Biosynthesis, chemical structure, and structure-activity relationship of orfamide lipopeptides produced by Pseudomonas protegens and related species

Orfamide type cyclic lipopeptides (CLPs) are biosurfactants produced by Pseudomonas and involved in lysis of oomycete zoospores, biocontrol of Rhizoctonia and insecticidal activity against aphids. In this study, we compared the biosynthesis, structural diversity, in vitro and in planta activities of orfamides produced by rhizosphere-derived Pseudomonas protegens and related Pseudornonas species. Genetic characterization together with chemical identification revealed that the main orfamide compound produced by the P. protegens group is orfamide A, while the related strains Pseudomonas sp. CMR5c and CMR12a produce orfamide B. Comparison of orfamide fingerprints led to the discovery of two new orfamide homologs (orfamide F and orfamide G) in Pseudornonas sp. CMR5c. The structures of these two CLPs were determined by nuclear magnetic resonance (NMR) and mass spectrometry (MS) analysis. Mutagenesis and complementation showed that orfamides determine the swarming motility of parental Pseudomonas sp. strain CMR5c and their production was regulated by luxR type regulators. Orfamide A and orfamide B differ only in the identity of a single amino acid, while orfamide B and orfamide G share the same amino acid sequence but differ in length of the fatty acid part. The biological activities of orfamide A, orfamide B, and orfamide G were compared in further bioassays. The three compounds were equally active against Magnaporthe oryzae on rice, against Rhizoctonia solani AG 4-HGI in in vitro assays, and caused zoospore lysis of Phytophthora and Pythium. Furthermore, we could show that orfamides decrease blast severity in rice plants by blocking appressorium formation in M. oryzae. Taken all together, our study shows that orfamides produced by P protegens and related species have potential in biological control of a broad spectrum of fungal plant pathogens

Comparison of explant responses treated with leachate and leonardite sources of humic substances during in vitro rooting of woody plants.

As heterogeneous mixtures of compounds resulting from the physical, chemical and microbiological transformations of organic residues, humic substances (HS) are mostly recognized for their biostimulation of plant growth that firstly involve the root development and architecture before further putative improvement of nutrients uptakes. To avoid the interferences currently reported from external origins, the successive steps of rooting have been carried out using shoots and isolated leaves of birch and alder vitro-plants. Extracts issued from landfill leachate (LHS) has been compared to a stable formulation from leonardite ("Humifirst" 12% humic acid 3% and fulvic acid) commercialized by TRADECORP company's (HHS). Chemical analysis showed that LHS source typically contain much higher N (mainly as ammonium (93%) and chloride concentration than HHS. Used at low concentration (10 ppm) during root induction/initiation phase, both HS sources may be slightly unfavorable to the root formation (21% of reduction in primary root number) of alder but not of birch. While, in root elongation phase, there is an increase in the primary root length and lateral root number. The direct effects of HS on in vitro root development vary from one species to another depending on the root treatment stage. Results showed that both explants type response are equivalent in the development of a complete rooting system

Elicitors of Plant Immunity Triggered by Beneficial Bacteria

The molecular basis of plant immunity triggered by microbial pathogens is being well-characterized as a complex sequential process leading to the activation of defense responses at the infection site, but which may also be systemically expressed in all organs, a phenomenon also known as systemic acquired resistance (SAR). Some plant-associated and beneficial bacteria are also able to stimulate their host to mount defenses against pathogen ingress via the phenotypically similar, induced systemic resistance phenomenon. Induced systemic resistance resembles SAR considering its mechanistic principle as it successively involves recognition at the plant cell surface, stimulation of early cellular immune-related events, systemic signaling via a fine-tuned hormonal cross-talk and activation of defense mechanisms. It thus represents an indirect but efficient mechanism by which beneficial bacteria with biocontrol potential improve the capacity of plants to restrict pathogen invasion. However, according to our current vision, induced systemic resistance is specific considering some molecular aspects underpinning these different steps. Here we overview the chemical diversity of compounds that have been identified as induced systemic resistance elicitors and thereby illustrating the diversity of plants species that are responsive as well as the range of pathogens that can be controlled via this phenomenon. We also point out the need for further investigations allowing better understanding how these elicitors are sensed by the host and the diversity and nature of the stimulated defense mechanisms. © Copyright © 2020 Pršić and Ongena.Peer reviewe

