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Emilio STEFANI

Professore Associato
Dipartimento di Scienze della Vita sede ex-Agraria

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Pubblicazioni

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 - Commodity risk assessment of Prunus persica and P. dulcis plants from Türkiye [Articolo su rivista]
Bragard, Claude; Baptista, Paula; Chatzivassiliou, Elisavet; Gonthier, Paolo; Jaques Miret, Josep Anton; Justesen, Annemarie Fejer; Macleod, Alan; Magnusson, Christer Sven; Milonas, Panagiotis; Navas-Cortes, Juan A; Parnell, Stephen; Potting, Roel; Reignault, Philippe Lucien; Stefani, Emilio; Thulke, Hans-Hermann; Van der Werf, Wopke; Civera, Antonio Vicent; Zappalà, Lucia; Lucchi, Andrea; Gómez, Pedro; Urek, Gregor; Bernardo, Umberto; Bubici, Giovanni; Carluccio, Anna Vittoria; Chiumenti, Michela; Di Serio, Francesco; Fanelli, Elena; Kaczmarek, Agata; Marzachì, Cristina; Mosbach-Schulz, Olaf; Yuen, Jonathan
abstract

: The European Commission requested the EFSA Panel on Plant Health to prepare and deliver risk assessments for commodities listed in Commission Implementing Regulation (EU) 2018/2019 as 'High risk plants, plant products and other objects'. This Scientific Opinion covers plant health risks posed by plants of Prunus persica and P. dulcis, as budwood/graftwood, rooted or grafted on rootstocks of either P. persica, P. dulcis, P. armeniaca, P. davidiana or their hybrids, imported from Türkiye, taking into account the available scientific information, including the technical information provided by Türkiye. All pests associated with the commodity were evaluated against specific criteria for their relevance for this opinion. Four quarantine pests (peach rosette mosaic virus, tomato ringspot virus, Anoplophora chinensis, Scirtothrips dorsalis) and 14 non-regulated pests (Hoplolaimus galeatus, Lasiodiplodia pseudotheobromae, Neoscytalidium dimidiatum, Neoscytalidium novaehollandiae, Didesmococcus unifasciatus, Euzophera semifuneralis, Lepidosaphes malicola, Lepidosaphes pistaciae, Maconellicoccus hirsutus, Malacosoma parallela, Nipaecoccus viridis, Phenacoccus solenopsis, Pochazia shantungensis, Russellaspis pustulans) that fulfilled all relevant criteria were selected for further evaluation. For these 18 pests, the risk mitigation measures proposed in the technical Dossier from Türkiye were evaluated taking into account the possible limiting factors. For the selected pests, an expert judgement is given on the likelihood of pest freedom taking into consideration the risk mitigation measures acting on the pest, including uncertainties associated with the assessment. The degree of pest freedom varies among the pests evaluated, with fungi from Botryosphaeriaceae family (L. pseudotheobromae, N. dimidiatum and N. novaehollandiae) being the pests most frequently expected on the imported plants. The Expert Knowledge Elicitation indicated with 95% certainty that between 9,813 and 10,000 bundles (consisting of 10 or 25 plants each) per 10,000 would be free from the above-mentioned fungi in the Botryosphaeriaceae family.

2023 - Commodity risk assessment of ash logs from the US treated with sulfuryl fluoride to prevent the entry of the emerald ash borer Agrilus planipennis [Articolo su rivista]
Bragard, Claude; Baptista, Paula; Chatzivassiliou, Elisavet; Di Serio, Francesco; Jaques Miret, Josep Anton; Justesen, Annemarie Fejer; Macleod, Alan; Magnusson, Christer Sven; Milonas, Panagiotis; Navas-Cortes, Juan A; Parnell, Stephen; Potting, Roel; Reignault, Philippe Lucien; Stefani, Emilio; Thulke, Hans-Hermann; Van der Werf, Wopke; Vicent Civera, Antonio; Yuen, Jonathan; Zappalà, Lucia; Battisti, Andrea; Mas, Hugo; Faccoli, Massimo; Gardi, Ciro; Mikulová, Alžběta; Mosbach-Schulz, Olaf; Stancanelli, Giuseppe; Stergulc, Fabio; Gonthier, Paolo
abstract

The European Commission submitted to the EFSA Panel on Plant Health a dossier by USDA proposing to use sulfuryl fluoride on ash log shipments to treat Agrilus planipennis for phytosanitary certification. After collecting additional evidence from USDA APHIS, external experts and literature, the Panel performed a quantitative assessment on the likelihood of pest freedom from A. planipennis, at the point of entry in the EU, of two different commodities fumigated with sulfuryl fluoride: (a) ash logs with bark; and (b) debarked ash logs. An expert judgement is given on the likelihood of pest freedom taking into consideration the measures acting on the pest, including uncertainties associated with the assessment. The likelihood of pest freedom from A. planipennis is lower for ash logs with bark compared with debarked ash logs. With 95% certainty, the Panel concludes that between 9,740 and 10,000 containers of ash logs with bark per 10,000 and between 9,989 and 10,000 containers of debarked ash logs per 10,000 will be free from A. planipennis, when fumigated with sulfuryl fluoride at the specific treatment regime proposed by the USDA APHIS.

2023 - Efficacy of a postharvest treatment aiming at eradication of all developmental stages of Tecia solanivora in ware potatoes [Articolo su rivista]
Bragard, C.; Chatzivassiliou, E.; Di Serio, F.; Baptista, P.; Gonthier, P.; Miret, J. A. J.; Justesen, A. F.; Macleod, A.; Magnusson, C. S.; Milonas, P.; Navas-Cortes, J. A.; Parnell, S.; Potting, R.; Reignault, P. L.; Stefani, E.; Thulke, H. -H.; Van der Werf, W.; Vicent, A.; Zappala, L.; Karlsson, M. F.; Kaczmarek, A.; Mosbach-Schulz, O.; Yuen, J.
abstract

2023 - Pest categorisation of Coniella granati [Articolo su rivista]
Bragard, Claude; Baptista, Paula; Chatzivassiliou, Elisavet; Di Serio, Francesco; Gonthier, Paolo; Jaques Miret, Josep Anton; Justesen, Annemarie Fejer; Macleod, Alan; Magnusson, Christer Sven; Milonas, Panagiotis; Navas-Cortes, Juan A; Parnell, Stephen; Potting, Roel; Reignault, Philippe Lucien; Stefani, Emilio; Thulke, Hans-Hermann; Van der Werf, Wopke; Civera, Antonio Vicent; Yuen, Jonathan; Zappalà, Lucia; Migheli, Quirico; Vloutoglou, Irene; Maiorano, Andrea; Streissl, Franz; Reignault, Philippe Lucien
abstract

: The EFSA Plant Health Panel performed a pest categorisation of Coniella granati, a clearly defined fungus of the Order Diaporthales and the family Schizoparmaceae, described for the first time in 1876 as Phoma granatii and later named as Pilidiella granati. The pathogen mainly affects Punica granatum (pomegranate) and Rosa spp. (rose), causing fruit rot, shoot blight and cankers on crown and branches. The pathogen is present in North America, South America, as well as in Asia, Africa, Oceania and Eastern Europe and has also been reported in the EU (Greece, Hungary, Italy and Spain), where it is widespread in the major pomegranate growing areas. Coniella granati is not included in Commission Implementing Regulation (EU) 2019/2072 and there are no interceptions in the EU. This pest categorisation focused on those hosts for which the pathogen was detected and formally identified in natural conditions. Plants for planting, fresh fruits and as well as soil and other plant growing media are the main pathways for the further entry of the pathogen into the EU. Host availability and climate suitability factors occurring in parts of the EU are favourable for the further establishment of the pathogen. In the area of its present distribution, including Italy and Spain, the pathogen has a direct impact in pomegranate orchards as well as during post-harvest storage. Phytosanitary measures are available to prevent the further introduction and spread of the pathogen into the EU. Coniella granati does not satisfy the criteria that are within the remit of EFSA to assess for this species to be regarded as potential Union quarantine pest as it is present in several EU MSs.

2023 - Pest categorisation of Icerya aegyptiaca [Articolo su rivista]
Bragard, Claude; Baptista, Paula; Chatzivassiliou, Elisavet; Di Serio, Francesco; Gonthier, Paolo; Miret, Josep Anton Jaques; Justesen, Annemarie Fejer; Magnusson, Christer Sven; Milonas, Panagiotis; Navas-Cortes, Juan A; Parnell, Stephen; Potting, Roel; Reignault, Philippe Lucien; Stefani, Emilio; Thulke, Hans-Hermann; Van der Werf, Wopke; Vicent Civera, Antonio; Yuen, Jonathan; Zappalà, Lucia; Grégoire, Jean-Claude; Malumphy, Chris; Akrivou, Antigoni; Kertesz, Virag; Papachristos, Dimitrios; Macleod, Alan
abstract

: The EFSA Panel on Plant Health performed a pest categorisation of Icerya aegyptiaca (Hemiptera: Sternorrhyncha: Monophlebidae), the Egyptian fluted scale, for the EU. This insect is established in several countries in tropical and subtropical regions of the world. Within the EU, the pest has not been reported. It is not listed in Annex II of Commission Implementing Regulation (EU) 2019/2072. It is highly polyphagous, feeding on plants in 128 genera and 66 families, with some preference for avocado (Persea americana), banana (Musa sp.), citrus (Citrus spp.), coconut (Coccos nucifera), common pear (Pyrus communis), fig (Ficus spp.), guava (Psidium guajava), maize (Zea mays), mango (Mangifera indica), white mulberry (Morus alba), and grapevine (Vitis vinifera). It has also been recorded feeding on tomato (Solanum lycopersicum), as well as on ornamental plants. Plants for planting and fruits, vegetables and cut flowers are the main potential pathways for entry of I. aegyptiaca into the EU. Climatic conditions and availability of host plants in parts of the EU where there are very few days of frost each year would likely allow this species to successfully establish and spread. Economic impact in cultivated hosts including citrus, grapes, maize, peppers, sunflowers, tomatoes and ornamental crops is anticipated if establishment occurs. Phytosanitary measures are available to reduce the likelihood of entry and spread. I. aegyptiaca meets the criteria that are within the remit of EFSA to assess for this species to be regarded as a potential Union quarantine pest.

2023 - Pest categorisation of Lasiodiplodia pseudotheobromae [Articolo su rivista]
Bragard, Claude; Baptista, Paula; Chatzivassiliou, Elisavet; Di Serio, Francesco; Gonthier, Paolo; Jaques Miret, Josep Anton; Justesen, Annemarie Fejer; Macleod, Alan; Magnusson, Christer Sven; Milonas, Panagiotis; Navas-Cortes, Juan A; Parnell, Stephen; Potting, Roel; Stefani, Emilio; Thulke, Hans-Hermann; Van der Werf, Wopke; Civera, Antonio Vicent; Yuen, Jonathan; Zappalà, Lucia; Migheli, Quirico; Vloutoglou, Irene; Maiorano, Andrea; Streissl, Franz; Reignault, Philippe Lucien
abstract

: The EFSA Plant Health Panel performed a pest categorisation of Lasiodiplodia pseudotheobromae, a clearly defined fungus of the family Botryosphaeriaceae, which was first described in 2008 as a cryptic species within the L. theobromae complex. The pathogen affects a wide range of woody perennial crops and ornamental plants causing root rot, damping-off, leaf spots, twig blight, cankers, stem-end rot, gummosis, branch dieback and pre- and post-harvest fruit rots. Lasiodiplodia pseudotheobromae is present in Africa, Asia, North and South America and Oceania and has also been reported from Spain with a restricted distribution. However, there is uncertainty on the status of the pathogen worldwide and in the EU because in the past, when molecular tools (particularly multigene phylogenetic analysis) were not available, the pathogen might have been misidentified as L. theobromae. Lasiodiplodia pseudotheobromae is not included in Commission Implementing Regulation (EU) 2019/2072 and there are no interceptions in the EU. Because of the very wide host range of the pathogen, this pest categorisation focused on those hosts for which there is robust evidence that the pathogen was formally identified by a combination of morphology, pathogenicity and multilocus sequence analysis. Plants for planting, including seeds, fresh fruits and bark and wood of host plants as well as soil and other plant-growing media are the main pathways for the further entry of the pathogen into the EU. Host availability and climate suitability factors occurring in parts of the EU are favourable for the further establishment of the pathogen. In the area of its present distribution, including Spain, the pathogen has a direct impact on cultivated hosts. multilocus measures are available to prevent the further introduction and spread of the pathogen into the EU. Lasiodiplodia pseudotheobromae satisfies the criteria that are within the remit of EFSA to assess for this species to be regarded as potential Union quarantine pest.

2023 - Pest categorisation of Nipaecoccus viridis [Articolo su rivista]
Bragard, Claude; Baptista, Paula; Chatzivassiliou, Elisavet; Di Serio, Francesco; Gonthier, Paolo; Jaques Miret, Josep Anton; Justesen, Annemarie Fejer; Magnusson, Christer Sven; Milonas, Panagiotis; Navas-Cortes, Juan A; Parnell, Stephen; Potting, Roel; Reignault, Philippe Lucien; Stefani, Emilio; Thulke, Hans-Hermann; Van der Werf, Wopke; Vicent Civera, Antonio; Yuen, Jonathan; Zappalà, Lucia; Grégoire, Jean-Claude; Malumphy, Chris; Kertesz, Virag; Maiorano, Andrea; Macleod, Alan
abstract

: The EFSA Panel on Plant Health performed a pest categorisation of Nipaecoccus viridis (Hemiptera: Sternorrhyncha: Pseudococcidae), the spherical scale, for the EU. It is of Asian origin and occurs widely in southern Asia, Africa and tropical Australia. It has been introduced to a few countries in the Americas. In the Mediterranean basin it is found in Algeria, Egypt, Israel, Syria and Turkey, where it is limited to the Marmara region. It has not been reported within the EU. It is not listed in Annex II of Commission Implementing Regulation (EU) 2019/2072. It reproduces sexually, has three generations each year in citrus orchards in South Africa, and all stages can overwinter. First instar nymphs may move to neighbouring plants by crawling or be passively dispersed by wind or hitchhiking on clothing, equipment or animals. It is highly polyphagous, feeding on plants in 115 genera and 46 families. It is an important pest of citrus (Citrus spp.), cotton (Gossypium spp.), mango (Mangifera indica), avocado (Persea americana) and stored potatoes (Solanum tuberosum). It also feeds on a wide range of other fruit (apple Malus domestica, olive Olea europea, pear Pyrus communis and grape Vitis vinifera) and vegetable crops (tomato Solanum lycopersicum), and ornamental plants (roses, Rosa spp.) that are widely grown in the EU. Plants for planting, fruits, vegetables, and cut flowers are the main potential pathways for entry of N. viridis into the EU. Climatic conditions and availability of host plants in southern parts of the EU where there are few days of frost each year would likely allow this species to successfully establish and spread. Reductions in yield and quality of cultivated hosts including avocado, citrus, cotton and mango is anticipated if establishment occurs. Phytosanitary measures are available to reduce the likelihood of entry and spread. N. viridis meets the criteria that are within the remit of EFSA to assess for this species to be regarded as a potential Union quarantine pest.

2023 - Pest categorisation of Resseliella maxima [Articolo su rivista]
Bragard, Claude; Baptista, Paula; Chatzivassiliou, Elisavet; Di Serio, Francesco; Gonthier, Paolo; Jaques Miret, Josep Anton; Justesen, Annemarie Fejer; Magnusson, Christer Sven; Milonas, Panagiotis; Navas-Cortes, Juan A; Parnell, Stephen; Potting, Roel; Reignault, Philippe Lucien; Stefani, Emilio; Thulke, Hans-Hermann; Van der Werf, Wopke; Vicent Civera, Antonio; Yuen, Jonathan; Zappalà, Lucia; Grégoire, Jean-Claude; Malumphy, Chris; Kertesz, Virag; Maiorano, Andrea; Macleod, Alan
abstract

: The EFSA Panel on Plant Health performed a pest categorisation of Resseliella maxima (Diptera: Cecidomyiidae), the soybean gall midge, for the EU. This midge was first described in 2018 and is widespread in north-western United States. It is not listed in Annex II of Commission Implementing Regulation (EU) 2019/2072. Larvae feed on and develop in soybean (Glycine max, Fabaceae), and possibly in two other Fabaceae, sweet clover (Melilotus officinalis) and alfalfa/lucerne (Medicago sativa). Feeding damage results in dark brown or black areas on the stems which become weak and can break near the soil; heavy infestations can cause plant death. R. maxima adults live only a few days and adult females lay eggs within 24 h after emergence. Larvae of R. maxima overwinter in the soil as third instars in silken cocoons. The main natural dispersal stage is the adult, which can fly. Freshly cut host plants for animal feed contaminated with larvae provide a potential pathway for entry into the EU. However, there is great uncertainty as to whether such plants are imported from USA states where R. maxima occurs. Climatic conditions and host availability in central-western EU MS are favourable for outdoor establishment. Phytosanitary measures are available to reduce the likelihood of entry and spread. Except for the uncertainty concerning the likelihood of entry, R. maxima satisfies the other criteria that are within the remit of EFSA to assess for it to be regarded as a potential Union quarantine pest.

2023 - Pest categorisation of Urocerus japonicus [Articolo su rivista]
Bragard, Claude; Baptista, Paula; Chatzivassiliou, Elisavet; Di Serio, Francesco; Gonthier, Paolo; Jaques Miret, Josep Anton; Justesen, Annemarie Fejer; Magnusson, Christer Sven; Milonas, Panagiotis; Navas-Cortes, Juan A; Parnell, Stephen; Potting, Roel; Reignault, Philippe Lucien; Stefani, Emilio; Thulke, Hans-Hermann; Van der Werf, Wopke; Vicent Civera, Antonio; Yuen, Jonathan; Zappalà, Lucia; Grégoire, Jean-Claude; Malumphy, Chris; Kertesz, Virag; Maiorano, Andrea; Macleod, Alan
abstract

: The EFSA Panel on Plant Health performed a pest categorisation of Urocerus japonicus (Hymenoptera: Siricidae), the Japanese horntail, for the territory of the EU. U. japonicus is not listed in Annex II of Commission Implementing Regulation (EU) 2019/2072 but was identified as an actionable pest in a commodity risk assessment of Pinus thunbergii artificially dwarfed plants from Japan. U. japonicus occurs across Japan and on the Korean Peninsula. It attacks fallen or weakened Japanese cedars, Cryptomeria japonica and Japanese cypresses, Chamaecyparis obtusa. It has also been observed attacking Pinus spp., Abies spp., Larix kaempferi and Picea jezoensis. The females oviposit into the sapwood. Eggs are deposited together with a symbiotic basidiomycete fungus, Amylostereum laevigatum. The larvae feed on wood infected by the fungus. All immature stages live in the hosts sapwood. The lifecycle of the pest lasts 1 year, sometimes 2 years. The wood of the host trees is discoloured by the fungus and therefore loses much of its economic value. U. japonicus can be carried in conifer wood, solid wood packaging material (SWPM) or plants for planting. Wood from Japan is regulated by 2019/2072 (Annexes VII and XI) whilst SWPM is managed by ISPM 15. The pathway plants for planting is largely closed by prohibition, with the exception of Cryptomeria spp. and specified bonsai plants for planting. Climatic conditions in several EU Member States are conducive for establishment, but the main host plants are not very common in those areas, being only amenity trees, although the other hosts mentioned in the literature, Pinus spp., Abies spp., Picea spp. and Larix spp., are widespread. The introduction of U. japonicus is likely to decrease the quality of host wood, as in Japan. Phytosanitary measures are available to reduce the likelihood of entry and further spread, and there is a potential for biological control. U. japonicus satisfies all the criteria that are within the remit of EFSA to assess for it to be regarded as a potential Union quarantine pest.

2023 - Pest categorisation of Xylella taiwanensis [Articolo su rivista]
Bragard, Claude; Baptista, Paula; Chatzivassiliou, Elisavet; Di Serio, Francesco; Gonthier, Paolo; Jaques Miret, Josep Anton; Justesen, Annemarie Fejer; Macleod, Alan; Magnusson, Christer Sven; Milonas, Panagiotis; Navas-Cortes, Juan A; Parnell, Stephen; Potting, Roel; Stefani, Emilio; Thulke, Hans-Hermann; Van der Werf, Wopke; Civera, Antonio Vicent; Yuen, Jonathan; Zappalà, Lucia; Chen, Jianchi; Migheli, Quirico; Vloutoglou, Irene; Streissl, Franz; Reignault, Philippe Lucien
abstract

: The EFSA Plant Health Panel performed a pest categorisation of Xylella taiwanensis, a Gram-negative bacterium belonging to the Xanthomonadaceae. The pathogen is a well-defined taxonomic entity, and it is the causal agent of the pear leaf scorch. X. taiwanensis is present in subtropical and temperate areas of the island of Taiwan, where it affects low chilling pear cultivars of the species Pyrus pyrifolia (Asian pear). No other plant species are reported to be affected by the pathogen. The pathogen is not known to be present in the EU territory and it is not included in the Commission Implementing Regulation (EU) 2019/2072. The main pathway for the entry of the pathogen into the EU territory is host plants for planting (except seeds); another possible pathway might be represented by putative insect vectors, though their identity remains unknown. The cultivated area of P. pyrifolia in the EU territory is very limited. Conversely, the genetically related P. communis is widely cultivated in most EU Member States and there is no information so far on the susceptibility of its several cultivars. Should the pest establish in the EU, economic impact is expected, provided that suitable insect vectors are present and P. communis is as susceptible to infection as P. pyrifolia. Phytosanitary measures are available to prevent the introduction and spread of the pathogen into the EU, since plants for planting from Taiwan is a closed pathway; nonetheless, putative vectors, if confirmed and identified, may represent an additional risk of the pathogen's introduction and spread. The lack of knowledge on whether X. taiwanensis can infect P. communis, the identity and presence of suitable vectors in the EU lead to key uncertainties on entry, establishment, spread and impact. X. taiwanensis satisfies the criteria that are within the remit of EFSA to assess for this species to be regarded as a potential Union quarantine pest.

2023 - Pest categorisation of cowpea mosaic virus [Articolo su rivista]
Bragard, Claude; Baptista, Paula; Chatzivassiliou, Elisavet; Gonthier, Paolo; Jaques Miret, Josep Anton; Justesen, Annemarie Fejer; Macleod, Alan; Magnusson, Christer Sven; Milonas, Panagiotis; Navas-Cortes, Juan A; Parnell, Stephen; Potting, Roel; Stefani, Emilio; Thulke, Hans-Hermann; Van der Werf, Wopke; Civera, Antonio Vicent; Yuen, Jonathan; Zappalà, Lucia; Streissl, Franz; Carluccio, Anna Vittoria; Chiumenti, Michela; Di Serio, Francesco; Rubino, Luisa; Reignault, Philippe Lucien
abstract

The EFSA Panel on Plant Health conducted a pest categorisation of cowpea mosaic virus (CPMV) for the EU territory. The identity of CPMV, a member of the genus Comovirus (family Secoviridae), is established and detection and identification methods are available. The pathogen is not included in the Commission Implementing Regulation (EU) 2019/2072. It has been reported from the Americas, and several countries in Africa and Asia and it is not known to be present in the EU in natural conditions. CPMV is considered a major pathogen of cowpea on which it causes symptoms ranging from mild to severe mosaic, chlorosis and necrosis. The virus has been reported sporadically on some other cultivated species of the family Fabaceae, including soybean and some common bean varieties. CPMV is transmitted by cowpea seeds, with uncertainty on the transmission rate. There is uncertainty on seed transmission by other Fabaceae host species due to lack of information. CPMV is also transmitted by several beetle species, one of which, Diabrotica virgifera virgifera, is present in the EU. Seeds for sowing of cowpea are identified as the major entry pathway. The cultivated area and production of cowpea in the EU territory are mainly limited to local varieties cultivated at a small scale in Mediterranean EU Member States. Should the pest establish in the EU, an impact is expected on cowpea crops at local scale. There is high uncertainty on the potential impact that CPMV would cause on other natural hosts cultivated in the EU due to the lack of information from the areas of CPMV's current distribution. Despite the uncertainty concerning the potential impact on bean and soybean crops in the EU, CPMV satisfies the criteria that are within the remit of EFSA to assess for it to be regarded as a potential Union quarantine pest.

