High-resolution melting curve analysis: A detection assay for Ceratocystis eucalypticola and C. manginecans in infected Eucalyptus.

Eucalyptus spp. in plantations are negatively affected by canker and wilt diseases caused by several species of Ceratocystis, particularly those in the Latin American Clade (LAC). Ceratocystis eucalypticola and Ceratocystis manginecans are of particular concern where disease epidemics are reported globally, with recent outbreaks emerging in South African and Indonesian Eucalyptus plantations. Consequently, a rapid screening protocol is required for these pathogens. In this study, a high-resolution melting curve analysis (HRMA) was developed to detect C. eucalypticola and C. manginecans that bypasses time-consuming isolation and post-PCR procedures. Primers targeting a 172 bp region of the cerato-platanin (CP) gene were designed. Using these primers, the accuracy of HRMA to detect and distinguish between these two LAC species was assessed using pure fungal DNA, and DNA extracted directly from Eucalyptus samples naturally infected with C. eucalypticola. The assay accurately detected the presence of C. eucalypticola and C. manginecans and quantifies their DNA, both from cultures, and directly from wood samples. HRMA further differentiated these two species from all other tested LAC individuals. This assay was also able to detect the presence of all the tested LAC species and distinguish seven of these, including C. fimbriata, to species level. Ceratocystis polyconidia was the only non-LAC off-target species detected. Based on these results, the developed assay can be used to rapidly identify C. eucalypticola and C. manginecans directly from infected plant material or fungal cultures, with the potential to also screen for several other LAC species.

Susceptibility of Eucalyptus trees to defoliation by the Eucalyptus snout beetle, Gonipterus sp. n. 2, is enhanced by high foliar contents of 1,8-cineole, oxalic acid and sucrose and low contents of palmitic and shikimic acid.

Gonipterus sp. n. 2 (Coleoptera, Curculionidae) is an invasive, commercially important weevil that causes large-scale defoliation of Eucalyptus trees. The weevil specifically feeds on young leaves and new shoots, thus reducing tree growth. The weevil displays a very strong preference for certain Eucalyptus genotypes, however, this behaviour and the chemistry underlying it is poorly understood, thereby complicating the selection of resistant trees. To elucidate the feeding preference of Gonipterus sp. n. 2, we assessed the relative levels of susceptibility of 62 Eucalyptus genotypes from 23 species using a laboratory choice assay. This revealed large intraspecific variation in susceptibility to weevil feeding, which for certain species, exceeded the interspecific variation. A semiquantitative metabolite profile analysis on 13 genotypes revealed strong correlations of 10 metabolites to feeding damage. The behavioural effects of the identified compounds were assessed through an in vitro feeding preference assay using artificial diets as well as under field conditions. This revealed three phagostimulants (1,8-cineole, oxalic acid and sucrose) and two feeding deterrent compounds (shikimic acid and palmitic acid) for Gonipterus sp. n. 2. These chemical markers can be applied to tree breeding programmes for the selection of resistant genotypes to reduce damage caused by Gonipterus weevils.

Conifer-killing bark beetles locate fungal symbionts by detecting volatile fungal metabolites of host tree resin monoterpenes.

Outbreaks of the Eurasian spruce bark beetle (Ips typographus) have decimated millions of hectares of conifer forests in Europe in recent years. The ability of these 4.0 to 5.5 mm long insects to kill mature trees over a short period has been sometimes ascribed to two main factors: (1) mass attacks on the host tree to overcome tree defenses and (2) the presence of fungal symbionts that support successful beetle development in the tree. While the role of pheromones in coordinating mass attacks has been well studied, the role of chemical communication in maintaining the fungal symbiosis is poorly understood. Previous evidence indicates that I. typographus can distinguish fungal symbionts of the genera Grosmannia, Endoconidiophora, and Ophiostoma by their de novo synthesized volatile compounds. Here, we hypothesize that the fungal symbionts of this bark beetle species metabolize spruce resin monoterpenes of the beetle's host tree, Norway spruce (Picea abies), and that the volatile products are used as cues by beetles for locating breeding sites with beneficial symbionts. We show that Grosmannia penicillata and other fungal symbionts alter the profile of spruce bark volatiles by converting the major monoterpenes into an attractive blend of oxygenated derivatives. Bornyl acetate was metabolized to camphor, and α- and ß-pinene to trans-4-thujanol and other oxygenated products. Electrophysiological measurements showed that I. typographus possesses dedicated olfactory sensory neurons for oxygenated metabolites. Both camphor and trans-4-thujanol attracted beetles at specific doses in walking olfactometer experiments, and the presence of symbiotic fungi enhanced attraction of females to pheromones. Another co-occurring nonbeneficial fungus (Trichoderma sp.) also produced oxygenated monoterpenes, but these were not attractive to I. typographus. Finally, we show that colonization of fungal symbionts on spruce bark diet stimulated beetles to make tunnels into the diet. Collectively, our study suggests that the blends of oxygenated metabolites of conifer monoterpenes produced by fungal symbionts are used by walking bark beetles as attractive or repellent cues to locate breeding or feeding sites containing beneficial microbial symbionts. The oxygenated metabolites may aid beetles in assessing the presence of the fungus, the defense status of the host tree and the density of conspecifics at potential feeding and breeding sites.

