Paolo Ermacora

Transmission Characteristics of the European Stone Fruit Yellows Phytoplasma and its Vector Cacopsylla Pruni

A study was carried out on the transmission parameters of the European stone fruit yellows phytoplasma by the vector Cacopsylla pruni. In the greenhouse, using groups of psyllids, the minimum acquisition period was 2–4 days, the minimum latent period 2–3 weeks and the minimum inoculation period 1–2 days. The vectors retained infectivity until their death. Under natural conditions retention of infectivity in C. pruni lasts through the winter and the following spring, when the overwintering insects reach the stone fruit trees, they are already infected and infective. The research shows that the vector C. pruni transmits the European stone fruit yellows phytoplasma in a persistent manner.

Production of monoclonal antibodies against apple proliferation phytoplasma and their use in serological detection

Two monoclonal antibodies were obtained against the apple proliferation phytoplasma that provide easy, rapid, specific and sensitive serological detection. They reacted specifically by using ELISA and immunofluorescence techniques with apple proliferation-infected periwinkles and apple trees from different regions in northern Italy and Slovenia, but not with several other phytoplasma isolates. We did not observe any monoclonal antibody reaction even using phytoplasmas belonging to the same phylogenetic group such as European stone fruit yellows and pear decline. Two serological techniques, immunofluorescence and ELISA, were compared with DAPI staining and PCR. From July until leaf fall ELISA was as sensitive as PCR but was more rapid and convenient than PCR; immunofluorescence was useful for specific detection of apple proliferation phytoplasma on roots throughout the year. Serological techniques could be conveniently applied in the roots, stems and leaves of apple trees depending on specific phenological stages of the plants.

High tolerance of European plum varieties to plum leptonecrosis

A 13 year comparative study was carried out on the behaviour of four European and two Japanese plum varieties grown in adjacent rows in an area of northern Italy where plum leptonecrosis is epidemic. Within seven years, 100% of the Japanese plum trees became symptomatically infected. Nine years after planting, five trees of each of the European cvs, which were asymptomatic, were top-grafted with healthy buds of the cv Ozark Premier, which is an indicator for plum leptonecrosis. Based on the results of PCR analysis, DAPI staining and on the reaction of the top-grafted Ozark Premier indicators, 50% of the European plum trees, despite their healthy appearance, were shown to be infected with plum leptonecrosis. The detectable presence and graft transmissibility of the plum leptonecrosis phytoplasma in the asymptomatic European plum trees means that the European plum trees are not resistant to the infection but that they are tolerant. The active presence of a still unknown vector/s in the investigated area is stressed as well as the important role of Prunus domestica L. played in the conservation and spread of plum leptonecrosis.

Differentially-regulated defence genes in Malus domestica during phytoplasma infection and recovery

To improve knowledge about plant/phytoplasma interactions and, in particular, about the ‘recovery’ phenomenon in previously-infected plants, we investigated and compared expression levels of several defence-related genes (four pathogenesis-related proteins and three jasmonate-pathway marker enzymes) in apple plants showing different states of health: vigorous (healthy), phytoplasma-infected, and recovered. Real Time-PCR analyses demonstrated that genes are differentially expressed in apple leaf tissue according to the plants’ state of health. Malus domestica Pathogenesis-Related protein (MdPR) 1, MdPR 2 and MdPR 5 were significantly induced in leaves of diseased and symptomatic plants compared to leaves of those plants that were healthy or recovered. On the other hand, levels of all the jasmonate (JA)-pathway marker genes that we selected for this study, were up-regulated in the leaves of recovered plants compared to the diseased ones. In conclusion, our study demonstrated that two different sets of defence genes are involved in the interactions between apple plants and ‘Candidatus Phytoplasma mali’ (‘Ca. P. mali’) and that these genes are differentially expressed during phytoplasma infection or recovery.

