Multiple new cryptic pathogenic Phytophthora species from Fagaceae forests in Austria, Italy and Portugal.

During surveys of Phytophthora diversity in natural and semi-natural Fagaceae forests in Austria, Italy and Portugal, four new cryptic species were isolated from rhizosphere soil samples. Multigene phylogeny based on nuclear ITS, ß-tubulin and HSP90 and mitochondrial cox1 and NADH1 gene sequences demonstrated that two species, P. tyrrhenica and P. vulcanica spp. nov., belong to phylogenetic Clade 7a, while the other two species, P. castanetorum and P. tubulina spp. nov., clustered together with P. quercina forming a new clade, named here as Clade 12. All four new species are homothallic and have low optimum and maximum temperatures for growth and very slow growth rates at their respective optimum temperature. They differed from each other and from related species by a unique combination of morphological characters, cardinal temperatures, and growth rates. Pathogenicity of all Phytophthora species to the root system of their respective host species was demonstrated in soil infestation trials.

Tuber melosporum smooth spores: an anomalous feature in the genus Tuber.

This paper adds new insights on ecology and micromorphology of Tuber melosporum, a rare species with smooth spores. Eight T. melosporum ascomata collected in a 50 y old Pinus halepensis and Quercus ilex plantation in Sardinia, Italy, represent the first recovery of this species outside Spain. In comparison to the T. melosporum holotype, Italian specimens revealed differences in the number of spores in asci and spore shape. We propose an emended description of Tuber to include species without spore ornamentation.

Endemic and Emerging Pathogens Threatening Cork Oak Trees: Management Options for Conserving a Unique Forest Ecosystem.

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.

Characterization of Tuber borchii and Arbutus unedo mycorrhizas.

For the first time, arbutoid mycorrhizas established between Tuber borchii and Arbutus unedo were described. Analyzed mycorrhizas were from one T. borchii natural truffle ground, dominated by Pinus pinea, as well as synthesized in greenhouse conditions. A. unedo mycorrhizas presented some typical characteristics of ectomycorrhizas of T. borchii. However, as in arbutoid mycorrhizas, ramification was cruciform and intracellular colonization in epidermal cells was present. The ability of T. borchii to form ectomycorrhizas with A. unedo opens up the possibility to also use this fruit plant for truffle cultivation. This represents an important economic opportunity in Mediterranean areas by combining both the cultivation of precious truffles and the production of edible fruits which are used fresh or in food delicacies.

Diplodia quercivora sp. nov.: a new species of Diplodia found on declining Quercus canariensis trees in Tunisia.

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.

Studies on the ectomycorrhizal community in a declining Quercus suber L. stand.

This survey was carried out in a Quercus suber L. stand with many trees affected by the disease "oak decline". Its aim was to obtain information about both the belowground ectomycorrhizal fungal community in a declining Q. suber stand as a whole, and the ectomycorrhizal fungal community of individual tree (EFT) detected in healthy and diseased plants. To this end, we first categorized the trees into four different decline classes (one for healthy plants and three for diseased plants) and then, by using morphological and molecular tools, we identified the ectomycorrhizas isolated from samples collected near the trees with different declining classes. The ectomycorrhizal community as a whole was seen to be composed of numerous ectomycorrhizal fungal species, only some of which appeared to be dominant (Cenococcum geophilum, Lactarius chrysorrheus, and some species of Tomentella genus), while most occurred sporadically. Results show that all root tips observed are mycorrhized and that decline class does not influence the number of ectomycorrhizal root tips found in the EFTs, thus oak decline does not impact the investment in ectomycorrhizal symbiosis. However, some statistical differences can be observed in the values of evenness and taxonomic distinctness in the EFT associated with trees with different states of health. Finally, both the analysis of similarity test and the ordination technique highlight a compositional difference between the EFT associated with trees in different health conditions, but also suggest that other factors may play a role in causing these differences.

Cyclobotryoxide, a phytotoxic metabolite produced by the plurivorous pathogen Neofusicoccum australe.

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.

Afritoxinones A and B, dihydrofuropyran-2-ones produced by Diplodia africana the causal agent of branch dieback on Juniperus phoenicea.

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.

Occurrence and characterization of peptaibols from Trichoderma citrinoviride, an endophytic fungus of cork oak, using electrospray ionization quadrupole time-of-flight mass spectrometry.

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.

Pre-exposure to ozone predisposes oak leaves to attacks by Diplodia corticola and Biscogniauxia mediterranea.

