Control of Hymenoscyphus fraxineus,the causal agent of ash dieback,using composting

Viability of Hymenoscyphus fraxineus inocula following temperature treatments for different exposure times was examined in vitro and in aerated flask‐ and large‐scale composting tests using green waste. After an exposure for up to 10 days at 20°C, 97.3% of H. fraxineus mycelium and pseudosclerotia plate cultures remained viable. No viability was detected following a 3‐day exposure to 40°C or a 1‐day exposure to 45°C although pseudosclerotia were more tolerant than mycelium to an exposure to 35°C. Primordial apothecia of H. fraxineus emerged from 62%–100% of infected ash rachises collected from two infected sites and stored at 4°C for 0–5 months; exposure to compost for up to 10 days at 20°C did not affect this emergence. No emergence of H. fraxineus apothecia was observed from ash rachises that were exposed to compost at 45°C for 1 day or at 35°C or 40°C for 3 days in flasks or at 40°C for 1 day or at 30°C for 5 days in a large‐scale composting system. Based on a fitted model, estimates of the survival of H. fraxineus inoculum in infected ash rachises exposed to compost at 50°C for 1 day were 0.081% of that in the untreated H. fraxineus ash rachis inoculum. Increasing loss in viability of H. fraxineus inoculum in infected ash rachises during longer and warmer exposures to compost at 35°C–45°C corresponded with a reduced concentration of pathogen DNA detected in the rachises using real‐time PCR. However, exposure of rachises to compost at >53°C resulted in a smaller reduction in pathogen DNA detected than exposure to compost at lower temperatures, possibly due to the inhibition of enzymatic degradation of DNA at elevated temperatures.     

Do higher summer temperatures restrict the dissemination of Hymenoscyphus fraxineus in France?

Hymenoscyphus fraxineus is an invasive pathogen that severely affects European ashes, jeopardizing the use of this species in forestry. However, even aggressive invasive pathogens require environmental conditions conducive to disease development. Hence, H. fraxineus survival at temperatures above 35°C is limited, which could reduce its impact in southern Europe. This study examined the effect of environmental conditions, mainly summer temperatures, on ash dieback development in southeast France between 2015 and 2016. Fourteen sites were selected according to altitudinal and longitudinal gradients covering a wide range of mean summer temperatures. Disease severity, density of infected and healthy rachises in the litter and quantity of apothecia produced on infected rachises were measured. Results point to limited disease severity in this area because of the occurrence of high summer temperatures that are unfavourable to the pathogen. In a context of global warming, increases in temperatures could have a positive impact on ashes by limiting ash dieback development.     

Hymenoscyphus fraxineus mycelial growth on media containing leaf extracts of different Oleaceae

We assessed the mycelial growth rate of Hymenoscyphus fraxineus, the causal agent of ash dieback, on agar media containing leaf extracts of seven common Mediterranean species of the Oleaceae (Fraxinus excelsior, F. angustifolia, F. ornus, Ligustrum vulgare, Olea europaea, Phyllirea latifolia and Syringa vulgaris). The pathogen grew on all media, but growth rates showed significant differences among media and H. fraxineus isolates. Growth rates were highest on media containing F. excelsior and F. angustifolia, intermediate on media containing O. europaea and P. latifolia and lowest on those containing F. ornus and L. vulgare.     

A versatile method for assessing pathogenicity of Hymenoscyphus fraxineus to ash foliage

We describe a method for inoculating rachises of Fraxinus excelsior (European or common ash) with Hymenoscyphus fraxineus, which is faster than previous methods and allows associated foliar symptoms to be assessed on replicate leaves. A total of ten ash seedlings were inoculated with five isolates of H. fraxineus and lesion development assessed over four weeks. A five‐point disease progress scale of symptom development was developed from no lesion (0), lesion on rachis (1), “pre‐top dead,” with curling of distal leaflets and bending of the rachis (2), top dead, with wilting and death of distal leaflets (3) to leaf abscission (4). The method revealed variation in aggressiveness of H. fraxinus isolates and may be suitable for assessing the resistance of F. excelsior and other Fraxinus species to dieback. The in vitro growth rate of H. fraxineus isolates was highly correlated with both disease progress and the length of rachis lesions on susceptible plants, indicating that it can be used as a preliminary step in selecting isolates with high aggressiveness for use in resistance screening.     