A Robust Disease Scoring Method to Screen Eucalyptus for Resistance Against the Aggressive Leaf Pathogen Teratosphaeria destructans.

Shoot and leaf blight caused by Teratosphaeria destructans is one of the most devastating foliar diseases on Eucalyptus. Therefore, breeding for resistance to this disease is considered urgent. Differences in susceptibility to T. destructans have been observed in the field but a robust inoculation protocol has, until recently, been unavailable and a disease scoring method for precise phenotyping has not been established. A first objective of this study was to determine the optimal conidial concentration for T. destructans inoculations on a susceptible Eucalyptus host. This concentration was then used to determine differences in susceptibility of six genotypes of Eucalyptus grandis × E. urophylla to the pathogen by assessing the percentage of infected stomata using electron microscopy and the percentage of leaf area covered by lesions (PLACL) using image processing. In addition, we developed a disease susceptibility index (SI) of six categories ranging from highly resistant (SI = 0) to highly susceptible (SI = 1.5 to 2). The more resistant genotypes were moderately resistant, with an SI value of 0.49 to 0.54 and a PLACL of 6.5 to 9%. In contrast, the more susceptible genotype scored an SI of 1.52 and PLACL of 48%. Host susceptibility was also assessed relative to the sporulation of the pathogen. This showed that the percentage of sporulation was not significantly correlated with host resistance. Overall, the results provide the basis for rigorous screening and selection of resistant genotypes to the disease caused by T. destructans using artificial inoculation.

Identification and Characterization of a QTL for Growth of Fusarium circinatum on Pine-Based Medium.

Fusarium circinatum is an economically important pathogen of pine and resides in the Fusarium fujikuroi species complex. Here we investigated the molecular processes underlying growth in F. circinatum by exploring the association between growth and the nutritional environment provided by the pine host. For this purpose, we subjected a mapping population consisting of F. circinatum X F. temperatum hybrid progeny to an analysis of growth rate on a pine-tissue derived medium. These data, together with the available genetic linkage map for F. circinatum, were then used to identify Quantitative Trait Loci (QTLs) associated with growth. The single significant QTL identified was then characterized using the available genome sequences for the hybrid progeny's parental isolates. This revealed that the QTL localized to two non-homologous regions in the F. circinatum and F. temperatum genomes. For one of these, the F. circinatum parent contained a two-gene deletion relative to the F. temperatum parent. For the other region, the two parental isolates encoded different protein products. Analysis of repeats, G+C content, and repeat-induced point (RIP) mutations further suggested a retrotransposon origin for the two-gene deletion in F. circinatum. Nevertheless, subsequent genome and PCR-based analyses showed that both regions were similarly polymorphic within a collection of diverse F. circinatum. However, we observed no clear correlation between the respective polymorphism patterns and growth rate in culture. These findings support the notion that growth is a complex multilocus trait and raise the possibility that the identified QTL contains multiple small-effect QTLs, of which some might be dependent on the genetic backgrounds. This study improved our current knowledge of the genetic determinants of vegetative growth in F. circinatum and provided an important foundation for determining the genes and processes underpinning its ability to colonize its host environment.

The first report of Phytophthora multivesiculata causing black rot of Cymbidium and Ansellia africana from South Africa.

