculations. Eleven species were found to be pathogenic on stem: Biscogniauxia mediterranea, Botryosphaeria stevensii, Diatrype cf. stigma, Endothia gyrosa, Fusarium solani, Graphium sp., Ophiostoma quercus, Phomopsis sp., Phytophthora cinnamomi, Sporendocladia bactrospora and an unidentified Coelomycete. Three fungi showed pathogenic effects on leaves: Dendrophoma myriadea, Lembosia quercina and Phomopsis quercella. No clear pathogenic effects were detected in the root inoculation experiment. Trunk pathogens were differentiated into two groups according to the effects induced in the inoculated plants; B. stevensii, Phomopsis sp. and P. cinnamomi caused the death of the inoculated plants and induced the formation of large cankers and vascular necroses. The other pathogenic species also produced severe cankers and vascular lesions, but no significant mortality was detected. Water stress increased the lesions caused by B. mediterranea and Phomopsis sp., but limited those of P. cinnamomi and the rest of the inoculated fungi. However, water stress did not significantly affect the damage caused by B. stevensii, which was the most virulent of the species tested. Leaf pathogens only showed their effects if the leaf cuticle was previously damaged. Lembosia quercina caused small dark lesions whereas D. myriadea and P. quercella produced large necrotic areas in well‐watered plants. The lesions caused by the last two fungi were reduced by water stress.     

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.     

Geographical and seasonal influences on the distribution of fungal endophytes in Laurus nobilis

The species composition of the endophytic mycobiota in bark, leaves and twigs of Laurus nobilis were studied in Western Anatolia. Four sampling sites were selected; one of which was sampled twice, in spring and autumn. About 3892 fungal isolates obtained grouped into 85 morphological types. The 11 dominant species, with isolation frequencies >1.5%, were Alternaria alternata, Botryosphaeria sarmentorum, Colletotrichum dematium, C. gloeosporioides, Hendersonula sp., Paraconiothyrium sp., Phoma sp., three species of Phomopsis and Seimatosporium lichenicola. Numbers of fungal species isolated per tree differed between the sampled sites. Isolation frequencies of the dominant species, and other less frequent species, were dependent on the sampling site. The degree of endophytic infection and the diversity of fungal species were significantly higher in spring. Frequencies of all dominant species depended significantly on season, except for Stemphylium sp. and S. lichenicola. Analysis of the total endophytic mycoflora of the sampled trees suggested that geographical factors affects the endophytic distribution patterns more significantly than seasonal factors.     

Fungal colonization patterns in necrotic rootstocks and stem bases of dieback‐affected Fraxinus excelsior L.

European ash (Fraxinus excelsior) trees currently face the major threat of ash dieback caused by an invasive fungus, Hymenoscyphus fraxineus. Collar rots in F. excelsior have been increasingly associated with infections by this pathogen. However, the aetiology of the collar rots is still unclear and remains heavily debated. In contrast to most studies of this kind, entire rootstocks of four diseased ash trees were dug out to examine necrotic tissues in these rootstocks and stem bases in detail and to sample necrotic wood for fungal isolation. With the aid of morphological and molecular identification techniques, five to twelve fungal taxa were detected per tree. Members of the Nectriaceae family and Botryosphaeria stevensii, the causal agent of stem and branch cankers on many tree species, were frequently isolated from outer xylem. In contrast, H. fraxineus was the dominating species in interior wood layers. Microsatellite genotyping of 77 H. fraxineus isolates helped to identify up to six different genotypes per tree. The role of H. fraxineus and other isolated fungi in the aetiology of ash collar rots are discussed.     

Fungi associated with necrotic apical parts of Fraxinus excelsior shoots

Stem necrosis was investigated in 2–3‐year‐old seedlings of European ash Fraxinus excelsior, both self‐sown and in plantations within four Forest Divisions in eastern, northern and central Poland. Fungi isolated from the apical parts of shoots showing fresh necroses were identified and the pathogenicity of some of them was determined. Some species were isolated at all four sites; the most consistent members of this group were Cladosporium cladosporioides, Cytospora ambiens, Discula sp., Fusarium lateritium, Phoma sp., Phomopsis scobina and P. controversa. Diplodia mutila was isolated at all the sites, but with variable frequency. It induced necrotic lesions following wound inoculation of stems of 2‐month‐old and 2‐year‐old seedlings of F. excelsior in growth chamber and greenhouse conditions.     

