- Lead scientist
- Jenny Denton
- Start date
- 2007
- End date
- 2009
- Keywords
Armillaria, honey fungus, PCR, RFLP, IGS, EF 1-α
- Benefits to gardeners
By identifying cases of Armillaria down to the species level in conjunction with the other research that is being carried out at the RHS it will help us to tailor the advice that we can give to Members who have Armillaria infections in their gardens, as most of the species behave in slightly different ways, and therefore approach to control may vary.
- The problem
For many years it was believed that Armillaria consisted of just one species, A. mellea, which existed as a complex that was morphologically diverse. However with more work it was found that in fact several species existed. Therefore, identification of the different species of Armillaria is important as studies on the pathogenicity and distribution of the separate species are becoming more significant.
Previous work at the RHS by A. Pérez Sierra on a Polymerase chain reaction (PCR)-based identification method for Armillaria species, which used restriction fragment length polymorphism (RFLP) analysis based on various different enzyme digests of the intergenic spacer (IGS) region, provided the main molecular identification technique of Armillaria used by the RHS. The patterns created can be used to identify the species, although the picture below shows each species with a distinctly different pattern, there are other patterns that a species can have, for example A. gallica can have four possible banding patterns with an Alu I digest.
Although this method produced many clear results with several of the main species of Armillaria found in Europe, it did have some problems. One of the main problems came with the identification of some species which all have the same banding pattern when digested with the enzyme Alu I. Other species also have very similar banding patterns, and although in theory a little different the visualisation of this on a gel proved difficult to define exactly which species it was.
A. cepistipes (pattern 1), A. ostoyae and A. borealis (pattern 2) all possess the same banding pattern when digested with the enzyme Alu I, therefore further digests were used. However negative results, where no digestion occurred, were used to give a definite result, for example with the enzyme Nde I, no cut was used to indicate A. cepistipes, and a cut was either an A. ostoyae or an A. borealis. This issue is also exacerbated by the second main problem with this method, the loss of restriction sites. This can often occur from single nucleotide polymorphisms (SNPs), or longer sequence changes or losses. The altering of the DNA means that the enzyme cannot cut where the normal site would be, and so changes the fragment size, therefore the banding pattern is altered. This could possibly lead to an initial misidentification, and with Nde I, there is only one cutting site, and therefore the loss of this site would clearly lead to a misidentification due to the use of negative results as indicative of species.
- Approach
Currently work has been started using a different region of DNA, elongation factor 1-alpha (EF 1-α), it was hoped this would be more conserved and show species differences more effectively as this region has worked well when used with other genera, such as Phytophthora, for identification purposes. Various primers were tested and eventually a set decided, although some problems still exist. Due to the smaller size this region is not really suitable for enzyme digestion and the use of RFLP analysis, so once the region was isolated and amplified it was sequenced and placed into an alignment.
The use of this region has proven quite successful, although the distinctions between species are mostly due to single nucleotide changes, which could be seen as an unstable way to assess identification, however if the region is considered as a whole it is definitely possible to see clear and distinct patterns which separate the main European species. This is particularly clear when separating those that were ambiguous with the IGS region. Therefore the use of both molecular techniques together creates a much stronger identification process.
- Further information
Read more on honey fungus
View the Armillaria survey
Read the studies on the pathogenicity of Armillaria spp
Read about the behaviour of severed rhizomorphs of Armillaria mellea and A. gallica in growing media
To read about the effect of abiotic factors on Armillaria growth
Read about the growth and behaviour of Armillaria in mulches
- References
Pérez Sierra A, Whitehead, D S & Whitehead, M P (1999). Investigation of a PCR-based method for the routine identification of British Armillaria species. Mycological Research 103 (12), 1631-1636