Globally, various species and hybrids of Cymbidium are of horticultural importance. In January 2022, we visited a private orchidarium near Pretoria (25° 54' 30" S, 28° 24' 34" E). During this visit, the owner reported mortality among various pure and hybrid Cymbidium and Ansellia africana, an indigenous South African ornamental orchid. Phytophthora was identified as a possible causative agent based on an initial examination of the affected orchids. The infected orchids exhibited vascular wilting. Brown, water-soaked lesions covered the roots. The pseudobulbs showed black rot symptoms. Necrotic lesions were also visible towards the basal part of the leaves. For isolation of the causal agent, pieces of infected tissues from roots, pseudobulbs and leaves were surface sterilised using 70% ethanol and plated into Phytophthora selective medium, NARPH. All the plates were incubated at 21°C in darkness. After three days, Phytophthora-like mycelia emerged from all three tissue types. These colonies were transferred onto the PDA medium. For molecular identification, genomic DNA was extracted from four representative isolates (CMW58027-30) using a Zymo Research Fungal/Bacterial DNA MiniPrep kit. The complete ITS, and partial beta-tubulin (BT) and cytochrome oxidase 1 (COX1) gene regions were amplified using the primers DC6/ITS4 (Cooke et al. 2000; White et al. 1990), Btub_F1A/Btub_R1 (Blair et al. 2008; Kroon et al. 2004) and FM84/FM83 (Martin and Tooley 2003), respectively. BLAST searches in NCBI showed that the four isolates were from Phytophthora ITS Clades 2. ITS, BT, and COX1 datasets from Bose et al. (2021) were used for the phylogenetic identification of our isolates. Single gene and concatenated datasets were analysed using both maximum likelihood and Bayesian approaches, which confirmed the identity of the isolates as Phytophthora multivesiculata. All the sequences were submitted to the GenBank: ITS (OM967212-15), BT (OM966588-91), and COX1 (OM966592-95). Measurements of sporangia and gametangia overlapped with those from Ilieva et al. (1998): sporangia (28.3 - 56.3) 41.6 x 31.3 (21.5 -3 9.6) µm; L:B 1.42 (1.08 - 1.69); exit pore 11.2 (7.1 - 14.6) µm; oogonia 44.2 (24.5 - 56.3) µm; oospore 34.2 (21.6 - 53.2) µm; antheridia (5.8 - 14.6) 11.8 x 15.3 (6.2-14.9) µm. The pathogenicity trial was conducted following the protocol suggested by Ilieva et al. (1998). Five A. africana roots and the cut ends of seven Cymbidium leaves were immersed in separate beakers containing 100 ml of sterile distilled water and ten 5 mm agar discs excised from a 7-day-old culture of P. multivesiculata (CMW58027) grown on PDA. Sterile distilled water was used as the control, with an equal number of plants and leaves. All of the sets were incubated at 21°C. After seven days, the plants and leaves developed lesions similar to those observed on the symptomatic plants at the orchidarium. Trials were repeated once and the pathogen was reisolated from both trials and the identity was confirmed by amplifying the complete ITS gene region. Phytophthora multivesiculata has been previously reported from the Netherlands (Ilieva et al. 1998), Taiwan (Chern et al. 2011), Australia (Cunnington et al. 2009), New Zealand (Hill 2004) and elsewhere causing black rot of Cymbidium. However, this is the first report of P. multivesiculata causing black rot of Cymbidium and A. africana from Africa. We are now conducting follow-up surveys to determine the distribution range of this pathogen in South Africa.

Bark Beetle Attack History Does Not Influence the Induction of Terpene and Phenolic Defenses in Mature Norway Spruce (Picea abies) Trees by the Bark Beetle-Associated Fungus Endoconidiophora polonica.

