Molecular Identification of Fomitiporia mediterranea and Eutypa lata/Libertella blepharis in Platanus × acerifolia

The studies of woody organs‐affecting diseases of Platanus × acerifolia can be hampered by the finding of fungi whose identification is difficult with mycological techniques. In a previous study on Platanus × acerifolia affected by severe cankers, Fomitiporia mediterranea/punctata‐like fungi, not associated with fruit bodies and Libertella sp. were recovered. Due to the severity of the associated symptoms, a characterization of fungal ribosomal DNA genes was undertaken in the present study, aimed to specific identification of the pathogens. From DNA of Fomitiporia‐vegetative isolates and Fomitiporia‐fruit body isolates, included in the study for comparison (from fruit body on plane trees typical of F. mediterranea/punctata), and Libertella sp., DNA fragments were amplified in polymerase chain reaction (PCR) with the use of ITS5/ITS4 and 5.8S‐R/LR7 primer pairs. Sequence alignments showed that Fomitiporia‐vegetative/fruit body isolates had highly homologous ITS5/ITS4 sequences, with a nucleotide identity of 98–100%. All the Fomitiporia isolates from plane tree showed 97–100% and 91–94% of nucleotide identity respectively with ITS5/ITS4 sequences of known strains of F. mediterranea and F. punctata, extracted from GenBank. The strong similarity of these identity ranges with those obtained within F. mediterranea (98–100%) and between the two Fomitiporia species (90–94%) confirms the identification of the isolates from plane tree as F. mediterranea. Sequence comparison between Libertella sp. from plane tree and known strains of Eutypa lata/L. blepharis showed 94–99% of nucleotide identity. The comparison with eight additional species of Eutypa showed 90–93% of nucleotide identity. As previously reported for the different taxa within Diatrypaceae, also ITS5/ITS4 sequence of Libertella sp. from plane tree exhibited 11‐bp tandem repeats motifs. Results of sequence alignments, of phylogenetic trees, and of the putative restriction map, identify the Fomitiporia isolates of this work as F. mediterranea, and the isolates of Libertella sp. as E. lata/L. blepharis. For comparative purposes, ITS5/ITS4 sequences of Spongipellis pachyodon and Fomes fomentarius from plane tree, were also obtained in this work.     

A new species of Fomitiporia (Hymenochaetaceae, Basidiomycota) from China based on morphological and molecular characters

Fomitiporia punicata sp. nov., collected from Shaanxi Province, northern China, and so far exclusively on Punica granatum (Punicaceae), is described and illustrated, and its preliminary phylogenetic relationships are inferred based on sequence data from the ribosomal ITS-5.8S regions. Fomitiporia punicata belongs to the F. robusta–F. punctata complex, but is characterized by an effused–reflexed to pileate basidioma with triquetrous to ungulate pilei, pale yellowish brown to cinnamon brown pore surface, moderately thick-walled skeletal hyphae in the context, which are partly collapsed on drying, lack of setae, presence of fusoid to subulate cystidioles, and growth on Punica. It also forms a distinct clade within Fomitiporia, and is related to some species from the temperate or Mediterranean zone of the northern hemisphere. An identification key to the species of Fomitiporia recorded in China is provided. Fomitiporia tibetica is transferred to Pseudoinonotus.     

Physiological characteristics of the trunk sap rot pathogen Fomitiporia sp. on the “Sanbu-sugi” cultivar of Cryptomeria japonica

An unidentified Fomitiporia sp. initially identified as Fomitiporia punctata, causes severe trunk sap rot on Cryptomeria japonica cultivar “Sanbu-sugi”. We investigated the physiological characteristics of the mycelia of the causal fungus (F2, F6, and F43), in comparison with F. punctata (Fp) as a reference, in eight different experiments. The three unidentified isolates showed similar tendencies in change in mycelial growth during incubation, optimal growth temperatures (25 °C), optimal pH range (pH 5–6), glucose to yeast extract ratio (45), utilizable carbon sources (amylose, CM-cellulose, and pectin), utilizable nitrogen sources (yeast extract and polypepton), and water potential (?1.7 Mpa).     

