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Insects have mutualistic symbioses with a variety of microorganisms. However, the chemical signals that maintain these insect−microbe relationships are poorly known compared to those from insect−plant symbioses. The spruce bark beetle, Ips typographus, the most destructive forest pest in Europe, has a symbiotic relationship with several fungi that are believed to contribute to its successful invasion of Norway spruce. Here we tested the hypothesis that volatile organic compounds (VOCs) emitted from fungal symbionts could be cues for bark beetles to recognize and distinguish among members of its microbial community. Behavioral experiments with fungi showed that immature adults of I. typographus are attracted to food sources colonized by their fungal symbionts but not to saprophytic fungi and that this attraction is mediated by volatile cues. GC-MS measurements revealed that the symbionts emitted VOCs. Testing the activity of these compounds on beetle antennae using single sensillum recordings showed that beetles detect many fungal volatiles and possess olfactory sensory neurons specialized for these compounds. Finally, synthetic blends of fungal volatiles attracted beetles in olfactometer experiments. These findings indicate that volatile compounds produced by fungi may act as recognition signals for bark beetles to maintain specific microbial communities that might have impact on their fitness.

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.

Phenolic secondary metabolites are often thought to protect plants against attack by microbes, but their role in defense against pathogen infection in woody plants has not been investigated comprehensively. We studied the biosynthesis, occurrence, and antifungal activity of flavan-3-ols in black poplar (Populus nigra), which include both monomers, such as catechin, and oligomers, known as proanthocyanidins (PAs). We identified and biochemically characterized three leucoanthocyanidin reductases and two anthocyanidin reductases from P. nigra involved in catalyzing the last steps of flavan-3-ol biosynthesis, leading to the formation of catechin [2,3-trans-(+)-flavan-3-ol] and epicatechin [2,3-cis-(−)-flavan-3-ol], respectively. Poplar trees that were inoculated with the biotrophic rust fungus (Melampsora larici-populina) accumulated higher amounts of catechin and PAs than uninfected trees. The de novo-synthesized catechin and PAs in the rust-infected poplar leaves accumulated significantly at the site of fungal infection in the lower epidermis. In planta concentrations of these compounds strongly inhibited rust spore germination and reduced hyphal growth. Poplar genotypes with constitutively higher levels of catechin and PAs as well as hybrid aspen (Populus tremula × Populus alba) overexpressing the MYB134 transcription factor were more resistant to rust infection. Silencing PnMYB134, on the other hand, decreased flavan-3-ol biosynthesis and increased susceptibility to rust infection. Taken together, our data indicate that catechin and PAs are effective antifungal defenses in poplar against foliar rust infection.

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 β-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.

Carbon (C) allocation plays a central role in tree responses to environmental changes. Yet, fundamental questions remain about how trees allocate C to different sinks, for example, growth vs storage and defense. In order to elucidate allocation priorities, we manipulated the whole‐tree C balance by modifying atmospheric CO2 concentrations [CO2] to create two distinct gradients of declining C availability, and compared how C was allocated among fluxes (respiration and volatile monoterpenes) and biomass C pools (total biomass, nonstructural carbohydrates (NSC) and secondary metabolites (SM)) in well‐watered Norway spruce (Picea abies) saplings. Continuous isotope labelling was used to trace the fate of newly‐assimilated C. Reducing [CO2] to 120 ppm caused an aboveground C compensation point (i.e. net C balance was zero) and resulted in decreases in growth and respiration. By contrast, soluble sugars and SM remained relatively constant in aboveground young organs and were partially maintained with a constant allocation of newly‐assimilated C, even at expense of root death from C exhaustion. We conclude that spruce trees have a conservative allocation strategy under source limitation: growth and respiration can be downregulated to maintain ‘operational’ concentrations of NSC while investing newly‐assimilated C into future survival by producing SM.

