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

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 Ca2+ ions in the phloem of all the three plant groups, Ca2+ 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 upregulated 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 Ca2+-dependent processes, the present results suggest that an inward flux of Ca2+ across the phloem plasma membrane could act as a signal for activating defense reactions leading to recovery in phytoplasma-infected apple trees.L'articolo é disponibile sul sito dell'editore: http://www.apsjournals.apsnet.or

Phytoplasma infection in tomato is associated with re-organization of plasma membrane, ER stacks, and actin filaments in sieve elements

Phytoplasmas, biotrophic wall-less prokaryotes, only reside in sieve elements of their host plants. The essentials of the intimate interaction between phytoplasmas and their hosts are poorly understood, which calls for research on potential ultrastructural modifications. We investigated modifications of the sieve-element ultrastructure induced in tomato plants by ‘Candidatus Phytoplasma solani,’ the pathogen associated with the stolbur disease. Phytoplasma infection induces a drastic re-organization of sieve-element substructures including changes in plasma membrane surface and distortion of the sieve-element reticulum. Observations of healthy and stolbur-diseased plants provided evidence for the emergence of structural links between sieve-element plasma membrane and phytoplasmas. One-sided actin aggregates on the phytoplasma surface also inferred a connection between phytoplasma and sieve-element cytoskeleton. Actin filaments displaced from the sieve-element mictoplasm to the surface of the phytoplasmas in infected sieve elements. Western blot analysis revealed a decrease of actin and an increase of ER-resident chaperone luminal binding protein (BiP) in midribs of phytoplasma-infected plants. Collectively, the studies provided novel insights into ultrastructural responses of host sieve elements to phloem-restricted prokaryotes

Phytoplasma infection in tomato is associated with re-organization of plasma membrane, ER stacks, and actin filaments in sieve elements

Phytoplasmas, biotrophic wall-less prokaryotes, only reside in sieve elements of their host plants. The essentials of the intimate interaction between phytoplasmas and their hosts are poorly understood, which calls for research on potential ultrastructural modifications. We investigated modifications of the sieve-element ultrastructure induced in tomato plants by ‘Candidatus Phytoplasma solani’, the pathogen associated with the stolbur disease. Phytoplasma infection induces a drastic re-organization of sieve-element substructures including changes in plasma membrane surface and distortion of the sieve-element reticulum. Observations of healthy and stolbur-diseased plants provided evidence for the emergence of structural links between sieve-element plasma membrane and phytoplasmas. One-sided actin aggregates on the phytoplasma surface also inferred a connection between phytoplasma and sieve-element cytoskeleton. Actin filaments displaced from the sieve-element mictoplasm to the surface of the phytoplasmas in infected sieve elements. Expression analysis revealed a decrease of actin and an increase of ER-resident chaperone luminal binding protein (BiP) in midribs of phytoplasma-infected plants. Collectively, the studies provided novel insights into ultrastructural responses of host sieve elements to phloem-restricted prokaryotes

IL “RECOVERY” DA APPLE PROLIFERATION IN MELO È ASSOCIATO ALL' AUMENTO DELLA CONCENTRAZIONE DELLO IONE CALCIO NEL FLOEMA

5nonenoneMUSETTI R; TUBARO F; POLIZZOTTO R; ERMACORA P; OSLER R.Musetti, Rita; Tubaro, Franco; Polizzotto, Rachele; Ermacora, Paolo; Osler, R

Detection of Bois Noir phytoplasma in grapevine roots by reverse transcription-Real Time TaqMan assays

Bois noir (BN) is a phytoplasma disease of grapevine spread in the Mediterranean basin, causing relevant economic losses. BN phytoplasma diagnosis is currently carried out by detecting the pathogen DNA sequences in the leaf. Even if reliable and usable in routine analyses, molecular diagnosis is nevertheless restricted to the late summer, when BN symptoms become evident. This is because the above methods are not enough sensitive to carry out diagnosis in other periods of the year, when symptoms are not evident, or on other plant organs such as roots. This consideration suggests that more sensitive and focused detection methods for BN would be needed. Because of the lack of diagnostic methods for root tissues, it is not known where the pathogen overwinters inside the host. We aim at expanding diagnosis effectiveness to root tissues in BN-infected grapevine, to better understand phytoplasma/ grapevine interactions and to give new insights to BN epidemiology. Different root samples from healthy, BN-infected and recovered grapevines, have been collected and RNA was extracted. As the low concentration of template in the host, we performed a nested Real-Time PCR using a BN specific TaqMan probe, following the method already described by Margaria et al. (Plant Pathol. 58:838-845, 2009). Preliminary analyses showed positive signal in roots of symptomatic and recovered plants, whereas no amplification was observed in healthy samples. This result suggests that BN phytoplasma persists in the root phloem tissues of recovered individuals. The epidemiological significance of this finding will be discussed

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

Phloem-specific protein expression patterns in apple and grapevine during phytoplasma infection and recovery

Recovery - complete remission of disease symptoms - has been reported in plants affected by phytoplasmas. The physiological basis for this phenomenon is not yet understood, but it seems associated to ultrastructural and biochemical modification of the phloem, the tissue where phytoplasmas live and spread. In this work we compared asymptomatic, phytoplasma-infected and recovered apple and grapevine leaf tissues by means of ultrastructural and gene-expression analyses, focusing on a possible role of specific phloem proteins in the plant defense-related processes. Preliminary results indicate that different occlusion mechanisms could interact in the phloem during phytoplasma symptomatic status and/or recovery