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Riparian formations encompass a diverse suite of transitional zones between terrestrial and aquatic ecosystems. During the last decades, these formations have been impacted by several emerging diseases. The first outbreaks were detected on alder formations, but have progressively also been observed on other plant species such as Betula pubescens , Nerium oleander , Populus alba , Salix alpina , Salix purpurea and Tamarix gallica . Declining plants showed a plethora of symptoms (leaf spot, shoot blight, bleeding cankers and root rot) indicative of Phytophthora infections. Since there is little information about the aetiology of these pathosystems, from November 2019 to March 2023, an in-depth study was conducted in 46 riparian ecosystems spanning from the Mediterranean to Alpine regions. Overall, 744 symptomatic samples (stem bleeding cankers and root with rhizosphere) from 27 host species were collected for Phytophthora isolation. Based on morphology and DNA sequence data, 20 known Phytophthora species belonging to seven phylogenetic clades have been identified: P. plurivora (202 isolates), P. gonapodyides (156), P. pseudosyringae (84), P. lacustris (57), P. acerina (31), P. idaei (30), P. alpina (20), P. pseudocryptogea (19), P. cambivora (13), P. pseudotsugae (13), P. cactorum (9), P. honggalleglyana (6), P. pseudogregata (6), P. debattistii (4), P. multivora (4), P. cinnamomi (3), P. bilorbang (2) P. crassamura (2), P. ilicis (2) and P. inundata (2). In addition, 26 isolates of a new putative species obtained from Alnus incana and Pinus sylvestris are described here as Phytophthora heteromorpha sp. nov. The new species proved to be pathogenic on grey alder causing symptoms congruent with field observations. This study represents the most comprehensive investigation on the Phytophthora species associated with declining riparian vegetation in Italy and highlights that the polyphagous pathogen P. plurivora represents a growing threat to Mediterranean, temperate and alpine ecosystems.

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.

An undescribed disubstituted dihydrofuranone, named diplofuranoxin, was isolated, together with the six well known metabolites sphaeropsidins A and C, epi-sphaeropsidone, mellein and cis- and trans-4-hydroxymelleins, from the fungal species Diplodia subglobosa, an emerging pathogen involved in the ash dieback aetiology in Europe. Currently, the disease represents the main threat to European ash heritage and the wood associated industry. Diplofuranoxin, was characterized essentially by NMR and HRESIMS spectra as (3Z)-3-(2,3-dihydroxybutylidene)-5-methyldihydrofuran-2(3H)-one. Its relative and absolute configuration was determined by joining NOESY NMR experiments and computational analysis of electronic circular dichroism spectrum. All the metabolites were screened for phytotoxic, antioomycetes and zootoxic activities and only sphaeropsidin A and epi-sphaeropsidone were active in two out of three bioassays performed. In addition, sphaeropsidin A completely inhibited mycelium growth of Phytophthora cambivora, whereas the inhibition rate of epi-sphaeropsidone was less than 50% at the higher concentration used. Both metabolites were inactive in the Artemia salina assay. Results obtained in this study have allowed to characterize for the first time the main metabolites produced in vitro by D. subglobosa and to increase the knowledge on the metabolic profile of Botryosphaeriaceae for a correct taxonomic classification of the strains belonging to this family., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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.

Since 1999, an unusual Phytophthora species has repeatedly been found associated with stem lesions and root and collar rot on young olive trees in Southern Italy. In all cases, this species was obtained from recently established commercial plantations or from nursery plants. Morphologically, the Phytophthora isolates were characterized by the abundant production of caducous non-papillate conidia-like sporangia (pseudoconidia) and caducous papillate sporangia with a short pedicel, resembling P. palmivora var. heterocystica . Additional isolates with similar features were obtained from nursery plants of Ziziphus spina-christi in Iran, Juniperus oxycedrus and Capparis spinosa in Italy, and mature trees in commercial farms of Durio zibethinus in Vietnam. In this study, morphology, breeding system and growth characteristics of these Phytophthora isolates with peculiar features were examined, and combined mitochondrial and nuclear multigene phylogenetic analyses were performed. The proportion between pseudoconidia and sporangia varied amongst isolates and depended on the availability of free water. Oogonia with amphigynous antheridia and aplerotic oospores were produced in dual cultures with an A2 mating type strain of P. palmivora , indicating all isolates were A1 mating type. Phylogenetically, these isolates grouped in a distinct well-supported clade sister to P. palmivora ; thus, they constitute a separate taxon. The new species, described here as Phytophthora heterospora sp. nov., proved to be highly pathogenic to both olive and durian plants in stem inoculation tests.