First definite report of natural infection of Fraxinus ornus by Hymenoscyphus fraxineus

Hymenoscyphus fraxineus was isolated from four leaf rachises with necrotic lesions of flowering ash (Fraxinus ornus) seedlings, which had been planted on a forest site in Austria where they were exposed to a massive natural infection pressure. This represents the first definite report of natural infection of this ash species by the ash dieback pathogen.     

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.     

The introduction of Hymenoscyphus fraxineus to Northern Ireland and the subsequent development of ash dieback

Ash dieback caused by Hymenoscyphus fraxineus was first recorded in Northern Ireland (NI) in November 2012. The disease was observed only on recently (<6 years) planted trees. An in-depth case study in 2015 of an ash plantation with severe symptoms indicated that many of the trees were infected at the time of planting. Apothecia were observed developing from pseudosclerotia beneath the epidermis of dead branches still attached to the tree, suggesting a possible mechanism whereby H. fraxineus could be disseminated without leaf or rachises infection. Apothecia also formed on roots, indicating that infections may also occur in the soil. Often young trees were killed by the formation of large basal lesions which did not arise from stem infections higher up. On first detecting the disease on the island of Ireland the Governments of NI and the Republic of Ireland published an “All-Ireland Chalara Control Strategy.” Part of that strategy was a ban on the importation of ash plants from regions where the disease was known to be present, to prevent the introduction of further inoculum, and the implementation of an ‘eradication and containment’ policy with the aim of preventing the establishment and spread of the disease. While these measures may have slowed disease establishment, they were ultimately unsuccessful and by 2018 ash dieback was widespread and established throughout the whole of NI in plantations and in the wider environment.     

Can Hymenoscyphus fraxineus infect hardy members of the Oleaceae other than ash species?

The interaction between the ash dieback fungus, Hymenoscyphus fraxineus, and two commonly grown members of the Oleaceae (Forsythia × intermedia ‘Lynwood’ and Ligustrum vulgare) was investigated. Shoots were inoculated with three isolates of H. fraxineus and harvested after 2 and 10 weeks. DNA was extracted from tissue collected at the point of inoculation, as well as 15 mm below and above (distal), and analysed using fungal‐specific ITS primers and specific primers for H. fraxineus. The fungus was frequently detected in tissue at the point of inoculation, but rarely in distal samples. There was no evidence that H. fraxineus was pathogenic on either of the two oleaceous species over the duration of the experiment.     

The susceptibility of Asian,European and North American <Emphasis Type="Italic">Fraxinus</Emphasis> species to the ash dieback pathogen <Emphasis Type="Italic">Hymenoscyphus fraxineus</Emphasis> reflects their phylogenetic history

In Europe, common ash (Fraxinus excelsior) is being decimated because of the invasive fungus Hymenoscyphus fraxineus. In its native range in Asia this ascomycete is considered a harmless leaf associate of F. mandshurica and F. chinensis subsp. rhynchophylla. Field observations from Europe suggest that there is species-specific variation in disease susceptibility among European and North American Fraxinus species, but a wider comparison at the genus level has been missing so far. We assessed disease symptoms and pathogen apothecium development in 17 Fraxinus species from Asia, Europe and North America exposed to high infection pressure in a Danish arboretum. We also tested their susceptibility to pathogen infection through controlled stem and leaf inoculations and subsequently assessed the level of pathogen DNA by a qPCR assay. The results suggested the presence of a phylogenetic signal in disease susceptibility where closely related Asian, European and North American species in section Fraxinus had relatively high levels of H. fraxineus DNA in the leaves and supported high production of apothecia. Leaves from some North American species also contained relatively high levels of H. fraxineus DNA, supported moderate production of apothecia and developed lesions—stating the need to avoid introduction of H. fraxineus to North America.     

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.     

First report of Hymenoscyphus fraxineus on Fraxinus excelsior in Montenegro

During the monitoring of the mycological complex on different forest tree species in the Biogradska Gora National Park in north‐east Montenegro, symptoms indicative of ash dieback caused by Hymenoscyphus fraxineus were observed on young Fraxinus excelsior trees in the protected virgin forest, including dieback of plants and branches, wilting of leaves and shoots leading to a “flag‐like” habitus, premature shedding of leaves and longitudinal bark necroses. Using standard isolation methods, slow‐growing cultures with numerous phialides, typical of the asexual phase of the ash dieback fungus, were obtained. In addition, petioles with numerous characteristic apothecia were also recorded. This is the first report of H. fraxineus on common ash in Montenegro. Possible pathways of introduction and implications of the findings are discussed.     

