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In recent years, an unusual decline and mortality has been observed in Paulownia plantations throughout the Marche region (Central Italy). Given the economic importance of this emerging forest crop, a study was conducted to determine which pathogens are directly involved in this syndrome. Field surveys performed in two plantations revealed the widespread occurrence of severe disease symptoms such as leaf chlorosis, crown thinning, shoot and branch dieback, sunken cankers, epicormic shoots and root rot. Disease incidence was also assessed by aerial remote sensing (RS) technologies using drones. Symptomatic samples collected from both stem and root tissues yielded fungal and fungal-like colonies representing two distinct families: Botryosphaeriaceae and Peronosporaceae. Morphological and DNA sequence data revealed five distinct species, identified as Macrophomina phaseolina and Botryosphaeria dothidea (Botryosphaeriaceae), Phytophthora pseudocryptogea, P. citrophthora and P. erythroseptica (Peronosporaceae). Given that all species are reported here for the first time on Paulownia, Koch's postulates were satisfied inoculating the three Phytophthora species and two Botryosphaeriaceae at the collar of the stem of potted 1-year-old rooted cuttings in June 2023. Thirty days after inoculation, all plants showed the same symptoms as those observed in the field. [ABSTRACT FROM AUTHOR]

The rapid growth and environmental adaptability of Eucalyptus species has favored their global cultivation for pulpwood production. On the island of Sardinia, Italy, eucalypt plantations were established in the 20th century primarily in areas reclaimed from marshland, but the trees are now grown all over the island as ornamentals or windbreaks, and for timber, pulp and honey production. In recent years, an unusual decline and mortality of unknown etiology has been observed in Eucalyptus camaldulensis (river red gum) plantations throughout the island. Given the ecological and economic importance of eucalypt ecosystems in Sardinia, a survey was carried out in 2013 to determine which insect pests and fungal pathogens are directly involved in these phenomena. Field surveys throughout the island revealed severe infestations with the red gum lerp psyllid (Glycaspis brimblecombei) at all 12 surveyed sites, with the greatest numbers of pre-imaginal stages and adults occurring between May and July. The adult population reached its peak in July, followed 2 months later by the peak population of its specific parasitoid, Psyllaephagus bliteus. Symptoms of leaf chlorosis, crown thinning, shoot and branch dieback, sunken cankers, epicormic shoots and exudations of kino gum were also observed at the 12 field sites. Symptomatic woody samples yielded fungal isolates representing three distinct families: Botryosphaeriaceae, Diaporthaceae and Valsaceae. Morphological and DNA sequence data revealed seven distinct fungal species, namely Diaporthe foeniculina, Neofusicoccum australe, N. luteum, N. mediterraneum, N. parvum, N. vitifusiforme and Valsa fabianae. Two putative new species of Cytospora were also identified. Neofusicoccum australe was the only species recovered from all 12 sites, with isolation frequencies of 51-95%. Pathogenicity trials revealed that all Neofusicoccum species except N. vitifusiforme are directly involved in the etiology of the observed decline in the E. camaldulensis population on Sardinia.

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. [ABSTRACT FROM AUTHOR]

Recent field surveys conducted in five common alder ecosystems in Portugal have shown the occurrence of severe canopy dieback, bleeding canker and root rot symptoms indicative of Phytophthora infections. Isolations from symptomatic tissues, rhizosphere and water samples yielded a total of 13 Phytophthora species belonging to 6 phylogenetic clades, including P. lacustris (13 isolates), P. multivora (10), P. amnicola (9), P. chlamydospora (6), P. polonica (6), P. bilorbang (4), P. plurivora (4), P. cinnamomi (3), P. asparagi (2), P. cactorum (2), P. pseudocryptogea (2), P. gonapodyides (1) and P. rosacearum (1). Results of the pathogenicity test confirmed the complex aetiology of common alder decline and the additional risk posed by Phytophthora multivora to the riparian habitats in Portugal. At the same time, the diversity of Phytophthora assemblages detected among the investigated sites suggests that different species could contribute to causing the same symptoms on this host. Two species, P. amnicola and P. rosacearum, are reported here for the first time in natural ecosystems in Europe. [ABSTRACT FROM AUTHOR]