Systemic resistance and lipoxygenase-related defence response induced in tomato by Pseudomonas putida strain BTP1

BACKGROUND: Previous studies showed the ability of Pseudomonas putida strain BTP1 to promote induced systemic resistance (ISR) in different host plants. Since ISR is long-lasting and not conducive for development of resistance of the targeted pathogen, this phenomenon can take part of disease control strategies. However, in spite of the numerous examples of ISR induced by PGPR in plants, only a few biochemical studies have associated the protective effect with specific host metabolic changes. RESULTS: In this study, we showed the protective effect of this bacterium in tomato against Botrytis cinerea. Following treatment by P. putida BTP1, analyses of acid-hydrolyzed leaf extracts showed an accumulation of antifungal material after pathogen infection. The fungitoxic compounds thus mainly accumulate as conjugates from which active aglycones may be liberated through the activity of hydrolytic enzymes. These results suggest that strain BTP1 can elicit systemic phytoalexin accumulation in tomato as one defence mechanism. On another hand, we have shown that key enzymes of the lipoxygenase pathway are stimulated in plants treated with the bacteria as compared with control plants. Interestingly, this stimulation is observed only after pathogen challenge in agreement with the priming concept almost invariably associated with the ISR phenomenon. CONCLUSION: Through the demonstration of phytoalexin accumulation and LOX pathway stimulation in tomato, this work provides new insights into the diversity of defence mechanisms that are inducible by non-pathogenic bacteria in the context of ISR

Coregulation of the cyclic lipopeptides orfamide and sessilin in the biocontrol strain Pseudomonas sp. CMR12a

Cyclic lipopeptides (CLPs) are synthesized by nonribosomal peptide synthetases (NRPS), which are often flanked by LuxR-type transcriptional regulators. Pseudomonas sp. CMR12a, an effective biocontrol strain, produces two different classes of CLPs namely sessilins and orfamides. The orfamide biosynthesis gene cluster is flanked up- and downstream by LuxR-type regulatory genes designated ofaR1 and ofaR2, respectively, whereas the sessilin biosynthesis gene cluster has one LuxR-type regulatory gene which is situated upstream of the cluster and is designated sesR. Our study investigated the role of these three regulators in the biosynthesis of orfamides and sessilins. Phylogenetic analyses positioned OfaR1 and OfaR2 with LuxR regulatory proteins of similar orfamide-producing Pseudomonas strains and the SesR with that of the tolaasin producer, Pseudomonas tolaasii. LC-ESI-MS analyses revealed that sessilins and orfamides are coproduced and that production starts in the late exponential phase. However, sessilins are secreted earlier and in large amounts, while orfamides are predominantly retained in the cell. Deletion mutants in ofaR1 and ofaR2 lost the capacity to produce both orfamides and sessilins, whereas the sesR mutant showed no clear phenotype. Additionally, RT-PCR analysis showed that in the sessilin cluster, a mutation in either ofaR1 or ofaR2 led to weaker transcripts of the biosynthesis genes, sesABC, and putative transporter genes, macA1B1. In the orfamide cluster, mainly the biosynthesis genes ofaBC were affected, while the first biosynthesis gene ofaA and putative macA2B2 transport genes were still transcribed. A mutation in either ofaR1, ofaR2, or sesR genes did not abolish the transcription of any of the other two

Key impact of an uncommon plasmid on bacillus amyloliquefaciens subsp. plantarum S499 developmental traits and lipopeptide production

