Search

In June 2010, 1-year-old potted plants of cherry laurel (Prunus laurocerasus L.) cv. Novita showing leaf spot symptoms were collected in a commercial nursery in the district of Pistoia (Tuscany, central Italy). Red-purple necrotic lesions (measuring a few millimeters up to 1 cm) surrounded by a brilliant light green halo were observed on the abaxial surface of symptomatic leaves. With age, the necrotic areas drop out, leaving a “shot-hole” appearance. Microscopic observation revealed the absence of fungal structures, whereas bacteria were isolated from symptomatic tissue on nutrient sucrose agar medium. Purified single colonies appeared mucoid, convex, and yellow on yeast extract-dextrose-CaCO3 agar (YDCA) medium, were positive to the KOH test, and induced hypersensitive responses on tobacco (cv. Virginia Bright). Three isolates were selected arbitrarily for further analysis. A fragment of approximately 500 bp of the 16S rRNA gene was amplified via PCR with the universal primer pair 27f/519r and sequenced. Subsequent database searches in the INSD (GenBank, EMBL, and DDBJ) indicated that the resulting sequences had 100% identity over 490 bp with the corresponding gene of a Xanthomonas sp. The isolates were further identified as Xanthomonas arboricola pv. pruni on the basis of quinate metabolism and starch hydrolysis tests and by sequencing the PCR products obtained with the gyrB (4) and X. arboricola pv. pruni-specific (3) primer sets. Pathogenicity tests were conducted on cvs. Novita and Caucasica following the detached leaf bioassay procedure (1) and by injecting with a hypodermic needle a bacterial suspension (1 × 107 CFU/ml) in the leaf mesophyll of 1-year-old potted plants (three plants per cultivar and three leaves per isolate on each plant). Incubation was carried out at 25°C under fluorescent lights with a 16-h photoperiod. After seven (detached leaves) and four (potted plants) days, all leaves inoculated with X. arboricola pv. pruni isolates showed brown necrotic spots delimited by a chlorotic margin. Reisolated bacteria on YDCA showed the same colony morphology as described above and tested positive to the X. arboricola pv. pruni-specific primer set, confirming the causal agent of the disease. Leaf tissue inoculated with sterile distilled water remained symptomless. Bacterial leaf spot on cherry laurel was reported in Lombardy (northern Italy) by the local plant protection service in 2005 but without a confirmatory diagnosis of the causal agent (2). To our knowledge, this is the first confirmed report on the occurrence of X. arboricola pv. pruni on cherry laurel in Italy. The pathogen could have a significant impact on the commercial cherry laurel production in the district of Pistoia, which is the most important area for ornamental plants nurseries (4,536 ha of cultivated surface in 2005) in Italy. X. arboricola pv. pruni is included in the EPPO A2 list of pests recommended for regulation to the member countries. References: (1) Anonymous. EPPO Bull. 36:129, 2006. (2) EPPO Reporting Service. Online publication. Retrieved from archives.eppo.org/EPPOReporting/2006/Rse-0606.pdf , 2006. (3) M. C. Pagani. Ph.D. diss. North Carolina State University. Online publication. http://repository.lib.ncsu.edu/ir/bitstream/1840.16/4540/1/etd.pdf , 2004. (4) N. Parkinson et al. Int. J. Syst. Evol. Microbiol. 59:264, 2009.

Phytotoxic metabolites produced in liquid culture by six species of Lasiodiplodia isolated in Brazil and causing Botryosphaeria dieback of grapevine were chemically identified. As ascertained by LC/MS, L. brasiliense, L. crassispora, L. jatrophicola, and L. pseudotheobromae produced jasmonic acid, and L. brasiliense synthesized, besides jasmonic acid, also (3R,4S)-4-hydroxymellein. L. euphorbicola and L. hormozganensis produced some low molecular weight lipophilic toxins. Specifically, L. euphorbicola produced (-)-mellein, (3R,4R)-(-)- and (3R,4S)-(-)-4-hydroxymellein, and tyrosol, and L. hormozganensis synthesized tyrosol and p-hydroxybenzoic acid. This is the first report on the production of the above cited metabolites from L. euphorbicola and L. hormozganensis. The phytotoxic activity of the metabolites produced is also discussed and related to the symptoms these pathogens cause in the grapevine host plants.

