A novel technique using the Hendrickx centrifuge for extracting winter sporangia of Synchytrium endobioticum from soil

A zonal centrifugation method, known as the Hendrickx centrifuge technique, was tested for routine detection of winter sporangia of Synchytrium endobioticum in soil. In four experiments the ability of the Hendrickx centrifuge to extract the sporangia from soil was compared with a method used by the Dutch Plant Protection Service, which is a modification of the recommended EPPO method. Naturally and artificially contaminated soil samples were used to study the recovery percentage of and variation in numbers of winter sporangia. The effects of soil type and inoculum density were studied. The Hendrickx centrifuge method, developed originally for extraction of free living nematodes from soil, performed better than the method used by the Dutch Plant Protection Service. This was due to a better extraction recovery (60% higher), a lower measurement error (50% lower) and a lower detection level (down to 0.02 sporangia g(-16) soil). The Hendrickx centrifuge method is much less labour-intensive than the method used by the Dutch Plant Protection Service. It can be used to extract many different organisms from soil, and DNA can be subsequently extracted from the supernatant for further PCR analysis. Inclusion of the Hendrickx centrifuge method in the official EPPO diagnostic protocol for regulated pests is recommended as an alternative method for detection of sporangia in soi

NiaA, the structural nitrate reductase gene of Phytophthora infestans: isolation, characterization and expression analysis in Aspergillus nidulans

The nitrate reductase (NR) gene niaA of the oomycete Phytophthora infestans was selected from a gene library by heterologous hybridization. NiaA occurs as a single-copy gene ant its expression is regulated by the nitrogen source. The nucleotide sequence of niaA was determined and comparison of the deduced amino-acid sequence of 902 residues with NRs of higher fungi and plants revealed a significant homology, particularly within the three cofactor-binding domains for molybdenum, heme and FAD. The P. infestans niaA gene was used as a model gene to test whether oomycete genes are functional in the ascomycete Aspergillus nidulans, a fungus which is highly accessible for molecular genetic studies. The complete niaA gene was stably integrated into the genome of a nia- deletion mutant of A. nidulans. However, transformants containing one or more copies of the niaA gene were not able to complement the nia- mutant. This suggests that there is no functional expression of the introduced niaA gene in A. nidulans. In addition, the activity of two other oomycete gene promoters was analyzed in a transient expression assay. Plasmids containing chimaeric genes with the promoter of the P. infestans ubiquitin gene ubi3R, or the Bremia lactucae ham34 gene, fused to the coding sequence of the Escherichia coli beta-glucuronidase (GUS) reporter gene, were transferred to A. nidulans protoplasts. No significant GUS activity was detectable indicating that the ubi3R and ham34 promoters are not active in A. nidulans. Apparently, the regulatory sequences which are sufficient for gene activation in oomycetes are not functional in the ascomycete A. nidulans

Isolation of putative pathogenicity genes of the potato late blight fungus Phytophthora infestans by differential hybridization of a genomic library

Plant pathogens produce pathogenicity factors which enable them to parasitize and colonize their host. A strategy for identifying pathogenicity factors involves the isolation and characterization of genes encoding these factors. Potential pathogenicity genes are genes whose expression is induced during pathogenesis. In order to isolate such genes of the late blight Fungus Phytophthora infestans , a genomic library of P. infestans DNA was differentially hybridized using labelled first strand cDNA probes synthesized on mRNA isolated from P. infestans infected potato leaves and on mRNA isolated from the fungus grown on a basic medium in culture. In total, nine distinct in planta induced (ipi) genes were selected. Expression studies revealed that the mRNA levels of seven of these genes, ipiA, ipi C, ipiD, ipiJ1, ipiJ2, ipiN and ipi Q increased 5-10 fold during colonization. The two other genes, ipi B and ipiO, showed a transient expression pattern with the highest mRNA levels in the early stages of infection