Documentation of Siderophore Activity, Metal Binding, and its Effect on Symptomatology of Cercospora Leaf Blight Caused in Soybean by Cercosporin from Cercospora cf. flagellaris

Abstract

Cercospora leaf blight of soybean (Glycine max) is a serious problem in the mid-south of the United States and is present in most soybean-growing regions of the world. The causal organisms, Cercospora kikuchii, C. cf. flagellaris and C. cf. sigesbeckiae, utilize the photo-activated toxin cercosporin as the primary pathogenicity factor. The disease has historically been understood to progress from a purpling or bronzing of the leaves to a blight in which tissue dies. Previous literature showed possible binding of cercosporin to metals and previous work in our group showed possible use of minor element nutrition in the plant to reduce disease severity. Through the use of UV-vis spectrometry, nuclear magnetic resonance spectroscopy, and mass spectrometry evidence is given in support of cercosporin-metal binding. From this work, it is hypothesized that the metals bind at the phenol group of the cercosporin molecule, yielding a dark purple compound. Cercosporin appears to have higher affinity for iron, especially the Fe(III) form, over the other plant minor elements but appears to have some minor binding affinity for aluminum, copper, manganese, and zinc. Cercosporin and iron combinations show greatly reduced toxicity compared to cercosporin alone, in support of previous reports in the literature. Manganese added to cercosporin resulted in more necrosis of the soybean tissue. Soybean plants fertilized with iron sufficient to increase tissue concentrations by 20 mg/kg dry weight resulted in a significantly higher resistance to the toxin. The iron fertilization also resulted in a roughly 60 mg/kg dry weight reduction in manganese, which may also help account for the reduction in toxicity. The symptoms also were less similar to the typical blight symptoms and more similar to the purple leaf symptoms. Isolates of C. cf. flagellaris grown on split chrome azurol sulfate (CAS) plates elicited the signature color shift indicative of siderophore production. Cultures of C. cf. flagellaris also were grown in liquid minimal medium amended with cercosporin, iron, or both. In broths containing both cercosporin and iron, fungal growth was higher, total cercosporin production was lower, and iron content of the mycelium was significantly higher than that of controls. This demonstrates that the fungus is capable of up-taking and incorporating iron in this form. When only cercosporin was provided, mycelial concentrations of aluminum, copper, and boron were higher than other treatments. This indicates that cercosporin may be involved in other metal uptake and these metals may compete with iron. While previous work