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Made available in DSpace on 2022-05-01T15:46:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-01-01 Key message: Based on transcriptome and qPCR analysis, a phenotypic abnormality resulting from a Eucalyptus crossing is associated with overexpression of metabolic pathways related to defense responses against biotic and abiotic stress. Abstract: Genetic recombination by outcrossing, a strategy used in improvement programs, can present a low frequency of individuals within progeny with morphological and physiological abnormalities. An abnormality was identified in progeny from a controlled cross of Eucalyptus grandis. Applying RNA-Seq technology to leaf, stem, and root tissue of normal and abnormal seedlings grown under the same greenhouse conditions, we examined global gene expression changes and compared the transcriptional profiles of the two contrasting phenotypes. We also conducted functional enrichment and metabolic pathway analyses. Among the identified differentially expressed genes, most were upregulated in leaf and stem tissues of abnormal plants, with gene ontology (GO) terms associated with 'response to stress', 'response to stimulus', and 'immune system process' categories. Further, in differential gene expression and functional enrichment analyses, we observed overexpression of metabolic pathways related to defense response processes in abnormal plants. Our results suggest that the anomaly is associated with changes in the defense response to biotic and abiotic stresses. As all progeny were grown in a controlled environment and there was no evidence of pathogenic attacks, the defense response in abnormal plants may be due to an autoimmune mechanism caused by genetic incompatibility. Departamento de Ciências Químicas e Biológicas Instituto de Biociências Universidade Estadual Paulista (UNESP), SP Instituto de Biotecnologia Universidade Estadual Paulista (UNESP), SP Departamento de Biofísica e Farmacologia Instituto de Biociências Universidade Estadual Paulista (UNESP), SP Luxembourg Centre for Systems Biomedicine (LCSB) University of Luxembourg Empresa Suzano Papel e Celulose SA, Av. Dr. José Lembo 1010, SP Plant Pathology and Entomology Section Department of Agricultural Food Environmental and Forestry Science and Technology (DAGRI) University of Florence, Florence province School of Agriculture São Paulo State University (Unesp), SP Departamento de Ciências Químicas e Biológicas Instituto de Biociências Universidade Estadual Paulista (UNESP), SP Instituto de Biotecnologia Universidade Estadual Paulista (UNESP), SP Departamento de Biofísica e Farmacologia Instituto de Biociências Universidade Estadual Paulista (UNESP), SP School of Agriculture São Paulo State University (Unesp), SP

Paulownia species are fast growing trees native to China, which are being grown in managed plantings in several European countries for the production of wood and biomasses. In 2018, wilting, stunting, leaf yellowing, and collapse, as a consequence of root and crown rot, were observed in around 40% of trees of a 2-year-old planting of Paulownia elongata × P. fortunei in Calabria (Southern Italy). Two species of Phytophthora were consistently recovered from roots, basal stem bark, and rhizosphere soil of symptomatic trees and were identified as Ph. nicotianae and Ph. palmivora on the basis of both morphological characteristics and phylogenetic analysis of rDNA ITS sequences. Koch’s postulates were fulfilled by reproducing the symptoms on potted paulownia saplings transplanted into infested soil or stem-inoculated by wounding. Both Phytophthora species were pathogenic and caused root rot and stem cankers. Even though P. palmivora was the only species recovered from roots of naturally infected plants, in pathogenicity tests through infested soil P. nicotianae was more virulent. This is the first report of Phytophthora root and crown rot of a Paulownia species in Europe. Strategies to prevent this emerging disease include the use of healthy nursery plants, choice of well-drained soils for new plantations, and proper irrigation management.

Oaks are dominant and key tree species in Mediterranean forest ecosystems. However, in recent decades, oak forests have been heavily impacted by oak decline, a worldwide phenomenon exacerbated by climate change. The charcoal disease agent Biscogniauxia mediterranea is involved in the decline of Mediterranean oak formations in a variety of contexts. Here, we investigated the impact and role of B. mediterranea in the decline of oaks in Castelfidardo Forest, a relict wood of the late Holocene and a Site of Community Importance. We established five plots within which we recorded tree positions, any symptoms and signs of decline, association of B. mediterranea to declining trees, and deadwood and associated mycota. Of 471 oaks inspected, 7.0% showed brownish exudates on the stems, 46.9% showed epicormic shoots along the main trunk, and 24.4% showed black carbonaceous stromata on diseased branches and trunks. The decline was most severe for Quercus cerris, which comprised plots #4 and #5, at 50.0% (81/162 trees) and 29.0% (33/114), respectively, then for Quercus robur for plot #3, at 40.0% (38/95), and finally for Quercus pubescens for plots #1 and #2, at 13.7% (7/51) and 12.3% (6/49), respectively. Bark tissue was collected from trees with charcoal cankers via microscopy examination and identified by mycological and molecular methods. This investigation revealed a close association between oaks with pronounced reduction of vitality and incidence of B. mediterranea. Deadwood was equally distributed among the five plots, and was heavily colonized by Basidiomycota. The high incidence of the charcoal canker pathogen B. mediterranea appeared to be related to environmental stresses. However, the absence of silvicultural management, high competition among physiologically mature trees, and the geographic isolation of this residual forest may have predisposed oaks to decline.