The rhizobacterium Bacillus amyloliquefaciens subsp. plantarum S499 (S499) is particularly efficient in terms of the production of cyclic lipopeptides, which are responsible for the high level of plant disease protection provided by this strain. Sequencing of the S499 genome has highlighted genetic differences and similarities with the closely related rhizobacterium B. amyloliquefaciens subsp. plantarum FZB42 (FZB42). More specifically, a rare 8008 bp plasmid (pS499) harboring a rap-phr cassette constitutes a major distinctive element between S499 and FZB42. By curing this plasmid, we demonstrated that its presence is crucial for preserving the typical physiology of S499 cells. Indeed, the growth rate and extracellular proteolytic activity were significantly affected in the cured strain (S499 P-). Furthermore, pS499 made a significant contribution to the regulation of cyclic lipopeptide production. Surfactins and fengycins were produced in higher quantities by S499 P-, whereas lower amounts of iturins were detected. In line with the increase in surfactin release, bacterial motility improved after curing, whereas the ability to form biofilm was reduced in vitro. The antagonistic effect against phytopathogenic fungi was also limited for S499 P-, most probably due to the reduction of iturin production. With the exception of this last aspect, S499 P- behavior fell between that of S499 and FZB42, suggesting a role for the plasmid in shaping some of the phenotypic differences observed in the two strains. © 2017 Molinatto, Franzil, Steels, Puopolo, Pertot and Ongena

Pilot scale biotransformation of vegetal oil into natural green note flavor using sugar beet leaves as sources of hydroperoxide lyase

Natural green note aromas (GLVs) are highly attractive flavors commonly used in the food industry. These are produced in extremely low levels upon physiological stress in plant organs of any sort. This weak sporadic presence entails a very expensive extraction step to obtain pure GLVs. Therefore catalytic biotransformations of fatty acid sources, the initial substrate for GLVs, have been developed. Enzymatic defense pathways and particularly the LOX pathway produce the major part of GLVs. Unlike GLV molecules that are emitted in the atmosphere, the enzymes are extractible from the plant material. Thus, a combination of plant enzyme extracts and substrate preparations provides all the ingredients for GLV production. Besides, sugar beet leaves present high levels of hydroperoxide lyase among plant sources and are available in large amounts during three months. In this enzymatic pathway, fatty acids are successively transformed by lipase, lipoxygenase and hydroperoxide lyase into aldehydes and alcohols, final compounds of GLVs pathway. Limiting and problematic steps occur with the action of hydroperoxide lyase, when enzymatic catalysis is followed by an enzyme destabilization. Alternative substrates bind irreversibly to the heme group of the enzyme and end the reaction. This poster briefly describes the development of a complete bioprocess for natural GLV production, from hydrolysis to purification. A high level of biotransformation could be achieved using optimum experimental conditions and a cheap source of plant materials

Bacillus amyloliquefaciens GA1 as a source of potent antibiotics and other secondary metabolites for biocontrol of plant pathogens

Phytopathogenic fungi affecting crop and post-harvested vegetables are a amajor threat to food production and food storage. To face these drawbacks, producers have become increasingly dependent on agrochemicals. However, intensive use of these compounds has led to the emergence of pathogen resistance and severe negative environmental impacts. There are also a number of plant diseases for which chemical solutions are ineffective or non-existent as well as an increasing demand by consumers for pesticide-free food. Thus, biological control through the use of natural antagonistic microorganisms has emerged as a promising alternative to chemical pesticides for more rational and safe crop management. RESULTS: The genome of the plant-associated B. amyloliquefaciens GA1 was sample sequenced. Several gene clusters involved in the synthesis of biocontrol agents were detected. Four gene clusters were shown to direct the synthesis of the cyclic lipopeptides surfactin, iturin A and fengycin as well as the iron-siderophore bacillibactin. Beside these non-ribosomaly synthetised peptides, three additional gene clusters directing the synthesis of the antibacterial polyketides macrolactin, bacillaene and difficidin were identified. Mass spectrometry analysis of culture supernatants led to the identification of these secondary metabolites, hence demonstrating that the corresponding biosynthetic gene clusters are functional in strain GA1. In addition, genes encoding enzymes involved in synthesis and export of the dipeptide antibiotic bacilysin were highlighted. However, only its chlorinated derivative, chlorotetaine, could be detected in culture supernatants. On the contrary, genes involved in ribosome-dependent synthesis of bacteriocin and other antibiotic peptides were not detected as compared to the reference strain B. amyloliquefaciens FZB42. CONCLUSION: The production of all of these antibiotic compounds highlights B. amyloliquefaciens GA1 as a good candidate for the development of biocontrol agents