One-year-old cork oak (Quercus suber) and turkey oak (Q. cerris) seedlings were exposed to ozone (110 ppb, 5 h day(-1), for 30 days) and were inoculated with Diplodia corticola and Biscogniauxia mediterranea, respectively, by spraying a suspension of spores on the leaves. Both fungi are endophytic and may act as weak parasites, contributing to oak decline. Ozone exposure stimulated leaf attacks after inoculation, although the physiological, visible, and structural responses of both oaks to O3 exposure were weak. In fact, steady-state gas exchange, leaf waxes, and wettability were not significantly affected by O3. In Q. cerris, O3 altered the structure of stomata, as observed by scanning microscopy, and reduced the leaf relative water content. No hyphal entry through stomata or growth towards stomata was, however, observed. Inoculations were performed in a humid chamber at low light; stomata were likely to be closed. When Q. cerris was inoculated in natural conditions, i.e., in a forest infected by B. mediterranea, seedlings pre-exposed to the enhanced O3 regime had a higher number of B. mediterranea isolates than the controls. This suggests that pre-exposure to O3 predisposed Q. cerris leaves to attacks by B. mediterranea independent of stomata. The hyphae of both fungi were able to enter the leaf through the cuticle, either by gradual in-growth into the cuticle or erosion of a hollow in the cuticle at the point of contact. The primary cause of increased leaf injury in O3-exposed seedlings appeared to be higher germination of spores than on control leaves.

Diplobifuranylones A and B, 5'-monosubstituted tetrahydro-2H-bifuranyl-5-ones produced by Diplodia corticola, a fungus pathogen of cork oak.

Two new 5'-monosubstituted tetrahydro-2H-bifuranyl-5-ones, named diplobifuranylones A and B (1 and 2), were isolated from the culture filtrates of Diplodia corticola, the causal agent of a canker of cork oak (Quercus suber). The same fungus also produced eight known metabolites, namely, the diplopyrone, (3S,4R)-trans- and (3R,4R)-cis-4-hydroxymellein, sapinofuranone B and its (S,S)-enantiomer, and sphaeropsidins A-C. Diplobifuranylones A and B (1 and 2) were characterized, using spectroscopic and chemical methods, as two diastereomeric 5'-(1-hydroxyethyl)-3,4,2',5'-tetrahydro-2H-[2,2']bifuranyl-5-ones. While the relative stereochemistry of the two metabolites (1 and 2) was deduced by NOESY and ROESY experiments, the absolute stereochemistry of the chiral carbon of the hydroxyethyl side chain at C-5', determined by application of Mosher's method, proved to be S and R in 1 and 2, respectively. Assayed on a nonhost plant, diplobifuranylones A and B did not show phytotoxic activity. In an Artemia salina larvae lethality bioassay neither 1 nor 2 was toxic at the highest concentration tested (300 microg/mL).

Biscopyran, a phytotoxic hexasubstituted pyranopyran produced by Biscogniauxia mediterranea, a fungus pathogen of cork oak.

A new phytotoxic hexasubstituted pyranopyran, biscopyran (3), was isolated together with phenylacetic acid (2) and previously isolated 5-methylmellein (1) from the liquid culture filtrates of Biscogniauxiamediterranea, a major fungal pathogen involved in oak decline in Sardinia. Biscopyran was characterized by spectroscopic methods as a new (Z)-2-methoxy-1-[7-((Z)-2-methoxybut-2-enoyl)-3,4,5,6-tetramethyl-2H,7H-pyrano[2,3-b]pyran-2-yl]but-2-en-1-one. Biscopyran assayed at 0.26-0.026 mM concentration range caused epinasty on cork oak cuttings. On a nonhost plant, tomato, biscopyran caused wilting. Phenylacetic acid, assayed at the same concentration, was toxic to Q. suber, while on tomato cuttings it induced internal tissue collapse on the stem.

Diplopyrone, a new phytotoxic tetrahydropyranpyran-2-one produced by Diplodia mutila, a fungus pathogen of cork oak.

A new phytotoxic monosubstituted tetrahydropyranpyran-2-one, named diplopyrone (1), was isolated from the liquid culture filtrates of Diplodia mutila, a plant pathogenic fungus causing a form of canker disease of cork oak (Quercus suber). Diplopyrone was characterized, using spectroscopic and chemical methods, as 6-[(1S)-1-hydroxyethyl]-2,4a,6,8a-tetrahydropyran[3,2-b]pyran-2-one. The absolute stereochemistry of the chiral secondary hydroxylated carbon (C-9), determined by application of Mosher's method, proved to be S. Diplopyrone assayed at a 0.01-0.1 mg/mL concentration range caused necrosis and wilting on cork oak cuttings. On a nonhost plant, tomato, diplopyrone caused brown discoloration or stewing on the stem.