Plants release complex volatile blends after separate attack by herbivores and pathogens, which play many roles in interactions with other organisms. Large perennials are often attacked by multiple enemies, but the effect of combined attacks on volatile emission is rarely studied, particularly in trees. We infested Populus nigra trees with a pathogen, the rust fungus Melampsora laricipopulina, and Lymantria dispar caterpillars alone and in combination. We investigated poplar volatile emission and its regulation, as well as the behavior of the caterpillars towards volatiles from rust-infected and uninfected trees. Both the rust fungus and the caterpillars alone induced volatile emission from poplar trees. However, the herbivore-induced volatile emission was significantly reduced when trees were under combined attack by the herbivore and the fungus. Herbivory induced terpene synthase transcripts as well as jasmonate concentrations, but these increases were suppressed when the tree was additionally infected with rust. Caterpillars preferred volatiles from rust-infected over uninfected trees. Our results suggest a defense hormone crosstalk upon combined herbivore–pathogen attack in poplar trees which results in lowered emission of herbivore-induced volatiles. This influences the preference of herbivores, and might have other far-reaching consequences for the insect and pathogen communities in natural poplar forests.

Conifer forests worldwide are becoming increasingly vulnerable to attacks by bark beetles and their fungal associates due to the effects of global warming. Attack by the bark beetle Ips typographus and the blue-stain fungus it vectors (Endoconidiophora polonica) on Norway spruce (Picea abies) is well known to induce increased production of terpene oleoresin and polyphenolic compounds. However, it is not clear whether specific compounds are important in resisting attack. In this study, we observed a significant increase in dihydroflavonol and flavan-3-ol content after inoculating Norway spruce with the bark beetle vectored fungus. A bioassay revealed that the dihydroflavonol taxifolin and the flavan-3-ol catechin negatively affected both I. typographus and E. polonica. The biosynthesis of flavan-3-ols is well studied in Norway spruce, but little is known about dihydroflavonol formation in this species. A flavanone-3-hydroxylase (F3H) was identified that catalyzed the conversion of eriodictyol to taxifolin and was highly expressed after E. polonica infection. Down-regulating F3H gene expression by RNA interference in transgenic Norway spruce resulted in significantly lower levels of both dihydroflavonols and flavan-3-ols. Therefore F3H plays a key role in the biosynthesis of defense compounds in Norway spruce that act against the bark beetle-fungus complex. This enzyme forms a defensive product, taxifolin, which is also a metabolic precursor of another defensive product, catechin, which in turn synergizes the toxicity of taxifolin to the bark beetle associated fungus.

The 2-C-methylerythritol 4-phosphate (MEP) pathway supplies precursors for plastidial isoprenoid biosynthesis including carotenoids, redox cofactor side chains, and biogenic volatile organic compounds. We examined the first enzyme of this pathway, 1-deoxyxylulose 5-phosphate synthase (DXS), using metabolic control analysis. Multiple Arabidopsis (Arabidopsis thaliana) lines presenting a range DXS activities were dynamically labeled with ¹³CO₂ in an illuminated, climate-controlled, gas exchange cuvette. Carbon was rapidly assimilated into MEP pathway intermediates, but not into the mevalonate pathway. A flux control coefficient of 0.82 was calculated for DXS by correlating absolute flux to enzyme activity under photosynthetic steady-state conditions, indicating that DXS is the major controlling enzyme of the MEP pathway. DXS manipulation also revealed a second pool of a downstream metabolite, 2-Cmethylerythritol-2,4-cyclodiphosphate (MEcDP), metabolicaUy isolated from the MEP pathway. DXS overexpression led to a 3-to 4-fold increase in MEcDP pool size but to a 2-fold drop in maximal labeling. The existence of this pool was supported by residual MEcDP levels detected in dark-adapted transgenic plants. Both pools of MEcDP are closely modulated by DXS activity, as shown by the fact that the concentration control coefficient of DXS was twice as high for MEcDP (0.74) as for 1-deoxyxylulose 5-phosphate (0.35) or dimethylallyl diphosphate (0.34). Despite the high flux control coefficient for DXS, its overexpression led to only modest increases in isoprenoid end products and in the photosynthetic rate. Diversion of flux via MEcDP may partly explain these findings and suggests new opportunities to engineer the MEP pathway.

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.

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.