Impacts of Plant Growth-Promoting Rhizobacteria-based Biostimulants on Wheat Growth under Greenhouse and Field Conditions

Plant Growth-Promoting Rhizobacteria (PGPR) are one of the main biostimulant classes due to their capacity of stimulating root growth and enhancing soil mineral availability, hence increasing nutrient use efficiency in crops. The aim of this study is to screen commercially PGPR-containing products to enhance wheat growth and yield in combination with an optimized nitrogen (N) fertilizer application scheme. This could lead to a significant reduction of N fertilizer application without affecting the subsequent grain yields. The screened products collection includes (1) Mix1 (a mix of Azospirillum sp., Azorhizobium sp., and Azoarcus sp.), (2) Mix2 (a mix of Mix1 complemented with two strains of phosphorus-solubilizing Bacillus sp.), (3) Bacillus amyloliquefaciens a, (4) B. subtilis, and (5) B. amyloliquefaciens b. These biostimulants were screened under greenhouse and field conditions in 2014 by using spring and winter wheat varieties respectively. There was a significant increase in root dry weight and in root per shoot ratio of plants inoculated with Mix1. Under field conditions, the interaction between PGPR inoculation and N fertilizer application was assessed. The grain yield was negatively impacted by low N fertilizer applications. Under such conditions, the inoculation of the wheat rhizosphere with Bacillus subtilis increased the grain yield by 15% relative to the water control. However, in the field trial, the variability between plot replicates was high and lead to non-significant results. Based on those results, modified screening strategies for PGPR selection were set up for the 2015 trials to reduce field variability and possibly achieve higher yield increases

Reprogramming of fatty acid and oxylipin synthesis in rhizobacteria-induced systemic resistance in tomato

The rhizobacterium Pseudomonas putida BTP1 stimulates induced systemic resistance (ISR) in tomato. A previous work showed that the resistance is associated in leaves with the induction of the first enzyme of the oxylipin pathway, the lipoxygenase (LOX), leading to a faster accumulation of its product, the free 13-hydroperoxy octadecatrienoic acid (13-HPOT), 2 days after Botrytis cinerea inoculation. In the present study, we further investigated the stimulation of the oxylipin pathway: metabolites and enzymes of the pathway were analyzed to understand the fate of the 13-HPOT in ISR. Actually the stimulation began upstream the LOX: free linolenic acid accumulated faster in P. putida BTP1-treated plants than in control. Downstream, the LOX products 13-fatty acid hydroperoxides esterified to galactolipids and phospholipids were more abundant in bacterized plants than in control before infection. These metabolites could constitute a pool that will be used after pathogen attack to produce free fungitoxic metabolites through the action of phospholipase A2, which is enhanced in bacterized plants upon infection. Enzymatic branches which can use as substrate the fatty acid hydroperoxides were differentially regulated in bacterized plants in comparison to control plants, so as to lead to the accumulation of the most fungitoxic compounds against B. cinerea. Our study, which is the first to demonstrate the accumulation of an esterified defense metabolite during rhizobacteria-mediated induced systemic resistance, showed that the oxylipin pathway is differentially regulated. It suggests that this allows the plant to prepare to a future infection, and to respond faster and in a more effective way to B. cinerea invasion.Peer reviewe