Hymenoscyphus fraxineus
(ash dieback)

H. fraxineus is an anamorphic fungal pathogen that causes ash dieback. Due to the severity of ash dieback H. pseudoalbidus has been on the EPPO Alert list since 2007. It is not known what caused the emergence of this 'new' disease (NAPPO, 2009). Its spread in Europe is thought to be mainly by ascospores, but infected nursery saplings may carry the fungus to new areas. The entire natural range of known hosts, including North Africa, Russia and south-west Asia (USDA-ARS, 2009), is currently threatened by ash dieback, with large areas already affected (Pautasso et al., 2013). Little is known about the susceptibility of the other species of ash in temperate zones.

Ash dieback disease was first observed in North and Central Europe in the 1990s and since then H. pseudoalbidus has spread throughout much of Europe. It has been found on Fraxinus mandshurica in Japan and China, where it appears to be non-pathogenic on its native host (Zhao et al., 2013, Zheng and Zhuang, 2014). Combined with the lack of resistance in European ash species this suggests an Asian origin for H. fraxineus.

H. fraxineus could certainly be distributed from forest nurseries on infected saplings (Kirisits et al., 2009). Wind-blown ascospores could be dispersed locally, as occurs with the related pathogen Crumenulopsis sororia (Hayes, 1980). Ash seeds can be naturally infected with H. fraxineus, although the effect of infection on germination is not known (Cleary et al., 2013a). Despite trade in ash seed between Europe and North America the disease has not been reported in the USA. International concern with the invasive emerald ash borer beetle (Agrilus planipennis) (USDA/APHIS, 2009; CFIA, 2009; EPPO, 2009a) should enhance restrictions on, and inspection of, any ash (Fraxinus) logs or lumber that might carry the fungus over great distances.

Only two Fraxinus species, F. excelsior (Kowalski, 2006) and F. angustifolia (Kirisits et al., 2009), are definitely known to be susceptible to the pathogen. The third species of ash native to Europe, F. ornus, was found to be less susceptible to the disease following field inoculations (Krautler and Kirisits, 2012). Other species in the same section of the genus (USDA-ARS, 2009) include: F. mandshurica, the cultivated F. holotricha in Europe; F.pallisiae, native to southeastern Europe; F.sogdiana in central Asia; and F. nigra, an ash native to North America (USDA-ARS, 2009). In Asia, H. fraxineus has only been recorded on F. mandshurica and F. chinensis. On these native hosts the fungus is apparently hemi-biotrophic and does not cause disease (Zhao et al., 2013). However, F. mandschurica was found to be mildly affected by ash dieback in south east Estonia (Drenkhan and Hanso, 2010). Three species of ash native to America have been investigated for susceptibility: F. nigra is badly affected, F. pennsylvanica shows slightly less severe symptoms whilst F. americana is the least affected (Drenkhan and Hanso, 2010). Stem inoculations of Acer pseudoplatanus and Sambucus nigra did not result in necrotic lesions (Kowalski and Holdenrieder, 2009a).

Life Cycle

Both the conidial anamorph and the apothecial teleomorph have been described for this species, but their roles in the life cycle and spread of the pathogen have not been fully determined. Unlike H. fraxineus, H. albidus does not form an anamorphic stage and this is one way to distinguish between the species (Kirisits et al., 2013).

Conidia are produced in culture (Kowalski, 2006; Halmschlager and Kirisits, 2008) and sporulation has been found on the surface of lesions on inoculated young trees, but is rarely observed in the field (Kowalski and Holdenrieder, 2009b). Production of conidia in culture is enhanced at low temperatures, but some isolates do sporulate at 23 to 25°C (Halmschlager and Kirisits, 2008; Jankovský and Holdenrieder, 2009; Szabó, 2009).

The fungus overwinters in the leaf litter, forming a black pseudosclerotial layer on ash rachises. Apothecia are produced between July and October, although this will vary according to climatic conditions (Kowalski and Holdenrieder, 2009b, Hietala et al., 2013). Apothecia were also found occasionally on shoots of dead seedlings in nurseries (Kowalski and Holdenrieder, 2009b). Gross et al. (2012) observed that UV light and moisture appear to be important for apothecial maturation. Ascospores are released in a diurnal pattern with a peak between 6am and 8am, possibly to coincide with morning dew and to prevent desiccation (Timmermann et al., 2011).

Ascospores adhere to leaflets and penetrate the cuticle via an appresorium and germ tube (Cleary et al., 2013b). Hyphae rapidly proliferate and the mycelium may spread from the leaf lamina via the rachis to the stem. However, this is a dead end for the pathogen and the life cycle is completed in the leaf litter after infected leaves are shed.

Molecular characterization of the mating type locus has shown that H. fraxineus is heterothallic, unlike H. albidus which is structurally homothallic (Gross et al., 2012). A multiplex PCR is available for mating type determination. Parental analysis of individual apothecia on single rachises revealed that rachises may be colonized by multiple genotypes which can either cross fertilize or be fertilized by external genotypes. It is therefore possible that conidia act as spermatia, although the timing of fertilization is not yet known (Gross et al., 2012).

