Sweets for the foe - effects of nonstructural carbohydrates on the susceptibility of Quercus robur against Phytophthora quercina.

The root-rot pathogen Phytophthora quercina is a key determinant of oak decline in Europe. The susceptibility of pedunculate oak (Quercus robur) to this pathogen has been hypothesized to depend on the carbon availability in roots as an essential resource for defense. Microcuttings of Q. robur undergo an alternating rhythm of root and shoot growth. Inoculation of mycorrhizal (Piloderma croceum) and nonmycorrhizal oak roots with P. quercina was performed during both growth phases, that is, root flush (RF) and shoot flush (SF). Photosynthetic and morphological responses as well as concentrations of nonstructural carbohydrates (NSC) were analyzed. Infection success was quantified by the presence of pathogen DNA in roots. Concentrations of NSC in roots depended on the alternating root/shoot growth rhythm, being high and low during RF and SF, respectively. Infection success was high during RF and low during SF, resulting in a significantly positive correlation between pathogen DNA and NSC concentration in roots, contrary to the hypothesis. The alternating growth of roots and shoots plays a crucial role for the susceptibility of lateral roots to the pathogen. NSC availability in oak roots has to be considered as a benchmark for susceptibility rather than resistance against P. quercina.

Multivariate analysis of physiological parameters reveals a consistent O3 response pattern in leaves of adult European beech (Fagus sylvatica).

• Increasing atmospheric concentrations of phytotoxic ozone (O(3) ) can constrain growth and carbon sink strength of forest trees, potentially exacerbating global radiative forcing. Despite progress in the conceptual understanding of the impact of O(3) on plants, it is still difficult to detect response patterns at the leaf level. • Here, we employed principal component analysis (PCA) to analyse a database containing physiological leaf-level parameters of 60-yr-old Fagus sylvatica (European beech) trees. Data were collected over two climatically contrasting years under ambient and twice-ambient O(3) regimes in a free-air forest environment. • The first principal component (PC1) of the PCA was consistently responsive to O(3) and crown position within the trees over both years. Only a few of the original parameters showed an O(3) effect. PC1 was related to parameters indicative of oxidative stress signalling and changes in carbohydrate metabolism. PC1 correlated with cumulative O(3) uptake over preceding days. • PC1 represents an O(3) -responsive multivariate pattern detectable in the absence of consistently measurable O(3) effects on individual leaf-level parameters. An underlying effect of O(3) on physiological processes is indicated, providing experimental confirmation of theoretical O(3) response patterns suggested previously.

Assessment of functional and structural changes of soil fungal and oomycete communities in holm oak declined dehesas through metabarcoding analysis.

Forest decline is nowadays a major challenge for ecosystem sustainability. Dehesas, which consists of savannah-like mediterranean ecosystems, are threatened by the holm oak decline in the south-west of Iberian Peninsula. Phytophthora cinnamomi is considered the main agent of holm oak root rot, but little is known about the relationship between diversity of soilborne microbial community and the decline syndrome of holm oak. It would be hypothesized that the changes in the structure and functionality of the soil microbiome might influence tree health status through changes in richness and diversity of beneficial organisms such as mycorrhizal species, or fungal plant pathogens such as Fusarium spp. or Alternaria spp. Total DNA of soil samples from declined oak dehesas was extracted and analyzed through metabarcoding techniques, to evaluate the specific composition and diversity of the fungal and oomycete communities and their relationship with the disease symptoms. The fungal community included a wide range of pathogens and abundance of ectomycorrhizal key taxa related with low defoliation degree. Phytophthora cinnamomi and Pythium spiculum did not appear among the most abundant oomycetes, nor were they related directly to defoliation levels. Moreover, a particular taxon belonging to the genus Trichoderma was strongly correlated with the scarcity of pathogenic Phytophthora spp. The diversity and composition of fungal and oomycete communities were related to the severity of the decline symptoms. The metabarcoding study of microbiome represents a powerful tool to develop biocontrol strategies for the management of the holm oak root rot.

Photosynthetic and leaf water potential responses of Alnus glutinosa saplings to stem-base inoculaton with Phytophthora alni subsp. alni.

