Acquired Tolerance in Apricot Plants that Stably Recovered from European Stone Fruit Yellows.

European stone fruit yellows (ESFY) is one of the most destructive phytoplasma diseases of plum, apricot, and peach in Europe. Conventional preventive defense strategies have been ineffective. Because apricot cultivars with innate-constitutive resistance against ESFY are not available, the aim of this more than 20-year-long study was to seek acquired resistance or tolerance. In the first experiment, we surveyed an orchard with seven apricot cultivars for 12 years in an area of northern Italy with a high rate of natural occurrence of ESFY. Of the diseased plants, a few (8.7%) became completely symptomless but retained the phytoplasma, as confirmed by polymerase chain reaction (PCR). In the second experiment, we grafted buds from two stably recovered plants and from two nonrecovered plants onto 'Rubira' peach. Over the next 9 years in an orchard with a high rate of natural infection, 93.0% of the "nonrecovered clones" became diseased but only 1.5% of the plants grafted with the two "recovered clones" developed ESFY symptoms. According to PCR analyses, all of the exposed test plants were ESFY-infected, whether they were derived from recovered or nonrecovered mothers. This could indicate that epigenetic changes occurred in recovered plants due to a graft-transmissible memory. Based on the results attained from the two described experiments, we propose that an acquired tolerance that occurred in stably recovered apricot trees was graft transmitted from two tolerant apricot clones. In contrast, we did not demonstrate a cross-protection process based on protectant avirulent phytoplasma strains that suppress severe strains.

DNA-Dependent Detection of the Grapevine Fungal Endophytes Aureobasidium pullulans and Epicoccum nigrum.

Aureobasidium pullulans and Epicoccum nigrum are frequently reported as endophytes of various crops, including grapevine (Vitis vinifera). Because of their potential role as biological control agents against grapevine pathogens, we examined the occurrence of A. pullulans and E. nigrum in two grapevine varieties (Merlot and Prosecco) in Italian vineyards where spontaneous recovery from phytoplasma disease is recurrent. Species-specific primers for A. pullulans and two genetically distinct strains of E. nigrum were designed in variable regions of ITS1 and ITS2. Primer specificity was confirmed by polymerase chain reaction using purified DNA from other fungal endophytes that are usually encountered during isolation attempts from grapevine tissues and from several other strains of A. pullulans and E. nigrum isolated from other sources. In order to determine the occurrence of the two endophytes in grapevine plants, DNA was extracted from shoots of 44 grapevines collected in six vineyards from different localities of northeast Italy. Both endophytes were detected and their identity was confirmed by restriction fragment length polymorphism (RFLP) patterns obtained from reference strains. RFLP analyses confirmed the presence of two E. nigrum strains belonging to different RFLP groups in grapevine. The molecular methods described allowed a sensitive, specific, and reliable identification of the two endophytes in grapevine.

Antifungal activity of diketopiperazines extracted from Alternaria alternata against Plasmopara viticola: an ultrastructural study.

Three dipeptides, belonging to the family of diketopiperazines (DKPs), were extracted from broth culture of the grapevine endophyte Alternaria alternata, and were tested against Plasmopara viticola on leaves of grapevine plants grown in greenhouse. DKPs, used at different concentrations (10(-3), 10(-4), 10(-5) and 10(-6)M) both singularly and in mixtures, demonstrated real effectiveness in inhibiting P. viticola sporulation when applied 2 or 24h after pathogen inoculation. Moreover, no necrotic lesions or other phytotoxicity symptoms were observed on DKP-treated grapevine leaf tissues. Ultrastructural analysis performed on grapevine leaf tissues revealed that the DKPs used singularly and in mixture, at above reported concentrations, did not cause leaf tissue damages. By contrast, hyphae of P. viticola exhibited marked structural changes, similar to those induced by the endophyte A. alternata. This demonstrates the involvement of these metabolites in the relationship of P. viticola and the endophyte. Further experimental trials will be carried out in the next future in order to test the effectiveness of these molecules also under field conditions, and to better understand the mechanism of action involved in the pathogen inhibition.

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.

Ultrastructural analysis of Vitis vinifera leaf tissues showing atypical symptoms of Plasmopara viticola.

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.

[Dipropylacetamide in the treatment of manic-depressive psychosis]. / Le dipropylacétamide dans le traitement de la psychose maniaco-dépressive

Dipropylacetamide, which was first used as a treatment of epilepsy, turned out to be also a normalizing agent of the periodic avolution of manic-depressive psychosis. The therapeutic action of dipropylacetamide has several effects: 1) total suppression of the fits; 2) reduced intensity of these fits; 3) increasing lapse of time between two fits; 4) greatest response to anti-depressants and neuroleptics which can be prescribed in much smaller doses. More manic than depressive states respond to dipropylacetamide. The average doses are about 900 mg or so. Cautiousness if advisable at the beginning of the treatment on account of the use of sedatives; but the treatment is extremely well received and accepted by the patients during periods of several years without the requirement of biological controls. Associating lithium with dipropylacetamide often yields remarkable results.