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.

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.