2023 - Risk assessment of Citripestis sagittiferella for the EU [Articolo su rivista]
Bragard, Claude; Baptista, Paula; Chatzivassiliou, Elisavet; Di Serio, Francesco; Gonthier, Paolo; Jaques Miret, Josep Anton; Justesen, Annemarie Fejer; Macleod, Alan; Magnusson, Christer Sven; Milonas, Panagiotis; Navas-Cortes, Juan A; Parnell, Stephen; Potting, Roel; Reignault, Philippe Lucien; Stefani, Emilio; Thulke, Hans-Hermann; van der Werf, Wopke; Yuen, Jonathan; Zappalà, Lucia; Makowski, David; Maiorano, Andrea; Mosbach-Schulz, Olaf; Pautasso, Marco; Vicent Civera, Antonio
abstract

Following a request from the European Commission, the EFSA Panel on Plant Health performed a risk assessment of Citripestis sagittiferella (Lepidoptera: Pyralidae), the citrus pulp borer, an oligophagous pest reported from South-East Asia and restricted to Citrus spp. The entry risk assessment focused on the citrus fruit pathway. Two scenarios were considered: scenario A0 (current practice) and A2 (additional post-harvest cold treatment). Based on the outputs of the entry model obtained in scenario A0, the median number of founder populations in the EU citrus-growing area is estimated to be slightly less than 10 per year (90%-uncertainty interval between about one entry per 180 years and 1,300 entries per year). The risk of entry and the simulated numbers of founder populations are orders of magnitude lower for scenario A2 compared to scenario A0. The key uncertainties in the entry model include transfer, the cold treatment effectiveness, the disaggregation factor and sorting. The simulated numbers of established populations are only slightly lower than the numbers of founder populations. As the probability of establishment has little impact on the number of established populations, it is not a major source of uncertainty, despite the lack of data on the thermal biology of the pest. The median lag period between establishment and spread is estimated to be slightly more than 1 year (90%-uncertainty interval between about 2 months and 33 months). After the lag period, the median spread rate by natural means (flying) and due to transport of harvested citrus fruit from orchards to packinghouses is estimated at about 100 km/year (90%-uncertainty interval between about 40 and 500 km/year). The main sources of uncertainties affecting the spread rate include the extent to which environmental factors could hamper the build-up of the populations and the lack of data on the spread rate at the origin. The median impact of C. sagittiferella in the EU citrus-growing area is estimated at about 10% of infested fruits among the harvested citrus fruits (90%-uncertainty interval between about 2% and 25%). Uncertainties affecting the impact assessment include the susceptibility of different Citrus species and cultivars.

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

2022 - Commodity risk assessment of Acer palmatum plants grafted on Acer davidii from China [Articolo su rivista]
Bragard, C.; Baptista, P.; Chatzivassiliou, E.; Di Serio, F.; Jaques Miret, J. A.; Justesen, A. F.; Macleod, A.; Magnusson, C. S.; Milonas, P.; Navas-Cortes, J. A.; Parnell, S.; Potting, R.; Reignault, P. L.; Stefani, E.; Thulke, H. -H.; Van der Werf, W.; Vicent Civera, A.; Yuen, J.; Zappala, L.; Battisti, A.; Mas, H.; Rigling, D.; Faccoli, M.; Iacopetti, G.; Mikulova, A.; Mosbach-Schulz, O.; Stergulc, F.; Gonthier, P.
abstract

The European Commission requested the EFSA Panel on Plant Health to prepare and deliver risk assessments for commodities listed in Commission Implementing Regulation (EU) 2018/2019 as ‘High risk plants, plant products and other objects’. This Scientific Opinion covers plant health risks posed by 2-year-old bare rooted plants for planting of Acer palmatum grafted on rootstocks of Acer davidii imported from China to the EU, taking into account the available scientific information, including the technical information provided by China. All pests associated with the commodity were evaluated against specific criteria for their relevance for this Scientific Opinion. Twenty-two pests that fulfilled all relevant criteria were selected for further evaluation. For 20 pests, the risk mitigation measures described in the technical dossier from China were evaluated taking into account the possible limiting factors. For these pests, an expert judgement is given on the likelihood of pest freedom taking into consideration the risk mitigation measures acting on the pest, including uncertainties associated with the assessment. While the estimated degree of pest freedom varied among pests, Lopholeucaspis japonica was the pest most frequently expected on the commodity. The Expert Knowledge Elicitation indicated, with 95% certainty, that 9,336 or more bare rooted plants per 10,000 will be free from L. japonica. For Anoplophora chinensis and Anoplophora glabripennis, the Panel considers that China applies the relevant measures as specified in Commission Implementing Decision (EU) 2012/138 and Commission Implementing Decision (EU) 2015/893.

2022 - Commodity risk assessment of Jasminum polyanthum unrooted cuttings from Uganda [Articolo su rivista]
Bragard, C.; Chatzivassiliou, E.; Di Serio, F.; Baptista, P.; Gonthier, P.; Jaques Miret, J. A.; Fejer Justesen, A.; Macleod, A.; Magnusson, C. S.; Milonas, P.; Navas-Cortes, J. A.; Parnell, S.; Reignault, P. L.; Stefani, E.; Thulke, H. -H.; Van der Werf, W.; Vicent Civera, A.; Yuen, J.; Zappala, L.; Debode, J.; Manceau, C.; Gardi, C.; Mosbach-Schulz, O.; Potting, R.
abstract

The European Commission requested the EFSA Panel on Plant Health to prepare and deliver risk assessments for commodities listed in Commission Implementing Regulation EU/2018/2019 as ‘High risk plants, plant products and other objects’. This Scientific Opinion covers plant health risks posed by unrooted cuttings of Jasminum polyanthum that are imported from Uganda, taking into account the available scientific information, including the technical information provided by the NPPO of Uganda. The relevance of any pest for this opinion was based on evidence following defined criteria. Six species, two EU-regulated pests (Bemisia tabaci, non-European populations and Scirtothrips dorsalis) and four EU non-regulated pests (Coccus viridis, Diaphania indica, Pulvinaria psidii and Selenaspidus articulatus), fulfilled all relevant criteria and were selected for further evaluation. For these pests, the risk mitigation measures proposed in the technical dossier from Uganda were evaluated taking into account the possible limiting factors. For these pests, an expert judgement is given on the likelihood of pest freedom taking into consideration the risk mitigation measures acting on the pest, including uncertainties associated with the assessment. The estimated degree of pest freedom varies among the pests evaluated, with B. tabaci and S. dorsalis being the pests most frequently expected on the imported plants. The Expert Knowledge Elicitation indicated, with 95% certainty, that between 9,950 and 10,000 plants per 10,000 would be free of B. tabaci.

2022 - Commodity risk assessment of Malus domestica plants from Turkey [Articolo su rivista]
Bragard, C.; Baptista, P.; Chatzivassiliou, E.; Gonthier, P.; Jaques Miret, J. A.; Justesen, A. F.; Macleod, A.; Magnusson, C. S.; Milonas, P.; Navas-Cortes, J. A.; Parnell, S.; Potting, R.; Reignault, P. L.; Stefani, E.; Thulke, H. -H.; Van der Werf, W.; Civera, A. V.; Zappala, L.; Lucchi, A.; Gomez, P.; Urek, G.; Bernardo, U.; Bubici, G.; Carluccio, A. V.; Chiumenti, M.; Di Serio, F.; Fanelli, E.; Gardi, C.; Marzachi, C.; Mosbach-Schulz, O.; Yuen, J.
abstract

The European Commission requested the EFSA Panel on Plant Health to prepare and deliver risk assessments for commodities listed in Commission Implementing Regulation (EU) 2018/2019 as ‘High risk plants, plant products and other objects’. This Scientific Opinion covers plant health risks posed by dormant grafted plants, rootstocks, budwood and scions of Malus domestica imported from Turkey, taking into account the available scientific information, including the technical information provided by Turkey. All pests associated with the commodities were evaluated against specific criteria for their relevance for this opinion. Three quarantine pests (Anoplophora chinensis, Lopholeucaspis japonica and tomato ringspot virus), one protected zone quarantine pest (Erwinia amylovora) and eight non-regulated pests (Calepitrimerus baileyi, Cenopalpus irani, Cicadatra persica, Diplodia bulgarica, Hoplolaimus galeatus, Malacosoma parallela, Pratylenchus loosi and Pyrolachnus pyri) that fulfilled all relevant criteria were selected for further evaluation. For E. amylovora, special requirements are specified in Commission Implementing Regulation (EU) 2019/2072. Based on the information provided in the dossier, the specific requirements for E. amylovora were not met. For Anoplophora chinensis, special measures are specified in Commission Implementing Decision (EU) 2012/138. The exporting country does meet the requirement for a certificate regarding plants for planting that originate from Turkish provinces other than Istanbul. For the 10 remaining selected pests, the risk mitigation measures proposed in the technical dossier from Turkey were evaluated taking into account the possible limiting factors. For the selected pests an expert judgement is given on the likelihood of pest freedom taking into consideration the risk mitigation measures acting on the pest, including uncertainties associated with the assessment. The degree of pest freedom varies among the pests evaluated, with D. bulgarica being the pest most frequently expected on the imported plants. The expert knowledge elicitation indicated with 95% certainty that between 9,863 and 10,000 bundles (consisting of 10 or 25 plants each) per 10,000 would be free from D. bulgarica.

2022 - Commodity risk assessment of Prunus domestica plants from Ukraine [Articolo su rivista]
Bragard, C.; Chatzivassiliou, E.; Dehnen-Schmutz, K.; Baptista, P.; Gonthier, P.; Jacques, M. -A.; Jaques Miret, J. A.; Justesen, A. F.; Macleod, A.; Magnusson, C. S.; Milonas, P.; Navas-Cortes, J. A.; Parnell, S.; Potting, R.; Reignault, P. L.; Stefani, E.; Thulke, H. -H.; Van der Werf, W.; Vicent, A.; Zappala, L.; Lucchi, A.; Gomez, P.; Urek, G.; Bernardo, U.; Bubici, G.; Carluccio, A. V.; Chiumenti, M.; Di Serio, F.; Fanelli, E.; Marzachi, C.; Gardi, C.; Mosbach-Schulz, O.; de la Pena, E.; Yuen, J.
abstract

The European Commission requested the EFSA Panel on Plant Health to prepare and deliver risk assessments for commodities listed in Commission Implementing Regulation (EU) 2018/2019 as ‘High risk plants, plant products and other objects’. This Scientific Opinion covers plant health risks posed by plants of Prunus domestica grafted on Prunus cerasifera imported from Ukraine, taking into account the available scientific information, including the technical information provided by Ukraine. All pests associated with the commodity were evaluated against specific criteria for their relevance for this opinion. One quarantine pest (Lopholeucaspis japonica), two protected zone quarantine pests (Erwinia amylovora and Xanthomonas arboricola pv. pruni) and one non-regulated pest (Eotetranychus prunicola) that fulfilled all relevant criteria were selected for further evaluation. For these four pests, the risk mitigation measures proposed in the technical dossier from Ukraine were evaluated taking into account the possible limiting factors. For the selected pests, an expert judgement is given on the likelihood of pest freedom taking into consideration the risk mitigation measures acting on the pest, including uncertainties associated with the assessment. The degree of pest freedom varies among the pests evaluated, with Xanthomonas arboricola pv. pruni being the pest most frequently expected on the imported plants. The Expert Knowledge Elicitation indicated with 95% certainty that between 9,870 and 10,000 bundles (consisting of 10 plants each) per 10,000 would be free from Xanthomonas arboricola pv. pruni.

2022 - Commodity risk assessment of bonsai plants from China consisting of Pinus parviflora grafted on Pinus thunbergii [Articolo su rivista]
Bragard, C.; Baptista, P.; Chatzivassiliou, E.; Di Serio, F.; Jaques Miret, J. A.; Justesen, A. F.; Macleod, A.; Magnusson, C. S.; Milonas, P.; Navas-Cortes, J. A.; Parnell, S.; Potting, R.; Reignault, P. L.; Stefani, E.; Thulke, H. -H.; Van der Werf, W.; Vicent Civera, A.; Yuen, J.; Zappala, L.; Battisti, A.; Mas, H.; Rigling, D.; Faccoli, M.; Iacopetti, G.; Mikulova, A.; Mosbach-Schulz, O.; Stergulc, F.; Gonthier, P.
abstract

The European Commission requested the EFSA Panel on Plant Health to prepare and deliver a scientific opinion on the risk posed by bonsai plants from China consisting of Pinus parviflora grafted on Pinus thunbergii taking into account the available scientific information, including the technical information provided by China. All pests associated with P. parviflora and/or P. thunbergii were evaluated against specific criteria for their relevance for this Scientific Opinion. Forty-three pests that fulfilled all relevant criteria were selected for further evaluation. For 24 pests that are not quarantine in the EU, the risk mitigation measures described in the technical dossier from China were evaluated taking into account the possible limiting factors. For these pests, an expert judgement is given on the likelihood of pest freedom taking into consideration the risk mitigation measures acting on the pest, including uncertainties associated with the assessment. While the estimated degree of pest freedom varied among pests, Setoptus parviflorae was the pest most frequently expected on the commodity. The Expert Knowledge Elicitation indicated, with 95% certainty, that 9,114 or more bonsai plants per 10,000 will be free from Setoptus parviflorae. For 19 pests that are quarantine in the EU, the implementation of specific measures defined in point 30 and 31 of Annex VII of Commission Implementing Regulation (EU) 2019/2072 was evaluated. The requirements of point 31 are met, whereas those of point 30 are not completely fulfilled.

2022 - Commodity risk assessment of grafted plants of Malus domestica from Moldova [Articolo su rivista]
Bragard, C.; Baptista, P.; Chatzivassiliou, E.; Gonthier, P.; Jaques Miret, J. A.; Fejer Justesen, A.; Macleod, A.; Magnusson, C. S.; Milonas, P.; Navas-Cortes, J. A.; Parnell, S.; Potting, R.; Reignault, P. L.; Stefani, E.; Thulke, H. -H.; Van der Werf, W.; Vicent Civera, A.; Zappala, L.; Di Serio, F.; Gomez, P.; Urek, G.; Lucchi, A.; Carluccio, A. V.; Chiumenti, M.; Fanelli, E.; Bernardo, U.; Marzachi, C.; Bubici, G.; de la Pena, E.; Gardi, C.; Yuen, J.
abstract

The European Commission requested the EFSA Panel on Plant Health to prepare and deliver risk assessments for commodities listed in Commission Implementing Regulation (EU) 2018/2019 as ‘High risk plants, plant products and other objects’. This Scientific Opinion covers plant health risks posed by defoliated and in dormant phase, grafted bare rooted plants for planting of Malus domestica imported from Moldova, taking into account the available scientific information, including the technical information provided by the applicant country. A list of 1,118 pests potentially associated with the commodity species was compiled. The relevance of these pests was assessed following defined criteria and based on evidence. The EU-quarantine pest Xiphinema rivesi non-EU populations fulfilled these criteria and was selected for further evaluation. For this pest, the risk mitigation measures proposed in the technical dossier from Moldova were evaluated taking into account the possible limiting factors. For this pest, an expert judgement is given on the likelihood of pest freedom taking into consideration the risk mitigation measures acting on it, including uncertainties associated with the assessment. The Expert Knowledge Elicitation indicated, with 95% certainty, that between 9,991 and 10,000 plants per 10,000 would be free of X. rivesi.

2022 - Commodity risk assessment of specified species of Lonicera potted plants from Turkey [Articolo su rivista]
Bragard, C.; Chatzivassiliou, E.; Di Serio, F.; dos Santos Baptista, P. C.; Gonthier, P.; Jaques Miret, J. A.; Justesen, A. F.; Macleod, A.; Magnusson, C. S.; Milonas, P.; Navas-Cortes, J. A.; Parnell, S.; Reignault, P. L.; Stefani, E.; Thulke, H. -H.; Van der Werf, W.; Vicent Civera, A.; Yuen, J.; Zappala, L.; Debode, J.; Manceau, C.; Gardi, C.; Mosbach-Schulz, O.; Potting, R.
abstract

The European Commission requested the EFSA Panel on Plant Health to prepare and deliver risk assessments for commodities listed in the Commission Implementing Regulation (EU) 2018/2019 as ‘High-risk plants, plant products and other objects’. This Scientific Opinion covers plant health risks posed by potted plants (2–4 years old) of specified Lonicera species produced in nurseries and that are imported from Turkey, taking into account the available scientific information, including the technical information provided by the NPPO of Turkey. The relevance of any pest for this Opinion was based on evidence following defined criteria listed in Section 4.1. Three species, the EU-quarantine pests Lopholeucaspis japonica and Meloidogyne chitwoodi and the protected zone quarantine pest Bemisia tabaci, fulfilled these criteria and were selected for further evaluation. For these pests, the risk mitigation measures proposed in the technical dossier from Turkey were evaluated taking into account the possible limiting factors. For these pests, an expert judgement is given on the likelihood of pest freedom taking into consideration the risk mitigation measures acting on the pest, including uncertainties associated with the assessment. The estimated degree of pest freedom varies among the pests evaluated, with B. tabaci on evergreen species of Lonicera spp. being the pest most frequently expected on the imported plants. The Expert Knowledge Elicitation indicated, with 95% certainty, that between 9,293 and 10,000 plants per 10,000 would be free of B. tabaci.

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.

2022 - Integrating Science on Xanthomonas and Xylella for Integrated Plant Disease Management [Articolo su rivista]
Costa, Joana; Pothier, Joël F; Boch, Jens; Stefani, Emilio; Koebnik, Ralf
abstract

: Present, emerging or re-emerging plant diseases due to infection by bacteria of the Lysobacteraceae (syn: Xanthomonadaceae) family are continually challenging food security and cause significant losses to the economies of European countries each year [...].

2022 - Pest categorisation of Amathynetoides nitidiventris [Articolo su rivista]
Bragard, C.; Baptista, P.; Chatzivassiliou, E.; Di Serio, F.; Gonthier, P.; Jaques Miret, J. A.; Justesen, A. F.; Magnusson, C. S.; Milonas, P.; Navas-Cortes, J. A.; Parnell, S.; Potting, R.; Reignault, P. L.; Stefani, E.; Thulke, H. -H.; Van der Werf, W.; Vicent Civera, A.; Yuen, J.; Zappala, L.; Gregoire, J. -C.; Malumphy, C.; Kertesz, V.; Maiorano, A.; Macleod, A.
abstract

The EFSA Panel on Plant Health performed the pest categorisation of the ulluco weevil, Amathynetoides nitidiventris (Hustache), for the EU territory. This species is not included in EU Commission Implementing Regulation 2019/2072. However, its only substantiated host, ulluco (Ullucus tuberosus), is included in Annex I of Regulation EU 2018/2019 as a high risk plant prohibited from entering the EU, pending risk assessment. In its native Andean region, A. nitidiventris is univoltine, with a lifecycle highly synchronised with the phenology of its host, reproduction and development take place during the development of tubers. Oviposition occurs in the soil. Larvae feed by tunnelling into the tubers, which most of them abandon to pupate in the soil. A minority pupates in the tubers. Because adult A. nitidiventris are often found in other crops due to crop rotations and crop associations, this species has been mistakenly identified as a pest of other crops. In principle soil and tubers of ulluco could provide a pathway for A. nitidiventris into the EU. However, the soil pathway is closed and ulluco tubers are regulated as high risk plants. There are no EU records of interception. Should this weevil enter the EU, the rarity of its host, which is not known to be cultivated in EU MSs, would hamper establishment, spread, and impact. As a consequence, A. nitidiventris does not satisfy all the criteria that are within the remit of EFSA to assess for it to be regarded as a potential Union quarantine pest. The criteria that are not met are the potential for establishment, spread, and economic or environmental consequences in the EU.

2022 - Pest categorisation of Apium virus Y [Articolo su rivista]
Bragard, C.; Gonthier, P.; Jaques Miret, J. A.; Justesen, A. F.; Macleod, A.; Magnusson, C. S.; Milonas, P.; Navas-Cortes, J. A.; Parnell, S.; Potting, R.; Thulke, H. -H.; Van der Werf, W.; Civera, A. V.; Yuen, J.; Zappala, L.; Dehnen-Schmutz, K.; Migheli, Q.; Stefani, Emilio.; Vloutoglou, I.; Czwienczek, E.; Streissl, F.; Chiumenti, M.; Di Serio, F.; Rubino, L.; Reignault, P. L.
abstract

Following a request from the EU Commission, the EFSA Panel on Plant Health conducted a pest categorisation of Apium virus Y (ApVY) for the EU territory. The identity of the ApVY, a member of the genus Potyvirus (family Potyviridae), is well established and reliable detection methods are available. The pathogen is not included in EU Commission Implementing Regulation 2019/2072. ApVY, considered endemic in Australia, was reported also in New Zealand and USA. In the EU, the virus was identified in Germany and Slovenia. No information on adoption of official control measures is available. In natural conditions, ApVY infects plant species of the family Apiaceae (i.e. celery, coriander, dill, parsley, bishop’s weed) in which it generally induces leaf symptoms and/or stunting. In some hosts (i.e. parsley and poison hemlock), ApVY may be asymptomatic. The virus is transmitted in a non-persistent manner by the aphid Myzus persicae which is widespread in the EU. Although ApVY transmission through seeds has been experimentally excluded for some hosts (i.e. poison hemlock and celery), uncertainty exists for the other hosts because seed transmission is not uncommon for potyvirids. Plants for planting, including seeds for sowing, were identified as potential pathways for entry of ApVY into the EU. Cultivated and wild hosts of ApVY are distributed across the EU. Economic impact on the production of the cultivated hosts is expected if further entry and spread in the EU occur. Phytosanitary measures are available to prevent further entry and spread of the virus. Currently, ApVY does not fulfil the criterion of being absent or present with restricted distribution and under official control to be regarded as a potential Union quarantine, unless official control is implemented. This conclusion is associated with high uncertainty regarding the current virus distribution in the EU.