Research into the vegetative incompatibility system in H. fraxineus is ongoing (Brasier and Webber, 2013). Initial results suggest that the majority of isolates from the UK are incompatible with each other, even when isolated from a small sample area, indicating a high degree of heterogeneity in the vic gene loci. It remains to be determined whether the vic system is active or inactive during the initial infection process, which would result in competition or cooperation, respectively, between juvenile mycelia. The latter might be a mechanism enabling different isolates to collaborate to overcome host resistance. Further work is needed to establish whether viruses can migrate between incompatible genotypes as this has implications for the potential to control the fungus through the use of hypovirulence-causing mycoviruses.

Physiology and Phenology

Differences in cultural morphology of isolates were illustrated in Halmschlager and Kirisits (2008) and Kowalski and Bartnik (2010). Ten isolates of H. fraxineus (as Chalara fraxinea), most of them from Germany, varied in extracellular oxidase activity (Schumacher et al., 2009). Kowalski and Bartnik (2010) found that colonies grew from 5 to 30°C with optimal growth for most isolates being 20°C. In this study temperature strongly influenced colony morphology.

The phytotoxin viridiol and related secondary metabolites have been implicated in pathogenicity (Grad et al., 2009, Andersson et al., 2010) although their role in the infection process has been cast into doubt following the finding that H. albidus also produces viridiol (Junker et al., 2013).

Associations

Other fungi, some of which may be opportunistic pathogens invading the lesions caused by H. fraxineus, are readily isolated from necrotic bark (Kowalski and Holdenrieder, 2009b; Schumacher et al., 2010). Root-infecting Phytophthora species were not found to be involved in the disease of ash dieback in Sweden (Bakys et al., 2009a; Schumacher et al., 2010).

Environmental Requirements

Abiotic stresses considered to be associated with ash dieback are drought, frost and changing winter conditions (Schumacher et al., 2007). As canker growth has been observed to be greater in winter, the fungus appears to be adapted to cold weather (Jankovský and Holdenrieder, 2009).

This fungus cannot be reliably isolated in culture (Lygis et al., 2005; Bakys et al., 2009a; Kowalski and Holdenrieder, 2009a) and is slow-growing (Kowalski and Holdenrieder, 2009b; Schumacher et al., 2010). Other fungi may overgrow it in culture or invade necrotic bark tissues in its cankers (Bakys et al., 2009b). Isolation from necrotic lesions has a higher success rate in autumn and winter, although isolation from pseudosclerotial rachises may be the most straightforward method (Gross and Holdenrieder, 2013).

Three PCR techniques for the detection of the fungus in infected plant tissue have been published, each using primers for sequences in the ITS region of rDNA to amplify DNA specific to H. fraxineus (Chandelier et al., 2009; Ioos et al., 2009; Johansson et al., 2009). The protocol of Johansson et al. (2009) uses the sequence of an intron in the region unique to this species within the genus Hymenoscyphus, whereas those of Chandelier et al. (2009) and Ioos et al. (2009, 2011) were tested against a number of Chalara species and other fungi that may be present in or on the lesions. The sequences of ITS regions of rDNA for the teleomorph and the anamorph are available in GenBank for comparison (NCBI, 2009).

Recently, Thi Lam Huong et al. (2013) demonstrated that H. fraxineus could be successfully identified in vitro and in vivo in infected ash leaves by mass spectrometry using specific secondary metabolites produced by the pathogen as markers for its presence.

Trees can be observed for dieback symptoms, but these may be confused with those caused by other fungi or by insects, and H. fraxineus can be present in asymptomatic leaves (Bakys et al., 2009a,b). The other pathogens may be excluded from the diagnosis on the basis of the absence of their characteristic fruiting structures. However, the Chalara anamorph has seldom been observed sporulating in natural lesions (Kowalski and Holdenrieder, 2009a). The apothecia are produced on detached petioles in the leaf litter (Kowalski and Holdenrieder, 2009b), but may also occur on dead shoots (Kowalski and Holdenrieder, 2009a). Forestry Commission (2012) includes a pictorial guide and video of symptoms in the field.

The Chalara anamorph of H. fraxineus can be distinguished from other Chalara species (Nag Raj and Kendrick, 1975; McKenzie et al., 2002) by its small, short cylindrical, aseptate conidia (Kowalski, 2006). The teleomorph is differentiated from most other Hymenoscyphus species by its occurrence on Fraxinus and the dark superficial layer produced on the substrate at the base of the apothecium (Kowalski and Holdenrieder, 2009b).  H. fraxineus can be distinguished from H. albidus by the presence of croziers at the ascus base which are absent in the latter (Zhao et al., 2013); by the shape of crystals within the tissues of the stipe base; by the outer layer of hyphae over the flanks of the ectal excipulum, which are parallel in H. albidoides and interwoven in H. fraxineus; and by the fact that unlike H. fraxineus, H. albidus does not form an anamorphic stage and this is one way to distinguish between the species (Kirisits et al., 2013; Zheng and Zhuang, 2013).