A cork oak endophytic strain of Trichoderma citrinoviride, previously selected for its antagonistic potential against various fungal pathogens involved in oak decline, was screened for the production of bioactive secondary metabolites. From liquid culture a mixture of polypeptide antibiotics (peptaibols) belonging to the paracelsin family was isolated and characterized. This peptide mixture was purified by column chromatography and preparative TLC on silica gel, and separated by analytical HPLC. It was analysed by MALDI-TOF MS and nano-ESI-QTOF MS. Tandem mass experiments were performed to determine the amino acid sequences based on the fragmentation pattern of selected parent ions. The mixture comprised 20-residue peptides with C-terminal phenylalaninol and N-terminal acetylation. Twenty-eight amino acid sequences were identified, and amino acid exchanges were located in positions 6, 9, 12 and 17. Among them, seven sequences are new as compared to those reported in the database specifically for peptaibols and in the literature. In addition, we obtained experimental evidence suggesting the existence of non-covalent dimeric forms (homo-and hetero-) of the various peptaibol species. The peptide mixture showed strong antifungal activity toward seven important forest tree pathogens, and it was highly toxic in an Artemia salina (brine shrimp) bioassay. These results emphasize the cryptic role of endophytic fungi as a source of novel bioactive natural products and biocontrol agents. DOI 10.1099/mic.0.030916-0

Cryptic species are common in Diplodia , a genus that includes some well-known and economically important plant pathogens. Thus, species delimitation has been based on the phylogenetic species recognition approach using multigene genealogies. We assessed the potential of mating type ( MAT ) genes sequences as phylogenetic markers for species delimitation in the genus Diplodia . A PCR-based mating type diagnostic assay was developed that allowed amplification and sequencing of the MAT1-1-1 and MAT1-2-1 genes, and determination of the mating strategies used by different species. All species tested were shown to be heterothallic. Phylogenetic analyses were performed on both MAT genes and also, for comparative purposes, on concatenated sequences of the ribosomal internal transcribed spacer (ITS), translation elongation factor 1-alpha ( tef1-α ) and beta-tubulin ( tub2 ). Individual phylogenies based on MAT genes clearly differentiated all species analysed and agree with the results obtained with the commonly used multilocus phylogenetic analysis approach. However, MAT genes genealogies were superior to multigene genealogies in resolving closely related cryptic species. The phylogenetic informativeness of each locus was evaluated revealing that MAT genes were the most informative loci followed by tef1-α . Hence, MAT genes can be successfully used to establish species boundaries in the genus Diplodia . [ABSTRACT FROM AUTHOR]

The article analyzes the role of endemic and emerging pathogens that cause cork oak decline and mortality. It mentions that the research conducted on Phytophthora genus and of the Botryosphaeriaceae and Xylariaceae families had helped in expansion of epidemiology and management of pathogens in different oak forests. It also reveals management options for conserving a forest ecosystem.

The Mediterranean basin is recognized as a global biodiversity hotspot accounting for more than 25,000 plant species that represent almost 10% of the world’s vascular flora. In particular, the maquis vegetation on Mediterranean islands and archipelagos constitutes an important resource of the Mediterranean plant diversity due to its high rate of endemism. Since 2009, a severe and widespread dieback and mortality of Quercus ilex trees and several other plant species of the Mediterranean maquis has been observed in the National Park of La Maddalena archipelago (northeast Sardinia, Italy). Infected plants showed severe decline symptoms and a significant reduction of natural regeneration. First studies revealed the involvement of the highly invasive wide-host range pathogen Phytophthora cinnamomi and several fungal pathogens. Subsequent detailed research led to a better understanding of these epidemics showing that multiple Phytophthora spp. were involved, some of them unknown to science. In total, nine Phytophthora species were isolated from rhizosphere soil samples collected from around symptomatic trees and shrubs including Asparagus albus, Cistus sp., Juniperus phoenicea, J. oxycedrus, Pistacia lentiscus and Rhamnus alaternus. Based on morphological characters, growth-temperature relations and sequence analysis of the ITS and cox1 gene regions, the isolates were identified as Phytophthora asparagi, P. bilorbang, P. cinnamomi, P. cryptogea, P. gonapodyides, P. melonis, P. syringae and two new Clade 6 taxa which are here described as P. crassamura sp. nov. and P. ornamentata sp. nov. Pathogenicity tests supported their possible involvement in the severe decline that is currently threatening the Mediterranean maquis vegetation in the La Maddalena archipelago. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The induction of defense responses in Pinus sylvestris L. seedlings by methyl jasmonate (MeJA) was investigated in three experiments. Two different MeJA application methods were tested, and induction of defense responses was assayed by seedling inoculation with Heterobasidion annosum (Fr.) Bref. and Lophodermium seditiosum Minter, Staley and Millar. In the first experiment, five-year-old P. sylvestris ramets of one clone were directly treated with MeJA, followed by inoculation with H. annosum. In the second experiment, open-pollinated Scots pine seedlings were treated with MeJA by direct spraying and vaporization, and inoculation with H. annosum was done using a slightly modified protocol. In the third experiment, open-pollinated Scots pine seedlings were treated with MeJA by vaporization and inoculated with L. seditiosum. Direct application of MeJA induced seedling mortality, and in some cases, decreased resistance to inoculation with H. annosum. Application of MeJA by vaporization was less stressful for seedlings, and resulted in increased resistance to both H. annosum and L. seditiosum. In addition, an unforeseen Neodiprion sertifer (Geoffroy) and Hylobius abietis L. infestation provided anecdotal evidence of the efficacy of MeJA in inducing resistance to insect pests as well. Further studies are required on the induction of resistance to additional diseases and pests. Induced resistance could be used as a possible protective mechanism for Scots pine seedlings prior to planting during reforestation of stands to increase vitality and survival. [ABSTRACT FROM AUTHOR]

