The Impact of Biotic and Abiotic Stress Factors on Development of European Ash Tissue Cultures

Fraxinus excelsior L. is threatened by a variety of environmental factors causing a decline of the species. The most important biotic factors negatively affecting the condition of the F. excelsior population are fungi such as the pathogen Hymenoscyphus fraxineus. Abiotic factors with potentially harmful effect to the F. excelsior population are the accumulation of heavy metals and salinity in soils. Thus, the aim of this study was to investigate the impact of selected biotic and abiotic stress factors to determine which of them pose a threat to European ash. The study was conducted using in vitro techniques based on callus and seedlings regenerated via indirect organogenesis. Tissue cultures exclude the influence of other factors, including the environmental impact on ash extinction. The results confirmed very strong pathogenic potential of H. fraxineus in which after 14 days the callus tissue cells died as the tissue failed to activate its defense mechanisms. Experiments showed the high toxicity of cadmium in concentration of 0.027 mmol/L. Salinity caused the activity of oxidation enzymes to vary among seedlings and calluses in the control suggesting the enzymes play a role in controlling the morphogenetic development of tissue cultures.

The Occurrence of Apiognomonia hystrix and Its Pathogenicity towards Acer pseudoplatanus and Fraxinus excelsior under Field Conditions

Apiognomonia hystrix is an ascomycetous fungus within Diaporthales that is found on maples and to a lesser extent on other hardwood trees in Europe, Northern America and Asia. To date, varying opinions on the species’ status as a cause of plant diseases have been expressed. In this study, we present the results of analyses conducted from 2012–2017 at forest sites in Poland on the occurrence of A. hystrix on Acer pseudoplatanus and Fraxinus excelsior and the pathogenicity of this fungus towards both tree species. For the sycamore leaves, A. hystrix conidiomata were detected in connection with 19.2% of galls caused by Dasineura vitrina, 20.4% of galls caused by Drisina glutinosa and 67.9% of extensive vein-associated necroses. The A. hystrix colonization of galls caused by both midge species resulted in statistically significantly larger necroses. On European ash leaves, conidiomata of A. hystrix occurred in connection with 0.8% of Dasineura fraxinea galls. Perithecia of A. hystrix were detected on overwintered leaf petioles in 8.1% of A. pseudoplatanus and 1.2% of F. excelsior samples. Twelve representative cultures were characterized molecularly by barcoding three marker genes (ITS, ACT, CAL). Results of phylogenetic analyses indicate that A. hystrix isolates are genetically variable, and three lineages are distinguishable. Eight isolates, including four originating from sycamore and four from European ash, were used to determine A. hystrix pathogenicity. Among the 48 A. pseudoplatanus petioles inoculated with A. hystrix, 41 developed necrotic lesions after 8 weeks, with the average necrosis length caused by particular isolates ranging from 14.5 to 67.2 mm. None of the 48 inoculated F. excelsior petioles developed necrotic lesions. Finally, selected aspects of A. hystrix morphology on natural substrates and in vitro are discussed in this paper, as well as the species’ potential to cause disease symptoms.

Transformation of European Ash (Fraxinus excelsior L.) Callus as a Starting Point for Understanding the Molecular Basis of Ash Dieback

The population of European ash (Fraxinus excelsior L.) is currently facing the risk of collapse, mainly due to ash dieback, a disease caused by a pathogenic fungus, Hymenoscyphus fraxineus. To facilitate studies into the molecular basis of ash dieback and design breeding strategies for a generation of resistant trees, it is necessary to develop tools enabling the study of gene function in F. excelsior. Despite this, a method for the genetic engineering of F. excelsior is still missing. Here, we report the first successful genetic transformation of F. excelsior callus and a selection process enabling the formation of stable transgenic callus lines. The protocol relies on the use of Agrobacterium tumefaciens to transform callus tissue derived from embryos of F. excelsior. In our experiments, we used the β-glucuronidase (GUS) reporter system to demonstrate the transformation of callus cells and performed RT-PCR experiments to confirm the stable expression of the transgene. Since ash dieback threatens the long-term stability of many native F. excelsior populations, we hope that the transformation techniques described in this manuscript will facilitate rapid progress in uncovering the molecular basis of the disease and the validation of gene targets previously proposed to be linked to the resistance of trees to H. fraxineus pathogenicity.

