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Abstract Sphaeropsidins are iso-pimarane diterpenes produced by phytopathogenic fungi that display promising anticancer activities. Sphaeropsidin A, in particular, has been shown to counteract regulatory volume increase, a process used by cancer cells to avoid apoptosis. This study reports the hemi-synthesis of new lipophilic derivatives obtained by modifications of the C15,C16-alkene moiety. Several of these compounds triggered severe ER swelling associated with strong proteasomal inhibition and consequently cell death, a feature that was not observed with respect to mode of action of the natural product. Significantly, an analysis from the National Cancer Institute sixty cell line testing did not reveal any correlations between the most potent derivative and any other compound in the database, except at high concentrations (LC50). This study led to the discovery of a new set of sphaeropsidin derivatives that may be exploited as potential anti-cancer agents, notably due to their maintained activity towards multidrug resistant models.

Extensive collar rot, sunken and bleeding cankers, shoot blight, and fruit rot symptoms on olive trees have recently been observed in several orchards in Italy. Since there is little information about the etiology of these diseases and given the high economic relevance of this iconic crop, a study was conducted from autumn 2017 to summer 2022, in four Italian regions, to define the occurrence, distribution and impact of the main pathogens involved. A total of 1064 symptomatic olive samples were collected and processed. Based on colony appearance, micromorphological analysis and DNA sequence data, thirty-eight species, including eighteen Botryosphaeriaceae species belonging to five genera and fifteen Phytophthora species, were isolated and identified, thirteen of which, Diplodia africana, D. fraxini, D. subglobosa, Dothiorella omnivora, Do. sarmentorum, Do. sempervirentis, Sardiniella urbana (Botryosphaeriaceae), Phytophthora cactorum, P. cinnamomi, P. citricola, P. crassamura, P. niederhauserii and P. pseudocryptogea, are reported here for the first time in olive trees. Pathogenicity tests performed on unripe drupes and on potted olive seedlings completed Koch postulates and highlighted that several species of Botryosphaeriaceae and Phytophthora represent a growing threat to olive trees.

New and emerging Phytophthora-related diseases in small trees, shrubs and herbaceous plants typical of subalpine vegetation have recently been observed in Italy and Slovenia. Diseased plants showed a complex symptomatology including foliar necrosis, fruit rot, shoot blight and branch bleeding cankers. Since little information is available about the aetiology of these aerial Phytophthora diseases, from 2019 to 2022, field surveys were conducted in 54 sites to define the occurrence, distribution and impact of the Phytophthora species on mountain vegetation. A total of 360 Phytophthora isolates were obtained from 397 samples collected from 33 herbaceous and woody host species. Based on phylogenetic analysis and morphometric data, 17 Phytophthora species were identified: P. pseudosyringae (201 isolates), P. plurivora (54), P. gonapodyides (21), P. ilicis (20), P. alpina (17), P. acerina (11), P. cactorum (7), P. pseudocryptogea (6), P. cambivora (5), P. idaei (4), P. psychrophila (3), P. bilorbang (2), P. chlamydospora (2), P. hedraiandra (1), P. kelmanii (1), P. rosacearum (1) and P. syringae (1). In addition, three isolates of a new putative Phytophthora species obtained from Alnus viridis, Juniperus communis and Rhododendron ferrugineum are described here as Phytophthora pseudogregata sp. nov. Overall, the results highlighted an unexpectedly high diversity of Phytophthora species in mountain areas, with many species able to cause aerial infections due to the production of caducous sporangia.