Three-year-old Alnus glutinosa (L.) Gaertn. (alder) saplings were single or double inoculated at the stem base with Phytophthora alni subsp. alni Brasier & S.A. Kirk under natural climatic conditions. Lesion formation on the bark showed a biphasic pattern of development, with extension occurring at a moderate rate in spring, and more rapidly during late summer. However, large variability was encountered in pathogen development within the population of infected saplings, ranging from high susceptibility to almost complete resistance. Infection resulted in severe growth retardation, and death within two years of inoculation in 75% of the saplings. During disease development, rates of transpiration and CO(2) uptake were significantly reduced. Consequently, minimum leaf water potentials were less negative in infected saplings than in control saplings. Surviving saplings matched control trees in photosynthetic capacity, transpiration rate and water potential during the second year of infection. Leaf starch concentration of infected saplings was significantly higher than in control saplings, possibly indicating that the destruction of bark tissue by the pathogen impaired phloem transport from leaves to roots.

Quantification of mRNAs and housekeeping gene selection for quantitative real-time RT-PCR normalization in European beech (Fagus sylvatica L.) during abiotic and biotic stress.

Analyses of different plant stressors are often based on gene expression studies. Quantitative real-time RT-PCR (qRT-PCR) is the most sensitive method for the detection of low abundance transcripts. However, a critical point to note is the selection of housekeeping genes as an internal control. Many so-called 'housekeeping genes' are often affected by different stress factors and may not be suitable for use as an internal reference. We tested six housekeeping genes of European beech by qRT-PCR using the Sybr Green PCR kit. Specific primers were designed for 18S rRNA, actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH1, GAPDH2), a-tubulin, and ubiquitin-like protein. Beech saplings were treated with increased concentrations of either ozone or CO2. In parallel, the expression of these genes was analyzed upon pathogen infection with Phytophthora citricola. To test the applicability of these genes as internal controls under realistic outdoor conditions, sun and shade leaves of 60-year-old trees were used for comparison. The regulation of all genes was tested using a linear mixed-effect model of the R-system. Results from independent experiments showed that the only gene not affected by any treatment was actin. The expression of the other housekeeping genes varied more or less with the degree of stress applied. These results highlight the importance of undergoing an individual selection of internal control genes for different experimental conditions.

Knowledge and use of HIV pre-exposure prophylaxis among men who have sex with men in Berlin - A multicentre, cross-sectional survey.

BACKGROUND: HIV pre-exposure prophylaxis (PrEP) has likely contributed to large decreases in HIV incidence among men who have sex with men (MSM) in several major cities. Berlin has seen a smaller decline, and affordable PrEP has been accessible through formal channels in Germany only since autumn 2017. We aimed to investigate knowledge and use of PrEP among MSM in Berlin, and factors predictive of a desire to use PrEP and history of PrEP use. METHODS: Multicentre, paper-based, self-administered survey of adult MSM whose HIV status was negative or unknown at time of participation. Data were collected from 1 October 2017 to 2 April 2018. RESULTS: 473 of 875 questionnaires were returned (response rate 54.1%; mean age 37.4 years, range 18-79). 90.0% of participants were aware of PrEP and, of these, 48.2% felt well informed about it. Among the 17.2% of participants reporting PrEP use, 59.3% indicated obtaining some or all of it from informal sources. 23.7% of those with no history of PrEP use reported having condomless anal intercourse (CAI) with two or more partners over the past six months. Worries about side effects, cost, not having a doctor who prescribes it, and a lack of information were the most frequently reported barriers to PrEP use. A desire to use PrEP and history of PrEP use were associated in our multivariable model with having multiple CAI partners. A history of PrEP use was associated with having a university degree, one or two parents born outside Germany, or friends living with HIV. CONCLUSIONS: We found high awareness of PrEP among MSM in Berlin, but also a strong need for more education on its pros, cons and proper use. The frequency of informal PrEP use was also high, raising urgent individual and public health concerns. Policy makers need to consider recent calls to improve access to PrEP and PrEP education through regular health services.

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.

Molecular profiling of the Phytophthora plurivora secretome: a step towards understanding the cross-talk between plant pathogenic oomycetes and their hosts.

