Mogens Nicolaisen

While multi‐cropping systems, such as strip‐cropping, assemble complex microbial communities that contribute to ecosystem resilience, we have limited understanding of their assembly and functions at field scale. Here, we examined the dynamics and site‐specific assembly of soil and phyllosphere microbiomes in a rotational faba bean ( Vicia faba ) and spring barley ( Hordeum vulgare ) strip‐system during two seasons. Using bacterial 16S and fungal ITS amplicon sequencing, we analysed microbial communities across gradients from the centre towards strip edges. We found that phyllosphere microbiomes are more affected by strip‐cropping than soil microbiomes. Neighbouring crops harbour different microbial communities, but with increasing proximity, their phyllosphere microbiomes share more specific microbial taxa. Likewise, we observed gradient‐specific microbial distribution, including fungal pathogen taxa. For instance, Puccinia was more abundant in the centre of the spring barley strips and gradually declined towards the edge. Correlation and co‐occurrence network analyses revealed distinct microbial interaction patterns across soil and phyllosphere gradients. These networks were more resilient at the edge of the strips compared to central networks of spring barley. Altogether, our findings revealed neighbour‐effects modulating host‐associated microbiomes across strips that could be harnessed for designing optimal cropping systems, for example, to reduce leaf pathogens.

The plant microbiome has been proposed as ‘the second genome of the plant’, motivating interest in microbiome-assisted breeding. For microbiome-assisted breeding to become a reality, the plant genome must obviously have a role in regulation of the microbiome. However, agricultural fields are spatially heterogeneous, and it remains unclear how host genetics and soil heterogeneity jointly shape plant microbiomes under field conditions. Here, using 16S and ITS metabarcoding data from 2,400 root representing 386 barley lines across spring and winter field trials, we quantified host genetic effects on root-associated fungal and bacterial communities and their modulation by soil spatial heterogeneity. Barley genotype exerted a detectable and reproducible effect on overall microbial community composition and on a subset of bacterial and fungal biomarker taxa associated with plant biomass. However, these host genetic effects were strongly modulated by field-scale spatial heterogeneity. Spatial context influenced both whole-community structure and the abundance patterns of individual host-associated biomarkers. These biomarkers differed markedly in their occupancy consistency, spatial dependency, and host Single-Nucleotide Polymorphism (SNP)-by-soil spatial structure interaction patterns, supporting the view that host-associated microbiome recruitment is context dependent rather than a fixed filtering across field environments. Consistent with this, variation of SNP-associated positive biomarkers significantly correlated with barley biomass stability, but these associations were stronger at the level of total positive biomarker abundance than at the level of individuals. Together, our findings demonstrate that large-scale field experiments are essential for dissecting host-microbiome assembly patterns and reveal that host genetic control of the root microbiome in the field is detectable but strongly context dependent, with important implications for microbiome-assisted breeding.

Shoot branching is critical not only in breeding for yield but also for ornamentals’ architecture. In the ornamental plant poinsettia ( Euphorbia pulcherrima ), shoot branching has traditionally been induced by phytoplasma ( Candidatus Phytoplasma pruni) inoculation. This study aimed to identify regulatory genes that could be leveraged in future breeding-by–genetic engineering efforts to develop phytoplasma-free, branching poinsettia plants. To elucidate mechanisms of phytoplasma-induced shoot branching, we performed RNA-sequencing and assembled an axillary bud-specific transcriptome for expression analyses in phytoplasma-infected and -free poinsettia. Phenotyping and RNA-sequencing were also conducted on Arabidopsis mutants and wild-type lines to investigate the transcriptional regulatory effects of candidate genes. The transcription factors Ep MAF3 and Ep bZIP67 were highly de-regulated in phytoplasma-infected poinsettia. We also found a two-fold increase in primary-stem branching levels of the Arabidopsis maf 3 and bzip 67 mutants, suggesting the two transcription factors as potential shoot branching suppressors. AtTcp 1, a CYC-clade TCP transcription factor, was up-regulated (78x) in leaves of the maf 3 mutants. Analyzing previously reported protein-level interactions for the differentially expressed genes (e.g., AtClamt, AtGh 3.9/3.15, AtSaur 32/36, AtAbi 3, AtGamt 2, AtTcp 3, and AtDwf 4) in bzip 67 mutants shed light on other shoot branching regulators such as TCPs, PINs, ABIs, DWARF14, and BES1, highlighting two regulatory sub-networks including membrane transport and hormonal signaling. The results open the way to rational engineering of shoot branching in poinsettia by targeted mutagenesis of MAF3 and bZIP67. In that way, the tedious and viral-infection prone process of phytoplasma inoculation can be avoided and poinsettia plants would have more homogenous branching. One-sentence summary: Phytoplasma infection in poinsettia induces bud-specific repression of the transcription factors EpMaf 3 and EpbZip 67, consistent with the enhanced stem branching observed in Arabidopsis maf 3 and bzip 67 mutant lines.

This study highlights biologically meaningless negative contributions to heritability estimates and introduces SAFE-h² method and application tool for accurate genetic estimates in genome-wide association studies. By examining 74 phenotypes across eight species, SAFE-h² revealed an average negative contribution, i.e., downward bias, of 30 units to SNP heritability estimates. Furthermore, SAFE-h² improved the SNP heritability estimates by up to 56 units through capturing intra-locus (Ref-allele_SNPi × Alt-allele_SNPi) additive and non-additive effects within the framework of linear models across 50 phenotypes in six species. SNPs with potentially negative contributions to the heritability estimate should be excluded from the model. The widespread assumption that SNP-specific non-additive allelic effects are negligible does not rest on any inherent genetic or biological principles. The advantage of SAFE-h² allelic adjustment approach is its reliance on a linear association model that jointly capture additive, dominance, overdominance, and heterosis-like effects. SAFE-h² not only minimizes false-negative rates but also provides a safe p value interval to minimize false-positive SNP hits.

