Characterisation of cultural, biological and molecular variability of Phoma ligulicola isolates associated with ray blight disease of pyrethrum and chrysanthemum

Pyrethrum (Tanacetum cinerarafolium) is a perennial plant grown commercially in Tasmania, Australia for production of insecticidal pyrethrins. Tasmania produces over 30 % of the world's supply of natural pyrethrins. Ray blight disease caused by the fungal pathogen Phoma ligulicola var. inoxydablis, is a major limiting factor of pyrethrum production in Tasmania. It causes dieback of emerging stems in spring, and flower disease over summer. Most of the information regarding this pathogen is based on studies of the variety that causes ray blight on chrysanthemum, P. ligulicola var. ligulicola. The objectives of this project were to characterise the biological, cultural and genetic variability, and investigate the reproductive nature of the P. ligulicola var. inoxydablis from pyrethrum. This study also aimed to confirm the species identity and determine the variety of Phoma exigua from diseased pyrethrum plants. Assessment of morphological and cultural characteristics showed considerable variability among P. ligulicola isolates from pyrethrum (n = 116) and chrysanthemum (n =5). Due to the observed variability and divergence from published descriptions of the two P. ligulicola varieties it was not possible to reliably differentiate between the two varieties on the basis of morphological criteria alone. The most notable discrepancy was that P. ligulicola var. inoxydablis isolates did not produce the teleomorph (Didymella ligulicola var. inoxydablis) in culture. The two P. ligulicola varieties were differentiated on the basis of the presence of metabolite E and minor divergence in the ribosomal DNA region of the internal transcribed spacer (ITS) region. Phylogenetic analysis of the translation elongation factor (EF1-α), ITS and glyceraldehyde-3-phoshpate dehydrogenase (G3PD) regions confirmed the identity of P. ligulicola and P. exigua isolates to species level, and suggested that the P. ligulicola var. inoxydablis population from pyrethrum is largely clonal. The EF1-α and G3PD sequences did not clearly differentiate between P. ligulicola varieties, the host from which they were isolated or the geographic location from which they were sourced. Phoma ligulicola var. inoxydablis isolates from pyrethrum (n = 111) were tested in vitro for sensitivity to the DMI fungicide difenoconazole and had a low mean EC₅₀ of 0.136 μg a.i./ml. The continuous sensitivity distributions detected for the P. ligulicola populations indicate that resistance development for this pathosystem is likely to be a gradual process. The mean EC₅₀ of four P. exigua isolates was 50 times greater than for P. ligulicola var. inoxydablis isolates. A PCR assay for determination of mating type detected the HMG motif in the five P. ligulicola var. ligulicola isolates and in one of the 116 P. ligulicola var. inoxydablis isolates. Individual isolates of P. ligulicola var. ligulicola produced perithecia and ascospores on leaf amended agar, but none were detected for P. ligulicola var. inoxydablis. These results indicate that the former variety was homothallic, and the latter variety was heterothallic. The pathogenicity of 18 P. ligulicola and two P. exigua isolates to a single variety of pyrethrum was characterised in two separate experiments by inoculating detached leaves in vitro and glass-house grown plants. In the detached leaf assay significant differences were detected between isolates in lesion growth. There was a positive correlation between the lesion growth in the detached leaf assay and disease incidence in the greenhouse trial for both Phoma species. This is the first report that P. exigua var. exigua is pathogenic to commercial pyrethrum. Symptoms produced on eight week old pyrethrum plants in the greenhouse, inoculated with P. exigua conidial suspensions, were mid-brown leaf lesions up to 5 mm long, and occasional stem lesions.