DAVIDE GIOVANARDI - personale UniMoRe

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DAVIDE GIOVANARDI

Ricercatore t.d. art. 24 c. 3 lett. B
Dipartimento di Scienze della Vita sede ex-Agraria

Pubblicazioni

2024 - Exploiting the microbiome associated with normal and abnormal sprouting rice (Oryza sativa L.) seed phenotypes through a metabarcoding approach [Articolo su rivista]
Dongmo Nanfack, Albert; Nguefack, Julienne; Musonerimana, Samson; La China, Salvatore; Giovanardi, Davide; Stefani, Emilio
abstract

Rice germination and seedlings’ growth are crucial stages that influence crop establishment and productivity. These performances depend on several factors, including the abundance and diversity of seed microbial endophytes. Two popular rainfed rice varieties cultivated in Cameroon, NERICA 3 and NERICA 8, were used for investigating the seed-associated microbiome using the Illumina-based 16 S rRNA gene. Significant differences were observed in terms of richness index between normal and abnormal seedlings developed from sprouting seeds, although no significant species evenness index was assessed within either phenotype. Two hundred ninety-two bacterial amplicon sequence variants were identified in seed microbiome of the rice varieties, and principal coordinate analysis revealed that microbial communities formed two distinct clusters in normal and abnormal seedling phenotypes. Overall, 38 bacteria genera were identified, belonging to 6 main phyla. Furthermore, the core microbiome was defined, and the differential abundance of 28 bacteria genera was assessed. Based on the collected results, putative bacterial genera were directly correlated with the development of normal seedlings. For most genera that are recognised to include beneficial species, such as Brevundimonas, Sphingomonas, Exiguobacterium, Luteibacter, Microbacterium and Streptomyces, a significant increase of their relative abundance was found in normal seedlings. Additionally, in abnormal seedlings, we also observed an increased abundance of the genera Kosakonia and Paenibacillus, which might have controversial aspects (beneficial or pathogenic), together with the presence of some genera (Clostridium sensu stricto) that are commonly correlated to sick plants. The putative functional gene annotation revealed the higher abundance of genes related to the metabolic biosynthesis of soluble carbohydrates and starch, tryptophan, nucleotides and ABC transporters in normal seedlings. Data presented in this study may help in further understanding the importance of the seed endophyte microbiome for driving a correct development of rice plants at the early stages and to identify possible beneficial bacteria for technological applications aimed to increase seed quality and crop productivity.

2024 - Tomato and Pepper Seeds as Pathways for the Dissemination of Phytopathogenic Bacteria: A Constant Challenge for the Seed Industry and the Sustainability of Crop Production [Articolo su rivista]
Xhemali, Bekri; Giovanardi, Davide; Biondi, Enrico; Stefani, Emilio
abstract

The seed industry plays a crucial role in global food production but it faces a persistent challenge in ensuring the health and quality of seeds, particularly those of tomato and pepper seeds, which represent key seed commodities on the global market. Seeds can serve as potential pathways for the introduction and dissemination of seed-borne bacteria, which may have devastating effects on crop yield, farmers’ remunerability, and food security. Therefore, fungicides and other antimicrobial compounds are extensively used to disinfect the seeds, thus increasing the input of chemicals in the agri-environment. In this review, we address aspects that connect disease epidemiology with seed infection and health, including seed contamination, endophytic colonization, and seedborne infections. We focused on the main bacterial diseases affecting tomato and pepper seeds by discussing their official seed testing methods as requirements supporting a smooth seed trade. Moreover, we present a survey on the past and recent innovations for seed treatments, focusing on sustainable disinfection methods. Therefore, this review will be a short but indispensable guide for seed technologists and pathologists involved in the production of high-quality seeds, providing indications and suggestions to contrast seed-borne pathogen dissemination and avoid international controversies and complaints by phytosanitary authorities, extension services, and farmers.

2023 - A viability qPCR protocol to assess the efficacy of a heat treatment to sanitize carrot seeds from Candidatus Liberibacter solanacearum [Articolo su rivista]
Ben Othmen, Soukaina; Conti Nibali, Gianmarco; Cassanelli, Stefano; Giovanardi, Davide; Stefani, Emilio
abstract

Seeds may harbour plant pathogens and, therefore, may be regarded as a possible pathway for their dissemination along trade routes worldwide. Heat treatment appears to be a proper seed sanitation method to comply with the requirement of phytosanitary measures. Carrot seeds have been supposed as a pathway for the dissemination of Candidatus Liberibacter solanacearum, although vertical pathogen transmission is still under debate: nonetheless, such seeds may be conveniently heat treated as a sanitation method. We used a dry thermal treatment (50 ± 1 °C for 72 h) to sanitize seeds from Ca. Liberibacter solanacearum and, since this bacterium is not cultivable, we additionally implemented and evaluated a viability qPCR protocol able to detect only viable cells, therefore confirming seed sanitation. The population of the pathogen was estimated through a plasmid calibration curve. The infection of Ca. Liberibacter solanacearum was quantified in two positive samples at approx. 5 × 106 cells gram −1 of seed. Seed sample tested with monoazides (EMA and PMA) qPCR clearly indicated that seed-associated bacteria lost their viability during the heat treatment, thus confirming seed sanitation. Finally, such heat treatment did not affect seed viability and seed quality: conversely, a remarkable reduction of seed saprophytes was observed, together with a better vigour germination performance.

2023 - Anthracnose of pepper caused by Colletotrichum scovillei: first report in Europe [Abstract in Atti di Convegno]
Cortiello, M.; Xhemali, B.; Modica, F.; Stefani, E.; Giovanardi, D.
abstract

2023 - FIRST FINDING IN EUROPE OF COLLETOTRICHUM SCOVILLEI, A NEW AGENT OF PEPPER ANTHRACNOSE AND ASSESSMENT OF POTENTIAL BACTERIAL BIOCONTROL AGENTS [Abstract in Atti di Convegno]
Xhemali, Bekri; Cortiello, Marina; Gjinovci, Gazmend; Bresilla, Betim; Modica, Francesco; Stefani, Emilio; Giovanardi, Davide
abstract

Pepper (Capsicum annuum L.) is one of the most important vegetables grown in Kosovo. In September 2022, 40% of pepper fruits, in two commercial fields located in the municipalities of Peja and Rahovec (Kosovo), showed typical symptoms of anthracnose. Dark lesions developed on fruits, which later appeared sunken, necrotic and surrounded by brown haloes. After isolation onto PDA, colonies and single spores morphology of both isolates revealed to be consistent with the description of Colletotrichum sp. Amplification and phylotyping of ITS, GAPDH and TUB2 gene sequences of both isolates were performed and the fungus was identified as C. scovillei. To confirm Koch’s postulates, a conidial suspension from both isolates was inoculated into healthy pepper fruits and incubated in a humid chamber. Ten days after pathogen inoculation, typical anthracnose symptoms developed, from which the fungus was successfully reisolated. No symptom was ever observed on fruits inoculated with sterile water. To our best knowledge, this is the first report of anthracnose caused by C. scovillei in Europe. Given the economic importance of the crop and the significant quality and yield losses observed, measures are urgently needed aiming at pathogen eradication or, alternatively, to ensure an efficient disease control to avoid severe damage both in the field and in postharvest. Four bacterial isolates from the UNIMORE collection were tested in vitro against C. scovillei: preliminary data showed their effectiveness in reducing mycelium growth. Studies are ongoing to test such antagonists in planta to search for sustainable alternatives to the extensive use of fungicides.

2023 - First report of Colletotrichum scovillei causing anthracnose of pepper in Kosovo [Articolo su rivista]
Xhemali, B.; Cortiello, M.; Gjinovci, G.; Bresilla, B.; Stefani, E.; Giovanardi, D.
abstract

Colletotrichum scovillei is one of several species in the C. acutatum complex causing anthracnose on pepper, currently present in eastern Asia, Brazil and South Carolina (USA). A severe anthracnose infection on pepper fruits was identified in two commercial fields in Kosovo. Infection assays, morphological analyses and sequencing identified the causal agent as C. scovillei. It could be considered an emerging disease as the severe crop losses, together with the spread of the pathogen/disease to areas and regions where it was never reported in previous times. This is the first report of C. scovillei infecting pepper in Kosovo and Europe.

