The impact of benzoxazinoids on agroecological plant-soil feedbacks

Plants modulate their growth environment by changing their root surrounding soil, which in turn modifies the performance of the next plant growing in that soil. How such plant-soil feedbacks are affected by root exuded secondary metabolites is not well understood. In particular, we know very little about how secondary metabolite-mediated plant-soil feedbacks affect agricultural productivity and food quality in crop rotations, and how secondary metabolites could help to alleviate negative agroecological plant-soil feedbacks. In this thesis, I aim to assess the potential of benzoxazinoids, an important class of secondary metabolites that are produced by cereals, to improve crop rotations through plant-soil feedbacks. First, in a two-year field experiment, I demonstrated that maize benzoxazinoid soil conditioning improved the performance of three subsequently growing wheat varieties without compromising food quality. Cereal leaf beetle infestation was reduced in response to benzoxazinoid soil conditioning and wheat yield was increased by more than 4%, mostly caused by enhanced emergence and tillering. Second, in another two-year field experiment, I found that such benzoxazinoid-dependent plant-soil feedbacks depend on local soil parameters. Soil chemistry was closely associated with soil benzoxazinoid concentrations and rhizosphere microbial community composition. Soil chemistry also explained the magnitude and direction of the feedbacks on plant performance, resistance, and kernel quality. Further, in a climate chamber and an incubation experiment I elucidated how benzoxazinoid degradation, but not exudation, was influenced by soil chemistry. In both field experiments, benzoxazinoid soil conditioning modified soil benzoxazinoid concentrations and the community compositions of root-associated microbes. The differences in rhizosphere microbial communities were only transient, while the chemical fingerprint of benzoxazinoid degradation products persisted to the next crop. Third, in climate chamber experiments, I demonstrated that three out of five tested preceding crops suppressed growth of maize through negative plant-soil feedbacks, and that benzoxazinoid exudation reduced this growth suppression. This resistance to growth suppression was, at least partially, dependent on soil biota. Overall, the results of this thesis reveal several new facets of secondary metabolites in agroecological plant-soil feedbacks. Exuded secondary metabolites can enhance crop rotation productivity and confer resistance to negative plant-soil feedbacks, thus making them a promising breeding target to improve crop productivity in a sustainable manner

Effects of pea breeding history on root microbiome attributes under pea root rot stress

The pea root rot complex (PRRC) poses a major threat to pea (Pisum sativum), one of the most important crops for plant-based protein production. The co-occurrence of various soil-borne pathogens within a PRRC triggers soil fatigue, and thereby constrains cultivation. Even though resistant cultivars against single pathogens exist, the complexity of interactions among the pathogens can still lead to root infections. In order to make further breeding progress, it is necessary to consider this complexity and link to interactions of the host with the entire root microbial community, including the pathobiome and plant beneficial members. In a previous study, we characterised several known taxa involved in the PRRC (Wille et al., 2021). It is however not known how the PRRC interacts with other members of the microbial community and how these interactions were steered by pea breeding. To shed light on this, we compare the root microbiome of pea landraces and modern European breeding material grown in PRRC-affected field soil. For this, 250 pea genotypes consisting of 174 landraces of the USDA pea core collection, 31 registered cultivars from Europe, and 45 advanced breeding lines from Getreidezüchtung Peter Kunz (CH) were grown for 21 days under controlled conditions in a walk-in climate chamber before roots were harvested for microbiome analysis. Root bacteria and fungi were characterized by 16S- and ITS-amplicon sequencing, respectively. To evaluate potential effect of plant breeding on microbiome characteristics in response to soil infestation, we investigate species richness (alpha diversity), microbial community composition (beta diversity), and potentially network characteristics, such as network complexity. Furthermore, we present potential microbial hubs and individual OTUs associated with breeding history. This will provide valuable information about the selection effects of plant breeding on PRRC-related microbiome attributes and thus help to evaluate the potential of microbe-assisted breeding for disease resistance against pea root rot. In a next step, we aim to exploit genome-wide association studies (GWAS) approaches to seek genetic loci involved in microbe-mediated disease resistance. Markers linked to such loci will be validated in additional genetic material provided by the KWS breeding company. This work could pave the way to microbiome-smart breeding that harnesses beneficial plant-microbiome interactions to promote sustainable agriculture

Root volatiles in plant-plant interactions I: Characterization of root sesquiterpene emissions from Centaurea stoebe and their effects on other plants

