Chemical and biological characterization of phytotoxins produced by Diplodia species, fungi involved in forest plants diseases

Doctoral thesis Italian OPEN
Masi, Marco (2013)

In recent years, numerous studies have been initiated in order to understand what are the microorganisms involved in forest plants diseases and the role played by phytotoxins produced in the pathogenesis processes. The aim of the present thesis was to study the fungi and the phytotoxins associated with canker disease of the Italian cypress (Cupressus sempervirens L.) and the branch dieback of juniper (Juniperus phoenicea L.) which are plant diseases with noteworthy social and economical implications. Two phytotoxic dihydrofuropyran-2-ones, named afritoxinones A and B, were isolated from liquid culture of Diplodia africana, a fungal pathogen responsible for branch dieback of Juniperus phoenicea L. in Italy. Additionally, six others known metabolites were isolated from the same fungus and identified as: oxysporone, sphaeropsidin A, episphaeropsidone, R-(-)-mellein, (3R,4R)-4-hydroxymellein and (3R,4S)-4-hydroxymellein. The structures of afritoxinones A and B were established by spectroscopic and optical methods and determined to be as (3aS*,6R*,7aS)-6-methoxy-3a,7a-dihydro-3H,6H-furo[2,3-b]pyran-2-one and (3aR*,6R*,7aS)-6-methoxy-3a,7a-dihydro-3H,6H-furo[2,3-b]pyran-2-one, respectively. The phytotoxic activity of afritoxinones A and B and oxysporone was evaluated on host and non-host plants by cutting and leaf puncture assay. Oxysporone proved to be the most phytotoxic compound. Sphaeropsidone is a phytotoxic dimedone methyl ether produced by Diplodia cupressi the causal agent of a canker disease of cypress in the Mediterranean area together with episphaeropsidone which was also isolated from D. africana. Eight derivatives obtained by chemical modifications and two natural analogues were assayed for phytotoxic and antifungal activities, and a structure-activity relationship was examined. Each compound was tested on non-host plants and on five fungal pathogenic species belonging to the genus Phytophthora. The results provide insights into structure-activity relationships within these compounds. It was found that the hydroxy group at C-5, the absolute C-5 configuration, the epoxy group, and the C-2 carbonyl group appear to be structural features important in conferring biological activity. The conversion of sphaeropsidone into the corresponding 1,4-dione derivative led to a compound showing greater antifungal activity than its precursor. Furthermore white block shaped single crystals of sphaeropsidone were obtained and an X-ray diffraction study was carried out. From Diplodia cupressi were also isolated some unrearranged pimarane diterpenes named sphaeropsidins A-C. These latter and 14 derivatives obtained by their chemical modifications were assayed for antibacterial activity against Xanthomonas oryzae pv. oryzae, Pseudomonas fuscovaginae, and Burkholderia glumae, the causal agents of severe bacterial rice diseases. The results showed a strong and specific activity of sphaeropsidin A against X. oryzae pv. oryzae, while no activity was observed against the other two pathogens. The results of structure-activity relationships study showed that structural features important to impart this antibacterial activity are the presence of the C-7 carbonyl group and the hemiketalic lactone functionality. The C-13 vinyl group, the double bond of ring C and/or the tertiary C-9 hydroxyl group, as well as the pimarame arrangement of the tricylic carbon skeleton were also important for the antibacterial activity. This findings may be useful in devising novel compounds for practical application in agriculture.
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