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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., (Copyright © 2019 Ullah, Unsicker, Reichelt, Gershenzon and Hammerbacher.)

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.