Volatile organic compounds (VOCs) emitted by plant leaves can influence the physiology of neighboring plants. In contrast to interactions above ground, little is known about the role of VOCs in belowground plant-plant interactions. Here, we characterize constitutive root volatile emissions of the spotted knapweed (Centaurea stoebe) and explore the impact of these volatiles on the germination and growth of different sympatric plant species. We show that C. stoebe roots emit high amounts of sesquiterpenes, with estimated release rates of (E)-beta-caryophyllene above 3 ug g-1 dw h-1. Sesquiterpene emissions show little variation between different C. stoebe populations, but vary substantially between different Centaurea species. Through root transcriptome sequencing, we identify six root-expressed sesquiterpene synthases (TPSs). Two root-specific TPSs, CsTPS4 and CsTPS5, are sufficient to produce the full blend of emitted root sesquiterpenes. Volatile exposure experiments demonstrate that C. stoebe root volatiles have neutral to positive effects on the germination and growth of different sympatric neighbors. Thus, constitutive root sesquiterpenes produced by two C. stoebe TPSs are associated with facilitation of sympatric neighboring plants. The release of root VOCs may thus influence C. stoebe abundance and plant community structure in nature

Plant secondary metabolite-dependent plant-soil feedbacks can improve crop yield in the field.

Plant secondary metabolites that are released into the rhizosphere alter biotic and abiotic soil properties, which in turn affect the performance of other plants. How this type of plant-soil feedback affects agricultural productivity and food quality in the field in the context of crop rotations is unknown. Here, we assessed the performance, yield and food quality of three winter wheat varieties growing in field plots whose soils had been conditioned by either wild type or benzoxazinoid-deficient bx1 maize mutant plants. Following maize cultivation, we detected benzoxazinoid-dependent chemical and microbial fingerprints in the soil. The benzoxazinoid fingerprint was still visible during wheat growth, but the microbial fingerprint was no longer detected. Wheat emergence, tillering, growth, and biomass increased in wild type conditioned soils compared to bx1 mutant conditioned soils. Weed cover was similar between soil conditioning treatments, but insect herbivore abundance decreased in benzoxazinoid-conditioned soils. Wheat yield was increased by over 4% without a reduction in grain quality in benzoxazinoid-conditioned soils. This improvement was directly associated with increased germination and tillering. Taken together, our experiments provide evidence that soil conditioning by plant secondary metabolite producing plants can increase yield via plant-soil feedbacks under agronomically realistic conditions. If this phenomenon holds true across different soils and environments, optimizing root exudation chemistry could be a powerful, genetically tractable strategy to enhance crop yields without additional inputs

Soil composition and plant genotype determine benzoxazinoid-mediated plant–soil feedbacks in cereals

Plant–soil feedbacks refer to effects on plants that are mediated by soil modifications caused by the previous plant generation. Maize conditions the surrounding soil by secretion of root exudates including benzoxazinoids (BXs), a class of bioactive secondary metabolites. Previous work found that a BX-conditioned soil microbiota enhances insect resistance while reducing biomass in the next generation of maize plants. Whether these BX-mediated and microbially driven feedbacks are conserved across different soils and response species is unknown. We found the BX-feedbacks on maize growth and insect resistance conserved between two arable soils, but absent in a more fertile grassland soil, suggesting a soil-type dependence of BX feedbacks. We demonstrated that wheat also responded to BX-feedbacks. While the negative growth response to BX-conditioning was conserved in both cereals, insect resistance showed opposite patterns, with an increase in maize and a decrease in wheat. Wheat pathogen resistance was not affected. Finally and consistent with maize, we found the BX-feedbacks to be cultivar-specific. Taken together, BX-feedbacks affected cereal growth and resistance in a soil and genotype-dependent manner. Cultivar-specificity of BX-feedbacks is a key finding, as it hides the potential to optimize crops that avoid negative plant–soil feedbacks in rotations

Bacterial tolerance to host-exuded specialized metabolites structures the maize root microbiome.