Revised concept of Chrysoperla mediterranea (Hölzel), a green lacewing associated with conifers: courtship songs across 2800 kilometres of Europe (Neuroptera: Chrysopidae)

The Holarctic carnea-group of Chrysoperla green lacewings consists of numerous cryptic, sibling species, distinguished principally by vibrational courtship songs. A European member of this species-group, C. mediterranea (Hölzel), was originally diagnosed by its dark green coloration, red band across the front of the head, narrow wings, minute basal dilation of the pretarsus and ecological association with pines. Here we describe its courtship and mating song, which uniquely distinguishes the species from all other members of the carnea-group in Europe. Males and females both produce a single type of song, characterized by a long train of short, closely spaced volleys and nearly constant carrier frequency. Measured song features are nearly invariant across a large geographical range that extends well outside that previously known for C. mediterranea. Principal components analysis confirms the uniqueness of the mediterranea song in comparison with five other sympatric song species of the carnea-group. Populations which show the mediterranea song phenotype do not necessarily retain all of the traditional morphological features of the species, particularly in eastern parts of the range. Specifically, the green coloration may be less intense, the wings broader and the pretarsal basal dilation larger, resulting in significant morphological overlap with other song species. Larval morphology and ecophysiology also fail to distinguish C. mediterranea from other song species. Based on unique song phenotype, however, we conclude that C. mediterranea is a valid biological species, which can also be recognized in museum collections when a suite of physical attributes is used.     

Diversity of saproxylic basidiomycetes in Quercus ilex woodlands of central and insular Greece

The sclerophyllous forests of Quercus ilex (holm oak) possess a unique position in earth’s biosphere, existing only in countries of the Mediterranean basin. However, very little information exists about mushrooms associated with holm oak in east Mediterranean, including Greece, where Q. ilex appears mostly in relict forests and fragmented stands. A pertinent long-term investigation was undertaken in selected Q. ilex dominated habitats of Crete, Andros, Naxos and Ikaria islands (Aegean Archipelago) as well as in mountains of Attica. Specimens of xylotrophic basidiomycetes were collected and identified. As a result, 74 species were recorded; among them Hemimycena cephalotricha, Hyphoderma cremeoalbum, Hyphodontia radula, Irpex litschaueri, Mycena algeriensis, Phanerochaete martelliana, Phloeomana alba and Phlebia unica constitute new records for the Greek mycobiota, while 68 species are reported for the first time on Q. ilex in Greece. Moreover, the presence of Fomitiporia mediterranea on this host was evidenced through ITS sequencing, and comments are made about its relationship to F. punctata and F. pseudopunctata. The importance of findings is discussed in conjunction with their environmental value; five species could be assigned in the “Species of Special Interest” categories, and other five polypores are proposed as suitable indicators for Q. ilex habitat assessment and conservation.     

Isolation,molecular characterization and virome analysis of culturable wood fungal endophytes in esca symptomatic and asymptomatic grapevine plants

Europe is the world largest grape producer, but in recent years, the report of diseases due to infection by grapevine trunk pathogens (GTPs) is becoming one of the main constraints for viticulture. Among grapevine trunk diseases (GTDs), esca syndrome is one of the most complex, characterized by simultaneous infection of several fungi, which leads to important reduction in yield and quality. Previous characterization of fungal isolates associated with esca syndrome leads to the recognition of at least three important players: Phaeocremonium minimum, Phaemoniella chlamydospora and Fomitiporia mediterranea. Here we isolated and characterized molecularly fungal endophytes directly from field wood tissues of plants showing or not esca symptoms. In addition, to better characterize such collection, a deep RNA sequencing (100 M reads in paired-end) to screen for mycovirus presence was performed. Thirty-nine viral genomes were detected, 38 of which were putative new viral species; some of these viruses infected GTPs, including P. minimum and F. mediterranea. In this work, we reported for the first time a curated collection of grapevine fungal endophytes identifying the associated mycoviruses some of which could be employed in future biotechnological exploitation as biological control agents for sustainable plant protection.     

Variation in recombination rates across Vitis species

Recombination rate data are presented for three populations of grape based on framework genetic linkage maps developed with simple-sequence repeat markers. These linkage maps were constructed from different Vitis species and represent three genetic backgrounds. The first population is pure Vitis vinifera, derived from a cross of the European cultivars Riesling and Cabernet Sauvignon. The second is an interspecific cross between two commercially used rootstock cultivars of different North American Vitis species parentage, Ramsey (Vitis champinii) and Riparia Gloire (Vitis riparia). The third population, D8909-15 (Vitis rupestris × (Vitis arizonica/Vitis girdiana form)) × F8909-17 (V. rupestris × (V. arizonica/Vitis candicans form)), is an F1 from two half-sibs. Genome-wide and chromosome-wide recombination rates varied across the three populations and among the six Vitis parents. Global recombination rates in the parents of the third F1 population, with a complex Vitis background, were significantly reduced. In the first and third populations, the recombination rate was significantly greater in the male parent. Specific genome locations with frequent heterogeneity in recombination were identified, suggesting that recombination rates are not equal across the Vitis genome. The identification of regions with suppressed or high recombination will aid grape breeders and geneticists who rely on recombination events to introgress disease resistance genes from the genomes of wild Vitis species, develop fine-scale genetic maps, and clone disease resistance genes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A novel Fomitiporia species associated with esca on grapevine in South Africa

