The infection process of Colletotrichum graminicola (Ces.) Wils. on Sorghum bicolor L. and analysis of an extracellular DNase produced by the fungus

Beth Ann Snyder, Purdue University

Abstract

The interactions that occur between host and pathogen during the disease process involve a complex series of structural and biochemical reactions initiated by both organisms. Defense mechanisms of Sorghum bicolor L. produced in response to attempted penetration by Colletotrichum graminicola were examined as were several extracellular enzymes found within the conidial mucilage and the culture fluid of the pathogen. One of the enzymes, a DNase, was characterized and purified from the culture fluid. The 3-deoxyanthocyanidin flavonoid phytoalexins produced by sorghum in response to infection are visible pigments. The subcellular site of accumulation of phytoalexins was monitored in vivo without disruption to the processes involved in the plant's defense against a pathogen. Sorghum responded to infection by producing red spherical bodies within epidermal cells immediately under fungal attack. These bodies originated as small colorless inclusions which enlarged, moved toward the site of penetration and became pigmented as the infection process proceeded. HPLC analysis of extracts from infection sites showed accumulation of the red phytoalexin pigments corresponding to the appearance of the spherical, red pigmented inclusions within infected cells. This research suggests that these bodies are the site of accumulation of the sorghum phytoalexins. Analysis of the conidial mucilage of C. graminicola identified enzymes which could hydrolyze DNA, RNA, protein and phosphomonoesters. One of those enzymes, the DNase, is produced by vegetative hyphae and by sporulating mycelia. Isolates of Colletotrichum spp were screened for the presence of the enzyme within the conidial mucilage. The DNase was characterized according to pH and temperature optima, cation effects, substrate hydrolysis and inhibitors of the enzyme. C. graminicola secretes a DNase into culture medium when grown on potato sucrose broth. Maximal DNase activity occurred on this medium after 11 days of growth. The extracellular DNase was purified almost 1,600-fold by separation on DEAE-Sephadex, affinity chromatography on DNA-cellulose and by passage through a Sephadex G-100 column. Polyacrylamide gel electrophoresis of the iodinated DNase from the Sephadex G-100 column revealed a single band of radioactivity. The purified DNase exhibited optimum activity at pH 8.2 against single-stranded DNA and was free from RNase activity.

Degree

Ph.D.

Advisors

Nicholson, Purdue University.

Subject Area

Plant pathology

COinS