In this work, we rechecked, using species-specific Loop mediated isothermal AMPlification (LAMP) diagnostic assays followed by sequencing of fungal isolates at the beta-2-tubulin (tub2) gene region, a historical and never confirmed report of Dothistroma needle blight (DNB) in the introduced Monterey pine (Pinus radiata D. Don) in the mountains in the extreme tip of southern Italy. The report dates back to the mid-1970s, and predates the molecular-based taxonomic revision of the genus Dothistroma that defined the species accepted today. In the fall of 2019, symptomatic needles of Monterey pine and Corsican pine (Pinus nigra subsp. laricio (Poir.) Palib. ex Maire) were sampled in the area of the first finding. The applied diagnostic methods revealed the presence of Dothistroma septosporum (Dorogin) M. Morelet on both pine species. In this way, we: (i) confirmed the presence of the disease, (ii) clarified the taxonomic identity of the causal agent now occurring at that site, (iii) validated the species-specific LAMP diagnostic protocol we recently developed for Dothistroma for use on a portable field instrument, and (iv) showed that the pathogen now also attacks the native P. nigra subsp. laricio, a species particularly susceptible to the disease, indigenous to the mountains of Calabria, which is one of the very few areas where the species’ genetic resources are conserved. Comparative genetic analysis of the rare populations of D. septosporum found in the central Mediterranean region and in the native range of P. nigra subsp. laricio could help to clarify the history of the spread of the pathogen in southern Europe and better evaluate the risk it poses to the conservation of native pine species.

Oaks are a dominant and key tree species in Mediterranean forest ecosystems. However, in recent decades, oak forests have been heavily impacted by oak decline, a worldwide phenomenon exacerbated by climate change. The charcoal disease agent Biscogniauxia mediterranea is involved in the decline of Mediterranean oak formations in a variety of contexts. Here, we investigated the impact and role of B. mediterranea in decline of oaks in Castelfidardo Forest, a relict wood of the late Holocene and a Site of Community Importance. We established five plots within which we recorded the tree positions, any symptoms and signs of decline, association of B. mediterranea to declining trees, and the deadwood and associated mycota. Of 471 oaks inspected, 7.0% showed brownish exudates on the stems, 46.9% showed epicormic shoots along the main trunk, and 24.4% showed black carbonaceous stromata on diseased branches and trunks. The decline was most severe for Quercus cerris, which comprised plots #4 and #5, at 50.0% (81/162 trees) and 29.0% (33/114), respectively; then for Quercus robur for plot #3, at 40.0% (38/95); and finally for Quercus pubescens for plots #1 and #2, at 13.7% (7/51) and 12.3% (6/49), respectively. Bark tissue was collected from trees with charcoal cankers, with microscopy examination and identification by mycological and molecular methods. This investigation revealed close association between oaks with pronounced reduction of vitality and incidence of B. mediterranea. The deadwood was equally distributed among the five plots, and was heavily colonized by Basidiomycota. The high incidence of this oak-weakening pathogen B. mediterranea appears to be related to stress conditions (e.g., water scarcity, prolonged drought). However, absence of silvicultural management, high competition among physiologically mature trees, and the geographic isolation of this residual forest might have predisposed these oaks to decline.

Forest health worldwide is impacted by many invasive alien pathogens and pests (IAPPs) that cause significant harm, with severe economic losses and environmental alterations. Destructive tree pathogens and pests have in the past devastated our forests, natural landscapes and cityscapes and still continue to represent a serious threat. The main driver of pathogen and pest invasions is human activities, above all global trade, which allows these invasive species to overstep their natural distribution ranges. While natural transport occurs according to a regular, expected colonization pattern (based on the dispersive capacity of the organism), human-mediated transport takes place on a larger, unpredictable scale. In order for a pathogen or pest species to become invasive in a new territory it must overcome distinct stages (barriers) that strongly affect the outcome of the invasion. Early detection is crucial to enabling successful eradication and containment. Although sophisticated diagnostic techniques are now available for disease and pest surveillance and monitoring, few control and mitigation options are usable in forestry; of these, biological control is one of the most frequently adopted. Since invasion by pathogens and pests is an economic and ecological problem of supranational relevance, governments should endorse all necessary preventive and corrective actions. To this end, establishing and harmonizing measures among countries is essential, both for preventing new introductions and for diminishing the eventual range expansion of IAPPs present at a local scale. Research is fundamental for: (i) developing effective and rapid diagnostic tools; (ii) investigating the epidemiology and ecology of IAPPs in newly introduced areas; and (iii) supporting policymakers in the implementation of quarantine regulations.

Melampsoridium hiratsukanum is an alien rust fungus which has spread pervasively throughout several European countries following introduction into North Europe at the end of the 20th century. The authenticity of several records of the Melampsoridium species infecting alder (Alnus spp.) in the northern hemisphere is questionable, due to the misidentification and confusion that surround many of the older reports. Given this complicated taxonomic history, and since a M. hiratsukanum-like rust is strongly impacting Alnus incana stands in the Alps, probably affecting the bank protection role of this species along rivers, the unambiguous identification of this pathogen was a pressing epidemiological and ecological issue. In this study, field surveys, light (LM) and scanning electron microscopy (SEM), and molecular characterization were put together in an attempt to solve the conundrum. Field monitoring data, LM and SEM analyses of key taxonomic traits (length of ostiolar cells of uredinium, uredinio-spore shape and size, spore echinulation, number and position of germ pores) and ITS-rDNA sequence-based identification, convergently and unambiguously connected the rust that is causing the current epidemic to the non-native M. hiratsukanum. We documented the completion of the M. hiratsukanum life cycle on its two taxonomically unrelated broadleaf/conifer hosts. This is the first report of M. hiratsukanum from naturally infected Larix decidua in Europe.