Terpenes and phenolics are important constitutive and inducible conifer defenses against bark beetles and their associated fungi. In this study, the inducible defenses of mature Norway spruce (Picea abies) trees with different histories of attack by the spruce bark beetle, Ips typographus were tested by inoculation with the I. typographus-associated fungus Endoconidiophora polonica. We compared trees that had been under previous attack with those under current attack and those that had no record of attack. After fungal inoculation, the concentrations of mono-, sesqui-, and diterpenes in bark increased 3- to 9-fold. For the phenolics, the flavan-3-ols, catechin, and gallocatechin, increased significantly by 2- and 5-fold, respectively, while other flavonoids and stilbenes did not. The magnitudes of these inductions were not influenced by prior bark beetle attack history for all the major compounds and compound classes measured. Before fungal inoculation, the total amounts of monoterpenes, diterpenes, and phenolics (constitutive defenses) were greater in trees that had been previously attacked compared to those under current attack, possibly a result of previous induction. The transcript levels of many genes involved in terpene formation (isoprenyl diphosphate synthases and terpene synthases) and phenolic formation (chalcone synthases) were significantly enhanced by fungal inoculation suggesting de novo biosynthesis. Similar inductions were found for the enzymatic activity of isoprenyl diphosphate synthases and the concentration of their prenyl diphosphate products after fungal inoculation. Quantification of defense hormones revealed a significant induction of the jasmonate pathway, but not the salicylic acid pathway after fungal inoculation. Our data highlight the coordinated induction of terpenes and phenolics in spruce upon infection by E. polonica, a fungal associate of the bark beetle I. typographus, but provide no evidence for the priming of these defense responses by prior beetle attack.

Phenolic degradation by catechol dioxygenases is associated with pathogenic fungi with a necrotrophic lifestyle in the Ceratocystidaceae.

Fungal species of the Ceratocystidaceae grow on their host plants using a variety of different lifestyles, from saprophytic to highly pathogenic. Although many genomes of fungi in the Ceratocystidaceae are publicly available, it is not known how the genes that encode catechol dioxygenases (CDOs), enzymes involved in the degradation of phenolic plant defense compounds, differ among members of the Ceratocystidaceae. The aim of this study was therefore to identify and characterize the genes encoding CDOs in the genomes of Ceratocystidaceae representatives. We found that genes encoding CDOs are more abundant in pathogenic necrotrophic species of the Ceratocystidaceae and less abundant in saprophytic species. The loss of the CDO genes and the associated 3-oxoadipate catabolic pathway appears to have occurred in a lineage-specific manner. Taken together, this study revealed a positive association between CDO gene copy number and fungal lifestyle in Ceratocystidaceae representatives.

Comparative Genomic and Metabolomic Analysis of Termitomyces Species Provides Insights into the Terpenome of the Fungal Cultivar and the Characteristic Odor of the Fungus Garden of Macrotermes natalensis Termites.

Macrotermitinae termites have domesticated fungi of the genus Termitomyces as food for their colony, analogously to human farmers growing crops. Termites propagate the fungus by continuously blending foraged and predigested plant material with fungal mycelium and spores (fungus comb) within designated subterranean chambers. To test the hypothesis that the obligate fungal symbiont emits specific volatiles (odor) to orchestrate its life cycle and symbiotic relations, we determined the typical volatile emission of fungus comb biomass and Termitomyces nodules, revealing α-pinene, camphene, and d-limonene as the most abundant terpenes. Genome mining of Termitomyces followed by gene expression studies and phylogenetic analysis of putative enzymes related to secondary metabolite production encoded by the genomes uncovered a conserved and specific biosynthetic repertoire across strains. Finally, we proved by heterologous expression and in vitro enzymatic assays that a highly expressed gene sequence encodes a rare bifunctional mono-/sesquiterpene cyclase able to produce the abundant comb volatiles camphene and d-limonene. IMPORTANCE The symbiosis between macrotermitinae termites and Termitomyces is obligate for both partners and is one of the most important contributors to biomass conversion in the Old World tropic's ecosystems. To date, research efforts have dominantly focused on acquiring a better understanding of the degradative capabilities of Termitomyces to sustain the obligate nutritional symbiosis, but our knowledge of the small-molecule repertoire of the fungal cultivar mediating interspecies and interkingdom interactions has remained fragmented. Our omics-driven chemical, genomic, and phylogenetic study provides new insights into the volatilome and biosynthetic capabilities of the evolutionarily conserved fungal genus Termitomyces, which allows matching metabolites to genes and enzymes and, thus, opens a new source of unique and rare enzymatic transformations.

Comparison of the Infection Biology of Teratosphaeria destructans and Teratosphaeria epicoccoides on Eucalyptus.