The influence of altitude on endophytic mycobiota in Quercus acuta leaves collected in two areas 1000 km apart

Endophytic fungi were isolated from healthy leaves of the evergreen oak Quercus acuta on Mt Takao in eastern Honshu and on Mt Osuzu in Kyushu, Japan, to study the effect of altitude on endophyte assemblages. Phomopsis sp. 1, Discula sp. and an unidentified species (QA-b) were isolated most frequently. Comparisons of the nucleotide sequence of the rDNA ITS regions of QA-b revealed a close match with species of Tubakia. On Mt Takao, Phomopsis sp. 1 and QA-b were isolated frequently, whereas on Mt Osuzu, Discula sp. and QA-b were dominant. The isolation frequency of QA-b decreased as altitude increased, whereas the isolation frequencies of Phomopsis sp. 1 and Discula sp. increased with altitude. In vitro, QA-b did not grow at temperatures <15°C whereas Phomopsis sp. 1 and Discula sp. still grew at 5°C. These results suggest that Phomopsis sp. 1 and Discula sp. are better adapted to lower temperatures and consequently to higher altitude than QA-b.     

Temperature effect on Chalara fraxinea: heat treatment of saplings as a possible disease control method

Ash dieback is an emerging disease caused by the fungus Chalara fraxinea that severely affects Fraxinus excelsior and F. angustifolia stands in Europe. Previous studies have shown that this pathogen prefers temperatures around 20°C, while its growth in pure cultures at 30°C proved to be very limited. The purpose of this study was to determine the effects of temperature on the development and growth of C. fraxinea in pure cultures and in plant tissues, as well as to test the heat tolerance of F. excelsior saplings. The sensitivity of fungus to heat in ash tissues was higher than in pure cultures. Low isolation success rate from diseased ash tissue after a five‐hour hot water treatment at 36°C and the relatively high survival rate of ash saplings after hot water treatments at 36°C and 40°C indicate possibilities for the development of a C. fraxinea eradication method in ash saplings. Field monitoring showed that in hot weather periods, thermal conditions inside the ash tissues can be extreme enough to markedly decrease the viability of C. fraxinea in infected plant tissues.     

Fungal endophytes associated with Sonneratia (Sonneratiaceae) mangrove plants on the south coast of China

The aim of this study was to describe endophytic fungal communities residing in five mangrove species (Sonneratia caseolaris, Sonneratia hainanensis, Sonneratia ovata, Sonneratia Paracaseolaris, Sonneratia apetala) collected at the south coast of China. The colonization frequency (CF) of endophytes in roots, stems and leaves was measured and the structures of endophytic communities were investigated, using endophytic fungal diversity indices and similarity coefficients. We found high biodiversity and tissue specificity of endophytic fungi. A total of 391 fungal isolates were identified using morphological and molecular methods. We recovered a total of 39 distinct endophytic species, of which Cytospora, Diaporthe, Fusarium, Glomerella, Mycosphaerella, Phoma, Phomopsis and Stemphylium were the dominant fungal taxa. Most recovered species were found on more than one host species but the dominant species differed according to host and tissue type.     

Cryptic speciation in Hymenoscyphus albidus

Ash dieback caused by the mitosporic ascomycete Chalara fraxinea is a novel disease of major concern affecting Fraxinus excelsior and Fraxinus angustifolia in large parts of Europe. Recently, its teleomorph was detected and assigned to Hymenoscyphus albidus, which has been known from Europe since 1851. In this study, we present molecular evidence for the existence of two morphologically very similar taxa, H. albidus, which is lectotypified and Hymenoscyphus pseudoalbidus sp. nov. Differences were found between the species in the loci calmodulin, translation elongation factor 1‐α and the internal transcribed spacers of the rDNA genes, and strong differentiation was obtained with ISSR markers. It is likely that H. albidus is a non‐pathogenic species, whereas H. pseudoalbidus is a virulent species causing ash dieback. Genotyping herbarium specimens showed that H. pseudoalbidus has been present in Switzerland for at least 30 years prior to the outbreak of the epidemic.     

The teleomorph of Chalara fraxinea,the causal agent of ash dieback

Ash dieback, caused by the pathogen Chalara fraxinea, is an emerging lethal disease of Fraxinus excelsior, threatening the host species in large parts of Europe. The ascomycete Hymenoscyphus albidus (Helotiaceae, Helotiales) was identified as the teleomorph of C. fraxinea by culturing from ascospores, morphological comparison and nuclear ribosomal internal transcribed spacer (ITS) sequencing.     

