Rapid identification and detection of pine pathogenic fungi associated with mountain pine beetles by padlock probes

Fifteen million hectares of pine forests in western Canada have been attacked by the mountain pine beetle (Dendroctonus ponderosae; MPB), leading to devastating economic losses. Grosmannia clavigera and Leptographium longiclavatum, are two fungi intimately associated with the beetles, and are crucia...

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Bibliographic Details
Published in:Journal of microbiological methods 2010-10, Vol.83 (1), p.26-33
Main Authors: Tsui, Clement K.M., Wang, Bin, Khadempour, Lily, Alamouti, Sepideh Massoumi, Bohlmann, Jörg, Murray, Brent W., Hamelin, Richard C.
Format: Article
Language:English
Subjects:
Animals
Biological and medical sciences
Bst polymerase
Circular probe
Coleoptera - microbiology
Dendroctonus ponderosae
Diagnostics
DNA Primers - genetics
DNA, Fungal - genetics
DNA, Ribosomal - genetics
DNA, Ribosomal Spacer - genetics
Economics
Forestry
Fundamental and applied biological sciences. Psychology
Fungal plant pathogens
Isothermal amplification
Ligation
Lodgepole pine
Microbiology
Mycological methods and techniques used in mycology
Mycology
Ophiostoma
Ophiostomatales - genetics
Ophiostomatales - isolation & purification
Ophiostomatales - physiology
Phytopathology. Animal pests. Plant and forest protection
Pinus - microbiology
Pinus - parasitology
Plant Diseases - microbiology
Plant Diseases - parasitology
Polymerase Chain Reaction - methods
Real-time PCR
Sapstain
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Summary:Fifteen million hectares of pine forests in western Canada have been attacked by the mountain pine beetle (Dendroctonus ponderosae; MPB), leading to devastating economic losses. Grosmannia clavigera and Leptographium longiclavatum, are two fungi intimately associated with the beetles, and are crucial components of the epidemic. To detect and discriminate these two closely related pathogens, we utilized a method based on ligase-mediated nucleotide discrimination with padlock probe technology, and signal amplification by hyperbranched rolling circle amplification (HRCA). Two padlock probes were designed to target species-specific single nucleotide polymorphisms (SNPs) located at the inter-generic spacer 2 region and large subunit of the rRNA respectively, which allows discrimination between the two species. Thirty-four strains of G. clavigera and twenty-five strains of L. longiclavatum representing a broad geographic origin were tested with this assay. The HRCA results were largely in agreement with the conventional identification based on morphology or DNA-based methods. Both probes can also efficiently distinguish the two MPB-associated fungi from other fungi in the MPB, as well as other related fungi in the order Ophiostomatales. We also tested this diagnostic method for the direct detection of these fungi from the DNA of MPB. A nested PCR approach was used to enrich amplicons for signal detection. The results confirmed the presence of these two fungi in MPB. Thus, the padlock probe assay coupled with HRCA is a rapid, sensitive and reproducible method for the identification and detection of these ophiostomatoid fungi.
ISSN:0167-7012
1872-8359
DOI:10.1016/j.mimet.2010.07.016