Leaf blight caused by Teratosphaeria destructans is one of the most important diseases of Eucalyptus planted in the subtropics and tropics. In contrast, the better-known T. epicoccoides, though also a primary pathogen of Eucalyptus, causes less damage to trees in these areas. Although T. destructans is an aggressive pathogen, nothing is known about its infection biology. In this study, the conditions for infection and disease development caused by T. destructans and T. epicoccoides were evaluated and compared on a Eucalyptus grandis × E. urophylla hybrid clone. The optimal temperature for germination ranged from 25 to 30°C for T. destructans and 15 to 20°C for T. epicoccoides. The germination of these pathogens was favored under conditions of light and high levels of RH. Penetration by T. destructans and T. epicoccoides occurred via stomata, and the hyphae colonized the intercellular spaces of infected leaves. Symptoms were clearly visible 3 weeks after inoculation by both pathogens, and reproductive structures started to develop in substomatal cavities at 4 weeks after inoculation. The results of this study will facilitate the establishment of rapid screening trials based on artificial inoculations aimed at reducing the impact of disease caused by T. destructans.

African elephants can detect water from natural and artificial sources via olfactory cues.

Water is vital for mammals. Yet, as ephemeral sources can be difficult to find, it raises the question, how do mammals locate water? Elephants (Loxodonta africana) are water-dependent herbivores that possess exceptional olfactory capabilities, and it has been suggested that they may locate water via smell. However, there is no evidence to support this claim. To explore this, we performed two olfactory choice experiments with semi-tame elephants. In the first, we tested whether elephants could locate water using olfactory cues alone. For this, we used water from two natural dams and a drinking trough utilised by the elephants. Distilled water acted as a control. In the second, we explored whether elephants could detect three key volatile organic compounds (VOCs) commonly associated with water (geosmin, 2-methylisoborneol, and dimethyl sulphide). We found that the elephants could locate water olfactorily, but not the distilled water. Moreover, they were also able to detect the three VOCs associated with water. However, these VOCs were not in the odour profiles of the water sources in our experiments. This suggests that the elephants were either able to detect the unique odour profiles of the different water sources or used other VOCs that they associate with water. Ultimately, our findings indicate that elephants can locate water olfactorily at small spatial scales, but the extent to which they, and other mammals, can detect water over larger scales (e.g. km) remains unclear.

Storage of carbon reserves in spruce trees is prioritized over growth in the face of carbon limitation.

Climate change is expected to pose a global threat to forest health by intensifying extreme events like drought and insect attacks. Carbon allocation is a fundamental process that determines the adaptive responses of long-lived late-maturing organisms like trees to such stresses. However, our mechanistic understanding of how trees coordinate and set allocation priorities among different sinks (e.g., growth and storage) under severe source limitation remains limited. Using flux measurements, isotopic tracing, targeted metabolomics, and transcriptomics, we investigated how limitation of source supply influences sink activity, particularly growth and carbon storage, and their relative regulation in Norway spruce (Picea abies) clones. During photosynthetic deprivation, absolute rates of respiration, growth, and allocation to storage all decline. When trees approach neutral carbon balance, i.e., daytime net carbon gain equals nighttime carbon loss, genes encoding major enzymes of metabolic pathways remain relatively unaffected. However, under negative carbon balance, photosynthesis and growth are down-regulated while sucrose and starch biosynthesis pathways are up-regulated, indicating that trees prioritize carbon allocation to storage over growth. Moreover, trees under negative carbon balance actively increase the turnover rate of starch, lipids, and amino acids, most likely to support respiration and mitigate stress. Our study provides molecular evidence that trees faced with severe photosynthetic limitation strategically regulate storage allocation and consumption at the expense of growth. Understanding such allocation strategies is crucial for predicting how trees may respond to extreme events involving steep declines in photosynthesis, like severe drought, or defoliation by heat waves, late frost, or insect attack.

Combining QTL Mapping and Transcriptomics to Decipher the Genetic Architecture of Phenolic Compounds Metabolism in the Conifer White Spruce.

Conifer forests worldwide are becoming increasingly vulnerable to the effects of climate change. Although the production of phenolic compounds (PCs) has been shown to be modulated by biotic and abiotic stresses, the genetic basis underlying the variation in their constitutive production level remains poorly documented in conifers. We used QTL mapping and RNA-Seq to explore the complex polygenic network underlying the constitutive production of PCs in a white spruce (Picea glauca) full-sib family for 2 years. QTL detection was performed for nine PCs and differentially expressed genes (DEGs) were identified between individuals with high and low PC contents for five PCs exhibiting stable QTLs across time. A total of 17 QTLs were detected for eight metabolites, including one major QTL explaining up to 91.3% of the neolignan-2 variance. The RNA-Seq analysis highlighted 50 DEGs associated with phenylpropanoid biosynthesis, several key transcription factors, and a subset of 137 genes showing opposite expression patterns in individuals with high levels of the flavonoids gallocatechin and taxifolin glucoside. A total of 19 DEGs co-localized with QTLs. Our findings represent a significant step toward resolving the genomic architecture of PC production in spruce and facilitate the functional characterization of genes and transcriptional networks responsible for differences in constitutive production of PCs in conifers.

