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Striga hermonthica (Del.) Benth is a devastating parasitic weed in Sub-Saharan Africa (SSA) and its soil seedbank is the major factor contributing to its prevalence and persistence. To date, there is a little information on the Striga seedbank density in agricultural fields in SSA due to the lack of reliable detection and quantification methods. We developed a high-throughput method that combines density- and size-based separation techniques with quantitative polymerase chain reaction (qPCR)-based detection of Striga seeds in soil. The method was optimised and validated by introducing increasing numbers of Striga seeds in two physicochemically different Striga-free agricultural soils. The results showed that as little as one seed of S. hermonthica per 150 g of soil could be detected. This technique was subsequently tested on soil samples of 48 sorghum fields from different agro-ecological zones in Ethiopia to map the geospatial distribution of the Striga seedbank along a trajectory of more than 1500 km. Considerable variation in Striga seed densities was observed. Striga seeds were detectable in 75% of the field soils with densities up to 86 seeds per 150 g of soil. The Striga seed density in soil and the number of emerged Striga plants in the field showed a non-linear relationship. In conclusion, the method developed allows for accurate mapping of the Striga seedbank in physicochemically diverse SSA field soils and can be used to assess the impact of management strategies on Striga seedbank dynamics.

Various root-colonizing bacterial species can promote plant growth and trigger systemic resistance against aboveground leaf pathogens and herbivore insects. To date, the underlying metabolic signatures of these rhizobacteria-induced plant phenotypes are poorly understood. To identify core metabolic pathways that are targeted by growth-promoting rhizobacteria, we used combinations of three plant species and three rhizobacterial species and interrogated plant shoot chemistry by untargeted metabolomics. A substantial part (50-64%) of the metabolites detected in plant shoot tissue was differentially affected by the rhizobacteria. Among others, the phenylpropanoid pathway was targeted by the rhizobacteria in each of the three plant species. Differential regulation of the various branches of the phenylpropanoid pathways showed an association with either plant growth promotion or growth reduction. Overall, suppression of flavonoid biosynthesis was associated with growth promotion, while growth reduction showed elevated levels of flavonoids. Subsequent assays with twelve Arabidopsis flavonoid biosynthetic mutants revealed that the proanthocyanidin branch plays an essential role in rhizobacteria-mediated growth promotion. Our study also showed that a number of pharmaceutically and nutritionally relevant metabolites in the plant shoot were significantly increased by rhizobacterial treatment, providing new avenues to use rhizobacteria to tilt plant metabolism towards the biosynthesis of valuable natural plant products.

Aims Striga hermonthica is a devastating parasitic weed in Sub-Saharan Africa (SSA) and its persistent soil seedbank is the major contributing factor for its prevalence and persistence. So far, there is little to no information on the Striga seedbank density in agricultural fields in SSA due to the lack of reliable detection and quantification methods. Methods We developed a high-throughput method that combines density- and size-based separation techniques with quantitative polymerase chain reaction (qPCR)-based detection of Striga seeds in soil. The method was optimized and validated on two physicochemically different Striga-free Dutch agricultural soils by introducing increasing numbers of Striga seeds (0, 1, 3, 9, 27, 81 and 243 seeds). Results The results showed that as little as one seed of S. hermonthica per 150 g of soil can be detected. This technique was subsequently tested on soil samples of 48 sorghum fields from different agro-ecological zones in Ethiopia to map the geospatial distribution of the Striga seedbank along a trajectory of more than 1500 km. Considerable variation in Striga seed densities was observed for these soils: in 75% of the field soils, Striga seeds were detectable up to 86 seeds per 150 g of soil. Correlation analyses further revealed a significant non-linear relationship between the seed density and Striga incidence assessed in the same sorghum field soils at the time of soil sampling. Conclusions The method developed allows for high-through-put and accurate mapping of the Striga seedbank in physicochemically diverse field soils and can be used to predict Striga incidence and to assess the impact of management strategies on Striga seedbank dynamics.

