Biodiversity of endophytic fungi from seven herbaceous medicinal plants of Malnad region,Western Ghats,southern India

A total of 3611 fungal isolates were recovered from 4200 leaf segments incubated from 7 medicinal herbs during monsoon, winter and summer seasons. These fungal isolates belonged to teleomorphic Asco-mycota （23.5%）, anamorphic Ascomycota producing conidiomata （17.4%）, anamorphic Ascomycota without conidiomata （46.9%）, Zygo-mycota （1.42%） and sterile forms （10.6%）. Chaetomium globosum, As-pergillus niger, Aureobasidium pullulans, Curvularia lunata, Fusarium spp., Penicillium spp., Pestalotiopsis spp., Trichoderma viridae, Cladosporium cladosporioides, were frequently isolated from more than one host plant. The number of endophytic isolates was higher in winter than in monsoon and summer seasons.     

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.     

紫金山两种主要林型有机物层中丝状真菌多样性

本文对紫金山两种主要林型,栓皮栎林(Quercus variabilis)和马尾松-枫香(Pinus massoniana-Liguidam barformasana)混交林的有机物层(L, F, H层及土壤淋溶A层)可培养丝状真菌多样性进行了研究。通过分离鉴定,共得到真菌67种,其中接合菌3种、子囊菌5种、半知菌类56种和未确定种3种,半知菌类最为丰富。两种林型分解真菌优势种群为链格孢(Alternaria sp.)、曲霉(Aspergillus spp.)、枝孢(Cladosporium sp.)、毛霉(Mucor sp.)、青霉(Penicillium sp.)、木霉(Trichoderma spp.)、根霉(Rhizopus sp.)、粘帚霉(Gliocladium sp.)。混交林型中真菌的种类和数量明显高于栓皮栎林。两个林型均以F层真菌种类最多,但F层之间和L层之间真菌多样性差异较大。在混交林型中从马尾松针叶上分离的真菌种类和数量比从枫香上分离得到的少; 从马尾松针叶上分离的真菌丰富度随着有机物层深度增加而增加,枫香则反之。比较两个林型以及混交林的两种落叶上真菌种类发现,随着有机物层深度的增加,真菌的种类差异性却随之减少,即同时出现在两个林型或两种针叶上的真菌种类增多。真菌种类随着分解过程的进行具有明显的演替现象。混交林型中同一层内不同落叶上分解真菌多样性的差异,表明凋落物基质的差异是决定真菌种类和数量的重要因素     

Seasonal,tissue and age influences on frequency and biodiversity of endophytic fungi of Citrus sinensis in Iran

A total of 4,009 endophytic fungal isolates were recovered from healthy leaves, bark and xylem of Citrus sinensis in different seasons and age‐classes. Corresponding to those factors, the majorities of fungal isolates were recovered from leaves, 2‐yr‐old trees and winter, respectively. Fungal isolates were initially categorized based on cultural and morphological characters, and representatives of each morphotype were subjected to molecular identifications based on ITS‐rDNA and β‐tubulin sequences. All isolates obtained in this study belonged to the phyla Ascomycota and Basidiomycota assigned to 30 fungal taxa. The most abundant fungal species were Alternaria spp. Species diversity indices of Margalef richness and Shannon–Wiener revealed a high diversity of fungal taxa recovered from leaf and during winter. Frequency (%) of fungal endophytes was highest in winter followed by summer, autumn, and spring significantly, whereas the corresponding sequence was winter, autumn, summer and spring for the diversity of endophytic fungi. Overall, the results of this study based on the community and diversity of endophytic fungal species in C. sinensis showed that the type of plant tissue, season of sampling and age of tree play a driving role on their abundance while their diversity was mainly dependent on the type of plant tissue, season of sampling than on the age of tree.     

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.     

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.     

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.     