Two new bioactive trisubstituted furanones, named pinofuranoxins A and B ( 1 and 2 ), were isolated from Diplodia sapinea , a worldwide conifer pathogen causing severe disease. Pinofuranoxins A and B were characterized essentially by NMR and HRESIMS spectra, and their relative and absolute configurations were assigned by NOESY experiments and computational analyses of electronic circular dichroism spectra. They induced necrotic lesions on Hedera helix L., Phaseolus vulgaris L., and Quercus ilex L. Compound 1 completely inhibited the growth of Athelia rolfsii and Phytophthora cambivora , while 2 showed antioomycetes activity against P. cambivora . In the Artemia salina assay both toxins showed activity inducing larval mortality.

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.

A new tetrasubstituted octanoic acid, named hyfraxinic acid ( 1 ), was isolated together with known 1-deoxyviridiol ( 2 ), viridiol ( 3 ), nodulisporiviridin M ( 4 ), and demethoxyviridiol ( 5 ) from the organic extract of Hymenoscyphus fraxineus responsible for ash ( Fraxinus excelsior L.) dieback in Europe. Hyfraxinic acid ( 1 ) was characterized, using spectroscopic methods, as 2,4-dihydroxy-7-methyl-6-methyleneoctanoic acid. Furthermore, the advanced Mosher method was used to determine the absolute configuration (3 R ) of 1-deoxyviridiol. Nodulisporiviridin M ( 4 ) was isolated for the first time from H. fraxineus . The phytotoxicity of each compound was tested by a leaf puncture assay on Celtis australis L., Quercus suber L., Hedera elix L., Juglans regia L., and Fraxinus angustifolia L. leaves. Compounds 1 , 3 , and 5 exhibited remarkable phytotoxicity on all plants tested, inducing necrotic lesions at concentrations of 1.0 and 0.5 mg/mL, while compounds 2 and 4 were found to be inactive in this bioassay. These results could contribute to a deeper understanding of the pathogenicity of H. fraxineus .

In this study the production of secondary metabolites by a virulent strain of Sardiniella urbana , a recently described pathogen originally found on declining European hackberry trees in Italy, was investigated for the first time. Chemical analysis of the culture filtrate extracts led to the isolation of three well known compounds as R -(-)-mellein and (3 R ,4 R )-and (3 R ,4 S )-4-hydroxy melleins which were identified by spectroscopic methods (essentially NMR and ESIMS). The isolated compounds were tested for their phytotoxic, antifungal and zootoxic activities. Among them, only R -(-)-mellein was found to be active.

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., (Copyright © 2018 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

From the culture filtrates of Diaporthella cryptica, an emerging hazelnut pathogen, 2-hydroxy-3-phenylpropanoate methyl ester and its 3-(4-hydroxyphenyl) and 3-(1 H-indol-3-yl) analogues, named crypticins A-C, were isolated together with the well-known tyrosol. Crypticins A-C were identified by spectroscopic (essentially nuclear magnetic resonance and high-resolution electrospray ionization mass spectrometry) methods. The R absolute configuration (AC) of crypticin A was determined by comparing its optical rotation and electronic circular dichroism (ECD) spectrum with those of papuline, the methyl ester of (-)( S)-phenyllactic acid isolated as the main phytotoxin of Pseudomonas syringae pv.  papulans, responsible for apple blister spot. The ACs of crypticins B and C were determined by time-dependent density functional theory calculations of their ECD spectra. Papuline and the new metabolites herein isolated, except tyrosol, were tested at 1 mg/mL on cork oak, grapevine, hazelnut, and holm oak leaves using the leaf puncture assay. They were also tested on tomato cuttings at 0.5 and 0.05 mg/mL. In the leaf puncture assay, none of the compounds was found to be active. Crypticin C and papuline were active in the tomato cutting assay. Additionally, crypticin C displayed moderate inhibitory effect against Phytophthora cambivora.