The aim of this study was the characterization of constitutive and induced defence mechanisms in the bark tissues of Cupressus sempervirens before and after infection with the bark fungus Seiridium cardinale that is responsible for Cypress Canker Disease. The time-course development of polyphenolic parenchyma cells (PP cells) and phloem axial resin ducts PARDs(PARD) like structures in the phloem was investigated in two C. sempervirens clones, one resistant and one susceptible to the disease, through anatomycal and hystologycal observations carried out by light microscope during a 19 days trial. PP cells were constitutively more abundant in the canker resistant clone compared to the susceptible clone, while PARDsPARD-like structures were not present in the bark of untreated plants of both clones. PP cells increased in both clones as a response to infection, but in the resistant clone they were more abundant 5 and 12 days after inoculation. Following inoculation, PARDsPARD-like structures appeared in the phloem after 5 days in the resistant clone and only after 12 days in the susceptible clone. Even the number of secretory cells(surrounding the PARDsPARD-like structures) was higher in the R clone 5 and 12 days after inoculation compared to the S clone. These observations demonstrate a faster phloem response of the resistant clone in the early phase of the infection. This may slow down initial growth of the fungus contributing to the resistance mechanism.

Tree pathogens and pests are fundamental components of forest ecosystems. By killing and decomposing susceptible trees, they regulate the cycle of nutrients and energy flow, thus shaping the structure and composition of forest stands. However, ecosystems can be seriously disrupted when the population density of these parasites increases beyond their tolerance level. Ascertaining the origin of pathogen and pest outbreaks, recognizing their causal agents in a precise and unequivocal way, while understanding their reproductive and dispersive dynamics are all crucial for the implementation of effective control measures. The studies collected in this special issue cover a wide range of topics in the field of forest pathology and entomology. Investigations range from molecular diagnosis of pathogens and pests to their monitoring and quantification in the field, from measurements of their proliferation rate to the analysis of their genetic variability, from the assessment of the role of plant diversity and ecosystem heterogeneity on pathogen and pest impacts to disease and pest management. Specific case studies show how applied research conducted with innovative methods is key to solving taxonomic issues that were, until now, controversial. The variety of experimental approaches and the range of scientific issues addressed document the trends and topicality of modern forest health protection science.

Made available in DSpace on 2020-12-12T02:12:34Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-09-01 The ascomycete fungus Geosmithia morbida (GM) and the walnut twig beetle (WTB), Pityophthorus juglandis, constitute a recently discovered fungal–insect complex responsible for the harmful Thousand Cankers Disease (TCD) in walnut trees. Key aspects of the epidemiology and ecology of the fungus and its insect vector were investigated in a disease outbreak found in central Italy, currently the southernmost introduction area of TCD on the Old Continent. Walnut trees with symptoms of crown dieback and epicormic branching with beetle galleries were sampled for pathogen isolation, while funnel traps were used to catch beetles and obtain adult flight curves. Growth-temperature relationships were determined for the fungus; optimum growth temperature for GM was 25°C, but it thrived up to 37°C. The phloem-boring bark beetle showed a longer flight period than in northern Italy. The beetle haplotype identified in Tuscany (H1) differed from haplotypes H2 and H21 found in northern Italy, suggesting possible multiple anthropogenic introductions. The substantial high temperature tolerance shown by the two organisms suggests they will not be constrained by the high temperatures of warmer areas. As a consequence, the impact of TCD in southern Europe, where the native Juglans regia is widely cultivated for its nuts and wood, would potentially be disastrous to local economies. In fact, although J. regia is not considered as susceptible as J. nigra, it can nevertheless be attacked in conditions of high bark beetle population density, a harbinger of high fungus’ propagule pressure. Department of Agricultural Food Environmental and Forestry Science and Technology (DAGRI) Plant Pathology and Entomology Section University of Florence Plant Protection Department Faculdade de Ciências Agronômicas (FCA) Universidade Estadual Paulista (UNESP) Instituto de Biociências Departamento de Genética Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP) Plant Protection Department Faculdade de Ciências Agronômicas (FCA) Universidade Estadual Paulista (UNESP) Instituto de Biociências Departamento de Genética Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP)

