Inhibition of broomrape germination by 2,4-diacetylphloroglucinol produced by environmental Pseudomonas.

Parasitic weeds such as broomrapes (Phelipanche ramosa and Orobanche cumana) cause severe damage to crops and their development must be controlled. Given that phloroglucinol compounds (PGCs) produced by environmental Pseudomonas could be toxic towards certain plants, we assessed the potential herbicidal effect of the bacterial model Pseudomonas ogarae F113, a PGCs-producing bacterium, on parasitic weed. By combining the use of a mutagenesis approach and of pure PGCs, we evaluated the in vitro effect of PGC-produced by P. ogarae F113 on broomrape germination and assessed the protective activity of a PGC-producing bacteria on oilseed rape (Brassica napus) against P. ramosa in non-sterile soils. We showed that the inhibition of the germination depends on the PGCs molecular structure and their concentrations as well as the broomrape species and pathovars. This inhibition caused by the PGCs is irreversible, causing a brown coloration of the broomrape seeds. The inoculation of PGCs-producing bacteria limited the broomrape infection of P. ramosa, without affecting the host growth. Moreover, elemental profiling analysis of oilseed rape revealed that neither F113 nor applied PGCs affected the nutrition capacity of the oilseed rape host. Our study expands the knowledge on plant-beneficial Pseudomonas as weed biocontrol agents and opens new avenues for the development of natural bioherbicides to enhance crop yield.

Dimethylpolysulfides production as the major mechanism behind wheat fungal pathogen biocontrol, by Arthrobacter and Microbacterium actinomycetes.

IMPORTANCE: As the management of wheat fungal diseases becomes increasingly challenging, the use of bacterial agents with biocontrol potential against the two major wheat phytopathogens, Fusarium graminearum and Zymoseptoria tritici, may prove to be an interesting alternative to conventional pest management. Here, we have shown that dimethylpolysulfide volatiles are ubiquitously and predominantly produced by wheat-associated Microbacterium and Arthrobacter actinomycetes, displaying antifungal activity against both pathogens. By limiting pathogen growth and DON virulence factor production, the use of such DMPS-producing strains as soil biocontrol inoculants could limit the supply of pathogen inocula in soil and plant residues, providing an attractive alternative to dimethyldisulfide fumigant, which has many non-targeted toxicities. Notably, this study demonstrates the importance of bacterial volatile organic compound uptake by inhibited F. graminearum, providing new insights for the study of volatiles-mediated toxicity mechanisms within bacteria-fungus signaling crosstalk.

Two novel species isolated from wheat rhizospheres in Serbia: Pseudomonas serbica sp. nov. and Pseudomonas serboccidentalis sp. nov.

Pseudomonas strains IT-194P, IT-215P, IT-P366T and IT-P374T were isolated from the rhizospheres of wheat grown in soils sampled from different fields (some of them known to be disease-suppressive) located near Mionica, Serbia. Phylogenetic analysis of the 16S rRNA genes and of whole genome sequences showed that these strains belong to two potentially new species, one containing strains IT-P366T and IT-194P and clustering (whole genome analysis) next to P. umsongensis DSM16611T, and another species containing strains IT-P374T and IT-215P and clustering next to P. koreensis LMG21318T. Genome analysis confirmed the proposition of novel species, as ANI was below the threshold of 95% and dDDH below 70% for strains IT-P366T (compared with P. umsongensis DSM16611T) and IT-P374T (compared with P. koreensis LMG21318T). Unlike P. umsongensis DSM16611T, strains of P. serbica can grow on D-mannitol, but not on pectin, D-galacturonic acid, L-galactonic acid lactone and α-hydroxybutyric acid. In contrary to P. koreensis LMG21318T, strains of P. serboccidentalis can use sucrose, inosine and α-ketoglutaric acid (but not L-histidine) as carbon sources. Altogether, these results indicate the existence of two novel species for which we propose the names Pseudomonas serbica sp. nov., with the type strain IT-P366T (=CFBP 9060 T = LMG 32732 T = EML 1791 T) and Pseudomonas serboccidentalis sp. nov., with the type strain IT-P374T (=CFBP 9061 T = LMG 32734 T = EML 1792 T). Strains from this study presented a set of phytobeneficial functions modulating plant hormonal balance, plant nutrition and plant protection, suggesting a potential as Plant Growth-Promoting Rhizobacteria (PGPR).