Background: Fungi play an important role in terrestrial ecosystems interacting positively or negatively with plants. These interactions are complex and the outcomes are different depending on the fungal lifestyles, saprotrophic, mutualistic or pathogenic. Furthermore, fungi are well known for producing secondary metabolites, originating from different biosynthetic pathways, which possess biological properties of considerable biotechnological interest. Among the terrestrial ecosystems, temperate forests represent an enormous reservoir of fungal diversity. This review will highlight the goldmine of secondary metabolites produced by pathogenic and endophytic fungi of forest trees with focus on their biological activities., Methods: A structured search of bibliographic databases for peer-reviewed research literature was undertaken using a research discovery application providing access to a large and authoritative source of references. The papers selected were examined and the main results were reported and discussed., Results: Two hundred forthy-one papers were included in the review, outlined a large number of secondary metabolites produced by pathogenic and endophiltic fungi and their biological activities, including phytotoxic, antifungal, antioomycetes, antibacterial, brine shrimp lethality, mosquito biting deterrence and larvicidal, cytotoxic, antiproliferative and many other bioactivities., Conclusion: The findings of this review confirm the importance of secondary metabolites produced by pathogenic and endophytic fungi from forest plants growing in temperate regions as an excellent prospects to discover compounds with new bioactivities and mode of actions. In addition, the potential of some metabolites as a source of new drugs and biopesticides is underlined., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

A new isochromanone, named fraxitoxin, was isolated together with (-)-mellein and tyrosol from liquid cultures of Diplodia fraxini, a pathogen involved in the etiology of canker and dieback disease of Fraxinus spp. in Europe. It was characterized as 5-methoxy-3-methylisochroman-1-one using spectroscopic methods (essentially NMR and HR-EI-MS). Its absolute configuration (R) at C(3) was assigned by electronic circular dichroism (ECD) measurements and calculations. Phytotoxic activity of the compound was evaluated on ash, cork and holm oak leaves at concentration of 1 mg/ml by the leaf-puncture assay. Interestingly, fraxitoxin caused necrotic lesions only on ash leaves., (© 2017 Wiley-VHCA AG, Zurich, Switzerland.)

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.

Three new lactones and a new fatty acid ester, named sapinofuranones C and D, diplopyrone B, and diplobifuranylone C, respectively, were isolated from Diplodia corticola, together with sphaeropsidins A and C, diplopyrone, diplobifuranylones A and B, diplofuranone A, and the (S,S)-enantiomer of sapinofuranone B. Sapinofuranones C and D, diplopyrone B, and diplobifuranylone C were characterized as (5S)-5-((1,S-1,6-dihydroxyhexa-2,4-dienyl)-dihydrofuran-2-one, 4,5-dihydroxy-deca-6,8-dienoic acid methyl ester, (5S)-5-hydroxy-6-(penta-1,3-dienyl)-5,6-dihydro-pyran-2-one, and 5'-((1R)-1-hydroxyethyl)-2',5'-dihydro-2H-[2,2']bifuranyl-5-one by spectroscopic and chemical methods, respectively. The relative configuration of sapinofuranone C was assigned by X-ray diffraction analysis, whereas its absolute configuration was determined by applying the advanced Mosher's method to its 11-O-p-bromobenzoyl derivative. The same method was used to assign the absolute configuration to C-5 of diplopyrone B and to that of the hydroxyethyl of the side chain of diplobifuranylone C, respectively. The metabolites isolated were tested at 1 mg/mL on leaves of cork oak, grapevine cv. 'Cannonau', and tomato using the leaf puncture assay. They were also tested on tomato cuttings at 0.2, 0.1, and 0.05 mg/mL. Each compound was tested for zootoxic activity on Artemia salina L. larvae. The efficacy of sapinofuranone C and diplopyrone B on three plant pathogens, namely, Athelia rolfsii, Fusarium avenaceum, and Phytophthora nicotianae was also evaluated. In all phytotoxic assays only diplopyrone B was found to be active. It also showed strong inhibition on the vegetative growth of A. rolfsii and P. nicotianae. All metabolites were inactive in the assay performed for the zootoxic activity (A. salina) even at the highest concentration used (200 μg/mL). Diplopyrone B showed a promising antioomycete activity for the control of Phytophthora spp. also taking into account the absence of zootoxic activity.