Phaeomoniella chlamydospora (Pc) and Phaeoacremonium minimum (Pm) are among the main grapevine trunk disease pathogens affecting vines in the nursery. These pathogens can cause infections of the propagation material often appearing as dark wood streaking, and are associated with decline occurrence in young vineyards. The infection may occur during the grafting process, particularly during hydration. Promising preliminary results were obtained in the nursery using electrolyzed acid water (EAW) for the hydration of cuttings; EAW is characterized by low values of pH (2.5), high oxidation reduction potential (ORP>1000) and a certain amount of free chlorine. No differences in absorption kinetics were recorded between EAW and tap water, nor among 1103P, K5BB and SO4 rootstocks. In all combinations no significant additional absorption was observed after 7 h of immersion. In order to assess the characteristics of EAW after the contact with the inner plant tissues ORP and pH were measured from the liquid extracted from rootstocks after immersion in EAW. The values of the liquid, named “weakened” EAW (wEAW) (625–650 mV ORP and 3.1–3.3 pH), were significantly different from the values of both EAW and the liquid extracted after immersion in tap water. When the propagation material was hydrated for 7 h in EAW no significant effect on vegetating growth performance and percentage of certifiable plants was observed for SO4 or 1103P, whereas a reduction in plant vegetative growth was observed in K5BB hydrated vines at 12hr hydration. EAW and wEAW gave no significant reduction of in vitro mycelial growth, whereas a consistent decrease in conidial germination was observed in Pc and Pm. The three-year experiments carried out in the nursery on cuttings inoculated with Pc and hydrated in EAW showed a remarkable reduction of the infection level in the treated plants.

The ability to produce indole-3-acetic acid (IAA) through the indole-3-acetamide (IAM) pathway as well as cytokinins is a common trait of Pseudomonas savastanoi populations causing disease on oleander and olive. These phytohormones are required for the induction and development of an outgrowth of plant cell tissue termed a knot. However, in myrtle orchards of Sardinia (Italy), strains of P. savastanoi unable to produce cytokinins were found coexisting with cytokinin-producing strains. Data presented here show that the ability to produce IAA through the IAM pathway is also a variable trait within this population, raising questions on the exact role of these plant growth substances in the disease process on myrtle. Three P. savastanoi strains were selected based on their differential ability to produce phytohormones in vitro, and their interaction with the host was investigated over a period of 8 months using histological methods. All strains successfully invaded the infected twigs, moving systemically (unhalted by host defences) upward and downward from the inoculation point, both by completely degrading the cell walls and by taking advantage of the xylem vessels and intercellular spaces. Moreover, all strains induced the development of cankers, which slowly evolved into typical knots only on the twigs inoculated with the phytohormone-producing strains. This study further demonstrates that cytokinins and IAA are essential for knot development; moreover, it ascertains that bacterial production of cytokinins is not necessary for host colonization and for the expression of pathogenicity (i.e. the ability to cause disease) of P. savastanoi on myrtle.

Species of Diaporthe are considered important plant pathogens, saprobes, and endophytes on a wide range of plant hosts. Several species are well-known on grapevines, either as agents of pre- or post-harvest infections, including Phomopsis cane and leaf spot, cane bleaching, swelling arm and trunk cankers. In this study we explore the occurrence, diversity and pathogenicity of Diaporthe spp. associated with Vitis vinifera in major grape production areas of Europe and Israel, focusing on nurseries and vineyards. Surveys were conducted in Croatia, Czech Republic, France, Hungary, Israel, Italy, Spain and the UK. A total of 175 Diaporthe strains were isolated from asymptomatic and symptomatic shoots, branches and trunks. A multi-locus phylogeny was established based on five genomic loci (ITS, tef1, cal, his3 and tub2), and the morphological characters of the isolates were determined. Preliminary pathogenicity tests were performed on green grapevine shoots with representative isolates. The most commonly isolated species were D. eres and D. ampelina. Four new Diaporthe species described here as D. bohemiae, D. celeris, D. hispaniae and D. hungariae were found associated with affected vines. Pathogenicity tests revealed D. baccae, D. celeris, D. hispaniae and D. hungariae as pathogens of grapevines. No symptoms were caused by D. bohemiae. This study represents the first report of D. ambigua and D. baccae on grapevines in Europe. The present study improves our understanding of the species associated with several disease symptoms on V. vinifera plants, and provides useful information for effective disease management., This research was supported by the European COST Action FA1303 on Sustainable control of grapevine trunk diseases (ManaGTD). Surveys and fungal collection performed in Hungary were supported by Szechenyi 2020 programme, the European Regional Development Fund, the Hungarian Government (GINOP-2.3.2-15-2016-00061) and EU H2020 project no. 652601. We are grateful to Arien van Iperen (cultures), Mieke Starink-Willemse and Elias Jonk (DNA isolation, amplification, and sequencing) for their technical assistance.

