Benye Liu

Apple replant disease (ARD) is a problem in apple tree and fruit production worldwide, and its etiology is not fully understood. This study aimed to evaluate the tolerance of six apple genotypes, including the susceptible rootstock M.26; three potentially tolerant rootstocks, EMR.2, G.202, and G.935; and two wild-type accessions, Malus spectabilis MAL0130 and Malus sargentii MAL0739 in ARD soils from six sites. Using a greenhouse biotest, plants were grown for 56 days in ARD or the respective γ-irradiated soil. No genotype showed a consistent lack of growth reduction in all ARD soils, indicating that no universal tolerance existed within the tested set of genotypes. Three genotypes (M.26, MAL0130, and MAL0739) were selected for analyses of ARD indicator gene expression and phytoalexin contents in roots as measures of ARD response. Biphenyl synthase 3 expression and phytoalexin content strongly correlated with highest levels observed in roots of the susceptible rootstock M.26. MAL0739 showed the lowest response, which was not consistently reflected in improved plant growth across all soils. Analysis of the bacterial and fungal communities in three root-affected ARD soils revealed soil- and genotype-specific differences. Bacterial alpha diversity was higher in MAL0739 compared with M.26, whereas beta diversity was mainly shaped by the soil. Significant enrichment of Nectria was observed for M.26 grown in one ARD soil. Phytoalexin contents and potentially plant-beneficial microbial taxa correlated negatively. Our findings highlight that ARD tolerance in apple genotypes is linked to the soil microbiome and other soil properties, indicating the need for an integrated approach to managing ARD. [ ] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

SUMMARY: Reverse‐prenylated phenolic compounds are an abundant class of bioactive plant natural products. Hyperixanthone A, an inhibitor of multidrug‐resistant Staphylococcus aureus, is a polyprenylated xanthone carrying two forward geminal and one reverse prenyl group. Although prenyltransferases responsible for the forward prenylations were identified, the final reverse prenylation reaction remained elusive. No plant enzyme catalyzing reverse prenylation of an aromatic carbon has been described so far. Here we use metabolic profiling and transcriptomic information from Hypericum perforatum and H. sampsonii to identify homologous enzymes involved in the formation of reverse‐prenylated xanthones and characterize their functions using in vitro, in vivo, and in silico approaches. The identified enzymes are non‐canonical UbiA‐type prenyltransferases, which surprisingly catalyze both forward and reverse prenylations with different regioselectivities. Reconstruction of the enzyme cascade in Saccharomyces cerevisiae and Nicotiana benthamiana confirmed reverse‐prenylated hyperixanthone A as the major product. Molecular modeling and docking simulations supported by site‐directed mutagenesis suggest two distinct binding modes, which enable forward and reverse prenylations and provide a rationale for the preferred catalysis of the reverse prenyl transfer reaction. The identification of reverse prenylation augments the repertoire of reactions catalyzed by membrane‐bound UbiA‐type plant aromatic prenyltransferases. The insights also provide a new tool for the biotechnological modification of pharmaceutically valuable natural products.

Background: Apple replant disease (ARD) represents a dysbiotic rhizosphere condition potentially driven by root exudates including phytoalexins at the root–soil interface. A promising mitigation strategy could be the application of bioinoculants that reduce these compounds and foster a diverse microbiome. This study investigated the effects of Rhodococcus pseudokoreensis R79^T, a strain with benzoate-degrading capabilities and genetic potential to degrade biphenyls, on the rhizosphere microbiome of apple plantlets grown in ARD-affected soil in a greenhouse experiment. Results: We applied R79^T at 10⁶ to 10⁹ CFU/ml, assessing its impact on bacterial 16S rRNA diversity and abundance, as well as the abundance of biphenyl dioxygenase (bphd) genes. Eight weeks post-inoculation reads of strain R79^T persisted in the rhizosphere, particularly at higher inoculation levels. Inoculation enhanced bacterial diversity and bphd gene abundance, with significant shifts in community composition. Key responders included members of Gaiellales, which increased, and Streptomyces, which decreased. Co-occurrence network analysis revealed that inoculation promoted positive interactions, more homogeneous connectivity, and a higher degree of connections. Effects on bacterial community structure varied significantly with inoculation concentration. Conclusions: The fact that R79^T enhanced rhizosphere bacterial diversity and modulated community composition in ARD-affected soil highlights the potential of R79^T to reshape microbial interactions. Further research is needed to elucidate the mechanisms underlying these effects, including studies on in situ degradation of phytoalexins and inoculation of R79^T alongside bacteria for plant growth promotion (PGP) in synthetic communities for elevated efficiency against ARD.