Aqueous leaf extract of Ligustrum vulgare inhibits ascospore germination and mycelial growth of Hymenoscyphus fraxineus

Hymenoscyphus fraxineus, the causal agent of common ash dieback, possesses a low level of genetic diversity in Europe. The introduction of novel strains of this fungus must be prevented, due to the possible emergence of new virulence alleles, which could result in the infestation of the small proportion of hitherto resistant or tolerant ash trees. More comprehensive knowledge of the host spectrum of H. fraxineus is necessary for preventing further introductions. It is possible that H. fraxineus manifests itself in hosts beyond the genus Fraxinus, though this proposition has received little attention thus far. Two in vitro experiments were set up to investigate whether privet (Ligustrum vulgare) could serve as a host: germination rate of fresh H. fraxineus ascospores and colony growth of H. fraxineus were tested on agar media containing leaf extracts of privet, common ash (Fraxinus excelsior) and manna ash (Fraxinus ornus). Two different media with leaf extracts were tested, one with high (60%) and one with low (12%) extract content. Barely any significant differences were recorded in the case of the media with low extract content. Significant effects occurred only at the higher extract content level: germination was completely inhibited on the privet medium. Mycelial growth on the privet medium was slower than on both the common ash and manna ash media and, in addition, one of the three H. fraxineus strains was completely inhibited. These observations indicate the presence of inhibitors in privet. It is therefore unlikely to be a suitable host for H. fraxineus.     

ADA,a fast‐growth medium for Hymenoscyphus fraxineus

The growth rate of Hymenoscyphus fraxineus was evaluated on five different media under laboratory conditions. Statistically significant differences were found among them, where H. fraxineus demonstrated the highest growth rate on ADA medium (a newly designed nutritive medium). This study suggests that ADA is a suitable medium for growing H. fraxineus.     

Differences in susceptibility to ash dieback in Czech provenances of Fraxinus excelsior

The impact of ash dieback caused by Hymenoscyphus fraxineus on 17 provenances of Fraxinus excelsior and one provenance of Fraxinus angustifolia was studied in an extensive field trial established in the Czech Republic prior to the H. fraxineus invasion in 1999. A difference in the level of resistance to ash dieback between the species was found: F. angustifolia was significantly less affected by the disease than F. excelsior. Moreover, particular provenances of F. excelsior showed important differences in the level of resistance to H. fraxineus. A relationship between the impact of ash dieback and altitude was also discovered – the provenances from altitudes above 600 m a.s.l. were less affected by the pathogen than were the provenances from lower areas. No difference in the impact of the disease among provenances of F. excelsior from different ecotopes (ravine, calcareous ravine and alluvial) was found. Substantial among‐tree variability in resistance to H. fraxineus was observed throughout the trial – promising genotypes (with crown defoliation up to 5%) were identified in all 18 tested provenances. In regard to this finding, it appears that the main source of resistance to the pathogen is probably at the individual genotype level in the trial. A secondary but massive attack by Hylesinus fraxini was identified in the trees that had been greatly damaged by ash dieback, and the beetle caused their health to deteriorate significantly. A significant negative effect of the presence of collar necroses caused by H. fraxineus and browse damage was also identified.     

Direct evidence of Hymenoscyphus fraxineus infection pathway through the petiole‐shoot junction

The symptoms of ash dieback caused by the fungus Hymenoscyphus fraxineus include wilting of the foliage followed by dieback of shoots, twigs and branches. Necroses in shoots are assumed to develop after infection through leaf petioles; however, clear evidence of this infection pathway has not yet been provided. Considering the multiple pathogen genotypes in dead ash petioles, we aimed to obtain a spatial overview of all H. fraxineus genotypes colonizing individual shoots and their corresponding petioles before leaf shedding to acquire precise information about the infection biology of H. fraxineus and its ability to cross the petiole‐shoot junction. Individual genotypes of H. fraxineus were characterized by the analysis of microsatellites using DNA extracted directly from petiole segments or cultures isolated from the segments. We detected 150 different multilocus genotypes in 10 analysed shoots and their respective petioles; the highest number of genotypes was eight for a single petiole and three for a single shoot. The genotypes of most shoot lesions were identical to particular genotypes from the proximal segments of petioles, implicating the main pathway of shoot infections. To test whether the amount of colonized substrate or intraspecific competition have an effect on successful infection, genotypes that reached the most proximal end of the petioles were scored for the number of invaded petiole segments and for the number of other H. fraxineus genotypes co‐occurring in the segments. However, the extent of colonization of the scored genotypes and intraspecific competition with other H. fraxineus strains did not influence pathogen success in entering the shoot. This study confirms that the majority of ash shoot infections are caused by genotypes of H. fraxineus originating from petioles. Compared to petioles, the frequency of shoot colonization as well as number of H. fraxineus genotypes in shoots was much lower.     