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.

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.

In this study, a strain (BL 101) of a species of Lasiodiplodia, not yet formally described, which was isolated from declining grapevine plants showing wedge-shaped cankers, was investigated for its ability to produce in vitro bioactive secondary metabolites. From culture filtrates of this strain three jasmonic acid esters, named lasiojasmonates A-C and 16-O-acetylbotryosphaerilactones A and C were isolated together with (1R,2R)-jasmonic acid, its methyl ester, botryosphaerilactone A, (3S,4R,5R)-4-hydroxymethyl-3,5-dimethyldihydro-2-furanone and (3R,4S)-botryodiplodin. The structures of lasiojasmonates A-C were established by spectroscopic methods as (1R*,2R*,3'S*,4'R*,5'R*)-4-hydroxymethyl-3,5-dimethyldihydro-2-furanone, (1R*,2R*,3'S*,4'R*,5'R*,10'R*,12'R*,13'R*,14'S*) and (1R*,2R*,3'S*,4'R*,5'R*,10'S*,12'R*,13'R*,14'S*)-4-(4-hydroxymethyl-3,5-dimethyltetrahydro-furan-2-yloxymethyl)-3,5-dimethyldihydro-2-furanones jasmonates (1, 4 and 5). The structures of 16-O-acetylbotryosphaerilactones A and C were determined by comparison of their spectral data with those of the corresponding acetyl derivatives obtained by acetylation of botryosphaerilactone A. The metabolites isolated, except 4 and 5, were tested at 1mg/mL on leaves of grapevine cv. Cannonau and cork oak using the leaf puncture assay. They were also tested on detached grapevine leaves at 0.5mg/mL and tomato cuttings at 0.1mg/mL. In all phytotoxic assays only jasmonic acid was found to be active. All metabolites were inactive in the zootoxic assay at 50 μg/mL., (Copyright © 2014. Published by Elsevier Ltd.)