Esca disease of grapevine is a complex trunk disease consisting of several symptoms, one of which, white rot, has been found to be caused by various basidiomycetes within the order Hymenochaetales. During recent surveys of esca-related pathogens in South African vineyards, several unidentified basidiomycetes were isolated from white rot occurring in diseased vines. A new Fomitiporia species, F. capensis, is described based on fruit body morphology and combined internal transcribed spacer (ITS) and large-subunit (LSU) phylogeny, where it forms a clearly delineated and well-supported clade. Morphologically, F. capensis is similar to F. punctata in that both species essentially lack setae. Fomitiporia capensis, F. punctata and F. aethiopica produce similarly sized basidiospores but differ in terms of host range, pore size and, possibly, fruit body shape. Phylogenetically, F. capensis appears to be related to F. tenuis, though morphologically, the species differ significantly in that F. tenuis has smaller pores and smaller basidiospores. Fomitiporia capensis was found to occur widely as vegetative mycelium throughout the Western Cape Province, though fruit bodies were scarce in comparison. A vineyard with fruit bodies was also found in Limpopo in the north east of the country. Fruit bodies were found growing on the underside of the cordon of living vines displaying external symptoms typically associated with esca, or general decline and dieback symptoms together with internal white rot.     

Differences between sympatric populations of <Emphasis Type="Italic">Eotetranychus carpini</Emphasis> collected from <Emphasis Type="Italic">Vitis vinifera</Emphasis> and <Emphasis Type="Italic">Carpinus betulus</Emphasis>: insights from host-switch experiments and molecular data

Eotetranychus carpini (Oudemans) is an important pest of grapevine (Vitis vinifera L.) in southern Europe. This mite is also found on a number of different plants, including Carpinus betulus L., which commonly occurs in stands and hedgerows bordering vineyards, where it may serve as a potential mite reservoir. The economic importance of this pest has motivated a number of studies aimed at investigating whether the mites found on V. vinifera and C. betulus are conspecific. The results obtained to date have been inconclusive. In this study, we used biological and molecular approaches to investigate this issue. First, we conducted host-switch experiments to test the ability of E. carpini to develop on an alternative host plant, using mite populations originally collected on either C. betulus or V. vinifera plants from the same area. Second, we investigated DNA-based differentiation using nucleotide sequences of the ITS1-5.8S-ITS2 region of the ribosomal DNA of individual E. carpini from the populations examined in our host-plant experiments. We also analyzed sequences of individuals collected in other regions (Italy and Slovenia) to estimate species variation. The results from our host-switch experiments suggest the differentiation of mites collected on the two hosts. Mites collected from C. betulus did not survive and reproduce on V. vinifera and vice versa. Our molecular work revealed significant genetic differentiation between the mites collected from the two hosts, but no evidence of genetic variation among specimens collected from the same host species. Our results indicate the existence of host races of E. carpini.     

Historical isolation and Quaternary range expansion of divergent lineages in wild grapevine

Vitis vinifera subsp. silvestris, the wild subspecies of Vitis vinifera L., is a unique and valuable genetic resource for cultivated grapevines. At present, this wild form is rare and is spread from the Southern Atlantic coast of Europe to the Western Himalayas. In the present study, six nuclear microsatellite DNAs were analysed in 301 wild grape samples, using factorial correspondence analysis and the Bayesian model. The aim was to detect the distribution of genetic variability and admixture proportions and thus to identify the main routes of recolonization after the quaternary glaciation, as well as signals of secondary contacts in Europe. The results obtained show that some Caucasus and Italian areas played key roles as refugial areas. Moreover, the data suggest the existence of two main migration routes through central Europe during the post‐glacial or the inter‐glacial periods. The first started from Southern Italy and moved northward, whereas the second originated from refugia possibly located in eastern areas and migrated westwards. This scenario is most likely associated with the population expansion that followed the European Quaternary glaciations. In accordance with this assumption, central Europe is proposed as an admixture confluence of migration routes radiating from separate grapevine refugia. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95 , 611–619.     

