Inhibition of Sporulation and Ultrastructural Alterations of Grapevine Downy Mildew by the Endophytic Fungus Alternaria alternata

ABSTRACT One hundred twenty-six endophytic microorganisms isolated from grapevine leaves showing anomalous symptoms of downy mildew were tested on grapevine leaf disks as biocontrol agents against Plasmopara viticola. Among the 126 microorganisms, only five fungal isolates completely inhibited the sporulation of P. viticola; all of them were identified as Alternaria alternata. Ultrastructural analyses were carried out by transmission electron microscopy to observe cellular interactions between P. viticola and A. alternata in the grapevine leaf tissue. Cytological observations indicated that, even without close contact with A. alternata, the P. viticola mycelium showed severe ultrastructural alterations, such as the presence of enlarged vacuoles or vacuoles containing electron-dense precipitates. Haustoria appeared necrotic and irregularly shaped or were enclosed in callose-like substances. Therefore, a toxic action of A. alternata against P. viticola was hypothesized. To examine the production of toxic low-molecular-weight metabolites by A. alternata, we analyzed the fungal liquid culture by thin layer chromatography and proton magnetic resonance spectroscopy. The main low-molecular-weight metabolites produced by the endophyte were three diketopiperazines: cyclo(l-phenylalanine-trans-4-hydroxy-l-proline), cyclo(l-leucine-trans-4-hydroxy-l-proline), and cyclo(l-alanine-trans-4-hydroxy-l-proline). When applied at different concentrations to both grapevine leaf disks and greenhouse plants, a mixture of the three diketopiperazines was very efficacious in limiting P. viticola sporulation.