Ectomycorrhizal fungal community associated with naturally regenerating Pinus densiflora Sieb. et Zucc. seedlings on exposed granite slopes along woodland paths

We investigated the ectomycorrhizal (EcM) fungal community of naturally regenerating Pinus densiflora seedlings on exposed decomposed granite (DG) slopes along woodland paths in the Republic of Korea. We sampled three- to five-year-old seedlings from slopes where DG was exposed (habitat S) and from the surrounding forest edge or gaps (habitat F). We classified EcMs into morphological groups and counted the number of root tips. The fungal taxa were determined by sequencing the internal transcribed spacer region of their nuclear rDNA. We observed a total of 27 EcM fungal taxa. Atheliaceae and Rhizopogon were the dominant taxa on exposed DG slopes. The fungal species compositions were similar between habitats (0.59 as calculated by the Morishita–Horn index). Fourteen fungal taxa were common to both habitats, and these accounted for more than 90% of the relative abundance in each habitat. There were no significant differences in relative abundance and colonization frequency of each EcM fungal taxon between the habitats, except for Russula sp. 2, which was significantly more frequent in habitat F than in habitat S. Our results indicated that P. densiflora seedlings on exposed DG slopes associated with EcM fungi that were common in forests and not specific to the exposed DG slopes.     

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.     

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.     

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.     

The ectomycorrhizal status of European larch (Larix decidua Mill.) seedlings from bare-root forest nurseries

In this paper, we report molecular investigation of an ectomycorrhizal fungi (EMF) community of European larch (Larix decidua Mill.) seedlings grown in a bare-root nursery. In total, we surveyed 32 nurseries that were each active in supplying planting stocks to restock forests and for afforestation of post-agricultural land. Sequence-based approach was used to identify EMF taxa, quantify EMF richness, and document differences in the relative abundance of individual taxa. We identified seven fungal species that might contribute to the mycorrhizal community structure of 1 to 3-year-old L. decidua seedlings. The species richness in the examined larches varied between one and four fungal taxa, depending on both the nursery stock samples (NSS) and age class of the seedlings. The average was 1.4 for 1-year-old seedlings and 2.3 for 2- and 3-year-old plants. The dominance of Ascomycota over Basidiomycota and the prevalence of two species, Wilcoxina mikolae and Suillus grevillei, as EMF partners were characteristic features of nursery-grown L. decidua seedlings. S. grevillei was the only one basidiomycetes colonized roots of tested seedlings. The rest of the mycorrhizal pool from forest nurseries was typically dominated by pioneer fungal ascomycetes. W. mikolae was the most common mycorrhizal ascomycete present at a high frequency on NSS from both age classes and with very high abundance (average 90%) on 1-year-old seedlings. Some other ascomycetes (Pezizales 1, Pezizales 2 and Pezizales 3) appeared on tested larches at a low frequency, but sometimes in high abundance. Tuber spp. appeared at a low frequency and low abundance. The relative abundance of S. grevillei was positively correlated with the age of seedlings, while W. mikolae was negatively correlated with age. Tuber sp. 1 and 2, Pezizales 2, and W. mikolae were positively associated with the basic soil pH values. However, forward selection of the environmental variables showed that only the age of the larch seedlings contributed significantly (F = 11.45, P = 0.02) to the variance in the ECF community.     

Changes in nitrogen level and mycorrhizal structure of Scots pine seedlings inoculated with Thelephora terrestris

? We investigated the influence of Thelephora terrestris, an ectomycorrhizal fungus, on the concentration of nitrogen in needles of Pinus sylvestris L. seedlings and their mycorrhizal structure within four seasons of growth.

? Seedlings were grown in four treatments: (I) inoculated + 0.030 g N, (II) inoculated + 0.045 g N, (III non-inoculated + 0.030 g N, (IV) non-inoculated + 0.045 g N under laboratory conditions and later planted in post-agricultural land.

? For inoculated treatments, statistically significant differences in N concentration of needles were observed for two- and four-year-old seedlings. The number of ectomycorrhizae and concentration of N in needles were negatively correlated. After four seasons of growth the greatest numbers of ectomycorrhizae were present on seedlings from treatment II, and the smallest on seedlings from treatment IV.