2023 - First report of Stemphylium leaf blight of onion (Allium cepa) caused by Stemphylium vesicarium in Italy [Articolo su rivista]
Cortiello, M.; Prodi, A.; Stefani, E.; Giovanardi, D.
abstract

In July 2019, severe leaf symptoms were observed on onion plants (Allium cepa L. cv. Dorata di Parma) in a commercial field located in the municipality of Medicina (Bologna, Emilia-Romagna region), in northern Italy. Diseased leaves showed yellowish-pale-brown and oval-shaped lesions, which later coalesced in larger necrotic areas, and black leaf tips. As the disease progressed, conidia developed on the necrotizing leaves, until premature desiccation of the whole plants. Disease incidence of approximately 70% was calculated in the affected field, together with yield losses that were estimated to be above 30%. Symptomatic tissue fragments excised from the leaf lesions were surface disinfested with NaOCl 1% for 2 minutes, rinsed with sterile water and transferred onto potato dextrose agar (PDA). Fungi were consistently isolated after 5 days of incubation at 27 ± 1°C in the dark. Single spore isolation was performed on PDA to obtain 7 pure cultures, whose morphological characteristics were consistent with the description of Stemphylium vesicarium (Ellis 1971). DNA from a representative single spore isolate was extracted and the internal transcribed spacer region (ITS) of ribosomal DNA (rDNA) was amplified using the universal primers P-ITS1 and P-ITS4 (White et al. 1990). The PCR product was sequenced and deposited in GenBank (Accession No. OP144057). A BLAST search in CBS-KNAW collection bank (Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands) showed 100% identity for the ITS gene with the strain of S. vesicarium under accession number CBS 124749. Moreover, the PCR assay using the primer pair KES 1999 and KES 2000 (Graf et al. 2016) for the cytochrome b gene displayed the specific fragments of 420 bp for S. vesicarium. The isolate was tested for pathogenicity on onion (potted plants cv. Texas Early Gran, fourth leaf stage) by spraying 4 ml of a conidial suspension (1 × 104 conidia/ ml) per plant. Inoculated and non-inoculated (sprayed with sterile distilled water) plants were kept at 24 ± 1°C and 90% relative humidity with a 16-h photoperiod. Seven days after inoculation, disease assessment was performed. Inoculated plants showed typical Stemphylium leaf blight (SLB) symptoms, similar to those observed in the field. No symptoms developed on the water-inoculated plants. S. vesicarium was consistently reisolated from the artificially inoculated onion plants and identified using a PCR assay, according to Graf et al. (2016). The assay was repeated twice with the same results. SLB is currently reported worldwide and it is considered a re-emerging threat and a truly challenging fungal disease, which can result in yield and quality losses of up to 90% in onion crops (Hay et al. 2021). In Italy, S. vesicarium has been reported several years ago on pear (Ponti et al. 1982) and, more recently, on radish sprouts (Belisario et al. 2008), chili pepper (Vitale et al. 2017) and spinach (Gilardi et al. 2022). To our knowledge, this is the first report of S.vesicarium on onion in Italy. Our results stress that development and implementation of innovative Integrated Pest Management (IPM) strategies are urgently needed to ensure an effective control of SLB, since only a few moderately resistant onion varieties are available (Hay et al. 2021) and no fungicides are currently registered to specifically control SLB in Italy. Further studies are underway to elucidate the pathogen geographic distribution and assess the impact of this disease on the onion crop in Italy.

2023 - Ozone treatments: environmentally friendly and effective techniques for sanitation of seedborne pathogens on maize and corn salad [Abstract in Atti di Convegno]
Cafasso, M.; Cortiello, M.; Modica, F.; Messi, P.; Stefani, E.; Giovanardi, D.
abstract

2023 - Reduction of copper inputs in the management of key diseases of grapevine, olive and tomato by an innovative Zeo-biopesticide [Abstract in Atti di Convegno]
Modica, F.; Fagioli, L.; Cortiello, M.; Giovanardi, D.; Reyes, F.; Stefani, E.
abstract

2023 - The seed-associated microbiome: relationship with seedling development in two rainfed rice cultivars from Cameroon [Abstract in Atti di Convegno]
Nanfack, A. D.; Musonerimana, S.; La China, S.; Giovanardi, D.; Stefani, E.
abstract

2022 - "Microrganismi per la lotta biologica alle malattie delle piante" [Brevetto]
CONTI NIBALI, Gianmarco; Stefani, Emilio; Giovanardi, Davide
abstract

2022 - Identification, evaluation and selection of a bacterial endophyte able to colonize tomato plants, enhance their growth and control Xanthomonas vesicatoria, the causal agent of the spot disease [Articolo su rivista]
Vurukonda, S. S. K. P.; Giovanardi, D.; Stefani, E.
abstract

Beneficial endophytes are key factors in plant productivity and disease control; therefore, research is ongoing to characterize the potential of endophytes as microbial inoculants to promote plant growth and act as biocontrol agents. In this study, we searched for and identified a set of endophytes occurring naturally in a few important crops. Beneficial endophytes were shown to antagonize several important fungi and bacteria, in particular those affecting tomato (Solanum lycopersicum), including Rhizoctonia solani, Alternaria alternata, Clavibacter michiganensis pv. michiganensis, Ralstonia solanacearum, and Xanthomonas vesicatoria. We characterized the most promising as potential inoculants of tomato, aiming to foster plant growth and control the bacterial spot disease caused by Xanthomonas vesicatoria. Among the selected endophytes, we identified a novel streptomycetes, taxonomically related to Streptomyces avermitilis and a pseudomonad, identified as Pseudomonas granadensis. The beneficial effects of these two bacteria, used as single inoculants or as a combination of both, were seen in a significant increase in root and shoot length (approximately 31% and 34% for the streptomycetes and 18% and 16% for the pseudomonad) and dry root biomass (90% for the streptomycetes and 70% for the pseudomonad). Additionally, both inoculants reduced disease progression and severity following inoculation with X. vesicatoria. There was no significant difference between plants treated with single inoculants and plants treated with both. The penetration and efficient colonization of tomato tissues by a green fluorescent protein-tagged culture of the streptomycetes was observed by confocal microscopy, confirming its endophytic nature.

2018 - Characterisation of Pseudomonas syringae isolates from apricot orchards in north-eastern Italy. [Articolo su rivista]
Giovanardi, Davide; Ferrante, Patrizia; Scortichini, Marco; Stefani, Emilio
abstract

A set of 31 isolates were obtained from apricot (Prunus armeniaca L.) orchards showing symptoms of blast and gummosis in orchards located in north-eastern Italy, mostly during three subsequent growing seasons (2014–2016). These isolates were identified by means of their colony morphology, pathogenicity (hypersensitive reaction in tobacco leaves and the detection of both syringomycin and coronatine coding genes), genetic features (BLASTn analysis of the rpoD gene) and physiological tests (GATTa) as Pseudomonas syringae pv. syringae and P. syringae pv. morsprunorum race 1 and race 2 strains, respectively. Phylogenetic analysis based on the rpoD gene sequence showed that the two pathovars and two races are well separated and highlighted high intrapathovar/race variation for both P. syringae pv. syringae and P. syringae pv. morsprunorum race 2. Pseudomonas syringae pv. morsprunorum race 1 formed a unique clade that had high sequence homology. Repetitive element-based polymerase chain reaction fingerprinting analysis using the ERIC, REP and BOX primers again showed both high intra-pathovar variation within the P. syringae pv. syringae isolates, which grouped into six distinct clusters, and notable intra-race genetic diversity within both P. syringae pv. morsprunorum races. All 31 strains showed tolerance to copper at 200 ppm in vitro. Among these isolates, 13 were highly resistant (up to 500 ppm) and revealed the presence of the cusCBA gene cluster. All P. syringae pv. syringae strains showed ice nucleation activity, and nine were markedly active (up −3 °C) and characterised by the presence of the INAz gene. Finally, this study is the first report of the isolation of P. syringae pv. morsprunorum race 2 strains from apricot in Italy.

2018 - Factors influencing the detection of Acidovorax citrulli in naturally contaminated cucurbitaceous seeds by PCR-based assays. [Articolo su rivista]
Giovanardi, Davide; Sutton, Safira A.; Stefani, Emilio; Walcott, Ronald R.
abstract

The success of Acidovorax citrulli detection by seed health testing of commercial cucurbitaceous seeds depends on the efficiency of pathogen extraction. In the present study, different extraction techniques were compared to identify factors that influence A. citrulli detection in naturally contaminated cucurbit seeds. Embryo-infected watermelon seeds, produced by pistil inoculation, were mixed with non-infected seeds to generate samples (n = 1000 seeds) with low infection levels (approximately 104 cells per sample). Additionally, two naturally infested melon seed lots were tested. A. citrulli was extracted from seeds by soaking or crushing, followed by one or two centrifugation steps. Samples extracted by soaking seeds yielded better amplification efficiency (103%) compared with crushing (93%), as determined by quantitative real-time PCR analysis. This was most likely due to a reduction of the concentration of inhibitors present in the DNA samples. PCR assays using three different A. citrulli-specific primer sets highlighted that soaking followed by two centrifugation steps enhanced pathogen detection (100% of the samples) and the mean cycle threshold (Ct) value was significantly lower than those observed for the other pathogen extraction techniques. These results indicate that the optimised extraction protocol combined with PCR analysis can improve routine seed health testing for A. citrulli.

