Interactions between 'Candidatus Phytoplasma mali' and the apple endophyte Epicoccum nigrum in Catharanthus roseus plants.

AIMS: We investigated the ultrastructural and molecular interactions between 'Candidatus Phytoplasma mali' and the apple endophyte Epicoccum nigrum in the experimental host Catharanthus roseus to determine whether inoculation of endophyte could trigger defence reactions in the host. METHODS AND RESULTS: Apple proliferation (AP) symptom severity was evaluated in AP-grafted plants that were treated by E. nigrum and compared with untreated controls. Phytoplasma concentration was quantified by real-time PCR in treated and untreated plants. Ultrastructural observations revealed that in endophyte-treated periwinkles, modifications to phytoplasmas, such as irregular shape and cytoplasm confined to the periphery of the cell, and plant cytological changes, such as abundant callose depositions and P-protein aggregations in the sieve elements, occurred. AP-grafted plants that were treated by the endophyte (E. nigrum) showed a reduction in symptom severity; in particular, flowers appeared normal in shape and size, when compared with uninfected controls. Real-time PCR indicated that phytoplasma concentration in AP-grafted plants treated with E. nigrum was about 2·8 times lower than that in untreated ones. CONCLUSIONS: These results demonstrated that the inoculation with E. nigrum influenced phytoplasma infection in C. roseus plants; plant ultrastructural modifications allowed us to hypothesize an enhancing host defence response. SIGNIFICANCE AND IMPACT OF THE STUDY: At present, curative protocols against this phytoplasma are not available. Alternative approaches are thus required to reduce disease spread. Our study might represent a first step in the clarification of plant-phytoplasma-endophyte relationships to find possible strategies for the control of phytoplasma diseases.

Phloem cytochemical modification and gene expression following the recovery of apple plants from apple proliferation disease.

Recovery of apple trees from apple proliferation was studied by combining ultrastructural, cytochemical, and gene expression analyses to possibly reveal changes linked to recovery-associated resistance. When compared with either healthy or visibly diseased plants, recovered apple trees showed abnormal callose and phloem-protein accumulation in their leaf phloem. Although cytochemical localization detected Ca(2+) ions in the phloem of all the three plant groups, Ca(2+) concentration was remarkably higher in the phloem cytosol of recovered trees. The expression patterns of five genes encoding callose synthase and of four genes encoding phloem proteins were analyzed by quantitative real-time reverse transcription-polymerase chain reaction. In comparison to both healthy and diseased plants, four of the above nine genes were remarkably up-regulated in recovered trees. As in infected apple trees, phytoplasma disappear from the crown during winter, but persist in the roots, and it is suggested that callose synthesis/deposition and phloem-protein plugging of the sieve tubes would form physical barriers preventing the recolonization of the crown during the following spring. Since callose deposition and phloem-protein aggregation are both Ca(2+)-dependent processes, the present results suggest that an inward flux of Ca(2+) across the phloem plasma membrane could act as a signal for activating defense reactions leading to recovery in phytoplasma-infected apple trees.

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.

The effect of fetal movement counting on maternal attachment to fetus.

The effect of fetal movement counting on maternal attachment to fetus was investigated in 213 women with uncomplicated singleton pregnancies at 28 to 32 weeks' gestation. Women were randomized into those who counted fetal movements using the Sadovsky (n = 63) or Cardiff (n = 62) charts and controls (n = 88). After 1 month of fetal movement counting, the Cranley 24-item scale with five subscales was used as a measure of maternal-fetal attachment. Univariate analysis revealed a statistically significant increase in total attachment scores and in each of the five attachment subscales among women who counted fetal movements (p less than 0.0001). Turkey's studentized range test confirmed significant differences between each of the Sadovsky and Cardiff groups compared with controls (p less than 0.05). Our study suggests that fetal movement counting may enhance the maternal-fetal attachment process.