? Two-year-old seedlings had been colonized by at least six different fungal taxa, i.e. Cenococcum geophilum, Hebeloma crustuliniforme, Rhizopogon sp., Suillus bovinus, Thelephora terrestris and a non-identified one. Treatments I and II were dominated by the ectomycorrhizae of T. terrestris and their number was significantly greater than in treatments III or TV. On four-year-old seedlings ectomycorrhizae of T. terrestris were dominated in all treatments, while ectomycorrhizae of Hebeloma sp. were not found.

     

Soil impact and radiation dose to native plants in forest ecosystem

A radiation dose assessment model was applied to determine naturally occurring radionuclides ²³⁸U, ²¹⁰Po and ²³²Th in trees like Elaeocarpus oblongus, Evodia roxburghiana (Juice of the leaves that are used to treat fever), Vaccinium neilgherrense, Viburnum hebanthum and Michelia nilagirica and shrubs like Lasianthus coffeoiaes and Hedyotis stylosa in the agroforests of the Western Ghats, India and these areas were used for different agroforestry models of land development systems. The concentration of radionuclides in the biota and corresponding soil was measured using a gamma ray spectrometer and an alpha counter. The concentration ratios of these radionuclides varied substantially between the species, and E. oblongus showed a preferential uptake of all the radionuclides and hence useful for bio-indication of radionuclides in such soils. ERICA assessment tool was employed to provide an assessment of the potential doses to biota’s growing in agro forest E. oblongus appears to be much prone to radiation absorption from the dose risk point of view. The annual effective dose (AED) due to ingestion of these radionuclides in E. roxburghiana was also estimated, and was found to be lower than the world average.     

两种杨树树皮内生真菌多样性及优势种群动态变化

为了解健杨94(转基因抗虫杨94)、三倍体毛白杨2个杨树品种干部树皮内生真菌区系及其优势种群的季节变化情况,本研究利用组织分离法从2个杨树品种996块组织中分离内生真菌1 175株,健杨94和三倍体毛白杨分别分离612、563株.利用形态特征和分子生物学方法鉴定为15个属、35个分类单元,包括担子菌1个分类单元,子囊菌34个分类单元.2个杨树品种内生真菌优势种群包括链格孢、葡萄座腔菌、镰孢属真菌、间座壳属真菌等,其中,仅有链格孢、葡萄座腔菌和桑砖红镰孢是两品种共有的优势种群种类,而且优势种群会随季节变化而变化.在两品种的内生真菌中,链格孢、葡萄座腔菌是最为常见的优势种群.     

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.     

Fungal community succession following wildfire in a Mediterranean vegetation type dominated by Pinus pinaster in Northwest Spain

This study examined the succession of fungal communities following fire in a Mediterranean ecosystem dominated by Pinus pinaster Ait. in northwestern Spain. A large wildfire occurred in August 2002. During the autumn seasons from 2003 to 2006, fruiting bodies were collected and identified, production in burned (early stage) and unburned (late stage) areas was measured. For statistical analysis, data were grouped into the following four categories: saprotrophic, mycorrhizal; edible and inedible. A total of 115 fungal taxa were collected during the four sampling periods (85 in the late and 60 in the early stage). The number of mycorrhizal species increased from early to late succession and there were shifts in community composition. After fire, pyrophytic species such as Pholiota carbonaria, Peziza violacea, Rhizopogon luteolus and Rhizopogon sp. appeared. Fire strongly affected the production of fungal species in the studied area. Thus, yields in the early stage treatment were significantly lower than those observed in the late stage. Total fungal fresh weight decreased from 209.95 kg fw ha⁻¹ in the late stage to 162.45 kg fw ha⁻¹in the early stage when richness and production of mycorrhizal species and production of edible fungi were significantly lower. Fresh weight for saprotrophic and inedible species was higher than for mycorrhizal fungi in the early stage treatment. The results obtained can be useful to forest managers for optimization of management and harvesting of these increasingly appreciated non-timber resources.     

