Species and strains of subgenus Penicillium

November 27th, 2011

Jos A.M.P. Houbraken1, Flemming Lund2 , Angelina F.A. Kuijpers1, Vincent Robert1, Ellen Kirstine Lyhne2 and Robert A. Samson1, Jens C. Frisvad2

1 Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands

2 BioCentrum-DTU, Technical University of Denmark, Kgs. Lyngby, Denmark

1. Introduction

Penicillium subgenus Penicillium is an extremely widespread and important group of filamentous fungi spoiling foods and other materials or used positively in biotechnology including food technology. The species included have been taxonomically revised several times, but always with fuzzy species boundaries and resulting difficulties in identifying new isolates. We have used a polyphasic but practical approach revising these Penicillia in order to classify and cladify them and in order to produce user-friendly identification key. Experiments showed that a combination of traditionally used media with some nutritional and ecophysiological test media were superior to combinations of chemically defined media. Some species are differentiated primary in conidial and reverse colour. In compiling this key, we deliberately used rather broad colour categories. More detailed identification sometimes requires the use of a colour guide. Micromorphological structures were measured using the BioloMICS software. Profiles of extrolites (secondary metabolites) are not yet added to the data set; this will be done in due time.

As rDNA ITS sequencing has proven to be insufficient to cladify these Penicillia, beta-tubulin sequences are used in this key.

2. Using the key

2.1 Quantitative characters

Several characters, such as colony diameter on standard media, sizes of conidia, metulae, stipes and phialides, are readily measured. For these measurements, average values should be used, rather than extreme values.

2.1.1 Macroscopical features

The colony diameters on Czapek Yeast extract Agar (CYA), Yeast Extract Agar (YES), Malt Extract Agar (MEA), Creatine Agar (CREA), Czapek Agar (CZ) and Dichloran 18% glycerol agar (DG18) should be measured after 7 days of incubation at 25°C. The value should be stated in millimetres. The formulations of these media, used in our experiments, are available on request.

2.1.2 Microscopical features

Micromorphological structures were measured using the BioloMICS software and are therefore are very accurate. Use a calibrated microscope to measure the size of the conidia, phiades, metulae and width of the stipes. The preparations should be made form the MEA medium and mature structures should be measured.

2.1.3 Ecophysiological experiments

CYA30/CYA25: Ratio of colony diameter on CYA grown at 30°C divided by the colony diameter on CYA at 25°C.

CYA15/CYA25: Ratio of colony diameter on CYA grown at 15°C divided by the colony diameter on CYA at 25°C.

CYAS/CYA: Ratio of colony diameter on CYA with 5% NaCl grown at 25°C divided by the colony diameter on CYA at 25°C.

2.1.4 Ehrlich reaction

All isolates are examined for the production of cyclopiazonic acid and other alkaloids reacting with Ehrlich reagent. In our experiments the filter paper method, described by Lund, (1995) was used. Ehrlich reagent consists of 2 g of 4-dimethylaminobenzaldehyde in 96% ethanol (85 ml) added 15 ml 10N HCl. A 4 mm agar plug is cut out of the centre of a colony grown on CYA (incubated 7 days at 25°C) and a round piece (1 cm diam.) of wet filter paper is placed on the mycelium side of the plug. Penicillium isolates will give either a yellow, violet, pink/red ring or no reaction.

2.2 Qualitative characters

Many of the qualitative characters are difficult to determine, including the ornamentation of stipes and conidia, adpressedness and branching pattern of the conidiophore, growth characteristics on Creatine Agar (CREA) and colony texture and colours.

2.2.1 Reactions on Creatine Agar (CREA):

Weak growth, moderate acid production

Weak to moderate growth, no acid production

Moderate growth, hugh acid production

Moderate to good growth, moderate acid production

Good growth, good acid production

2.2.2 Reverse colours

The reverse colour is determined by the naked eye. Seven main “groups” are made:

1. Pale, 2. Yellow, 3. Orange, 4. Red, 5. Beige, light brown, crème brown, 6. Brown, 7. Dark brown / blackish green

Example 1:

If a Penicillium discolor isolate has a orange red reverse on YES agar, the groups “orange” and “red” should be set on ‘yes’, while the others should be on ‘No’.

Pale No

Yellow No

Orange Yes

Red Yes

Beige, light brown, crème brown No

Brown No

Dark brown / blackish green No

Example 2:

A Penicillium albocoremium isolate has an orange brown reverse on CYA agar:

Pale No

Yellow No

Orange Yes

Red No

Beige, light brown, crème brown No

Brown Yes

Dark brown / blackish green No

2.3 Remarks

Although this key is still in development, accurate identification based on solely macro- and micromorphological criteria remains difficult, though it is possible. The closely related species are difficult to separate and therefore the result of the identification should always be verified with the species description. Accurate descriptions are made and can be found within due time in Frisvad & Samson, Studies in Mycology (in progress).

Please send remarks, errors and tips for improvement to Jos Houbraken, Centraalbureau voor Schimmelcultures, the Netherlands, P.O. box 85167, 3508 AD Utrecht, houbraken@cbs.knaw.nl.




General Address:

Visiting address: CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT, Utrecht, The Netherlands

Postal address: CBS-KNAW Fungal Biodiversity Centre, P.O. Box 85167, 3508 AD, Utrecht, The Netherlands

Phone: +31 (0)30 21 22 600

Fax: +31 (0)30 25 12 097

Mycobank and nomenclature:

Dr. Joost Stalpers and Ir. Gerrit Stegehuis

E-mails: j.stalpers@cbs.knaw.nl

Software, websites and bioinformatics:

Dr. Vincent Robert and Kasper Luijsterburg

E-mail: v.robert@cbs.knaw.nl