Fungal endophytes in Fraxinus excelsior petioles and their in vitro antagonistic potential against the ash dieback pathogen Hymenoscyphus fraxineus.

Fungal endophytes were isolated from 250 asymptomatic leaf petioles of Fraxinus excelsior collected from trees showing symptoms of ash dieback in five forest sites in southern Poland. Fungal isolations yielded 1646 colonies representing 97 taxa, including 92 Ascomycota and 5 Basidiomycota species. The most common Ascomycota comprised Nemania serpens (38.0 % of colonized petioles), Diaporthe eres (33.6 %), Venturia fraxini (26.4 %), Diaporthe sp. 1 (20.4 %), Alternaria sp. 1 (14.8 %), Colletotrichum acutatum (14.8 %), Nemania diffusa (14.0 %), Colletotrichum gloeosporioides (12.4 %) and Colletotrichum sp. (12.4 %). The occurrence of all these taxa except Alternaria sp. 1 was significantly different between the studied forest sites. Two yeast species, Vishniacozyma foliicola (4.8 %) and Cystobasidium pinicola (2.8 %), dominated among the Basidiomycota endophytes detected. All the fungal endophytes were tested in dual culture antagonistic assays against two strains of Hymenoscyphus fraxineus, resulting in the development of four interaction types. The interactions included the physical contact of co-partners' mycelia (41.8 %), development of an inhibition zone (47.4 %), growth of endophyte mycelia over H. fraxineus colonies (9.3 %) and growth of H. fraxineus mycelia over endophyte colonies (1.5 %). The strongest antibiotic activity against H. fraxineus, measured by the width of the inhibition zone, was observed for Cytospora pruinosa, Fusarium lateritium, Phoma sp. 2, Pleosporales sp. 2 and Thielavia basicola. A variety of morphophysiological deformations of H. fraxineus hyphae were observed under endophyte pressure: spiral twist of the hyphae, formation of cytoplasmic extrusions, development of torulose hyphae and excessive lateral branching of the hyphae. The strongest antagonistic effects, coupled with the potential to overgrow H. fraxineus colonies, was shown by Clonostachys rosea, Nemania diffusa, N. serpens, Peniophora cinerea, Rosellinia corticium and Xylaria polymorpha. Some of these species were able to attack H. fraxineus hyphae in a mycoparasitic manner. The antagonistic activities included the physical penetration of H. fraxineus hyphae, dissolution of hyphal cell walls, disappearance of pigmentation, disintegration of hyphae and degradation of other fungal structures. In contrast, one of the most commonly detected endophytes in ash leaves, Venturia fraxini, did not show in vitro antagonistic potential against H. fraxineus. Finally, we discuss the potential of the detected fungal endophytes to combat H. fraxineus invasion, the cause of ash decline in Europe.

Fungicolous Fungi on Pseudosclerotial Plates and Apothecia of Hymenoscyphus fraxineus and Their Biocontrol Potential.

In the present work, research tasks were carried out in the search for fungi with potential biocontrol possibilities in relation to the ash dieback pathogen, Hymenoscyphus fraxineus. In the years 2012-2021, dead petioles of F. excelsior and F. mandshurica were collected, on which morphological structures of H. fraxineus showed unusual symptoms of dying (apothecia) and signs of colonization by other fungi (pseudosclerotial plates). Based on morphological and molecular phylogenetic data, 18 fungal taxa were identified. Thirteen of them belong to Ascomycota: Clonostachys rosea, Cl. solani, Cordyceps sp., Minimidochium sp., Nemania diffusa, Fusarium sp., Pestalotiopsis sp., Trichoderma atroviride, T. harzianum, T. polysporum, T. rodmanii, T. tomentosum, Trichoderma sp., and five other taxa are represented by Basidiomycota: Corticiales sp., Cyathus olla, Efibula sp., Gymnopus sp. and Polyporales sp. In 108 dual cultures in vitro, three different types of interactions were distinguished: (i) physical colony contact (5.6%), (ii) presence of an inhibition zone between the colonies (0.9%), and (iii) copartner overgrowth of H. fraxineus colonies and partial or complete replacement of the pathogen (93.5%). In the dual cultures, various morphological deformations of H. fraxineus hyphae were observed: the development of apical or intercalary cytoplasmic extrusions, development of internal hyphae of the test fungi in pathogens' hyphae, the deformation and disruption of significant sections of H. fraxineus hyphae via lysis and mycoparasitism, complete desolation of H. fraxineus cells and breakdown of hyphae into short fragments, and disappearing of pigment in the affected hyphae of H. fraxineus. The inoculation tests performed in vivo or in glass Petrie dishes showed that all the identified taxa were able to lead to pathological changes in H. fraxineus apothecia, and the mycelium of some of them completely covered pseudosclerotial plates of H. fraxineus. It was emphasized in the discussion that such activity of these fungi in forest stands may contribute to the reduction in the H. fraxineus inoculum reservoir.