Wheat Metabolite Interferences on Fluorescent Pseudomonas Physiology Modify Wheat Metabolome through an Ecological Feedback.

Plant roots exude a wide variety of secondary metabolites able to attract and/or control a large diversity of microbial species. In return, among the root microbiota, some bacteria can promote plant development. Among these, Pseudomonas are known to produce a wide diversity of secondary metabolites that could have biological activity on the host plant and other soil microorganisms. We previously showed that wheat can interfere with Pseudomonas secondary metabolism production through its root metabolites. Interestingly, production of Pseudomonas bioactive metabolites, such as phloroglucinol, phenazines, pyrrolnitrin, or acyl homoserine lactones, are modified in the presence of wheat root extracts. A new cross metabolomic approach was then performed to evaluate if wheat metabolic interferences on Pseudomonas secondary metabolites production have consequences on wheat metabolome itself. Two different Pseudomonas strains were conditioned by wheat root extracts from two genotypes, leading to modification of bacterial secondary metabolites production. Bacterial cells were then inoculated on each wheat genotypes. Then, wheat root metabolomes were analyzed by untargeted metabolomic, and metabolites from the Adular genotype were characterized by molecular network. This allows us to evaluate if wheat differently recognizes the bacterial cells that have already been into contact with plants and highlights bioactive metabolites involved in wheat-Pseudomonas interaction.

A Cross-Metabolomic Approach Shows that Wheat Interferes with Fluorescent Pseudomonas Physiology through Its Root Metabolites.

Roots contain a wide variety of secondary metabolites. Some of them are exudated in the rhizosphere, where they are able to attract and/or control a large diversity of microbial species. In return, the rhizomicrobiota can promote plant health and development. Some rhizobacteria belonging to the Pseudomonas genus are known to produce a wide diversity of secondary metabolites that can exert a biological activity on the host plant and on other soil microorganisms. Nevertheless, the impact of the host plant on the production of bioactive metabolites by Pseudomonas is still poorly understood. To characterize the impact of plants on the secondary metabolism of Pseudomonas, a cross-metabolomic approach has been developed. Five different fluorescent Pseudomonas strains were thus cultivated in the presence of a low concentration of wheat root extracts recovered from three wheat genotypes. Analysis of our metabolomic workflow revealed that the production of several Pseudomonas secondary metabolites was significantly modulated when bacteria were cultivated with root extracts, including metabolites involved in plant-beneficial properties.

Secondary metabolites from plant-associated Pseudomonas are overproduced in biofilm.

Plant rhizosphere soil houses complex microbial communities in which microorganisms are often involved in intraspecies as well as interspecies and inter-kingdom signalling networks. Some members of these networks can improve plant health thanks to an important diversity of bioactive secondary metabolites. In this competitive environment, the ability to form biofilms may provide major advantages to microorganisms. With the aim of highlighting the impact of bacterial lifestyle on secondary metabolites production, we performed a metabolomic analysis on four fluorescent Pseudomonas strains cultivated in planktonic and biofilm colony conditions. The untargeted metabolomic analysis led to the detection of hundreds of secondary metabolites in culture extracts. Comparison between biofilm and planktonic conditions showed that bacterial lifestyle is a key factor influencing Pseudomonas metabolome. More than 50% of the detected metabolites were differentially produced according to planktonic or biofilm lifestyles, with the four Pseudomonas strains overproducing several secondary metabolites in biofilm conditions. In parallel, metabolomic analysis associated with genomic prediction and a molecular networking approach enabled us to evaluate the impact of bacterial lifestyle on chemically identified secondary metabolites, more precisely involved in microbial interactions and plant-growth promotion. Notably, this work highlights the major effect of biofilm lifestyle on acyl-homoserine lactone and phenazine production in P. chlororaphis strains.

Feedback Regulation of N Fixation in Frankia-Alnus Symbiosis Through Amino Acids Profiling in Field and Greenhouse Nodules.

