Effects of the nematode Litylenchus crenatae subsp. mccannii and beech leaf disease on leaf fungal and bacterial communities on Fagus grandifolia (American beech).

Beech leaf disease (BLD) is a newly emerging disease in North America that affects American beech (Fagus grandifolia). It is increasingly recognized that BLD is caused by a subspecies of the anguinid nematode Litylenchus crenatae subsp. mccannii (hereafter L. crenatae), which is likely native to East Asia. How nematode infestation of leaves affects the leaf microbiome and whether changes in the microbiome could contribute to BLD symptoms remain uncertain. In this study, we examined bacterial and fungal communities associated with the leaves of F. grandifolia across nine sites in Ohio and Pennsylvania that were either symptomatic or asymptomatic for BLD and used qPCR to measure relative nematode infestation levels. We found significantly higher levels of infestation at sites visibly symptomatic for BLD. Low levels of nematode infestation were also observed at asymptomatic sites, which suggests that nematodes can be present without visible symptoms evident. Bacterial and fungal communities were significantly affected by sampling site and symptomology, but only fungal communities were affected by nematode presence alone. We found many significant indicators of both bacteria and fungi related to symptoms of BLD, with taxa generally occurring in both asymptomatic and symptomatic leaves, suggesting that microbes are not responsible for BLD but could act as opportunistic pathogens. Of particular interest was the fungal genus Erysiphe, which is common in the Fagaceae and is reported to overwinter in buds-a strategy consistent with L. crenatae. The specific role microbes play in opportunistic infection of leaves affected by L. crenatae will require additional study. IMPORTANCE: Beech leaf disease (BLD) is an emerging threat to American beech (Fagus grandifolia) and has spread quickly throughout the northeastern United States and into southern Canada. This disease leads to disfigurement of leaves and is marked by characteristic dark, interveinal banding, followed by leaf curling and drop in more advanced stages. BLD tends to especially affect understory leaves, which can lead to substantial thinning of the forest understory where F. grandifolia is a dominant tree species. Understanding the cause of BLD is necessary to employ management strategies that protect F. grandifolia and the forests where it is a foundation tree species. Current research has confirmed that the foliar nematode Litylenchus crenatae subsp. mccannii is required for BLD, but whether other organisms are involved is currently unknown. Here, we present a study that investigated leaf-associated fungi and bacteria of F. grandifolia to understand more about how microorganisms may contribute to BLD.

Beech tree masting explains the inter-annual variation in the fall and spring peaks of Ixodes ricinus ticks with different time lags.

BACKGROUND: The tick Ixodes ricinus is an important vector of tick-borne diseases including Lyme borreliosis. In continental Europe, the nymphal stage of I. ricinus often has a bimodal phenology with a large spring peak and a smaller fall peak. There is consensus about the origin of the spring nymphal peak, but there are two alternative hypotheses for the fall nymphal peak. In the direct development hypothesis, larvae quest as nymphs in the fall of the same year that they obtained their larval blood meal. In the developmental diapause hypothesis, larvae overwinter in the engorged state and quest as nymphs one year after they obtained their larval blood meal. These two hypotheses make different predictions about the time lags that separate the larval blood meal and the density of questing nymphs (DON) in the spring and fall. METHODS: Inter-annual variation in seed production (masting) by deciduous trees is a time-lagged index for the density of vertebrate hosts (e.g., rodents) which provide blood meals for larval ticks. We used a long-term data set on the masting of the European beech tree and a 15-year study on the DON at 4 different elevation sites in western Switzerland to differentiate between the two alternative hypotheses for the origin of the fall nymphal peak. RESULTS: Questing I. ricinus nymphs had a bimodal phenology at the three lower elevation sites, but a unimodal phenology at the top elevation site. At the lower elevation sites, the DON in the fall was strongly correlated with the DON in the spring of the following year. The inter-annual variation in the densities of I. ricinus nymphs in the fall and spring was best explained by a 1-year versus a 2-year time lag with the beech tree masting index. Fall nymphs had higher fat content than spring nymphs indicating that they were younger. All these observations are consistent with the direct development hypothesis for the fall peak of I. ricinus nymphs at our study site. Our study provides new insight into the complex bimodal phenology of this important disease vector. CONCLUSIONS: Public health officials in Europe should be aware that following a strong mast year, the DON will increase 1 year later in the fall and 2 years later in the spring. Studies of I. ricinus populations with a bimodal phenology should consider that the spring and fall peak in the same calendar year represent different generations of ticks.

