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Phomopsis cane and leaf spot (PCLS) disease, affecting grapevines (Vitis vinifera and Vitis spp.), has been historically associated with Diaporthe ampelina. Typical disease symptoms, comprising bleaching and black pycnidia, have also been associated with other Diaporthe spp. In this study, we conducted a molecular identification of the Diaporthe isolates isolated from grapevine canes from different geographic areas of southern Europe showing PCLS symptoms. Then, we investigated their morphological characteristics (including mycelium growth and production of pycnidia and alpha and beta conidia) in response to temperature. Finally, we artificially inoculated grapevine shoots and leaves with a subset of these isolates. Based on our results, PCLS etiology should be reconsidered. Though D. ampelina was the most crucial causal agent of PCLS, D. eres and D. foeniculina were also pathogenic when inoculated on green shoots and leaves of grapevines. However, D. rudis was not pathogenic. Compared to D. ampelina, D. eres and D. foeniculina produced both pycnidia and alpha conidia at lower temperatures. Thus, the range of environmental conditions favorable for PCLS development needs to be widened. Our findings warrant further validation by future studies aimed at ascertaining whether the differences in temperature requirements among species are also valid for conidia-mediated infection since it could have substantial practical implications in PCLS management.

Destructive Xylella fastidiosaXf

The use of molecular tools to identify insect pests is a critical issue, especially when rapid and reliable tests are required. We proposed a protocol based on qPCR with SYBR Green technology to identify Philaenus italosignus (Hemiptera, Aphrophoridae). The species is one of the three spittlebugs able to transmit Xylella fastidiosa subsp. pauca ST53 in Italy, together with Philaenus spumarius and Neophilaenus campestris. Although less common than the other two species, its identification is key to verifying which role it can play when locally abundant. The proposed assay shows analytical specificity being inclusive with different populations of the target species and exclusive with non-target taxa, either taxonomically related or not. Moreover, it shows analytical sensibility, repeatability, and reproducibility, resulting in an excellent candidate for an official diagnostic method. The molecular test can discriminate P. italosignus from all non-target species, including the congeneric P. spumarius.

Quantitative PCR protocols for phytoplasma detection were used to monitor grapevine yellows (GY) in 373 vineyards located in nine Tuscan districts. Among more than 70,000 plants visually monitored, 1.867 plants were sampled and “flavescence dorée” phytoplasmas (FD) were detected in 122 plants and mainly identified as trains belonging to 16SrV-C subgroup. The “bois noir” (BN) phytoplasma was found in 734 samples, with prevalence of tufB type-b strains. The 2013–2015 monitoring program was strongly influenced by the first survey (2012) in which FD was found consistently in the North West (15 samples), whereas only a few cases were observed in the East territory (2 samples). Both areas were thoroughly monitored in the following years: few foci were found in the East (2 in 2014, 1 in 2015), while several infected areas were found in the North West (6, 10 and 22 foci in 2013, 2014 and 2015, respectively). Definitely, the novel FD foci detected in the survey (17, 6, 12 and 23 in each year of survey) and the widespread of BN, suggest a dangerous distribution of GY in Tuscany.

Xylella fastidiosa causing disease on different plant species has been reported in several European countries, since 2013. Based on multilocus sequence typing (MLST) results, there is evidence of repeated introductions of the pathogen in Spain and France. In contrast, in the Salento area of Apulia (Puglia) in Southern Italy, the existence of a unique Apulian MLST genotype of X. fastidiosa, causing the olive quick decline syndrome (OQDS; also referred to as “CoDiRO” or “ST53”) was proven, and this was tentatively ascribed to X. fastidiosa subsp. pauca. In order to acquire information on intra population diversity European Food Safety Authority (EFSA) has strongly called for the characterization of X. fastidiosa isolates from Apulia to produce the necessary data to better understand strain diversity and evolution. In this work, for the first time the existence of sub-variants within a set of 14 “ST53” isolates of X. fastidiosa collected from different locations was searched using DNA typing methods targeting the whole pathogen genome. Invariably, VNTR, RAPD and rep-PCR (ERIC and BOX motifs) analyses indicated that all tested isolates possessed the same genomic fingerprint, supporting the existence of predominant epidemiological strain in Apulia. To further explore the degree of clonality within this population, two isolates from two different Salento areas (Taviano and Ugento) were completely sequenced using PacBio SMRT technology. The whole genome map and sequence comparisons revealed that both isolates are nearly identical, showing less than 0.001% nucleotide diversity. However, the complete and circularized Salento-1 and Salento-2 genome sequences were different, in genome and plasmid size, from the reference strain 9a5c of X. fastidiosa subsp. pauca (from citrus), and showed a PCR-proved large genome inversion of about 1.7 Mb. Genome-wide indices ANIm and dDDH indicated that the three isolates of X. fastidiosa from Salento (Apulia, Italy), namely Salento-1, Salento-2, and De Donno, whose complete genome sequence has been recently released, share a very recent common ancestor. This highlights the importance of continuous and extensive monitoring of molecular variation of this invasive pathogen to understand evolution of adaptive traits, and the necessity for adoption of all possible measures to reduce the risk of new introductions that may augment pathogen diversity.