Pinus radiata D. Don is a forest tree species native to the Monterey Baja in California. Due to its rapid growth and desirable lumber and pulp qualities, between 1960 and 1980, about 12,000 ha of P. radiata were planted in Sardinia, Italy. The only disease reported on this conifer species has been Diplodia pinea, which causes tip and branch dieback (3). In January 2012, dieback and mortality of 25-year-old radiata pine trees were observed in a reforestation area of about 20 ha located in northern Sardinia (40°43'N, 9°22'E, 600 m a.s.l.). Symptoms included chlorosis, reddish-brown discoloration of the whole crown or dieback starting in the upper crown and progressing downward through the crown, and necrotic bark tissues at root collar. Approximately 25% of the trees were affected. In a first attempt, a Phytophthora species was consistently isolated from the rhizosphere of 23 symptomatic trees, which included necrotic fine roots using oak leaves as bait (4). Afterwards, it was also isolated from phloem samples taken from the margins of fresh lesions at the stem base and upper roots of affected trees using synthetic mucor agar medium (1). Isolation from soil samples of six healthy pine trees randomly selected in the site did not yield any Phytophthora isolate. On carrot agar (CA), Phytophthora colonies were stellate to slightly radiate with limited aerial mycelium. Sporangia were obpiryform, non-papillate, and non-caducous, measuring 46.9 to 51.2 × 29.1 to 32.6 μm (l:b ratio 1.9). Hyphal swellings were formed in chains or clusters; chlamydospores were not observed. These isolates had cardinal temperatures of <5°C, 25°C, and 35°C, respectively. Their morphological and cultural features were typical of Phytophthora cryptogea Pethybridge & Lafferty. They were heterothallic and produced oogonia with amphyginous antheridia when paired with an A2 mating type tester strain of P. cryptogea. This identity was corroborated by sequence analysis of the internal transcribed spacer (ITS) region of the rDNA. BLAST searches showed 99% homology with sequences of P. cryptogea available in GenBank (DQ479410 and HQ697245). The ITS sequence of a representative isolate (PH101) was submitted to GenBank (Accession Nos. KC603895). The strain PH101 was stored in the culture collection of the Department of Agriculture at the University of Sassari. Pathogenicity of isolate PH101 was verified by inoculating five freshly cut logs of radiata pine (1 m long and 15 cm diam.) with a 5-mm agar plug taken from the margin of 4-day-old culture grown on CA (4). The plug was inserted in a 5-mm hole made through the bark with a cork borer. Five control logs were inoculated with sterile CA. All logs were incubated in a growth chamber at 20°C. Phloem lesion sizes were assessed after 1 month and measured 9.7 ± 5.5 cm 2 (average ± standard deviation). Control logs had no lesions. The pathogen was re-isolated from the lesions, thus fulfilling Koch's postulates. P. cryptogea has been previously reported in Australia, causing decline of radiata pine trees in wet and flooded soils (2). To our knowledge, this is the first report of P. cryptogea on P. radiata trees in Europe. References: (1) C. M. Brasier and S. A. Kirk. Plant Pathol. 50:218, 2001. (2) M. Bumbieris. Aust. J. Bot. 24:703, 1976. (3) A. Franceschini et al. Informatore Fitopatologico 1:54, 2006. (4) B. Scanu et al. For. Pathol. 43:340, 2013.

During a study of the species of Botryosphaeriaceae associated with oak decline in Tunisia, a large collection of Diplodia strains were isolated from Quercus afares, Q. canariensis and Q. suber trees showing a progressive dieback of shoots and branches, trunk canker and exudates and collar rot. Most of the isolates were identified as Diplodia corticola, while two isolates from Q. canariensis were morphologically and phylogenetically (ITS and tef1-α sequences data) distinct from all other known species of Diplodia. They are described here as Diplodia quercivora sp. nov. In addition, phylogenetic analyses showed for the first time the existence of two distinct lineages within D. corticola. In artificial inoculation experiments, D. quercivora caused necrotic lesions on bark and wood of three Mediterranean oak species, Q. ilex, Q. pubescens and Q. suber. In particular, among the oak species tested, Q. pubescens was the most susceptible.

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.