2018 - Impact of bacterial spot outbreaks on the phytosanitary quality of tomato and pepper seeds [Articolo su rivista]
Giovanardi, Davide; Biondi, Enrico; Ignjatov, Maja; Jevtic, Radivoje; Stefani, Emilio
abstract

The impact of disease outbreaks on the phytosanitary quality of seeds was investigated for two pathosystems: tomato– Xanthomonas vesicatoria and pepper–Xanthomonas euvesicatoria. This study, which was performed in Italy and Serbia, aimed to evaluate the season-to-season transmission of phytopathogenic regulated bacteria associated with phytosanitary risks posed by seeds produced in areas where bacterial infections are possible. For each pathosystem, field plots were experimentally inoculated to simulate an initial infection rate of 1%, 5% and 15%. The area under the disease progress curve (AUDPC) was calculated for each field plot, the seeds produced were analysed to determine the contamination level and rate, and the plant-to-seed transmission was evaluated by a seedling grow-out (SGO) assay. To investigate transmission under field conditions, a second-year experiment was performed, wherein seeds collected from the first year were used to establish new field plots. During the first growing season, AUDPC values were positively correlated with the percentages of initial infection for each pathosystem. Seed contamination levels in pepper ranged from 34 to 100 CFU g 1, and the contamination rate ranged from 1.50% up to 3.17% for X. euvesicatoria, whereas processing and fresh market tomato seeds produced both in Italy and Serbia were not infected by X. vesicatoria. During SGO assays and the second cropping year, no symptoms were observed in either tomato or pepper plants. Therefore, the calculated pepper seed contamination rate for X. euvesicatoria appeared to be less than the threshold necessary to initiate a disease outbreak. Finally, all seeds obtained during the second cropping year were uninfected.

2018 - Performance of diagnostic tests for the detection and identification of Pseudomonas syringae pv. actinidiae (Psa) from woody samples [Articolo su rivista]
Loreti, Stefania; Cunty, Amandine; Pucci, Nicoletta; Chabirand, Aude; Stefani, Emilio; Abelleira, Adela; Balestra, Giorgio M.; Cornish, Deirdre A.; Gaffuri, Francesca; Giovanardi, Davide; Gottsberger, Richard A.; Holeva, Maria; Karahan, Aynur; Karafla, Charikleia D.; Mazzaglia, Angelo; Taylor, Robert; Cruz, Leonor; Lopez, Maria M.; Vanneste, Joel L.; Poliakoff, Françoise
abstract

The aim of this study was to characterise the performance of new molecular methods for the detection and identification of Pseudomonas syringae pv. actinidiae (Psa) and to provide validation data in comparison to the assays mentioned in official diagnostic protocols and being currently used. Eleven molecular tests for the Psa detection were compared in an interlaboratory comparison where each laboratory had to analyse the same panel of samples consisting of thirteen Psa-spiked kiwifruit wood extracts. Laboratories had to perform also isolation from the wood extracts. Data from this interlaboratory test performance study (TPS) was statistically analysed to assess the performance of each method. In order to provide complete validation data, both for detection and identification, this TPS was supplemented by a further study of identification from pure culture of phylogenetically closely related Pseudomonas spp., Psa, and bacterial strains associated with kiwifruit. The results of both these studies showed that simplex-PCRs gave good results, whereas duplex-PCR and realtime PCR were the most reliable tools for detection and identification of Psa. Nested and multiplex-PCR gave false-positive results. The use of the most reliable detection test is suggested for routine analyses, but when Psa-free status needs to be accurately assessed, it is recommended that at least two detection tests are used. This work provides a wide comparison of the available diagnostic methods, giving new information for a possible revision of the official diagnostic protocols (e.g. European and Mediterranean Plant Protection Organization (EPPO) protocol PM7/120 for the detection of Psa).

2018 - Plant Growth Promoting and Biocontrol Activity of Streptomyces spp. as Endophytes [Articolo su rivista]
Vurukonda, Sai; Giovanardi, Davide; Stefani, Emilio
abstract

There has been many recent studies on the use of microbial antagonists to control diseases incited by soilborne and airborne plant pathogenic bacteria and fungi, in an attempt to replace existing methods of chemical control and avoid extensive use of fungicides, which often lead to resistance in plant pathogens. In agriculture, plant growth-promoting and biocontrol microorganisms have emerged as safe alternatives to chemical pesticides. Streptomyces spp. and their metabolites may have great potential as excellent agents for controlling various fungal and bacterial phytopathogens. Streptomycetes belong to the rhizosoil microbial communities and are efficient colonizers of plant tissues, from roots to the aerial parts. They are active producers of antibiotics and volatile organic compounds, both in soil and in planta, and this feature is helpful for identifying active antagonists of plant pathogens and can be used in several cropping systems as biocontrol agents. Additionally, their ability to promote plant growth has been demonstrated in a number of crops, thus inspiring the wide application of streptomycetes as biofertilizers to increase plant productivity. The present review highlights Streptomyces spp.-mediated functional traits, such as enhancement of plant growth and biocontrol of phytopathogens.

2017 - BIOCONTROL OF THE BACTERIAL BLIGHT OF WALNUT: IS THERE A CHANCE TO REDUCE COPPER INPUTS INTO WALNUT GROVES? [Abstract in Atti di Convegno]
Giovanardi, Davide; Fagioli, Luca; Gilli, Lorenzo; Stefani, Emilio
abstract

The bacterial blight is a re-emerging disease, severely affecting the productivity of walnut groves. Disease symptoms are observed on all aerial parts of the host plant, but the most damaging are the necrotic spots developing on fruits, leading to massive fruit drops, mainly before endocarp hardening. The causal agent is Xanthomonas arboricola pv. juglandis (Xaj), a gram-negative rod with a worldwide distribution. The population structure of Xaj includes several morphotypes, at least five sequence types, and other features confirming its genomic heterogeneity. Copper resistance is widespread and very effective among Xaj populations: therefore, disease management based on copper sprays is not particularly effective, even in cases of 10-14 treatments during the growing season. The recent, severe outbreaks reported in Italy are due to two concomitant events: i) the rapid increase of walnut acreage, especially in the northern part of the Country and ii) the development of Xaj populations showing high resistance to copper. Since most of the cultivated walnut varieties are either highly susceptible or susceptible to the disease, we tried to implement control strategies based on the use of a single antagonist or the field application of microbial consortia. Additionally, innovative agrochemicals, with a reduced copper content, have also been used. Results showed that: i) microbial biocontrol agents were able to significantly reduce the disease in the field; ii) innovative agrochemicals may reduce the bacterial blight, but might enhance phytotoxicity; iii) a significant reduction of copper inputs is possible, coupled with an effective disease control in walnut groves.

2017 - In vitro characterization of plant growth promoting and biocontrol activity of beneficial microorganisms [Abstract in Atti di Convegno]
Vurukonda, Sai Shiva Krishna Prasad; Giovanardi, Davide; Stefani, Emilio
abstract

Plant roots are associated with numerous and diverse types of beneficial and pathogenic microorganisms. Among them, plant growth–promoting (rhizo)bacteria (PGPB or PGPR) are isolated from plants crops worldwide, and many of them are used as agricultural inoculants. Agricultural biofertilization and biocontrol of pathogens are eco-friendly alternatives to chemical usage and have less energy, environmental, and economic costs. PGPB isolation and evaluation are essentials steps for determining bacteria that could improve plant development and productivity. In the present study three Streptomyces sp. strains SB14, SA51 & SL81, two Pseudomonas sp. strains PT65 & PN53, an Agrobacterium sp. strain AR39 and an internal control (IC) Pseudomonas synxantha were evaluated in vitro for different plant growth promoting and biocontrol activities. The results were aimed to identify possible antagonists able to inhibit different plant bacterial (Xanthomonas vesicatoria, Clavibacter michiganensis subsp. michiganensis, Clavibacter michiganensis subsp. sepedonicus, Acidovorax citrulli and Ralstonia solanacearum) and fungal (Rhizoctonia solani, Sclerotium sp., Fusarium oxysporum, Alternaria solani and Monilia laxa) pathogens. All the strains were screened for biocontrol activity on three different media’s and AIA (average inhibition area) was calculated. Among the isolates, each strain showed different ability to inhibit the pathogens: Streptomyces sp. strain SA51 was found to be most active. The most prospective strains SA51, AR39 and DLS65 were further evaluated in the field, as possible biocontrol agents for the tomato spot disease (X. vesicatoria), singularly and as a consortium. Results will improve our understanding on the use of such microbial biocontrol agents and will implement innovative biocontrol strategies to bacterial diseases.