Symbiosis established between actinorhizal plants and Frankia spp., which are nitrogen-fixing actinobacteria, promotes nodule organogenesis, the site of metabolic exchange. The present study aimed to identify amino acid markers involved in Frankia-Alnus interactions by comparing nodules and associated roots from field and greenhouse samples. Our results revealed a high level of citrulline in all samples, followed by arginine (Arg), aspartate (Asp), glutamate (Glu), γ-amino-n-butyric acid (GABA), and alanine (Ala). Interestingly, the field metabolome approach highlighted more contrasted amino acid patterns between nodules and roots compared with greenhouse samples. Indeed, 12 amino acids had a mean relative abundance significantly different between field nodule and root samples, against only four amino acids in greenhouse samples, underlining the importance of developing "ecometabolome" approaches. In order to monitor the effects on Frankia cells (respiration and nitrogen fixation activities) of amino acid with an abundance pattern evocative of a role in symbiosis, in-vitro assays were performed by supplementing them in nitrogen-free cultures. Amino acids had three types of effects: i) those used by Frankia as nitrogen source (Glu, Gln, Asp), ii) amino acids stimulating both nitrogen fixation and respiration (e.g., Cit, GABA, Ala, valine, Asn), and iii) amino acids triggering a toxic effect (Arg, histidine). In this paper, a N-metabolic model was proposed to discuss how the host plant and bacteria modulate amino acids contents in nodules, leading to a fine regulation sustaining high bacterial nitrogen fixation.

Identification of a small set of genes commonly regulated in rice roots in response to beneficial rhizobacteria.

Rhizosphere bacteria, whether phytopathogenic or phytobeneficial, are thought to be perceived by the plant as a threat. Plant Growth-Promoting Rhizobacteria (PGPR), such as many strains of the Azospirillum genus known as the main phytostimulator of cereals, cooperate with host plants and favorably affect their growth and health. An earlier study of rice root transcriptome, undertaken with two rice cultivars and two Azospirillum strains, revealed a strain-dependent response during the rice-Azospirillum association and showed that only a few genes, including some implicated in plant defense, were commonly regulated in all tested conditions. Here, a set of genes was selected from previous studies and their expression was monitored by qRT-PCR in rice roots inoculated with ten PGPR strains isolated from various plants and belonging to various genera (Azospirillum, Herbaspirillum, Paraburkholderia). A common expression pattern was highlighted for four genes that are proposed to be markers of the rice-PGPR interaction: two genes involved in diterpenoid phytoalexin biosynthesis (OsDXS3 and OsDTC2) and one coding for an uncharacterized protein (Os02g0582900) were significantly induced by PGPR whereas one defense-related gene encoding a pathogenesis-related protein (PR1b, Os01g0382000) was significantly repressed. Interestingly, exposure to a rice bacterial pathogen also triggered the expression of OsDXS3 while the expression of Os02g0582900 and PR1b was down-regulated, suggesting that these genes might play a key role in rice-bacteria interactions. Integration of these results with previous data led us to propose that the jasmonic acid signaling pathway might be triggered in rice roots upon inoculation with PGPR.

A common metabolomic signature is observed upon inoculation of rice roots with various rhizobacteria.

Plant growth-promoting rhizobacteria (PGPR), whose growth is stimulated by root exudates, are able to improve plant growth and health. Among those, bacteria of the genus Azospirillum were shown to affect root secondary metabolite content in rice and maize, sometimes without visible effects on root architecture. Transcriptomic studies also revealed that expression of several genes involved in stress and plant defense was affected, albeit with fewer genes when a strain was inoculated onto its original host cultivar. Here, we investigated, via a metabolic profiling approach, whether rice roots responded differently and with gradual intensity to various PGPR, isolated from rice or not. A common metabolomic signature of nine compounds was highlighted, with the reduced accumulation of three alkylresorcinols and increased accumulation of two hydroxycinnamic acid amides (HCAA), identified as N-p-coumaroylputrescine and N-feruloylputrescine. This was accompanied by the increased transcription of two genes involved in the N-feruloylputrescine biosynthetic pathway. Interestingly, exposure to a rice bacterial pathogen triggered a reduced accumulation of these HCAA in roots, a result contrasting with previous reports of increased HCAA content in leaves upon pathogen infection. Accumulation of HCAA, that are potential antimicrobial compounds, might be considered as a primary reaction of plant to bacterial perception.

Disruption of phenylalanine hydroxylase reduces adult lifespan and fecundity, and impairs embryonic development in parthenogenetic pea aphids.

