D.W. Parry

Phylogenetic analysis of EF-1 alpha gene sequences from isolates of Microdochium nivale leads to elevation of varieties majus and nivale to species status

Degenerate PCR primers were designed based on EF-1 alpha (EF-1alpha) gene sequences of several filamentous fungi retrieved from sequence databases. These primers were used to isolate a partial sequence, approximately 830 bp in length of the EF-1alpha from isolates of Microdochium nivale obtained from various geographic locations across the world. Two distinct groups of isolates were evident among those isolates examined. Sequence homology for comparisons within group was 99.7% for group A and 99.8% for group B. Primers specific to either group A or group B sequences were designed and tested on isolates from around the world. Comparisons were made with primers previously reported for the two varieties of M. nivale and revealed that Group A type isolates correlated with M. nivale var. majus and group B isolates with M. nivale var. nivale. The primers from this study and those previously reported were in agreement for all isolates with the exception of one isolate (NRRL 3289) which failed to amplify with previously published M. nivale primers. Sequence analysis of NRRL 3289 suggested that it was an isolate of M. nivale var. nivale as indicated by the EF-1alpha based primers developed in this study. This study provides sequence based phylogenetic evidence of two species and when taken together with biological differences reported, leads to the recognition of M. majus comb. nov. (syn. Fusarium nivale var. majus). Descriptions of the two species are provided.

Development of stem-base pathogens on different cultivars of winter wheat determined by quantitative PCR

The progress of development of stem-base pathogens in crops of second winter wheat was plotted in nine experiments in three years. The amount of each pathogen present was determined by quantitative PCR. Where Tapesia yallundae was present in quantifiable amounts, it usually developed earlier than the other eyespot pathogen, T. acuformis. Both species were usually present in greater amounts on cultivars which are more susceptible to eyespot. The sharp eyespot pathogen, Rhizoctonia cerealis, developed more erratically than either of the Tapesia spp. and there were no consistent effects on different cultivars. Fusarium spp., the cause of brown foot rot, were rarely present in quantifiable amounts, but Microdochium nivale was usually present as one or both of the varieties nivale and majus. Late-season (after anthesis) decreases in M. nivale suggest that any brown foot rot symptoms attributable to this fungus would have fully developed earlier. Cultivar differences in amounts of M. nivale were most clear in stems during internode extension and when relatively large amounts of DNA were present. Such differences approximately reflected eyespot susceptibility, cv. Soissons containing most and cv. Lynx containing least DNA. The results emphasise the difficulty in relating diagnoses, by quantitative PCR or other means, at early growth stages when decisions to apply fungicides against stem-base disease are made, to later disease severity.

Analysis of variation within Microdochium nivale from wheat: evidence for a distinct sub-group.

Random amplified polymorphic DNA (RAPD) assay revealed polymorphisms among isolates of Microdochium nivale from wheat which indicated the presence of a distinct sub-group. Average conidial length and average number of septa were distributed continuously among the isolates and did not correlate with the groupings indicated by RAPDs. However, conidial width fell into two broad groups which correlated closely with the groups revealed by RAPD analysis. The sub-group identified by RAPDs showed a correlation with M. nivale var. majus as distinguished by conidial morphology, and the remaining isolates largely corresponded to M. nivale var. nivale. The perithecial state of M. nivale occurred in vitro, indicating that the majority of isolates were homothallic, but not all isolates produced perithecia.

Quantitative Fusarium spp. and Microdochium spp. PCR assays to evaluate seed treatments for the control of Fusarium seedling blight of wheat

Aims:  To develop sensitive quantitative PCR assays for the two groups of pathogens responsible for Fusarium seedling blight in wheat: Fusarium group (Fusarium culmorum and Fusarium graminearum) and Microdochium group (Microdochium nivale and Microdochium majus); and to use the assays to assess performance of fungicide seed treatments against each group.