2022 - Pest categorisation of Arboridia kakogawana [Articolo su rivista]
Bragard, C.; Baptista, P.; Chatzivassiliou, E.; Di Serio, F.; Gonthier, P.; Jaques Miret, J. A.; Justesen, A. F.; Magnusson, C. S.; Milonas, P.; Navas-Cortes, J. A.; Parnell, S.; Potting, R.; Reignault, P. L.; Stefani, E.; Thulke, H. -H.; Van der Werf, W.; Civera, A. V.; Yuen, J.; Zappala, L.; Gregoire, J. -C.; Malumphy, C.; Kertesz, V.; Maiorano, A.; Macleod, A.
abstract

The EFSA Panel on Plant Health performed a pest categorisation of the Japanese grape leafhopper, Arboridia kakogawana (Matsumura, 1932) (Hemiptera: Cicadellidae), for the EU territory. This species is not included in the EU Commission Implementing Regulation 2019/2072. Adults of A. kakogawana overwinter in broad-leaved and mixed forests and move to vineyards in the spring where there may be up to four generations, before adults move back to forests during late summer–early autumn to overwinter, possibly under diapause. A. kakogawana has a restricted host range (Vitis spp. and Parthenocissus quinquefolia). It is native to Eastern Asia, from where it moved westwards reaching southern Russia in 1999, and subsequently Ukraine, Romania, Bulgaria and Serbia in 2020. A. kakogawana develops on the abaxial side of the leaves causing chlorotic spots that reduce grape quality. Plants for planting of Vitis L. are banned from entering the EU except from Switzerland, where A. kakogawana is not known to occur. Therefore, this can be considered as a closed entry pathway. However, other plants for planting including the host P. quinquefolia and many broad-leaved trees where overwintering takes place, as well as isolated bark and wood with bark provide potential pathways which are partly regulated but remain open. There are no EU records of interception. Additional introductions and further spread of A. kakogawana into/within the EU, coupled with the ample availability of grapevines and the climatic conditions would most probably allow successful establishment in most EU member states. Should this happen, economic impact in table and wine grapes is anticipated. A. kakogawana satisfies all the criteria that are within the remit of EFSA to assess for it to be regarded as a potential Union quarantine pest (UQP).

2022 - Pest categorisation of Atalodera andina [Articolo su rivista]
Bragard, C.; Baptista, P.; Chatzivassiliou, E.; Di Serio, F.; Gonthier, P.; Jaques Miret, J. A.; Justesen, A. F.; Macleod, A.; Magnusson, C. S.; Milonas, P.; Navas-Cortes, J. A.; Parnell, S.; Potting, R.; Stefani, E.; Thulke, H. -H.; Van der Werf, W.; Civera, A. V.; Yuen, J.; Zappala, L.; Migheli, Q.; Vloutoglou, I.; Maiorano, A.; Streissl, F.; Reignault, P. L.
abstract

The EFSA Panel on Plant Health performed a pest categorisation of Atalodera andina (Nematoda: Heteroderidae) for the European Union (EU) territory. A. andina belongs to the order Rhabditida, subfamily Ataloderinae. This species has not been reported from the EU. It is not included in the EU Commission Implementing Regulation 2019/2072. It is present in the area of the Lake Titicaca of both Peru and Bolivia and in valleys of the region. There is a report in literature stating that specimens were obtained from Chile and identified as A. andina but details on their geographical origin were not given. The identity of A. andina is well established and methods of its identification are available. Natural hosts include the tuber crops Ullucus tuberosus, Oxalis tuberosa and the Andean potato (Solanum tuberosum subsp. andigenum). Experimental hosts include plants of the genus Brassica (such as B. oleracea, B. napus, B. campestris), sugar beet, tomato and clover. Pathways of entry are host plants for planting including seed tubers, subterranean parts of plants intended for consumption, soil as such or attached to plants for planting, machinery or footwear, soil in packaging (bags). Suitable climates exist in the EU but their extent is uncertain and depends on assumptions made on the occurrence of the pest around Lake Titicaca. In the EU, potato, which is grown on about 1,500,000 ha annually, is expected to be the main host of the nematode. Soil and plants for planting are prohibited from import to the EU from third countries where the pest is known to occur. However, this does not cover hosts of A. andina other than species of Solanaceae. The nematode has been reported to damage Andean potato crops, although this has not been quantified. Following its introduction in the EU, A. andina is expected to cause impacts on potato (S. tuberosum subsp. tuberosum), although there is uncertainty on the magnitude of this impact. Also damage on other hosts cannot be excluded. Therefore, the Panel concludes that A. andina satisfies all the criteria that are within the remit of EFSA to assess for it to be regarded as a potential Union quarantine pest.

2022 - Pest categorisation of Aulacaspis tubercularis [Articolo su rivista]
Bragard, C.; Baptista, P.; Chatzivassiliou, E.; Di Serio, F.; Gonthier, P.; Jaques Miret, J. A.; Justesen, A. F.; Magnusson, C. S.; Milonas, P.; Navas-Cortes, J. A.; Parnell, S.; Potting, R.; Reignault, P. L.; Stefani, E.; Thulke, H. -H.; Van der Werf, W.; Civera, A. V.; Yuen, J.; Zappala, L.; Gregoire, J. -C.; Malumphy, C.; Antonatos, S.; Kertesz, V.; Maiorano, A.; Papachristos, D.; Macleod, A.
abstract

The EFSA Panel on Plant Health performed a pest categorisation of Aulacaspis tubercularis (Hemiptera: Diaspididae), the white mango scale, for the EU. A. tubercularis is a tropical species that originates from Asia but is now established in several tropical and subtropical regions throughout the world. It also occurs within the EU and is established in Italy, Portugal and Spain. A. tubercularis is not listed in Commission Implementing Regulation (EU) 2019/2072. It is polyphagous, feeding on plants in more than 37 genera in 23 families and is most frequently reported on mango (Mangifera indica). Indeed, it is considered one of the key pests of mango crops around the world. No evidence was found indicating damage to crops other than mango. A. tubercularis is established in southern Spain (Andalusia) with four overlapping generations and two population peaks, one in summer and another in autumn. Andalusia is the main mango producing area of the EU and A. tubercularis can cause losses through downgrading of fruit. The main natural dispersal stage is the first instar, which crawls over the host plant or may be dispersed further by wind and animals. Plants for planting and fruits provide potential pathways for further entry and spread. Climatic conditions and availability of host plants in southern EU countries are conducive for establishment. Phytosanitary measures are available to reduce the likelihood of further entry and further spread. A. tubercularis satisfies the criteria that are within the remit of EFSA to assess for it to be regarded as a potential Union quarantine pest.

2022 - Pest categorisation of Bagrada hilaris [Articolo su rivista]
Bragard, C.; Baptista, P.; Chatzivassiliou, E.; Di Serio, F.; Gonthier, P.; Jaques Miret, J. A.; Justesen, A. F.; Magnusson, C. S.; Milonas, P.; Navas-Cortes, J. A.; Parnell, S.; Potting, R.; Reignault, P. L.; Stefani, E.; Thulke, H. -H.; Van der Werf, W.; Civera, A. V.; Yuen, J.; Zappala, L.; Gregoire, J. -C.; Malumphy, C.; Kertesz, V.; Maiorano, A.; Macleod, A.
abstract

The EFSA Panel on Plant Health performed a pest categorisation of Bagrada hilaris (Hemiptera: Pentatomidae) for the EU territory. B. hilaris, known as the bagrada bug or painted bug, is a polyphagous pest feeding on at least 25 plant families including several economically important brassica crops such as broccoli, cabbage and cauliflower. Other economically important hosts suffering impacts include beans (Fabaceae), wheat and maize (Poaceae). Young plants are particularly vulnerable to adults and nymphs feeding on tender leaves and growing points, which can cause yield losses. B. hilaris occurs in Africa and Asia and has spread to North America (USA and Mexico) and South America (Chile) where there are multiple generations per year. It is not widely distributed in the EU but has been established in Malta and on the Italian island of Pantelleria, south west of Sicily, since the 1970s where it is an economically important pest of capers. The reasons why it has not spread further within southern Europe are unknown. B. hilaris is not a regulated pest in the EU. It could further enter and spread within the EU via the import and movement of host plants or as a hitchhiking species forming aggregations in conveyances and amongst non-plant traded goods. Host availability and climate suitability suggest that, in addition to Malta and Pantelleria, southern areas of the EU around the Mediterranean would also be suitable for B. hilaris establishment. The introduction of B. hilaris to other Mediterranean areas of the EU would likely cause impacts in a range of crops, particularly brassicas. Measures to prevent entry and spread are available. B. hilaris satisfies all of the criteria that are within the remit of EFSA to assess for it to be regarded as a potential Union quarantine pest.

2022 - Pest categorisation of Capsicum chlorosis virus [Articolo su rivista]
Bragard, C.; Baptista, P.; Chatzivassiliou, E.; Gonthier, P.; Jaques Miret, J. A.; Justesen, A. F.; Macleod, A.; Magnusson, C. S.; Milonas, P.; Navas-Cortes, J. A.; Parnell, S.; Potting, R.; Reignault, P. L.; Stefani, E.; Thulke, H. -H.; Van der Werf, W.; Vicent Civera, A.; Yuen, J.; Zappala, L.; Dehnen-Schmutz, K.; Migheli, Q.; Vloutoglou, I.; Czwienczek, E.; Streissl, F.; Carluccio, A. V.; Chiumenti, M.; Di Serio, F.; Rubino, L.; Reignault, P. L.
abstract

The EFSA Panel on Plant Health conducted a pest categorisation of Capsicum chlorosis virus (CaCV) for the EU territory. The identity of CaCV, a member of the genus Orthotospovirus (family Tospoviridae), is established and reliable detection and identification methods are available. The pathogen is not included in the EU Commission Implementing Regulation 2019/2072. CaCV has been reported in Australia, China, India, Iran, Taiwan, Thailand and USA (Hawaii). In the EU, it has been reported once in Greece (Crete Island). The NPPO of Greece reported that CaCV is no longer present in Greece. CaCV infects plant species in the family Solanaceae (i.e. pepper, tomato) and several species of other families, including ornamentals. It may induce severe symptoms on its hosts, mainly on leaves and fruits, which may become unmarketable. The virus is transmitted in a persistent propagative mode by the thrips Ceratothripoides claratris, Frankliniella schultzei, Microcephalothrips abdominalis and Thrips palmi. C. claratris and T. palmi are EU quarantine pests. M. abdominalis is known to be present in several EU member states and it is not regulated in the EU. Plants for planting, parts of plants, fruits and cut flowers of CaCV hosts, and viruliferous thrips were identified as the most relevant pathways for the entry of CaCV into the EU. Cultivated and wild hosts of CaCV are distributed across the EU. Should the pest enter and establish in the EU territory, impact on the production of cultivated hosts is expected. Phytosanitary measures are available to prevent entry and spread of the virus in the EU. CaCV fulfils the criteria that are within the remit of EFSA to assess for it to be regarded as a potential Union quarantine pest.

2022 - Pest categorisation of Colletotrichum aenigma, C. alienum, C. perseae, C. siamense and C. theobromicola [Articolo su rivista]
Bragard, C.; Baptista, P.; Chatzivassiliou, E.; Di Serio, F.; Gonthier, P.; Jaques Miret, J. A.; Justesen, A. F.; Macleod, A.; Magnusson, C. S.; Milonas, P.; Navas-Cortes, J. A.; Parnell, S.; Potting, R.; Reignault, P. L.; Stefani, E.; Thulke, H. -H.; Van der Werf, W.; Vicent Civera, A.; Yuen, J.; Zappala, L.; Migheli, Q.; Vloutoglou, I.; Czwienczek, E.; Maiorano, A.; Streissl, F.; Reignault, P. L.
abstract

The EFSA Plant Health Panel performed a pest categorisation of Colletotrichum aenigma, C. alienum, C. perseae, C. siamense and C. theobromicola, five clearly defined fungi of the C. gloeosporioides complex causing anthracnose. The pathogens are widely distributed in at least three continents. C. aenigma and C. siamense are reported from Italy and C. alienum from Portugal, including the Madeira Islands, with a restricted distribution. C. perseae and C. theobromicola are not known to be present in the EU. However, there is uncertainty on the status of the pathogens worldwide and in the EU because of the taxonomic re-evaluation of the genus Colletotrichum and the lack of specific surveys. The pathogens are not included in Commission Implementing Regulation (EU) 2019/2072 and there are no reports of interceptions in the EU. With the exception of C. perseae, which has a very limited number of hosts, the other four Colletotrichum species have relatively wide host ranges. Therefore, this pest categorisation focused on those hosts for which there is robust evidence that the pathogens were formally identified by a combination of morphology, pathogenicity and multilocus sequence analysis. Host plants for planting and fresh fruits are the main entry pathways into the EU. Host availability and climate suitability factors occurring in some parts of the EU are favourable for the establishment of the pathogens. No yield losses have been reported so far in the EU but in non-EU areas of their current distribution, the pathogens have a direct impact on cultivated hosts that are also relevant for the EU. Phytosanitary measures are available to prevent the further introduction and spread of C. aenigma, C. alienum and C. siamense into the EU as well as the introduction and spread of C. perseae and C. theobromicola. C. aenigma, C. alienum, C. perseae, C. siamense and C. theobromicola satisfy the criteria that are within the remit of EFSA to assess for these species to be regarded as potential Union quarantine pests.

2022 - Pest categorisation of Dendrolimus superans [Articolo su rivista]
Bragard, C.; Baptista, P.; Chatzivassiliou, E.; Di Serio, F.; Gonthier, P.; Jaques Miret, J. A.; Justesen, A. F.; Magnusson, C. S.; Milonas, P.; Navas-Cortes, J. A.; Parnell, S.; Potting, R.; Reignault, P. L.; Stefani, E.; Thulke, H. -H.; Van der Werf, W.; Vicent Civera, A.; Yuen, J.; Zappala, L.; Gregoire, J. -C.; Malumphy, C.; Kertesz, V.; Maiorano, A.; Macleod, A.
abstract

The EFSA Panel on Plant Health performed a pest categorisation of Dendrolimus superans Butler (Lepidoptera: Lasiocampidae), the larch caterpillar, for the EU territory. D. superans is a major pest of conifer forests in Japan, northeast China and non-European Russia. However, reports of damage are to conifer species not grown in EU forestry. Larix gmelinii and Pinus pumila are regarded as major hosts. Eggs are laid on host needles and developing larvae feed on host foliage. Larvae overwinter in the soil. In its native range, D. superans usually takes one or two years to develop. In principle, host plants for planting and plant products, such as cut branches and wood with bark, could provide pathways into the EU. However, prohibitions on the import of Abies, Cedrus, Larix, Picea, Pinus and Tsuga from areas where D. superans occurs closes such pathways. Nevertheless, a derogation for specific dwarfed Pinus plants from Japan exists. Climates similar to those in some of its existing range occur in the EU. Norway spruce (Picea abies) is a known host in Japan although reports of any impact are lacking. Experiments on the related species D. sibiricus indicated that larvae were able to develop on forestry conifer species occurring in the EU, but which are found outside the native range of D. sibiricus. Were D. superans to be introduced into the EU, impacts on P. abies are possible and it is conceivable that D. superans could expand its host range, as seems possible with D. sibiricus. However, this remains uncertain. Other hosts are grown in the EU as ornamentals or amenity trees. D. superans satisfies all the criteria that are within the remit of EFSA to assess for it to be regarded as a potential Union quarantine pest. Some uncertainty exists over the magnitude of potential environmental and economic impacts.

2022 - Pest categorisation of Fusarium oxysporum f. sp. cubense Tropical Race 4 [Articolo su rivista]
Bragard, C.; Baptista, P.; Chatzivassiliou, E.; Di Serio, F.; Gonthier, P.; Jaques Miret, J. A.; Justesen, A. F.; Macleod, A.; Magnusson, C. S.; Milonas, P.; Navas-Cortes, J. A.; Parnell, S.; Potting, R.; Stefani, E.; Thulke, H. -H.; Van der Werf, W.; Civera, A. V.; Yuen, J.; Zappala, L.; Migheli, Q.; Vloutoglou, I.; Maiorano, A.; Streissl, F.; Reignault, P. L.
abstract

The EFSA Plant Health Panel performed a pest categorisation of Fusarium oxysporum f. sp. cubense Tropical Race 4 (Foc TR4), an ascomycete fungus causing Fusarium wilt (Panama disease) on Musa spp. Foc TR4 is pathogenic to the commercial banana varieties including those of the ‘Cavendish’ group and is considered as the most destructive among Foc haplotypes. Uncertainty exists on the host range of Foc TR4, since it has not been demonstrated whether it can infect plant species other than Musa spp., which were previously reported as hosts of other Foc races. Foc TR4 is morphologically and physiologically identical to other representatives of the Fusarium oxysporum Species Complex (FOSC), but all Foc TR4 isolates belong to a single clonal lineage within the vegetative compatibility groups 01213-01216. Several PCR protocols are described in the literature, but their specificity has been questioned as they may generate false positives. The pathogen is not included in EU Commission Implementing Regulation 2019/2072 and is not reported as present in the EU territory. Several potential entry pathways and means of spread were identified, including host plants for planting other than vitroplants, fresh fruits and leaves of host plants, soil and other substrates originating in infested third countries. Host availability and climate suitability occurring in some areas of the EU are favourable for the establishment of Foc TR4. Being a soil-borne pathogen, eradication of Foc TR4 once it enters a new area is very difficult. Therefore, effective quarantine measures are essential in pathogen-free areas. Although not specifically targeting against Foc TR4, phytosanitary measures are currently available to prevent the introduction of the pathogen into the EU. Considering that banana-growing EU countries account for over 12% of the EU banana supply, it is expected that the economic impact of Foc TR4 on the European banana production areas would be devastating. Foc TR4 satisfies the criteria that are within the remit of EFSA to assess for this pathogen to be regarded as a potential Union quarantine pest.

2022 - Pest categorisation of Fusarium pseudograminearum [Articolo su rivista]
Bragard, C.; Baptista, P.; Chatzivassiliou, E.; Di Serio, F.; Gonthier, P.; Jaques Miret, J. A.; Justesen, A. F.; Macleod, A.; Magnusson, C. S.; Milonas, P.; Navas-Cortes, J. A.; Parnell, S.; Potting, R.; Stefani, E.; Thulke, H. -H.; Van der Werf, W.; Civera, A. V.; Yuen, J.; Zappala, L.; Migheli, Q.; Vloutoglou, I.; Czwienczek, E.; Maiorano, A.; Streissl, F.; Reignault, P. L.
abstract

The EFSA Plant Health Panel performed a pest categorisation of Fusarium pseudograminearum O'Donnell & T. Aoki. F. pseudograminearum is a soil-borne fungal pathogen, able to cause a disease known as Fusarium crown rot (FCR, also known as foot and root rot) and occasionally Fusarium head blight on small grain cereals, particularly Triticum aestivum L., Triticum turgidum L. spp. durum (Dest.), Hordeum vulgare L. and triticale (xTriticosecale). In addition, F. pseudograminearum has been isolated from soybean (Glycine max L.) and from some grass genera, such as Phalaris, Agropyron and Bromus, which represent potentially important inoculum reservoirs. This pathogen has been reported in arid and semi-arid cropping regions in Australia, New Zealand, North and South America, northern Africa and South Africa, the Middle East and Asia. In the EU, it has been reported in Italy since 1994 and later in Spain on field-grown durum wheat, but uncertainty remains regarding the actual distribution of the pathogen in the EU. The pathogen is not included in the EU Commission Implementing Regulation 2019/2072. Seeds of host plants and soil and other substrates are the main pathways for the entry and spread of the pathogen into the EU. There are no reports of interceptions of F. pseudograminearum in the EU. Host availability and climate suitability occurring in the EU favour establishment of the pathogen and allow it to establish in areas from which it has not been reported. Phytosanitary measures are available to prevent the introduction of the pathogen into the EU, and additional measures are available to mitigate the risk of spread. In the non-EU areas of its present distribution, the pathogen has a direct impact on cultivated hosts (e.g. wheat, barley, triticale and soybean) that are also relevant for the EU. However, no crop losses have been reported so far in the EU. The Panel concludes that F. pseudograminearum satisfies all the criteria to be regarded as a potential Union quarantine pest.

2022 - Pest categorisation of High Plains wheat mosaic virus [Articolo su rivista]
Bragard, C.; Baptista, P.; Chatzivassiliou, E.; Di Serio, F.; Gonthier, P.; Jaques Miret, J. A.; Justesen, A. F.; Macleod, A.; Magnusson, C. S.; Milonas, P.; Navas-Cortes, J. A.; Parnell, S.; Potting, R.; Reignault, P. L.; Stefani, E.; Thulke, H. -H.; Van der Werf, W.; Vicent Civera, A.; Yuen, J.; Zappala, L.; Dehnen-Schmutz, K.; Migheli, Q.; Vloutoglou, I.; Streissl, F.; Chiumenti, M.; Di Serio, F.; Rubino, L.; Reignault, P. L.
abstract

The EFSA Panel on Plant Health conducted a pest categorisation of High Plains wheat mosaic virus (HPWMoV) for the EU territory. The identity of HPWMoV, a member of the genus Emaravirus (family Fimoviridae), is well established and reliable identification methods are available. The pathogen is not included in the EU Commission Implementing Regulation 2019/2072. HPWMoV has been reported from Argentina, Australia, Canada, Ukraine and USA, and it is not known to be present in the EU. HPWMoV infects plant species of the family Poaceae (i.e. wheat, maize and several other cultivated or wild Poaceae species). It is the causal agent of High Plains disease of wheat and maize, inducing symptoms ranging from mild to severe mosaic, chlorosis and necrosis in wheat, and chlorotic streaks in maize plants. The virus is transmitted by the wheat curl mite Aceria tosichella, which is present in the EU. HPWMoV transmission via seeds was reported to occur in sweet corn. Sweet corn seeds for sowing were identified as the most relevant pathway for entry of HPWMoV into the EU. Seeds from other hosts and viruliferous wheat curl mites were identified as entry pathways associated with uncertainties. Machinery not appropriately cleaned may move infected seeds and/or parts of cereals infested by viruliferous mites. Cultivated and wild hosts of HPWMoV are distributed across the EU. Would the pest enter and establish in the EU territory, economic impact on the production of cultivated hosts is expected. Phytosanitary measures are available to prevent entry and spread of the virus in the EU. HPWMoV fulfils the criteria that are within the remit of EFSA to assess for it to be regarded as a potential Union quarantine pest.