Research Highlights: Protected natural areas are a reservoir of Phytophthora species and represent the most suitable sites to study their ecology, being less disturbed by human activities than other environments. Background and Objectives: The specific objective of this study was to correlate the diversity and distribution of Phytophthora species with the vegetation in aquatic, riparian and terrestrial habitats within a protected area in Eastern Sicily, Southern Italy. Materials and Methods: Environmental samples (water and soil) were sourced from two streams running through the reserve and six different types of vegetation, including Platano-Salicetum pedicellatae, the Sarcopoterium spinosum community, Myrto communis-Pistacietum lentisci, Pistacio-Quercetum ilicis,Oleo-Quercetum virgilianae and a gallery forest dominated by Nerium oleander (Natura 2000 classification of habitats). Phytophthora species were recovered from samples using leaf baiting and were classified on the basis of morphological characteristics and sequencing of internal transcribed spacer (ITS) regions of ribosomal DNA (rDNA). Results: As many as 11 Phytophthora species, within five different ITS clades, were identified, including P. asparagi, P. bilorbang, P. cryptogea, P. gonapodyides, P. lacustris, P. multivora, P. nicotianae, P. oleae, P. parvispora, P. plurivora and P. syringae. No Phytophthora species were found in the Sarcopoterium spinosum comm. Phytophthora asparagi, P. lacustris and P. plurivora were the prevalent species in the other five plant communities, but only P. plurivora was present in all of them. Overall aquatic species from clade 6 (100 out of 228 isolates) were the most common, they were recovered from all five types of vegetation, streams and riparian habitats. Phytophthora populations found in the Platano-Salicetum pedicellatae and Oleo-Quercetum virgilianae show the highest diversity, while no correlation was found with the physicochemical characteristics of the soil. Conclusions: The vegetation type and the aquatic or terrestrial habitat were identified as major environmental factors correlated with the diversity of Phytophthora communities in this reserve.

Background Obesity and type 2 diabetes mellitus, which are widespread throughout the world, require therapeutic interventions targeted to solve clinical problems (insulin resistance, hyperglycaemia, dyslipidaemia and steatosis). Several natural compounds are now part of the therapeutic repertoire developed to better manage these pathological conditions. Cladosporols, secondary metabolites from the fungus Cladosporium tenuissimum, have been characterised for their ability to control cell proliferation in human colon cancer cell lines through peroxisome proliferator-activated receptor gamma (PPARγ)-mediated modulation of gene expression. Here, we report data concerning the ability of cladosporols to regulate the differentiation of murine 3T3-L1 preadipocytes. Methods Cell counting and MTT assay were used for analysing cell proliferation. RT-PCR and Western blotting assays were performed to evaluate differentiation marker expression. Cell migration was analysed by wound-healing assay. Results We showed that cladosporol A and B inhibited the storage of lipids in 3T3-L1 mature adipocytes, while their administration did not affect the proliferative ability of preadipocytes. Moreover, both cladosporols downregulated mRNA and protein levels of early (C/EBPα and PPARγ) and late (aP2, LPL, FASN, GLUT-4, adiponectin and leptin) differentiation markers of adipogenesis. Finally, we found that proliferation and migration of HT-29 colorectal cancer cells were inhibited by conditioned medium from cladosporol-treated 3T3-L1 cells compared with the preadipocyte conditioned medium. Conclusions To our knowledge, this is the first report describing that cladosporols inhibit in vitro adipogenesis and through this inhibition may interfere with HT-29 cancer cell growth and migration. General significance Cladosporols are promising tools to inhibit concomitantly adipogenesis and control colon cancer initiation and progression.

The cultivation of walnuts (Juglans sp.) in Europe retains high economic, social, and environmental value. The recent reporting of the Thousand Cankers Disease (TCD) fungus, Geosmithia morbida, and of its vector, Pityophthorus juglandis, in walnut trees in Italy is alarming the whole of Europe. Although Italy is at present the only foothold of the disease outside North America, given the difficulties inherent in traditional identification of both members of this beetle/fungus complex, a rapid and effective protocol for the early detection and identification of TCD organisms is an absolute priority for Europe. Here we report the development of an effective and sensitive molecular tool based on simplex/duplex qPCR assays for the rapid, accurate and highly specific detection of both the bionectriaceous fungal pathogen and its bark-beetle vector. Our assay performed excellently, detecting minute amounts of target DNA without any non-specific amplification. Detection limits from various and heterogeneous matrices were lower than other reported assays. Our molecular protocol could assist in TCD organism interception at entry points, territory monitoring for the early identification and eradication of outbreaks, delineation of quarantine areas, and tracing back TCD entry and dispersal pathways.