Barley grown in temperate regions is often challenged by powdery mildew disease. An effective solution is mildew resistance locus o (mlo)-based resistance, which is monogenic, durable, and broad-spectrum. While the pleiotropic effects of mlo mutations on above-ground tissues are well documented, their impact on the root-associated microbiome remains underexplored. We utilized CRISPR/Cas9 to generate novel mlo mutant lines and evaluated their resistance to causal fungus Blumeria hordei. We further examined if mlo knockout has any impact on the overall root microbiome diversity and composition under field-like conditions and applied DESeq2 to compare the abundance of microbial taxa between mutants and wild type. We created five novel resistant mlo lines, including the first mutants with amino acid alterations in the protein’s extracellular region. Mutant lines showed significantly reduced B. hordei colony formation (0.5–5%). While microbial alpha and beta diversity were not significantly altered, a few microbial taxa displayed time-dependent shifts in abundance. Overall, our study demonstrates the effectiveness of CRISPR/Cas9 in generating mlo-based resistance. Moreover, the study revealed functionally important residues in the protein’s extracellular region. Finally, we present the first evidence of limited mlo-associated effects on root microbiome diversity and relative abundance of microbial taxa.

Rice synthesises diterpenoid phytoalexins (DPs) which are known to operate in defence against foliar microbial pathogens and the root‐knot nematode Meloidogyne graminicola . Here, we examined the role of DPs in shaping rice‐associated root microbiomes in nematode‐infested field soil. Further, we assessed how DPs affect interactions between the root microbiomes and M. graminicola . We used 16S and ITS2 rRNA gene amplicon analysis to characterise the root‐ and rhizosphere‐associated microbiomes of DP knock‐out rice mutants and their wild‐type parental line, at an early (17 days) and late (28 days) stage of plant development in field soil. Disruption of DP synthesis resulted in distinct changes in the composition and structure of microbial communities both relative to the parental/wild‐type line but also between individual mutants, indicating specificity in DP‐microbe interactions. Moreover, the abundance of nematode‐suppressive microbial taxa, including Streptomyces , Stenotrophomonas and Enterobacter was negatively correlated with that of Meloidogyne . Differential enrichment of microbial taxa in the roots of rice DP knock‐out mutants versus wild‐type suggests that DPs modulate specific taxa in the rice root microbiome. These findings indicate a role for DPs in plant‐microbiome assembly and nematode interactions, further underscoring the potential of leveraging phytoalexins for sustainable management of crop diseases.

Phytopathogenic Sclerotinia minor Jagger causes lettuce drop, a destructive soil-borne disease. As potential biocontrol agents for this disease, 2 of 31 bacterial strains isolated from soil samples from fields containing S. minor Jagger were identified using in vitro antagonistic assays against S. minor Jagger. Bioactivity experiments showed that Bac20 had higher inhibitory activity against S. minor Jagger than Bac45. Based on 16S rRNA sequences and phylogenetic analysis of a combination of sequences from gyrA, rpoB, purH, polC, and groEL, Bac20 and Bac45 were identified as Bacillus velezensis and Bacillus subtilis, respectively. Lipopeptide compounds produced by each strain were identified using matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI–TOF MS) analysis. Both strains produced three types of lipopeptides, namely surfactins, iturins, and fengycins, whereas Bac20 showed the strongest intensity in its production of iturins, more than that of Bac45. Bac20 inhibited oxalic acid formation in early-stage lettuce leaves infected with S. minor Jagger, delaying pathogen infestation. Greenhouse experiments for controlling lettuce drop demonstrated that inoculation with Bac20 controlled lettuce drop by 71.7%. In conclusion, this study revealed that B. velezensis Bac20 has high potential for use as a biocontrol agent for controlling the lettuce drop caused by S. minor Jagger.

Rice synthesizes diterpenoid phytoalexins (DPs) are known to operate in defense against foliar microbial pathogens and root-knot nematode Meloidogyne graminicola . Here, we examined the role of DPs in shaping rice⍰associated root microbiomes in nematode-infested rice paddy soils. Further, we assessed how DPs affect interactions between the root microbiomes and nematode communities, particularly rice root-knot nematodes from the Meloidogyne genus. We used 16S and ITS2 rRNA gene amplicon analysis to characterize the rice root-and rhizosphere-associated microbiomes of DP knock-out mutants and their wild-type parental line, at an early (17 days) and late (28 days) stage of plant development in field soil. Disruption of DP synthesis resulted in distinct composition and structure of microbial communities both relative to the parental/wild-type line but also between individual mutants, indicating specificity in DP-microbe interactions. Moreover, nematode-suppressive microbial taxa, including Streptomyces , Stenotrophomonas , Enterobacter , Massilia , and Acidibacter , negatively correlated with Meloidogyne . Comparative analysis revealed differential enrichment of microbial taxa in the roots of rice diterpenoid phytoalexin (DP) knock-out mutants versus wild-type, suggesting that DPs modulate specific taxa in the rice root microbiome. These findings indicate DPs role in plant-microbiome assembly and nematode interactions, further underscoring the potential of leveraging phytoalexins for sustainable management of crop diseases.