2022 - Pest categorisation of Maconellicoccus hirsutus [Articolo su rivista]
Bragard, C.; Baptista, P.; Chatzivassiliou, E.; Di Serio, F.; Gonthier, P.; Jaques Miret, J. A.; Justesen, A. F.; Sven Magnusson, C.; Milonas, P.; Navas-Cortes, J. A.; Parnell, S.; Potting, R.; Reignault, P. L.; Stefani, E.; Thulke, H. -H.; Van der Werf, W.; Civera, A. V.; Yuen, J.; Zappala, L.; Gregoire, J. -C.; Malumphy, C.; Antonatos, S.; Kertesz, V.; Maiorano, A.; Papachristos, D.; Macleod, A.
abstract

The EFSA Panel on Plant Health performed a pest categorisation of Maconellicoccus hirsutus (Hemiptera: Pseudococcidae), the pink hibiscus mealybug, for the EU. M. hirsutus is native to Southern Asia and has established in many countries in tropical and subtropical regions throughout the world. Within the EU, the pest has been reported from Cyprus and Greece (Rhodes). M. hirsutus is not listed in Annex II of Commission Implementing Regulation (EU) 2019/2072. It is highly polyphagous, feeding on plants assigned to 229 genera in 78 plant families, and shows some preference for hosts in the families Malvaceae, Fabaceae and Moraceae. Economically important crops in the EU such as cotton (Gossypium spp.), citrus (Citrus spp.), ornamentals (Hibiscus spp.), grapes (Vitis vinifera), soybean (Glycinae max), avocado (Persea americana) and mulberry trees (Morus alba) may be significantly affected by M. hirsutus. The lower and upper developmental temperature threshold of M. hirsutus on Hibiscus rosa-sinensis are 14.5 and 35.0°C, respectively, with optimal female development estimated to be at 29.0°C. There are about 10 generations a year in the subtropics but as many as 15 may occur under optimal conditions. Plants for planting, fruits, vegetables and cut flowers provide potential pathways for entry into the EU. Climatic conditions in EU member states around the Mediterranean Sea and host plant availability in those areas are conducive for establishment. The introduction of M. hirsutus is expected to have an economic impact in the EU through damage to various ornamental plants, as already observed in Cyprus and Greece, and reduction in yield and quality of many significant crops. Phytosanitary measures are available to reduce the likelihood of entry and further spread. Some uncertainties include the area of establishment, whether it could become a greenhouse pest, impact, and the influence of natural enemies. M. hirsutus meets the criteria that are within the remit of EFSA to assess for it to be regarded as a potential Union quarantine pest.

2022 - Pest categorisation of Malacosoma disstria [Articolo su rivista]
Bragard, C.; Baptista, P.; Chatzivassiliou, E.; Di Serio, F.; Gonthier, P.; Jaques Miret, J. A.; Justesen, A. F.; Magnusson, C. S.; Milonas, P.; Navas-Cortes, J. A.; Parnell, S.; Potting, R.; Reignault, P. L.; Stefani, E.; Thulke, H. -H.; Van der Werf, W.; Vicent Civera, A.; Yuen, J.; Zappala, L.; Gregoire, J. -C.; Malumphy, C.; Kertesz, V.; Maiorano, A.; Macleod, A.
abstract

The European Commission requested the EFSA Panel on Plant Health to conduct a pest categorisation of Malacosoma disstria Hübner (Lepidoptera: Lasiocampidae), commonly known as the forest tent caterpillar, for the territory of the EU. M. disstria is a North American polyphagous leaf-eating pest primarily feeding on deciduous trees belonging to the genera Acer, Malus, Populus, Prunus, Quercus and Tilia. It is a univoltine species. Eggs are laid on twigs and branches. Larvae emerge in the spring to feed on buds and fresh leaves. Host plants can be completely defoliated although they often refoliate and recover within a few weeks. Nevertheless, three consecutive years of heavy defoliation or repeated periods of defoliation combined with drought can cause extensive tree mortality. As such, M. disstria is regarded as one of the most serious hardwood forestry insect pests in North America. Population upsurges leading to outbreaks are cyclical, generally nine to 13 years apart and can last 2–3 years. Outbreaks have been reported in eastern North America since the late 18th century. Outbreaks in western Canada have spanned up to 200,000 km2. Plants for planting, cut branches and isolated bark provide pathways for entry. Host availability and climate suitability suggest that large parts of the EU would be suitable for establishment. The pest could spread naturally by flight within the EU. Eggs on plants for planting could also facilitate spread. The introduction of M. disstria into the EU could lead to serious outbreaks causing significant damage to forest, orchard and amenity trees and shrubs. Phytosanitary measures are available to inhibit the entry and spread of this species. M. disstria satisfies the criteria that are within the remit of EFSA to assess for it to be regarded as a potential Union quarantine pest.

2022 - Pest categorisation of Oligonychus perseae [Articolo su rivista]
Bragard, C.; Baptista, P.; Chatzivassiliou, E.; Di Serio, F.; Gonthier, P.; Jaques Miret, J. A.; Justesen, A. F.; Magnusson, C. S.; Milonas, P.; Navas-Cortes, J. A.; Parnell, S.; Potting, R.; Reignault, P. L.; Stefani, E.; Thulke, H. -H.; Van der Werf, W.; Vicent Civera, A.; Yuen, J.; Zappala, L.; Gregoire, J. -C.; Malumphy, C.; Kertesz, V.; Maiorano, A.; Macleod, A.
abstract

The EFSA Panel on Plant Health performed a pest categorisation of Oligonychus perseae (Acari: Prostigmata: Tetranychidae), the persea mite, for the EU. O. perseae is a tropical species that originated in Mesoamerica and has now spread and established in California, Florida, Hawaii, Morocco, southern Europe and Israel. Within the EU, it is established in Italy, Portugal and Spain. O. perseae is not listed in Commission Implementing Regulation (EU) 2019/2072. It is polyphagous, feeding on plants in 20 genera in 17 families. It is most frequently reported on avocado (Persea americana), where it is considered a key pest. No evidence was found indicating damage to other crops. O. perseae live on leaves and do not attack the fruit. Populations usually grow exponentially at the beginning of summer and decline at the end of this season. High population densities can cause severe defoliation, resulting in downgrading of fruit through sunburn. However, this type of damage is common only if trees additionally suffer from water stress. The lack of additional avocado pests in the EU, which facilitates the production of organic avocados, is jeopardised by the occurrence of this mite, as it may require pesticide applications. This is why O. perseae is considered an important pest of avocados in Spain, where more than 80% of EU avocado production occurs. Natural dispersal is restricted to neighbouring trees. However, human-assisted movement can result in long-distance spread. Plants for planting provide potential pathways for further entry and spread, including O. perseae-free EU MS where avocados are grown (i.e. Cyprus, France, Greece). Climatic conditions and availability of host plants in southern EU countries are conducive for establishment. Phytosanitary measures are available to reduce the likelihood of further entry and spread. O. perseae satisfies with no key uncertainties the criteria that are within the remit of EFSA to assess for it to be regarded as a potential Union quarantine pest.

2022 - Pest categorisation of Platypus apicalis [Articolo su rivista]
Bragard, C.; Baptista, P.; Chatzivassiliou, E.; Di Serio, F.; Gonthier, P.; Jaques Miret, J. A.; Justesen, A. F.; Magnusson, C. S.; Milonas, P.; Navas-Cortes, J. A.; Parnell, S.; Potting, R.; Reignault, P. L.; Stefani, E.; Thulke, H. -H.; Van der Werf, W.; Vicent Civera, A.; Yuen, J.; Zappala, L.; Gregoire, J. -C.; Malumphy, C.; Kertesz, V.; Maiorano, A.; Macleod, A.
abstract

The EFSA Panel on Plant Health performed a pest categorisation of Platypus apicalis (Coleoptera: Curculionidae: Platypodinae), an ambrosia beetle, also known as a pinhole borer, for the EU territory. P. apicalis is a polyphagous pest native to New Zealand. The majority of its life cycle is spent inside tree wood, but it does not directly feed on plant tissue, instead larvae and adults feed on a symbiotic fungus (Sporothrix nothofagi which is pathogenic to Nothofagus spp.) vectored by adults and introduced when they bore tunnels into the host. P. apicalis feeds within a wide range of live, often stressed trees, in dead or dying hardwood and softwood trees, and fallen or felled trees. Successful reproduction can occur inside a number of living tree species including Castanea sativa, Pinus spp. and Ulmus spp. P. apicalis is not known to have established outside of New Zealand although findings have been reported in Australia. Whilst there are no records of interceptions of this species in the EU, platypodines are intercepted with solid wood packing material (SWPM) and Platypus species, but not P. apicalis, have been intercepted with wooden logs in Japan. Host plants for planting also provide a potential pathway. Hosts are grown widely across the EU in areas with climates comparable to those in New Zealand where the pest occurs suggesting that conditions in the EU are suitable for its establishment. If introduced into the EU, adults could disperse naturally by flight, perhaps tens or hundreds of metres. The movement of infested wood and host plants for planting within the EU could facilitate spread. Economic impacts in forestry and timber industries would result from the galleries created by P. apicalis and from wood staining caused by the symbiotic fungus. Phytosanitary measures are available to inhibit the entry of P. apicalis. P. apicalis satisfies the criteria that are within the remit of EFSA to assess for it to be regarded as a potential Union quarantine pest.

2022 - Pest categorisation of Plicosepalus acaciae [Articolo su rivista]
Bragard, C.; Baptista, P.; Chatzivassiliou, E.; Di Serio, F.; Gonthier, P.; Jaques Miret, J. A.; Fejer Justesen, A.; Macleod, A.; Magnusson, C. S.; Milonas, P.; Navas-Cortes, J. A.; Parnell, S.; Potting, R.; Stefani, E.; Thulke, H. -H.; Van der Werf, W.; Vicent Civera, A.; Yuen, J.; Zappala, L.; Dehnen-Schmutz, K.; Migheli, Q.; Vloutoglou, I.; Maiorano, A.; Streissl, F.; Reignault, P. L.
abstract

The EFSA Panel on Plant Health performed a pest categorisation of Plicosepalus acaciae (Zuccarini) Wiens & Polhill), the acacia strap flower, a hemiparasitic plant of the family Loranthaceae parasitising woody plants. Host plants include several species of the genera Vachellia, Tamarix and Ziziphus and various fruit crops. P. acaciae is present in the Middle East and Eastern Africa and is not known to occur in the EU. P. acaciae has a long flowering period of about 10 months, from June to April the following year, during which flowers are pollinated by insects and birds. P. acaciae produces single seeded red berries that are eaten by birds, which then disseminate the seeds. The only known bird observed to disseminate the seeds of P. acaciae is Pycnonotus xanthopygos, which has been recorded just once (Spain) but it is not established in the EU. P. acaciae could enter into the EU with host plants for planting. Host plants are present and suitable climatic conditions occur in parts of the EU. If a suitable bird would adapt to transfer the seeds, establishment and spread of P. acaciae within the EU would be possible. If P. acaciae would be able to establish and spread, impacts on some crop plants (e.g. Juglans regia, Ficus carica, Punica granatum, Pistacia vera), ornamental plants, as well as native vegetation could occur. P. acaciae fulfils the criteria that are within the remit of EFSA to assess for this species to be regarded as a potential Union quarantine pest. Uncertainty remains on bird species other than P. xanthopygos transferring P. acaciae, the magnitude of potential impacts and the host range.

2022 - Pest categorisation of Pseudococcus cryptus [Articolo su rivista]
Bragard, C.; Baptista, P.; Chatzivassiliou, E.; Di Serio, F.; Gonthier, P.; Jaques Miret, J. A.; Justesen, A. F.; Magnusson, C. S.; Milonas, P.; Navas-Cortes, J. A.; Parnell, S.; Potting, R.; Reignault, P. L.; Stefani, E.; Thulke, H. -H.; Van der Werf, W.; Vicent Civera, A.; Yuen, J.; Zappala, L.; Gregoire, J. -C.; Malumphy, C.; Antonatos, S.; Kertesz, V.; Maiorano, A.; Papachristos, D.; Macleod, A.
abstract

The EFSA Panel on Plant Health performed a pest categorisation of Pseudococcus cryptus Hempel (Hemiptera: Pseudococcidae), the citriculus mealybug, for the EU. P. cryptus originates from Southeast Asia but is now established in East Africa, the Middle East and South America. The pest is not currently known to occur in the EU (there was a record once, in 2006, in a zoo/botanical garden from southern Spain). P. cryptus is not listed in Commission Implementing Regulation (EU) 2019/2072. It is polyphagous, feeding on plants in more than 90 genera in 51 families, and exhibits a preference for citrus (Citrus spp.) and palms (especially Cocos nucifera, Elaeis guineensis and Areca catechu). It is an important pest of citrus in Japan and parts of the Middle East, although in Israel, it is controlled by natural enemies. It is sexually reproductive, has six overlapping generations each year in Israel, and each female lays up to approximately 150 eggs, depending on temperature and host species. The main natural dispersal stage is the first instar, which crawls over the host plant or may be dispersed further by wind and animals. Plants for planting, fruits, vegetables and cut flowers provide potential pathways for entry into the EU. Climatic conditions in EU member states around the Mediterranean Sea where there is host plant availability, especially citrus, are conducive for establishment. The introduction of P. cryptus is expected to have an economic impact in the EU through reduction in yield and quality of important crops (mainly citrus) and damage to various ornamental plants. Phytosanitary measures are available to reduce the likelihood of entry and further spread. P. cryptus meets the criteria that are within the remit of EFSA to assess for this species to be regarded as a potential Union quarantine pest.

2022 - Pest categorisation of Pulvinaria psidii [Articolo su rivista]
Bragard, C.; Baptista, P.; Chatzivassiliou, E.; Di Serio, F.; Gonthier, P.; Jaques Miret, J. A.; Justesen, A. F.; Magnusson, C. S.; Milonas, P.; Navas-Cortes, J. A.; Parnell, S.; Potting, R.; Reignault, P. L.; Stefani, E.; Thulke, H. -H.; Van der Werf, W.; Vicent Civera, A.; Yuen, J.; Zappala, L.; Gregoire, J. -C.; Malumphy, C.; Akrivou, A.; Kertesz, V.; Maiorano, A.; Papachristos, D.; Macleod, A.
abstract

The EFSA Panel on Plant Health performed a pest categorisation of Pulvinaria psidii (Hemiptera: Coccidae), the green shield scale, for the EU. P. psidii was originally described from Hawaii on Psidium sp. and it is now established in many countries in tropical and subtropical regions of the world. Within the EU, the pest has been reported from mainland Spain and the Canary Islands. P. psidii is not listed in Annex II of Commission Implementing Regulation (EU) 2019/2072. It is highly polyphagous, feeding on 230 plant species belonging to more than 70 botanical families with preference for avocado (Persea americana), citrus (Citrus spp.), coffee (Coffea sp.), guava (Psidium guajava), litchi (Litchi chinensis), mango (Mangifera indica), mulberry (Morus sp.) and pomegranate (Punica granatum). It has also been recorded feeding on some solanaceous plants: tomato (Solanum lycopersicum) and pepper (Capsicum annuum), as well as on ornamental plants. Climatic conditions and availability of host plants in southern EU countries would most probably allow this species to successfully establish and spread. Economic impact in cultivated hosts including citrus, mangoes, mulberries, as well as vegetable and ornamental crops is anticipated if establishment occurs. Indeed, P. psidii has already been reported causing damage to Melia azedarach, a widely used ornamental tree that lines streets in Valencia. There is contradictory information regarding impact in mangoes in Spain. This could be due to the relatively recent establishment of the pest. Phytosanitary measures are available to reduce the likelihood of entry and further spread. P. psidii meets the criteria that are within the remit of EFSA to assess for this species to be regarded as a potential Union quarantine pest.

2022 - Pest categorisation of Russellaspis pustulans [Articolo su rivista]
Bragard, C.; Baptista, P.; Chatzivassiliou, E.; Di Serio, F.; Gonthier, P.; Jaques Miret, J. A.; Justesen, A. F.; Macleod, A.; Magnusson, C. S.; Milonas, P.; Navas-Cortes, J. A.; Parnell, S.; Potting, R.; Reignault, P. L.; Stefani, E.; Thulke, H. -H.; Van der Werf, W.; Civera, A. V.; Yuen, J.; Zappala, L.; Gregoire, J. -C.; Malumphy, C.; Kertesz, V.; Maiorano, A.; Macleod, A.
abstract

The EFSA Panel on Plant Health performed a pest categorisation of Russellaspis pustulans (Hemiptera: Asterolecaniidae), the oleander pit scale, for the EU. R. pustulans occurs widely in tropical and subtropical areas of the world and is restricted to indoor plantings in cooler temperate regions. Within the EU, it has been reported in some literature from Cyprus, Italy and Malta though not confirmed by the NPPOs. R. pustulans is not listed in Commission Implementing Regulation (EU) 2019/2072. It is very polyphagous, feeding on plants in 69 families and exhibits a preference for fig (Ficus carica) and oleander (Nerium oleander). R. pustulans was observed completing up to three generations per year in Egypt, with peaks of presence in June, October and December. The main natural dispersal stage is the first instar, which crawls over the host plant or may be dispersed further by wind and animals. Plants for planting, cut branches and fruits provide potential pathways for entry into the EU. Climatic conditions in some parts of southern EU countries are favourable and host plants are available in those areas to support establishment. However, the magnitude of impact following introduction is uncertain. Phytosanitary measures are available to reduce the likelihood of entry and further spread. R. pustulans does meet the criteria that are within the remit of EFSA to assess for it to be regarded as a potential Union quarantine pest.

2022 - Pest categorisation of Sirex nitobei [Articolo su rivista]
Bragard, C.; Baptista, P.; Chatzivassiliou, E.; Di Serio, F.; Gonthier, P.; Jaques Miret, J. A.; Justesen, A. F.; Magnusson, C. S.; Milonas, P.; Navas-Cortes, J. A.; Parnell, S.; Potting, R.; Reignault, P. L.; Stefani, E.; Thulke, H. -H.; Van der Werf, W.; Vicent Civera, A.; Yuen, J.; Zappala, L.; Gregoire, J. -C.; Malumphy, C.; Kertesz, V.; Maiorano, A.; Macleod, A.
abstract

The EFSA Panel on Plant Health performed a pest categorisation of Sirex nitobei (Hymenoptera: Siricidae), the nitobe horntail, for the territory of the EU. S. nitobei is not listed in Annex II of Commission Implementing Regulation (EU) 2019/2072 but was identified as a potential regulated pest in a commodity risk assessment of Pinus thunbergii artificially dwarfed plants from Japan. This species is present in Japan (except Hokkaidō), the Republic of Korea and 13 Chinese provinces. S. nitobei attacks several Pinus species and has been reported less frequently on Abies firma and Larix spp., including L. leptolepis. The females oviposit into the sapwood. Eggs are deposited together with a phytotoxic mucus and a symbiotic fungus, Amylostereum areolatum or A. chailletii. The combined action of the venom and the fungus results in the death of the host trees. The fungus degrades the lignocellulosic components of the wood, and the larvae feed on the liquid fraction of the digested residues left by the fungus. All immature stages live in the hosts sapwood. The lifecycle of the pest lasts 1 year. S. nitobei can travel with conifer wood, wood packaging material or plants for planting, but these pathways from third countries are closed by prohibition. However, a derogation exists for artificially dwarfed Japanese black pine (Pinus thunbergii) from Japan, which therefore provides a potential pathway. Climatic conditions in several EU member states and host plant availability in those areas are conducive for establishment. The introduction of S. nitobei is potentially damaging for pines. Phytosanitary measures are available to reduce the likelihood of entry and further spread, and there is a potential for biological control. S. nitobei satisfies all the criteria that are within the remit of EFSA to assess for it to be regarded as a potential Union quarantine pest.

2022 - Pest categorisation of Tetraleurodes perseae [Articolo su rivista]
Bragard, C.; Baptista, P.; Chatzivassiliou, E.; Di Serio, F.; Gonthier, P.; Jaques Miret, J. A.; Justesen, A. F.; Magnusson, C. S.; Milonas, P.; Navas-Cortes, J. A.; Parnell, S.; Potting, R.; Reignault, P. L.; Stefani, E.; Thulke, H. -H.; Van der Werf, W.; Vicent Civera, A.; Yuen, J.; Zappala, L.; Gregoire, J. -C.; Malumphy, C.; Kertesz, V.; Maiorano, A.; Macleod, A.
abstract

The EFSA Panel on Plant Health performed a pest categorisation of Tetraleurodes perseae (Hemiptera: Aleyrodidae), the red-banded whitefly, for the territory of the EU. T. perseae is a tropical and subtropical species that originated in the Neotropical region and has now spread and established in the USA (California and Florida), Israel and Lebanon. T. perseae is not listed in Commission Implementing Regulation (EU) 2019/2072. It is oligophagous on Lauraceae and most frequently reported on avocado (Persea americana), on which it is considered a minor or secondary pest. No evidence was found indicating damage to other plants. T. perseae larvae develop on the foliage and don’t attack the fruit. The number of generations per year varies between one and ten. High populations may promote the growth of black sooty moulds on the foliage and fruit, and adults feeding on the buds can lead to deformed immature leaves and premature leaf drop. However, T. perseae populations are usually effectively controlled by hymenopteran parasitoids, at least one of which (Cales noacki) is widespread in the EU. The producers of organic avocados in the EU could encourage the use of C. noacki, although occasional outbreaks of T. perseae could temporarily impact the fruit quality. Adults disperse naturally by flying and all stages can be moved over long distances by the trade of infested plant material. Plants for planting provide potential pathways for entry and spread in the EU. Climatic conditions and availability of host plants in southern EU countries are conducive for establishment. Phytosanitary measures are available to reduce the risk. T. perseae satisfies all of the criteria that are within the remit of EFSA to assess for it to be regarded as a potential Union quarantine pest. However, this conclusion has a high uncertainty regarding magnitude of potential impact as the insect is a minor and sporadic pest in its current area of distribution.

2022 - Pest categorisation of Thecodiplosis japonensis [Articolo su rivista]
Bragard, C.; Baptista, P.; Chatzivassiliou, E.; Di Serio, F.; Gonthier, P.; Jaques Miret, J. A.; Justesen, A. F.; Magnusson, C. S.; Milonas, P.; Navas-Cortes, J. A.; Parnell, S.; Potting, R.; Reignault, P. L.; Stefani, E.; Thulke, H. -H.; Van der Werf, W.; Civera, A. V.; Yuen, J.; Zappala, L.; Gregoire, J. -C.; Malumphy, C.; Kertesz, V.; Maiorano, A.; Macleod, A.
abstract

The EFSA Panel on Plant Health performed a pest categorisation of Thecodiplosis japonensis (Diptera: Cecidomyiidae) for the EU territory. This species is not included in the EU Commission Implementing Regulation 2019/2072. T. japonensis Uchida & Inouye is a well-defined species, native to a large part of Japan, which was introduced to the Republic of Korea and eastern China: Fujian and Shandong. It attacks Pinus densiflora, P. thunbergii and P. luchuensis in Japan; P. densiflora and P. thunbergii in Korea; and P. massoniana in China, and has been observed to attack other two-needle pine species, including species present in the EU. The pest is univoltine and the adults emerge between May and August. The adults live only for 1 day. Each female oviposits in batches on developing needles. The neonate larvae crawl to the base of the needle fascicle and create a gall in which they feed gregariously by sap sucking. The third-instar larvae leave the galls in November, overwinter in a cocoon in the soil and pupate at the end of the winter. Degree day models have been developed to predict adult emergence. Survival of overwintering stages is poor below 15°C and above 30°C. The pest can be detected by its symptoms (stunted or dead needles, galls at the base of infected needle fascicles), and identified using morphological characters or the mitochondrial COI gene. T. japonensis is one of the major forest pests in the Republic of Korea, where 1.7 million trees were cut to control it in 2014–2015. It flies uneasily (a few hundred metres) but can be transported in galls on Pinus plants for planting, including artificially dwarfed plants, or with cut branches. Climate matching and host tree distribution suggest that T. japonensis would be able to establish and have an impact in the EU territory. T. japonensis satisfies all the criteria that are within the remit of EFSA to assess for it to be regarded as a potential Union quarantine pest.

