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Eucalyptus globulus, an exotic species in Portugal, is one of the dominant and intensively managed forest species in the country. A disease syndrome characterised by leaf necrosis, stem girdling and cutting dieback in eucalyptus and associated with pestalotioid fungi has been detected in nurseries and young plantations in the last years. Twenty-seven isolates were recovered from diseased plants. Phylogenetic analysis based on internal transcribed spacers, partial translation elongation factor 1-α gene and partial β-tubulin gene sequence data grouped the isolates in five separate clades. Combining morphological, cultural and molecular data, five new species of Neopestalotiopsis are described, namely, Neopestalotiopsis eucalyptorum, Neopestalotiopsis hispanica, Neopestalotiopsis iberica, Neopestalotiopsis longiappendiculata and Neopestalotiopsis lusitanica.

Research Highlights: Pestalotiopsis pini sp. nov. is an emerging pathogen on stone pine, Pinus pinea L., in Portugal. Background and Objectives: Stone pine is one of the most important forest tree species in Portugal and in the whole Mediterranean basin. Pestalotiopsis species are common endophytes, saprobes or pathogens in a variety of hosts and environments. The objective of the present study was to identify the Pestalotiopsis species associated with the symptomatic stone pine trees. Materials and Methods: Samples of stone pine trees showing shoot blight and stem necrosis were obtained from stone pine orchards and urban areas in Portugal, and the isolated Pestalotiopsis species were identified based on morphology and combined ITS, TEF and TUB DNA sequence data. Artificial inoculations on one-year-old stone pine seedlings were performed with the two species most frequently found in association with shoot blight disease. Results: Five Pestalotiopsis spp. were isolated. A taxonomic novelty, Pestalotiopsis pini is described, representing a new pathogen for stone pine. Conclusions: Pestalotiopsis species may represent a threat to the health of pine forests in the Mediterranean basin. Future research should be done in order to increase our knowledge about the potential impact of pestalotioid species in stone pine, in order to develop management strategies against these pathogens.

Eucalyptus globulus, a non-native species, is currently the most abundant forest species in Portugal. This economically important forest tree is exploited mainly for the production of pulp for the paper industry. The community of Botryosphaeriaceae species occurring on diseased and healthy E. globulus trees was studied on plantations throughout the country. Nine species from three different genera were identified, namely Botryosphaeria (B. dothidea), Diplodia (D. corticola and D. seriata) and Neofusicoccum (N. australe, N. algeriense, N. eucalyptorum, N. kwambonambiense, N. parvum and Neofusicoccum sp.). Of these, N. algeriense, D. corticola and D. seriata are reported for the first time on E. globulus, while N. algeriense, N. eucalyptorum and N. kwambonambiense correspond to first reports in Portugal. The genus Neofusicoccum was clearly dominant with N. australe and N. eucalyptorum being the most abundant species on both diseased and healthy trees. In artificial inoculation trials representative isolates from all nine species were shown to be pathogenic to E. globulus but there were marked differences in aggressiveness between them. Thus, D. corticola and N. kwambonambiense were the most aggressive while B. dothidea and D. seriata were the least aggressive of the species studied.

Summary The Eucalyptus pathogen Quambalaria eucalypti has been reported from several subtropical and tropical countries of the Southern Hemisphere. During the course of a nationwide monitoring programme aimed at the detection of pathogens in commercial eucalypt plantations in Portugal, Q. eucalypti was found affecting Eucalyptus globulus. The identity of the pathogen was confirmed by sequence analyses of the internal transcribed spacer (ITS) region of the rDNA operon, as well as morphological characteristics. Quambalaria eucalypti is widespread throughout the eucalypt-producing areas of Portugal. Pathogenicity studies revealed that different host genotypes showed various degrees of susceptibility to the pathogen. To our knowledge, this is the first report of Q. eucalypti on Eucalyptus spp. in Portugal and in the Northern Hemisphere.

