Monthly Archives: April 2013

Primordial fruiting bodies of H. pseudoalbidus identified on material collected from Ashwellthorpe lower wood

The contributors

Anne Edwards at JIC

The material

We used DNA from primordial fruit bodies growing on dead ash raciness collected from Ashwellthorpe Lower Wood on 10th February 2013 and incubated at room temperature on moist filter paper for 10 weeks. The ITS region of 18S rDNA was amplified using universal primers: 5’-TCCGTAGGTGAACCTGCGG-3’ and 5’-TCCTCCGCTTATTGATATGC-3’ and Chalara specific primers 5’-AGCTGGGGAAACCTGACTG-3’ and 5’-ACACCGCAAGGACCCTATC-3’

The analysis

The DNA sequences (in the github repository at this link and this link) were blasted against the NCBI nucleotide database.

The interpretation

The structures are the primordial fruit bodies of Hymenoscyphus pseudoalbidus

 _DSC1546

Both mating types of Hymenoscyphus pseudoalbidus (Chalara fraxinea) are present at nursery, recent planting and natural spread sites

The contributors

Gavin Hunter, Joan Webber, Stephen Hendry and Clive Brasier at Forest Research

Background

The ash dieback pathogen Hymenoscyphus pseudoalbidus is now to be considered established in eastern Britain (see distribution in this map) and samples regularly come into the laboratory at Forest Research for morphological and DNA-based molecular diagnosis. This has formed the nucleus of a culture collection of the pathogen which we are characterising, including analysis of mating type.

Analysis

We used the multiplex PCR method of Gross et al (2012) to identify the MAT 1-1 and MAT 1-2 idiomorphs or sexual mating types of H. pseudoalbidus. Sequences of the primer products were in all cases homologous to the MAT sequences reported by Gross et al (2012).
The mating types were determined for six isolates from individual trees at a single nursery in Lincolnshire; six isolates from individual trees at a single recent ash planting site in Leicestershire; and nine isolates from individual trees at seven different ‘natural spread’ or woodland sites in Suffolk and Norfolk.

Results

As shown in the Tables linked at the bottom, both mating types, MAT1-1 and MAT1-2, were present in each site type. The overall ratio of MAT1-1: MAT1-2types in the 21 isolates was 11 : 10.

Interpretation

  1. Ash nursery stock entering the UK can carry both mating types of H. pseudoalbidus onto a nursery site.
  2. Out-planting of ash nursery stock can also carry both mating types onto a new site.
  3. As already indicated by Diane Saunders (January 28) for Kenninghall Wood and Ashwellthorpe Wood (Norfolk), both mating types are present across ‘natural spread’ or woodland sites in eastern England.
  4. Across all samples the frequency of MAT1-1 and MAT1-2 types was close to 1:1. This is consistent with the previous report of MAT ratios of 1:1 in H. pseudoalbidus by Gross et al. (2012); and with what is expected for a regularly sexually outcrossing ascomycete.It is also consistent with reports of high genetic variability in H. pseudoalbidus elsewhere in Europe i.e. H. pseudoalbidus now being found in the UK is also likely to be regularly sexually outcrossing.

nornex_isolates_1

nornex_isolates_2

nornex_isolates_3

nornex_isolates_4

see also GitHub HP01 .. HP036