2022 - Pest categorisation of Toumeyella parvicornis [Articolo su rivista]
Bragard, C.; Baptista, P.; Chatzivassiliou, E.; Di Serio, F.; Gonthier, P.; Jaques Miret, J. A.; Fejer Justesen, A.; Magnusson, C. S.; Milonas, P.; Navas-Cortes, J. A.; Parnell, S.; Potting, R.; Reignault, P. L.; Stefani, E.; Thulke, H. -H.; Van der Werf, W.; Vicent Civera, A.; Yuen, J.; Zappala, L.; Gregoire, J. -C.; Malumphy, C.; Kertesz, V.; Maiorano, A.; Macleod, A.
abstract

The EFSA Panel on Plant Health performed a pest categorisation of Toumeyella parvicornis (Cockerell) (Hemiptera: Coccidae) for the EU territory. This species is not included in EU Commission Implementing Regulation 2019/2072. T. parvicornis is a soft-scale insect native to North America and has been introduced to the Caribbean region and the EU. It has been present in Italy since 2014 (Abruzzo, Campania, Lazio, and Apulia regions) and in France since 2021 (Provence–Alpes–Côte d'Azur region) and is under official control. It develops on Pinus spp. (Pinaceae), feeding on the needles and twigs, especially on new growth. It is sexually reproductive, has one or more generations each year (three in southern Italy), and adult females overwinter on the Pinus needles. It has a high fecundity, up to 1,014 eggs per female in Italy, with an average of 199 eggs for the summer generation and 730 for the overwintering generation. The main natural dispersal stage is the first instar, which crawls over the plant or may be dispersed further by wind and animals. The species can be transported over longer distances with plants for planting. Large populations cause yellowing, needle loss, reduction in growth and recruitment, flagging, dieback and tree mortality. It has had a significant impact to P. pinea (stone pine) in Italy and caused a catastrophic decline of P. caribbea var. bahamensis (Caribbean pine) in the Turks and Caicos Islands. Adult and immature T. parvicornis could enter the EU with Pinus plants for planting; however, the import of Pinus from third countries where the scale is found is prohibited. Host availability and climate suitability indicate that most of the EU would be suitable for establishment. Phytosanitary measures are available to inhibit further introductions and slow the spread within the EU. T. parvicornis satisfies all the criteria that are within the remit of EFSA to assess for it to be regarded as a potential Union quarantine pest.

2022 - Pest categorisation of Zaprionus indianus [Articolo su rivista]
Bragard, C.; Baptista, P.; Chatzivassiliou, E.; Di Serio, F.; Gonthier, P.; Jaques Miret, J. A.; Justesen, A. F.; Magnusson, C. S.; Milonas, P.; Navas-Cortes, J. A.; Parnell, S.; Potting, R.; Reignault, P. L.; Stefani, E.; Thulke, H. -H.; Van der Werf, W.; Vicent Civera, A.; Yuen, J.; Zappala, L.; Gregoire, J. -C.; Malumphy, C.; Kertesz, V.; Maiorano, A.; Macleod, A.
abstract

The EFSA Panel on Plant Health performed a pest categorisation of Zaprionus indianus (Diptera: Drosophilidae), the African fig fly for the territory of the EU. This species successfully colonised the Indian subcontinent more than four decades ago, and more recently South and North America. Within the EU, the pest occurs in Cyprus, Malta, Portugal (Madeira) and Spain (Canary Islands and Andalusia). Z. indianus is not listed in Annex II of Commission Implementing Regulation (EU) 2019/2072. The larvae of this fly feed on more than 80 plant species both cultivated and non-cultivated. Females produce around 60–70 eggs. Egg laying mostly occurs in decaying fruit or fruit with injuries or mechanical damage. However, Z. indianus can oviposit on undamaged healthy fruit such as figs, strawberries and guavas which provide a potential pathway for entry into the EU. Lower temperature thresholds are around 9–10°C. Optimum development occurs at 28°C. The number of generations per year varies from 12 to 16. Climatic conditions in many EU member states and host plant availability in those areas are conducive for establishment. The introduction of Z. indianus is expected to have an economic impact in the EU especially on fig and strawberry production. Damage caused by other fruit flies (Drosophilidae and Tephritidae) could be increased by mixed infestations. Phytosanitary measures are available to reduce the likelihood of entry and further spread. Z. indianus satisfies all of the criteria that are within the remit of EFSA to assess for it to be regarded as a potential Union quarantine pest.

2021 - Bacteriophage-mediated control of phytopathogenic xanthomonads: A promising green solution for the future [Articolo su rivista]
Stefani, E.; Obradovic, A.; Gasic, K.; Altin, I.; Nagy, I. K.; Kovacs, T.
abstract

Xanthomonads, members of the family Xanthomonadaceae, are economically important plant pathogenic bacteria responsible for infections of over 400 plant species. Bacteriophage-based biopesticides can provide an environmentally friendly, effective solution to control these bacteria. Bacteriophage-based biocontrol has important advantages over chemical pesticides, and treatment with these biopesticides is a minor intervention into the microflora. However, bacteriophages’ agricultural application has limitations rooted in these viruses’ biological properties as active sub-stances. These disadvantageous features, together with the complicated registration process of bacteriophage-based biopesticides, means that there are few products available on the market. This review summarizes our knowledge of the Xanthomonas-host plant and bacteriophage-host bacterium interaction’s possible influence on bacteriophage-based biocontrol strategies and provides examples of greenhouse and field trials and products readily available in the EU and the USA. It also details the most important advantages and limitations of the agricultural application of bacteriophages. This paper also investigates the legal background and industrial property right issues of bacteriophage-based biopesticides. When appropriately applied, bacteriophages can provide a promising tool against xanthomonads, a possibility that is untapped. Information presented in this review aims to explore the potential of bacteriophage-based biopesticides in the control of xanthomonads in the future.

2021 - Chemical characterization of an aqueous extract and the essential oil of Tithonia diversifolia and their biocontrol activity against seed-borne pathogens of rice. [Articolo su rivista]
Dongmo, Albert Nanfack; Nguefack, Julienne; Dongmo, Joseph Blaise Lecagne; Fouelefack, François Romain; Azah, Rene Udom; Nkengfack, Ephrem Augustin; Stefani, Emilio
abstract

The high cost of chemical pesticides and their negative impact on the environment prompted the search for natural pesticides from plants. The objective of our study was to control rice seed pathogenic fungi and bacteria using aqueous extract and essential oil from Tithonia diversifolia leaves. We obtained aqueous extract and essential oil, respectively, by maceration and hydrodistillation; the antimicrobial activities were determined in vitro on a solid medium by the food poisoning method. The secondary metabolites were determined by qualitative and quantitative assays; the chemical composition of the essential oil obtained from Titonia diversifolia was studied using gas-chromatography coupled with mass spectrometry. The results showed that phenols, tannins, flavonoids, alkaloids, terpenoids, sugars and saponins were present in the aqueous extract. The essential oil contained mainly hydrocarbonated, oxygenated monoterpenes, terpenoids and sesquiterpenes. α-terpineol (20.3%), eucalyptol (14.6%), camphor (14.3%) and α-pinene (13.5%) as the main compounds. Regarding the antimicrobial activity, all tested bacteria were sensitive to aqueous extract and essential oil. The activity of the aqueous extract on the tested fungi showed an inhibitory concentration 50 (IC50) of 50 mg/mL against Bipolaris oryzae and Fusarium moniliforme. The activity of the essential oil on bacteria and fungi showed MIC of 125 μg/mL (Xanthomonas oryzae pv. oryzae and Pseudomonas fuscovaginae) and MFC of 5000 μg/mL (Bipolaris oryzae and Fusarium moniliforme). These results allow us to consider Tithonia diversifolia as a potential source of natural biopesticides against rice seed-borne pathogens.

2021 - Growth Promotion and Biocontrol Activity of Endophytic Streptomyces spp. [Capitolo/Saggio]
Vurukonda, Sai Shiva Krishna Prasad; Giovanadri, 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.

2021 - Integrating science on Xanthomonadaceae for sustainable plant disease management in Europe [Articolo su rivista]
Costa, J.; Pothier, J. F.; Boch, J.; Stefani, E.; Jacques, M. -A.; Catara, V.; Koebnik, R.
abstract

2021 - Pest categorisation of Xanthomonas citri pv. viticola [Articolo su rivista]
Bragard, C.; Di Serio, F.; Gonthier, P.; Jaques Miret, J. A.; Justesen, A. F.; MacLeod, A.; Magnusson, C. S.; Milonas, P.; Navas-Cortes, J. A.; Parnell, S.; Potting, R.; Thulke, H. -H.; Van der Werf, W.; Civera, A. V.; Yuen, J.; Zappala, L.; Migheli, Q.; Stefani, E.; Vloutoglou, I.; Czwienczek, E.; Maiorano, A.; Streissl, F.; Reignault, P. L.
abstract

The EFSA Plant Health Panel performed a pest categorisation of Xanthomonas citri pv. viticola (Nayudu) Dye, a Gram-negative bacterium belonging to the Xanthomonadaceae family. The pathogen is a well-defined taxonomic unit and is the causal agent of the leaf spot and bacterial canker of Vitis vinifera. This bacterium is present in India and Brazil, where it affects table grape cultivation; the same pathogen is able to cause a disease on Azadirachta indica and on some weed species. Reports indicate that the bacterium is present in Thailand as well. The pathogen has never been reported from the EU territory and it is not included in EU Commission Implementing Regulation 2019/2072. The pathogen can be detected on its host plants using direct isolation, serological or PCR-based methods. Its identification is achieved using biochemical and nutritional assays, together with a multilocus sequence analysis based on seven housekeeping genes. The main pathway for the entry of the pathogen into the EU territory is plant propagation material. In the EU, there is large availability of host plants, with grapevine being one of the most important crops in Europe and more specifically in its Mediterranean areas. Since X. citri pv. viticola is only reported in tropical and subtropical areas (BSh and Aw climatic zones according to the Köppen–Geiger classification), there is uncertainty whether the climatic conditions in the EU territory are suitable for its establishment. Nevertheless, due to the great importance of grapevine for the EU agriculture, any disease outbreak may have a high-economic impact. Phytosanitary measures are available to prevent the introduction of the pathogen into the EU. X. citri pv. viticola satisfies the criteria that are within the remit of EFSA to assess for this species to be regarded as a potential Union quarantine pest.

2021 - Pest categorisation of Xylotrechus chinensis [Articolo su rivista]
Bragard, C.; Baptista, P.; Chatzivassiliou, E.; Di Serio, F.; Gonthier, P.; Jaques Miret, J. A.; Justesen, A. F.; Magnusson, C. S.; Milonas, P.; Navas-Cortes, J. A.; Parnell, S.; Potting, R.; Reignault, P. L.; Stefani, E.; Thulke, H. -H.; Van der Werf, W.; Vicent Civera, A.; Yuen, J.; Zappala, L.; Gregoire, J. -C.; Malumphy, C.; Kertesz, V.; Maiorano, A.; Macleod, A.
abstract

The EFSA Panel on Plant Health performed a pest categorisation of Xylotrechus chinensis (Coleoptera: Cerambycidae) for the EU territory. This species is not included in the EU Commission Implementing Regulation 2019/2072. X. chinensis is native to China, Japan, the Korean peninsula and Taiwan. It has recently been reported from Spain (Catalonia; Region of Valencia), Greece (Athens; Crete) and France (Hérault; Gironde). X. chinensis attacks and kills Morus spp. in Europe and is also a pest of Malus domestica, Pyrus sp. and Vitis vinifera in Asia. This last species, however, was not confirmed as a host in an experimental study in Spain. The pest is univoltine. The adults are 1.5–2.5 cm long; they emerge between May and August. Each female produces approximately 80 eggs which are laid on the bark. The larvae live in the phloem and tunnel into the xylem where they pupate. Infested trees show injuries including longitudinal slits in the bark, caused by larval activity next to the surface and round exit holes from which frass emerges. The females respond to a male sex pheromone, which has not been developed into a detection method. The adults spread by flight as suggested by the local expansion of damage in Europe. However, wood packaging material and wooden objects can also be a pathway as suggested by interceptions in Germany and the USA. In Greece and Spain, hundreds of Morus trees have already been attacked within a few years, and often killed. The infested area has been observed to expand from 44 to 380 km2 within 2 years in Spain (Catalonia). Phytosanitary measures are available to inhibit further introductions and slow the spread within the EU. X. chinensis satisfies all the criteria that are within the remit of EFSA to assess for it to be regarded as a potential Union quarantine pest.

2021 - Pest categorisation of carrot thin leaf virus [Articolo su rivista]
Bragard, C.; Gonthier, P.; Jaques Miret, J. A.; Justesen, A. F.; Macleod, A.; Magnusson, C. S.; Milonas, P.; Navas-Cortes, J. A.; Parnell, S.; Potting, R.; Thulke, H. -H.; Van der Werf, W.; Vicent Civera, A.; Yuen, J.; Zappala, L.; Dehnen-Schmutz, K.; Migheli, Q.; Stefani, E.; Vloutoglou, I.; Czwienczek, E.; Streissl, F.; Chiumenti, M.; Di Serio, F.; Rubino, L.; Reignault, P. L.
abstract

Following a request from the EU Commission, the EFSA Panel on Plant Health conducted a pest categorisation of carrot thin leaf virus (CTLV) for the EU territory. The identity of CTLV, a member of the genus Potyvirus (family Potyviridae), is well established and reliable detection methods are available. The pathogen is not included in the EU Commission Implementing Regulation 2019/2072. CTLV has been reported from the USA and Colombia. In the EU, the virus was reported in Germany and Slovenia and the NPPO of both countries confirmed these reports. No official national measures have been taken so far. In 2018, CTLV was reported from Greece on Torilis arvensis subsp. arvensis. Since then, no other reports exist. According to the NPPO, the virus did not establish in Greece. In natural conditions, CTLV infects plant species of the family Apiaceae (i.e., carrot, coriander, parsley and several wild weed species). The virus is transmitted in a non-persistent manner by the aphids Myzus persicae and Cavariella aegopodii, which are widely distributed in the EU. CTLV has been reported not to be transmitted by carrot seeds, while no information is available for the other hosts. Since transmission through seeds is not uncommon for potyvirids, it cannot be excluded that CTLV can be seed transmitted for some hosts. Plants for planting, including seeds for sowing, were identified as potential pathways for entry of CTLV into the EU. Cultivated and wild hosts of CTLV are distributed across the EU. Economic impact on the production of cultivated hosts is expected if further entry and spread in the EU occur. Phytosanitary measures are available to prevent further entry and spread of the virus on its cultivated hosts. Currently, CTLV does not fulfil the criterion of being absent or present with restricted distribution and under official control to be regarded as a potential Union quarantine pest, unless official control is implemented. This conclusion is associated with high uncertainty regarding the current virus distribution in the EU.

2021 - Xanthomonas arboricola pv. juglandis and pv. corylina: Brothers or distant relatives? Genetic clues, epidemiology, and insights for disease management [Articolo su rivista]
Kaluzna, Monika; Fischer-Le Saux, Marion; Pothier, Joel F; Jacques, Marie-Agnes; Obradivic, Aleksa; Tavares, Fernando; Stefani, Emilio
abstract

Background The species Xanthomonas arboricola comprises up to nine pathovars, two of which affect nut crops: pv. juglandis, the causal agent of walnut bacterial blight, brown apical necrosis, and the vertical oozing canker of Persian (English) walnut; and pv. corylina, the causal agent of the bacterial blight of hazelnut. Both pathovars share a complex population structure, represented by different clusters and several clades. Here we describe our current understanding of symptomatology, population dynamics, epidemiology, and disease control. Taxonomic status Bacteria; Phylum Proteobacteria; Class Gammaproteobacteria; Order Lysobacterales (earlier synonym of Xanthomonadales); Family Lysobacteraceae (earlier synonym of Xanthomonadaceae); Genus Xanthomonas; Species X. arboricola; Pathovars: pv. juglandis and pv. corylina. Host range and symptoms The host range of each pathovar is not limited to a single species, but each infects mainly one plant species: Juglans regia (X. arboricola pv. juglandis) and Corylus avellana (X. arboricola. pv. corylina). Walnut bacterial blight is characterized by lesions on leaves and fruits, and cankers on twigs, branches, and trunks; brown apical necrosis symptoms consist of apical necrosis originating at the stigmatic end of the fruit. A peculiar symptom, the vertical oozing canker developing along the trunk, is elicited by a particular genetic lineage of the bacterium. Symptoms of hazelnut bacterial blight are visible on leaves and fruits as necrotic lesions, and on woody parts as cankers. A remarkable difference is that affected walnuts drop abundantly, whereas hazelnuts with symptoms do not. Distribution Bacterial blight of walnut has a worldwide distribution, wherever Persian (English) walnut is cultivated; the bacterial blight of hazelnut has a more limited distribution, although disease outbreaks are currently more frequently reported. X. arboricola pv. juglandis is regulated almost nowhere, whereas X. arboricola pv. corylina is regulated in most European and Mediterranean Plant Protection Organization (EPPO) countries. Epidemiology and control For both pathogens infected nursery material is the main pathway for their introduction and spread into newly cultivated areas; additionally, infected nursery material is the source of primary inoculum. X. arboricola pv. juglandis is also disseminated through pollen. Disease control is achieved through the phytosanitary certification of nursery material (hazelnut), although approved certification schemes are not currently available. Once the disease is present in walnut/hazelnut groves, copper compounds are widely used, mostly in association with dithiocarbamates; where allowed, antibiotics (preferably kasugamycin) are sprayed. The emergence of strains highly resistant to copper currently represents the major threat for effective management of the bacterial blight of walnut. Useful websites https://gd.eppo.int/taxon/XANTJU, https://gd.eppo.int/taxon/XANTCY, https://www.euroxanth.eu, http://www.xanthomonas.org

2020 - Draft genome sequence of plant growth-promoting Streptomyces sp. strain SA51, isolated from olive trees [Articolo su rivista]
Shiva Krishna Prasad Vurukonda, Sai; MANDRIOLI, Mauro; D'APICE, GRETA; STEFANI, Emilio
abstract

A streptomycete was isolated from the rhizosphere of olive trees in autumn 2004. Its molecular characterization showed the presence of metabolic pathways promoting plant growth and additional properties, thus indicating such strain as a prospective agent for future biocontrol applications in planta. We report here the whole genome sequence of Streptomyces avermitilis, strain SA51.

2020 - First report of bacterial spot caused by Xanthomonas cucurbitae on pumpkin in Italy [Articolo su rivista]
Altin, Irem; Casoli, Luca; Stefani, Emilio
abstract

In 2018, a disease outbreak was observed on pumpkin (Cucurbita moschata cv. Violina) in the municipality of Reggio Emilia (Northern Italy). Symptoms were observed on leaves and fruits.

2020 - Le batteriosi delle drupacee, un problema di non facile soluzione [Articolo su rivista]
Stefani, Emilio; Altin, Irem
abstract

Da diversi anni, però, i frutticoltori italiani lamentano serie difficoltà nel mantenere la produttività degli impianti a causa di frequenti scoppi epidemici di alcune malattie batteriche che sono responsabili della comparsa di sintomi a carico dei frutti, foglie e parti legnose degli alberi.

2020 - Strategie per contenere la batteriosi delle drupacee [Articolo su rivista]
Ceredi, Gianni; Fava, Giuseppe; Ventrucci, Daniele; Fagioli, Luca; Franceschelli, Fabio; Bugiani, Riccardo; Tommasini, Maria Grazia; Stefani, Emilio
abstract

CONTRO XANTHOMONAS arboricola pv pruni sono stati provati tra il 2012 e il 2019 non solo diversi agrofarmaci, ma anche concimi fogliari e un corroborante che agiscono positivamente contro la batteriosi. Il rame resta l’elemento che garantisce maggiori risultati. È comunque necessaria una strategia più ampia che comprenda non solo il rame, che può dare fenomeni di fitotossicità, ma anche altri formulati, valutando però se sono accessibili all’impiego (mancanza di una etichetta specifica) o quanto meno utilizzabili in qualità di agrofarmaci.

2019 - Activity of extracts from three tropical plants towards fungi pathogenic to tomato (Solanum lycopersicum) [Articolo su rivista]
Mekam, Pascal Noel; Martini, Serena; Nguefack, Julienne; Tagliazucchi, Davide; Mangoumou, Ghislaine Ndonkeu; Stefani, Emilio
abstract

Antifungal properties were assessed of water and ethanol extracts from the pan-tropical plants Oxalis barrelieri L., Stachytarpheta cayennensis L., and Euphorbia hirta L. against Fusarium oxysporum f. sp. vasinfectum, Alternaria solani Sorauer, and Rhizoctonia solani Kuhn. The plant extracts inhibited fungal growth in vitro at 1.25-20 mg mL-1, and the degrees of inhibition increased in a dose-dependent manner. Ethanol extracts from the plants inhibited fungal growth by 80-100%, while water extracts showed less antifungal activity, with maximum growth inhibition of 62%. Growth inhibition from ethanol extracts was two- to three-fold greater than for water extracts at equivalent concentrations. Antifungal activity of the extracts varied with their content and composition of phenolics, flavonoids, tannins, and alkaloids. In greenhouse experiments, spraying tomato plants (Solanum lycopersicum L.) with ethanol extract from E. hirta at 2.5 mg mL-1 did not cause phytotoxicity, and increased plant size, when compared to untreated plants. Spraying E. hirta ethanol extract on tomato plants infected by R. solani reduced disease severity up to 80%, when compared to non-sprayed plants. These results demonstrate potential of leaf extracts from E. hirta, O. barrelieri, and S. cayennensis as biofungicides for the control of R. solani, A. solani, and F. oxysporum, which are among the most important causal agents of tomato diseases.

2019 - Biological control of plant bacterial pathogens by a beneficial Streptomyces sp. [Abstract in Atti di Convegno]
Vurukonda, Sai Shiva Krishna Prasad; Altin, Irem; Stefani, Emilio
abstract

In the present study a set of beneficial microorganisms were evaluated for their in vitro antimicrobial activity on various phytopathogenic bacteria, like Xanthomonas vesicatoria, Clavibacter michiganensis subsp. michiganensis (Cmm), Clavibacter michiganensis subsp. sepedonicus (Cms), Acidovorax citrulli and Ralstonia solanacearum. A prospective antagonist, strain SA51 (Streptomyces sp.) was selected based on its remarkable in vitro antibacterial activity and was further studied under greenhouse conditions on tomato (Solanum lycopersicum) seedlings as a model plant against X. vesicatoria. Fromin vitro results, it was clearly evident that SA51 was very active against bacteria like Cmm, Cms, R. solanacearum and X. vesicatoria compared to other antagonists. Tomato plantlets were inoculated with SA51 at the roots and, thereafter, they were sprayed with X. vesicatoria. Protection against X. vesicatoria by the bacterized tomato plants was confirmed in the greenhouse: disease was reduced by approximately 96%. Additionally, plants bacterized by strain SA51 showed significant plant growth, particularly in aerial parts as compared to un-bacterized controls. Finally, benefit was seen in inoculated healthy plants in terms of a significant increase in dry weight and length of roots and shoots, as compared to the uninoculated controls. A GFP mutant of strain SA51 was produced to study its endophytic colonisation in tomato plants: results confirmed that SA51 was able to efficiently colonise tomato endophytically, from the roots to the leaves. Field experiments confirmed the ability of strain SA51 to act as plant growth promoting agent: such promoting activity was also reflected into an increase of fruit production by approximately 7%.