Two phytotoxic dihydrofuropyran-2-ones, named afritoxinones A and B, were isolated from liquid culture of Diplodia africana, a fungal pathogen responsible for branch dieback of Phoenicean juniper in Italy. Additionally, six others known metabolites were isolated and characterized: oxysporone, sphaeropsidin A, epi-sphaeropsidone, R-(-)-mellein, (3R,4R)-4-hydroxymellein and (3R,4S)-4-hydroxymellein. The structures of afritoxinones A and B were established by spectroscopic and optical methods and determined to be as (3aS(*),6R(*),7aS)-6-methoxy-3a,7a-dihydro-3H,6H-furo[2,3-b]pyran-2-one and (3aR(*),6R(*),7aS)-6-methoxy-3a,7a-dihydro-3H,6H-furo[2,3-b]pyran-2-one, respectively. The phytotoxic activity of afritoxinones A and B and oxysporone was evaluated on host (Phoenicean juniper) and non-host plant (holm oak, cork oak and tomato) by cutting and leaf puncture assay. Oxysporone proved to be the most phytotoxic compound. This study represents the first report of secondary metabolites produced by D. africana. In addition, the taxonomic implications of secondary metabolites in Botryosphaeriaceae family studies are discussed., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Lentisk (Pistacia lentiscus L., Anacardiaceae) is an evergreen shrub that is widespread over the Mediterranean Region. The species is also cultivated as an ornamental plant in Italy. In August 2008, a survey carried out in a forest nursery in Sardinia (39°57'N, 9°13'E) revealed the presence of symptoms such as wilting and desiccation of foliage associated with root and collar rot on 1- to 3-year-old potted seedlings of lentisk. Approximately 30% of 1,500 potted plants were affected. A Phytophthora sp. was consistently isolated from infected roots on synthetic mucor agar medium. Colonies on carrot agar (CA) were stellate to slightly radiate with low aerial mycelium. Growth occurred from 6 to 38°C, with an optimum around 30°C (mean radial growth rate was 11.8 mm per day). Sporangia were produced abundantly in unsterile pond water; they were nonpapillate, persistent, ellipsoid to obpyriform, (57.8-) 80.5 (-100.5) × (30.2-) 39.3 (-51.5) μm, with a length/breadth ratio of 2.0:1, proliferating internally or externally. Hyphal swellings were spherical to irregular and frequently produced in chains. Chlamydospores were not observed. Isolates were heterothallic and produced oogonia with amphigynous antheridia when paired with A2 mating type of Phytophthora drechsleri and P. cryptogea. Cultural and morphological features were in close agreement with those recently published for Phytophthora sp. "niederhauserii" (4). The rDNA internal transcribed spacer (ITS) sequence (ITS1-5.8S-ITS2) of a representative isolate (LEN1) was submitted to GenBank (Accession No. GU119914) and BLAST searches showed 100% similarity with sequences of P. sp. "niederhauserii" deposited in GenBank (Accession Nos. GQ848201 and EU244850). The strain LEN1 was stored in the culture collection of the Department of Plant Protection at the University of Sassari. Its pathogenicity was verified by inoculating 10 1-year-old lentisk seedlings grown in pots. A mycelial plug (3 to 4 mm 2 ) taken from the margin of a 4-day-old culture grown on CA was put in a shallow wound (~3 mm) made by a sterile scalpel at the root collar of each seedling. All plants were kept in a greenhouse at 25°C in natural daylight. After 20 days, inoculated plants began to show symptoms similar to those observed on naturally infected plants. Five control plants inoculated with sterile CA plugs did not develop any disease symptoms. The pathogen was reisolated from infected tissues, thus fulfilling Koch's postulates. P. sp. "niederhauserii" has not been formally described, however, so far there have been several reports of this species in Europe (1,3). Previously, other Phytophthora spp. were reported associated with lentisk root rot in Italy (2). To our knowledge, this is the first report of P. sp. "niederhauserii" on Pistacia lentiscus and it emphasizes the susceptibility of the Mediterranean species to this new pathogen. References: (1) A. Józsa et al. Plant Pathol. 59:1166, 2010. (2) G. Magnano Di San Lio et al. Micol. Ital. 21:3, 1992. (3) E. Moralejo et al. Plant Pathol. 58:100, 2009. (4) A. Pérez-Sierra et al. Plant Dis. 94:534, 2010.