Several root-colonizing bacterial species can simultaneously promote plant growth and induce systemic resistance. How these rhizobacteria modulate plant metabolism to accommodate the carbon and energy demand from these two competing processes is largely unknown. Here, we show that strains of three Paraburkholderia species, P. graminis PHS1 (Pbg), P. hospita mHSR1 (Pbh), and P. terricola mHS1 (Pbt), upon colonization of the roots of two Broccoli cultivars led to cultivar-dependent increases in biomass, changes in primary and secondary metabolism and induced resistance against the bacterial leaf pathogen Xanthomonas campestris. Strains that promoted growth led to greater accumulation of soluble sugars in the shoot and particularly fructose levels showed an increase of up to 280-fold relative to the non-treated control plants. Similarly, a number of secondary metabolites constituting chemical and structural defense, including flavonoids, hydroxycinnamates, stilbenoids, coumarins and lignins, showed greater accumulation while other resource-competing metabolite pathways were depleted. High soluble sugar generation, efficient sugar utilization, and suppression or remobilization of resource-competing metabolites potentially contributed to curb the tradeoff between the carbon and energy demanding processes induced by Paraburkholderia-Broccoli interaction. Collectively, our results provide a comprehensive and integrated view of the temporal changes in plant metabolome associated with rhizobacteria-mediated plant growth promotion and induced resistance.

Genome-wide analysis of plant-growth-promoting Pseudomonas fluorescens strain SS101 (PfSS101) followed by site-directed mutagenesis previously suggested that sulfur assimilation may play an important role in growth promotion and induced systemic resistance in Arabidopsis. Here, we investigated the effects of sulfur metabolism in PfSS101 on growth, defense, and shoot metabolomes of Arabidopsis and the Brassica crop, Broccoli. Root tips of seedlings of Arabidopsis and two Broccoli cultivars were treated with PfSS101 or with a mutant disrupted in the adenylsulfate reductase cysH, a key gene in cysteine and methionine biosynthesis. Phenotyping of plants treated with wild-type PfSS101 or its cysH mutant revealed that sulfur assimilation in PfSS101 was associated with enhanced growth of Arabidopsis but with a reduction in shoot biomass of two Broccoli cultivars. Untargeted metabolomics revealed that cysH-mediated sulfur assimilation in PfSS101 had significant effects on shoot chemistry of Arabidopsis, in particular on chain elongation of aliphatic glucosinolates (GLSs) and on indole metabolites, including camalexin and the growth hormone indole-3-acetic acid. In Broccoli, PfSS101 sulfur assimilation significantly upregulated the relative abundance of several shoot metabolites, in particular, indolic GLSs and phenylpropanoids. These metabolome changes in Broccoli plants coincided with PfSS101-mediated suppression of leaf infections by Xanthomonas campestris. Our study showed the metabolic interconnectedness of plants and their root-associated microbiota.

Covering: 1981-2017Plants are colonized by an astounding number of microorganisms that can reach cell densities much greater than the number of plant cells. Various plant-associated microorganisms can have profound beneficial effects on plant growth, development, physiology and tolerance to (a)biotic stress. In return, plants release metabolites into their direct surroundings, thereby feeding the microbial community and influencing their composition, gene expression and the production of secondary metabolites. Similarly, microbes living on and in plant tissue may induce known and yet unknown biosynthetic pathways in plants leading to diverse alterations in the plant metabolome. Here, we provide an overview of the impact of beneficial microbiota on plant chemistry, with an emphasis on bacteria living on or inside root tissues. We will also provide new perspectives on deciphering the yet untapped potential of microbe-mediated alteration of plant chemistry as an alternative platform to discover new pathways, genes and enzymes involved the biosynthesis of high value natural plant products.

Pseudomonas fluorescens strain SS101 (Pf.SS101) promotes growth of Arabidopsis thaliana, enhances greening and lateral root formation, and induces systemic resistance (ISR) against the bacterial pathogen Pseudomonas syringae pv. tomato (Pst). Here, targeted and untargeted approaches were adopted to identify bacterial determinants and underlying mechanisms involved in plant growth promotion and ISR by Pf.SS101. Based on targeted analyses, no evidence was found for volatiles, lipopeptides and siderophores in plant growth promotion by Pf.SS101. Untargeted, genome-wide analyses of 7488 random transposon mutants of Pf.SS101 led to the identification of 21 mutants defective in both plant growth promotion and ISR. Many of these mutants, however, were auxotrophic and impaired in root colonization. Genetic analysis of three mutants followed by site-directed mutagenesis, genetic complementation and plant bioassays revealed the involvement of the phosphogluconate dehydratase gene edd, the response regulator gene colR and the adenylsulfate reductase gene cysH in both plant growth promotion and ISR. Subsequent comparative plant transcriptomics analyses strongly suggest that modulation of sulfur assimilation, auxin biosynthesis and transport, steroid biosynthesis and carbohydrate metabolism in Arabidopsis are key mechanisms linked to growth promotion and ISR by Pf.SS101.

