Sequential infections by 32 isolates of

Abstract

Context Studies of Phoma black stem and leaf spot disease (caused by Phoma medicaginis) in annual medics (Medicago spp.) normally involve a ‘once-only’ inoculation not reflecting multiple pathogen infection and phytoestrogen production cycles in the field. Phytoestrogen production by plants can result in lower ovulation rates in grazing animals. Aims We aimed to determine whether sequential infections by P. medicaginis increase production of phytoestrogens in annual medics, and to measure the genetic diversity of isolates. Methods In a greenhouse experiment, pathogenicity and virulence were investigated across 32 isolates of P. medicaginis following one, two or three rounds of inoculation of M. polymorpha var. brevispina. Production of the phytoestrogens coumestrol and 4′-O-methyl coumestrol was measured, and correlation with disease parameters assessed. DNA sequencing using ITS, β-tubulin, calmodulin and P. medicaginis-specific EFNI-1α was applied for phylogenetic analysis of isolates from Western Australia and elsewhere. Key results Across isolates, highest leaf disease incidence was 76%, petiole disease incidence 61%, leaf disease severity 52% and petiole disease severity 53%. Stem coumestrol content range was 45–1247 mg kg−1, and 4′-O-methyl coumestrol 0–344 mg kg−1. All measures were highest after three rounds of inoculation. Overall, there was a positive correlation of leaf disease incidence with coumestrol content (P < 0.05) and of both leaf and petiole disease incidence with 4′-O-methylcoumestrol content (P < 0.01, P < 0.05, respectively). Phylogenetic analysis revealed a high degree of genetic similarity among Western Australian isolates, generally grouping into a single separate cluster across the four markers, and genetically distinct from isolates sourced outside Australia. Conclusions Leaf disease incidence was the best discriminating disease parameter for coumestrol and 4′-O-methylcoumestrol content. Western Australian isolates of P. medicaginis were genetically similar and unique, possibly due to geographic separation. Implications The study emphasised the importance of sequential inoculations when screening annual Medicago genotypes towards developing cultivars with superior disease resistance and enhanced animal reproductive outcomes.