2019 - Cancro batterico dell’albicocco: dalla diagnosi alla difesa [Articolo su rivista]
Fagioli, Luca; Franceschelli, Franco; Ceredi, Gianni; Bugiani, Riccardo; Tommasini, Maria Grazia; Stefani, Emilio
abstract

Le infezioni di Pseudomonas syringae pv. su albicocco causano dapprima deprezzamento della produzione e successivamente il deperimento dell’intera pianta. Scelta varietale, materiale vivaistico sano e ambienti con limitata frequenza di gelate sono i principi alla base di una corretta prevenzione della malattia, mentre a oggi il rame resta l’unico agrofarmaco ammesso in grado di garantire effi cacia battericida

2019 - Criticità e tecniche di controllo delle principali avversità fitosanitarie [Articolo su rivista]
Fagioli, Luca; Stefani, Emilio
abstract

Bacterial blight, caused by Xanthomonas arboricola pv juglandis and anthracnose, caused by the ascomycete Marssonina juglandis (teleomorph: Gnomonia leptostyla) are considered the major diseases for walnut species. Other well-known diseases of minor impact are the shallow bark necrosis caused by the Gram-negative rod Brenneria nigrifluens, the Armillaria root rot, caused by the basidiomycete Armillaria mellea and the crown and root rot, caused by a few oomycetes belonging to the genus Phytophthora. Many insect pests may also damage the crop and the main ones are the codling moth (Cydia pomonella), the walnut husk fly (Rhagoletis completa) and aphids (Callaphis juglandis e Chromaphis juglandicola). Other insect pests, defined as minor threats are: the leopard moth (Zeuzera pyrina), the carpenter moth (Cossus cossus), scales and mites.

2019 - From transnational research collaboration to regional standards [Articolo su rivista]
Giovani, Baldissera; Cellier, Gilles; Mcmullen, Madeleine; Saponari, Maria; Stefani, Emilio; Petter, Françoise
abstract

Reliable and rapid diagnostic methods are essential to support inspection activities conducted by National Plant Protection Organizations (NPPOs) in the framework of their official mandate, and to evaluate the efficacy of control measures taken. Since 1998 the European and Mediterranean Plant Protection Organization (EPPO) has been supporting the harmonization of diagnostic methods for regulated pests in the EPPO region through the development of technical standards. In order to increase active collaboration among the organizations involved in plant health research activities at the national and regional levels, Euphresco (European Phytosanitary Research Coordination) was established in 2006 and funded by the EU as an ERA- NET project. Euphresco has subsequently evolved into a self-sustaining international network hosted by EPPO. This paper describes the EPPO diagnostic programme. It will also provide some examples of research projects funded through Euphresco that have provided valuable support for the development of pest-specific diagnostic protocols and will show how NPPOs can shape the research agenda of research funders and help to identify gaps to be addressed through transnational collaboration.

2019 - Le principali malattie del noce da frutto: aspetti epidemiologici e tecniche di controllo [Relazione in Atti di Convegno]
Stefani, Emilio; Altin, Irem
abstract

Well-known bacterial and fungal diseases affecting walnut trees represent a limiting factor in intensively cultivated areas. Nowadays, the most important bacterial and fungal diseases are: the bacterial blight, caused by Xanthomonas arboricola pv juglandis, and the anthracnose, caused by the ascomycete Marssonina juglandis (teleomorph: Gnomonia leptostyla). Other well-known diseases, although of minor impact, are the shallow bark necrosis caused by the Gram-negative rod Brenneria nigrifluens, the Armillaria root rot, caused by the basidiomycete Armillaria mellea and the crown and root rot, caused by a few oomycetes belonging to the genus Phytophthora. Two more diseases appear to be a possible future threat in walnut production: the blackline disease, caused by the Cherry Leaf Roll Virus (CLRV) and the thousand cankers disease, caused by Geosmithia morbida, a fungus that is disseminated by the walnut twig beetle, Pityophthorus juglandis.

2019 - Phenolic compounds profile of water and ethanol extracts of Euphorbia hirta L. leaves showing antioxidant and antifungal properties [Articolo su rivista]
Mekam, Pascal Noel; Martini, Serena; Nguefack, Julienne; Tagliazucchi, Davide; Stefani, Emilio
abstract

The bioactive chemical constituents of water and ethanol extracts of Euphorbia hirta L. leaves have been identified and quantified using an un-targeted mass spectrometric approach. The study allowed the tentative identification of 123 individual phenolic compounds and 18 non-phenolic phytochemicals, most of them described in Euphorbia hirta L. leaves for the first time. Gallotannins, hydroxybenzoic and hydroxycinnamic acids were the most abundant phenolic classes in Euphorbia hirta L. leaves, representing together the 71.5% (26.3%, 25.2% and 20%, respectively) of the total amount of identified phenolics. The main phenolic compounds detected were tri-O-galloyl-glucose isomers, feruloyl-coniferin, tetra-O-galloyl-glucose isomers, di-O-galloyl-glucose isomers, ethyl-gallic acid, protocatechuic acid-O-pentoside-O-hexoside, 5-O-caffeoyl-quinic acid trans isomer and digalloyl-quinic acid. Feruloyl-coniferin was found to be approximately six times more concentrated in the ethanol extract with respect to the water extract. The ethanol extract exhibited higher ABTS (1338.3 ± 85.3 and 802.3 ± 91.0 μmol ascorbic acid equivalent/gram of dry extract, respectively) and superoxide anion (2014.6 ± 78.6 and 1528.0 ± 111.7 μmol ascorbic acid equivalent/gram of dry extract, respectively) scavenging abilities than the water extract. Additionally, the ethanol extract also showed a remarkable anti-fungal effect against Fusarium oxysporum f. sp. vasinfectum, Alternaria solani and Rhizoctonia solani. This study provides new information about Euphorbia hirta L., offering reasons to promote this plant species as rich source of phenolics and an excellent source of antifungal molecules that might have a prospective use in controlling fungal diseases of vegetable crops.

2019 - Postharvest biocontrol ability of Pseudomonas synxantha against Monilinia fructicola and Monilinia fructigena on stone fruit [Articolo su rivista]
Aiello, Dalia; Restuccia, Cristina; Stefani, Emilio; Vitale, Alessandro; Cirvilleri, Gabriella
abstract

The biocontrol properties of the endophyte Pseudomonas synxantha DLS65 were tested in vitro and in vivo against Monilinia fructicola and Monilinia fructigena, causal agents of postharvest brown rot of stone fruit. P. synxantha cells significantly reduced the mycelial growth of both pathogens on Potato Dextrose Agar (PDA), and strongly inhibited the Monilinia fructicola growth on Peach Extract Agar (PEA). Cell-free culture filtrates inhibited the pathogens on PDA and PEA to lesser extent. The production of volatile organic compounds (VOCs), with in vitro inhibitory effects on mycelial growth, was also observed. P. synxantha significantly reduced brown rot incidence and severity on peach fruit artificially inoculated with M. fructicola after 5 d at 25 °C. Moreover, P. synxantha more significantly reduced incidence and severity after 10 d at 10 °C and after 20 d in cold storage at 0 °C in comparison to control fruit, even if its activity was never comparable to that of the synthetic fungicide Scholar® (fludioxonil). Similarly, P. synxantha exhibited an excellent antagonistic activity against M. fructigena on fruit at 10 and at 0 °C, and a weak biocontrol activity at 25 °C. Competition for nutrients and space, production of diffusible toxic metabolites and VOCs may play a role in the antagonism of P. synxantha toward M. fructicola and M. fructigena, especially at the lowest temperatures of storage. For that reason, this strain of P. synxantha could be suggested as active ingredient for the setting up of bioformulates against Monilinia species representing a limiting factor for stone fruit production.

2019 - Xylella fastidiosa: il patogeno, le malattie e l’attuale situazione fitosanitaria [Articolo su rivista]
Stefani, Emilio; Boscia, Donato
abstract

Xylella fastidiosa è un batterio fitopatogeno Gram negativo che appartiene alla famiglia delle Xanthomonadaceae. È l’agente causale di numerose malattie d’importanti colture arboree, erbacee, forestali e ornamentali e, a differenza di quanto molta parte dell’opinione pubblica crede, non è un problema di recente comparsa, ma ha una lunga storia che affonda le sue radici ancora alla fine del diciannovesimo secolo. L’etimologia e la semantica del nome assegnato al patogeno da parte dei ricercatori che per primi lo isolarono in coltura pura svelano due aspetti fondamentali della sua biologia: Xylella rivela la nicchia biologica del patogeno all’interno delle sue piante ospiti, cioè lo xilema; fastidiosa indica la grande difficoltà che il batteriologo incontra durante il processo d’isolamento e purificazione della coltura pura.

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 - Endophytic colonization of tomato plants by plant growth promoting rhizobacteria [Abstract in Atti di Convegno]
Vurukonda, Sai Shiva Krishna Prasa; Stefani, Emilio
abstract

Several microorganisms, part of the microbial communities' present in soil and plant (as endophytes or epiphytes), play a significant role in agro-environments as plant growth promoters, control of plant diseases, facilitation of nutrients uptake by plants. Understanding the mode of action of such microbes (bacteria, yeasts, fungi) and their relationship with crop plants will help to focus on selected microorganisms, with the scope to develop innovative products (Microbials) to be used in sustainable crop production, gaining a positive impact on agricultural environments in terms of microbial diversity and stability. The research focuses on searching microbial candidates in agricultural and natural environments suitable for crop management as biocontrol agents or plant growth promoters. The Streptomyces sp. strain SA51 and Pseudomonas sp. strain PT65 used in the present study were previously studied extensively to evaluate their in vitro plant growth promoting (PGP) and bio-control activity against various bacterial and fungal phytopathogens (AMI-2017, pp 222). Here, we characterized both the strains SA51 and PT65 for their colonization ability, plant growth promotion and protection against tomato spot disease caused by Xanthomonas vesicatoria on tomato (Solanum lycopersicum) as model plant. In this study, direct inhibitory action against X. vesicatoria by the bacterized tomato plants showed significantly good plant growth, particularly in aerial parts as compared to un-bacterized controls. In addition, benefit was seen in inoculated healthy plants in terms of increase in dry weight and length of roots and shoots as compared to the uninoculated controls. The mechanism of biocontrol also involved induction of plant defense response. Endophytic colonization of the streptomycetes was confirmed by both PCR amplification applied to different plant segments and its visual observation with a confocal microscopy, after its transformation with green fluorescent protein (gfp); gfp tagged Streptomyces strain SA51 showed profuse colonization in roots, collar and aerial parts of tomato plants.

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 - ANTIFUNGAL ACTIVITY OF AQUEOUS AND ETHANOL EXTRACTS OF TROPICAL PLANTS AGAINST TOMATO FUNGAL PATHOGENES. [Abstract in Atti di Convegno]
Mekam, Pascal Noel; Martini, Serena; Tagliazucchi, Davide; Nguefack, Julienne; Stefani, Emilio
abstract

Several plant extracts may have a strong antifungal activity that can be exploited in the management of fungal diseases as an alternative to synthetic fungicides, the abuse of which may have consequences on the environment and health. Ethanol extracts (EE) and water extracts (WE) of three tropical plants, Stachytarpheta cayennensis (Verbenaceae), Oxalis barrelieri (Oxalidaceae) and Euphorbia hirta (Euphorbiaceae) were obtained and screened for their antifungal activity against three major phytopathogenic fungi of tomato: Fusarium sp., Alternaria sp. and Colletotrichum sp. The phytopathogenic fungi used across the experiments were isolated in Cameroon on severely affected tomatoes and their molecular identification and characterization is ongoing. The antifungal activity was checked in vitro on different media supplemented with different concentrations of the extracts. O. barrelieri EE, at the concentration of 25 mg/ml, inhibited the mycelium growth of Fusarium sp. by 26.8% and remarkably modified its morphology. WE and EE of the same O. barrelieri inhibited the mycelium growth of Colletotrichum sp. by 43.7% and 50.3% respectively, but showed no inhibition on Alternaria sp. The phytochemical analysis of these plant extracts revealed that EE of S. cayennensis was the richest in polyphenols and flavonoids. EE from O. barrelieri was particularly rich in alkaloids. The inhibitory effects on the phytopathogenic fungi were possibly related to the amount of polyphenols and alkaloids obtained through the extraction. Field experiments are being conducted on tomato to confirm the action of such extracts in vivo. These findings may contribute to develop new biofungicides to protect tomato from some fungal pathogens.

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 - CRITICAL FEATURES OF THE DIAGNOSIS OF CLAVIBACTER MICHIGANENSIS subsp. MICHIGANENSIS FROM TOMATO SEED [Abstract in Atti di Convegno]
Pucci, Nicoletta; Catara, Vittoria; Scortichini, Marco; Stefani, Emilio; Pereg, G; Loreti, Stefania
abstract

Bacterial canker, caused by Clavibacter michiganensis subsp. michiganensis (Cmm) is one of the most important bacterial disease of tomato (Solanum lycopersicum) worldwide. Seeds are the main pathway for the transmission of the bacterium, listed as an A2 quarantine pest, consequently the reliability of seed detection tests represents a critical point to prevent the introduction and spread of the pathogen. In this study some conventional (isolation and immunofluorescence) and molecular (end-point PCR) methods for the detection of Cmm from tomato seed samples were compared by an inter-laboratory comparison (ITC). Several Italian laboratories, belonging to different institutions, analysed the same panel consisting of 11 Cmm-spiked tomato seed samples for the evaluated methods. The obtained results showed that end-point PCR gave acceptable performance values, even if the choice of enzyme in the PCR reaction was crucial. Currently the EPPO protocol (Standard PM7/42 2) does not contemplate a preliminary screening phase based on molecular methods, but suggests two parallel flow diagrams that use, as first step, the isolation and immunofluorescence, respectively. Since these latter techniques showed low values of performance criteria, the study highlighted the necessity to integrate the EPPO protocol with a preliminary molecular screening test. It is advisable to validate new molecular methods more specific and sensitive than end-point PCR, based on systems such as real time PCR, LAMP and digital PCR.

2017 - EU-COST ACTION CA16107 - EUROXANTH: INTEGRATING SCIENCE ON XANTHOMONADACEAE FOR INTEGRATED PLANT DISEASE MANAGEMENT IN EUROPE. [Abstract in Atti di Convegno]
Stefani, E; Catara, V; Emeriau, E; Koebnik, R
abstract

Bacteria of the family Xanthomonadaceae, including species of Xanthomonas and Xylella fastidiosa, are devastating plant pathogens. Many are quarantine organisms in the EU and their study is of uttermost importance. These pathogens infect all kinds of crop plants. The COST Action CA16107 “EuroXanth” aims at creating an interdisciplinary network in order to develop strategies for sustainably protecting plants and prevent yield losses. COST (European Cooperation in Science and Technology) is a funding agency for research and innovation networks. COST Actions help connect research initiatives and enable scientists to grow and share ideas with their peers. Specifically, this COST Action addresses key aspects of the pathogen-vector-host interactions, from the cellular to the population level. A better insight into population structures and virulence mechanisms of the pathogens, together with the exploration of the molecular mechanisms underlying disease resistance, will enable development of durably resistant plant cultivars and exploitation of bio-control schemes tailored to population and pathogen. This COST Action has duration of 4 years (March 2017-March 2021) and will generate a platform that gathers experts from different disciplines, such as molecular diagnostics, molecular host-microbe interactions, plant resistance breeding, etc. The network includes 43 working groups from 21 different countries. Joining their efforts will help to develop and implement effective plant protection schemes, be it via resistant crop cultivars or via other control mechanisms. This goal will be achieved by mobilizing and training scientists from major European institutions, regulatory bodies and commercial companies working on the various aspects of this complex of problems.

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.

2017 - Surveys of potato-growing areas and surface water in Lebanon for potato brown and ring rot pathogens. [Articolo su rivista]
Choueiri, Elia; Jreijiri, Fouad; Wakim, Samer; EL KHOURY, M. i. c. h. e. l. Issa; Valentini, Franco; Dubla, Nino; Galli, Daniele; Habchy, Rosine; Khalil, Akl; Stefani, Emilio
abstract

Field surveys were carried out over three growing seasons (2013–2015), in the main potato growing areas of Lebanon, to assess the occurrence of potato brown rot caused by Ralstonia solanacearum and potato ring rot caused by Clavibacter michiganensis subsp. sepedonicus. A total of 232 potato samples were collected from Bekaa valley and 145 samples from Akkar plain, which are the largest Lebanese areas cropped with potatoes. Composite samples of 200 potato tubers were randomly collected from each field, following procedures laid down in EU legislation. Twelve potato demonstration fields were established in Akkar plain and designed for potato export to European markets: these were also surveyed using the same strategy. Furthermore, a network of 40 sampling sites in Bekaa and 19 sites in Akkar was established to collect surface water. GPS coordinates of potato fields and water sampling sites were recorded to map specific sampling points using Geographic Information System. All samples gave negative results for R. solanacearum and C. michiganensis subsp. sepedonicus in potatoes and R. solanacearum in water, as indicated using the official EU methods for detection and diagnosis for these pathogens. A monitoring system for R. solanacearum and C. michiganensis subsp. michiganensis has been set up in Lebanon. This will increase the phytosanitary quality of potatoes and provide access to broader international markets.

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 - BIOSAFETY AND ENVIRONMENTAL CONCERNS RELATED TO THE USE OF BACTERIAL BIOCONTROL AGENTS IN CROP PROTECTION [Abstract in Atti di Convegno]
Stefani, Emilio
abstract

The use of microorganisms in agriculture for the biological control of plant pathogens is increasing, due to a rising awareness among citizens for sustainability of agricultural production systems, coupled with a deeper knowledge of the relationships among the microbial communities in agricultural environments. In the EU, 54 microorganisms are currently approved for a possible use in plant protection and many more are under approval/authorization. Regulating the use of microbials in agriculture is a challenge: several countries regulate them as chemical pesticides, without taking into consideration their biological properties. Challenges might refer to: i) identification, characterization and biological properties; ii) toxicology and environmental risks; iii) residues of microorganisms and their metabolites on food crops; iv) level of efficacy. Microorganisms may pose risks to the environment, as they are able to survive, proliferate and disseminate. Registration data requirements for biocontrol agents are currently concerning their toxicity, pathogenicity and/or infectivity. Environmental safety is assessed according a case-by-case evaluation. Mensik & Scheepmaker (2007) proposed a procedure, adopted by the OECD. The decision scheme starts with data on microbial characterization, followed by assessment on contamination/exposure in soil/surface water, together with fate and behavior of inoculum. Environmental toxicology is done on terrestrial and aquatic organisms, checking any adverse effects and, eventually, mitigation options. The environmental risk assessment terminates in “RISK ACCEPTABLE” or “RISK NOT ACCEPTABLE”. The regulatory authority may, nonetheless, consider those biocontrol agents, whose risk is assessed as “not acceptable”, if mitigation of adverse effects are possible or their use will replace a toxic pesticide.

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 - Le malattie batteriche emergenti o riemergenti delle colture agrarie in Italia e nel bacino del Mediterraneo [Articolo su rivista]
Stefani, Emilio; Catara, Vittoria
abstract

Vengono descritte alcune malattie batteriche emergenti, o riemergenti, che potrebbero diventare un serio fattore limitante le produzioni ortofrutticole italiane e mediterranee.

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 - Sampling seeds in storage facilities: hunting for Fusarium infection [Abstract in Rivista]
DAL PRA', Mauro; Alberti, Ilaria; Tonti, Stefano; Montanari, Massimo; Stefani, Emilio
abstract

Fusarium graminearum is one of the most studied fungal pathogens in the world. A great number of scientific publications are available that describe morphology, physiology, toxicology and genome of this species. On the other hand, F. poae is less widely studied but its importance as toxigenic fungus has been recently recognized. In Italy, F. poae is rapidly becoming one of the most widespread Fusarium species on small seeds cereals heads. Soft wheat is often colonised by these species. So far, Fusarium Head Blight (FHB) symptoms have been always investigated on soft wheat heads or on seeds collected in the field during harvesting. This poses a problem, as the seed industry and the national seed certification services would be interested to know the percentage of infection of seeds stored into facilities after processing and how infection is distributed inside seed lots. In order to study these aspects, we performed a mycological screening on soft wheat seed samples collected inside a facility, sampling directly from bags intended for seed trading. Sampling was performed during the years 2013 and 2014. The screening, carried out on Potato Dextrose Agar (PDA), was limited to organic soft wheat coming from Northern and Central Italy. Putative F. graminearum and F. poae colonies were selected and single spores purified and confirmed through microscope observations and through a molecular identification. Seed samples resulted to be infected at a very low level. Though some lack of homogeneity was observed among the lots, this was sufficiently small to allow statistical studies.

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 - Candidatus Liberibacter solanacearum - eine neue Gefahr für den Kartoffel- und Tomatenanbau [Articolo su rivista]
G., Schrader; P., Müller; Stefani, Emilio
abstract

The bacterium Candidatus Liberibacter solanacearum, originating in North America, can massively damage potatoes, tomatoes and bell peppers. Also damage on carrots has been observed. So far, the bacterium has not been detected in Solanaceae in Europe, however, in Finland, Spain and France it has been found on carrots. It is transmitted to potatoes by Bactericera cockerelli (Sulc) and probably also other psyllid species and probably also with planting material. Up to now, B. cockerelli is not present in Germany. In case the infested vector is introduced, establishment and spread of the bacterium are possible. If also other psyllids, that are already present in Germany or the EU, could act as vectors, there is an increased risk of the spread of the bacterium. There is some evidence that also the psyllids Trioza apicalis Förster and B. trigonica Hodkinson, both present in the EU, can transmit the pathogen, however, as far as known, only to carrots. The bacterium is not yet listed in the EU plant quarantine directive 2000/29/EC, but is, as well as its vector B. cockerelli, listed on the EPPO A1 List. A JKI pest risk analysis in particular for Germany and an EPPO pest risk analysis for the whole EPPO region are available. Due to its high damage potential, Ca. L. solanacearum presents an essential phytosanitary risk to Germany and other EU member states. In particular for the cultivation of potatoes in areas with very suitable conditions like the Mediterranean basin, high yield losses could be expected. The control of the vector is difficult (monitoring with yellow sticky traps, massive application of pesticides). According to the risk analysis, it can be assumed that the bacterium could establish in Germany or other EU member states and that it could cause important damage, in case its vector is also introduced or present. Therefore, measures according to § 4a of the German plant protection order should be taken to prevent the introduction of the bacterium as well as its vector. Symptoms in cultivation and production of host plants should be noted and verified. If infestation is suspected, it should be confirmed in the laboratory (plant protection service or JKI) and in case of positive findings notified to the JKI. Infested lots should be destroyed and vectors be controlled.