Identification of a cyanidin-3-O-ß-galactoside in gall tissue induced by midges on Japanese beech (Fagus crenata Blume).

Insect gall structures have many characteristic forms and colors, which are distinguishable from host plants. In this study, we identified an anthocyanin from red color insect galls and revealed that the anthocyanin biosynthesis of plants was induced by the gall extracts. The galling insects presumably regulate the anthocyanin biosynthesis of host plants to protect their larvae from environmental stresses.

Metatranscriptomics reveals unsuspected protistan diversity in leaf litter across temperate beech forests, with Amoebozoa the dominating lineage.

Forest litter harbors complex networks of microorganisms whose major components are bacteria, fungi and protists. Protists, being highly selective consumers of bacteria and fungi could influence decomposition processes by shifting competitive microbial interactions. We investigated the eukaryotic diversity from 18 samples of one-year beech (Fagus sylvatica) leaf litter by RNA-based high-throughput sequencing of the small-subunit ribosomal RNA gene. By applying a metatranscriptomics approach, we avoided biases inherent to PCR-based methods, and could therefore focus on elusive protistan groups. We obtained 14 589 eukaryotic assembled sequences (contigs) representing 2223 unique taxa. Fungi dominated the eukaryotic assemblage, followed by an equal proportion of protists and plants. Among protists, the phylum Amoebozoa clearly dominated, representing more than twice the proportion of Alveolata (mostly ciliates) and Rhizaria (mostly Cercozoa), which are often retrieved as the dominant protistan groups in soils, revealing potential primer biases. By assigning functional traits to protists, we could assess that the proportion of free-living and heterotrophs was much higher than that of parasites and autotrophs, opening the way to a better understanding of the role played by the protistan communities and how biodiversity interacts with decomposition processes.

Draft Genome Sequence for the Tree Pathogen Phytophthora plurivora.

Species from the genus Phytophthora are well represented among organisms causing serious diseases on trees. Phytophthora plurivora has been implicated in long-term decline of woodland trees across Europe. Here we present a draft genome sequence of P. plurivora, originally isolated from diseased European beech (Fagus sylvatica) in Malmö, Sweden. When compared with other sequenced Phytophthora species, the P. plurivora genome assembly is relatively compact, spanning 41 Mb. This is organized in 1,919 contigs and 1,898 scaffolds, encompassing 11,741 predicted genes, and has a repeat content of approximately 15%. Comparison of allele frequencies revealed evidence for tetraploidy in the sequenced isolate. As in other sequenced Phytophthora species, P. plurivora possesses genes for pathogenicity-associated RXLR and Crinkle and Necrosis effectors, predominantly located in gene-sparse genomic regions. Comparison of the P. plurivora RXLR effectors with orthologs in other sequenced species in the same clade (Phytophthora multivora and Phytophthora capsici) revealed that the orthologs were likely to be under neutral or purifying selection.

Three new Leptographium spp. (Ophiostomatales) infecting hardwood trees in Norway and Poland.

Species of Leptographium are characterized by mononematous or synnematous conidiophores and are commonly associated with different arthropods. Some of them also produce a sexual state characterised by globose ascomata with elongated necks. Compared to investigations on coniferous trees, the occurrence of Leptographium species on hardwood trees has been poorly studied in Europe. During a survey of ophiostomatoid fungi on various hardwood tree species in Norway and Poland, three unusual species, which fit in the broader morphological description of Leptographium spp., were found in association with Trypodendron domesticum, Trypodendron signatum and Dryocoetes alni, and from wounds on a variety of hardwoods. Phylogenetic analyses of sequence data for six different loci (ITS1-5.8 S-ITS2, ITS2-LSU, ACT, ß-tubulin, CAL, and TEF-1α) showed that these Leptographium species are phylogenetically closely related to the species of the Grosmannia olivacea complex. The first species forms a well-supported lineage that includes Ophiostoma brevicolle, while the two other new taxa resided in a separate lineage; possibly affiliated with Grosmannia francke-grosmanniae. All the new species produce perithecia with necks terminating in ostiolar hyphae and orange-section shaped ascospores with cucullate, gelatinous sheaths. These species also produce dark olivaceous mononematous asexual states in culture. In addition, two of the newly described species have a second type of conidiophore with a short and non-pigmented stipe. The new Leptographium species can be easily distinguished from each other by their appearance and growth in culture. Based on novel morphological characters and distinct DNA sequences, these fungi were recognised as new taxa for which the names Leptographium tardum sp. nov., Leptographium vulnerum sp. nov., and Leptographium flavum sp. nov. are provided.