2017 - Population features of Xanthomonas arboricola pv. pruni from Prunus spp. orchards in northern Italy [Articolo su rivista]
Giovanardi, Davide; Dallai, Davide; Stefani, Emilio
abstract

Bacterial leaf/fruit spot and canker of stone fruits, caused by Xanthomonas arboricola pv. pruni, is a recurrent disease in Italy. A set of 23 strains has been isolated in peach and plum orchards in an intensively stone fruit cultivated area located in north-eastern Italy. They were all identified as X. arboricola pv. pruni by means of phytopathological and serological features: hypersensitive reaction on bean pods, pathogenicity test on immature peach or plum fruitlets, identification by immunofluorescence assay and conventional PCR. Phylogenetic analysis based on sequencing of the gyrB housekeeping gene of the isolates showed that they formed a unique clade, well characterised and separated from other xanthomonads. An insight into the genetic population features was attempted by rep-PCR analysis, using the ERIC, REP and BOX primers. The combined rep-PCR fingerprints showed a slight intra-pathovar variation within our isolates, which grouped in five close clusters. Copper resistance has been assessed in vitro for our whole X. arboricola pv. pruni collection, highlighting that two isolates show a level of resistance in vitro up to 200 ppm of copper. Nonetheless, the copLAB gene cluster, present in many other species of Xanthomonads, was not detected in any isolate, confirming the presence of a still unknown mechanism of copper detoxification in our Xanthomonads arboricola pv. pruni tolerant/resistant strains.

2017 - SYMBIOTIC AGRICULTURE: PLANT GROWTH PROMOTIO AND BIOCONTROL ACTIVITY OF BENEFICIAL MICROORGANISMS. [Abstract in Atti di Convegno]
Vurukonda, Sskp; Giovanardi, Davide; Stefani, Emilio
abstract

Symbiotic microbial inoculation is emerging as a potential technology for sustainable agriculture. Towards a sustainable agricultural vision, crops need to be equipped with disease resistance to various biotic and abiotic stresses, together with better nutritional value. To fulfil the above desired crop properties, one possibility is to use soil microorganisms (bacteria, fungi, algae, etc.) in order to enhance microbial biodiversity. Among these potential soil microorganisms, bacteria known as plant growth promoting rhizobacteria (PGPR) are the most promising. In the present study three Streptomyces sp. strains SB14, SA51 and SL81, two Pseudomonas sp. strains PT65 & PN53 and one Agrobacterium sp. strain AR39 were evaluated in vitro for different plant growth promoting and biocontrol activities. Our aim is to identify possible antagonists able to inhibit different plant bacterial pathogens like Xanthomonas vesicatoria, Clavibacter michiganensis subsp. michiganensis, Clavibacter michiganensis subsp. sepedonicus, Acidovorax citrulli and Ralstonia solanacearum. All the strains were screened for biocontrol activity on three different media ISP – 2 (International streptomyces project), PDA (Potato Dextrose Agar) and HPDA (Half Strength PDA) and AIA (average inhibition area) was calculated. Among the isolates, each strain showed different ability to inhibit the pathogens: Streptomyces sp. strain SA51 was found to be most active. The most prospective strains were further evaluated in the field, as possible biocontrol agents for the tomato spot disease (X. vesicatoria), singularly and as a consortium. Results will improve our understanding on the use of such microbial biocontrol agents and will implement innovative biocontrol strategies to bacterial diseases.

2016 - Actinobacteria: isolation, identification, characterisation and preliminary experiments for their possible use against Clavibacter michiganensis subsp. michiganensis. [Abstract in Atti di Convegno]
Ferrari, Michele; Kaewla, Onuma; Franco, Christopher; Giovanardi, Davide; Stefani, Emilio
abstract

In recent years, new actinobacteria species have been isolated as endophytes from plants and are sought after for the role of bio-control inoculants for sustainable agriculture. In particular, our studies focus on the isolation of some endophytic actinobacteria from tomato healthy plants, with a potential antagonistic activity against the causal agent of bacterial canker of tomato: Clavibacter michiganensis subsp. michiganensis (Cmm). Cmm is a plant-pathogenic bacterium belonging to the order of Actinomycetales. It infects tomato plants, spreads through the xylem and causes bacterial wilt and canker which can be considered to be the most important bacterial disease of tomato causing substantial economic losses worldwide. A total of 50 endophytic actinobacteria strains were isolated from tomato healthy plants collected from Adelaide Province.

2016 - ELICITATION OF RESISTANCE TO FIRE BLIGHT BY A MICROBIAL CONSORTIUM: SEARCH FOR AN EVIDENCE THROUGH A TRANSCRIPTOMIC APPROACH [Abstract in Atti di Convegno]
Giovanardi, Davide; Catalano, Valentina; Verzelloni, Elena; Dondini, Luca; Stefani, Emilio
abstract

Fire blight is the most destructive bacterial disease of pome fruits: it is caused by Erwinia amylovora and its management remains cumbersome. Nowadays, treatments with several beneficial microorganisms can lead to an affective integrated pest management (IPM): nevertheless, their specific activity in enhancing plant defence mechanisms is only partially understood. An extensive 3-years study in a commercial pear orchard was carried out, to verify the efficacy of a commercial microbial consortium (Micosat F®, CCS Aosta srl, Italy) to control a fire blight outbreak. In parallel, we have used a dHPLC (denaturing High Performance Liquid Chromatography) and automated DNA fragment collection using the WAVE System to analyse and recover cDNA-AFLP fragments. This transcriptomic approach was applied to understand which complex transcriptional changes these microorganisms may have elicited in the plant-pathogen interaction. In the commercial orchard, the beneficial effects of microbial consortium were confirmed by a significant disease reduction starting from the first year of application, as compared with other treatments commonly used by orchardists in IPM strategies. Among the eighty-five transcriptderived fragments (TDFs) collected and identified through the cDNA-AFLP-dHPLC approach, fourteen were found involved in systemic acquired resistance (SAR) according to the available literature. The transcriptomic approach developed in this study has been a robust and user-friendly mRNA fingerprinting method for the identification of differentially expressed genes, where prior knowledge of specific gene sequences is not a prerequisite. Finally, we confirmed the activity of such the microbial consortium as resistance inducer.

2016 - Elicitation of resistance to bacterial canker of stone fruits by humic and fulvic acids (glucohumates): a cDNA-AFLP-dHPLC approach [Articolo su rivista]
Giovanardi, Davide; Dallai, Davide; Dondini, Luca; Mantovani, Vilma; Stefani, Emilio
abstract

Bacterial leaf/fruit spot and canker of stone fruits, caused by Xanthomonas arboricola pv. pruni is a challenging disease in Italian orchards. In recent years, novel molecules such as biostimulants or/and resistance-inducers have been developed and used to implement an effective Integrated Pest Management. An extensive study in vitro, in glasshouse and in a peach orchard was carried out to verify the efficacy of commercial glucohumates to control bacterial spot/canker outbreaks and to understand their mode of action. A transcriptomic approach was implemented to study the complex transcriptional changes that these biomolecules may possibly elicit in the plant-pathogen interaction. The cDNA-AFLP analysis of differential gene expression in treated plant tissue was made. Discrimination of differentially expressed sequences was made with denaturing High Performance Liquid Chromatography (dHPLC), and functional annotation of such transcripts was assigned based on similarity search on public genome databases. The results highlighted the activity of glucohumates in controlling Xanthomonas arboricola pv. pruni both in vitro and in vivo. Beneficial effects of humic substances towards this pathogen were confirmed by a significant disease reduction (up to 78%) in the commercial orchard. On the other hand, cDNA-AFLPdHPLC analysis allowed the collection of fourteen up-regulated transcript-derived fragments belonging to peach genes and putatively involved in the defence response. In particular, the activation of these genes within 24 h after treatment supposedly triggered the early-induced resistance, notoriously involved in maintaining a protection state in plants against biotic stresses.