Disease-suppressive soils are ecosystems in which plants suffer less from root infections due to the activities of specific microbial consortia. The characteristics of soils suppressive to specific fungal root pathogens are comparable to those of adaptive immunity in animals, as reported by Raaijmakers and Mazzola (Science 352:1392–3, 2016), but the mechanisms and microbial species involved in the soil suppressiveness are largely unknown. Previous taxonomic and metatranscriptome analyses of a soil suppressive to the fungal root pathogen Rhizoctonia solani revealed that members of the Burkholderiaceae family were more abundant and more active in suppressive than in non-suppressive soils. Here, isolation, phylogeny, and soil bioassays revealed a significant disease-suppressive activity for representative isolates of Burkholderia pyrrocinia, Paraburkholderia caledonica, P. graminis, P. hospita, and P. terricola. In vitro antifungal activity was only observed for P. graminis. Comparative genomics and metabolite profiling further showed that the antifungal activity of P. graminis PHS1 was associated with the production of sulfurous volatile compounds encoded by genes not found in the other four genera. Site-directed mutagenesis of two of these genes, encoding a dimethyl sulfoxide reductase and a cysteine desulfurase, resulted in a loss of antifungal activity both in vitro and in situ. These results indicate that specific members of the Burkholderiaceae family contribute to soil suppressiveness via the production of sulfurous volatile compounds.

There is increasing demand to bring the level of metabolomics analyses down to the tissue or cell level. Significant progress has been made involving the use of in situ metabolomics imaging techniques where no tissue collection or extraction is needed prior to analysis. In this chapter we describe a relatively new method which is simple and easy to use. No ectopic matrix or vacuum is required, and analyses are performed with living plant materials directly from (or even still attached to) the plant. Although relatively straightforward, there are still a few caveats as regards this method which are described at the end of the chapter.

There is increasing demand to bring the level of metabolomics analyses down to the tissue or cell level. Significant progress has been made involving the use of in situ metabolomics imaging techniques where no tissue collection or extraction is needed prior to analysis. In this chapter we describe a relatively new method which is simple and easy to use. No ectopic matrix or vacuum is required, and analyses are performed with living plant materials directly from (or even still attached to) the plant. Although relatively straightforward, there are still a few caveats as regards this method which are described at the end of the chapter.

Laser-ablation electrospray ionization (LAESI)-mass spectrometry imaging has been applied to contrasting plant organs to assess its potential as a procedure for performing in vivo metabolomics in plants. In a proof-of-concept experiment, purple/white segmented Phalaenopsis spp. petals were first analyzed using standard liquid chromatography-mass spectrometry analyses of separate extracts made specifically from the purple and white regions. Discriminatory compounds were defined and putatively annotated. LAESI analyses were then performed on living tissues, and these metabolites were then relocalized within the LAESI-generated data sets of similar tissues. Maps were made to illustrate their locations across the petals. Results revealed that, as expected, anthocyanins always mapped to the purple regions. Certain other (nonvisible) polyphenols were observed to colocalize with the anthocyanins, whereas others were found specifically within the white tissues. In a contrasting example, control and Cladosporium fulvum-infected tomato (Solanum lycopersicum) leaves were subjected to the same procedures, and it could be observed that the alkaloid tomatine has clear heterogeneous distribution across the tomato leaf lamina. Furthermore, LAESI analyses revealed perturbations in alkaloid content following pathogen infection. These results show the clear potential of LAESI-based imaging approaches as a convenient and rapid way to perform metabolomics analyses on living tissues. However, a range of limitations and factors have also been identified that must be taken into consideration when interpreting LAESI-derived data. Such aspects deserve further evaluation before this approach can be applied in a routine manner.

