Sampling and PCR method for detecting pathogenic Fusarium oxysporum strains in onion harvest.

Fusarium basal rot is a worldwide disease problem in onions, and causes substantial losses in onion production, both during the growing season and in the storage. To minimize the post-harvest losses, a protocol for screening of latent infections with pathogenic Fusarium oxysporum strains from harvested onions was developed. This protocol is based on a dual PCR test with primers specific for the fungal species and new SIX3 primers specific for the onion-pathogenic F. oxysporum strains. A pooled sample containing pieces from 50 harvested symptomless onions was prepared for the dual PCR using microwave disruption of the filamentous Fusarium fungi and Whatman FTATM filter paper matrix technology, or as a reference protocol, by extracting DNA with a commercial kit. The two sample preparation protocols gave consistent results with the tested onion samples. Detection limit of the dual PCR protocol was 100 pg of F. oxysporum DNA, in a mixture with onion DNA, when the FTA card was applied. The new protocol reported here is simple and sensitive enough for routine testing, enabling the detection of latent infections in harvest lots even at the infection levels under 10%. SIGNIFICANCE AND IMPACT OF THE STUDY: Fusarium basal rot causes serious problems in onion production. To minimize post-harvest losses, a simple protocol based on FTATM technology and a dual PCR test with Fusarium oxysporum species-specific and pathogenicity-specific primers was developed. By testing pooled onion samples using this method, latent infections with F. oxysporum can be screened from a representative sample of the harvest. This screening method could be a useful tool to manage the post-harvest losses caused by latent infections with F. oxysporum and, with modification of the PCR protocol, with other Fusarium species pathogenic to onion.

Phenotypic variation within a clonal lineage of Phytophthora infestans infecting both tomato and potato in Nicaragua.

Late blight caused by Phytophthora infestans (Mont.) de Bary is a constraint to both potato and tomato crops in Nicaragua. The hypothesis that the Nicaraguan population of P. infestans is genotypically and phenotypically diverse and potentially subdivided based on host association was tested. A collection of isolates was analyzed using genotypic markers (microsatellites and mitochondrial DNA haplotype) and phenotypic markers (mating type, virulence, and fungicide sensitivity). The genotypic analysis revealed no polymorphism in 121 of 132 isolates of P. infestans tested. Only the Ia haplotype and the A2 mating type were detected. Most of the tested isolates were resistant to metalaxyl. The virulence testing showed variation among isolates of P. infestans. No evidence was found of population differentiation among potato and tomato isolates of P. infestans based on the genotypic and phenotypic analysis. We conclude that the Nicaraguan population of P. infestans consists of a single clonal lineage (NI-1) which belongs to the A2 mating type and the Ia mitochondrial DNA haplotype. Moreover, based on the markers used, this population of P. infestans does not resemble the population in countries from which potato seed is imported to Nicaragua or the population in neighboring countries. The data presented here indicate that the NI-1 clonal lineage is the primary pathogen on both potato and tomato, and its success on both host species is unique in a South American context.

Snow-mold-induced apoplastic proteins in winter rye leaves lack antifreeze activity

During cold acclimation, winter rye (Secale cereale L.) plants secrete antifreeze proteins that are similar to pathogenesis-related (PR) proteins. In this experiment, the secretion of PR proteins was induced at warm temperatures by infection with pink snow mold (Microdochium nivale), a pathogen of overwintering cereals. A comparison of cold-induced and pathogen-induced proteins showed that PR proteins accumulated in the leaf apoplast to a greater level in response to cold. The PR proteins induced by cold and by snow mold were similar when separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and examined by immunoblotting. Both groups of PR proteins contained glucanase-like, chitinase-like, and thaumatin-like proteins, and both groups exhibited similar levels of glucanase and chitinase activities. However, only the PR proteins induced by cold exhibited antifreeze activity. Our findings suggest that the cold-induced PR proteins may be isoforms that function as antifreeze proteins to modify the growth of ice during freezing while also providing resistance to the growth of low-temperature pathogens in advance of infection. Both functions of the cold-induced PR proteins may improve the survival of overwintering cereals.