ABSTRACT
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.
ABSTRACT
In an abandoned farm in Tuscany a year by year regression of downy mildew disease on grapevines was observed and a decrease in virulence as well as vigor and fertility of the causal fungus, Plasmopara viticola. Anomalous spots of the fungus (i.e. atypical coloration of leaves or mosaic) on leaf tissues of a sensitive Vitis vinifera grapevine were observed. The anomalous symptoms were often associated with the typical 'oil spots' and were present under environmental conditions favourable for a normal development of the disease. An ultrastructural study was carried out on leaf tissues of grapevine plants aimed at clarifying the cause of this phenomenon and detecting whether there were alterations in P. viticola mycelium and endophytes present. ELISA was also performed to check the presence of grapevine viruses in the plants. TEM results demonstrated that characteristic P. viticola was present in leaf samples showing oil spots, while, both the fungus and the host tissues showed cytological alterations in leaves with mosaic symptoms. Finally, hyphae were absent in leaf tissues without downy mildew spots, but showing severe ultrastructural modifications. Several plant virus infections were found in these grapevines. Literature reports that the development and sporulation of some phytopathogenic fungi inside their hosts can be limited by virus infections. Further experimental approaches are required to determine if resistance to P. viticola can be induced by viral infections in grapevines.
