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.

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.

Ribosomal protein gene-based phylogeny for finer differentiation and classification of phytoplasmas.

Extensive phylogenetic analyses were performed based on sequences of the 16S rRNA gene and two ribosomal protein (rp) genes, rplV (rpl22) and rpsC (rps3), from 46 phytoplasma strains representing 12 phytoplasma 16Sr groups, 16 other mollicutes and 28 Gram-positive walled bacteria. The phylogenetic tree inferred from rp genes had a similar overall topology to that inferred from the 16S rRNA gene. However, the rp gene-based tree gave a more defined phylogenetic interrelationship among mollicutes and Gram-positive walled bacteria. Both phylogenies indicated that mollicutes formed a monophyletic group. Phytoplasmas clustered with Acholeplasma species and formed one clade paraphyletic with a clade consisting of the remaining mollicutes. The closest relatives of mollicutes were low-G+C-content Gram-positive bacteria. Comparative phylogenetic analyses using the 16S rRNA gene and rp genes were performed to evaluate their efficacy in resolving distinct phytoplasma strains. A phylogenetic tree was constructed based on analysis of rp gene sequences from 87 phytoplasma strains belonging to 12 16Sr phytoplasma groups. The phylogenetic relationships among phytoplasmas were generally in agreement with those obtained on the basis of the 16S rRNA gene in the present and previous works. However, the rp gene-based phylogeny allowed for finer resolution of distinct lineages within the phytoplasma 16Sr groups. RFLP analysis of rp gene sequences permitted finer differentiation of phytoplasma strains in a given 16Sr group. In this study, we also designed several semi-universal and 16Sr group-specific rp gene-based primers that allow for the amplification of 11 16Sr group phytoplasmas.

Oligonucleotides as hybridization probes to localize phytoplasmas in host plants and insect vectors.

ABSTRACT Protocols have been developed using 20- to 24-mer oligodeoxynucleotides, originally designed as polymerase chain reaction primers, as hybridization probes for the nonradioactive detection of Italian clover phyllody (ICPh) phytoplasma in plant (Chrysanthemum carinatum) and leafhopper (Euscelidius variegatus) tissue. In situ hybridization of paraffin-embedded tissue sections was carried out using oligodeoxynucleotides 5' end-labeled with either Cy5 fluorochrome, biotin, or digoxigenin. The Cy5-labeled oligonucleotide probes that hybridized to phytoplasmas present in plant tissue were visualized by confocal microscopy. The biotin- and digoxigeninlabeled probes were detected in both plant and insect tissue using a chromogenic alkaline phosphatase-nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phosphate reaction. An enhancement of a signal was observed in plant tissue when a tyramide signal-amplification procedure was incorporated into the biotin or digoxigenin detection systems. The results obtained using these techniques with the ICPh phytoplasma system showed that they can provide a rapid means of confirming vector status in insects. Due to the potential ability of short, labeled, oligonucleotide probes to specifically distinguish between different phytoplasmas present in multiple infections, this technique should provide a powerful new tool for epidemiological and vector ecology studies.