First report of Diplodia fraxini and Diplodia subglobosa causing canker and dieback of Fraxinus excelsior in Slovenia

Over the last decades the vitality and productivity of European ash trees in Slovenia has been seriously impacted by the onset of canker and dieback disease symptoms on young and old trees, primarily identified as ash dieback caused by Hymenoscyphus fraxineus. Given the limited information available about the aetiology of this emerging disease, a study was carried out to isolate, identify and characterize the fungal species involved in the observed ash symptoms. Field surveys were conducted in five forest sites where 50 symptomatic branch samples were collected. All samples were inspected and used for fungal isolation. Based on morphology, colony appearance and DNA sequence data of the internal transcribed spacer region (ITS), 125 fungal colonies belonging to five species were isolated and identified. Only a few symptomatic ash samples yielded colonies of H. fraxineus, whereas Botryosphaeriaceae species were isolated with a high frequency, with Diplodia fraxini as the dominant species. A pathogenicity test proved that all isolated species were pathogenic on European ash, causing bark lesions and wood discoloration. All Botryosphaeriaceae species isolated in this study are reported for the first time on European ash in Slovenia.

A Novel Plant Resistance Inducer for the Protection of European Ash (Fraxinus excelsior L.) against Hymenoscyphus fraxineus—Preliminary Studies

Ash tree disease is caused by an ascomycete fungus Hymenoscyphus fraxineus, which first emerged in 1992, eastern Poland. Site factors, genetic predispositions, and resistance to the pathogen have not been fully described yet. The general aim of the study undertaken was to check the effect of using a new active substance representing benzothiadiazoles, a BTH derivative, namely, N-methyl-N-methoxyamide-7-carboxybenzo(1.2.3)thiadiazole (BTHWA), on ash saplings. A total of 41 ash saplings, aged three to five years, were subjected to this experiment in six variants of treatment. The results of the inoculation with H. fraxineus indicated that the treatment with BTHWA resulted in the limitation of the size of necrotic phloem lesions. Although the lesions were detectable in the cross section, the plants showed no visible signs of infection. The results suggest that H. fraxineus development in ash saplings can be slowed down or even completely stopped through triggering plant resistance by BTHWA.

European-wide forest monitoring substantiate the neccessity for a joint conservation strategy to rescue European ash species (Fraxinus spp.)

European ash (Fraxinus excelsior) and narrow-leafed ash (F. angustifolia) are keystone forest tree species in Europe with a broad ecological amplitude and significant economic importance. Besides global warming both species are currently under significant thread by an invasive fungal pathogen that has been progressively spreading throughout the continent for almost three decades. Ash dieback caused by the invasive ascomycete Hymenoscyphus fraxineus is capable of damaging ash trees of all age classes and often leads to the ultimate death of a tree after years of progressively developing crown defoliation. While studies at national and regional level already suggested rapid decline of ash populations as a result of ash dieback, a comprehensive survey at European level with harmonized crown assessment data across countries could shed more light into the population decline from a pan-European perspective and could also pave the way for a new conservation strategy beyond national boarders. Here, we present data from the ICP Forests Level I crown condition monitoring including 27 countries, covering the timespan from 1987-2020. In total, 407 survey plots randomly distributed across these countries were analyzed resulting in >36,000 individual observations. We found a substantial increase in defoliation and mortality over time indicating that crown defoliation has almost doubled during the last three decades. Hotspots of mortality are currently situated in southern Scandinavia and north-eastern Europe, well corresponding to the fact that the disease spread fast from north-east to north-west. Overall survival probability after nearly 30 years of infection has already reached a critical value of 0.51, but with large differences among regions (0.00-0.907). Both a Cox proportional hazard model as well as an Aalen additive regression model strongly suggest that survival of ash is significantly lower in locations with excessive water regime and which experienced more extreme precipitation events during the last two decades. Our results underpin the necessity for fast governmental acting and joint rescue efforts beyond national boarders since overall mean defoliation will likely reach 50% as early as 2030 as suggested by time series forecasting . We strongly recommend to develop a pan-European conservation strategy before the decline will reach its tipping point resulting into non-reversible loss of diversity in the European forest landscape.