Descriptions of two new notodontid species from the relic Fagus forests in northeastern Taiwan (Lepidoptera, Notodontidae).

Two new Fagus-feeding notodontids, i.e. Syntypistis taipingshanensis Wu & Hsu sp. n. and Pheosiopsis seni Wu & Hsu sp. n. are recently discovered from the relic Fagus forests in northeastern Taiwan. Based on the genitalia structures, the closely relatives of two new species are S. melana Wu & Fang, 2003 and P. albalienata Kishida & Kobayashi, 2005, respectively, both occurring in the Fagus forests of Southern China.

Mechanisms Driving Galling Success in a Fragmented Landscape: Synergy of Habitat and Top-Down Factors along Temperate Forest Edges.

Edge effects play key roles in the anthropogenic transformation of forested ecosystems and their biota, and are therefore a prime field of contemporary fragmentation research. We present the first empirical study to address edge effects on the population level of a widespread galling herbivore in a temperate deciduous forest. By analyzing edge effects on abundance and trophic interactions of beech gall midge (Mikiola fagi Htg.), we found 30% higher gall abundance in the edge habitat as well as lower mortality rates due to decreased top-down control, especially by parasitoids. Two GLM models with similar explanatory power (58%) identified habitat specific traits (such as canopy closure and altitude) and parasitism as the best predictors of gall abundance. Further analyses revealed a crucial influence of light exposure (46%) on top-down control by the parasitoid complex. Guided by a conceptual framework synthesizing the key factors driving gall density, we conclude that forest edge proliferation of M. fagi is due to a complex interplay of abiotic changes and trophic control mechanisms. Most prominently, it is caused by the microclimatic regime in forest edges, acting alone or in synergistic concert with top-down pressure by parasitoids. Contrary to the prevailing notion that specialists are edge-sensitive, this turns M. fagi into a winner species in fragmented temperate beech forests. In view of the increasing proportion of edge habitats and the documented benefits from edge microclimate, we call for investigations exploring the pest status of this galling insect and the modulators of its biological control.

Tanzawana flavomaculata (Hymenoptera, Ichneumonidae, Ctenopelmatinae), a new genus and species of parasitoid of Fagineura crenativora (Tenthredinidae, Nematinae), a serious pest of beech tree.

We describe a new genus, and a new species, of parasitoid--Tanzawana flavomaculata Watanabe & Kasparyan (Hymenoptera: Ichneumonidae: Ctenopelmatinae)--based on material collected in Honshu, Japan. As T. flavomaculata is found on Fagineura crenativora Vikberg & Zinovjev, 2000 (Hymenoptera: Tenthredinidae), a serious pest of beech tree, this parasitoid is an important natural enemy of F. crenativora that can be used for the biological control of this pest.

First insight into dead wood protistan diversity: a molecular sampling of bright-spored Myxomycetes (Amoebozoa, slime-moulds) in decaying beech logs.

Decaying wood hosts a large diversity of seldom investigated protists. Environmental sequencing offers novel insights into communities, but has rarely been applied to saproxylic protists. We investigated the diversity of bright-spored wood-inhabiting Myxomycetes by environmental sequencing. Myxomycetes have a complex life cycle culminating in the formation of mainly macroscopic fruiting bodies, highly variable in shape and colour that are often found on decaying logs. Our hypothesis was that diversity of bright-spored Myxomycetes would increase with decay. DNA was extracted from wood chips collected from 17 beech logs of varying decay stages from the Hainich-Dün region in Central Germany. We obtained 260 partial small subunit ribosomal RNA gene sequences of bright-spored Myxomycetes that were assembled into 29 OTUs, of which 65% were less than 98% similar to those in the existing database. The OTU richness revealed by molecular analysis surpassed that of a parallel inventory of fruiting bodies. We tested several environmental variables and identified pH, rather than decay stage, as the main structuring factor of myxomycete distribution.