Oleaster (Olea europaea subsp. europaea var. sylvestris) is a sclerophyllous forest tree occurring in the Mediterranean region including Sardinia (Italy). Oleaster suffers from few major diseases, but since 2017 a new and unusual disease leading to the death of both young and old trees has been observed in several woodlands of high ecological value in north-eastern Sardinia. Declining trees showed a variety of symptoms including leaf chlorosis, dieback and sunken canker on trunk and branches. The cankers often exuded a white to orange sap, giving them the appearance of bleeding, which gradually dried to a whitish gluey mass on the bark. Symptomatic wood samples yielded fungal isolates belonging to five fungal species: Diplodia olivarum, Dothiorella sarmentorum, Neofusicoccum parvum, Rosellinia corticium and unidentified Pestalotiopsis sp. In pathogenicity trials Neofusicoccum parvum proved to be the most aggressive species. Our results provide the first evidence for a combined involvement of different Botryosphaeriaceae species in the aetiology of a new oleaster disease.

A new cleistanthane nor-diterpenoid, named olicleistanone (1), was isolated as a racemate from the culture filtrates of Diplodia olivarum, an emerging pathogen involved in the aetiology of branch canker and dieback of several plant species typical of the Mediterranean maquis in Sardinia, Italy. When the fungus was grown in vitro on Czapek medium, olicleistanone was isolated together with some already known phytotoxic diterpenoids identified as sphaeropsidins A, C, and G, and diplopimarane (2-5). Olicleistanone was characterized as 4-ethoxy-6a-methoxy-3,8,8-trimethyl-4,5,8,9,10,11-hexahydrodibenzo[de,g]chromen-7(6aH)-one. When D. olivarum was grown on mineral salt medium it produced (-)-mellein (6), sphaeropsidin A and small amounts of sphaeropsidin G and diplopimarane. Olicleistanone (1) exhibited strong activity against the insect Artemia salina L. (100% larval mortality) at 100 μg mL-1 but did not exhibit phytotoxic, antifungal or antioomycete activity. Among the metabolites isolated (1-6), sphaeropsidin A (2) was active in all bioassays performed exhibiting strong phytotoxicity on leaves of Phaseolus vulgaris L., Juglans regia L. and Quercus suber L. at 1 mg mL-1. Sphaeropsidin A (2) also completely inhibited mycelium growth of Athelia rolfsii, Diplodia corticola, Phytophthora cambivora and P. lacustris at 200 μg per plug, and was active in the Artemia salina assay. Also in this assay, diplopimarane (5) and sphaeropsidin G (4) were active (100% larval mortality). Diplopimarane also showed antifungal and antioomycete activities. Athelia rolfsii was the most sensitive species to diplopimarane. Sphaeropsidin C (3) and (-)-mellein (6) were inactive in all bioassays. These results expand knowledge on the metabolic profile of Botryosphaeriaceae, and embody the first characterization of the main secondary metabolites secreted by D. olivarum.

Field surveys conducted on nine farms over a 2-year period showed the widespread presence of Phytophthora-related diseases on globe artichoke plants in the main growing area in Sardinia (Italy). Characteristic symptoms included wilting and necrosis of the outermost leaves and dark brown discoloration of stem tissues, as well as root rot. A total of 18 Phytophthora colonies belonging to three species were isolated and characterized. Based on morphological features and ITS sequence data, Phytophthora isolates were identified as P. crassamura (eight isolates) and P. cactorum (four isolates). Six isolates could not be assigned to any formally described species of Phytophthora and are therefore described here as Phytophthora marrasii sp. nov. The ITS phylogeny places P. marrasii in a terminal clade basal to the sister taxa (P. foliorum, P. hibernalis, P. lateralis, and P. ramorum) of the clade 8c. In particular, P. marrasii is phylogenetically related to P. foliorum, a species from which it differs in 62 nucleotides in the ITS region. At the same time, it can easily be distinguished morphologically from P. foliorum mainly because of the low minimum temperature for growth, the bigger and persistent non-papillate sporangia, and smaller oogonia. Pathogenicity tests confirmed that all three Phytophthora species are pathogenic on globe artichokes, which represent a new host for these pathogens.