Pseudomonas fluorescens strain SS101 (Pf.SS101) promotes growth of Arabidopsis thaliana, enhances greening and lateral root formation, and induces systemic resistance (ISR) against the bacterial pathogen Pseudomonas syringae pv. tomato (Pst). Here, targeted and untargeted approaches were adopted to identify bacterial determinants and underlying mechanisms involved in plant growth promotion and ISR by Pf.SS101. Based on targeted analyses, no evidence was found for volatiles, lipopeptides and siderophores in plant growth promotion by Pf.SS101. Untargeted, genome-wide analyses of 7488 random transposon mutants of Pf.SS101 led to the identification of 21 mutants defective in both plant growth promotion and ISR. Many of these mutants, however, were auxotrophic and impaired in root colonization. Genetic analysis of three mutants followed by site-directed mutagenesis, genetic complementation and plant bioassays revealed the involvement of the phosphogluconate dehydratase gene edd, the response regulator gene colR and the adenylsulfate reductase gene cysH in both plant growth promotion and ISR. Subsequent comparative plant transcriptomics analyses strongly suggest that modulation of sulfur assimilation, auxin biosynthesis and transport, steroid biosynthesis and carbohydrate metabolism in Arabidopsis are key mechanisms linked to growth promotion and ISR by Pf.SS101.

The Cf-5 gene of tomato confers resistance to strains of the fungal pathogen Cladosporium fulvum carrying the avirulence gene Avr5. Although Cf-5 has been cloned, Avr5 has remained elusive. We report the cloning of Avr5 using a combined bioinformatic and transcriptome sequencing approach. RNA-Seq was performed on the sequenced race 0 strain (0WU; carrying Avr5), as well as a race 5 strain (IPO 1979; lacking a functional Avr5 gene) during infection of susceptible tomato. Forty-four in planta–induced C. fulvum candidate effector (CfCE) genes of 0WU were identified that putatively encode a secreted, small cysteine-rich protein. An expressed transcript sequence comparison between strains revealed two polymorphic CfCE genes in IPO 1979. One of these conferred avirulence to IPO 1979 on Cf-5 tomato following complementation with the corresponding 0WU allele, confirming identification of Avr5. Complementation also led to increased fungal biomass during infection of susceptible tomato, signifying a role for Avr5 in virulence. Seven of eight race 5 strains investigated escape Cf-5-mediated resistance through deletion of the Avr5 gene. Avr5 is heavily flanked by repetitive elements, suggesting that repeat instability, in combination with Cf-5-mediated selection pressure, has led to the emergence of race 5 strains deleted for the Avr5 gene.

· α-Tomatine is an antifungal glycoalkaloid that provides basal defense to tomato (Solanum lycopersicum). However, tomato pathogens overcome this basal defense barrier by the secretion of tomatinases that degrade α-tomatine into the less fungitoxic compounds β-tomatine and tomatidine. Although pathogenic on tomato, it has been reported that the biotrophic fungus Cladosporium fulvum is unable to detoxify α-tomatine. · Here, we present a functional analysis of the glycosyl hydrolase (GH10), CfTom1, which is orthologous to fungal tomatinases. · We show that C. fulvum hydrolyzes α-tomatine into tomatidine in vitro and during the infection of tomato, which is fully attributed to the activity of CfTom1, as shown by the heterologous expression of this enzyme in tomato. Accordingly, ∆cftom1 mutants of C. fulvum are more sensitive to α-tomatine and are less virulent than the wild-type fungus on tomato. · Although α-tomatine is thought to be localized in the vacuole, we show that it is also present in the apoplast, where it is hydrolyzed by CfTom1 on infection. The accumulation of tomatidine during infection appears to be toxic to tomato cells and does not suppress defense responses, as suggested previously. Altogether, our results show that CfTom1 is responsible for the detoxification of α-tomatine by C. fulvum, and is required for full virulence of this fungus on tomato.