DNA-based approaches were used to characterize a strain (Salento-1) of Xylella fastidiosa obtained from an olive plant suffering from the syndrome of quick decline in Apulia (South Italy). Salento-1 was indistinguishable from strain CoDiRO previously isolated from olive in Apulia and assigned to X. fastidiosa subsp. pauca. Based on our results and comparative analysis with reported data, the subspecies pauca, multiplex, and fastidiosa may invade olive throughout the world (California, Italy, Argentina and Brazil). The strain Salento-1 has been deposited in the National Collection of Plant Pathogenic Bacteria (NCPPB), England, and in the Belgian Coordinated Collections of Microorganisms (BCCM), Belgium.

Wild herbaceous dicotyledonous plants (dicots) showing symptoms ascribable to phytoplasma disorders were found to be widely distributed in organic vineyards in central Tuscany (Italy) affected by bois noir, a grapevine yellows disease caused by “Candidatus Phytoplasma solani”. In 2010 symptomatic dicots were tentatively identified to species level and the incidence of symptoms estimated in two selected vineyards in the province of Florence. Incidence ranged from 2 to 77%, and was not related to the relative abundance of hosts since very common species as well as relatively rare ones were consistently found to be symptomatic. PCR indexing and 16S rRNA sequence analyses indicated that two phytoplasmas co-existed in the vineyards: “Ca. P. solani”, infecting the root systems of 17 taxa, and a phytoplasma closely related to “Ca. P. phoenicium”, infecting 11 taxa, and occasionally co-infecting the same plant. Regardless of the high frequency of both pathogens in the vineyards, only “Ca. P. solani” could be detected in the grapevines. Population screening by means of tuf sequence analyses revealed the presence of only the tuf-b “Ca. P. solani” type both in dicot hosts and grapevine. This supports current notions of bois noir epidemiology, indicating that some infected dicots act as sources of “Ca. P. solani” inoculum whereas others are dead-end hosts. When the same specimens were screened by sequence analysis of the vmp1 gene, evidence was found that different phytoplasma genotypes may be predominant in grapevines and dicots.

Severe decline of olive trees was observed in the Lecce province, Apulia (Italy), and received the name ‘complesso del disseccamento rapido dell’olivo’ (olive rapid decline complex). Affected plants showed leaf scorch symptoms and dieback of twigs, branches and even of the whole plant. Similar symptoms, unusual for the area, have also been observed in other Apulian localities (Cerignola, Foggia, Canosa di Puglia, and Andria). Three fungal species were associated with the symptoms: Phaeoacremonium aleophilum, Neofusicoccum parvum, and Pleurostomophora richardsiae. The latter is the first report of this fungal species infecting olives. In the Lecce province, the bacterium Xylella fastidiosa also was detected from affected olive trees. Xylella fastidiosa is a quarantine agent in Europe that had been previously reported in the Mediterranean region, but did not spread probably because of the lack of a vector. Present findings suggest that this fundamental condition has now been met.

Four vineyards in Tuscany [GTFI at Gambassi Terme, cv. Sangiovese; CBSI-1, 2, 3 at Castelnuovo Berardenga, cv. Sangiovese (1), cv. Trebbiano (2) and various cultivars (3)] were examined for esca over periods of 4 (CBSI-1, 2, 3) or 6 years (GTFI). A high level of discontinuity in the symptoms expression of each diseased plant was observed from year to year. The cumulated disease incidence, calculated by counting all plants exhibiting symptoms at least once during the entire 4 to 6 year test period, was 49.09% at Gambassi Terme and an average of 13% for the 3 vineyards at Castelnuovo Berardenga. Analysis of the field data by three indices of dispersion (Lloyd’s index of patchiness, variance-to-mean ratio and Morisita’s index) and ordinary runs tests showed occasional aggregation of diseased vines only in vineyards with higher disease incidence (GTFI and CBSI-3). The results of two-dimensional distance class and correlation analyses (2DCLASS and 2DCORR) indicated a significant spatial correlation of infected- infected plant pairs in the GTFI vineyard both along and across columns. For CBSI-3 (19 columns, one cultivar for column), however, the results indicated a tendency for infected vines to be aggregated along columns. The remaining 2 vineyards (CBSI-1 and 2) consistently exhibited a random spatial pattern of diseased vines. This result suggests that the down-column contagion found for CBSI-3 may merely be a byproduct of cultivar dependent susceptibility to disease. On the whole, the results obtained suggest that in the vineyards examined esca was spread by airborne spores from distant and/or internal sources rather then by contaminated pruning tools along the vine columns.