Since December 2008, a severe outbreak of ink disease has been observed in a chestnut grove in the Sardinia Region in Italy (40°01'N, 9°13'E, 1,200 m above sea level). Trees have shown symptoms such as microphylly and yellowish foliage as well as necrosis on the main roots and collar. Isolations were made from infected roots and soil using green apples as baits. Small pulp pieces were cut from the lesions that developed in the apples and plated on Phytophthora selective medium (1). In addition to Phytophthora cambivora, another Phytophthora sp. was detected from 60% of 25 symptomatic trees sampled. Colonies subcultured onto carrot agar (CA) were generally appressed and stellate. Growth occurred from 2 to 26°C with an optimum at 20°C (mean radial growth rate of 4.5 mm/day). Sporangia were produced abundantly in unsterile pond water; they were semipapillate, rarely bipapillate, limoniform or ovoid, occasionally caducous with short pedicels (<5 μm), and 35.2 to 58.1 (46.3) × 22.1 to 35.3 (31.9) μm, with a length/breadth ratio of 1.5:1. Catenulate hyphal swellings were frequently present, whereas no chlamydospores were observed. Isolates produced numerous homothallic oogonia with diameters from 23.7 to 31.7 (27.3) μm. Antheridia were predominantly paragynous. Cultural and morphological features were in close agreement with those described for P. pseudosyringae (2). Identity was confirmed by analysis of the internal transcribed spacer region (ITS1-5.8S-ITS2) of rDNA. BLAST searches at GenBank showed 100% identity with reference sequences of P. pseudosyringae (Accession Nos. AY230190 and EU074793). The representative sequence of one P. pseudosyringae strain (CST2A), stored in the culture collection of the Department of Plant Protection-University of Sassari, was submitted to GenBank (Accession No. GU460375). Koch's postulates were fulfilled by inoculating 10 5-month-old chestnut seedlings grown in pots. One shallow cut was made into the bark on the main stem and an agar plug colonized by P. pseudosyringae was inserted beneath the flap. Seedlings were kept at the laboratory at temperatures varying from 16 to 22°C and watered as necessary. After 20 days, extensive, sunken, necrotic lesions measuring 27.2 ± 1.9 mm (mean + standard error) developed around the inoculation sites. Control plants inoculated with sterile CA plugs did not show any disease symptoms. The pathogen was consistently reisolated from infected tissues. P. pseudosyringae has recently been reported as the causal agent of stem necroses on chestnut seedlings in a nursery in Spain (3). To our knowledge, this is the first report of P. pseudosyringae on Castanea sativa in Italy. References: (1) C. M. Brasier and S. A. Kirk. Plant Pathol. 50:218, 2001. (2) T. Jung et al. Mycol. Res. 107:772, 2003. (3) C. Pintos Varela et al. Plant Dis. 91:1517, 2007.

Strawberry tree, family Ericaceae, is an evergreen shrub or small tree that grows in the Mediterranean Region. In spring 2009, a survey was conducted to study the fungi associated with canker and branch dieback of strawberry tree in a natural stand located on Caprera Island (41°12'N, 9°27'E), Italy. Fungal isolates obtained from live twigs and branches showing sunken necrotic bark lesions were identified as Diplodia scrobiculata J. de Wet, Slippers & M.J. Wingf. on the basis of morphological features (1). On potato dextrose agar (PDA) at 25°C, D. scrobiculata isolates developed white colonies with appressed mycelium that became dark gray after 4 to 6 days and formed pycnidia after 2 weeks on sterile Pinus radiata needles placed on the PDA surface. The conidia were dark brown with zero to three septa, clavate with truncate base, and measured 31.1 to 41.9 (36.8) × 11.3 to 16.3 (12.6) μm, with a length/width ratio of 2.9 ± 0.1 (mean ± standard error) (n = 50). Identity was confirmed by analysis of the internal transcribed spacer region (ITS1-5.8S-ITS2) of rDNA. BLAST searches in GenBank showed 100% similarity with reference sequences of D. scrobiculata (GenBank Nos. AY160200, EU220438, EU220444, and EU392283). The representative sequence of one D. scrobiculata strain (BL5), stored in the culture collection of the Department of Plant Protection at the University of Sassari, was deposited in GenBank (Accession No. GU722102). Pathogenicity of strain BL5 was tested by stem inoculation on eight 2-year-old strawberry tree seedlings maintained in a greenhouse at 18 to 26°C. A mycelial plug (3 to 4 mm 2 ) taken from the margin of an actively growing colony on PDA was put in a shallow wound (~3 mm) made by a scalpel on the basal part of the stem of each seedling. Four weeks after inoculation, the seedlings displayed dark brown-to-black discoloration, measuring 1.7 ± 0.6 cm (mean ± standard error) of the bark and wood tissues of the stems. The pathogen was successfully reisolated from symptomatic stem tissues, thus fulfilling Koch's postulates. Three control seedlings inoculated with sterile PDA plugs remained asymptomatic. These results demonstrate the active role played by D. scrobiculata in the aetiology of the canker and branch dieback observed on strawberry tree. D. scrobiculata is generally recognized as a weak pathogen of gymnosperms worldwide (2), however, it has recently been reported on olive in Italy (3). To our knowledge, this is the first report of D. scrobiculata on strawberry tree. Currently, further investigations are in progress to determine the possible role of biotic and abiotic factors in the development of this disease. References: (1) J. De Wet et al. Mycol. Res. 107:557, 2003. (2) J. De Wet et al. Mol. Phylogenet. Evol. 46:116, 2008. (3) C. Lazzizera et al. Fungal Divers. 31:63, 2008.

