Forster, B.; Knizek, M.; Grodzki, W. (eds.) 1999: Methodology of Forest Insect and Disease Survey in Central Europe.
Proceedings ofthe Second Workshop ofthe IUFRO WP 7.03.10, Apri120-23, 1999, Sion-Chiiteauneuf, Switzerland.
Birmensdorf, Swiss Federal Institute for Forest, Snow and LandscapP. Research (WSL) 279-280.
Characterisation of a Global Collection of Dothistroma Pini Isolates.
R. J. Ganley and R. E. Bradshaw.
Introduction.
Dothistroma pini Hulbary (teleomorph Mycosphaerella pini) is a filamentous fungus which causes needle blight in conifers, particularly in pines. Dothistromin is a fungal toxin which is produced in large quantities by D. pini and appears to be directly implicated in the development of disease symptoms in vitro.
This pathogen is a serious forest disease in many Central European forests as well as in other countries world-wide, including New Zealand. An international collection of D. pini isolates was accumulated with isolates from the Bavarian Alps, Brazil, Canada, Guatemala, France, Minnesota, Nebraska, New Zealand, Oregon and Slovakia. A total of 28 viable strains were obtained, from these 12 'core' strains (ALP3, BRZl, CAN3, FRAI, GUAI, MIN11, NEBl, NEB6, NEBS, NZEI, ORE12, SLVI) were chosen as being representative of the entire collection.
Morphology, Growth Rates and Spore Size.
Individual colonies of each strain were obtained by streak plating a spore suspension on Dothistroma medium. Strains were morphologically diverse with differences in colony appearances between strains from the same country as well as between those from different countries.
The relative rates of growth of all strains were measured either by radial growth on plates or by dry weight yield from liquid cultures used for dothistromin assays. In general, the NEB, FRA and NZE strains were the fastest growing in plate cultures with the ALP, SLV and CAN isolates significantly slower growing than most other strains.
Spore were made from confluent growth of strains on Dothistroma sporulation medium.
Spores were examined microscopically and the lengths of up to 100 spores per strain were measured using an eyepiece micrometer. All strains tested produced a wide range of different spore lengths, with ranges for all strain overlapping. There was no correlation between spore length and growth rate. The strains were unable to be segregated into defmed groups on the basis of differences in spore length.
ITS Sequence
The ribosomal ITS 1 region was PCR amplified, yeilding products of approximately 250bp , which were directly sequenced. All strains shared a similar ribosomal ITS 1 sequence which supports the classification of all isolates as D. pini (Fig 1 ).
Fig 1 : Ribosomal ITS 1 sequence of D. pini strains.
Group1 Group2
1 CTGAGTGAGG GCGAAAGCCC GACCTCCAAC CCTTTGTGAA CCAACTCTGT
Ctloupl 51 TGCTTCGGGG GCCACCCCGC CGTTTCGGCG ACGGCGCCCC CGGAGGTCAT Group2 . . . T . . . .
Group1 101 CAAACACTGC ATCTTTGCGT CGGAGTCTTA AAGTAAATTT AAAC Group2 . . .
A... . . . . .. .
Dots indicate identical sequence in group 1 and 2 strains.
Group! strains: ALP3-6, BRZl, CAN3-4, FRAI, GUA, NZEI, ORE12, SLVl Group2 strains: MINI!, NEB 1-9
279
Dothistromin Production
Flasks containing liquid Dothistroma medium were inoculated with suspensions of macerated mycelium of each isolate, in triplicate, and incubated at 230C, 220 rpm for either 7 or 14 days. Total flask contents were harvested by filtration or centrifugation. Mycelium was freeze-dried for determination of dry weight. Filtrates were tested for dothistromin content using a modification of the competitive ELISA assays described by Jones et al. (1993).
The ALP3 strain was an outstandingly high producer, producing more than ten times as much dothistromin per mg mycelium (Fig 2) and three times as much dothistromin per ml medium (results not shown) than any of the other 'core' strains tested. Other ALP strains tested also showed high levels of dothistromin production (results not shown). No correlation was found between radial growth rates on plates and dothistromin production levels in liquid culture.
Fig 2: Dothistromin Production by D. pini (ug per mg dry wt. mycelium )
D. pini isolate
Future Work.
fCi7dl
14d
7 d Days incubation
Investigation of the effects of environmental factors (i.e. light, shaking, liquid medium) on the production of dothistromin in vitro.
Development of a robust DNA fingerprinting method, using microsatellite markers, to generate a distinct genetic fmgerprint which will be used to analyse the genetic diversity among the isolates.
References.
Jones, W . T., Harvey, D., Jones, S. D., Fielder, S., Debnam, P., and Reynolds, P. H. S. (1993).
Competitive ELISA Employing Monoclonal Antibodies Specific for Dothistromin. Food and Agricultural Immunology 5: 187-197.
Bradshaw, R. E., Ganley, R . J., Jones, W. T., and P . Dyer. (1999). Characterisation of a Global Collection of Dothistroma pini Isolates. Mycological Research, submitted for publication.
280