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

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

Pathogenicity of fungi associated with ash dieback symptoms of one‐year‐old Fraxinus excelsior in Montenegro

In addition to Hymenoscyphus fraxineus, two fungi identified as Diaporthe eres aff. and Fusarium sambucinum aff. were also isolated from necrotic bark lesions on declining one‐year‐old Fraxinus excelsior in a forest stand in Montenegro. To examine their involvement in ash decline, a pathogenicity test was performed using under bark inoculations on one‐year‐old Fraxinus excelsior. Hymenoscyphus fraxineus was included as comparison. All three fungal species proved highly pathogenic towards one‐year‐old seedlings although lesion sizes differed significantly between the different species. Hymenoscyphus fraxineus was most aggressive, followed by F. sambucinum aff., while D. eres aff. caused the smallest lesions. This study demonstrates for the first time the ability of isolates in the D. eres and F. sambucinum species complexes to cause decline on one‐year‐old common ash seedlings.     

Comparative analyses of fungal biota carried by the pine shoot beetle (Tomicus piniperda L.) in northern and southern Finland

The pine shoot beetle, Tomicus piniperda, is a secondary colonizer of pines and other conifers. Although it is necessary to understand interactions of this insect with other organisms, few studies have focused on its fungal associates. This study focused on the effect of geographical distance on the occurrence of fungi carried by the beetle. Adult beetles were collected from Scots pine (Pinus sylvestris) in North (Rovaniemi) and South (Hyytiälä) Finland. The mycota was isolated and identified using a combination of morphological and molecular data. The results revealed a great diversity of fungal taxa isolated from T. piniperda, with a total of 3073 isolates representing 20 taxonomic units. The most frequently isolated fungi in the bark beetles from North Finland were Beauveria bassiana, Kuraishia sp., whereas Penicillium velutinum was mostly observed in the insects from South Finland. Ophiostoma canum and Ophiostoma minus were also isolated from the beetles from both North and South Finland. The number of fungi isolates per insect was 2.58 in the North and 3.74 in the South, respectively. Significant differences were found between South and North Finland in fungal taxa isolated from the beetles. The highest richness and diversity of the fungal taxa was observed in the South. However, the overall fungal diversity index analysis revealed that the mycobiota was under-sampled which suggests that a proportion of the fungal species may have remained undetected.     

Root-associated fungi of healthy-looking Pinus sylvestris and Picea abies seedlings in Swedish forest nurseries

The aim of this study was to assess fungal communities in roots of healthy-looking Pinus sylvestris L. and Picea abies (L.) Karst. seedlings in nine forest nurseries in Sweden using a combination of traditional culturing and direct sequencing of internal transcribed spacer of fungal ribosomal RNA (ITS rRNA) from the roots. Culturing from 1800 surface-sterilised root segments resulted in 2387 fungal cultures representing 42 different taxa. Direct sequencing from 180 root segments resulted in 119 ITS rRNA sequences representing 25 different taxa. In total, 55 different fungal taxa were detected using both methods. Although direct sequencing was more efficient than culturing in detecting different fungal taxa, both methods provided complementary information about fungal communities in roots since each detected rather different groups of fungi. The most dominant taxa detected by culturing were Trichoderma viride Pers. (19.5%), Phoma mucivora Davey & Currah (19.1%), Phialocephala fortinii Wang & Wilcox (17.4%) and Meliniomyces variabilis Hambl. & Sigler (10.2%), while Thelephora terrestris Ehrh. (26.1%), Unidentified sp. NS126 (25.2%) and Heliotales sp. C20 (10.1%) were most commonly detected by direct sequencing. In conclusion, results showed that forest nurseries in Sweden harbour diverse communities of fungi associated with the roots of healthy-looking P. sylvestris and P. abies seedlings. Although fungal communities were often dominated by saprotrophs and endophytes, several facultative pathogens were also detected indicating that under suitable conditions they may be a potential threat to the plants.     

Pathogenicity of Chalara fraxinea

The mitosporic fungus Chalara fraxinea (Ascomycota) is associated with dieback of common ash, an emerging disease of Fraxinus excelsior (Oleaceae) in Europe. The pathogenicity of C. fraxinea was demonstrated by field inoculations on young trees.