2016 - Isolation of bacterial endophytes from Actinidia chinensis and preliminary studies on their possible use as antagonists against Pseudomonas syringae pv. actinidiae [Articolo su rivista]
Tontou, Rodanthi; Giovanardi, Davide; Ferrari, Michele; Stefani, Emilio
abstract

Abstract:BACKGROUND:The bacterial canker, caused by Pseudomonas syringae pv. actinidiae, is the most severe disease of cultivated Actinidia spp. The pathogen is systemic and not easily controlled by agrochemical means. OBJECTIVE:Our aim was to search, select and identify, among kiwifruit bacterial endophytes, possible antagonists able to control Pseudomonas syringae pv. actinidiae. METHODS:Several kiwifruit production areas were inspected, in order to find host plants without any disease symptom inside severely affected orchards. From those plants, endophytes were isolated, selected, tested for their ability to inhibit the growth of the pathogen and identified. RESULTS:A set of 65 different bacterial endophytes was isolated and tested: several of them were able to inhibit the growth of Pseudomonas syringae pv. actinidiae in vitro. None of the antagonists proved to possess either ice nucleation activity or transferable copper resistance. Taxonomically, antagonists belonged to the families of Pseudomonadaceae and Enterobacteriaceae. CONCLUSIONS:Effective bacterial antagonists were found as endophytes in kiwifruit plants and bearing features of safety of use and negligible risk for the crop. Such finding makes it possible to select the most prospective of them, in order to develop efficient biopesticides able control the bacterial canker in commercial orchards.

2016 - Molecular characterisation of an endophyte showing a strong antagonistic activity against Pseudomonas syringae pv. actinidiae [Articolo su rivista]
Tontou, Rodanthi; Gaggia, Francesca; Baffoni, Loredana; Devescovi, Giulia; Venturi, Vittorio; Giovanardi, Davide; Stefani, Emilio
abstract

Abstract Background and aims Endophytic bacteria have been often studied as biological control agents of plant pathogens and many of their secondary metabolites involved in antagonism are Non Ribosomal Peptides (NRPs). In this study, the molecular basis of the biocontrol properties of the endophyte Pseudomonas synxantha, isolated from Actinidia chinensis, against Pseudomonas syringae pv. actinidiae (Psa), the causal agent of bacterial kiwi canker, has been investigated. Methods Antagonism-deficient mutants of P. synxantha strain DLS65 were generated by insertion of minitransposon mTn5-GNm in its genome. Southern blot analysis allowed the selection of single transposon insertion-mutants. Amplification of the transposon flanking regions by means of arbitrary and single primer PCR in selected mutants was perfomed to obtain amplicons for sequencing purposes. Results Sequencing results of the amplicons obtained from three antagonism-deficient mutants led to the localization of the transposonin three genes, which implies their involvement in the antagonism of P. synxantha: an acyl-homoserine lactone acylase gene (pvdQ), a glucose-6-phosphate dehydrogenase gene (zwf) and an mbtH-like gene were identified. It is known from the literature that these three genes are involved directly or indirectly in NRPs synthesis. Conclusions We suggest that a molecule with antibiotic properties, produced by NRP synthetases, contributes to the antagonistic activity of P. synxantha. Keywords

2016 - Morphological and genotypic features of Xanthomonas arboricola pv. juglandis populations from walnut groves in Romagna region, Italy [Articolo su rivista]
Giovanardi, Davide; Bonneau, Sophie; Gironde, Sophie; Saux, Marion Fischer Le; Manceau, Charles; Stefani, Emilio
abstract

Seventy-seven Xanthomonas arboricola pv. juglandis isolates, originating from a small region (Romagna, Italy) within 4 years, were phenotypically typed, in order to study their population features. Assessment of phenotypes resulted in the identification of three different groups of morphotypes, in the assessment of different virulence on walnut fruitlets, and in the evidence that all isolates were able to grow on Mannitol-glutamate-yeast agar containing 50 ppm of copper sulphate. Moreover, several isolates showed to be highly copper resistant in vitro, up to 500 ppm. Forty-one isolates, selected considering year/origin of plant material and phenotypic features, were molecularly studied by rep-PCR fingerprinting using BOXA1R primer. These strains showed a clear intra-pathovar variation by the presence of eight different haplotypes. Twenty isolates, representative of different BOX profile, were studied by means of variable number of tandem repeats (VNTR) on the locus TR5b. Such analysis highlighted five different sequence types. Eight polymorphic strains on this flanking region isolated in between 2007 and 20 and one isolated in 2010 were subject to multilocus sequence analysis (MLSA) using atpD, dnaK, efP, fyuA, glnA, gyrB, and rpoD housekeeping genes. A diversity level in Italian isolates was highlighted in the same range as in reference strains from a worldwide origin. Finally, the gene cluster copLAB presence was confirmed for all isolates. In this study, the high phenotype and genotype variability inside Xanthomonas arboricola pv. juglandis, was explained by the different origin of the propagation material. Information provided in this study on an Italian Xanthomonas arboricola pv. juglandis collection allows a better understanding of the walnut bacterial blight epidemiology.

2016 - Pero: consorzi microbici contro il colpo di fuoco [Articolo su rivista]
Verzelloni, Elena; Catalano, Valentina; Giovanardi, Davide; Dondini, Luca; Stefani, Emilio
abstract

La lotta al colpo di fuoco delle pomacee è oggi basata sulla scelta di materiale di propagazio- ne certifi cato, su corretti interventi agronomici e sull’uso di prodotti a base rameica. Sul mercato sono disponibili alcuni prodotti a base microbica, classificati come fertilizzanti naturali, contenenti un ampio consorzio micro- bico utili per migliorare la produttività di numerose colture. Le prove condotte in un triennio di sperimentazione in campo e at- traverso uno studio di espressione genica in pero hanno indaga- to le potenzialità che il consorzio microbico offre nella lotta alla grave batteriosi evidenziando un’ottimale capacità di contenere il batterio e una possibile induzione di resistenza.

2015 - Detection and identification of Xanthomonas arboricola pv. pruni from symptomless plant material: results of an Italian test performance study [Articolo su rivista]
Pucci, Nicoletta; Loreti, Stefania; Perez, Grazia; Catara, Vittoria; Scortichini, Marco; Bella, Patrizia; Ferrante, Patrizia; Giovanardi, Davide; Stefani, Emilio
abstract

ABSTRACT. Xanthomonas arboricola pv. pruni, the causal agent of bacterial spot disease of stone fruits, is a regulated quarantine pathogen in the European Union, listed as an A2 pest by the European and Mediterranean Plant Protection Organization (EPPO). Because detection and identification of this pathogen is key for its management and to ensure the production of pest free propagation material, it should be based on reliable tests, in particular when dealing with symptomless material. The current EPPO diagnostic Standard (PM 7/64) does not provide specific molecular methods for detection of this pest. The present paper summarizes the results of a test-performance study (TPS) to validate, at a national level, a detection procedure for this bacterium. A working group was established in order to evaluate the performance criteria for tests included in the current EPPO Standard, and for a conventional PCR. On the basis of the obtained performance criteria, a diagnostic procedure was elaborated and then applied to perform an inter-laboratory comparison. Screening tests for the detection of the bacterium on symptomless plant material based on IF and/or PCR were proposed, in parallel with isolation on agar media. For identification two methods were suggested: a molecular test or IF. This paper reports on the results of the TPS and proposes a flow diagram for the detection and identification of X. arboricola pv. pruni.

2015 - Impact of climatic conditions on outbreaks of bacterial spot on tomato and pepper caused by Xanthomonas vesicatoria and Xanthomonas euvesicatoria [Relazione in Atti di Convegno]
Giovanardi, Davide; Biondi, Enrico; Ignjatov, Maja; Gasic, Katarina; Ferrari, Michele; Perez, Set; Jevtic, Radivoje; Stefani, Emilio
abstract

Seed-borne bacterial pathogens of tomato and pepper are of major concern worldwide. Xanthomonas vesicatoria (Xv) and Xanthomonas euvesicatoria (Xe), the causal agents of bacterial leaf spot, and Clavibacter michiganensis subsp. michiganensis (Cmm), the causal agent of tomato bacterial canker, are worldwide distributed, but the occurrence of the latter is usually erratic. In order to evaluate the risk of seed transmission and the relationship between seed contamination and disease outbreak, an extensive field trial has been put in place in 2013 for each pathosystem. Three artificially contamination levels were considered, composed of 100 seedlings each. Disease outbreaks were weekly monitored during the growing season until harvesting and disease was quantified by means of AUDPC. Seeds were produced from each plot and analysed in order to assess their contamination level. Preliminary results of our studies showed that disease quantity caused by Xv, Cmm or Xe was directly correlated to the percentage of initial infection, according to AUDPC values obtained. Contamination rate of seed produced in diseased fields was not always correlated with disease quantity observed. A microbial consortium, a bacterial antagonist and a plant polyphenol were assayed to assess their potential efficacy in seed disinfection: naturally contaminated tomato and pepper seeds were treated and sown. Pepper and tomato seedlings were inspected and analysed for the presence of bacterial spot. Preliminary results obtained show that none of the above mentioned treatments was able to eradicate the pathogen from seeds.

