Kjøller, R., Rosendahl, S. 1999. Detection of arbuscular mycorrhizal fungi (Glomales) in roots by nested PCR (Polymerase Chain Reaction) and SSCP (Single Stranded Conformation Polymorphism). Plant and Soil. in press.
PCR amplification of a region of the large subunit ribosomal DNA sequence with Glomus specific primers was used to detect arbuscular mycorrhizal fungi in root tissue of four plant species. The primers were specific to Glomus mosseae, Glomus caledonium, Glomus geosporum, Glomus coronatum, Glomus fragilistratum and Glomus constrictum but did not recognise sequences from Glomus claroideum. Sequence differences between isolates could be revealed by Single Stranded Conformation Polymorphisms (SSCPs) in polyacrylamide gels under non- denaturing conditions. Isolates of G. mosseae, G. caledonium, and G. coronatum could be separated by their SSCP patterns while three isolates of G. geosporum showed no variation. Specific SSCP patterns from isolates of G. mosseae and G. caledonium allowed detection of both fungi in the same root segment. Sequence differences leading to variations in SSCP patterns were confirmed by direct sequencing.Kjøller, R.,Rosendahl, S. 1998. Enzymatic activity of the mycelium compared with oospore development during infection of pea roots by Aphanomyces euteiches. Phytopathology 88:992-996.
To describe the disease cycle of the root pathogen Aphanomyces euteiches, enzymatic activity in the mycelium was compared with the development of oospores in pea roots. Plants were inoculated with two zoospore concentrations to achieve different disease levels. Hyphae were stained for fungal alkaline phosphatase activity in the roots. Additionally, enzyme activity was measured after electrophoresis of an A. euteiches- specific glucose-6-phosphate isozyme. Development of oospores in the roots was measured after staining the oospores with trypan blue. In plants inoculated with the higher zoospore concentration, the enzymatic activity of the pathogen mycelium peaked 10 to 14 days after inoculation, when oospore formation was initiated. Oospore formation was associated with a gradual increase in disease symptoms. At the last harvest, plants inoculated with the higher zoospore concentration had died. In these plants, oospores were found in 90% of the root length, while the enzymatic activity of the mycelium was low. This suggests that the pathogen mycelium is only active on living plants and does not grow saprophytically on dead plant material.Kjøller, R., Rosendahl, S. 1997. Polyacrylamide gel electrophoresis (PAGE) and densitometric measurement of enzyme activity of the pea root pathogen Aphanomyces euteiches in pea roots. Journal of Phytopathology 145: 253-256.
A new method to measure enzyme activity of the fungal root pathogen Aphanomyces euteiches in pea roots is described. The specific enzymes of the fungus and the host were separated by polyacrylamide gel electrophoresis (PAGE) and the activity of fungal Glucose-6-phosphate dehydrogenase and Phosphoglucomutase were quantified by densitometry. Fungal activity could be correlated to the percentage infected root length and to the disease symptoms of the plants. The activity of A. euteiches was studied in a time course experiment with increasing levels of zoospore inoculum. The results indicated that an increase in inoculum level resulted in a faster disease development in the plants. The relation between fungal enzyme activity and infection level is discussed.