Detection and characterization of phytoplasmas in diseased stone fruits and pear by PCR-RFLP analysis in Turkey

During the late summer-early autumn of 2002, surveys were carried out in Turkey to determine the presence of phytoplasma diseases in fruit trees. Phytoplasmas were detected and characterized by PCR-RFLP analysis and TEM technique in stone fruit and pear trees in the eastern Mediterranean region of the country. Six out of 24 samples, including almond, apricot, peach, pear and plum, gave positive results in PCR assays. RFLP analysis usingSspI andBsaAI enzymes of PCR products obtained with primer pair f01/r01 enabled identification of the phytoplasmas involved in the diseases. Stone fruit trees, including a local apricot variety (‘Sakıt’) and a pear sample, were found to be infected with European stone fruit yellows (ESFY, 16SrX-B) and pear decline (PD, 16SrX-C) phytoplasmas, respectively. This is the first report in Turkey of PD phytoplasma infecting pear and of ESFY phytoplasma infecting almond, apricot, myrobalan plum and peach; ESFY phytoplasma infecting Japanese plum was previously reported.

Transmissible tolerance to European stone fruit yellows (ESFY) in apricot: cross-protection or a plant mediated process?

European Stone Fruit Yellows (ESFY) is an emerging disease caused by ‘Candidatus Phytoplasma prunorum’ (‘Ca. P. prunorum’) affecting stone fruits, as apricots. Resistant apricot cultivars are unknown, but it has been demonstrated that individual plants can recover from the disease, behaving as completely tolerant to ESFY. The status of tolerance is transmissible by grafting to successive apricot individuals, but it is not clear whether recovery corresponds to a transmissible tolerance that depends on a plant–mediated reaction or if it is due to a cross-protection promoted by a transmissible protective agent i.e. hypovirulent strain/s of ‘Ca. P. prunorum’. Results achieved after prolonged field experiments support the first hypothesis. Two groups of apricot plants derived from a common recovered mother (one ‘Ca. P. prunorum’-free after heat-treatment and the second not heat-treated, i.e. harbouring potential protective strain/s of the phytoplasma), behaved similarly: no plants from either of the two groups developed stable ESFY symptoms after natural infections. Corresponding groups of plants, derived from symptomatic mothers, developed a high percentage of diseased plants after natural infection. No potential protective ‘Ca. P. prunorum’ hypovirulent strains were detected in the asymptomatic apricot plants. The summarized evidence supports a host-defence induction, likely of epigenetic feature. The present long-term study in apricot represents an uncommon empiric proof supporting the theory of inducible resistance to pathogens in plants.

Genomic Structural Variations Affecting Virulence During Clonal Expansion of Pseudomonas syringae pv. actinidiae Biovar 3 in Europe

Pseudomonas syringae pv. actinidiae (Psa) biovar 3 caused pandemic bacterial canker of Actinidia chinensis and Actinidia deliciosa since 2008. In Europe, the disease spread rapidly in the kiwifruit cultivation areas from a single introduction. In this study, we investigated the genomic diversity of Psa biovar 3 strains during the primary clonal expansion in Europe using single molecule real-time (SMRT), Illumina and Sanger sequencing technologies. We recorded evidences of frequent mobilization and loss of transposon Tn6212, large chromosome inversions, and ectopic integration of IS sequences (remarkably ISPsy31, ISPsy36, and ISPsy37). While no phenotype change associated with Tn6212 mobilization could be detected, strains CRAFRU 12.29 and CRAFRU 12.50 did not elicit the hypersensitivity response (HR) on tobacco and eggplant leaves and were limited in their growth in kiwifruit leaves due to insertion of ISPsy31 and ISPsy36 in the hrpS and hrpR genes, respectively, interrupting the hrp cluster. Both strains had been isolated from symptomatic plants, suggesting coexistence of variant strains with reduced virulence together with virulent strains in mixed populations. The structural differences caused by rearrangements of self-genetic elements within European and New Zealand strains were comparable in number and type to those occurring among the European strains, in contrast with the significant difference in terms of nucleotide polymorphisms. We hypothesize a relaxation, during clonal expansion, of the selection limiting the accumulation of deleterious mutations associated with genome structural variation due to transposition of mobile elements. This consideration may be relevant when evaluating strategies to be adopted for epidemics management.