Emerald Ash Borer Approaches the Borders of the European Union and Kazakhstan and Is Confirmed to Infest European Ash

Emerald ash borer (EAB), Agrilus planipennis, native to East Asia, is an invasive pest of ash in North America and European Russia. This quarantine species is a threat to ash trees all over Europe. Survey in ten provinces of European Russia in 2019–2020 showed that EAB had spread faster and farther than was previously thought. The new infested sites were first detected in St. Petersburg (110–120 km from the EU border: Estonia, Finland) and Astrakhan Province (50 km from the Kazakhstan border). The current range of EAB in Europe includes Luhansk Province of Ukraine and 18 provinces of Russia: Astrakhan, Belgorod, Bryansk, Kaluga, Kursk, Lipetsk, Moscow, Orel, Ryazan, Smolensk, St. Petersburg, Tambov, Tula, Tver, Vladimir, Volgograd, Voronezh, and Yaroslavl. Within these, only seven quarantine phytosanitary zones in five provinces are declared by the National Plant Protection Organization of Russia. EAB was not found in the regions along the Middle Volga: Mari El, Chuvash and Tatarstan republics, Nizhny Novgorod, Samara and Saratov provinces. The infested sites in St. Petersburg and in the Lower Volga basin are range enclaves separated from the core invasion range by 470 and 370 km, correspondingly. It is possible that new enclaves can appear in the cities of Eastern Europe and Kazakhstan far from the current known range. All previously known infestations in European Russia were in green ash (Fraxinus pennsylvanica), which was introduced from North America, and individual trees of European ash (F. excelsior). A first confirmed case of mass decline of several thousand of EAB-infested European ash trees in Moscow province is provided. Therefore, there is no more doubt that under certain conditions EAB can seriously damage native ash trees in European forests.

Ash dieback and contributing factors of forest weakening in provenance tests in the Sumy region

The aim of this study was to evaluate the health condition of Fraxinus excelsior L. in provenance tests, with special focus on ash dieback (ADB), but taking into account also other causes of its decline. The research was carried out in the provenance tests of F. excelsior in the forest-steppe part of the Sumy region. ADB symptoms were revealed in all provenance tests. For 2012–2019 the health condition index, ADB incidence and severity increased for all provenances except the Steppe. Collar rot was present in all ash provenances. Fungi species were isolated from the stem parts of ash at all provenances. Hymenoscyphus fraxineus induced longest necrotic lesions following wound inoculation of stems of 7–10-years-old plants of European ash in the forest while inoculation with both Cytospora sp. and Diplodia sp. resulted in smaller necroses. The conclusion from other regions about the coincidence the damage of European ash by ADB and collar rots as well as the coincidence the damage of European ash by collar rot and tree colonization by Hylesinus crenatus (Fabricius, 1787) is supported.

Mycobiome of Fraxinus excelsior With Different Phenotypic Susceptibility to Ash Dieback

For the last two decades, large-scale population decline of European ash (Fraxinus excelsior) has occurred in Europe because of the introduction of the alien fungal pathogen, Hymenoscyphus fraxineus, from East Asia. Since European ash is a keystone species having critical importance for biodiversity, and only a small percentage of the ash population appears to show some tolerance against the pathogen, the loss of ash trees means that other associated organisms, especially those with high or obligate associations to ash, are at risk of further species declines. In this study, we used high throughput DNA sequencing and multivariate analysis to characterize: (i) the mycobiome in aerial tissues (i.e., leaf, bark, and xylem) of ash trees showing different phenotypic response to ash dieback, (ii) the temporal variation in fungal communities across the growing season, and (iii) the similarity in fungal community structure between ash and other common trees species that may serve as an ecological niche substitute for ash microfungi. Results showed that fungal communities differed among the three tissue types, susceptibility classes, in time and between sites. Trophic analysis of functional groups using the FUNGuild tool indicated a higher presence of pathotrophic fungi in leaves than in bark and xylem. The share of pathotrophic fungi increased along a gradient of low to high disease susceptibility in both bark and xylem tissue, while the proportion of symbiotrophic fungi correspondingly decreased in both tissue types. Neighboring, alternative host trees did not share all the fungal species found in ash, however, most microfungi uniquely associated to ash in this study are generalists and not strictly host specific. The progressive disappearance of ash trees on the landscape imposes a high risk for extinction of Red-listed macrofungal species, and breeding for resistance against ash dieback should help sustain important biodiversity associated to ash. Microfungal diversity though may be less prone to such demise since most ash-associated endophytes appear to occur on a broad range of host species.