Botryosphaeriaceae-related diseases occur worldwide in a wide variety of plant hosts. The number of studies targeting the distribution, diversity, ecology, and pathogenicity of Botryosphaeriaceae species are consistently increasing. However, with the lack of consistency in species delimitation, the name of hosts, and the locations of studies, it is almost impossible to quantify the presence of these species worldwide, or the number of different host–fungus interactions that occur. In this review, we collected and organized Botryosphaeriaceae occurrences in a single cured dataset, allowing us to obtain for the first time a complete perspective on species' global diversity, dispersion, host association, ecological niches, pathogenicity, communication efficiency of new occurrences, and new host–fungus associations. This dataset is freely available through an interactive and online application. The current release (version 1.0) contains 14,405 cured isolates and 2989 literature references of 12,121 different host–fungus interactions with 1692 different plant species from 149 countries. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In recent decades the vitality and productivity of European ash trees in Slovenia have been reduced by the onset of canker and dieback disease symptoms on young and old trees, identified primarily as ash dieback caused by Hymenoscyphus fraxineus . Given the limited information available about the etiology of this emerging disease, a study was carried out to isolate, identify, and characterize the fungal species involved in the observed ash symptoms. Field surveys were conducted in five forest sites where 50 symptomatic branch samples were collected. All samples were inspected and used for fungal isolation. Based on morphology, colony appearance, and DNA sequence data of the internal transcribed spacer region, 125 fungal colonies belonging to five species were isolated and identified. Only a few symptomatic ash samples yielded colonies of H. fraxineus , whereas Botryosphaeriaceae species were isolated with a high frequency, with Diplodia fraxini as the dominant species. A pathogenicity test proved that all isolated species were pathogenic on European ash, causing bark lesions and wood discoloration. All Botryosphaeriaceae species isolated in this study are reported for the first time on European ash in Slovenia.