Phenylalanine hydroxylase (PAH) is a key tyrosine-biosynthetic enzyme involved in neurological and melanin-associated physiological processes. Despite extensive investigations in holometabolous insects, a PAH contribution to insect embryonic development has never been demonstrated. Here, we have characterized, for the first time, the PAH gene in a hemimetabolous insect, the aphid Acyrthosiphon pisum. Phylogenetic and sequence analyses confirmed that ApPAH is closely related to metazoan PAH, exhibiting the typical ACT regulatory and catalytic domains. Temporal expression patterns suggest that ApPAH has an important role in aphid developmental physiology, its mRNA levels peaking at the end of embryonic development. We used parental dsApPAH treatment to generate successful knockdown in aphid embryos and to study its developmental role. ApPAH inactivation shortens the adult aphid lifespan and considerably affects fecundity by diminishing the number of nymphs laid and impairing embryonic development, with newborn nymphs exhibiting severe morphological defects. Using single nymph HPLC analyses, we demonstrated a significant tyrosine deficiency and a consistent accumulation of the upstream tyrosine precursor, phenylalanine, in defective nymphs, thus confirming the RNAi-mediated disruption of PAH activity. This study provides first insights into the role of PAH in hemimetabolous insects and demonstrates that this metabolic gene is essential for insect embryonic development.

Physiological effects of major up-regulated Alnus glutinosa peptides on Frankia sp. ACN14a.

Alnus glutinosa has been shown previously to synthesize, in response to nodulation by Frankia sp. ACN14a, an array of peptides called Alnus symbiotic up-regulated peptides (ASUPs). In a previous study one peptide (Ag5) was shown to bind to Frankia nitrogen-fixing vesicles and to modify their porosity. Here we analyse four other ASUPs, alongside Ag5, to determine whether they have different physiological effects on in vitro grown Frankia sp. ACN14a. The five studied peptides were shown to have different effects on nitrogen fixation, respiration, growth, the release of ions and amino acids, as well as on cell clumping and cell lysis. The mRNA abundance for all five peptides was quantified in symbiotic nodules and one (Ag11) was found to be more abundant in the meristem part of the nodule. These findings point to some peptides having complementary effects on Frankia cells.

Alnus peptides modify membrane porosity and induce the release of nitrogen-rich metabolites from nitrogen-fixing Frankia.

Actinorhizal plant growth in pioneer ecosystems depends on the symbiosis with the nitrogen-fixing actinobacterium Frankia cells that are housed in special root organs called nodules. Nitrogen fixation occurs in differentiated Frankia cells known as vesicles. Vesicles lack a pathway for assimilating ammonia beyond the glutamine stage and are supposed to transfer reduced nitrogen to the plant host cells. However, a mechanism for the transfer of nitrogen-fixation products to the plant cells remains elusive. Here, new elements for this metabolic exchange are described. We show that Alnus glutinosa nodules express defensin-like peptides, and one of these, Ag5, was found to target Frankia vesicles. In vitro and in vivo analyses showed that Ag5 induces drastic physiological changes in Frankia, including an increased permeability of vesicle membranes. A significant release of nitrogen-containing metabolites, mainly glutamine and glutamate, was found in N2-fixing cultures treated with Ag5. This work demonstrates that the Ag5 peptide is central for Frankia physiology in nodules and uncovers a novel cellular function for this large and widespread defensin peptide family.

Insects recycle endosymbionts when the benefit is over.

Symbiotic associations are widespread in nature and represent a driving force in evolution. They are known to impact fitness, and thereby shape the host phenotype. Insects subsisting on nutritionally poor substrates have evolved mutualistic relationships with intracellular symbiotic bacteria (endosymbionts) that supply them with metabolic components lacking in their diet. In many species, endosymbionts are hosted within specialized host cells, called the bacteriocytes, and transmitted vertically across host generations. How hosts balance the costs and benefits of having endosymbionts, and whether and how they adjust symbiont load to their physiological needs, remains largely unexplored. By investigating the cereal weevil Sitophilus association with the Sodalis pierantonius endosymbiont, we discover that endosymbiont populations intensively multiply in young adults, before being rapidly eliminated within few days. We show that young adults strongly depend on endosymbionts and that endosymbiont proliferation after metamorphosis matches a drastic host physiological need for the tyrosine (Tyr) and phenylalanine (Phe) amino acids to rapidly build their protective exoskeleton. Tyr and Phe are precursors of the dihydroxyphenylalanine (DOPA) molecule that is an essential component for the cuticle synthesis. Once the cuticle is achieved, DOPA reaches high amounts in insects, which triggers endosymbiont elimination. This elimination relies on apoptosis and autophagy activation, allowing digestion and recycling of the endosymbiont material. Thus, the weevil-endosymbiont association reveals an adaptive interplay between metabolic and cellular functions that minimizes the cost of symbiosis and speeds up the exoskeleton formation during a critical phase when emerging adults are especially vulnerable.