A technique to screen American beech for resistance to the beech scale insect (Cryptococcus fagisuga Lind.).

Beech bark disease (BBD) results in high levels of initial mortality, leaving behind survivor trees that are greatly weakened and deformed. The disease is initiated by feeding activities of the invasive beech scale insect, Cryptococcus fagisuga, which creates entry points for infection by one of the Neonectria species of fungus. Without scale infestation, there is little opportunity for fungal infection. Using scale eggs to artificially infest healthy trees in heavily BBD impacted stands demonstrated that these trees were resistant to the scale insect portion of the disease complex(1). Here we present a protocol that we have developed, based on the artificial infestation technique by Houston(2), which can be used to screen for scale-resistant trees in the field and in smaller potted seedlings and grafts. The identification of scale-resistant trees is an important component of management of BBD through tree improvement programs and silvicultural manipulation.

Phosphite protects Fagus sylvatica seedlings towards Phytophthora plurivora via local toxicity, priming and facilitation of pathogen recognition.

Phytophthora plurivora causes severe damage on Fagus sylvatica and is responsible for the extensive decline of European Beech throughout Europe. Unfortunately, no effective treatment against this disease is available. Phosphite (Phi) is known to protect plants against Phytophthora species; however, its mode of action towards P. plurivora is still unknown. To discover the effect of Phi on root infection, leaves were sprayed with Phi and roots were subsequently inoculated with P. plurivora zoospores. Seedling physiology, defense responses, colonization of root tissue by the pathogen and mortality were monitored. Additionally the Phi concentration in roots was quantified. Finally, the effect of Phi on mycelial growth and zoospore formation was recorded. Phi treatment was remarkably efficient in protecting beech against P. plurivora; all Phi treated plants survived infection. Phi treated and infected seedlings showed a strong up-regulation of several defense genes in jasmonate, salicylic acid and ethylene pathways. Moreover, all physiological parameters measured were comparable to control plants. The local Phi concentration detected in roots was high enough to inhibit pathogen growth. Phi treatment alone did not harm seedling physiology or induce defense responses. The up-regulation of defense genes could be explained either by priming or by facilitation of pathogen recognition of the host.

Tree litter and forest understorey vegetation: a conceptual framework to understand the effects of tree litter on a perennial geophyte, Anemone nemorosa.

BACKGROUND AND AIMS: Litter is a key factor in structuring plant populations, through positive or negative interactions. The litter layer forms a mechanical barrier that is often strongly selective against individuals lacking hypocotyle plasticity. Litter composition also interacts with plant growth by providing beneficial nutrients or, inversely, by allowing harmful allelopathic leaching. As conspicuous litter fall accumulation is often observed under deciduous forests, interactions between tree litter and understorey plant populations are worthy of study. METHODS: In a 1-year ex-situ experiment, the effects of tree litter on the growth of Anemone nemorosa, a small perennial forest geophyte, were investigated. Three 'litter quantity' treatments were defined, representative of forest floor litter (199, 356·5 and 514 g m(-2)), which were crossed with five 'litter composition' treatments (Quercus petraea, Fagus sylvatica, Carpinus betulus, Q. petraea + F. sylvatica and Q. petraea + C. betulus), plus a no-litter control. Path analysis was then used to investigate the pathways linking litter characteristics and components of adult plant growth. KEY RESULTS: As expected, the heavier the litter, the longer the petiole; rhizome growth, however, was not depreciated by the litter-induced petiole lengthening. Both rhizome mass increment and number of initiated buds marginally increased with the amount of litter. Rhizome mass increment was in fact determined primarily by leaf area and leaf life span, neither of which was unequivocally correlated with any litter characteristics. However, the presence of litter significantly increased leafing success: following a late frost event, control rhizomes growing in the absence of litter experienced higher leaf mortality before leaf unfolding. CONCLUSIONS: The study questions the role of litter as a physical or chemical barrier to ground vegetation; to better understand this role, there is a need for ex-situ, longer-term experiments coupled with in-situ observations in the forest.