Purification of four strains of endophytic fungi from Astragalus and their optimized liquid fermentations

This study was designed to isolate endophytic fungi from A. mongholicus(growing in northeast China) to determine whether they can produce bioactive metabolites. Four strains of endophytic fungi(strains 16, 17, 23 and 75) were successfully isolated from A. mongholicus using the surface disinfection method. According to ITS-rDNA sequences analysis, strains 16 and 75 were identified as Fusarium oxysporum, and strains 17 and 23 were identified as Bionectria ochroleuca. We applied the Box-Behnken design(BBD) to optimize the liquid fermentation conditions and obtain the maximum cell dry weight(CDW) yield. Optimal parameters were obtained under the following experimental conditions: temperature of 28°C, potato dextrose agar(PDA) liquid medium of 80 mL and rotation speed of 150 rpm. The four isolated endophytic fungi did not produce astragalosides I–IV, flavonoids or polysaccharides. Isolation of additional species of endophytic fungi from A. mongholicus and determination of their capacity to produce biologically active substances are subjects in need of further research.     

Meroterpenes and azaphilones from marine mangrove endophytic fungus Penicillium 303#

Three new metabolites (compounds 1–2 and 6), one azaphilone, and two meroterpenes, together with eleven known compounds have been isolated from a mangrove endophytic fungus, Penicillium 303#. Structure elucidation was achieved by 1D and 2D NMR spectroscopy. The absolute configurations of 1 and 2 were determined by electronic circular dichroism (ECD). Cytotoxic activities of new compounds 1–2 and 6 and compound 7were evaluated in vitro against human cancer lines MDA-MB-435, HepG2, HCT-116, and A549. Those compounds showed weak to moderate cytotoxic activities.     

The effect of bagging branches on levels of endophytic fungal infection in Japanese beech leaves

The species composition of the endophytic mycobiota in leaves of Japanese beech trees (Fagus crenata) and the sources for leaf infections were studied in a forest reserve situated in central eastern Honshu, Japan. To clarify the mechanism of infection of leaves, half of the branches were covered with polyethylene bags and species composition and levels of endophytic fungal infection were then compared with those of unbagged controls. Isolations were carried out from the leaves, petioles, and current‐year twigs of both, bagged and unbagged branches. Additionally, species composition was detected in overwintered terminal buds of beech trees and in the leaves of potted seedlings that had been placed in the field in different seasons. The species assemblage of the unbagged leaves, petioles, and current‐year twigs was dominated by Mycosphaerella buna, Ascochyta fagi, Periconiella sp., and Tritirachium sp. Other frequently recovered species were Xylaria sp., Phomopsis sp., and Tubakia dryina. Mycosphaerella buna and A. fagi were never isolated from leaves on bagged branches. A. fagi was, however, detected on both bagged and unbagged petioles and current‐year twigs at comparatively low isolation frequencies. The detection of Periconiella sp. on all occasions in both bagged and unbagged leaves was a characteristic feature that differs from those of the other three dominant endophytic fungi. The fungus was also detected without significant differences in bagged and unbagged petioles and current‐year twigs on most sampling dates. Furthermore, Periconiella sp. was isolated from immature twigs inside the bud scales. Tritirachium sp. was frequently detected in unbagged leaves and petioles and in both bagged and unbagged current‐year twigs, and rarely in bagged leaves and petioles, but was never recovered from terminal buds. The results of the potted seedling experiments revealed that all four dominant species had airborne inocula. The infection of leaves by M. buna occurs exclusively by airborne propagules, i.e. ascospores in spring and conidia in autumn. In Periconiella sp. hyphal growth of the fungus from immature twigs inside the buds into the leaf tissues was suggested in addition to infection by airborne inocula. Tritirachium sp. hyphae were suggested to grow from previous‐ to current‐year twigs. Ascochyta fagi was present in the outermost scales of overwintered terminal buds, but no systemic growth of the fungus into the petioles and current‐year twigs was observed. Our technique of covering the branches before new leaves unfolded was effective in preventing infection by airborne inocula of endophytic fungi.