In plant-associated Pseudomonas species, the production of several secondary metabolites and exoenzymes is regulated by the GacS/GacA two-component regulatory system (the Gac-system). Here, we investigated if a mutation in the GacS sensor kinase affects the production of volatile organic compounds (VOCs) in P. fluorescens SBW25 (Pf.SBW25) and how this impacts on VOCs-mediated growth promotion and induced systemic resistance of Arabidopsis and tobacco. A total of 205 VOCs were detected by Gas Chromatography Mass Spectrometry (GC-MS) for Pf. SBW25 and the gacS-mutant grown on 2 different media for 3 and 6 days. Discriminant function analysis followed by hierarchical clustering revealed 24 VOCs that were significantly different in their abundance between Pf.SBW25 and the gacS-mutant, which included three acyclic alkenes (3-nonene, 4-undecyne, 1-undecene). These alkenes were significantly reduced by the gacS mutation independently of the growth media and of the incubation time. For Arabidopsis, both Pf.SBW25 and the gacS-mutant enhanced, via VOCs, root and shoot biomass, induced systemic resistance against leaf infections by P. syringae and rhizosphere acidification to the same extent. For tobacco, however, VOCs-mediated effects on shoot and root growth were significantly different between Pf.SBW25 and the gacS-mutant. While Pf.SBW25 inhibited tobacco root growth, the gacS-mutant enhanced root biomass and lateral root formation relative to the non-treated control plants. Collectively these results indicate that the sensor kinase GacS is involved in the regulation of VOCs production in Pf.SBW25, affecting plant growth in a plant species-dependent manner.

There is increasing evidence that volatile organic compounds (VOCs) play an important role in the interactions between fungi and bacteria, two major groups of soil inhabiting microorganisms. Yet, most of the research has been focused on effects of bacterial volatiles on suppression of plant pathogenic fungi whereas little is known about the responses of bacteria to fungal volatiles. In the current study we performed a metabolomics analysis of volatiles emitted by several fungal and oomycetal soil strains under different nutrient conditions and growth stages. The metabolomics analysis of the tested fungal and oomycetal strains revealed different volatile profiles dependent on the age of the strains and nutrient conditions. Furthermore, we screened the phenotypic responses of soil bacterial strains to volatiles emitted by fungi. Two bacteria, Collimonas pratensis Ter291 and Serratia plymuthica PRI-2C, showed significant changes in their motility, in particular to volatiles emitted by Fusarium culmorum. This fungus produced a unique volatile blend, including several terpenes. Four of these terpenes were selected for further tests to investigate if they influence bacterial motility. Indeed, these terpenes induced or reduced swimming and swarming motility of S. plymuthica PRI-2C and swarming motility of C. pratensis Ter291, partly in a concentration-dependent manner. Overall the results of this work revealed that bacteria are able to sense and respond to fungal volatiles giving further evidence to the suggested importance of volatiles as signaling molecules in fungal-bacterial interactions.

The hydraulic conductivity of cut flower stems decreases 4-8 h after cutting, possibly because of up-regulation of L-phenylalanine ammonia-lyase (PAL). To get more insight into the processes linking wounding with the increase in enzyme activity we explored the movement of reactive oxygen species (principally H2O2) in the xylem of Arabidopsis. Following wounding there is a rapid rise in the production of H2O2. The peroxide persists for a long time in the xylem, making feasible its role as a long distance signalling molecule. The link between wound-induced H2O2 in the xylem, up-regulation of PAL activity and decreases in hydraulic conductance postharvest needs further exploration

The hypersensitive response (HR) is considered to be the hallmark of the resistance response of plants to pathogens. To study HR-associated transcriptome and metabolome reprogramming in tomato (Solanum lycopersicum), we used plants that express both a resistance gene to Cladosporium fulvum and the matching avirulence gene of this pathogen. In these plants, massive reprogramming occurred, and we found that the HR and associated processes are highly energy demanding. Ubiquitin-dependent protein degradation, hydrolysis of sugars, and lipid catabolism are used as alternative sources of amino acids, energy, and carbon skeletons, respectively. We observed strong accumulation of secondary metabolites, such as hydroxycinnamic acid amides. Coregulated expression of WRKY transcription factors and genes known to be involved in the HR, in addition to a strong enrichment of the W-box WRKY-binding motif in the promoter sequences of the coregulated genes, point to WRKYs as the most prominent orchestrators of the HR. Our study has revealed several novel HR-related genes, and reverse genetics tools will allow us to understand the role of each individual component in the HR.