One vineyard at Castelnuovo Berardenga in the province of Siena (Italy) was surveyed from 1995 to 1999 to determine the incidence of esca. The vineyard had been established in 1982 using various cultivars (17) and rootstocks (6). In July and September of each year 19 columns, comprising 44% of the total number of vines, were inspected for esca, and symptom severity of diseased vines was recorded. At the end of the survey period, maps showing the cumulative incidence of esca were drawn and the incidence for each cultivar and for each cultivar/ rootstock combination was calculated. Data expressed on a binomial scale were analysed with the c2 goodness of fit test. Esca incidence differed significantly among cultivars (P

An analysis of symptom expression in esca infected grapevines was performed by focusing on the dynamics of each plant. A parametric statistical model was proposed to evaluate the probability that a plant would show esca symptoms at given values for a relevant set of factors (year, presence of symptoms in the previous year, presence of plants with symptoms in the close neighborhood). The statistical tests of the hypotheses revealed that the considered factors explained a large amount of the observed variability. In particular, the state of plants in the close vicinity is one of those factors. Thus we found evidence that there was an association between plant vicinity and esca symptoms. Future developments of our model will include the factors field column and weather.

The incidence of esca in 5 vineyards (SCFI, GTFI, CBSI-1, 2, 3) in Tuscany was monitored from 1992 to 1994 in SCFI, from 1993 to 1998 in GTFI, and from 1995 to 1998 in CBSI-1, 2 and 3. Disease incidence varied from vineyard to vineyard and from year to year. SCFI was the vineyard most affected with an incidence of 63% in 1993, 20% in 1994, and 46% in 1992. However, symptom expression in individual plants was characterized by very marked discontinuity from year to year. As a result, only 8% of diseased plants in SCFI had foliar symptoms in all 3 years of the survey period. The percentage of consistently diseased plants went down to less than 1% in GTFI and CBSI-3 and was nil in CBSI-1 and 2. The cumulative disease incidence, calculated by counting all the plants that showed foliar symptoms at least once in the years surveyed, was 82, 50, 19, 12, and 10% in SCFI, GTFI, CBSI-3, 1 and 2 respectively. An examination of rainfall and air temperature parameters in relation to esca incidence did not detect any weather conditions especially conducive to esca. However, it seemed that a fresh, rainy summer is more favorable to the chronic form of esca while a hot, dry summer is more conducive to the acute form.

Polygalacturonase and polymethylgalacturonase activity was detected in 13 strains of Phaeomoniella chlamydospora as well as in filtrates of isolates of some related fungi (Phaeoacremonium aleophilum, P. inflatipes, P. rubrigenum). Tests consisted of growth and colorimetric assays on media designed to evaluate different pectic enzymes. Phaeomoniella isolates on the same medium did not differ greatly in their morphological and cultural characters, but pectinolytic activity differed among isolates.

Young esca was monitored on 3 to 7-year-old grapevines in two vineyards in the Riverland region of Australia over two seasons (1999–’00 and 2000–’01). The affected cultivars were own-rooted Shiraz, own-rooted Cabernet Sauvignon and Merlot grafted onto Kober 5BB. Some of the Cabernet Sauvignon vines began to show symptoms as young as two-years-old. Phaeomoniella chlamydospora was the only esca-associated fungus consistently isolated from symptomatic vines, suggesting that P. chlamydospora alone is responsible for esca symptoms. Spatial analysis was unable to reveal any consistent pattern of symptom distribution.

Abstract Pseudomonas savastanoi pv. savastanoi, the causal agent of olive knot disease, has for a long time been included in subgroup 1b of phytopathogenic, fluorescent Pseudomonas species by the LOPAT determinative tests (production of levan, oxidase, pectinolytic and arginine dihydrolase activity, tobacco hypersensitivity). Pseudomonas savastanoi pv. savastanoi differs from the Pseudomonads in subgroup 1a only in being levan-negative. However, in 1990, during a survey on the spread of olive knot in Tuscany, levan-positive isolates of P. savastanoi pv. savastanoi were isolated from knots on two olive trees in an orchard in the province of Florence (Bagno a Ripoli). Some years later, to assess the further spread of levan production in populations of P. savastanoi pv. savastanoi, the survey was extended to 39 other orchards randomly scattered across Tuscany, and levan-positive bacteria were found in approximately 38% of these orchards. Phenotypic, genotypic and pathogenic characterisation allowed these levan-positive isolates to be assigned to P. savastanoi pv. savastanoi. The data suggest that in Tuscan olive orchards, levan-positive and levan-negative subpopulations of this phytopathogenic bacterium can coexist on the same plant. On the basis of the results obtained we suggest that subgroups 1a and 1b of the LOPAT determinative scheme should be combined, and that P. savastanoi should be considered a bacterial species that can be either levan-negative or levan-positive. [ABSTRACT FROM AUTHOR]