2015 - Seed transmission of Acidovorax citrulli: implementation of detection in watermelon seeds and development of disinfection methods [Relazione in Atti di Convegno]
Giovanardi, Davide; Ferrari, Michele; Stefani, Emilio
abstract

Acidovorax citrulli is a seed-borne pathogen and the causal agent of bacterial fruit blotch of cucurbits. It is listed as an A1 quarantine pathogen by EPPO. Seed certification is based on the availability of a sensitive and specific pathogen detection in seed lots: this is a must for an effective disease management strategy. Therefore, an effective DNA extraction and purification procedure is a critical issue to ensure a robust PCR analysis. Pathogen detection in seed lots has been implemented by testing different known contamination levels by Acidovorax citrulli. Initially, two different sample preparation methods have been tested: a) Overnight soaking; b) Hammering of dry seeds, followed by three different manual DNA extraction. Each DNA sub-sample obtained has been analysed with two different primers sets, SEQID3/SEQID4 and WFB1/WFB2, to evaluate the capability to detect the pathogen. Results showed that a DNA extraction and purification procedure, based on soaking the seeds, followed by the use of the DNeasy Plant Mini kit (Qiagen) on the washing fluids gave the highest amount of DNA, sufficient to increase the detection threshold of the pathogen. This will allow the improvement of current detection procedures. Furthermore, naturally contaminated watermelon seeds were treated through different methods, in order to achieve a possible sanitation or eradication of Acidovorax citrulli: a bacterial antagonist, a microbial consortium, a plant polyphenol. Our results showed that treated seeds were only partially disinfected, and the pathogen was not eradicated after any of the methods used.

2015 - Serbian-Italian Cooperation through researchers mobility within Erasmus Mundus Program [Relazione in Atti di Convegno]
Katarina, Gašic; Enrico, Biondi; Giovanardi, Davide; Assunta, Bertaccini; Stefani, Emilio; Aleksa, Obradovic
abstract

Mutual interests in agricultural research provide continuous opportunity for cooperation among Serbian and Italian institutions. Recently, bilateral cooperation between Serbia and Italy was implemented through the postdoctoral research of Dr. Katarina Gašic, research associate from the Institute for Plant Protection and Environment, Belgrade, Serbia. Dr. Gašic spent nine months on a postdoctoral scholarship (2014-2015), received from the Erasmus Mundus Action 2 - Join EU-SEE IV Program, at the University of Bologna, Department of Agricultural Sciences, Laboratory of Phytobacteriology, Italy, under the supervision of Prof. Assunta Bertaccini. During her stay, Dr. Gašic also visited the Department of Life Sciences of the University of Modena and Reggio Emilia and participated to a research on the management of seed transmitted bacteria, that was part of the ongoing project: EU-FP7 Project „Development of seed testing methods for pests and pathogens of plant health concern (TESTA; www.seedtesta.eu)“, under the supervision of Prof. Emilio Stefani; and III46008 project “Development of integrated management of harmful organisms in plant production in order to overcome resistance and to improve food quality and safety”, coordinator Prof. Aleksa Obradovic, financed by Ministry of Education, Science and Technological Development, Republic of Serbia.

2015 - USE OF MICROBIAL CONSORTIA FOR AN ECO-SUSTAINABLE MANAGEMENT OF SOME TOMATO DISEASES [Abstract in Atti di Convegno]
Catalano, V.; Verzelloni, E.; Giovanardi, D.; Ferrari, M.; Casagrande Biasuz, E.; Prodi, A.; Nipoti, P.; Stefani, E.
abstract

The use of beneficial microbial population in agriculture has proven to be important for organic productions and enhances biodiversity in agro-ecosystems. This study was aimed to evaluate the beneficial effects of commercial microbial consortia (Micosat F®) on the growth and productivity of tomato plants and, in particular, the ability of such consortia to elicit an endogenous protection against the bacterial spot, (Xanthomonas vesicatoria), and the root and crown rot (Rhizoctonia solani). Experiments were conducted under field conditions on tomato for industrial processing during two seasons, following a randomised plot design and in duplicate for a statistical evaluation of data. Micosat F® was applied 5 times fortnightly, from first fruit set to three weeks before harvest. Phytopathometrical assessments were done on plants during the whole season and the tomato production was also assessed. Results highlighted that Micosat F® consistently enhanced plant size by an average increase of 13.04%. In case on plots inoculated with R. solani, the use of Micosat F® led to an increase of tomato production by 18.3%, when compared with the untreated controls. In case of plots inoculated with X. vesicatoria, no significant difference was seen between treated and untreated plants, as regards the harvested fruits. The induction of a protective status in tomato plants, elicited by the use of Micosat F®, was evaluated by measuring the increased activity of antioxidant enzymes (CAT, POD, SOD). Preliminary data are very promising and are indicating that microbial consortia, such as those contained in Micosat F®, might have a significant role in both the biological and integrated tomato production.

2015 - XANTHOMONAS EUVESICATORIA IN PEPPER SEEDS: IMPLEMENTATION OF ITS DETECTION AND PRELIMINARY STUDY ON ITS GENETIC FINGERPRINTS. [Abstract in Atti di Convegno]
Ferrari, M.; Xhemali, B.; Giovanardi, D.; Valentini, F.; Ignjatov, M.; Jevtić, R.; Stefani, E.
abstract

The bacterial fruit blotch is an important disease of cucurbits, caused by the Gram negative rod Acidovorax citrulli. The disease has a great potential to cause significant losses, especially on watermelon. There are only two reports of the disease in Italy, but both the seed industry and farmers are particularly concerned, since the pathogen has been already intercepted in imported watermelon seed lots. In order to evaluate the risk of seed transmission and the relationship between seed contamination, disease outbreak and seed quality, an extensive field trial has been put in place in 2013. The highly sensitive watermelon cultivar Charleston Gray was used for the experiments. Three seed contamination levels were considered (1%, 5% and 15%) and for each contamination level a plot with 100 seedlings was transplanted. Outbreaks were monitored during the growing season until harvesting and disease was quantified by means of AUDPC. The harvested watermelons were used to produce seed with a natural contamination and such seed was used to implement a detection protocol for A. citrulli. The results obtained showed a significant correlation between initial contamination rate and disease severity in the field, with an AUDPC score of 1.312, 7.315 and 32.970 for the three contamination levels considered. The disease was observed on leaves and vines, but not on fruits. Nonetheless, seed obtained was contaminated by the pathogen, indicating that A. citrulli efficiently progressed along the vascular system, into the fruits and seeds. Pathogen detection in seed lots has been implemented by using samples of 5,000 seeds, extraction of bacterial DNA and its amplification with a RealTime PCR protocol.

2014 - Pollen as a possible pathway for the dissemination of Pseudomonas syringae pv. actinidiae and bacterial canker of kiwifruit [Articolo su rivista]
Tontou, Rodanthi; Giovanardi, Davide; Stefani, Emilio
abstract

Pollen collected in a kiwifruit orchard with symptoms of bacterial canker and naturally contaminated by Pseudomonas syringae pv. actinidiae (Psa), was used to pollinate an experimental orchard, in order to confirm its role, under commercial orchard conditions, in disseminating the pathogen and, possibly, contributing to disease spread. A pollen lot, certified free from Psa, was used with the same methods as a control. Two pollination techniques were used: dusting (dry pollen) and spraying (pollen suspension in water). The orchard was monitored during 2 years from experimental pollination, with regular sampling of flowers, fruits, leaves, and vines, to check for Psa as an epiphyte or endophyte, and for bacterial canker symptoms. Psa was recovered from flowers, fruitlets and leaves during the first season, mainly in plots where contaminated pollen had been sprayed in water suspension. From early August until harvesting time (mid-October), Psa detection was possible only on leaves. No symptoms developed during the first season after pollination. No endophytic Psa was detected in pruned vines in the following winter. During the second season, detection and isolation of Psa was erratic, but direct isolation was achieved from four plots. During the second season after pollination, typical leaf symptoms were observed on a few vines, and Psa was isolated and identified. Our results suggest that Psa could be disseminated via contaminated kiwifruit pollen as a pathway for spread of bacterial canker. However, further pollination experiments are needed to establish, beyond any doubt, whether contaminated pollen may contribute to possible disease outbreaks.