In autumn 2018, during a study on the pathogens involved in the etiology of chestnut nut rot symptoms observed in three of the main sweet chestnut (Castanea sativa) growing areas in Sardinia (Site 1: 39°56'55"N/09°11'45"E; site 2: 39°58'20"N/09°09'41"E; site 3: 40°52'50"N/09°08'45"E), Gnomoniopsis smithogilvyi was found to be the main causal agent. In addition to G. smithogilvyi, 15 out of 450 nuts processed, yielded on potato dextrose agar (PDA, 39 g/L) at 22°C white colonies with dense aerial mycelium becoming dark grey after 4 to 7 days. Pycnidia were produced within 4 weeks in half-strength PDA incubated at room temperature under natural daylight. The hyaline, ellipsoid to fusiform and aseptate conidia measured 13.4-19.2 × 4.8-7.7 μm (n = 50). All morphological characters matched those reported for Neofusicoccum parvum by Phillips et al. (2013). Identity of isolates was confirmed by DNA sequence analysis of the internal transcribed spacer region (ITS) and part of the translation elongation factor 1-alpha gene (tef1-α). DNA extraction, PCR amplification reactions and DNA sequencing were carried out according to Linaldeddu et al. (2016). In the phylogenetic analysis based on combined ITS and tef1-α gene sequences the N. parvum isolates clustered within two well-supported subclades. In the first subclade (ML bootstrap = 88%) three isolates clustered together with the ex-type culture of N. parvum (CMW9081) while in the second subclade (ML bootstrap = 95%) three isolates clustered together with the ex-type culture of Neofusicoccum algeriense (CBS 137504), a species recently synonymised with N. parvum by Lopes et al. (2016). Sequences of six representative isolates were deposited in GenBank (MK968559-MK968564 and MT010339-MT010344 for ITS and tef1-α, respectively). The pathogenicity of six isolates, belonging to the two haplotypes, was undertaken by inoculating five asymptomatic nuts per isolate. After disinfecting the nut surface with 70% ethanol and removing a piece of shell (5 mm diameter) with a sterile cork borer, the nuts were inoculated with a same-sized agar-mycelium plug cut from the margin of a 5-day-old PDA colony. Ten control nuts were inoculated with a sterile PDA plug applied as described above. Inoculated nuts were kept in thermostat at 22 °C in the dark for 18 days. All nuts inoculated with N. parvum showed light-brown to dark necrosis of kernel associated with loss of tissue consistency. The symptoms were congruent with those observed in nature. All N. parvum isolates were successfully reisolated from all the inoculated nuts, fulfilling Koch's postulates. No lesions were observed on controls. N. parvum is recognized as an emerging plant pathogen worldwide. In particular, several studies report N. parvum as a growing threat to agricultural and forest ecosystems in the Mediterranean area (Larignon et al., 2015; Manca et al., 2020). This is the first report of N. parvum causing chestnut nut rot in Italy.

In this study a new 20-nor-ent-pimarane, named diplopimarane, was isolated together with sphaeropsidins A (9) and C (10), and (+)-epiepoformin (11) from organic crude extracts of Diplodia quercivora, a recently described oak pathogen originally found on declining Quercus canariensis trees in Tunisia. Diplopimarane was characterized as (1S,2R)-2,8,8-trimethyl-2-vinyl-1,2,3,4,5,6,7,8-octahydrophenanthrene-1,9,10-triol by spectroscopic, X-ray, optical, and chemical methods. It exhibited a wide range of activities including remarkable phytotoxicity on nonhost plants such as tomato cuttings, moderate antifungal activity against important plant pathogens, and moderate zootoxicity against Artemia salina. Its derivatives (2-4 and 6) were also tested for their phytotoxic and zootoxic activities. All these derivatives proved to be active against A. salina at 200 μg/mL, while 2 and 6 were also active on tomato cuttings. The other secondary metabolites (9, 10, and 11) herein reported for D. quercivora exhibited phytotoxic, antifungal, and zootoxic activity. This is the first report on the secondary metabolites secreted in vitro by this oak pathogen that could be key components of its adaptative strategies.

Two isolates of Neofusicoccum australe belonging to ITS haplotypes H4 and H1 and associated with grapevine cordon dieback and branch dieback of Phoenicean juniper, respectively, have been shown to produce in vitro structurally different secondary metabolites. From the strain BOT48 of N. australe (haplotype H4) a new cyclohexenone oxide, namely, cyclobotryoxide, was isolated together with 3-methylcatechol and tyrosol. Cyclobotryoxide was characterized as (1S,5R,6S)-5-hydroxy-3-methoxy-4-methyl-7-oxabicyclo[4.1.0]hept-3-en-2-one by spectroscopic, optical, and chemical methods. The strain BL24 (haplotype H1) produced tyrosol along with botryosphaerone D and (3S,4S)-3,4,8-trihydroxy-6-methoxy-3,4-dihydro-1(2H)-naphthalenone. The metabolites obtained from both strains were tested at four concentrations on leaves of grapevine cv. Cannonau, holm oak, and cork oak by the leaf puncture assay. Cyclobotryoxide proved to be the most phytotoxic compound. Tyrosol and cyclobotryoxide were also tested on detached grapevine leaves at concentrations of 0.25 and 0.5 mg/mL. Only cyclobotryoxide was found to be active in this bioassay.