Genomic-assisted characterisation of Pseudomonas sp. strain Pf4, a potential biocontrol agent in hydroponics

ABSTRACT In an attempt to select potential biocontrol agents against Pythium spp. and Rhizoctonia spp. root pathogens for use in soilless systems, 12 promising bacteria were selected for further investigations. Sequence analysis of the 16S rRNA gene revealed that three strains belonged to the genus Enterobacter, whereas nine strains belonged to the genus Pseudomonas. In in vitro assays, one strain of Pseudomonas sp., Pf4, closely related to Pseudomonas protegens (formerly Pseudomonas fluorescens), showed noteworthy antagonistic activity against two strains of Pythium aphanidermatum and two strains of Rhizoctonia solani AG 1-IB, with average inhibition of mycelial growth >80%. Strain Pf4 was used for in vivo treatments on lamb’s lettuce against R. solani root rot in small-scale hydroponics. Pf4-treated and untreated plants were daily monitored for symptom development and after two weeks of infection, a significant protective effect of Pf4 against root rot was recorded. The survival and population density of Pf4 on roots were also checked, demonstrating a density above the threshold value of 105 CFU g−1 of root required for disease suppression. Known loci for the synthesis of antifungal metabolites, detected using PCR, and draft-genome sequencing of Pf4 demonstrated that Pseudomonas sp. Pf4 has the potential to produce an arsenal of secondary metabolites (plt, phl, ofa and fit-rzx gene clusters) very similar to that of the well-known biocontrol P. protegens strain Pf-5.

Symptoms of Phytoplasma Diseases

Phytoplasmas are associated with diseases in several hundreds of cultivated herbaceous and woody plants. Their impact in agriculture and the periodical outbreak of worrying epidemics make very important, besides precise laboratory-based diagnosis, the direct in-field recognition of phytoplasma disease symptoms. Even if some symptoms are typical of this kind of pathogens, in-field diagnosis requires the knowledge of the host plant, strong field experience, and awareness of the symptom variability of the various organs of the plant during different seasons and under various environmental conditions. It is therefore very important to be familiar with factors like environmental conditions, agronomical features, and disease progression that influence symptom expression. Therefore, a satisfactory diagnosis should be based on repeated and complete observations scored over the entire plant and across different times of the year. A more suitable diagnosis is possible if the observer is able to recognize and distinguish the symptoms of other biotic or abiotic diseases. A general rule is to observe three different symptoms, at least, and to seek input from the grower about the initial development, frequency, diffusion, and particular characteristics of the disease.After a short introduction the following symptoms are presented: the most common and representative symptoms caused by phytoplasmas; the most common symptoms of phytoplasma diseases occurring in particular plant organs, with some references to specific diseases; phytoplasma symptoms on the model plant periwinkle (Vinca rosea or Catharanthus roseus); the main factors influencing phytoplasma symptoms expression; and several practical procedures that should be followed for suitable diagnosis. A series of original photos have been included to illustrate typical symptoms.

Localization and subcellular association of Grapevine Pinot Gris Virus in grapevine leaf tissues.

Despite the increasing impact of Grapevine Pinot gris disease (GPG-disease) worldwide, etiology about this disorder is still uncertain. The presence of the putative causal agent, the Grapevine Pinot Gris Virus (GPGV), has been reported in symptomatic grapevines (presenting stunting, chlorotic mottling, and leaf deformation) as well as in symptom-free plants. Moreover, information on virus localization in grapevine tissues and virus-plant interactions at the cytological level is missing at all. Ultrastructural and cytochemical investigations were undertaken to detect virus particles and the associated cytopathic effects in field-grown grapevine showing different symptom severity. Asymptomatic greenhouse-grown grapevines, which tested negative for GPGV by real time RT-PCR, were sampled as controls. Multiplex real-time RT-PCR and ELISA tests excluded the presence of viruses included in the Italian certification program both in field-grown and greenhouse-grown grapevines. Conversely, evidence was found for ubiquitous presence of Grapevine Rupestris Stem Pitting-associated Virus (GRSPaV), Hop Stunt Viroid (HSVd), and Grapevine Yellow Speckle Viroid 1 (GYSVd-1) in both plant groups. Moreover, in every field-grown grapevine, GPGV was detected by real-time RT-PCR. Ultrastructural observations and immunogold labelling assays showed filamentous flexuous viruses in the bundle sheath cells, often located inside membrane-bound organelles. No cytological differences were observed among field-grown grapevine samples showing different symptom severity. GPGV localization and associated ultrastructural modifications are reported and discussed, in the perspective of assisting management and control of the disease.