2014 - ISOLATION AND CHARACTERIZATION OF AN ENDO- PHYTE FROM ACTINIDIA SP. SHOWING A STRONG ANTAGONISTIC ACTIVITY AGAINST PSEUDOMONAS SYRINGAE PV. ACTINIDIAE [Relazione in Atti di Convegno]
Tontou, Rodanthi; Gaggia, Francesca; Baffoni, Loredana; Venturi, Vottorio; 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 pathogen grows inside the trunk, vines and leaves of its host plants, causing wilting and formation of cankers, with subsequent death of the plant. During the past three years, samples have been collected from various parts of asymptomatic Actinidia spp. within infected kiwi orchards and many endophytic bacteria have been isolated. Among them, a few isolates were identified and proved to be able to strongly inhibit Psa namely an isolate of Pantoea agglomerans, two pseudomonads belonging to the fluorescens/putida group, and one novel Pseudomonas sp., which proved to be highly effective in inhibiting, in vitro, several important phytopathogenic bacteria. This last isolate was chosen for further studies. Concentrated supernatant of its liquid culture in LB medium could inhibit Psa, indicating that the active compound produced by the antagonist is excreted from the cell into the environment. The nature of that biotoxin is not proteic, and its size is smaller than 3 kDa, as a result of its partial purification. Moreover, a bank of mutants deficient in their antagonistic activity has been constructed by triparental mating, in order to search and identify genes involved in antagonism against Psa. Fifty six prospective mutants have been already obtained: twenty two of them were subject to Southern blotting to verify a single transposon insertion. Gene identification is under way.

2014 - Occurrence of Fusarium langsethiae on oat kernels in Italy [Relazione in Atti di Convegno]
DAL PRA', Mauro; Tonti, Stefano; Montanari, Massimo; Stefani, Emilio; Alberti, Ilaria
abstract

Fusarium langsethiae Torp & Niremberg, a recently described fungus, is a high-level producer of T-2 and HT-2 toxins. These trichotecenes are related to alimentary toxic aleukia in humans and haematotoxicity and immunotoxicity in animals. F. langsethiae has been reported in oats, wheat and barley in Central and Northern Europe, but it is more problematic in the former crop rather than the latter two. Little is known about the occurrence of this fungus in Italy, where the demand for oat products is rising. During a 4-year survey, 50 organic seed samples were tested for the presence of F. langsethiae. Four hundred seeds per sample were surface sterilized and then placed in 90 mm Petri dishes containing potato dextrose agar (PDA) and incubated 7 days at 21°C. All Fusarium strains were single-spore purified and characterized at species level on the basis of their morphological features on Spezieller Nährstoffarmer Agar (SNA) plates. Morphological identification was confirmed by translation elongation factor 1-α (TEF) gene sequencing. The most represented Fusarium species was F. poae (Peck) Wollenw. F. langsethiae was recovered from thirteen samples and was usually associated with low levels of infected seeds. Interestingly the main agents of Fusarium Head Blight (FHB), F. graminearum and F. culmorum, were almost absent. In order to detect the presence of T-2 and HT-2 toxins on samples, oat seeds were grinded to a fine powder in a blender. After extraction of the toxins from 10 g of the grounded samples with 70% methanol, the extracts were analysed by Rapid One Step Assay (ROSA) T2-HT2 Quantitative Test (Charm Sciences, Inc., USA). Seed samples resulted to be contaminated by T-2 and HT-2 toxins, but it was impossible to find a statistical correlation between F. langsethiae occurrence, infection levels and toxin contamination.

2014 - PM 7/120 (1) Pseudomonas syringae pv. actinidiae [Articolo su rivista]
Stefani, Emilio; Loreti, Stefania
abstract

This Standard describes a diagnostic protocol for Pseudomonas syringae pv. actinidiae

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

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.

2013 - Operational Certification Manual for the Export of Lebanese Ware Potatoes [Monografia/Trattato scientifico]
Stefani, Emilio
abstract

Potato production is considered one of the most important in Lebanon. Potato is mainly produced in the Bekaa valley in the eastern part of the Country and in the northern Akkar plain. A big portion of potato production is consumed locally while the rest is exported to Syria, Jordan, Egypt and the gulf countries. In 2006 Lebanon signed a commercial agreement with the European Union which would allow it to export 50.000 tons of ware potatoes implemented yet for plant quarantine reasons. This ware potatoes export certification operational manual is destined to be used by field and border ware potatoes phytosanitary inspection units and inspectors to fulfill import requirements of trading partners, specifically of the European Union. The steps below describe the phytosanitary measures taken by the Plan Protection and the Export-Import and Quarantine services (TMPPO) of the Ministry of Agriculture in Lebanon to maintain Lebanese ware potatoes production free from quarantine pests of EU concern, specifically Brown Rot (Ralstonia solanacearum) and Ring Rot (Clavibacter michiganensis subsp. sepedonicus). The Plant Protection service and the Export-Import and Quarantine Service in cooperation with the Lebanese Agricultural Research Institute (LARI) are undertaking the below described measures to fulfill the import phytosanitary requirements of the European Union for ware potatoes.

2013 - PM 7/110 (1) Xanthomonas spp. (Xanthomonas euvesicatoria, Xanthomonas gardneri, Xanthomonas perforans, Xanthomonas vesicatoria) causing bacterial spot of tomato and sweet pepper [Articolo su rivista]
M., Scortichini; Stefani, Emilio; J., Elphinstone; M., Bergsma Vlami
abstract

This standard describes a diagnostic protocol for Xanthomonas spp. causing bacterial spot of tomato and sweet pepper (Xanthomonas euvesicatoria, Xanthomonas gardneri, Xanthomonas perforans, Xanthomonas vesicatoria).

2013 - Protocollo diagnostico per Xanthomonas arboricola pv. pruni (Xap) [Articolo su rivista]
G., Perez; V., Catara; N., Pucci; M., Scortichini; Stefani, Emilio; S., Loreti
abstract

Un protocollo per la diagnosi ed identificazione di Xanthomonas arboricola pv. pruni, agente causale del cancro batterico delle drupacee, è stato messo a punto e validato. Tale protocollo sarà utilizzato per la certificazione delle drupacee da frutto.

2013 - TESTA-SEED HEALTH: Development of seed treatment methods, evidence for seed transmission and assessment of seed health. [Abstract in Rivista]
Henry, Christine; Aveling, Terry; Bonants, Peter; Stefani, Emilio; Et, A. l.
abstract

A wide range of diseases and pests are carried by seeds. Seeds may carry pest already present in the European Community, but also introduce new ones via this route. Under the EU 7th Framework Programme a collaborative project was granted to a research Consortium, in order to face open questions related to seed pathology, such as pathogen transmission by seed, development, implementation and validation of sampling and detection methods, development of effective disinfection protocols for contaminated seeds. The TESTA project will develop a range of novel methods to underpin the control of these diseases, including faster, more accurate methods to assess the mode of seed transmission, economic and practical sampling approaches for the detection of pathogens present in low levels in large seed lots, novel and efficient generic detection methodologies, non-destructive testing methods and improved, effective and sustainable disinfection procedures. Several target crops and disease combinations have been identified in consultation with EPPO, ISHI-ISF and ISTA. Among them, most relevant are following: Tomato/pepper and their bacterial and viral pathogens, such as Clavibacter michiganensis subsp. michiganensis, Xanthomonads, and a number of pospiviroids; cucurbits and their most important bacterial pathogen, Acidovorax citrulli; brassicas and other crucifers with Xanthomonas campestris pv. campestris and Phoma lingam; wheat and its fungal pathogens Tilletia spp. and Fusaria, parasitic nematodes of legumes. Important aims of TESTA are: (i) enhance knowledge of the biology of seed transmission, both by developing novel methods based on labelled microorganisms and through extensive field trials; (ii) establish a comprehensive web-based database as a global resource, detailing all known pests and diseases of crop plants transmitted by seed; (iii) develop novel methods for assessing levels of seed transmission and their relevance to disease levels in crops; (iv) improve sampling strategies and methodologies for seed lots, in order to generate representative samples for lab testing; (v) establish generic platforms for seed testing methods, together with the assessment of innovative protocols, possibly using non-destructive methodologies; (vi) develop novel disinfection/sanitation procedures for seed, and assessing their proficiency; (vii) disseminate results to national policy stakeholders, testing laboratories and inspection services. Complete information is available on the website: www.seedtesta.eu.

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.

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 - A TRANSCRIPTOMIC APPROACH TO UNDERSTAND INDUCED RESISTANCE TO BACTERIAL CANKER OF STONE FRUITS ELICITED THROUGH TREATMENTS WITH COPPER GLUCOHUMATES. [Abstract in Rivista]
Dallai, Davide; Dondini, Luca; Stefani, Emilio
abstract

Bacterial canker of stone fruits, caused by Xanthomonas arboricola pv. pruni, is a recurrent disease in Italian peach and plum orchards. Several field and glasshouse trials on peach and plum were done, with the aim to effectively control the disease by using some novel molecules, such as glucohumates. The most remarkable results were obtained with copper glucohumates (with a reduction of the disease by ca. 80%). 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. In order to study and understand the effect of the biomolecules used, untreated and glucohumate-treated peach plants were subject to further molecular analyses in order identify possible genes/sequences involved in the induction of disease resistance. A transcriptomic approach was developed for detecting the transcripts present in plant tissues, after elicitation of an induced protection state. Total RNA was extracted, retro-transcribed and c-DNA-AFLP was done to identify different sequence fingerprints in the protected plant tissue. Discrimination of newly expressed sequences was performed with DHPLC, and comparison of transcripts was done on the complete peach genome database in order to identify the genes or sequences involved in the elicitation of induced resistance. Preliminary data showed, in treated plants, the presence of nine putative genes like a putative senescence protein, already described as being involved in the induced resistance to fire blight in other Rosaceae. Thus, copper glucohumates might be considered possible candidates for the elicitation of resistance to bacterial diseases.

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 - Economic significance and control of bacterial spot/canker of stone fruits caused by Xanthomonas arboricola pv. pruni [Articolo su rivista]
Stefani, Emilio
abstract

Bacterial spot and canker of stone fruits is a multifaceted disease caused by ...

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.

2010 - QBOL: a new EU project focusing on DNA barcoding of quarantine organisms. [Articolo su rivista]
P. Bonants, P.; E., Groenewald; J. Y., Rasplus; M., Maes; P., Devos; J., Frey; N., Boonham; M., Nicolaisen; A., Bertaccini; V., Robert; I., Baker; L., Kox; M., Ravnikar; K., Tomankova; D., Caffier; M., Li; K., Armstrong; J., Freitas Astua; Stefani, Emilio; J., Cuberoand; L., Mostert
abstract

In 2009 a new three year EU funded project (QBOL) started on DNA barcoding of important plant pests. An international consortium of 20 partners (universities, research institutes, and phytosanitary organizations) from around the world, coordinated by Plant Research International (Wageningen, the Netherlands), will collect DNA barcodes from many plant pathogenic quarantine organisms, store these sequences in a database accessible over the internet, develop a DNA bank and train end-users. All these activities should help National Plant Protection Services in the correct identification and detection of plant pathogenic quarantine organisms.

2009 - Gene expression in grapevine cultivars in response to BOIS NOIR phytoplasma infection [Articolo su rivista]
Albertazzi, Giorgia; Milc, Justyna Anna; Caffagni, Alessandra; Francia, Enrico; Roncaglia, Enrica; F., Ferrari; Tagliafico, Enrico; Stefani, Emilio; Pecchioni, Nicola
abstract

Bois Noir phytoplasma is an emerging disease of Vitis vinifera in several regions of the world. No completely resistant grapevine cultivars are known and the physiology of disease remains still poorly understood so far. Affymetrix GeneChip® oligonucleotide arrays have been used to identify differentially expressed genes between infected and recovered samples from cv. Chardonnay and between infected and healthy samples from cv. Manzoni Bianco. In the field, cv. Manzoni showed reduced symptoms,while cv. Chardonnay was highly susceptible to the disease. Results showed that expression levels of few hundreds genes were altered in infected plants, both common and specific for each cultivar, with effects on various metabolic pathways. In cv. Chardonnay a serious inhibition of whole photosynthetic chain and photosystem I activity, Calvin-cycle enzymes transcription, lipid metabolism and phenylpropanoid biosynthesis was observed. Increasing physical barriers to limit phytoplsma spread in the plant was observed in both Chardonnay and Manzoni infected plants, with the repression of genes responsible for cell wall degradation and the induction of genes involved in cell wall reinforcement. Interestingly, specifically in cv. Manzoni the expression of a Myb transcription factor, belonging to a gene family that has a role in defense response, was induced.This is the first analysis of gene expression profiling in a grapevine-phytoplasma interaction using Affymetrix GeneChip® array. Presented data provide an interesting picture of the transcriptional response of grapevine to Bois Noir and allowed the selection of several candidate genes for future functional analysis.

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.

2008 - Il cancro batterico delle drupacee [Articolo su rivista]
Stefani, Emilio
abstract

Il cancro batterico è una malattia ricorrente e particolarmente dannosa delle drupacee, soprattutto pesco e susino .....

2008 - Use of a bacterial antagonist for the biological control of bacterial leaf/fruit spot of stone fruits. [Abstract in Atti di Convegno]
E., Biondi; Dallai, Davide; A., Brunelli; Stefani, Emilio
abstract

The possibility of bio-controlling the bacterial canker of stone fruit is highlighted. The use of bacterial antagonists to control the pathogen Xanthomonas arboricola pv. pruni proved to be very efficient.

2007 - Genetic variations among Xanthomonas arboricola pv. pruni populations from peach orchards in Romagna [Abstract in Rivista]
Dallai, Davide; Stefani, Emilio
abstract

The genetic variability among populations of Xanthomonas arboricola pv. pruni, the causal agent of the bacterial canker of stone fruits, was studied. Results highlighted the uniformity of most bacterial populations isolated in different geographical areas and in different years. This implies the possibility to efficiently control the pathogen by using a suitable antagonist.

2006 - PCR-RFLP and multiplex PCR assay for the specific detection of Ralstonia solanacearum race 3/biovar 2 [Abstract in Atti di Convegno]
Martini, Marta; Stefani, Emilio
abstract

A sensitive and reliable method for the molecular detection of Ralstonia solanacearum, race 3, biovar 2 is presented.

2005 - Detection of latent infections of Ralstonia solanacearum biovar 2, race 3 in tomato crops [Articolo su rivista]
Stefani, Emilio; Giosue, Stefana; Mazzucchi, Umberto
abstract

A protocol was set up to detect latent infections of Ralstonia solanacearum biovar 2, race 3 in field tomatoes. For two growing seasons, healthy tomato plants were inoculated with a virulent strain, and monitored for between 7 and 55 days. Final extracts used for direct isolation on Kelman's medium and for indirect immunofluorescence staining were prepared from 1 cm segments collected from the base of the lowest side shoots. Reisolations were identified by colony morphology, PCR, WAS, and pathogenicity tests on tomato plantlets. Reisolation was successful from 18 days onwards, with a frequency that constantly increased in the following weeks. Samples prepared by mixing one latently infected segment with increasing numbers of healthy segments revealed a sensitivity threshold of 1: 999. This non-destructive protocol was shown to be appropriate for monitoring in the open field.

2004 - Deperimento dll'albicocco: come difendere la pianta [Articolo su rivista]
Stefani, Emilio; Mazzucchi, Umberto
abstract

Si descrive una nuova malattia dell'albicocco, causata dal batterio fitopatogeno Pseudomonas syringae, e si suggeriscono le strategie di controllo.

2004 - Il deperimento dell'albicocco in Romagna: ulteriori indagini e prove di lotta [Relazione in Atti di Convegno]
Bugini, Simona; Mazzucchi, Umberto; Pirazzini, Paola; Stefani, Emilio
abstract

Nel 2003 si è avuta una recrudescenza della malattia, che ha interessato numerose varietà , ma soprattutto Aurora, Carmene Vitillo. Sono stati isolati nuovi e numerosi ceppi batteri fitopatogeni da cncri rameali attivi e indeterminati. Tre ceppi isolati delle annate passate sono stati inoculati su piante di albicocco della cv. Aurora, tramite tagli di potatura e attraverso le ferite da sgemmatura / caduta delle foglie; è stato possibile riprodurre i cancri della malattia dai tagli di potatura e in un solo caso da ferite di sgemmatura/caduta delle foglie. Sono stati inoltre approntati tre campi sperimentali nei pressi di Imola, Faenza e Cesena al fine di valutare interventi agronomici atti a limitare la progressione del deperimento: questi interventi si basano sulla individuazione del periodo ottimale per la potatura bruna, sulla messa a punto di appropriati interventi basati sull'utilizzo di prodotti rameici e su misure atte alla riduzione o eliminazione delle sorgenti di inoculo primario e secondario. I primi dati sperimentali sono promettenti ed hanno messo in evidenza la possibilità di intervenire positivamente per il risanamento degli albicoccheti colpiti dalla sindrome.

2004 - Problemi emergenti nella difesa delle colture: malattie batteriche [Articolo su rivista]
IACOBELLIS N., S; C., Bazzi; Stefani, Emilio
abstract

Sono descritte le principali avversità batteriche emergenti, o riemergenti, delle colture agrarie in Italia.

2004 - Prohexadione-ca: uno stimolo per le piante a difendersi dai patogeni. [Relazione in Atti di Convegno]
Bazzi, Carlo; Stefani, Emilio; Bini, Federica; Messina, Christian; Betti, Federica; Biondi, Enrico; Brunelli, Agostino; Gianati, Paolo
abstract

Il Prohexadione-Ca è un nuovo composto multifunzionale, ritardante la crescita vegetale, attualmente in uso su pomacee in diversi paesi, Italia compresa. Questo composto chimico che agisce da inibitoe diossigenasico nella biosintesi delle gibberelline, può costituire una valida alternativa all'uso di antibiotici e/o di altri antiparassitari nella profilassi del colpo di fuoco batterico delle rosacee da Erwinia amylovora e della ticchiolatura del melo da Venturia inaequalis. Inoltre l'applicazione programmata di tale composto chimico, privo di attività antibatterica e antifungina diretta, si è rivelato efficace nello stimolare significative risposte di difesa anche in importanti specie coltivate (pomodoro, vite) contro diversi patogeni batterici e fungini.

2003 - Control of pathogen incidence in pome fruits and other horticultural crop plants with prohexadione-Ca [Articolo su rivista]
Bazzi, C.; Messina, Ch.; Tortoreto, L.; Stefani, E.; Bini, F.; Brunelli, A.; Andreotti, C.; Sabatini, E.; Spinelli, F.; Costa, G.; Hauptmann, S.; Stammler, G.; Doerr, S.; Marr, J.; Rademacher, W.
abstract

Products containing Prohexadione-Ca represent a considerable alternative to antibiotics for the contgrol of secundary fireblight (Erwinia amylovora) in pear as well as in apple.

2003 - Investigations on the possible use of abiotic and biotic elicitors in defence-related responses in plants [Articolo su rivista]
Bazzi, Carlo; Messina, Christian; Tortoreto, Lucia; Bini, Federica; CECCA Gian, Stefano; Stefani, Emilio
abstract

Experiments were conducted to determine the protective effect triggered by the plant growth retardant prohexadione-Ca (Regalis®), bacterial lipopolysaccharides (LPS) and oligosaccharides (OLS) in different host-pathogen systems. These molecules did not show any direct antibacterial activity in vitro. On tobacco leaves, LPS infiltration (100 μg ml-1) prevented hypersensitive reaction (HR), whereas OLS at different concentrations did not give constant and reliable responses. On micropropagated pear plantlets in vials, only Regalis® and LPS (100 μg ml-1) gave a significant defence-related response against fire blight (Erwinia amylovora): in particular, plant-derived antimicrobial compounds were related to flavan-3-ols and appeared to be newly synthesized after LPS application. Regalis®, applied at different concentrations, significantly lowered progression of bacterial wilt (Ralstonia solanacearum) and reduced leaf lesions of bacterial speck (Pseudomonas syringae pv. tomato) and spot (Xanthomonas vesicatoria) on tomato plants in the greenhouse. OLS application (100 μg ml-1) significantly protected tomato leaves from bacterial speck, but not from bacterial spot. Two treatments with Regalis®, at a twelve-day interval and at different concentrations (75+50 ppm), gave a valuable protection of grapevine leaves to downy mildew (Plasmopara viticola) in the greenhouse. When Regalis® was sprayed at different concentrations and at different times on potted plum trees kept outdoors it was apparently ineffective against bacterial spot/canker (Xanthomonas arboricola pv. pruni). These findings further support the potential of prohexadione-Ca and of other biotic molecules as tools for plant defence responses against different pathogens, in the perspective of integrated, eco-friendly and sustainable control programmes.

2002 - Applicazione del modello previsionale Maryblyt per la valutazione dei rischi di infezione di colpo di fuoco batterico (Erwinia amylovora nelle aree frutticole marchigiane [Relazione in Atti di Convegno]
Stefani, Emilio; L. FLAMINI AND C., Bazzi
abstract

Nell'arco di un quadriennio si sono svolte indagini per la valutazione dei rischi di infezione di colpo di fuoco batterico in aree marchigiane coltivate a melo e a pero. Sono state effettuate ispezion mirate, analisi batteriologiche ed un'ampia raccolta di dati fenologici e meteorologici che sono stati inseriti nel modello di previsione Maryblyt 4.2. I risultati ottenuti hanno permesso di differenziare nell'ampia area sottoposta ad indagine delle microaree caratterizzate sia da diverso potenziale d'inoculo epifita che da diverso rischio di infezioni da Erwinia amylovora. L'elaborazione dei dati con il modello previsionale e l'analisi dei risultati ottenuti hanno permesso di ottenere informazioni utili per l'individuazione delle zone a basso rischio d'infezione come pure dettagliate indicazioni per la gestione dei meleti e pereti esistenti.

2002 - Molecular methods for the detection of Ralstonia solanacearum (race3/biovar2) and for biovar differentiation. [Articolo su rivista]
Martini, Marta; Stefani, Emilio
abstract

Ralstonia solanacearum can be separated into 4 races and 5 biovars. R. solanacearum race3/biovar2 infects primarily potato, causing brown rot disease, and has never been reported to infect potato in the US. Brown rot disease is spread by latently infected seed potatoes; therefore a highly sensitive diagnostic method is required for quarantine purposes. In the present study, primer pairs designed from a novel repetitive insertion sequence were developed for PCR detection of R. solanacearum race3/biovar2. This PCR method provided a reliable and sensitive tool for the specific detection of all the R. solanacearum race3/biovar2 strains tested, including strains isolated from geranium in the US. Southern hybridization and rep-PCR analyses showed that the geranium strains are very closely related to the potato strains. A PCR/RFLP method based on the ribosomal protein operon was also developed for the molecular differentiation of 5 biovars, which are currently distinguished according to different biochemical properties .

2002 - Un grave deperimento dell'albicocco associato alla presenza di pseudomonadi fitopatogene [Abstract in Atti di Convegno]
Stefani, Emilio; Gozzi, Rossella; Spada, Gianluigi
abstract

Viene presentata una nuova malattia dell'albicocco, il deperimento batterico, causata da Pseudomonas syringae pv. syringae.