A strain of Fusarium oxysporum was isolated from grapevine showing heavy decline disease in a vineyard of Veneto region in Italy. The fungus showed to produce phytotoxic metabolites when grown in liquid culture. The main metabolite was identified as fusaric acid produced for the first time as a phytotoxin by a strain of F. oxysporom isolated from diseased grapevine plants. Its quantification in the fungus cultures filtrates was accomplished by HPLC. When tested on tobacco by leaf-puncture assay fusaric acid at 0.5 mg/mL induced the formation of extensive necrosis.

A type II restriction–modification system was found in a native plasmid of Pseudomonas savastanoi pv. savastanoi MLLI2. Functional analysis of the methyltransferase showed that the enzyme acts by protecting the DNA sequence CTGCAG from cleavage. Restriction endonuclease expression in recombinant Escherichia coli cells resulted in mutations in the REase sequence or transposition of insertion sequence 1A in the coding sequence, preventing lethal gene expression. Population screening detected homologous RM systems in other P. savastanoi strains and in the Pseudomonas syringae complex. An epidemiological survey carried out by sampling olive and oleander knots in two Italian regions showed an uneven diffusion of carrier strains, whose presence could be related to a selective advantage in maintaining the RM system in particular environments or subpopulations. Moreover, carrier strains can coexist in the same orchards, plants, and knot tissues with non-carriers, revealing unexpected genetic variability on a very small spatial scale. Phylogenetic analysis of the RM system and housekeeping gene sequences in the P. syringae complex demonstrated the ancient acquisition of the RM systems. However, the evolutionary history of the gene complex also showed the involvement of horizontal gene transfer between related strains and recombination events.

In March 2014, in a ‘Trebbiano’ vineyard in the province of Florence (Tuscany, Italy), 15% of the grapevines (Vitis vinifera L.) showed 1-year old canes with bleached areas, sometimes surrounded by dark margins, irregular dark blotches, and several dead buds. Canes were covered by black pycnidia, and occasionally cracks on the cortex were evident. Woody tissues under the bark were necrotic and canes broke easily. Fifteen symptomatic canes were collected from different vines and cane segments of 20 cm length were placed in a moist chamber and incubated at 25°C. After 72 h, a cream-white cirri mass of conidia was observed from pycnidia and it was plated onto potato dextrose agar (PDA) plates. After one week incubation at 22 ± 1°C in the dark, grown fungal colonies showed a white mycelium with overlapping regions of denser mycelium with rosulate margins. From collected pycnidia, length and width of 100 conidia of each type (alpha and beta) were measured, and the 95% confidence interval, mean, and standard deviation were calculated. Alpha conidia were unicellular, hyaline, fusiform, with one or both ends tapered, not guttulated or unibiguttulate (sometimes with more than two guttules), measuring (5.85) 7.71 to 8.21 (13.56) μm × (1.48) 2.12 to 2.20 (2.70) μm (mean 7.96 ± 1.29 μm × 2.16 ± 0.22 μm). Beta conidia were hyaline, aseptate, filiform, from curved to hamate, measuring (26.61) 25.70 to 26.71 (32.86) μm × (1.03) 1.34 to 1.39 (1.64) μm, mean 26.15 ± 2.33 μm × 1.37 ± 0.14 μm. Based on the current revisions of the genus Diaporthe Nitschke (Gomes et al. 2013; Udayanga et al. 2014), a multigene phylogenetic analysis was carried out. Internal transcribed spacer (ITS1-5.8S-ITS2) region and parts of the translation elongation factor 1-α (TEF1) and β-tubulin (TUB) genes of two isolates were sequenced (GenBank Accession No. KT369109 to KT369114). ITS, TEF1, and TUB partial gene alignments were generated by including the corresponding alleles of 22 strains of D. eres along with 27 most closely related Diaporthe species (Gomes et al. 2013; Udayanga et al. 2014) retrieved from the NCBI databases. Maximum likelihood analyses were performed on individual and concatenated data sets using MEGA6 (Tamura et al. 2013). According to the morphological and phylogenetic characters, the two isolates were identified as belonging to the species D. eres Nitschke. A pathogenicity test was carried out on excised, lignified, one-year old canes collected from grapevines cv. Sangiovese, cut into 20-cm-long segments. Canes were surface sterilized with 2% sodium hypochlorite, rinsed, dried, and wounded with a sterile scalpel creating a 1-cm-long incision on an internode. A 4-mm agar disc with mycelium grown on PDA at 25°C for 7 days was placed into each wound and sealed with Parafilm. Sterile PDA plugs were used as a negative control. Ten canes were inoculated with each strain and placed in sterile, glass petri dishes containing wet, sterile paper towels. After one month of incubation at 25°C, necrotic lesions of the wood had developed on the inoculated canes with an average length of 5.42 ± 1.66 cm. Negative control canes remained symptomless. Diaporthe eres strains were reisolated from inoculated canes while no fungal colonies were recovered from the control canes. To the best of our knowledge, this is the first report of D. eres causing cane blight of grapevine in Italy.