The understanding of molecular mechanisms underlying host-pathogen interactions in plant diseases is of crucial importance to gain insights on different virulence strategies of pathogens and unravel their role in plant immunity. Among plant pathogens, Phytophthora species are eliciting a growing interest for their considerable economical and environmental impact. Plant infection by Phytophthora phytopathogens is a complex process coordinated by a plethora of extracellular signals secreted by both host plants and pathogens. The characterization of the repertoire of effectors secreted by oomycetes has become an active area of research for deciphering molecular mechanisms responsible for host plants colonization and infection. Putative secreted proteins by Phytophthora species have been catalogued by applying high-throughput genome-based strategies and bioinformatic approaches. However, a comprehensive analysis of the effective secretome profile of Phytophthora is still lacking. Here, we report the first large-scale profiling of P. plurivora secretome using a shotgun LC-MS/MS strategy. To gain insight on the molecular signals underlying the cross-talk between plant pathogenic oomycetes and their host plants, we also investigate the quantitative changes of secreted protein following interaction of P. plurivora with the root exudate of Fagus sylvatica which is highly susceptible to the root pathogen. We show that besides known effectors, the expression and/or secretion levels of cell-wall-degrading enzymes were altered following the interaction with the host plant root exudate. In addition, a characterization of the F. sylvatica root exudate was performed by NMR and amino acid analysis, allowing the identification of the main released low-molecular weight components, including organic acids and free amino acids. This study provides important insights for deciphering the extracellular network involved in the highly susceptible P. plurivora-F. sylvatica interaction.

Seasonal changes of plasma membrane H(+)-ATPase and endogenous ion current during cambial growth in poplar plants.

The plasma membrane H(+)-ATPase (PM H(+)-ATPase), potassium ions, and endogenous ion currents might play a fundamental role in the physiology of cambial growth. Seasonal changes of these parameters were studied in twigs of Populus nigra and Populus trichocarpa. Monoclonal and polyclonal antibodies against the PM H(+)-ATPase, x-ray analysis for K(+) localization and a vibrating electrode for measurement of endogenous ion currents were used as probes. In dormant plants during autumn and winter, only a slight immunoreactivity against the PM H(+)-ATPase was found in cross sections and tissue homogenates, K(+) was distributed evenly, and the density of endogenous current was low. In spring during cambial growth, strong immunoreactivity against a PM H(+)-ATPase was observed in cambial cells and expanding xylem cells using the monoclonal antibody 46 E5 B11 F6 for fluorescence microscopy and transmission electron microscopy. At the same time, K(+) accumulated in cells of the cambial region, and strong endogenous current was measured in the cambial and immature xylem zone. Addition of auxin to dormant twigs induced the formation of this PM H(+)-ATPase in the dormant cambial region within a few days and an increase in density of endogenous current in shoot cuttings within a few hours. The increase in PM H(+)-ATPase abundance and in current density by auxin indicates that auxin mediates a rise in number and activity of an H(+)-ATPase in the plasma membrane of cambial cells and their derivatives. This PM H(+)-ATPase generates the necessary H(+)-gradient (proton-motive force) for the uptake of K(+) and nutrients into cambial and expanding xylem cells.

Phytophthora pseudosyringae sp. nov., a new species causing root and collar rot of deciduous tree species in Europe.

In several studies of oak decline in Europe, a semi-papillate homothallic Phytophthora taxon was consistently isolated, together with other Phytophthora species, from rhizosphere soil samples. It was also found associated with necrotic fine roots and stem necroses of Fagus sylvatica and Alnus glutinosa. Due to morphological and physiological similarities, the semi-papillate isolates were previously identified as P. syringae by various authors. The morphology, physiology and pathogenicity against fine roots of Quercus robur, Q. petraea and F. sylvatica, bark of A. glutinosa, leaves of Ilex aquifolium and apple fruits of this Phytophthora species are described and compared with those of related and similar Phytophthora species, namely P. ilicis, P. psychrophila, P. quercina, P. citricola and P. syringae. The phylogenetic placement on the basis of ITS and mtDNA sequence data was also examined. Isolates of this taxon produce colonies with stellate to rosaceous growth patterns and limited aerial mycelium on various agar media. Antheridia are predominantly paragynous. In water culture catenulate hyphal swellings and semi-papillate caducous sporangia, that are usually limoniform, ellipsoid or ovoid, are formed abundandly, mostly in lax or dense sympodia. This taxon is a moderately slow growing, low temperature species with optimum and maximum temperatures around 20 and 25 degrees C, respectively. Tested isolates are moderately aggressive to fine roots of oaks and beech, highly aggressive to holly leaves and apple fruits, and slightly pathogenic to alder bark. Thirteen tested isolates had an identical and distinct ITS sequence which was more similar to that of P. ilicis and P. psychrophila than any other known taxa. On the basis of their unique combination of morphological characters, colony growth patterns, cardinal temperatures for growth, growth rates, pathogenicity to oaks, beech, alder, apple and holly, their host range, and ITS and mtDNA sequences the semi-papillate caducous Phytophthora isolates from oaks, beech and alder are clearly separated from related and similar Phytophthora spp., and described as a new species, P. pseudosyringae sp. nov.