Stilbene biosynthesis and gene expression in response to methyl jasmonate and continuous light treatment in Vitis vinifera cv. Malvasia del Lazio and Vitis rupestris Du Lot cell cultures

Vitis rupestris is used as rootstock or to obtain hybrids with Vitis vinifera, due to its resistance to certain pathogens. Its resistance mechanisms are poorly understood, while it is known that stilbene neo‐synthesis is a central defense strategy in V. vinifera. In the present study, the response to methyl jasmonate (MeJa) and light treatment in terms of stilbene biosynthesis and the expression of genes involved in polyphenol biosynthesis was investigated in V. vinifera and V. rupestris cells. The two species exhibited a similar constitutive stilbene content [2.50–2.80 mg g^?1 dry weight (DW)], which greatly increased in response to elicitation (8.97–11.90 mg g^?1 DW). In V. vinifera, continuous light treatment amplified the effect of MeJa, with a stilbene production that had never previously been obtained (26.49 mg g^?1 DW). By contrast, it suppressed the effect of MeJa in V. rupestris. Gene expression was consistent with stilbene production in V. vinifera, whereas discrepancies were recorded in V. rupestris that could be explained by the synthesis of stilbenes that had never before been analyzed in this species.     

Characterization of genome-wide long terminal repeat retrotransposons provide insights into trait evolution of four grapevine species

Grapevine is one of the most economically important crops in the world. Although long terminal repeat (LTR) retrotransposons are thought to have played an important role in plants, its distribution in grapevine is not clear. Here, we identified genome-wide intact LTR retrotransposons in a total of six high-quality grapevine genomes from Vitis vinifera L., Vitis sylvestris C.C. Gmel., Vitis riparia Michx. and Vitis amurensis Rupr. with an average of 2938 per genome. Among them, the Copia superfamily (particularly for Ale) is a major component of the LTR retrotransposon in grapevine. Insertion time and copy number analysis revealed that the expansion of 70% LTR retrotransposons concentrating on approximately 2.5 Ma was able to drive genome size variation. Phylogenetic tree and syntenic analyses showed that most LTR retrotransposons in these genomes formed and evolved after species divergence. Furthermore, the function and expression of genes inserted by LTR retrotransposons in V. vinifera (Pinot noir) and V. riparia were explored. The length and expression of genes related to starch metabolism and quinone synthesis pathway in Pinot noir and environmental adaptation pathway in V. riparia were significantly affected by LTR retrotransposon insertion. The results improve the understanding of LTR retrotransposons in grapevine genomes and provide insights for its potential contribution to grapevine trait evolution.     

Remarks on mixotrophic and autotrophic carbon nutrition of Vitis plantlets cultured in vitro

Two Vitis species were cultured in vitro under photoautrophic (sucrose-free culture medium) and photomixotrophic (sucrose 15 g l^-1) conditions during the period following microcutting rooting (day 34 to day 120). Several parameters were measured at the end of the culture: growth, plant dry weight, carbohydrate uptake from the medium and rates of photosynthesis and dark respiration. The two species behaved very differently. Under photoautotrophic conditions, dark respiration, net photosynthesis and daily CO₂ fixation were higher in Vitis vinifera than in Vitis rupestris. Culture under mixotrophic conditions caused increase in growth, respiration and photosynthesis in Vitis rupestris. In contrast, photosynthesis decreased in Vitis vinifera under the same conditions.     

Use of in vitro method to evaluate some grapevine varieties for tolerance and susceptibility to sodium bicarbonate-induced chlorosis

Nodal shoot segments of four grapevine genotypes well known for their Fe-chlorosis characteristic [Vitis vinifera × Vitis berlandieri Fercal, resistant; V. berlandieri × Vitis rupestris 1103 P, mid-resistant; Solonis (Vitis riparia × V. rupestris × Vitis candicans) × Othello (Vitis labrusca × V. riparia × V. vinifera) 1613 C, susceptible; V. vinifera L. cv. Perlette, resistant] were cultured in vitro. The effects of three levels of iron sodium ethylene-diaminotetraacetate (FeNaEDTA; 9, 18, 36 mg l⁻¹) and three mixtures of iron and 840 mg l⁻¹ NaHCO₃ (sodium bicarbonate) in the Murashige and Skoog (MS) medium supplemented 4.9 μM indole-3-butyric acid (IBA) were compared. We assayed the chlorosis rating of leaves, total chlorophyll of leaves, dry shoot weights of the plantlets, and active and total Fe content of the leaves. The most suitable concentration in determining the reaction of genotypes to iron chlorosis was 9 mg l⁻¹ FeNaEDTA. Bicarbonate addition had negative effects on the iron intake and development of plants. While all genotypes were affected by non-ferrous conditions, Fercal and Perlette were found to be the most resistant genotypes and 1613 C rootstock as the most susceptible. The chlorosis rating of the tested genotypes ranked according to their known degree of tolerance and susceptibility to lime-induced chlorosis. The results of this study showed that the in vitro technique could successfully be used in viticulture to get results in shorter times in the studies, aiming at breeding new rootstocks and varieties suitable to calcareous soil conditions and determining the reactions of existing genotypes to Fe chlorosis.     