Unlike most other dieback and canker-causing pathogens known to affect Fraxinus (Sinclair and Lyon, 2005), this fungus does not sporulate in pycnidia or perithecia and produces no obvious stromata in or on infected stems or branches. Complicating the diagnosis, some of those other fungi, including Fusarium species and Botryosphaeriastevensii, may be isolated from necrotic bark lesions caused by H. fraxineus (Bakys et al., 2009a,b; Kowalski and Holdenrieder, 2009a; Schumacher et al., 2010).

Symptoms of ash dieback are similar to those caused by the emerald ash borer (Agrilus planipennis) (NAPPO, 2009), but the beetle is known to attack a wider range of Fraxinus species, and the larvae create S-shaped galleries in the sapwood, whereas emerging adults leave characteristic holes in the bark (APHIS, 2009; CFIA, 2009).

Due to the variable regulations around (de)registration of pesticides, your national list of registered pesticides or relevant authority should be consulted to determine which products are legally allowed for use in your country when considering chemical control. Pesticides should always be used in a lawful manner, consistent with the product's label.

Prevention

SPS Measures

As ash (Fraxinus) saplings may be infected without showing symptoms, quarantines may be necessary to prevent additional distribution from affected nurseries in Europe. Restriction of the movement of other ash material may be useful or necessary due to the possible role of vectors (NAPPO, 2009).

Importation of Fraxinus species from European countries to the USA was prohibited already due to the occurrence of another pathogen, Pseudomonas savastanoi, that causes cankers and dwarfing (CFR, 2008a), and importation of ash plants from other countries was later prohibited in order to prevent further introductions of the emerald ash borer (Agrilus planipennis) (CFR, 2008b).

Control

Cultural Control and Sanitary Measures

Not enough is known of the biology of this pathogen to indicate the usefulness of particular methods. Avoidance of wounding and destruction of infected plants or plant parts are control measures suggested for other dieback and canker-causing fungi (Kile, 1993). Leaf scars have served as infection courts for artificial inoculation of saplings (Talgø et al., 2009).

Since the host can tolerate higher temperatures than the fungus, hot water treatments have been suggested for small plants (Hauptman et al., 2013). However, masking disease symptoms may facilitate long distance dispersal of the disease when treated seedlings are planted out. Infected trees should only be destroyed when there is limited recently introduced infected material and the surrounding wider environment is disease free. Otherwise trees should be left in place in order to identify potentially resistant stock.

Host Resistance

Variation in tolerance to disease has been found amongst clones and half-sib progeny in a number of sites (McKinney et al., 2011; Pliura, 2011; Kjær et al., 2012; Stener, 2012). These differences have a genetic basis and are heritable, suggesting the potential to breed a resistant ash population. However, tolerant trees have been found in low numbers in Denmark, where only 1% of trees in natural populations expected to produce tolerant offspring (Kjær et al., 2012). Other ash species in the section Fraxinus or other sections are being tested as sources of resistance (Drenkhan and Hanso, 2010).

A correlation has been found between early leaf senescence and resistance (McKinney et al., 2011, Stener, 2012). This could be an escape mechanism, indicating that site conditions influencing phenology are important. However, early senescing clones also develop smaller lesions, suggesting they possess a genetically determined defence mechanism (McKinney et al., 2012).

As Schumacher et al. (2009) indicated, more research is needed concerning the source of inoculum, possible vectors, points of infection and the conditions of the infection process itself. Information on these may yield clues concerning the reason for the emergence of this disease (Kowalski and Holdenrieder, 2009b; NAPPO, 2009).

Variation in virulence amongst different strains of the pathogen requires further investigation, but see Bakys et al. (2009), Husson et al. (2011) and Kowalski and Holdenrieder (2009a). Furthermore, studies into disease severity along environmental gradients could offer insight into the role of the environment in determining disease outcome.

Virus mediated hypovirulence in European Castanea dentata has enabled this host to coexist with the otherwise highly destructive Chestnut canker pathogen, Cryphonectia parasitica. Work is therefore underway to identify viruses in H. fraxineus (Pliura et al., 2013).

A better understanding of the mechanism enabling the fungus to switch from hemi-biotrophic in its natural range to pathogenic on non-native hosts is needed (Gross et al., 2013). This would be facilitated by more detailed studies of the fungus in its natural range.

Genomic projects are underway to gain insights into host-pathogen interactions (MacLean et al., 2013). Some progress has already been made in identifying putative virulence factors. Clones of the known susceptible species and other Fraxinus species should be tested by inoculation to identify sources of resistance as well as to establish the possible host range for the pathogen in Europe and elsewhere.

The use of molecular methods to test nursery trees for infection may validate these molecular methods and provide data for determination of the need for quarantines to prevent spread of the fungus from nurseries.

12/12/13 Reviewed by:

Jessica Needham, Consultant, UK and Joan Webber, Forest Research, UK

02/04/10 Original text by: