A petunia ABC protein controls strigolactone-dependent symbiotic signalling and branching.

Strigolactones were originally identified as stimulators of the germination of root-parasitic weeds that pose a serious threat to resource-limited agriculture. They are mostly exuded from roots and function as signalling compounds in the initiation of arbuscular mycorrhizae, which are plant-fungus symbionts with a global effect on carbon and phosphate cycling. Recently, strigolactones were established to be phytohormones that regulate plant shoot architecture by inhibiting the outgrowth of axillary buds. Despite their importance, it is not known how strigolactones are transported. ATP-binding cassette (ABC) transporters, however, are known to have functions in phytohormone translocation. Here we show that the Petunia hybrida ABC transporter PDR1 has a key role in regulating the development of arbuscular mycorrhizae and axillary branches, by functioning as a cellular strigolactone exporter. P. hybrida pdr1 mutants are defective in strigolactone exudation from their roots, resulting in reduced symbiotic interactions. Above ground, pdr1 mutants have an enhanced branching phenotype, which is indicative of impaired strigolactone allocation. Overexpression of Petunia axillaris PDR1 in Arabidopsis thaliana results in increased tolerance to high concentrations of a synthetic strigolactone, consistent with increased export of strigolactones from the roots. PDR1 is the first known component in strigolactone transport, providing new opportunities for investigating and manipulating strigolactone-dependent processes.

Globally distributed root endophyte Phialocephala subalpina links pathogenic and saprophytic lifestyles.

BACKGROUND: Whereas an increasing number of pathogenic and mutualistic ascomycetous species were sequenced in the past decade, species showing a seemingly neutral association such as root endophytes received less attention. In the present study, the genome of Phialocephala subalpina, the most frequent species of the Phialocephala fortinii s.l. - Acephala applanata species complex, was sequenced for insight in the genome structure and gene inventory of these wide-spread root endophytes. RESULTS: The genome of P. subalpina was sequenced using Roche/454 GS FLX technology and a whole genome shotgun strategy. The assembly resulted in 205 scaffolds and a genome size of 69.7 Mb. The expanded genome size in P. subalpina was not due to the proliferation of transposable elements or other repeats, as is the case with other ascomycetous genomes. Instead, P. subalpina revealed an expanded gene inventory that includes 20,173 gene models. Comparative genome analysis of P. subalpina with 13 ascomycetes shows that P. subalpina uses a versatile gene inventory including genes specific for pathogens and saprophytes. Moreover, the gene inventory for carbohydrate active enzymes (CAZymes) was expanded including genes involved in degradation of biopolymers, such as pectin, hemicellulose, cellulose and lignin. CONCLUSIONS: The analysis of a globally distributed root endophyte allowed detailed insights in the gene inventory and genome organization of a yet largely neglected group of organisms. We showed that the ubiquitous root endophyte P. subalpina has a broad gene inventory that links pathogenic and saprophytic lifestyles.

MAT--gene structure and mating behavior of Hymenoscyphus fraxineus and Hymenoscyphus albidus.

Recently, different reproductive modes were proposed between the emerging forest pathogen Hymenoscyphus fraxineus and its closely related avirulent sister species, Hymenoscyphus albidus. In the present study, inter- and intraspecific crosses were performed to experimentally assess the reproduction barriers between the two species and to verify H. albidus' putative haploid-selfing reproductive mode. By means of H. fraxineus-specific microsatellite markers, no hybridization was observed in 29 apothecia that emerged from inter-specific crosses, suggesting reproduction barriers are well-established. In a similar experimental setup, we used two newly developed polymorphic H. albidus-specific microsatellites to show that haploid-selfing is H. albidus' only reproductive mode (N=17 apothecia). Further to this, the reproductive modes of both species were investigated under natural conditions. Microsatellite allele-segregation studies of H. fraxineus' single-spore progeny of apothecia (N=31) from field samples suggest that often more than two paternal nuclei are involved in mating. In contrast, analysis of single-spore progeny of field-collected H. albidus apothecia (N=21) confirmed the solely haploid-selfing reproductive mode detected in vitro. Furthermore, we present the complete mating type 1-1 locus of H. fraxineus and report the finding of three additional genes within this region; the as yet unobserved typical mating type gene MAT1-1-1, a DNA polymerase zeta catalytic subunit-like gene and a pre-mRNA-splicing factor SLU7-like gene. The same genes were also detected in the homothallic mating type locus of H. albidus. Further analysis confirmed the expression of all typical mating type genes (MAT1-2-1, MAT1-1-3, MAT1-1-1) in both species. Interestingly, the MAT1-1-3 gene of homothallic H. albidus is expressed despite three stop codons interrupting the coding sequence. Overall, our findings highlight vital differences in the reproduction systems of the two species and suggest that interspecific hybridization is not possible.