In July 2010, extensive decline of English ash (Fraxinus excelsior L.) was observed near Chiusi della Verna, Tuscany, in central Italy (altitude 956 m a.s.l.; lat. 43° 41′ 54″ N, long. 11° 56′ 9″E). The symptoms on the tree trunks and leaves included lengthwise bark cracks, detached bark, withering of the crown where the bark was detached, and extensive microphyllia. In September 2010, perithecial stromata were observed in the parts of the tree that had lost their bark. They were applanate and black and fruiting bodies were recognized as Biscogniauxia mediterranea (De Not.) Kuntze (basionym Sphaeria mediterranea (De Not.), Ascomycotina, Xylaraceae), a fungus causing charcoal canker that attacks oaks, Acer spp., Castanea sativa Miller, Fagus sylvatica L., and Platanus acerifolia Willd. The biometric characteristics of 30 stromata were examined. The stromata were slightly convex, ellipsoid, and elongate, 7.2 to 20.5 × 3.5 to 4.2 cm. The perithecia were ovoid to tubular, 0.74 to 0.80 × 0.12 to 0.15 mm; the asci were short and stipitate, 7.9 to 10.0 × 120.2 to 170.4 μm. The ascospores were ovoid, brownish-black, with narrowed and roundish ends, 6.9 to 9.1 × 14.6 to 20.0 μm. Colonies grown on PDA at 25°C for 5 days were grey viewed from the top and black viewed from the dish underside. A comparison with the data in the literature (1) confirmed the macro- and microscopic identification. Traditional identification was further confirmed by sequence information from the rDNA ITS region. A BLAST search of the ITS sequence of our B. mediterranea isolate (GenBank Accession No. JX262798) revealed an exact match (100%) with several reference sequences of the fungus present in the database, mainly from oak hosts. Four branches each of five English ash trees growing in a natural environment were inoculated at the trunk junction with a 10–5 ml ascospore suspension. Control consisted of a branch per tree inoculated with an identical volume of sterile water only. After 25 days, the bark became detached and after a further 15 days the typical black stromata appeared. The pathogen was reisolated from the lesions, confirming Koch's postulates. No symptoms were observed on control branches, which presented healed wounds. B. mediterranea was first detected in 1986 on Quercus cerris L. and Q. pubescens Willd. in the Circeo natural park in central Italy (altitude 50 m a.s.l; lat. 41° 27′ 55″44 N, long. 12° 53′ 53″52 E). From there it spread north to other parts of the country. All oak species in Italy appear to be susceptible. Its northward expansion is likely associated with the high temperature and water stress that have been affecting the Italian peninsula for the last few years (4). Most recently, B. mediterranea has also been reported on Q. cerris in the Karst region of Slovenia (lat. 45° 43′ 03″7 N, long. 13° 45′ 20″ 4 E). This confirms its current spread to the more northerly territories, most likely because of ongoing changes in the climate that are creating optimal conditions for its survival in areas that were previously unsuitable to it. References: (1) P. Capretti and L. Mugnai, Inform. Fitopatol. 37:39, 1987. (2) D. Jurc and N. Ogris. Plant Pathol. 55:299, 2006. (3) A. Ragazzi. Micologia Italiana 1:29, 2009. (4) A. Vannini and R. Valentini. Tree Physiol. 14:129, 1994.

High mortality was noticed in a 10-year-old stand of Pinus pinea in the Alberese area (Grosseto, central Italy, elev. 40 m, 42° 39′ 46″ N, 11° 06′ 25″ E) in July 2010. Aerial symptoms of trees included chlorosis, crown thinning, stunted growth, bark lesions at the stem base with resinous exudations, and extensive necroses of the underlying xylem tissue. Woody roots of two uprooted trees exhibited bark necroses and a high proportion of fine roots was destroyed. Soil around necrotic roots was baited using apple fruits (cv. Gala). After 1 week of incubation at 24°C, typical firm fruit rot developed and small tissue samples were transferred to clarified V8 agar (V8A) amended with 5 ml/l PARPNH and incubated at 24°C. After 7 days, stellate to rosaceous, finely lobed cottony colonies arose that were transferred to FPM medium and incubated at 24°C. Within 7 days, spherical oogonia with a smooth surface and predominantly paragynous antheridia formed; sporadic amphyginous antheridia could be observed. Colony squares (1 cm2) were then placed in filtered and sterilized pond water. After 48 h, ovoid, obpyriform, or clavate, nonpapillate, persistent sporangia with internal nested and extended proliferation were formed. Fifty oogonia and 30 sporangia were measured. The diameter of the 50 spherical oogonia varied from 33.6 to 44.9 μm (avg. 39 μm); dimensions of the 30 sporangia were 42.6 to 59.8 × 28.9 to 47.8 μm (avg. 52.95 × 38.98 μm; 1:b ratio 1.37). The isolate was identified as Phytophthora humicola W. H. Ko & Ann on the basis of colony type, size and morphology of oogonia and sporangia, average length/width ratio of sporangia, the homothallic formation of oogonia (4), and ITS rDNA sequence information (GenBank Accession No. JQ757060). A BLAST search of the ITS sequence of P. humicola isolate B33 revealed a 99% identity with the Phytophthora ITS Clade 6 species P. humicola and P. inundata (2). This latter species could be ruled out, however, since it is self-sterile, whereas our isolate B33 was self-fertile (3). A strain of P. humicola was deposited in the CBS-KNAW Fungal Biodiversity Centre, strain no. CBS129249. Pathogenicity tests were conducted on 10 one-year-old twigs of Pinus pinea. A bark portion was removed aseptically and a V8A disc (0.5 cm diam.) of P. humicola mycelium was placed on the wound. Control twigs (3) received sterile V8A discs. Inoculated and control twigs were incubated at 20°C in the dark. Clearly noticeable necrotic lesions (avg. length 2.2 × 0.68 cm) were observed after 15 days on inoculated twigs. Control twigs showed no symptoms. Reisolations on selective V8-PARPNH-agar confirmed P. humicola as the causal agent. P. humicola is mainly associated with woody horticultural crops (1, 3), while the other taxa grouped with this species in Clade 6 are mainly found in forest and riparian ecosystems (1). These aquatic Phytophthora species normally have a saprophytic lifestyle, but under favourable environmental circumstances can act as opportunistic pathogens, attacking susceptible trees and causing scattered mortality in forest stands and natural ecosystems (3). To our knowledge, this is the first report of P. humicola from a pine stand. It is supposed that the pathogen reached the stand through infected plant material or infested soil introduced into the stand. References: (1) C. M. Brasier et al. Mycol. Res. 107:277, 2003. (2) D. E. L. Cooke et al. Fungal Genet. Biol. 30:17, 2000. (3) T. Jung et al. Persoonia 26:13, 2011. (4) W. H. Ko and P. J. Ann. Mycologia 77:631, 1985.