Summary Teratosphaeria gauchensis and T. zuluensis both cause a stem canker of eucalypts that leads to serious damage in various parts of the world. Until recently, this disease was unknown in Portugal. Nevertheless, severe damage to Eucalyptus globulus has been observed in Portugal since 2006 when symptoms appeared as necrotic spots on young green stems and leaves, twig lesions, and dark oval-shaped lesions on stems and trunks. The isolates from affected E. globulus tree plantations were identified using sequence data of the ITS1-5.8S-ITS2 and EF-1α clusters, together with morphological characteristics. Based on these results, the causal organism was identified as T. gauchensis, which represents the first report of this pathogen from Portugal.

Short Communication The fungus Sydowia polyspora is frequently isolated from conifers worldwide and is considered a pathogen on several hosts. Stone pine (Pinus pinea) is one of the most important forestry species throughout the Mediterranean basin due to the value of the edible pine nut. Stone pines showing tip dieback, needles with tan- to yellowcoloured lesions and shoot death, observed in stands in Portugal, were sampled for analysis. Fungal colonies covered with cream-coloured spore masses, were consistently obtained. Morphological and phylogenetic analyses of the ITS rDNA region enabled identification of these isolates as S. polyspora. Inoculation tests showed that the fungus caused lesions on excised P. pinea shoots. The symptoms observed might have a negative effect on pine nut production, and thus, evaluation of the impact of this disease is of relevance to future research. This paper is the first to report S. polyspora causing disease on P. pinea info:eu-repo/semantics/publishedVersion

Summary Cryphonectria parasitica, the causal agent of chestnut blight, has been responsible for the decline in chestnut in Portugal for the last two decades. In order to improve understanding of C. parasitica diversity, a total of 617 isolates from all affected chestnut-growing areas in continental Portugal, Madeira and the Azores archipelagos were studied. Nine vegetative compatibility (vc) types were identified among the isolates. EU-11 was the most widespread vc type comprising 80.2% of the isolates, followed by EU-12 (7.1%) and EU-66 (6.6%). Two of the Portuguese vc types could not be assigned to a known European vc type. The diversity of vc types was low in the Portuguese populations of C. parasitica, but comparable with other areas where C. parasitica was introduced recently. The frequent occurrence of perithecia and both mating types of C. parasitica indicates that sexual reproduction of the chestnut blight fungus is common in Portugal. One C. parasitica isolate from Tras-os-Montes showed a white culture morphology and contained dsRNA, indicating the presence of hypovirulence in this area.

In a recent study intended to assess the distribution of Cryphonectria parasitica in Portugal, 22 morphologically atypical orange isolates were collected in the Midwestern regions. Eleven isolates were recovered from Castanea sativa, in areas severely affected by chestnut blight and eleven isolates from Quercus suber in areas with cork oak decline. These isolates were compared with known C. parasitica and Cryphonectria radicalis isolates using an integrated approach comprising morphological and molecular methods. Morphologically the atypical isolates were more similar to C. radicalis than to C. parasitica. Phylogenetic analyses based on internal transcribed spacer (ITS) and β-tubulin sequence data grouped the isolates in a well-supported clade separate from C. radicalis. Combining morphological, cultural, and molecular data Cryphonectria naterciae is newly described in the Cryphonectria-Endothia complex. Microsatellite-primed PCR fingerprinting with (GACA)(4) primer discriminated between C. naterciae, C. radicalis, and C. parasitica.