Changes in susceptibility of beech (Fagus sylvatica) seedlings towards Phytophthora citricola under the influence of elevated atmospheric CO2 and nitrogen fertilization.

The growth-differentiation balance hypothesis (GDBH) predicts changes in susceptibility of plants against herbivores with changing resource availability. In the presented study we tested the validity of the GDBH for trees infected with a root pathogen. For this purpose Fagus sylvatica seedlings grown under different atmospheric CO(2)- and soil nitrogen regimes were infected with the root pathogen Phytophthora citricola. High nitrogen supply increased total biomass of beech regardless of the CO(2)-treatment, whereas elevated CO(2) enhanced biomass only in the high nitrogen treatment. The responses of beech under the different growing regimes to the Phytophthora root infection were not in line with the predictions of the GDBH. Enhanced susceptibility of beech against P. citricola was found in seedlings grown under elevated CO(2) and low nitrogen supply. Fifteen months after inoculation these plants were characterized by enhanced water use efficiency, by altered root-shoot ratios, and by enhanced specific root tip densities.

Comparative proteomic analysis of responses to pathogen infection and wounding in Fagus sylvatica.

Defense responses of Fagus sylvatica seedlings elicited by infection with the root pathogen Phytophthora citricola and root or leaf wounding were compared at local and systemic levels in differential display experiments using two-dimensional gel electrophoresis followed by homology-driven mass spectrometric identification of proteins. A total of 68 protein spots were identified representing 51 protein functions related to protein synthesis and processing, energy, primary and secondary metabolism, as well as signal transduction, stress and defense. Changes in the abundance of root and leaf proteins partly overlapped between plant responses to the different stressors. The response to pathogen infection was rather late, weak and unspecific and accompanied by adjustments of the energy and primary metabolism which suggested either a lack of recognition or a suppression of host's defense reaction by the invading pathogen. The response to wounding involved changes in the basal metabolism as well as activation of defense mechanisms. Both types of changes were largely specific to the wounded organ. Similarities between the defense mechanisms activated by root infection and root wounding were also observed.

Establishment success of sooty beech scale insects, Ultracoelostoma sp., on different host tree species in New Zealand.

The sooty beech scale insect (Ultracoelostoma sp.) (Hemiptera: Margarodidae) exhibits a highly patchy distribution at local and regional scales. A major factor driving this common distributional phenomenon in other phloem-feeding insects is aggregation and local adaptation. The aim of this study was to determine if Ultracoelostoma was locally adapted to its natal host trees, by contrasting the establishment rates of first instar "crawlers" in reciprocal transfers to natal versus novel hosts. Although there are two closely-related species of sooty beech scale insect, the morphological characters of crawlers in this study were intermediate between those of U. assimile and U. brittini. However, all of the voucher specimens examined had consistent morphology, indicating that they belong to one species which we refer to as Ultracoelostoma sp. Reciprocal transfers of crawlers were carried out between individual red beech (Nothofagus fusca), as well as between mountain beech (N. solandri) and red beech trees, to ascertain if insects had become locally adapted to their individual host tree or to host species. In total, 480 crawlers were placed in enclosures on their natal and novel host trees, of which only 32 (6.7 %) became established. No evidence for local adaptation, either to individual host trees or to host tree species, was found. There was also no difference in crawler establishment between natal and novel hosts. However, crawlers originating from mountain beech trees had significantly higher establishment rates on both natal mountain beech and novel red beech hosts, than did crawlers originating from red beech trees. The superior ability of mountain beech crawlers to become established, even on novel red beech trees, suggests that scale insects on mountain beech trees have higher individual fitness (possibly due to maternal effects mediated by differences in host nutritional quality, defensive compounds or growth rate). This increased fitness may result in crawlers being better provisioned to search for appropriate establishment sites. The results of this study indicate that beech scale insects perform better on mountain beech at this site, although crawlers did not preferentially establish on mountain beech.