In spring 2013, pot-grown Viburnum tinus plants shipped to an ornamental nursery in Pescia (Pistoia, central Italy, 287 m a.s.l., 43°54′0″ N, 10°41′0″ E) from another local nursery were found to bear disease symptoms. Symptoms included brown to black foliar lesions, later expanding into larger blotches; necrosis of the petioles; shoot wilting and folding; browning of the stems; and necrosis of the cambium. Infected leaves, shoots, and entire plants eventually died. Tissue samples (2 mm2) were cut at the edge of active lesions from tissue of the phloem, the xylem, and the leaves and plated on selective PARPNH V8 agar (V8A) (1). Rose-shaped and finely lobed cottony colonies arose in 2 to 3 days. Mono-hyphal colonies were isolated and transferred to V8A. Square colony pieces (1 cm2) from isolates SB05a and SB05b were placed in filtered pond water after 5 to 7 days. Semipapillate, caducous sporangia with a rounded or conical base were produced within 24 h, individually or in pairs, on each sporangiophore. Sporangia (n = 30 per isolate) were examined: they were 56.2 ± 9.5 × 29.3 ± 4.3 μm (l:b ratio 1.9 ± 0.3). Exit pores averaged 7.0 ± 1.0 μm. Sporangia were ellipsoid (30%), lemon-shaped (28.3%), ovoid (20%), obovoid (16.7%), ampulliform (3.3%), or “peanut-like” (1.7%). Globose chlamydospores, borne intercalarly or terminally, were abundant on both V8A and carrot agar (CA), and were on average 54.7 ± 8.5 μm. Mono-hyphal isolates incubated for 7 days at 23°C were also transferred to CA, corn meal agar (CMA), malt extract agar (MEA), potato dextrose agar (PDA), and V8A. Colonies on these media were identical in shape and appearance to those described in previous reports (2,4). Isolates were identified as Phytophthora ramorum Werres, De Cock & Man in't Veld (4) on the basis of colony type; size, the average l:b ratio and shape of sporangia; and the type and size of the chlamydospores. Isolates were found to be the A1 mating type by pairing them with P. cryptogea BBA 63651 (mating type A2). PCR-amplification of the rDNA ITS region with specific primers Ph1/Ph4 (3) gave fragments of the expected size (GenBank Accession Nos. KF181162 and KF181163). A BLAST search of these ITS sequences in the database found that isolates of P. ramorum were the closest phylogenetically with 100% homology (YQ653034 and HM004221). Pathogenicity tests were conducted on 16 detached V. tinus leaves. A small cut was made aseptically on each of the leaf surfaces and a V8A disc (0.5 cm Ø) with mycelium was placed over the wounds. Control leaves received only sterile V8A discs. Inoculated and control leaves were incubated at 23°C in the dark. Necrotic areas (average 3.5 ± 1.3 cm2) arose on inoculated leaves after 6 days. Control leaves had no symptoms. Re-isolations on PARPNH V8A confirmed P. ramorum as the causal agent. P. ramorum was reported in Italy in 2003 on the exotic Rhododendron yakushimanum (2). This is the first report of the pathogen on a native species (V. tinus) in this country. The Pistoia area is important for nursery gardens and flowers. P. ramorum, which probably arrived on infected plant material, could compromise the export/import trade in stock plants. For this reason, the plant protection services were promptly alerted and the infected plants were destroyed. References: (1) Y. Balci et al. Plant Dis. 91:705, 2007. (2) C. Gullino et al. Inf. Agrar. 19:87, 2003. (3) K. J. Hayden et al. Phytopathology 94:1075, 2004. (4) S. Werres et al. Mycol. Res. 105:1155, 2001.

Cork oak (Quercus suber) forests are economically and culturally intertwined with the inhabitants of the Mediterranean basin and characterize its rural landscape. These forests cover over two million hectares in the western Mediterranean basin and sustain a rich biodiversity of endemisms as well as representing an important source of income derived from cork production. Currently cork oak forests are threatened by several factors including human-mediated disturbances such as poor or inappropriate management practices, adverse environmental conditions (irregular water regime with prolonged drought periods), and attacks of pathogens and pests. All these adverse factors can interact, causing a complex disease commonly known as “oak decline.” Despite the numerous investigations carried out so far, decline continues to be the main pathological problem of cork oak forests because of its complex etiology and the resulting difficulties in defining suitable control strategies. An overview of the literature indicates that several pathogenic fungi and oomycota can play a primary role in the etiology of this syndrome. Therefore, the aim of this review is to analyze the recent advances achieved regarding the bio-ecology of the endemic and emerging pathogens that threaten cork oak trees with particular emphasis on the species more directly involved in oak decline. Moreover, the effect of climate change on the host-pathogen interactions, a task fundamental for making useful decisions and managing cork oak forests properly, is considered.

Findings of a study extending over a number of years on some key aspects of the biology and ecology of Phomopsis quercina in Mediterranean oak forests are reported. The main biometric parameters and physiological requirements of this significant endophytic fungus were determined in the laboratory. The microorganism was also studied in the field to explore its lifestyle in natural oak stands. The isolation frequencies of the fungus in various oak species were also related to the vitality of the trees (healthy or declining) and this showed that the fungus is involved in the widespread phenomenon of oak decline. In planta studies have proved the pathogenic activity of P. quercina in oak, and have found that it remains latent for a long time in the inner wood of oaks, but may turn into an aggressive and harmful coloniser of the tree if this is weakened by some stress factors. An important part of the study concerns an examination of the antagonism that some other endophytes, which colonise the same oak tissues and organs as P. quercina, display against this fungus. The increasing presence and abundance of P. quercina in declining (and especially drought-stressed) oaks suggests that climate change is having a significant role in destabilising the precarious balance between the host tree and the fungus, to the benefit of the latter. Lastly, some practical suggestions are offered on ways to counter the epidemic spread of this latent pathogen.

