Root-knot nematodes (Meloidogyne spp.) are among the most aggressive phytonematodes. While moving through soil to reach the roots of their host, specific microbes attach to the cuticle of the infective second-stage juveniles (J2). Reportedly, the attached microorganisms affect nematodes and reduce their performance on the host plants. We have previously shown that some non-parasitic bacterial strains isolated from the cuticle of Meloidogyne hapla in different soils affected J2 mortality, motility, hatching, and root invasion. Here we tested whether cuticle-attached microbes trigger plant defenses upon penetration of J2. In in vitro assays, M. hapla J2-attached microbes from a suppressive soil induced pathogenassociated molecular pattern-triggered immunity (PTI) in tomato roots. All tested PTIresponsive defense genes were upregulated after root invasion of J2 with attached microbes, compared to surface-sterilized J2, particularly the jasmonic acid-mediated PTI marker genes TFT1 and GRAS4.1. The strain Microbacterium sp. K6, that was isolated from the cuticle, significantly reduced root invasion when attached to the J2. Attached K6 cells supported plant defense and counteracted suppression of plant basal defense in roots by invaded J2. The plant response to the J2-attached K6 cells was stronger in leaves than in roots, and it increased from 1 to 3 days post inoculation (dpi). At 1 dpi, the plant responded to J2-attached K6 cells by ameliorating the J2-triggered down-regulation of defense genes mostly in roots, while at 3 dpi this response was systemic and more pronounced in leaves. In a reactive oxygen species (ROS) assay, the compounds released from J2 with attached K6 cells triggered a stronger ROS burst in tomato roots than the compounds from nematodes without K6, or the metabolites released from strain K6 alone. Leaves showed a 100 times more sensitive response than roots, and the metabolites of K6 with or without J2 induced strong ROS bursts. In conclusion, our results suggest the importance of microorganisms that attach to M. hapla in suppressive soil, inducing early basal defenses in plants and suppressing nematode performance in roots.

Biosurfactants are amphiphilic compounds of microbial origin with a wide range of industrial applications. Rhamnolipids (RLs) offer a broad range of potential applications as biosurfactants in industry and agriculture. Several studies report a high capacity for controlling different plant pests and pathogens and consider RLs as promising candidates for bio-based plant protection agents. RLs are a class of glycolipids consisting of one (mRLs) or two (dRLs) rhamnose moieties linked to a single or branched β fatty acid. Due to the diversity of RLs, so far little is known about the relation between molecular structure and biological activity. Engineering the synthesis pathway allowed us to differentiate between the activities of mixtures of pure mRL and of pure dRL congeners and elaborated HPLC techniques further enabled to analyse the activity of single congeners. In a model system with the plant Arabidopsis thaliana and the plant-parasitic nematode Heterodera schachtii we demonstrate that RLs can significantly reduce infection, whereas their impact on the host plant varies depending on their molecular structure. While mRLs reduced plant growth even at low concentration, dRLs showed no or a beneficial impact on plant development. In addition, the effect of RLs on plant H2O2 production was measured as an indicator of plant defense activity. Treatment with mRLs or dRLs at a concentration of 50 ppm increased H2O2 production. Lower concentrations of up to 10 ppm were used to stimulate plants prior to a treatment with water or flagellin (flg22), a bacterial inducer of plant defense responses. At 10 ppm both mRLs and dRLs fostered an increased response to flg22. However, mRLs also led to an increased response to water, the non-inducing negative control, indicating a generally elevated stress level. Neither mRLs nor dRLs induced expression of plant defense marker genes of salicylic acid, jasmonic acid and ethylene pathways within a 1 hour and 48 hours treatment. Due to the negative effect of mRLs on plants further studies were concentrated on dRLs. Treatment of pre-parasitic infective juveniles of H. schachtii revealed that dRLs did not increase mortality even at a very high concentration of 755 ppm. In order to analyse the effect of single dRL congeners nematode infection assays were performed. While dRL congeners with a C10-C8 acyl chain increased nematode infection, dRLs with C10-C12 and C10-C12:1 acyl chains reduced nematode infection even at concentrations below 2 ppm. Plant growth was not reduced by C10-C8 dRLs, but by C10-C12 and C10-C12:1 dRLs at concentrations of 8.3 ppm. H2O2 production was increased compared to the water control upon treatment with C10-C8 dRLs at a concentration of 200 ppm, while C10-C12 and C10-C12:1 dRLs triggered the same effect already at 50 ppm. Our experiments show a clear structure-effect relation. In conclusion, functional assessment and analysis of mode of action of RLs in plants require careful consideration of their molecular structure and composition.

SummaryCoumarins can fight pathogens and are thus promising for crop protection. Their biosynthesis, however, has not yet been engineered in crops. We tailored the constitutive accumulation of coumarins in transgenic Nicotiana benthamiana, Glycine max and Arabidopsis thaliana plants, as well as in Nicotiana tabacum BY‐2 suspension cells. We did so by overexpressing A. thaliana feruloyl‐CoA 6‐hydroxylase 1 (AtF6’H1), encoding the key enzyme of scopoletin biosynthesis. Besides scopoletin and its glucoside scopolin, esculin at low level was the only other coumarin detected in transgenic cells. Mechanical damage of scopolin‐accumulating tissue led to a swift release of scopoletin, presumably from the scopolin pool. High scopolin levels in A. thaliana roots coincided with reduced susceptibility to the root‐parasitic nematode Heterodera schachtii. In addition, transgenic soybean plants were more tolerant to the soil‐borne pathogenic fungus Fusarium virguliforme. Because mycotoxin‐induced accumulation of reactive oxygen species and cell death were reduced in the AtF6’H1‐overexpressors, the weaker sensitivity to F. virguliforme may be caused by attenuated oxidative damage of coumarin‐hyperaccumulating cells. Together, engineered coumarin accumulation is promising for enhanced disease resilience of crops.