Petunia hybrida PDR2 is involved in herbivore defense by controlling steroidal contents in trichomes.

As a first line of defense against insect herbivores many plants store high concentrations of toxic and deterrent secondary metabolites in glandular trichomes. Plant Pleiotropic Drug Resistance (PDR)-type ABC transporters are known secondary metabolite transporters, and several have been implicated in pathogen or herbivore defense. Here, we report on Petunia hybrida PhPDR2 as a major contributor to trichome-related chemical defense. PhPDR2 was found to localize to the plasma membrane and be predominantly expressed in multicellular glandular trichomes of leaves and stems. Down-regulation of PhPDR2 via RNA interference (pdr2) resulted in a markedly higher susceptibility of the transgenic plants to the generalist foliage feeder Spodoptera littoralis. Untargeted screening of pdr2 trichome metabolite contents showed a significant decrease in petuniasterone and petuniolide content, compounds, which had previously been shown to act as potent toxins against various insects. Our findings suggest that PhPDR2 plays a leading role in controlling petuniasterone levels in leaves and trichomes of petunia, thus contributing to herbivory resistance.

Forest health in a changing world.

Forest pathology, the science of forest health and tree diseases, is operating in a rapidly developing environment. Most importantly, global trade and climate change are increasing the threat to forest ecosystems posed by new diseases. Various studies relevant to forest pathology in a changing world are accumulating, thus making it necessary to provide an update of recent literature. In this contribution, we summarize research at the interface between forest pathology and landscape ecology, biogeography, global change science and research on tree endophytes. Regional outbreaks of tree diseases are requiring interdisciplinary collaboration, e.g. between forest pathologists and landscape ecologists. When tree pathogens are widely distributed, the factors determining their broad-scale distribution can be studied using a biogeographic approach. Global change, the combination of climate and land use change, increased pollution, trade and urbanization, as well as invasive species, will influence the effects of forest disturbances such as wildfires, droughts, storms, diseases and insect outbreaks, thus affecting the health and resilience of forest ecosystems worldwide. Tree endophytes can contribute to biological control of infectious diseases, enhance tolerance to environmental stress or behave as opportunistic weak pathogens potentially competing with more harmful ones. New molecular techniques are available for studying the complete tree endobiome under the influence of global change stressors from the landscape to the intercontinental level. Given that exotic tree diseases have both ecologic and economic consequences, we call for increased interdisciplinary collaboration in the coming decades between forest pathologists and researchers studying endophytes with tree geneticists, evolutionary and landscape ecologists, biogeographers, conservation biologists and global change scientists and outline interdisciplinary research gaps.

A case study on the application of spore sampling for the monitoring of macrofungi.

Fungi play a vital role in ecosystem functioning, yet significant knowledge gaps persist in understanding their diversity and distribution leading to uncertainties about their threat status and extinction risk. This is partly owed to the difficulty of monitoring fungi using traditional fruiting body surveys. The present study evaluates airborne environmental DNA (eDNA) sampling as a monitoring tool with a focus on grassland macrofungi. We applied active and passive air sampling methods, complemented by extensive field surveys of waxcap and clavarioid fungi-species groups of high relevance for conservation. Twenty-nine species were recorded during the field surveys, 19 of which were also detectable by ITS2 metabarcoding of the collected samples. An additional 12 species from the studied genera were identified exclusively in air eDNA. We found that the patterns of species detection and read abundance in air samples reflected the abundance and occurrence of fruiting bodies on the field. Dispersal kernels fitted for the three dominant species predicted rapidly decreasing spore concentrations with increasing distance from fruitbodies. Airborne assemblages were dominated by a high diversity of common species, while rare and threatened red-listed species were under-represented, which underscores the difficulty in detecting rare species, not only in conventional surveys. Considering the benefits and drawbacks of air sampling and fruitbody surveys, we conclude that air sampling serves as a cost- and time-efficient tool to characterize local macrofungal communities, providing the potential to facilitate and improve future fungal monitoring efforts.

The Endophytic Mycobiome of European Ash and Sycamore Maple Leaves - Geographic Patterns, Host Specificity and Influence of Ash Dieback.

The European ash (Fraxinus excelsior) is threatened by the introduced ascomycete Hymenoscyphus fraxineus, the causal agent of ash dieback. Endophytic fungi are known to modulate their host's resistance against pathogens. To understand possible consequences of ash dieback on the endophytic mycobiome, F. excelsior leaves were collected in naturally regenerated forests and the fungal communities analyzed by classic culture and Illumina amplicon sequencing using a newly developed and validated fungal-specific primer. Collections were done in the area infested by ash dieback north of the Alps, and in the disease free area on the south side. Sycamore maple (Acer pseudoplatanus) was additionally collected, as well as the flowering ash (F. ornus), which occurs naturally in the south and shows tolerance to ash dieback. Both cultivation and amplicon sequencing revealed characteristic endophytic fungal communities dominated by several strictly host specific Venturia species. On A. pseudoplatanus, a hitherto undescribed Venturia species was identified. Due to its dominance on F. excelsior, V. fraxini is unlikely to go extinct in case of reduced host densities. A majority of species was not strictly host specific and is therefore likely less affected by ash dieback in the future. Still, shifts in community structure and loss of genetic diversity cannot be excluded. The potentially endangered endophyte Hymenoscyphus albidus was rarely found. In addition to host specificity, species with preferences for leaf laminae or petioles were found. We also detected considerable geographical variation between sampling sites and clear differences between the two sides of the Alps for endophytes of F. excelsior, but not A. pseudoplatanus. Since sycamore maple is not affected by an epidemic, this could point toward an influence of ash dieback on ash communities, although firm conclusions are not possible because of host preferences and climatic differences. Furthermore, the mycobiota of F. excelsior trees with or without dieback symptoms were compared, but no clear differences were detected. Besides methodical refinement, our study provides comprehensive data on the ash mycobiome that we expect to be subject to changes caused by an emerging disease of the host tree.

Analysis of the Phialocephala subalpina Transcriptome during Colonization of Its Host Plant Picea abies.

BACKGROUND: Phialocephala subalpina belongs to the Phialocephala fortinii s.l.-Acepphala applanata species complex (PAC) forming one of the major groups belonging to the dark septate endophytes (DSE). Depending on the strain, PAC was shown to form neutral to pathogenic associations with its host plant Picea abies. To understand PACs lifestyle we investigated the effect of presence/absence of Picea abies on the transcriptome of strain 6_70_1. MATERIALS AND METHODS: PAC strain 6_70_1 was grown in liquid Pachlewski media either induced by its host plant Picea abies or without host plant as a control. Mycelia were harvested in a time course (1, 2, 3, 4, 7, 11, 18 days) with and without induction by the host plant and the fungal transcriptome revealed by Illumina sequencing. Differential gene expression analysis over the time course comparing control and treatment at each time point using the 'edgeR glm approach' and a gene enrichment analysis using GO categories were performed. RESULTS: The three main functional groups within differentially expressed genes were 'metabolism', 'transport' and 'cell rescue, defense and virulence'. Additionally, genes especially involved in iron metabolism could be detected by gene set enrichment analysis. CONCLUSION: In conclusion, we found PAC strain 6_70_1 to be metabolically very active during colonization of its host plant Picea abies. A major shift in functional groups over the time course of this experiment could not be observed but GO categories which were found to be enriched showed different emphasis depending in the day post induction.

Effects of endophytic fungi on the ash dieback pathogen.

While Hymenoscyphus fraxineus causes dieback of the European ash (Fraxinus excelsior), flowering ash (F. ornus) appears resistant to the pathogen. To date, contributions of endophytic fungi to host resistance are unknown. The following hypotheses were tested: (i) endophytic fungi enhance the resistance of F. excelsior to the pathogen; (ii) resistance of F. ornus relies on its community of endophytic fungi. Two experiments were performed. (i) The effect of exudates of ash endophytes on the germination rate of H. fraxineus ascospores was studied in vitro Isolates of abundant Fraxinus leaf endophytes, such as Venturia fraxini, Paraconiothyrium sp., Boeremia exigua, Kretzschmaria deusta and Neofabraea alba inhibited ascospore germination. (ii) Ash seedlings inoculated in a climate chamber, with fungi sporulating on the previous year's leaf litter, were exposed to natural infections by the pathogen present in the forest. Non-inoculated seedlings were used as controls. Venturia spp. dominated the inoculated endophyte 'communities'. Subsequent exposure to H. fraxineus led to infection of F. excelsior leaves by the pathogen, but no differences in health status between pre-inoculated and non-inoculated seedlings were detected. Fraxinus ornus leaves experienced a low infection rate, independent of their colonization by endophytic fungi. These results did not support either hypothesis.