Plant–soil feedback and soil microbial legacies play crucial roles in replanting success of apple. This study investigated how different soil amendment strategies influence these factors in replant disease-affected soil. Two approaches were tested: (i) the preculture and amendment of catch crops—either a single species, Tagetes patula, or a diverse catch crop mixture (CCM), and (ii) the inoculation of plant-beneficial microbes—bacteria, arbuscular mycorrhizal fungi, or their combination (SynC). Apple rootstock M.26 was grown for seven weeks in a greenhouse, and plant growth, soil nutrients, root phytoalexins, and microbial communities in rhizosphere and root-affected soil were analyzed. Catch crop amendments but not microbial inoculations, significantly altered soil nutrients. Root length increased significantly under CCM, and in tendency in Tagetes and SynC. Phytoalexin contents were lowest in Tagetes and highest in CCM, both differing from the control in specific compounds. Microbiome analysis revealed that catch crops strongly modulated fungal communities in rhizosphere and root-affected soil, favoring potentially beneficial Linnemannia and Mortierella, while microbial inoculations predominantly modulated bacterial/archaeal rhizosphere communities. Our results suggest that catch crops and microbial inoculants induced distinct shifts in soil–plant–microbe interactions under replanting conditions.

Apple replant disease (ARD) is a serious soilborne disease in apple nurseries and orchards worldwide. ARD is the result of an unbalanced soil microbiome in which multiple soilborne plant-pathogenic fungi, oomycetes, and nematodes form a disease complex. Biphenyl and dibenzofuran phytoalexins are found in greater quantities in the roots of apple plants grown in ARD soil compared with disinfected ARD soil. However, the contribution of these compounds to plant health or disease is not yet understood. Here, the antimicrobial activity of 14 chemically synthesized biphenyl and dibenzofuran phytoalexins was tested against eight selected microorganisms isolated from either rhizosphere soils or apple roots. These included five potentially beneficial bacteria ( Rhodococcus pseudokoreensis strain R79^T, R. koreensis strain R85, Streptomyces pulveraceus strain ES16, S. ciscaucasicus GS2, and Priestia megaterium strain B1), two ARD fungal pathogens ( Ilyonectria robusta H131 and Dactylonectria torresensis N3), and one oomycete ( Globisporangium terrestre). Two phytoalexin mixtures reflecting the percentages of the individual compounds inside the roots (mixture A) and the root exudate (mixture B) were also tested. The two phytoalexin mixtures demonstrated higher antimicrobial activity than the individual phytoalexins, suggesting a synergistic effect. The minimum inhibitory concentration and the half-maximal effective concentration values determined to be active against the eight microbes were within a range of 2.5-fold the ecologically relevant phytoalexin concentration (approximately 33 and 24 µg ml⁻¹ in roots and exudate, respectively). The results contribute to our understanding of the impact of apple root phytoalexins on ARD and suggest potential strategies for disease management.

Introduction: Apple replant disease (ARD) causes reduced growth and fruit yield and affects orchards and tree nurseries worldwide. A number of pathogens have been consistently identified as causal agents of ARD; however factors affecting disease-severity are not fully understood. Aims: We examined five soils from German tree nurseries and apple orchards featuring different soil characteristics and replant histories. We aimed to link the plant-soil interaction to replant disease severity. Methods: In a greenhouse experiment, young apple plants were grown for eight weeks on untreated and disinfected (control) soils. Growth parameters were recorded to evaluate the severity of ARD. The defence response of the plants was examined by expression analysis of ARD indicator genes (BIS3, B4H and ERF1B) and GC–MS-based detection of phytoalexins. The fungal and bacterial rhizosphere communities were investigated by ITS and 16S rRNA amplicon sequencing, respectively. Results: After eight weeks, ARD symptoms were observed on all soils. Growth depression was highest on soils that had faced intensive apple cultivation and lowest on a soil with only one year of apple cultivation prior to the experiment. These results correlated with increases in the BIS3 expression level and the phytoalexin content in the roots. No bacteria and fungi commonly found in increased abundance in ARD soils were consistently detected in all soils. Conclusions: Replant history influenced disease severity more than soil characteristics. ARD symptoms correlated with BIS3 expression and phytoalexin (PA) formation. PA exudation increased the relative abundance of bacterial genera with the potential ability to degrade phenolic compounds.