Infection of Vitis vinifera (cv Chardonnay) Inflorescences by Colletotrichum acutatum and Greeneria uvicola

Detached Vitis vinifera (cv Chardonnay) inflorescences were inoculated with spore suspensions of either Colletotrichum acutatum or Greeneria uvicola at 25°C, and a combination of light microscopy, scanning electron microscopy and plating out of inoculated flowers on to dichloran rose bengal chloramphenicol agar was used to investigate the time frame for infection. Colletotrichum acutatum infection commenced within 2 h of inoculation, while infection by G. uvicola commenced between 12 and 18 h postinoculation. All parts of the flowers were infected by both fungi.     

Morphological characterization and optimization of conditions for conidial production of Elsinoë ampelina,the causal organism of grapevine anthracnose

Grape anthracnose, which is caused by Elsinoë ampelina, is a disease that negatively affects grape production. This study aimed to investigate the effects of aeration, temperature, light, and preculture period on the formation of E. ampelina conidia and conidial germination and virulence. The colony morphology on potato dextrose agar (PDA) plates was more diverse than that in PDA bottles. The assessment of different culture methods, temperatures, light conditions, and preculture periods revealed that optimal conidial production occurred on 25‐day‐old colonies grown in PDA bottles at 21°C for 24 hr in the dark. The cultures in PDA bottles consistently produced approximately 5.0 × 10⁶ conidia under these conditions. No conidial formation occurred when the cultures were kept at 25°C in the dark. The highest germination rate of E. ampelina was 80% at 25°C after 24 hr, whereas no germination was observed at 17°C after 12 hr. Pathogenicity tests revealed that symptoms of the disease were observed 4 days postinoculation (dpi) on leaves of Vitis vinifera cv. Red Globe. New conidia were observed on the lesions at 8 dpi. This study provides an effective method for the conidial production of E. ampelina that may also be applicable for other Elsinoë fungal species.     

Effects of Thermotherapy Treatments on Systemic Agrobacterium vitis in Dormant Grape Cuttings

Agrobacterium tumefaciens biovar 3 (A. vitis) was eradicated or reduced below the level of detection in dormant cuttings of grape [Vitis vinifera cultivar (cv.) Thompson seedless] and rootstock NAZ3 (V. vinifera × V. rupestris) by hot‐water treatment (exposure to 50°C for 30 min). Strains of A. vitis varied in their sensitivity to heat, but were generally more sensitive than strains of A. tumefaciens biovar 1 or biovar 2. Populations of about 10³ CFU/ml in broth were killed by a 30‐min treatment. Biovar 1 strains were apparently unaffected by 50°C, even when exposed for 30 min. Non‐tumorigenic biovar 1 strains were recovered from hot‐water‐treated cuttings. An assessment was also made of the effect of treatment on growth parameters of cuttings in a field nursery after 9 months. The effect of hot‐water treatment on the vitality and growth of vines varied with different cultivars or rootstocks. The number and length of canes, root dry weight and length and diameter of trunks, increased in most instances. Treatment and time usually did affect bud survival and, in most cases, increased the level of callus formation at the base of the cuttings. Hot‐water treatment may offer a simple, effective, economical and environmentally safe means of eradicating A. vitis from dormant grape cuttings.     

Botrytis cinerea: Activité Antifongique dans les Jeunes Grappes de Vitis vinifera, Varieté Gamay

Botrytis cinerea: fungicidal activity in young grape berries of Vitis vinifera (Gamay) Extracts of young grape berries of Vitis vinifera (Gamay) inhibit the germination of conidia of Botrytis cinerea. This inhibitory activity is present in extracts obtained from clusters at bloom stage and is maintained until a developmental stage close to the maturity. These extracts ruptured the cellular membranes.     

Effect of temperature on lipid,starch and enzymes of starch metabolism in grape,tomato and broad bean leaves

Grapevine (Vitis vinifera cv Cabernet Sauvignon) leaves have previously been shown to accumulate starch at temperatures of ca 18/13° (day/nig