Monitoring surveys of Phytophthora related diseases in four forest nurseries in Italy revealed the occurrence of fourteen Phytophthora species to be associated with collar and root rot on fourteen plants typical of Mediterranean and alpine regions. In addition, a multilocus phylogeny analysis based on nuclear ITS and ß-tubulin and mitochondrial cox1 sequences, as well as micromorphological features, supported the description of a new species belonging to the phylogenetic clade 7c, Phytophthora mediterranea sp. nov. Phytophthora mediterranea was shown to be associated with collar and root rot symptoms on myrtle seedlings. Phylogenetically, P. mediterranea is closely related to P. cinnamomi but the two species differ in 87 nucleotides in the three studied DNA regions. Morphologically P. mediterranea can be easily distinguished from P. cinnamomi on the basis of its smaller sporangia, colony growth pattern and higher optimum and maximum temperature values. Data from the pathogenicity test showed that P. mediterranea has the potential to threaten the native Mediterranean maquis vegetation. Finally, the discovery of P. cinnamomi in alpine nurseries, confirms the progressive expansion of this species towards cold environments, probably driven by climate change.

In Italy, after the first report in Friuli-Venezia Giulia along the border with Slovenia in 2009, ash dieback has successively been reported in Veneto, Tuscany and Trentino-Alto Adige. Given its alarming expansion in European ash formations along the sub-montane belt of north-eastern Italy and the limited information about the associated fungal microorganisms; since 2017, a study has been conducted in order to isolate and characterize the fungal species involved in the aetiology of the disease. The surveys were conducted in six ash–maple forests distributed along the Veneto and Friuli-Venezia Giulia pre-Alpine regions (Italy). In each site, the health status of ash trees was assessed and a sample (shoot or branch) with the typical symptoms of the disease was taken from ten trees to isolate the associated pathogens. The fungal colonies developed were identified using morphological features and DNA sequences. The 60 samples processed yielded a total of 109 fungal isolates belonging to 9 families including: Botryosphaeriaceae (62 isolates), Diaporthaceae (18), Nectriaceae (10), Didymellaceae (9), Helotiaceae (5), Diatrypaceae (2), Didymosphaeriaceae (1), Phaeosphaeriaceae (1) and Valsaceae (1). In particular, three species—Diplodia subglobosa, Diplodia fraxini and Diaporthe eres—were isolated with high frequency, while Hymenoscyphus fraxineus was isolated from only five plants distributed in four sites. The pathogenicity tests, conducted on 3-year-old seedlings, detached branches (3–4 cm diameter), and leaves of Fraxinus excelsior, showed that Diplodia fraxini is the most virulent species and the only one able to reproduce the symptoms observed in nature. Overall, the results obtained emphasize that several fungal pathogens are involved in the aetiology of the disease, many of which belong to the Botryosphaeriaceae family.

Extensive decline and mortality events of alder trees have recently been observed in several riparian ecosystems in Italy. Since there is little information about the aetiology of this disease and given the high ecological relevance of riparian ecosystems, an in-depth study was conducted in three sites spanning from the Mediterranean to Alpine regions. From spring 2019 to spring 2020, 261 samples of bleeding cankers, rhizosphere soil and leaves used as baits along waterways were collected and used for Phytophthora isolation. Based on morphology, colony appearance and DNA sequence data, 10 species belonging to 6 clades were identified. These included P. plurivora (84 isolates), P. pseudocryptogea (50), P. hydropathica (18), P. gonapodyides (14), P. bilorbang (13), P. pseudosyringae (12), P. lacustris (11), P. acerina (7), P. cactorum (1) and one isolate of the hybrid Phytophthora ×serendipita. In addition, two new Phytophthora species, one of which is described here as Phytophthora alpina sp. nov., were isolated. The pathogenicity of P. alpina and other species obtained from samples collected in the green alder stand was assessed on 3-year-old seedlings. All species proved to be pathogenic on green alder causing symptoms congruent with field observations. Results obtained have allowed us to expand knowledge about alder decline aetiology. The diversity of pathogenicity of Phytophthora species associated with symptomatic alder trees suggested that no single agent is responsible for the disease, but that it is the result of multiple infections of different Phytophthora species, variable in assemblages among sites.