Here, we use transcriptomic data from seeds of Musella lasiocarpa to identify five enzymes involved in the formation of dihydrocurcuminoids. Characterization of the substrate specificities of the enzymes reveals two distinct dihydrocurcuminoid pathways leading to phenylphenalenones and linear diarylheptanoid derivatives, the major seed metabolites. Furthermore, we demonstrate the stepwise conversion of dihydrobisdemethoxycurcumin to the phenylphenalenone 4 '-hydroxylachnanthocarpone by feeding intermediates to M. lasiocarpa root protein extract.

Medicinal compounds from plants include bicyclo[3.3.1]nonane derivatives, the majority of which are polycyclic polyprenylated acylphloroglucinols (PPAPs). Prototype molecules are hyperforin, the antidepressant constituent of St. John’s wort, and garcinol, a potential anticancer compound. Their complex structures have inspired innovative chemical syntheses, however, their biosynthesis in plants is still enigmatic. PPAPs are divided into two subclasses, named type A and B. Here we identify both types in Hypericum sampsonii plants and isolate two enzymes that regiodivergently convert a common precursor to pivotal type A and B products. Molecular modelling and substrate docking studies reveal inverted substrate binding modes in the two active site cavities. We identify amino acids that stabilize these alternative binding scenarios and use reciprocal mutagenesis to interconvert the enzymatic activities. Our studies elucidate the unique biochemistry that yields type A and B bicyclo[3.3.1]nonane cores in plants, thereby providing key building blocks for biotechnological efforts to sustainably produce these complex compounds for preclinical development.

Modulating the soil microbiome by applying microbial inoculants has gained increasing attention as eco-friendly option to improve soil disease suppressiveness. Currently, studies unraveling the interplay of inoculants, root-associated microbiome, and plant response are lacking for apple trees. Here, we provide insights into the ability of Bacillus velezensis FZB42 or Pseudomonas sp. RU47 to colonize apple root-associated microhabitats and to modulate their microbiome. We applied the two strains to apple plants grown in soils from the same site either affected by apple replant disease (ARD) or not (grass), screened their establishment by selective plating, and measured phytoalexins in roots 3, 16, and 28 days post inoculation (dpi). Sequencing of 16S rRNA gene and ITS fragments amplified from DNA extracted 28 dpi from different microhabitat samples revealed significant inoculation effects on fungal β-diversity in root-affected soil and rhizoplane. Interestingly, only in ARD soil, most abundant bacterial amplicon sequence variants (ASVs) changed significantly in relative abundance. Relative abundances of ASVs affiliated with Enterobacteriaceae were higher in rhizoplane of apple grown in ARD soil and reduced by both inoculants. Bacterial communities in the root endosphere were not affected by the inoculants but their presence was indicated. Interestingly and previously unobserved, apple plants responded to the inoculants with increased phytoalexin content in roots, more pronounced in grass than ARD soil. Altogether, our results indicate that FZB42 and RU47 were rhizosphere competent, modulated the root-associated microbiome, and were perceived by the apple plants, which could make them interesting candidates for an eco-friendly mitigation strategy of ARD. Key points: • Rhizosphere competent inoculants modulated the microbiome (mainly fungi) • Inoculants reduced relative abundance of Enterobacteriaceae in the ARD rhizoplane • Inoculants increased phytoalexin content in roots, stronger in grass than ARD soil

Phenylphenalenones (PPs) are complex polycyclic natural products that play an important role in the chemical defense system of banana and plantain (Musaceae). Although suggestions for how plants synthesize the PP scaffold were first proposed more than 50 years ago, no biosynthetic information is yet available at the enzyme level. Here, we use transcriptomic data from seeds of Musella lasiocarpa , the Chinese dwarf banana, to identify five biosynthetic genes involved in the formation of dihydrocurcuminoids. Characterization of the substrate specificities of the enzymes reveals two distinct dihydrocurcuminoid pathways leading to the two types of major aromatic seed metabolites, the PPs and the linear diarylheptanoid derivatives. Furthermore, through multiple rounds of feeding potential intermediates to M. lasiocarpa root protein extract, followed by high-resolution mass spectrometry profiling, product isolation, and NMR-based elucidation, we demonstrate the stepwise conversion of a dihydrocurcumin-type precursor to the PP 4’-hydroxylachnanthocarpone. In contrast to the commonly hypothesized Diels–Alder cyclization mechanism, we propose an unexpected two-step cyclization route to the PP scaffold.