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.

A survey was carried out in the spring of 2003 to study the fungi associated with declining trees in a cork oak (Quercus suber L.) forest located in Sassari Province, Sardinia, Italy (40°52'N, 9°01'E) at an altitude of 150 m (above sea level). Several isolates obtained from live twigs and branches showing sunken necrotic bark lesions were identified as Fusicoccum parvum Pennycook & Samuels (teleomorph Botryosphaeria parva Pennycook & Samuels). Neither pycnidia nor ascomata were observed on the symptomatic samples collected. On potato dextrose agar (PDA) at 25°C, the isolates developed an aerial and compact mycelium, initially white but becoming gray after 4 to 6 days, and produced pycnidia after 1 month on sterile cork oak twigs placed on the surface of PDA. Conidia from culture were hyaline, ellipsoidal to fusiform, externally smooth, thin walled, nonseptate, 12 to 19 (15.5) × 5.5 to 8.5 (6.5) μm, with length/width ratio of 2.4 ± 0.1 (mean ± standard error). Identity was confirmed by analysis of the nucleotide sequences of the internal transcribed spacer (ITS) from the rRNA repeat and the translation elongation factor 1-alpha (EF1-α), as done elsewhere (1,4). BLAST searches at GenBank showed a high identity with reference sequences (ITS: >99%; EF1-α: 100%). Representative sequences of both regions were deposited at GenBank (ITS: Accession No. DQ487157; EF1-α: Accession No. DQ487158). Pathogenicity tests were carried out on seven 2-year-old cork oak seedlings maintained in a greenhouse at 14 to 26°C with the B. parva strain CBS 119937 obtained in this study. A mycelial plug (3 to 4 mm 2 ) taken from the margin of an actively growing colony on PDA was put in a shallow wound made by a scalpel on the basal part of the stem of each seedling. Sterile PDA plugs were placed into similar wounds on three control seedlings. The inoculation points were wrapped in Parafilm to retain moisture for 1 week. After 4 weeks, all seedlings inoculated with B. parva died and showed a collapse of the stem cortical tissues associated with dark brown discolorations and vascular necrosis measuring 10.9 ± 0.4 cm. No symptoms were visible in the control seedlings. The pathogen was reisolated from all the inoculated seedlings, thus fulfilling Koch's postulates. The results confirm the virulence of this fungus and point to its possible involvement in the aetiology of cork oak decline. B. parva is a cosmopolitan, plurivorous pathogen causing disease in several hosts of economic importance, such as grapevine (3), kiwi (2), and Eucalyptus spp. trees (1). To our knowledge, this is the first report of B. parva causing canker disease on cork oak trees. References: (1) A. Gezahgne et al. S. Afr. J. Bot. 70:241, 2004. (2) S. R. Pennycook and G. J. Samuels. Mycotaxon 24:445, 1985. (3) A. J. L. Phillips. Phytopathol. Mediterr. 41:3, 2002. (4) B. Slippers et al. Mycologia 96:83, 2004.