Chalara spp., the anamorphic state of H. fraxineus, have known insect vectors. There are no reports of an insect vector for H. fraxineus at this time.

Method	Instructions	NAPIS Survey Method
Visual	Collect symptomatic plant material.	3031 - General Visual Observation

A heavily melonized pseudothecial layer may form on petioles. Visual detection of this layer is a very good way to detect the pathogen in the earliest stages of the epidemic when symptoms on leaves and branches are still rare. On the leaves, the veinal necrosis is a characteristic symptom.

No specific signs are present.

Initial symptoms include leaf spotting, vein necrosis towards the petiole, necrosis on petioles and partial or complete leaf wilting. Later on, small necrotic spots (without exudate) appear on the stems and branches. These necrotic lesions then enlarge and might the girdle shoot resulting in wilting and premature shedding of leaves, dieback of branches, shoots, and twigs, and particularly in the death of the top of the crown. A brownish to grayish discoloration of the wood that often extends in a longitudinal direction beyond necrotic areas in the bark has been observed in Austria.

Affected trees show prolific formation of epicormic shoots (shoots that grow from epicormic buds found under the bark of trunks, stems or branches). Epicormic buds lie dormant beneath the bark, their growth suppressed by hormones from active shoots higher up the plant. Under certain conditions, they develop into active shoots, such as when damage occurs to higher parts of the plant or light levels are increased following removal of nearby plants.

In addition to the aforementioned symptoms caused by H. fraxineus, collar necrosis near the tree base has shown to be common in infected host strands and is likely to accelerate the declining process of ash dramatically. Fungi from the species Armillaria are also commonly found on collar rot of ash hosts along with H. fraxineus and may act as a secondary agent of mortality. It has not yet been excluded that Armillaria spp. causes the initial collar necrosis in ash hosts and H. fraxineus infects the tree base as a secondary pathogen.

ID/Diagnostic: Multiple

Morphological: Most frequently isolated on malt extract agar (supplemented with streptomycin sulfate) from the leading edge of discolored wood and necrotic leaf petioles, but rarely from necrotic bark.

Kowalski (2006) notes that C. fraxinea differs from other species of Chalara by its small, short, cylindrical phialoconidia extruded in chains or in slimy droplets, morphological features of the phialophores, and by colony characteristics.

Molecular methods are being validated by the CPHST Beltsville laboratory. The approved methods will be updated after the validation is completed.

Chalara species

Molecular: Bakys et al. (2009) combined PCR amplification of the ITS region of the ribosomal genes with terminal restriction fragment length polymorphism (T-RFLP) to determine which pathogens existed on Fraxinus excelsior in Sweden. In order to identify taxa in the T-RFLP profiles, PCR products were cloned, enabling sequencing of the ITS region of the rDNA genes for individual taxa within the mixed sample, and subsequent identification by database comparison.

Johansson et al. (2010) developed a set of species-specific PCR primers, based on ITS sequences, to detect C. fraxinea in plant (Fraxinus excelsior) tissue.

Real-time PCR: Ioos et al. (2009) developed a real-time PCR for detection of C. fraxinea in planta . Species-specific polymorphisms observed within the ITS region were used to design a primer pair and a dual-labeled probe for use in a real-time PCR assay for the detection of C. fraxinea. In silico and in vitro assessments showed that the PCR could detect as little as 20 fg of C. fraxinea DNA.

Chandelier et al. (2010) developed a real-time PCR for the detection of C. fraxinea from woody tissues. A procedure for DNA extraction from woody tissues using an electric drill yielded DNA of appropriate quality for DNA extraction. PCR primers and Taqman probes were developed based on the internal transcribed spacer (ITS) region of a multi-copy gene rDNA. The primers amplified an 81 bp fragment from C. fraxinea. The limit of detection was 5 pg of genomic DNA per PCR.

The ash dieback pathogen has been on the CAPS prioritized pest list for several years. It was initially listed as Chalara fraxinea (the anamorphic/ asexual stage). In 2014, we added Hymenoscyphus pseudoalbidus (the teleomorphic/sexual stage) to the name. As a result of a large international effort to assign one name to each fungus, the suggested name has changed to Hymenoscyphus fraxineus (Baral et al., 2014).