Taxonomy, molecular and pathogenic variability of Phoma medicaginis Malbr. & Roum. isolates from Minnesota on alfalfa (Medicago sativa L.) and visualization of the infection process of Phoma medicaginis Malbr. & Roum. on alfalfa (Medicago sativa L.) leave

Alfalfa (Medicago sativa L.) is the most widely grown perennial legume species in the United States and is an important forage crop for animal production in temperate regions. Spring black stem and leaf spot of alfalfa caused by Phoma medicaginis Malbr. & Roum. (Pm) is one of the most important diseases of alfalfa. The fungus is able to infect different tissues of the alfalfa plant. An adequate level of resistance to the disease is lacking in alfalfa varieties. The taxonomic placement of Pm has been controversial. It was reported that Pm isolates show morphological, cultural, and pathogenic variability. Few molecular studies have been used to identify and characterize this pathogen. In addition, no studies have been done to characterize the infection process on alfalfa genotypes with differing degrees of resistance. A collection of Pm isolates collected from different organs of alfalfa plants collected mostly in Minnesota, and a few from Wisconsin, Kansas and Pennsylvania were characterized using the internal transcribed spacer regions of the nuclear rDNA and by AFLP fingerprinting. Sequencing of the ITS1, ITS2 and 5.8S subunit of the nuclear rDNA showed that these regions are highly conserved and thus useful for taxonomic identification, but failed to reveal variation among isolates. AFLP analysis using the Dice coefficient revealed a moderate amount of variation among the isolates with no correlation by area of collection or plant organ. Virulence tests of a subset of isolates representing diversity found by AFLP analysis resulted in different virulence patterns.; Time course experiments were done using light and scanning electron microscopy to study the infection and colonization process of an isolate of Pm from the time of spore deposition on the leaf surface to pycnidia formation. In older alfalfa leaves spore germination, fungal penetration and infection, as well as development and maturation of pycnidia were delayed approximately 24 h on the moderately resistant alfalfa genotype compared to the susceptible genotype. The delay in symptom development occurred until 192 h after inoculation, after which the amount of chlorosis in both genotypes was similar. Chlorosis appeared to precede infection, and requires additional investigation into its causes.