Many pathogens involved in human infection have rapidly increased their antibiotic resistance, reducing the effectiveness of therapies in recent decades. Most of them can form biofilms and effective drugs are not available to treat these formations. Natural products could represent an efficient solution in discovering and developing new drugs to overcome antimicrobial resistance and treat biofilm-related infections. In this study, 20 secondary metabolites produced by pathogenic fungi of forest plants and belonging to diverse classes of naturally occurring compounds were evaluated for the first time against clinical isolates of antibiotic-resistant Gram-negative and Gram-positive bacteria. epi-Epoformin, sphaeropsidone, and sphaeropsidin A showed antimicrobial activity on all test strains. In particular, sphaeropsidin A was effective at low concentrations with Minimum Inhibitory Concentration (MIC) values ranging from 6.25 μg/mL to 12.5 μg/mL against all reference and clinical test strains. Furthermore, sphaeropsidin A at sub-inhibitory concentrations decreased methicillin-resistant S. aureus (MRSA) and P. aeruginosa biofilm formation, as quantified by crystal violet staining. Interestingly, mixtures of sphaeropsidin A and epi-epoformin have shown antimicrobial synergistic effects with a concomitant reduction of cytotoxicity against human immortalized keratinocytes. Our data show that sphaeropsidin A and epi-epoformin possess promising antimicrobial properties.

The black-banded oak borer, Coraebus florentinus, is an emerging pest of oak trees in the western Mediterranean region. Larvae of the insect are xylophagous and progressively excavate an annular gallery that interrupts sap flow, resulting in the death of the attacked branches. Until now, limited information has been available regarding the ecological interactions between C. florentinus and the main plant pathogenic fungi involved in the etiology of oak decline. Knowledge of these interactions is important in understanding their impact in natural ecosystems and developing appropriate management strategies. Therefore, in this study, we characterized the fungal communities occurring in the exoskeleton of adults and larvae of C. florentinus and associated with the necrotic wood tissues surrounding the branch galleries of declining oak trees. A total of 29 fungal species were identified based on DNA sequence data and morphological features, of which 14 were from symptomatic woody tissues, six from insect exoskeleton, and nine from both insects and symptomatic wood tissues. The most frequent fungal species, Cryphonectria naterciae (15.9% of isolates), Dothiorella iberica (11.3%), and Diplodia corticola (9.9%), were isolated from both insect and gallery systems. All three species are well-known oak pathogens and are reported here, for the first time, to be associated with C. florentinus. At the same time, 89.6% of the fungal taxa were isolated from one or two sites, highlighting the site-dependence of fungal community assemblages.

Shoot blight was observed on Erica arborea L., in a natural growing area on Caprera Island (Italy), during 2011 and 2012. Fungal isolates obtained from 324 symptomatic shoots were identified as Neofusicoccum luteum by analysis of morphological and cultural characteristics, as well as DNA sequence data of the Internal Transcribed Spacer (ITS) of the ribosomal DNA and part of the translation Elongation Factor 1-alpha (EF-1α) gene. Pathogenicity of the fungus was verified by stem inoculation of 3-year-old seedlings of E. arborea. This is the first report of N. luteum as a pathogen of E. arborea.

Branch dieback was observed on Phoenicean juniper trees, in a natural growing area on Caprera Island (Italy), during 2009 and 2010. Fungal isolates obtained from symptomatic twigs and branches show­ing dieback and bark necrosis were identified as Diplodia africana by analysis of morphological features and genomic DNA sequences of the ITS region and translation elongation factor 1-α gene. Pathogenicity was verified by stem inoculation of 3-year-old saplings of Phoenicean juniper. This is the first report of D. africana in the northern hemisphere, and of this fungus as a pathogen of Phoenicean juniper