The present-day phytosanitary disasters caused by biological invasions are afflicting urban and peri-urban forest stands worldwide, as well as the varied services they normally provide. In Europe, we are witnessing an alarming situation due to an increasing introduction of infectious diseases and pests. The authors present an up-to-date list of alien microbial pathogens and insect pests affecting urban greening that have been accidentally imported in Italy or that are likely to be introduced. Information about the biology, epidemiology, ethology, and control of these invasive organisms is provided. For each species, the current geographical distribution, including newly-colonized areas, is also given, as well as the chronological progression of its occurrence. Particular detail is used for describing symptoms—the key diagnostic elements for appropriate and timely phytosanitary management. This paper will benefit urban forest management, which is a crucial factor in maintaining the social and ecological viability of urban green spaces, as well as ecosystem services. The importance of engaging citizens in community-based monitoring of urban greenspaces for tracking the location, abundance, and pathways of invasive pathogens and pests will also be touched upon.

Many biotic and abiotic factors contribute to the onset of oak decline. Among biotic agents, a variety of fungi and insects cause extensive disease and insect outbreaks in oak forests. To date, research on fungus-insect interactions in Mediterranean forest ecosystems is still scarce and fragmentary. In this study, we investigated the assemblage of endophytic mycobiota and insect pests occurring in a declining oak stand, with the aim to explore if, and to what extent, the insect species were active vectors of fungal propagules. It emerged that some known latent pathogens of the Botryosphaeriaceae family, namely Botryosphaeria dothidea, Diplodia corticola, Diplodia seriata, Dothiorella sarmentorum, and Neofusicoccum parvum were isolated at high frequency from physiologically-impaired trees. In addition, propagules of these fungi were isolated from five insects, two of which (Cerambyx welensii and Coraebus fasciatus) are main oak pests. The life-history strategies of these fungi and those of wood-boring beetles were strikingly interconnected: both the fungi and beetles exploit drought-stressed trees and both occur at high frequency during hot, dry periods. This synchronicity increased their chance of co-occurrence and, consequently, their probability of jointly leading to oak decline. If these interactions would be confirmed by future studies, they could help to better understand the extensive decline/dieback of many Mediterranean forest ecosystems.

Cladosporols are secondary metabolites from Cladosporium tenuissimum characterized for their ability to control cell proliferation. We previously showed that cladosporol A inhibits proliferation of human colon cancer cells through a PPARγ-mediated modulation of gene expression. In this work, we investigated cladosporol B, an oxidate form of cladosporol A, and demonstrate that it is more efficient in inhibiting HT-29 cell proliferation due to a robust G0/G1-phase arrest and p21(waf1/cip1) overexpression. Cladosporol B acts as a PPARγ partial agonist with lower affinity and reduced transactivation potential in transient transfections as compared to the full agonists cladosporol A and rosiglitazone. Site-specific PPARγ mutants and surface plasmon resonance (SPR) experiments confirm these conclusions. Cladosporol B in addition displays a sustained proapoptotic activity also validated by p21(waf1/cip1) expression analysis in the presence of the selective PPARγ inhibitor GW9662. In the DMSO/H2O system, cladosporols A and B are unstable and convert to the ring-opened compounds 2A and 2B. Finally, docking experiments provide the structural basis for full and partial PPARγ agonism of 2A and 2B, respectively. In summary, we report here, for the first time, the structural characteristics of the binding of cladosporols, two natural molecules, to PPARγ. The binding of compound 2B is endowed with a lower transactivation potential, higher antiproliferative and proapoptotic activity than the two full agonists as compound 2A and rosiglitazone (RGZ).

[EN] An analysis of incidence of Phytophthora spp. in 732 European nurseries producing forest transplants, larger specimen trees, landscape plants and ornamentals, plus 2525 areas in which trees and shrubs were planted, is presented based on work conducted by 38 research groups in 23 European countries between 1972 and 2013. Forty-nine Phytophthora taxa were recorded in 670 nurseries (91.5%); within these nurseries, 1614 of 1992 nursery stands (81.0%) were infested, although most affected plants appeared healthy. In forest and landscape plantings, 56 Phytophthora taxa were recovered from 1667 of 2525 tested sites (66.0%). Affected plants frequently showed symptoms such as crown thinning, chlorosis and dieback caused by extensive fine root losses and/or collar rot. Many well-known highly damaging host¿Phytophthora combinations were frequently detected but 297 and 407 new Phytophthora¿host associations were also observed in nurseries and plantings, respectively. On average, 1.3 Phytophthora species/taxa per infested nursery stand and planting site were isolated. At least 47 of the 68 Phytophthora species/taxa detected in nurseries and plantings were exotic species several of which are considered well established in both nurseries and plantings in Europe. Seven known Phytophthora species/taxa were found for the first time in Europe, while 10 taxa had not been previously recorded from nurseries or plantings; in addition, 5 taxa were first detections on woody plant species. Seven Phytophthora taxa were previously unknown to science. The reasons for these failures of plant biosecurity in Europe, implications for forest and semi-natural ecosystems and possible ways to improve biosecurity are discused., The authors are grateful to all public and private owners of nurseries and plantings who contributed to this extensive study. T. Jung acknowledges the support by the Regione Autonoma della Sardegna, Visiting Professor Program at the University of Sassari, Italy. The authors also thank the COST Office and the European Council for providing the European COST Actions FP0801 (http://www.cost.eu/domains_actions/fps/Actions/FP0801) and FP 1002 (http://www.cost.eu/domains_actions/fps/Actions/FP1002), the EU projects FOR-THREATS and ISEFOR, and BiodivERsA project RESIPATH as platforms for stimulating discussions on the nursery pathway and possible management solutions.