2014 - SEED TRANSMISSION OF ACIDOVORAX CITRULLI AND ITS DETECTION IN CUCURBIT SEED LOTS [Abstract in Atti di Convegno]
Giovanardi, D.; Ferrari, M.; Stefani, E.
abstract

2013 - The epiphitic life of Pseudomonas syringae pv. actinidiae on kiwifruit and other cultivated and spontaneous plants. [Abstract in Rivista]
Tontou, Rodanthi; Giovanardi, Davide; Facchini, Carlo; Stefani, Emilio
abstract

Bacterial canker of kiwifruit is the most destructive disease of cultivated Actinidia spp. The causal agent is the Gram-negative bacterium Pseudomonas syringae pv. actinidiae. Several surveys have been carried out in kiwi orchards in Romagna (northern Italy) and proved that Psa may have a significant epiphytic life, mainly on flowers, pollen and leaves. On the fruits of affected orchards Psa was detectable until early August. In Romagna, kiwifruit is cultivated in areas where also other fruit crops are grown, often side by side. A two-year survey confirmed that Psa is present as a leaf epiphyte in neighbouring stone fruit orchards (apricot and plum), when an affected kiwi orchard is nearby. Several other spontaneous plants and weeds present in kiwifruit orchards have been checked for the epiphytic presence of Psa. Stinging nettle was constantly associated with the epiphytic presence of Psa, thus providing good conditions for bacterial growth and survival. Psa was also detected on, or isolated from other weeds, like amaranth, common mallow, purslane, dandelion and smartweed. The time range during which Psa was detected/isolated on other plants was the same as Psa could be traced on kiwi plants. These results suggest that Psa, like other pathogenic and non pathogenic pseudomonads, might spend a significant lapse of time on non-host plants. The role in the epidemiology of the bacterial canker of kiwifruit of such epiphytic population is not known. As a good practice for kiwifruit orchards, control of weeds, especially in case of Psa presence, is particularly advised.

2013 - The epiphytic life of Pseudomonas syringae pv. actinidiae on kiwifruit and on other cultivated and spontaneous plants. [Abstract in Rivista]
Tontou, R.; Giovanardi, D.; Facchini, C.; Stefani, E.
abstract

Bacterial canker of kiwifruit is the most destructive disease of cultivated Actinidia spp. The causal agent is the Gram-negative bacterium Pseudomonas syringae pv. actinidiae (Psa). Several surveys have been carried out in kiwi orchards in Romagna (northern Italy) and proved that Psa may have a significant epiphytic life, mainly on flowers, pollen and leaves. On the fruits of affected orchards Psa was detectable until early August. In Romagna, kiwifruit is cultivated in areas where also other fruit crops are grown, often side by side. A two-year survey confirmed that Psa is present as a leaf epiphyte in neighbouring stone fruit orchards (apricot and plum), when an affected kiwi orchard is nearby. Several other spontaneous plants and weeds present in kiwifruit orchards have been checked for the epiphytic presence of Psa. Stinging nettle was constantly associated with the epiphytic presence of Psa, thus providing good conditions for bacterial growth and survival. Psa was also detected on, or isolated from other weeds, like amaranth, common mallow, purslane, dandelion and smartweed. The time range during which Psa was detected/isolated on other plants was the same as Psa could be traced on kiwi plants. These results suggest that Psa, like other pathogenic and non pathogenic pseudomonads, might spend a significant lapse of time on non-host plants. The role in the epidemiology of the bacterial canker of kiwifruit of such epiphytic population is not known. As a good practice for kiwifruit orchards, control of weeds, especially in case of Psa presence, is particularly advised.

2012 - Endophytes and epiphytes from Actinidia spp. as potential agents for the biocontrol of the bacterial canker of kiwifruit. [Abstract in Rivista]
Giovanardi, Davide; Tontou, Rodanthi; C., Facchini; Stefani, Emilio
abstract

The bacterial canker of kiwifruit is the most destructive disease of cultivated Actinidia spp. The causal agent is the Gram- negative bacterium, Pseudomonas syringae pv. actinidiae (Psa). The two commonly grown host species, Actinidia deliciosa (green fleshed) and Actinidia chinensis (yellow fleshed) show different susceptibility to the disease: the yellow kiwifruit is far more susceptible than the green one. The pathogen has significant epiphytic and endophytic phases: it penetrates the host through natural openings and thorough wounds. The control of the disease is difficult, due to the lack of effective chemicals that might be applied in the orchards. Copper compounds alone did not yield a satisfactory control of the disease. For endophyte and epiphyte isolation, orchards were chosen where the disease had the highest incidence and severity. Inside orchard plots, where most kiwi plants were dead, occasional symptomless plants were observed, cut and taken to the lab. Epiphytes were isolated and purified from washings of the leaves and endophytes were obtained from the washings of woody disks, taken at different levels along the trunk. A selection of ca. 60 isolates was chosen and subjected to preliminary genetic characterisation with rep-PCR, using the BOX primer. The selected endophytes and epiphytes were assayed in vitro for their ability to inhibit a set of phytopathogenic Gram-negative and Gram-positive bacteria, among them Psa. The results of the experiments showed that at least a dozen of endophytes and epiphytes were able to strongly inhibit both Psa and other important phytopathogenic bacteria.

2011 - AN INSIGHT IN SOME POPULATION FEATURES OF XANTHOMONAS ARBORICOLA pv. JUGLANDIS [Abstract in Rivista]
Giovanardi, Davide; Dallai, Davide; Bonneau, Sophie; Le Saux Fisher, Marion; Manceau, Charles; Stefani, Emilio
abstract

Xanthomonas arboricola pv. juglandis (Xaj) is the causal agent of bacterial blight of walnut, an emerging disease, which has the potential to severely affect walnut orchards. An Italian strain collection of Xaj, obtained during the past 3 years from affected orchards in Romagna, was first assayed with conventional PCR with XajF/XajR primer pair developed to confirm strain identity. The population structure of the collection of Xaj isolates, confirms the presence of different genetic groups identified by rep-PCR (using Italian collection are currently being analysed by MLSA (multi locus sequence analysis), using 7 primers for 7 different housekeeping genes, with the purpose to better characterise the Italian isolates for phylotyping. The study of copper resistance on a wide collection of over 150 Xaj strains frequently showed high resistance (up to 500 ppm Cu++): two strains have been further studied confirming the presence of chromosomal genes copA and copB involved in the general copABCD copper resistance structure, as described for Pseudomonas syringae. Sequencing and comparing with other Xanthomonads were done. The elucidation of Xaj population structure may help to deeper investigate some additional aspects of the molecular epidemiology of the disease, thus allowing a better control strategy in the field. the REP, BOX and ERIC primers) and by multilocus sequence typing (MLST) and multilocus variable number analysis of tandem repeat (MLVA). Xaj and Xaj-like bacterial isolates from the

2011 - An insight of some population features of Xanthomonas arboricola pv. juglandis. [Abstract in Rivista]
Giovanardi, D.; Dallai, D.; Bonneau, S.; Lesaux-Fischer, M.; Manceau, C.; Stefani, E.
abstract

Xanthomonas arboricola pv. juglandis (Xaj) is the causal agent of bacterial blight of walnut, an emerging disease, which has the potential to severely affect walnut orchards. An Italian strain collection of Xaj, obtained during the past 3 years from affected orchards in Romagna, was first assayed with conventional PCR with XajF/XajR primer pair developed to confirm strain identity. The population structure of the collection of Xaj isolates, confirms the presence of different genetic groups identified by rep-PCR (using the REP, BOX and ERIC primers) and by multilocus sequence typing (MLST) and multilocus variable number analysis of tandem repeat (MLVA). Xaj and Xaj-like bacterial isolates from the Italian collection are currently being analysed by MLSA (multi locus sequence analysis), using 7 primers for 7 different housekeeping genes, with the purpose to better characterise the Italian isolates for phylotyping. The study of copper resistance on a wide collection of over 150 Xaj strains frequently showed high resistance (up to 500 ppm Cu++): two strains have been further studied confirming the presence of chromosomal genes copA and copB involved in the general copABCD copper resistance structure, as described for Pseudomonas syringae. Sequencing and comparing with other Xanthomonads were done. The elucidation of Xaj population structure may help to deeper investigate some additional aspects of the molecular epidemiology of the disease, thus allowing a better control strategy in the field.