2001 - Phytoplasma infection in peach and cherry in Italy. [Articolo su rivista]
Paltrinieri, Samantha; Martini, Marta; Stefani, Emilio; Pondrelli, Massimo; Fideghelli, Carlo; Bertaccini, Assunta
abstract

A survey to verify phytoplasma presence was carried out in North-Central Italy on samples from peach and cherry orchards using the same PCR/RFLP methodology for both species. Direct and nested PCR with primers amplifying phytoplasma ribosomal or ribosomal plus spacer DNA were applied. Primers specific for different phytoplasma groups already detected in fruit trees, as well as primers amplifying a short (500 bp) ribosomal phytoplasma DNA fragment, were used to increase detection sensitivity. RFLP analysis for phytoplasma identification was then performed. Three nectarine cultivars showing some trees with delay in flowering and in fruit production were tested together with asymptomatic plants in April/May 1999. Only nested PCR with phytoplasma group specific or M1/M2 primers provided positive results. RFLP analyses identified 16SrX-A, 16SrI, and 16SrXII-A phytoplasmas. In one plant a mixed infection of 16SrI and 16SrX-A phytoplasmas was detected. No clear relationship was found among symptoms, peach variety and phytoplasma presence. The sweet cherry variety Prime Giant showed a decline shortly after plantation; in samples tested during spring and summer phytoplasmas of 16SrI-B, 16SrX-B; 16SrX-C, 16SrXII-A and 16SrIII groups were often detected therefore a relationship between cherry decline and phytoplasmas could be possible.

2000 - Protocollo armonizzato per la diagnosi di Ralstonia Solanacearum in piante e tuberi di patata. [Relazione in Atti di Convegno]
Messina, Christian; Stefani, Emilio; Mazzucchi, Umberto
abstract

Ralstonia solanacearum è un organismo nocivo a tolleranza fitosanitaria zero, oggetto della Direttiva UE di lotta del Consiglio delle Comunità Europee 98/57. La razza 3 (biovar 2) patogena per patat, pomodoro e melanzana è di preminente interesse per gli Stati dell'UE e per i Paesi circummediterranei. Piante spontanee, solanacee o non, possono essere ospiti secondari. L'introduzione e la disseminazione in nuove aree ha luogo principalmente attraverso il commercio dei tuberi di patata, da tavola e da seme, dove le infezioni possono essere palesi o latenti. Rischi di disseminazione è offerto anche dai mezzi di trasporto contaminati. La Direttiva prescrive che i Servizi Fitosanitari degli Stati UE attuino un sistematico monitoraggio delle proprie produzioni di patata e del loro territorio incluse le acque superficiali e i liquidi di scarico delle industrie che lavorano e trasformano le patate. In questo lavoro si descrive il protocollo che i Servizi Fitosanitari Europei devono seguire per effettuare diagnosi corrette delle infezioni palesi o latenti conformemente agli allegati della Direttiva UE.

2000 - Standardization of methods for detection of Ralstonia solanacearum in potato [Articolo su rivista]
Elphinstone, John; Stead, David; Caffier, David; Janse, Jaap; López, Maria Milagro; Mazzucchi, Umberto; Müller, Petra; Persson, Paula; Sousa Santos, Madalena; Stefani, Emilio; van Vaerenbergh, Johan
abstract

In support of EU Council Directive 98/57/EC on the control of potato brown rot disease, caused by Ralstonia solanacearum, a ring test was conducted to evaluate and standardize currently recommended methods for official testing of potato tubers. Test tubers were either naturally infected (symptomatic) or vacuum-infiltrated with different R. solanacearum suspensions or water. Testing was performed independently in 19 EU plant health laboratories using standardized protocols based on those annexed to the Directive. With high pathogen concentrations, fully reliable results were obtained with dilution plating on a selective medium, immunofluorescent antibody staining (IFAS), enzyme-linked immunosorbent assay (ELISA), fluorescent in situ hybridization and a tomato bioassay. The reliability of diagnoses across all laboratories decreased with inoculum concentration in asymptomatic tubers, irrespective of the methods used. Comparable results were obtained with 3 commercial antisera in IF AS or with 2 antisera in ELISA but the IFAS method was slightly more reliable. In several laboratories, diagnoses obtained using polymerase chain reaction (PCR) protocols were less reliable than those using the other methods. Independent optimization of PCR conditions in each laboratory and the introduction of internal positive PCR controls are recommended.

1999 - Biological control of Agrobacterium vitis using non-tumorigenic agrobacteria [Articolo su rivista]
Bazzi, C.; Alexandrova, M.; Stefani, E.; Anaclerio, F.; Burr, T. J.
abstract

The potential use of non-tumorigenic agrobacteria for the biological control of grapevine crown gall in Italy was investigated. Four Agrobacterium strains belonging to the species radiobacter and vitis were used to protect the susceptible cv. Malvasia Istriana grafted on the rootstock 420 A. Moreover, the effect of each treatment on grapevine vitality and growth was assessed, including the percentage of marketable vines, as determined by industry standards. Treatments with the antagonists clearly reduced tissue colonization by the pathogen, with a drop of more than 100-fold in pathogen populations in the samples collected at the graft point. Another important effect was the reduction of internal necrosis possibly induced by the high concentration of the nopaline strain CG 49 used in the experiments. According to viticultural and commercial parameters, treatments with the antagonists improved the quality of the vines, with fewer discards and a high percentage of marketable material. Therefore, these antagonists can be considered beneficial for grapevine.

1998 - The economic impact of the bacterial blight of soybean under European agroclimatic conditions [Articolo su rivista]
Stefani, E.; Caffier, D.; Fiore, N.
abstract

The economic impact of the bacterial blight of soybean caused by Pseudomonas syringae pv. glycinea has been investigated in three countries belonging to the European Union: Italy, France and Spain. Weather and growing conditions have been monitored over three years of field experiments (1992-1994) and the data analysed in order to evaluate possible yield losses and in view of the production of pathogen free seed. In Italy and France, using different cultivars and seed with a contamination level of 0.5-20% no significant yield losses were found. In Italy the initial seed contamination level was positively correlated with the contamination of the harvested seed by the pathogen; both in Italy and in France on some cultivars, it was possible to correlate seed contamination level with the epiphytic population of the pathogen and the intensity of symptoms affecting plants in the field. No epiphytic contamination by the pathogen was observed in Spain, even at the highest seed contamination rate (20%), and there was no disease in the field and no yield reduction. The pathogen seemed not to become systemic since no contamination was observed on seed aseptically taken in the field just before harvest. The experiments highlighted the low impact of soybean bacterial blight under European climatic conditions, but suggested the choice of dry and warm regions for the production of quality seed to prevent the accumulation of effective inoculum on the seed year by year.

1997 - Protein electrophoretograms for the identification of ralstonia solanacearum in potato tubers [Articolo su rivista]
Stefani, E.; Mazzucchi, U.
abstract

Densitometric analysis was performed on the whole cell proteins of 53 strains of Ralstonia solanacearum (Rs) isolated from different host plants and geographical areas. This study also included 18 unidentified, endophytic potato bacteria, non-pathogenic on tomato and eggplant, forming colonies similar to Rs (Rs-like bacteria) on differential or selective media. The comparison of the electrophoretograms was performed by calculating the correlation coefficient (r) and by principal component analysis. Visual comparison generally made it possible to distinguish between the Rs strains and nonpathogenic Rs-like bacteria. Densitometric analysis of the electrophoretograms revealed a main group of strains (r=0.76) including 4 subgroups. Subgroup 1 (r=0.92) included potato and tomato strains attributable to race 3 biovar 2, including all those of Mediterranean origin. Principal component analysis again included the Mediterranean strains in a single group characterised by just one of the two bands identified as differential in the Rs profile. Principal component analysis revealed that one Rs-like bacterium, with a markedly higher correlation coefficient (r=0.83), was clearly distinct from Rs. The results confirmed the reliability of the European diagnostic protocol as a basis for identification at the species level, that must be based on the pathogenicity test combined with whole cell protein profiles when these are used as a single phenotypic fingerprint.

1997 - Pseudomonas syringae pv. aptata HRP mutants which fail to produce periplasmic oligosaccharides [Relazione in Atti di Convegno]
Stefani, Emilio; Minardi, Paola
abstract

Periplasmic oligosaccharides, extracted from Pseudomonas syringae pv. aptata and purified by column chromatography, modify the homologous interaction between tobacco leaves and P. syringae pv. tabaci. The osmoregulating oligosaccharides can act as signal molecules to plant cells, but the molecular basis of this function is not known. Genetical analysis of pathogenicity determinants in P. s. pv. aptata strain NCPPB2664 allowed the identification of hrp genes which are essential in governing the ability of the bacteria to cause disease on host plants (such as sugar beet, lettuce), and the ability to induce the hypersensitive response on non-host plants (such as tobacco). The current study addresses whether a correlation between the pathogenicity and the production of oligosaccharides exists. To test the hypothesis that in the early stages of plant-bacteria interactions the hrp genes might be involved in the production of the periplasmic oligosaccharides, the latter has been studied in P. syringae pv. aptata Hrp- mutants.

1996 - Maryblyt 4.2: Prediction of fire blight outbreaks in Italy (Po valley) [Relazione in Atti di Convegno]
Bazzi, C.; Merighi, M.; Stefani, E.; Gambin, E.; Saccardi, A.; Calzolari, A.; Perugini, C.
abstract

A project (1993-1996) is under way to assess infection risks by analyzing the phenological-climatological data using the MARYBLYT model and monitoring Erwinia amylovora in asymptomatic pear, apple and hawthorn flowers collected in different Veneto and Emilia-Romagna fruit-growing areas. Analyses of bulk samples of intact open flowers with brown anthers were all negative and conventional and molecular methods did not reveal E. amylovora in any of the suspect bacterial isolates. In Veneto, data for MARYBLYT processing were collected from a "Conference" pear orchard. In 1993 the model revealed high risk conditions in the last week of bloom and in the post-bloom period with 3 and 1 infection cycles of blossom blight symptom (BBS) and shoot blight symptom (SBS). No secondary bloom was recorded. In 1994, BBS were hindered by low temperatures during bloom; delay in SBS was due to the absence of blossom infections. The model predicted trauma blight symptoms (TBS) caused by hail at the end of August but not BBS on secondary blossoms in October. In 1995 no BBS were forecasted during primary bloom. In Emilia-Romagna, the model was assessed in 1994-95 in 2 and 4 stations. The risk index in 1994 was medium-low with no BBS on "Williams" and "Conference" pears or "Golden" apples. Four infections during secondary bloom (April-May) were only predicted in one station. In 1995 the weather was very conducive to BBS during secondary bloom due to higher temperatures and abundant rain. MARYBLYT seemed to provide an accurate prediction of infection periods and/or symptom development.

1995 - Testing for Pseudomonas syringae pv. atrofaciens and Xanthomonas campestris pathovars on cereals in Italy [Articolo su rivista]
Alexandrova, M.; Zaccardelli, M.; Stefani, E.; Bazzi, C.
abstract

During recent years, recurrent attacks of basal glume rot/leaf blight attributed to Pseudomonas syringae pv. atrofaciens have been observed on cultivars of durum wheat grown in the Po valley (IT). The main aim of this study was to test for this pathogen and Xanthomonas campestris pathovars in commercial seed lots of cereals produced in different regions of Italy, as well as in symptomatic plants collected in the field. None of the analyses led to the detection of xanthomonads. In testing for P.s. atrofaciens, representative bacterial isolates were selected and characterized; this was done by combining conventional identification tests and computerized densitometric analysis of electrophoretic patterns of cell proteins obtained using the SDS‐PAGE technique. P.s. atrofaciens was detected in seed samples (barley, wheat and durum wheat) and symptomatic plants (durum wheat) grown in northern and central Italy. The possible pathogenic role of other unrelated fluorescent pseudomonads in this interaction was also examined. Difficulties in discriminating pvs syringae and atrofaciens of P. syringae are discussed and the uncertain taxonomic relationship between these two pathovars is emphasized. Copyright © 1995, Wiley Blackwell. All rights reserved

1994 - Modification of the Pseudomonas syringae pv. tabaci -tobacco leaf interaction by bacterial oligosaccharides [Articolo su rivista]
Stefani, E.; Bazzi, C.; Mazzucchi, U.
abstract

Cell-associated oligosaccharides were extracted with trichloroacetic acid treatment from Pseudomonas syringae pv. aptata, purified by column chromatography, analysed by GC-MS and fast atom bombardment-mass spectrometry, and bioassayed on tobacco leaves. The material was shown to be a mixture of neutral oligosaccharides, with a mol. wt of 800-1800, degree of polymerization 5-11, and linear glucose and mannose chains. Local infiltrations of the mixture, in concentrations of 1 mg, 100, 10 and 1 μg ml-1, did not cause tobacco tissue necrosis within 1 week. When the mixture was infiltrated in the interveinal leaf panels, in concentrations of 1 μg, 100, 10 and 1 ng ml-1, 48 h before challenge inoculation, it delayed or prevented normosensitive necrosis by P. syringae pv. tabaci but not hypersensitive necrosis by P. syringae pv. aptata. Five days after inoculation with P. syringae pv. tabaci, inhibition of normosensitive necrosis, as compared with the control tissue, was 80·55, 77·21, 56·36 and 45·46 % for the four concentrations. In the bioassay with P. syringae pv. tabaci the efficacy threshold was assessed at approx. 1 ng ml-1. Pre-treatment with oligosaccharides (1 μg ml-1) significantly inhibited the growth of P. syringae pv. tabaci between 3 and 5 days after inoculation and, between day 6 and 8, it delayed the death of bacteria when the tissue was not subject to necrosis. An interval of 6 h was necessary between the infiltration of oligosaccharides (1 μg ml-1) and inoculation of P, syringae pv. tabaci for a clear inhibition of normosensitive necrosis. These oligosaccharides acted as signal molecules by modifying, at nanomolar concentrations, the P. syringae pv. tabaci-tobacco leaf interaction, but they were not elicitors of the hypersensitive reaction. © 1994 Academic Press, Inc.

1994 - SDS‐PAGE: a tool to discriminate Xylella fastidiosa from other endophytic grapevine bacteria [Articolo su rivista]
Bazzi, C.; Stefani, E.; Zaccardelli, M.
abstract

There is a real risk of introducing Xylella fastidiosa into the EPPO region where this quarantine pest has not yet been found and constitutes a serious threat to the Mediterranean grapevine industry. Direct isolation of endophytic bacteria from imported propagation material, combined with one‐dimensional SDS‐PAGE of the total soluble proteins of the bacterial cell envelope, can be considered as a test to be used in grapevine certification schemes. The gel electrophoretic pattern of Xylella fastidiosa is characterized by a constant distribution of 14 major diagnostic bands. Even without gel scanning, this relatively simple technique allows discrimination between grapevine strains of X. fastidiosa and other xylem‐inhabiting, slow‐growing bacteria. Copyright © 1994, Wiley Blackwell. All rights reserved

1993 - Ultrastructure in Bean leaves infiltrated with bacterial extracellular polysaccharides [Articolo su rivista]
Stefani, Emilio; Bonatti, Piera
abstract

Ultrastructural observations were carried out in bean leaf tissue infiltrated with bacterial extracellular polysaccharides. These investigations revealed changes in the cell fine structure, especially related with chloroplast organization. Up to 24 h after EPS infiltration alterations consisted in irregularly running thylakoids and distortions of stacked regions. Invaginations of the plastid envelope and appearance of cytoplasm pockets inside the stroma were also noted. The most severe alterations, consisting in stroma dilations and envelope infoldings, were noted 24 h after EPS infiltration, paralleling the yellowing of the treated leaf areas; chloroplast shrinkage and collapse of thylakoid system were also sometimes observed. Chloroplast ultrastructure generally recovered 48 h after treatment; at this time local detachment of the plasma membrane and vesicle formation in the periplasmic space were observed, resembling a non specific, localized cellular response. The observed, permanent chlorosis and ultrastructural alterations suggest an interference of the EPS in the global metabolism of the bean mesophyll cell.

1989 - Induced Resistance in Bean Leaves Pretreated with Extracellular Polysaccharides from Phytopathogenic Bacteria [Articolo su rivista]
Stefani, E.; Rudolph, K.
abstract

Bean leaves were infiltrated with bacterial extracellular polysaccharides (= EPS) in order to study their effect on a subsequent (48 h later) inoculation with bacteria during compatible and incompatible interactions. Three different EPS‐preparations from the pathovars pisi and phaseolicola of Pseudomonas syringae were tested: pisi‐EPS (S) contained predominantly levan, phaseolicola‐EVS (Go) predominantly alginate, and phaseolicola‐EPS (S) contained about equal proportions of levan and alginate. Bean leaves showed a distinct chlorosis 48 hours after treatment with phaseolicola‐EPS. The susceptible cultivar ‘Red Kidney’ was more sensitive then the resistant breeding line ‘02’. The latter sometimes developed necrotic reactions after EPS‐treatment. Pisi‐EVS induced only a very weak chlorosis. No chlorosis appeared after treatment with commercial alginate (obtained from seaweed). Other plant species revealed a decreasing sensitivity towards EPS in the following order: ‘Red Kidney’ bean, tobacco, soybean, tomato, cotton. Chlorosis caused by phaseolicola‐EPS was obviously not due to phaseolotoxin, since EPS‐preparations did not inhibit E. coli, and since the concentration of ornithine in affected bean leaves increased only very slightly. EPS‐treatment of ‘Red Kidney’ bean leaves induced a “resistance” towards a subsequent bacterial inoculation in the compatible (P. phaseolicola) as well as in the incompatible (P. pisi) combination. The latter case may more precisely be termed “protection”. A similar effect was observed in the resistant bean ‘02’, that is a protection towards HR (= hypersensitive reaction), which normally develops after inoculation with high bacterial concentrations. Several differences in the reactions of the tested cultivar‐EPS combinations were observed. In general, bacterial EPS in bean leaves induced a more effective protection against P. pisi than against P. phaseolicola. In the cultivar ‘Red Kidney’ the induced resistance against P. phaseolicola was more pronounced with phaseolicola‐EPS than with pisi‐EPS. Induzierte Resistenz in Bohnenblättern, die mit extrazellulären Polysacchariden phytopathogener Bakterien vorbehandelt wurden Bohnenblätter wurden mit bakteriellen, extrazellulären Polysacchariden (EPS) infiltriert, um deren Wirkung auf eine 48 Stunden später erfolgende Bakterieninokulation bei kompatibler und inkompatibler Interaktion zu untersuchen. Drei verschiedene EPS‐Präparate der Pseudomonas syringae Pathovare pisi und phaseolicola wurden eingesetzt: pisi‐EPS (S) enthielt überwiegend Lävan, phaseolicola‐EPS (Go) vornehmlich Alginat und phaseolicola‐EPS (S) bestand zu etwa gleichen Anteilen aus Lävan und Alginat. Auf Bohnenblättern entwickelte sich 48 h nach Behandlung mit phaseolicola‐EPS eine deutliche Chlorose. Die anfällige Sorte ‘Red Kidney’ war empfindlicher als die resistente Zuchtlinie ‘02’. Auf letzterer entstanden manchmal nekrotische Reaktionen nach EPS‐Behandlung. P. pisi‐EPS rief nur eine sehr schwache Chlorose hervor. Nach Behandlung mit einem Handelspräparat von Alginat (aus Meeresalgen gewonnen) erschien keine Chlorose. Andere Pflanzenarten zeigten gegenüber EPS eine abnehmende Empfindlichkeit in folgender Reihenfolge: ‘Red Kidney’ Bohnen, Tabak, Sojabohnen, Tomaten, Baumwolle. Die durch phaseolicola‐E'PS hervorgerufene Chlorose wurde offenbar nicht durch Phaseolotoxin verursacht, da EPS‐Präparationen E. coli nicht hemmten, und da die Konzentration von Ornithin im behandelten Blatt nur sehr schwach anstieg. EPS‐Behandlung von ‘Red Kidney’‐Bohnenblättern induzierte eine Resistenz gegenüber einer 48 Stunden später folgenden Bakterieninokulation, sowohl in der kompatiblen (P. phaseolicola) wie in der inkompatiblen (P. pisi) Kombination. Der zweite Fall müßte zutreffender als “Schutzeffekt” (= p

1986 - Ultrastructural Evidence for Cellular Response in Tobacco Leaves Following Infiltration with Protein‐Lipopolysaccharide Complexes of Pseudomonas syringae pv. tabaci) [Articolo su rivista]
Bonatti, P. M.; Stefani, E.; Mazzucchi, U.
abstract

The ultrastructure of tobacco leaves cv. White Burley infiltrated in the intercellular spaces with protein‐lipopolysaccharide (pr‐LPS) complexes of a virulent strain of Pseudomonas syringae pv. tabaci was studied. Light and electron microscope observations were made immediately and 4, 6, 8 and 24 h after infiltration. For the light microscope semithin sections were treated with the periodic acid‐Schiff (PAS) method to reveal polysaccharides. For electron microscopy ultrathin sections of 24 h samples were cytochemically treated to reveal the plasmalemma, the proteins and the β → 4 polysaccharides. Light microscopy only revealed the presence of intercellular PAS positive areas in the treated tissue from 6 h onwards. Electron microscopy revealed a localized cellular response (L.C.R.) on the walls where pr‐LPS complexes had formed deposits. The L.C.R., barely visible after 4 h, became well established at 24 h. It involved the plasmalemma, which retracted, and the underlying cytoplasm, where there was an increase in the endoplasmic reticulum. Details of the ultrastructural alterations were interpreted on the basis of the cytochemical visualization. During the L.C.R. the plasmalemma appeared to be site of an active transport process. The results show that the virulent strain of P. syringae pv. tabaci possesses surface macromolecules containing cognons involved in recognition by tobacco leaf cells. Die Ultrastruktur von Tabakblättern der Sorte ‘White Burley’ wurde nach der Infiltration von Protein‐Lipopolysaccharid (pr‐LPS)‐Komplexen eines virulenten Stammes von Pseudomonas syringae pv. tabaci in die Interzellularräume untersucht. Licht‐ und elektronenmikroskopische Beobachtungen wurden 4, 6, 8 und 24 Std. nach Infiltration durchgeführt. Für die lichtmikroskopischen Studien wurden halbdünne Schnitte nach der Schiff'schen Perjodsäure (PAS)‐Methode behandelt, um Polysaccharide sichtbar zu machen. Für die Elektronenmikroskopie wurden Ultradünnschnitte von 24 Std. pi‐Proben cytochemisch aufbereitet, um Plasmalemma, Proteine und die β1→4 Polysaccharide kenntlich zu machen. Die lichtmikroskopischen Untersuchungen ergaben erst ab 6 Std. pi im behandelten Gewebe bei Gegenwart von interzellulärer PAS positive Zonen. Im Elektronenmikroskop war eine lokalisierte zelluläre Reaktion (L.C.R.) an den Zellwänden sichtbar, an denen die pr‐LPS‐Komplexe Ablagerungen gebildet hatten. Die L.C.R. war nach 4 Std. kaum sichtbar, nach 24 Std. deutlich ausgebildet und führte zu einer Kontraktion des Plasmalemmas und im darunter liegenden Cytoplasma zu einer Zunahme des endoplasmatischen Reticulums. Einzelheiten der ultrastrukturellen Veränderungen wurden auf der Basis der cytochemischen Sichtbarmachung interpretiert. Während der L.C.R.‐Reaktion war das Plasmalemma offenbar der Ausgangspunkt für den aktiven Transportprozeß. Die Ergebnisse zeigen, daß der virulente Stamm von P. syringae pv. tabaci an den Oberflächen Makromoleküle besitzt, die sog. Cognon enthalten, und von den Tabak‐Blattstellen als solche wieder erkannt werden. 1985 Paul Parey Scientific Publishers, Berlin and Hamburg