Plants exude specialized metabolites from their roots, and these compounds are known to structure the root microbiome. However, the underlying mechanisms are poorly understood. We established a representative collection of maize root bacteria and tested their tolerance against benzoxazinoids (BXs), the dominant specialized and bioactive metabolites in the root exudates of maize plants. In vitro experiments revealed that BXs inhibited bacterial growth in a strain- and compound-dependent manner. Tolerance against these selective antimicrobial compounds depended on bacterial cell wall structure. Further, we found that native root bacteria isolated from maize tolerated the BXs better compared to nonhost Arabidopsis bacteria. This finding suggests the adaptation of the root bacteria to the specialized metabolites of their host plant. Bacterial tolerance to 6-methoxy-benzoxazolin-2-one (MBOA), the most abundant and selective antimicrobial metabolite in the maize rhizosphere, correlated significantly with the abundance of these bacteria on BX-exuding maize roots. Thus, strain-dependent tolerance to BXs largely explained the abundance pattern of bacteria on maize roots. Abundant bacteria generally tolerated MBOA, while low abundant root microbiome members were sensitive to this compound. Our findings reveal that tolerance to plant specialized metabolites is an important competence determinant for root colonization. We propose that bacterial tolerance to root-derived antimicrobial compounds is an underlying mechanism determining the structure of host-specific microbial communities

Genetic basis of microbiome recruitment in pea roots challenged by root rot disease

Legumes play a crucial role in the shift towards more sustainable protein production, but root rot complexes can cause massive yield losses in many legume crops such as pea. The pea root rot complex (PRRC) is caused by various soil borne pathogens that likely act synergistically and influence the composition of the rhizosphere microbiome (Wille et al., 2021). As there is genotypic variation in the abundance of key PRRC taxa and disease susceptibility, we aimed to investigate the genotype effect on the root microbiome composition affecting plant health. This crucial interaction between the plant genotype and its associated microbiome, also known as the holobiont, has the potential to lead to increased resistance to PRRC

Root volatiles in plant-plant interactions II: Root terpenes from Centaurea stoebe modify Taraxacum officinale root chemistry and root herbivore growth

Volatile organic compounds (VOCs) emitted by plant roots can influence the germination and growth of neighboring plants. However, little is known about the effects of root VOCs on plant-herbivore interactions. The spotted knapeed (Centaurea stoebe) constitutively releases high amounts of sesquiterpenes into the rhizosphere. Here, we examine the impact of C. stoebe root VOCs on primary and secondary metabolites of sympatric Taraxacum officinale plants and the resulting plant-mediated effects on a generalist root herbivore, the white grub Melolontha melolontha. We show that exposure of T. officinale to C. stoebe root VOCs does not affect the accumulation of defensive secondary metabolites, but modulates carbohydrate and total protein levels in T. officinale roots. Furthermore, VOC exposure increases M. melolontha growth on T. officinale plants. Exposure of T. officinale to a major C. stoebe root VOC, the sesquiterpene (E)-β-caryophyllene, partially mimics the effect of the full root VOC blend on M. melolontha growth. Thus, releasing root VOCs can modify plant-herbivore interactions of neighboring plants. The release of VOCs to increase the susceptibility of other plants may be a form of plant offense

Root-exuded benzoxazinoids can alleviate negative plant–soil feedbacks

- Plants can suppress the growth of other plants by modifying soil properties. These negative plant–soil feedbacks are often species-specific, suggesting that some plants possess resistance strategies. However, the underlying mechanisms remain largely unknown. - Here, we investigated whether benzoxazinoids, a class of dominant secondary metabolites that are exuded into the soil by maize and other cereals, allow maize plants to cope with plant–soil feedbacks. - We find that three out of five tested crop species reduce maize (Zea mays L.) performance via negative plant–soil feedbacks relative to the mean across species. This effect is partially alleviated by the capacity of maize plants to produce benzoxazinoids. Soil complementation with purified benzoxazinoids restores the protective effect for benzoxazinoid-deficient mutants. Sterilization and reinoculation experiments suggest that benzoxazinoid-mediated protection acts via changes in soil biota. Substantial variation of the protective effect between experiments and soil types illustrates context dependency. - In conclusion, exuded plant secondary metabolites allow plants to cope with plant–soil feedbacks. These findings expand the functional repertoire of plant secondary metabolites and reveal a mechanism by which plants can resist negative effects of soil feedbacks. The uncovered phenomenon may represent a promising avenue to stabilize plant performance in crop rotations

Fördert die Mikrobendiversitätdie pflanzliche Resistenz?Mögliche Rolle von Wurzel-assoziierten Mikrobiomen bei der Züchtung

Mikroorganismen: Ein vitales und vielfältiges Bodenleben ist wichtig für eine gesunde Kultur. Die Partnerschaft mit nützlichen Mikroorganismen kann unter anderem die Widerstandsfähigkeit von Pflanzen gegen Krankheiten erhöhen. Die Züchtungsgruppe am FiBL erforscht, wie dieses Wissen bei Nutzpflanzen angewendet werden kann