Fraxinus excelsior seed is not a probable introduction pathway for Hymenoscyphus fraxineus

We investigated the transmission of Hymenoscyphus fraxineus from infested seed to germinating seedlings of common ash (Fraxinus excelsior) in order to determine the potential risk associated with intra‐ and intercontinental movement of seed. Neither fungal isolations from necrotic or healthy embryos nor PCR testing with H. fraxineus‐specific primers detected the pathogen. Similarly, H. fraxineus was not detected in axenically grown seedlings generated from infested seed lots. The results help clear up prior confusion of the pathogen being seed‐borne. Any remaining surface contamination by pathogen spores could be washed off seeds as a quarantine measure.     

Ascocarp formation of Hymenoscyphus fraxineus on several‐year‐old pseudosclerotial leaf rachises of Fraxinus excelsior

The ash dieback pathogen Hymenosycphus fraxineus can form ascocarps on pseudosclerotial leaf rachises of Fraxinus excelsior not only in the year after leaf fall but also on older rachises, at least up to five growing seasons after the leaves have been shed. The significance of this finding for the epidemiology of ash dieback is discussed.     

Individual resistance of Fraxinus angustifolia clones to ash dieback

The presumed resistance of individual ash trees to ash dieback caused by invasive pathogen Hymenoscyphus fraxineus is an important issue for the maintenance of ash in European forests. All known studies regarding the resistance of ash trees to ash dieback were conducted in plantations and stands of F. excelsior; however, no such data exist for F. angustifolia. Crown damage assessments were performed over four consecutive years between 2009 and 2012 at a F. angustifolia clonal plantation in Hra??ica, Slovenia. Inoculation of H. fraxineus into the branches of the most and least damaged clones of F. angustifolia and leaf phenology assessments was performed to verify the presence of defence mechanisms that limit fungal growth or promote disease escape. Additionally, root collars of selected clones were inspected for fungal infections. The crown damage assessments showed considerable differences among F. angustifolia clones, indicating genetic variability in susceptibility to ash dieback. Crown dieback progressed significantly over the 4‐year time period; the mean crown damage of individual clones in 2012 varied between 16.7% and 83.8%. Significant differences among F. angustifolia clones were found in the inoculation trials and leaf phenology assessments. However, defence mechanisms such as early leaf flushing, early leaf shedding and the ability to inhibit pathogen growth in host tissues were not confirmed. High frequency of Armillaria spp. and H. fraxineus root collar infection demonstrated the need for whole tree inspection to determine causal agent of damages on individual ash trees. Armillaria spp. may be highly associated with ash decline epidemiology.     

On the longevity of Hymenoscyphus pseudoalbidus in petioles of Fraxinus excelsior

Air‐dried pseudosclerotia produced by the ash dieback pathogen Hymenoscyphus pseudoalbidus (anamorph: Chalara fraxinea) can survive up to 3 months. Under field conditions, they are capable of postponing fructification for 1 year. The implications of this observation for epidemiology and control of ash dieback are discussed.     

Hymenoscyphus fraxineus can directly infect intact current‐year shoots of Fraxinus excelsior and artificially exposed leaf scars

Hymenoscyphus fraxineus, the causal agent of ash dieback, was inoculated onto intact, unwounded current‐year shoots and leaf scars of 4‐year‐old, potted Fraxinus excelsior seedlings. Pieces of ash wood colonized by the fungus were used as inoculum. Three of 25 (12%) of the inoculated intact shoots and nine of 25 (36%) of the inoculated leaf scars were infected by H. fraxineus and developed typical symptoms of ash dieback, including necrotic lesions on the shoot surface and wood discoloration as well as shoot and leaf wilting distal to the inoculation site. No symptoms occurred on control seedlings, which had been inoculated in the same way but with sterile wood pieces. Visible necrotic lesions on shoots and wood discoloration were statistically significantly longer in proximal than in distal direction from the inoculation site, a pattern which resembles symptoms after natural infection. The ash dieback pathogen was re‐isolated from nine of 12 (75%) of the symptomatic seedlings. These results provide indirect supportive evidence that the fungus infects shoots via leaves and shows that it is able, under experimental conditions using a massive mycelial inoculum, to directly infect intact, unwounded current‐year shoots of its main host in Europe.