by Vikki Warren, Identification Services Manager
Species level identification of fungi has long been considered challenging and, in contrast to bacteria, fungal isolates obtained from environmental monitoring programs are often identified to genus rather than species level. More detailed identification of environmental isolates can be desirable, so what are the difficulties when it comes to species level identification of fungi?
Sequencing options
Two distinct approaches have emerged for genetic identification of fungi, and both are based on sequencing sections of ribosomal DNA. Fungal ribosomes have a large and a small subunit, and the ribosomal RNA (rRNA) operon, that is the DNA that codes for ribosomal RNA, has three rRNA sequences and two Internal transcribed spacer regions: ITS1 and ITS2. The two methods used for fungal identification involve either sequencing of the D2 region of the large subunit ribosomal DNA (D2 LSU) or sequencing either one or both of the internal transcribed spacer regions (ITS). D2 LSU sequencing is probably the most widely used approach for fungal identification at present.
Validated and public databases
Similarly to 16S rDNA sequencing for bacteria, data obtained by D2 LSU sequencing can be analysed using a validated commercial database that has been built using reference strains, as well as publicly available but non validated sources such as the EMBL-EBI (European Molecular Biology Laboratory – European Bioinformatics Institute) database. The ITS approach however, generally relies more heavily on the use of data obtained from unvalidated public databases.
Challenges
While I find it is usually possible to obtain a species level identification of yeasts using D2 LSU sequencing, we sometimes can’t get that level of identification for moulds, but in these cases it is often possible to obtain species level identification using ITS sequencing.
This can be attributed to the level of variation that exists in the sequences of these sections. Generally, there is a lower level of differentiation in the ribosomal DNA sequenced for the D2 LSU method than is found in ITS sequences. Unlike ribosomal DNA, ITS sequences have no functional role, and consequently have accumulated a greater level of mutation, which aids identification.
Example 1: Penicillium identification
A good example of moulds that are difficult to identify to species level using D2 LSU comes from the genus Penicillium – a commonly occurring genus amongst environmental isolates. I have found Penicillium camemberti, Penicillium clavigerum, Penicillium commune, Penicillium corylophilum and Penicillium crustosum have all matched a single isolate sequence at 100% similarity. However, when ITS sequencing has subsequently been undertaken, a species level result has been achieved. In one specific example the isolate was identified as Penicillium crustosum with a 100% match.
Example 2: Cladosporium or Mycosphaerella?
The above example is an illustration of where D2 LSU sequencing cannot differentiate between different species of the same genera, but in some cases, D2 LSU sequencing alone cannot distinguish between different, but closely related, genera.
For example, I have found several species of Cladosporium and Mycosphaerella have all matched to the same isolate sequence. Cladosporium is a large genus that has been reported to be the most common fungal component isolated from air, and is therefore also quite commonly found in the course of environmental monitoring programs.
Again, I have found ITS sequencing to be successful in providing a species level identification – one isolate which matched to Cladosporium cladosporioides, Cladosporium herbarum, Cladosporium oxysporum, Mycosphaerella aronici and Mycosphaerella tassiana, was identified as Cladosporium cladosporioides when ITS was used.
Example 3: closely related genera
In another example, we found that it was not possible to differentiate between an even larger group of closely related genera using D2LSU sequencing. Searching the non-validated EMBL database for D2 LSU sequences failed to distinguish between Saccothecium sepincola, Mycosphaerella sojae, Pithomyces chartarum, Pleospora gaeumannii, Leptosphaerulina saccharicola, Heterophoma adonidis, Nothophoma quercina and Leptosphaerulina australis.
These eight species, from seven different genera, all matched a single D2 LSU isolate sequence at 100% similarity. In this case however, ITS sequencing did not result in a species level match either – the isolate matched to Leptosphaerulina saccharicola, Leptosphaerulina australis, Leptosphaerulina chartarum, and Leptosphaerulina trifolii. It was however, successful in narrowing the identification down to Leptosphaerulina species rather than several different genera – a substantial improvement on the initial result obtained.
What is the best approach?
As many common fungal isolates do match very well to the validated D2 LSU database, at present, we recommend the use of D2 LSU sequencing in the first instance. If a match is not found using the validated database, we would analyse the results against the non-validated EMBL database, before considering whether to follow up with ITS sequencing.
We always refer to any relevant published papers for additional supporting information when using unvalidated databases for identification purposes.
The decision on whether to progress to ITS sequencing if a species or genus level identification cannot be obtained using D2 LSU sequencing is really dependent on the individual circumstances and whether family, genus or species level identification is required. For example, it may be requested when investigating excursions from normal populations or contamination issues. While ITS does not always provide a species level match, the examples above illustrate that it has been successful in doing so with some common isolates, and where a species level match has not been found it has given an improved result. Validated databases of ITS sequences are being developed and in future, our experience suggests that this may result in increased popularity of the method.
If you would like to find out more about our fungal identification services, visit our fungal identification web page, or get in touch!
About the author
Vikki Warren joined NCIMB in 2005. She leads a team of scientists responsible delivering NCIMB’s Identification Services and sequencing new deposits to the UK’s National Collection of Industrial Food and Marine Bacteria. Vikki holds a BSC
(hons) degree in Applied Biosciences and Management, and an MSC in Instrumental Analytical Techniques; DNA Analysis, Proteomics and Metabolomics from the Robert Gordon University in Aberdeen.