Investigation of the extracts of a culture of Cladosporium tenuissimum , a known hyperparasite of several rust fungi, gave rise to the isolation of cladosporols B–E ( 2 – 5 ). Their structure and stereochemistry were elucidated on the basis of 1 H and 13 C NMR evidence and CD measures. Cladosporols 1 – 5 were active in inhibiting the urediniospore germination of the bean rust agent Uromyces appendiculatus .

Interactions between the mycoparasite Cladosporium tenuissimum and the bean rust Uromyces appendiculatus were studied through light and electron microscopy in vitro at the host-parasite interface. Urediniospore germination decreased on contact with ungerminated C. tenuissimum conidia, possibly due to antibiosis mechanisms. C. tenuissimum grew towards the bean rust spores and coiled around their germ tubes. Penetration of the urediniospores occurred either enzymatically and/or mechanically, through appressorium or infection cushion structures, from which a thin penetrating hypha was generated. Enzyme production by the mycoparasite was suggested by the loosening of the matricial components of the spore wall, which sometimes left chitin fibrils visible. Mycoparasite hyphae grew within the host spore, emptied its content, and emerged profusely forming conidiophores and conidia. C. tenuissimum was able to grow on media containing laminarin, suggesting the ability of producing glucanases, but not when chitin was used as the sole carbon source. Conidia that had been grown on a sugar-rich medium, filtered, and extracted with organic solvents, were found to contain cladosporol and related compounds. Complete control of the bean rust disease was achieved by application of C. tenuissimum culture filtrates but not by conidial suspensions. This is the first report of parasitism by C. tenuissimum on U. appendiculatus. These investigations provide additional observations on a genus besides Melampsora and Cronartium from which this fungus has been isolated and tested to date. The possible role of environmental factors for the exploitation of this organism as a biocontrol agent is also mentioned.

Summary The composition of the endophytic fungal assemblages in Quercus cerris, Q. pubescens and Q. robur, three oak species most susceptible to decline, was investigated in Fagare ` (Padova), Ulignano (Pisa), and Radda in Chianti (Firenze). The endophytic assemblages were studied as a function of health status of the whole tree and of twigs and leaves. The effect of station altitude was tested on Q. cerris. Samples were gathered from healthy and declining trees. In April (bud opening time) 20 twigs and 10 leaves were collected from each tree. Fungi were isolated from twig segments and leaf portions. A total of 23 fungal genera with 27 different species were isolated, in addition to a few sterile mycelia. Eleven species were found to be common to all three oak species, while other species were found only on one oak species, suggesting host specificity. Among the fungal species isolated, some were known to be pathogenic (Apiognomonia quercina, Colpoma quercinum, Diplodia mutila, Phomopsis quercina). The colonization frequency (CF) of pathogenic species varied between 0.9% for A. quercina in Q. cerris and 60.2% for P. quercina in Q. robur. Analysis of variance showed a statistically significant difference among the oak species tested. The CF was higher in declining trees in comparison with healthy trees, and also in twigs vs. leaves. The CF was found to be higher for Q. cerris trees growing at sea level as compared with those situated at an elevation of 350‐400 m. In stands where, on account of particular ecological conditions, pathogenic behaviour is displayed simultaneously by more than one endophytic fungal species, trees accentuate their decline.

The first axenic culture of a free living saprophytic stage of the exotic rust fungus Melampsoridium hiratsukanum is reported. Colonies were obtained from one-celled, dikaryotic urediniospores on eight nutrient media out of twelve. Modified Harvey and Grasham (HG) and Schenk and Hildebrandt (HS) media HG1 and SH1 and their bovine serum albumin (BSA)-enriched derivatives gave abundant mycelial growth, but modified Murashige and Skoog (MS) QMS media and their BSA-enriched modifications performed poorly, colony growth being low on QMS-1 and QMS1+BSA, and nil on QMS-5 and QMS-6, with or without BSA. Colonies initially grew poorly when subcultured for one month in purity, but much better after re-transfer to fresh media later: presumably because only the most exploitative genotypes survived, best able to cope with an uncongenial medium. Stabilised cultures survived, and remained vegetative, but only few reproductive colonies produced spore-like bodies. Though the agarised medium remains an inhospitable environment for this biotrophic parasite, it is shown that non-living media can nevertheless sustain the growth and sporulation of this fungus outside its natural hosts and habitat. Axenic culture promises important advances in basic and applied research on this rust, leading to a better understanding of its nutrition, metabolism, diversity and pathogenicity.