Essential oils from foliage, bark and heartwood of Cryptomeria japonica D. Don from Azores Archipelago (Portugal) were analyzed by GC and GC-MS. Two populations, of black and reddish heartwood color, were studied. The main compounds found in the foliage of both populations were α-pinene (9.6–29.5%), (+)-phyllocladene (3.5–26.5%), ent-kaur-16-ene (0.2–20.6%), sabinene (0.5–19.9%) and limonene (1.4–11.5%), with a large variation in individual compounds from each population. Heartwood oils were characterized by a high content of cubebol (2.8–39.9%) and epi-cubebol (4.1–26.9%) isomers, which were absent in the foliage. Elemol and eudesmol isomers were found in the foliage and heartwood oils, while (+)-phyllocladene was absent in heartwood. Black and reddish bark oils were composed of the diterpenes dehydroferruginol (1.9–5.1%) and ferruginol (2.6–11.5%), along with the sesquiterpenes δ-cadinene (10.4–15.9%), α-muurolene (3.3–5.4%), epi-zonarene (4.0–5.0%), cubenol (9.3–14.0%), τ-muurolol (4.8–10.7%), β-eudesmol (3.0–9.9%), γ-eudesmol (1.9–7.0%) and hedycariol (1.4–6.2%). Azorean C. japonica oils exhibited significant chemical differences compared with native plants from Asia. The essential oils showed moderate antimicrobial activity against the pathogenic fungus Cryptococcus neoformans and human pathogenic bacteria (especially against multidrug-resistant Mycobacterium tuberculosis). The antimicrobial activity of the essential oils may be attributed to compounds such as ent-kaur-16-ene, (+)-phyllocladene, ferruginol and elemol, which are present in different proportions within the complex oil mixture. These results suggest a potential use for C. japonica oils obtained from wood industry leftovers.

In November of 2007, dieback symptoms (basal needle dieback, wilting, and dieback of terminal shoot) were observed on plant groups of Pinus radiata and P. pinaster in a tree nursery located in Anadia in the central region of Portugal (40°26′N, 08°23′W). Two containers with a total of 112 plants per pine species (with and without symptoms) were collected. Small pieces (5 mm long; two from the roots, stem at the soil level, and the aerial part, totaling six pieces) of 20 symptomatic plants were sterilized with 3% sodium hypochlorite, and isolations were performed on potato dextrose agar (PDA) supplemented with 0.5 mg/ml of streptomycin sulfate. A species of Fusarium was isolated from all infected tissues and pure cultures were obtained by single hyphal tip transfers on PDA and Spezieller Nährstoffarmer agar and incubated at 25°C for 10 days with a 12-h photoperiod. The species was identified as Fusarium circinatum Nirenberg & O'Donnell (= F. subglutinans Wollenweb & Reinking) on the basis of morphological and cultural characteristics (2). They produced white, aerial mycelia, violet pigment, typically three-septate macroconidia with slightly curved walls, single-celled microconidia, and characteristic sterile, coiled hyphae. Microconidia were ovoid or allantoid and born in false heads on aerial polyphialides. The identification was confirmed by PCR with specific primers CIRC1A/CIRC4A, resulting in a 360-bp DNA fragment of the two nuclear ribosomal intergenic spacer regions (3). Pathogenicity tests were performed by inoculating 5- and 9-month-old P. pinaster and P. radiata seedlings, respectively. Plants belonging to P. pinea species (8-month-old), the second most important pine in the country, were also included in the tests. Small strips of bark (10 × 1 mm) were cut from the stems and similar-sized pieces of PDA colonized by two isolates of F. circinatum were placed in contact with the open wounds and covered with Parafilm. Ten seedlings for each pine species, isolate, and control (with sterile PDA) were provided in a total of 90 plants. First symptoms, basal needle and shoot dieback, were observed in P. radiata 8 days after inoculation. One month later, all P. radiata and 70% of the P. pinaster plants were dead. In all P. pinea plants, needles turned red along the main stem, from center to periphery, but only 2% of these plants presented wilting of the terminal shoot after 1 month. No symptoms were observed on control seedlings. F. circinatum was reisolated from symptomatic plants of the three species tested. To our knowledge, this is the first report of F. circinatum in Portugal. Pitch canker, caused by Gibberella circinata (anamorph F. circinatum), is one of the most aggressive pathogens on several pine species in the world (1). In 2005, the fungus was detected in the European continent affecting P. radiata and P. pinaster in northern Spain. References: (1) E. Landeras et al. Plant Dis. 89:1015, 2005. (2) H. I. Niremberg and K. O'Donnell. Mycologia 90:434, 1998. (3) W. Schweigkofler et al. Appl. Environ. Microbiol. 70:3512, 2004.