Detection of a pheomelanin-like pigment by EPR spectroscopy in the mycelium of Plenodomus biglobosus.

Melanin occurrence in Plenodomus biglobosus was investigated using electron paramagnetic (spin) resonance (EPR, ESR) spectroscopy. The fungus was isolated from living and dead leaves of European ash (Fraxinus excelsior L.). Dark pigmentation of P. biglobosus mycelium in vitro, especially on the reverse, was observed. The black coloration intensified with the age of the culture and inspired us to check if the analyzed fungus species synthesizes melanin. Melanin contains unpaired electrons, thus, EPR spectroscopy was applied, as a specific technique, to verify its presence in P. biglobosus. The EPR spectrum of the mycelium showed a very strong melanin signal, revealing pheomelanin-like features. Thus, the black pigment of P. biglobosus was clearly identified as melanin. However, no melanin was detected in the apparently dark culture medium even when zinc (II) acetate was added to increase the sensitivity of detection. Pheomelanin has many unusual biological functions but it is not commonly found in fungi. Detection of this type of melanin in P. biglobosus, which can be both endophytic or pathogenic, suggests a closer examination of the potential role of this melanin in host-parasite interaction.

The genetic relationships within Apiognomonia errabunda and related species.

Species of Apiognomonia are some of the most ubiquitous leaf-associated fungi of broad-leaved trees of the northern temperate zone. Especially widespread and diverse is Apiognomonia errabunda, found mostly on beech, oak, and linden. This species and its closest relatives are known for their complicated phylogenetic relationships that have caused considerable confusion in the past. In the present paper, we describe the results of a three-gene-based reconstruction of phylogenetic relationships between A. errabunda, A. veneta, and A. hystrix for 373 isolates. Using combined information from the nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS barcode) and intron regions of actin (ACT) and calmodulin (CAL) genes for 263 isolates, we confirmed the occurrence of host-associated lineages within A. errabunda. However, the clustering of isolates by host species was incomplete: some isolates occurred on the "wrong" hosts and a number of isolates carried mixed genetic profile, indicating substantial level of inter-host group recombination. In addition, a number of isolates were identified as putative, pending further verification, interspecies hybrids between the much more divergent A. errabunda and A. hystrix.

Virulence of Hymenoscyphus albidus and H. fraxineus on Fraxinus excelsior and F. pennsylvanica.

European ash (Fraxinus excelsior) is currently battling an onslaught of ash dieback, a disease emerging in the greater part of its native area, brought about by the introduction of the ascomycete Hymenoscyphus fraxineus (= Hymenoscyphus pseudoalbidus). The closely-related fungus Hymenoscyphus albidus, which is indigenous to Europe, is non-pathogenic when in contact with F. excelsior, but could pose a potential risk to exotic Fraxinus species. The North American green ash (Fraxinus pennsylvanica) is planted widely throughout Europe and regenerates naturally within this environment but little is known about the susceptibility of this species to ash dieback. We performed wound inoculations with both fungi (nine strains of H. fraxineus and three strains of H. albidus) on rachises and stems of F. excelsior and F. pennsylvanica under field conditions in Southern Poland. Necrosis formation was evaluated after two months on the rachises and after 12 months on the stems. After inoculation of H. albidus, only small lesions (of up to 1.3 cm in length) developed on the F. excelsior and F. pennsylvanica rachises, but with no significant distinction from the controls. Hymenoscyphus albidus did not cause necrotic lesions on the stems of either Fraxinus species. In contrast, H. fraxineus induced necroses on all inoculated rachises of both ash species with mean lengths of 8.4 cm (F. excelsior) and 1.9 cm (F. pennsylvanica). Necroses also developed on all of the inoculated F. excelsior stems (mean length 18.0 cm), whereas on F. pennsylvanica such lesions only occurred on about 5% of the stems (mean length 1.9 cm). The differences between strains were negligible. No necroses were observed on the control plants. Reisolations of H. albidus were only successful in around 8-11% of the cases, while H. fraxineus was reisolated from 50-70% of the inoculated organs showing necrotic lesions. None of the Hymenoscyphus species were isolated from the control plants. Our data confirm H. fraxineus' high virulence with regards to F. excelsior and demonstrate a low virulence in relation to F. pennsylvanica under field conditions in Poland. Hymenoscyphus albidus did not express any perceivable pathogenicity on both host species.