Tyrosine pathway regulation is host-mediated in the pea aphid symbiosis during late embryonic and early larval development.

BACKGROUND: Nutritional symbioses play a central role in insects' adaptation to specialized diets and in their evolutionary success. The obligatory symbiosis between the pea aphid, Acyrthosiphon pisum, and the bacterium, Buchnera aphidicola, is no exception as it enables this important agricultural pest insect to develop on a diet exclusively based on plant phloem sap. The symbiotic bacteria provide the host with essential amino acids lacking in its diet but necessary for the rapid embryonic growth seen in the parthenogenetic viviparous reproduction of aphids. The aphid furnishes, in exchange, non-essential amino acids and other important metabolites. Understanding the regulations acting on this integrated metabolic system during the development of this insect is essential in elucidating aphid biology. RESULTS: We used a microarray-based approach to analyse gene expression in the late embryonic and the early larval stages of the pea aphid, characterizing, for the first time, the transcriptional profiles in these developmental phases. Our analyses allowed us to identify key genes in the phenylalanine, tyrosine and dopamine pathways and we identified ACYPI004243, one of the four genes encoding for the aspartate transaminase (E.C. 2.6.1.1), as specifically regulated during development. Indeed, the tyrosine biosynthetic pathway is crucial for the symbiotic metabolism as it is shared between the two partners, all the precursors being produced by B. aphidicola. Our microarray data are supported by HPLC amino acid analyses demonstrating an accumulation of tyrosine at the same developmental stages, with an up-regulation of the tyrosine biosynthetic genes. Tyrosine is also essential for the synthesis of cuticular proteins and it is an important precursor for cuticle maturation: together with the up-regulation of tyrosine biosynthesis, we observed an up-regulation of cuticular genes expression. We were also able to identify some amino acid transporter genes which are essential for the switch over to the late embryonic stages in pea aphid development. CONCLUSIONS: Our data show that, in the development of A. pisum, a specific host gene set regulates the biosynthetic pathways of amino acids, demonstrating how the regulation of gene expression enables an insect to control the production of metabolites crucial for its own development and symbiotic metabolism.

Biological and biochemical characteristics for quality control of Harmonia axyridis (Pallas) (Coleoptera, Coccinellidae) reared on a liver-based diet.

Biological and biochemical parameters of a flightless strain of Harmonia axyridis, fed on a pork liver-based artificial diet and on Ephestia kuehniella eggs as controls, were compared. The diet-grown larvae showed a significantly longer developmental time and a lower adult emergence rate compared to control larvae. The weights of the newly emerged adults were significantly higher for adults fed E. kuehniella eggs during their larval stages than fed the artificial diet. In contrast, larval food source had no effect on the duration of the pre-oviposition period or adult longevity. For adults fed on E. kuehniella eggs as larvae, a significantly longer pre-oviposition period, lower daily weight gain and fecundity were found for the diet-fed females compared to those fed on E. kuehniella eggs throughout the life span. The adult food source had no significant effect on longevity and fertility. Lower amino acid and fatty acid contents (in particular C16:1 and C18:3n-3) were found for the prepupae and newly emerged females obtained from diet-reared larvae compared to controls. Deficiencies in fatty acids C16:1 and C18:3n-3 were also observed in females obtained from E. kuehniella egg-reared larvae and fed on diet from adult emergence. The analyses of the foods showed deficiencies in artificial diet, especially for some amino and fatty acids. The results suggest a non-optimal composition of the artificial diet and some possibilities for its improvement. However, this polyphagous predator could be reared from first instar larvae to fully reproductive adults on a pork liver-based artificial diet.