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.