Characterization of the Ergosterol Biosynthesis Pathway in Ceratocystidaceae.

Terpenes represent the biggest group of natural compounds on earth. This large class of organic hydrocarbons is distributed among all cellular organisms, including fungi. The different classes of terpenes produced by fungi are mono, sesqui, di- and triterpenes, although triterpene ergosterol is the main sterol identified in cell membranes of these organisms. The availability of genomic data from members in the Ceratocystidaceae enabled the detection and characterization of the genes encoding the enzymes in the mevalonate and ergosterol biosynthetic pathways. Using a bioinformatics approach, fungal orthologs of sterol biosynthesis genes in nine different species of the Ceratocystidaceae were identified. Ergosterol and some of the intermediates in the pathway were also detected in seven species (Ceratocystis manginecans, C. adiposa, Huntiella moniliformis, Thielaviopsis punctulata, Bretziella fagacearum, Endoconidiophora polonica and Davidsoniella virescens), using gas chromatography-mass spectrometry analysis. The average ergosterol content differed among different genera of Ceratocystidaceae. We also identified all possible terpene related genes and possible biosynthetic clusters in the genomes used in this study. We found a highly conserved terpene biosynthesis gene cluster containing some genes encoding ergosterol biosynthesis enzymes in the analysed genomes. An additional possible terpene gene cluster was also identified in all of the Ceratocystidaceae. We also evaluated the sensitivity of the Ceratocystidaceae to a triazole fungicide that inhibits ergosterol synthesis. The results showed that different members of this family behave differently when exposed to different concentrations of triazole tebuconazole.

Canditate metabolites for ash dieback tolerance in Fraxinus excelsior.

Ash dieback, a forest epidemic caused by the invasive fungus Hymenoscyphus fraxineus, threatens ash trees throughout Europe. Within Fraxinus excelsior populations, a small proportion of genotypes show a low susceptibility to the pathogen. We compared the metabolomes from a cohort of low-susceptibility ash genotypes with a cohort of high-susceptibility ash genotypes. This revealed two significantly different chemotypes. A total of 64 candidate metabolites associated with reduced or increased susceptibility in the chemical families secoiridoids, coumarins, flavonoids, phenylethanoids, and lignans. Increased levels of two coumarins, fraxetin and esculetin, were strongly associated with reduced susceptibility to ash dieback. Both coumarins inhibited the growth of H. fraxineus in vitro when supplied at physiological concentrations, thereby validating their role as markers for low susceptibility to ash dieback. Similarly, fungal growth inhibition was observed when the methanolic bark extract of low-susceptibility ash genotypes was supplied. Our findings indicate the presence of constitutive chemical defense barriers against ash dieback in ash.

The phytopathogenic fungus Sclerotinia sclerotiorum detoxifies plant glucosinolate hydrolysis products via an isothiocyanate hydrolase.

Brassicales plants produce glucosinolates and myrosinases that generate toxic isothiocyanates conferring broad resistance against pathogens and herbivorous insects. Nevertheless, some cosmopolitan fungal pathogens, such as the necrotrophic white mold Sclerotinia sclerotiorum, are able to infect many plant hosts including glucosinolate producers. Here, we show that S. sclerotiorum infection activates the glucosinolate-myrosinase system, and isothiocyanates contribute to resistance against this fungus. S. sclerotiorum metabolizes isothiocyanates via two independent pathways: conjugation to glutathione and, more effectively, hydrolysis to amines. The latter pathway features an isothiocyanate hydrolase that is homologous to a previously characterized bacterial enzyme, and converts isothiocyanate into products that are not toxic to the fungus. The isothiocyanate hydrolase promotes fungal growth in the presence of the toxins, and contributes to the virulence of S. sclerotiorum on glucosinolate-producing plants.

Herbivory meets fungivory: insect herbivores feed on plant pathogenic fungi for their own benefit.