In April 2012 the presence of hyperplastic outgrowths on trunks, branches, and twigs of sweet olive plants, Osmanthus fragrans Lour (Fam. Oleaceae), was recorded in two ornamental hedges made up of five and four plants, respectively, in the city center of Montecatini (Pistoia-Italy). All sweet olive plants were seriously affected by the disease with outgrowths appearing either singly or close together, often forming a single mass that could extend up to 20 cm along the stems, occasionally surrounding the entire circumference. The symptoms observed on O. fragrans closely resembled those induced by the bacterium Pseudomonas savastanoi on Olea europea (common olive) and other plant species. Suspecting a bacterial origin of the disorder, young knots were collected from four diseased plants and used for bacterial isolation with standard techniques on nutrient sucrose agar medium (1). After 3 days of incubation at 26°C, non-levan forming colonies about 3 mm in diameter that were circular, convex, smooth, and cream colored with entire margins appeared on the surface of the agar medium. Purified isolates were gram negative, levan negative, oxidase negative, potato rot negative, arginine dihydrolase negative, showed a tobacco hypersensitive reaction, and tested positive to PCR screening for the presence of the iaaM (tryptophan-2-monooxygenase), iaaH (indoleacetamide hydrolase), ptz (isopentenyl transferase) (1) and iaaL (IAA-lysine synthethase) (3) genes. Three isolates were selected arbitrarily and further characterized by sequencing a fragment of the housekeeping genes rpoD (sigma factor 70) and pgi (phosphoglucose isomerase) (2). All sequenced gene fragments, of 620 bp and 552 bp for the rpoD and pgi genes, respectively, were identical to those of P. savastanoi pv. savastanoi strain NCPPB3335. The pathogenicity of the three isolates was verified on three O. fragrans plants and three Olea europea (cv. Frantoio) plants. Per each isolate, three 1-cm wounds were made on the branches of each plant using a sterile scalpel dipped in a bacterial suspension (1 × 108 CFU/ml). P. savastanoi pv. savastanoi PVFi-t2b isolated from olive was also inoculated as reference strain. After 30 days, all isolates including the reference strain induced typical knots on both plant species while no symptoms were observed on wounds inoculated with sterile water. Bacteria were reisolated from induced knots and Koch's postulates were confirmed. On the basis of biochemical tests, PCR screening, pathogenicity testing, and sequence analyses, the causal agent of knot disease on O. fragrans was identified as P. savastanoi. The potential susceptibility of O. aquifolium Sieb. to the causal agent of olive knot disease has been demonstrated in the past by means of artificial inoculations but interestingly, in the same trials, O. fragrans had tested negative (4). To the best of our knowledge, this is the world's first report of O. fragrans as natural host of P. savastanoi, which extends the growing list of cultivated and ornamental plant species affected by this phytopathogenic bacterium. References: (1) G. Marchi et al. Eur J. Plant Pathol. 112:101, 2005. (2) N. Parkinson et al. Plant Pathol. 60:338, 2011. (3) R. Penyalver et al. Appl. Environ. Microbiol. 66:2673, 2000. (4) C. O. Smith. Phytopathology 12:271, 1922.

This paper reports the isolation and chemical and biological characterization of exopolysaccharides produced by phytopathogenic fungi belonging to different genera and inducing various diseases on grapevine. Their role in the phytopathogenic processes is also discussed.