2011 - Detection of Pseudomonas syringae pv. actinidiae in pollen samples [Articolo su rivista]
J. L., Vanneste; Giovanardi, Davide; J., Yu; D. A., Cornish; C., Kay; F., Spinelli; Stefani, Emilio
abstract

Presence of Pseudomonas syringae pv. actinidiae (Psa), the causal agent of bacterial canker of kiwifruit, in pollen samples collected from infected and non infected orchards in Italy and in New Zealand was determined by polymerase chain reaction (PCR) and by direct bacterial isolation. Psa was isolated only from pollen samples collected in Italy, including pollen collected from two uninfected orchards, which the following year showed signs of infection. Psa was also detected in pollen collected from male and female vines in an Italian infected orchard. Pollen samples from Italy, but not from New Zealand, were collected with a vacuum device. Psa could not be isolated from any of the 25 New Zealand pollen samples analysed. This is the first report of Psa being associated with pollen. There is currently no evidence that artificial pollination leads to increased infection or that pollen has been responsible for the introduction of Psa in a previously Psa-free area

2011 - Dissemination of Pseudomonas syringae pv. actinidiae through pollen and its epiphytic life on leaves and fruit [Articolo su rivista]
Stefani, Emilio; Giovanardi, Davide
abstract

The role of pollen in disseminating Pseudomonas syringae pv. actinidiae (Psa) in kiwifruit orchards was investigated and the survival of the pathogen as an epiphyte on leaves and fruits was followed, from pollination time until the pre-harvest season. Pollen with natural inoculum was obtained from an infected orchard and pollen harvested without any contamination by Psa was experimentally inoculated at approximately the same contamination level. Two pollination techniques were used in glasshouse experiments: dusting and spraying. In parallel, field trials in commercial orchards were carried out: two plots were designed in two orchards, where bacterial canker was present at low incidence. From petal fall to 3 weeks before harvesting, leaf and fruit samples were taken and analysed for the presence of Psa, using two different PCR protocols and direct isolation. Results confirmed the dissemination of Psa through pollen, especially when using the aqueous suspensions. Both in glasshouse experiments and in the orchards Psa was found as an epiphyte for several weeks after pollination. Pathogen populations on leaves were, initially, 10 to 100 times less than on fruitlets. As the summer continued, the epiphytic contamination levels of fruits decreased constantly, being no more detectable from early August, whereas Psa was present at detectable levels on leaves until early October, approx. 20 days before harvesting time. Our results confirmed the role of pollen in disseminating Psa, the long epiphytic survival of the pathogen on kiwifruit leaves and the increasing unsuitability of fruits to harbour detectable, epiphytic populations of the bacterium through the summer season in commercial orchards with low disease incidence, when they are reaching the final development stage. Thus, kiwifruit surfaces do not appear to be a suitable niche for a long term survival of Psa as an epiphyte and, therefore, kiwifruits should not represent a pathway for Psa dissemination and pose a negligible risk for the introduction of the pathogen into new areas.

2010 - Population features of Xanthomonas arboricola pv. juglandis and epidemiology of walnut blight in Romagna (Italy) [Abstract in Atti di Convegno]
Giovanardi, D.; Dallai, D.; Stefani, E.
abstract

Bacterial blight of walnut caused by Xanthomonas arboricola pv. juglandis (Xaj) is an emerging disease, which has the potential to severely affect walnut orchards (Mulrean and Schroth, 1981). Symptoms are visible on all aerial parts of the host plant and particularly on leaves and nuts; the disease develops more rapidly during spring, causing spots on leaves and immature fruits, followed by the formation of small cankers on leaf petioles and twigs. Affected fruits fall down throughout the growing season, with a peak from mid-May to mid-June. Primary inoculum is released early in spring, from small overwintering cankers present on twigs. Primary inoculum is spread by wind-driven rain droplets and by pollen. Our study confirms the spread by pollen, but catkins seems to become infected during their spring development from bacteria oozing out from small twig cankers. Female flowers are not contaminated before pollination, and become infected during pollination and/or during spring rain. The source of primary inoculum appears to be the small overwintering cankers developing near the fruiting buds. The population structure of a broad collection of Xaj isolates, obtained from affected orchards in Romagna, confirms the presence of different genetic groups, identified by rep-PCR (using the REP, BOX and ERIC primers) and by multilocus sequence typing (MLST) and multilocus variable number analysis of tandem repeats (MLVA). Copper resistance was studied on a wide collection of over 150 Xaj strains isolated in Romagna walnut orchards during 2007-2009: 83% of the collection strains proved to be tolerant to copper, whereas 36% proved to be highly resistant. Control strategies are difficult to implement and are based on the timely effective use of copper compounds with an emphasis on spring treatments. The Walnut Blight Forecast Model “Xanthocast”, developed in California (Adaskaveg et al., 2004), is under evaluation in European walnut orchards. In order to avoid the development of copper resistance, the use of possible resistance inducers is under evaluation, coupled with a reduced use of copper. Glucohumates (active humic and fulvic acids, obtained from leonardite and gluconic acid) were able to control lesion development on walnuts in vitro and reduce disease incidence in field experiments.

2009 - Population studies of Xanthomonas arboricola pv. pruni and new strategy of control in peach orchards. [Abstract in Atti di Convegno]
Dallai, D.; Parkinson, N.; Giovanardi, D.; Stefani, E.
abstract

The population structure of Xanthomonas arboricola pv. pruni (Xap), the causal agent of the bacterial canker/leaf and fruit spots of stone fruits, was investigated in order to achieve a deeper insight in this taxon, thus enabling an improvement of the current diagnostic protocols and a more effective control of the disease. We confirmed the homogeneity of a vast collection of strains (over 100), isolated from stone fruit orchards during the last decade in Romagna (Northern Italy). Protein profiling and repPCR genetic fingerprints showed no significant differences among the strains: population homogeneity was also confirmed by phylotyping analysis. Copper resistant strains are present, but not widespread among the whole population. Xap is regulated (Directive 2000/29/EC, Annex A, Part II, Section II) and is included in the EPPO A2 list. An effective control of it is based both on analysis of propagation material and use of appropriate control strategies in the field. Based on available DNA sequences, we designed primers and attempted to developed a PCR protocol to be used in certification schemes. We also conducted field and glasshouse trials on peach, with the aim to effectively control the disease by using novel molecules, such as Glucohumates or antagonistic bacteria. Results are very promising and suggest the possibility to implement effective control strategies, where copper compounds and novel molecules are both used in commercial orchards.

2009 - Recent avances on the epidemiology and control of bacterial blight of walnut, incited by Xanthomonas arboricola pv. juglandis [Abstract in Rivista]
Giovanardi, D.; Dallai, D.; Cozzolino, C.; Stefani, E.
abstract

The bacterial blight of walnut caused by Xanthomonas arboricola pv. juglandis (Xaj) is an emerging disease, which can severely affect walnut orchards. Symptoms are visible on all aerial parts of the host, particularly on leaves and nuts. The disease develops more rapidly in spring, causing spots on the leaves and immature fruits, followed by the formation of small cankers on leaf petioles and twigs. Affected fruits fall down throughout the growing season, with a peak within mid-May and mid-June. The primary source of the inoculum is available very early in spring, since evasion is likely to occur from small overwintering cankers present on twigs as soon as the new vegetation busts. The primary inoculum is spread by wind-driven rain droplets and pollen. Our study confirms the spread by pollen, but catkins seems to become infected during their spring development from bacteria oozing from small twig cankers. Female flowers are not contaminated before pollination, and become infected during pollination and/or during spring rain. Copper resistance has been studied on a collection of over 150 strains isolated in Romagna during 2007- 2009. A high percentage (83%) of the collection proved tolerant to copper, whereas 36% was highly resistant. Control strategies are difficult to implement and are based on the timely and effective use of copper compounds with emphasis on spring treatments. In order to bypass copper resistance the use of alternative molecules as resistance inducers is under evaluation, coupled with a reduced use of copper.

2004 - DNA-based analysis for authenticity assessment of monovarietal pasta [Articolo su rivista]
Terzi, V.; Morcia, C.; Giovanardi, D.; D'Egidio, M. G.; Stanca, A. M.; Faccioli, P.
abstract

Because of the presence on the market of high-value monovarietal pasta, the aim of this work was to evaluate the potential of DNA profiling to test its authenticity. AFLP profiles obtained from eight monovarietal pasta samples and from the corresponding varieties were compared. The possibility of identifying the variety of an unknown monovarietal pasta by referring to a durum wheat molecular markers database is proposed. © Springer-Verlag 2004.

Università degli studi di Modena e Reggio Emilia

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