Plants are regularly colonised by fungi and bacteria, but plant-inhabiting microbes are rarely considered in studies on plant-herbivore interactions. Here we show that young gypsy moth (Lymantria dispar) caterpillars prefer to feed on black poplar (Populus nigra) foliage infected by the rust fungus Melampsora larici-populina instead of uninfected control foliage, and selectively consume fungal spores. This consumption, also observed in a related lepidopteran species, is stimulated by the sugar alcohol mannitol, found in much higher concentration in fungal tissue and infected leaves than uninfected plant foliage. Gypsy moth larvae developed more rapidly on rust-infected leaves, which cannot be attributed to mannitol but rather to greater levels of total nitrogen, essential amino acids and B vitamins in fungal tissue and fungus-infected leaves. Herbivore consumption of fungi and other microbes may be much more widespread than commonly believed with important consequences for the ecology and evolution of plant-herbivore interactions.

Tree defence and bark beetles in a drying world: carbon partitioning, functioning and modelling.

Drought has promoted large-scale, insect-induced tree mortality in recent years, with severe consequences for ecosystem function, atmospheric processes, sustainable resources and global biogeochemical cycles. However, the physiological linkages among drought, tree defences, and insect outbreaks are still uncertain, hindering our ability to accurately predict tree mortality under on-going climate change. Here we propose an interdisciplinary research agenda for addressing these crucial knowledge gaps. Our framework includes field manipulations, laboratory experiments, and modelling of insect and vegetation dynamics, and focuses on how drought affects interactions between conifer trees and bark beetles. We build upon existing theory and examine several key assumptions: (1) there is a trade-off in tree carbon investment between primary and secondary metabolites (e.g. growth vs defence); (2) secondary metabolites are one of the main component of tree defence against bark beetles and associated microbes; and (3) implementing conifer-bark beetle interactions in current models improves predictions of forest disturbance in a changing climate. Our framework provides guidance for addressing a major shortcoming in current implementations of large-scale vegetation models, the under-representation of insect-induced tree mortality.

Accumulation of Catechin and Proanthocyanidins in Black Poplar Stems After Infection by Plectosphaerella populi: Hormonal Regulation, Biosynthesis and Antifungal Activity.

Flavan-3-ols including the monomeric catechin and the polymeric proanthocyanidins (PAs) are abundant phenolic metabolites in poplar (Populus spp.) previously described to protect leaves against pathogen infection. However, it is not known whether stems are also defended in this way. Here we investigated flavan-3-ol accumulation, activity, and the regulation of formation in black poplar (P. nigra) stems after infection by a newly described fungal stem pathogen, Plectosphaerella populi, which forms canker-like lesions in stems. We showed that flavan-3-ol contents increased in P. populi-infected black poplar stems over the course of infection compared to non-infected controls. Transcripts of leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR) genes involved in the last steps of flavan-3-ol biosynthesis were also upregulated upon fungal infection indicating de novo biosynthesis. Amending culture medium with catechin and PAs reduced the mycelial growth of P. populi, suggesting that these metabolites act as anti-pathogen defenses in poplar in vivo. Among the hormones, salicylic acid (SA) was higher in P. populi-infected tissues compared to the non-infected controls over the course of infection studied, while jasmonic acid (JA) and JA-isoleucine (JA-Ile) levels were higher than controls only at the early stages of infection. Interestingly, cytokinins (CKs) were also upregulated in P. populi-infected stems. Poplar saplings treated with CK showed decreased levels of flavan-3-ols and SA in stems suggesting a negative association between CK and flavan-3-ol accumulation. Taken together, the sustained upregulation of SA in correlation with catechin and PA accumulation suggests that this is the dominant hormone inducing the formation of antifungal flavan-3-ols during P. populi infection of poplar stems.

Bark Beetle Population Dynamics in the Anthropocene: Challenges and Solutions.

Tree-killing bark beetles are the most economically important insects in conifer forests worldwide. However, despite >200 years of research, the drivers of population eruptions and crashes are still not fully understood and the existing knowledge is thus insufficient to face the challenges posed by the Anthropocene. We critically analyze potential biotic and abiotic drivers of population dynamics of an exemplary species, the European spruce bark beetle (ESBB) (Ips typographus) and present a multivariate approach that integrates the many drivers governing this bark beetle system. We call for hypothesis-driven, large-scale collaborative research efforts to improve our understanding of the population dynamics of this and other bark beetle pests. Our approach can serve as a blueprint for tackling other eruptive forest insects.