Pyrrolnitrin Research Articles

Pyrrolnitrin in Pseudomonas chlororaphis strain AFS009 metabolites reduces constitutive and DMI-induced MfCYP51 gene expression in Monilinia fructicola.

Brown rot, caused by Monilinia fructicola, is one of the most economically important diseases of peach. Demethylation inhibitor (DMI) fungicides play an important part in managing brown rot in the southeastern U.S., but over the last 20 years, reduced efficacy to DMIs has been reported in field isolates overexpressing the DMI target enzyme encoding MfCYP51 gene. Metabolites of the biocontrol agent (BCA) Pseudomonas chlororaphis strain AFS009 suppressed the MfCYP51 gene in sensitive and resistant M. fructicola isolates previousely, but it is not known what molecule was responsible. The goals of this study were to determine the presence and role of pyrrolnitrin (PRN), a common metabolite of P. chlororaphis and chemical analogue to fludioxonil with antifungal activity, in the suppression of the MfCYP51 gene and to investigate if MfCYP51 expression can also be suppressed by Bacillus subtilis. High-performance liquid chromatography (HPLC) detected pyrrolnitrin at 1.75 µg/mg in P. chlororaphis metabolites formulated as Howler EVO (Howler). PRN at 0.1 µg/ml, fludioxonil at 0.1 µg/ml, and Howler applied at a dose that contained 0.1 µg/ml PRN significantly reduced the MfCYP51 gene expression at similar levels in DMI-resistant isolates. Furthermore, MfCYP51 expression in DMI-sensitive and three DMI-resistant isolates treated with Howler (88.1 µg/ml), Theia (209.5 µg/ml), propiconazole (0.3 µg/ml), or the mixture of either Howler or Theia + propiconazole revealed that Howler significantly reduced the MfCYP51 target gene expression in two of three sensitive and all three resistant M. fructicola isolates. On the other hand, Theia showed no suppressive effect and even increased the MfCYP51 gene expression level in two of three resistant isolates. In detached fruit assays on apple with a DMI resistant isolate, only the mixture of Howler + 50 µg/ml propiconazole resulted in synergism. The results indicate that suppression of MfCYP51 target gene is BCA-dependent and can be induced by pyrrolnitrin.

Identification of an Endogenous Strong Promoter in Burkholderia sp. JP2-270.

Burkholderia is the second largest source of natural product bacteria after Actinomyces and can produce many secondary metabolites including pyrrolnitrin (PRN). Natural products of microbial origin are usually found in trace amounts, so in metabolic engineering, promoter engineering is often used to regulate gene expression to increase yield. In this study, an endogenous strong promoter was identified based on RNA-seq to overexpress biosynthetic genes to increase the production of PRN. By analyzing the transcriptomic data of the antagonistic bacterium Burkholderia sp. JP2-270 in three different development periods, we screened 50 endogenous promoters with high transcriptional activity, nine of which were verified by an obvious fluorescent signal via fluorescence observation. Then, combined with RT-qPCR analysis, Php, the promoter of a hypothetical protein, was found to be significantly expressed in all three periods. In order to increase the suitability of endogenous promoters, the promoter Php was shortened at different lengths, and the results show that a sequence length of 173 bp was necessary for its activity. Moreover, this promoter was used to overexpress the PRN biosynthesis genes (prnA, prnB, prnC and prnD) in JP2-270, resulting in a successful increase in gene expression levels by 40-80 times. Only the overexpression of the prnB gene successfully increased PRN production to 1.46 times that of the wild type. Overall, the endogenous strong promoters screened in this study can improve gene expression and increase the production of secondary metabolites in JP2-270 and other strains.

Eco-friendly Management of Plant Pathogens through Secondary Metabolites Released by Fluorescent Pseudomonads

Concerning sustainable agriculture, plant growth promoting rhizobacteria (PGPR), which are a subgroup of “fluorescent pseudomonads,” are crucial. They are widely known for supporting plant health through a variety of methods. The use of fluorescent pseudomonads in agri-biotechnology has gained traction due to their potential for safeguarding plants from a variety of phytopathogens. Fluorescent pseudomonads being commercialized as bioinoculants for the treatment of various plant diseases is currently regarded as highly successful on a global scale. Fluorescent pseudomonads are being employed as efficient bio-control agents (BCAs) against an array of phytopathogens. Due to their capacity to generate a wide range of secondary metabolites, they offer enormous promise as BCA. Thus, this review’s goal is to outline and evaluate the functions of fluorescent pseudomonads’ secondary metabolites in reducing phytopathogens and improving plant health. Prominent secondary metabolites linked to biocontrol through fluorescent pseudomonads include phenazines (PHZ), 2, 4-diacetylphloroglucinol (DAPG), pyoluteorin (PLT), pyrrolnitrin (PRN), cyclic lipopeptides (CLPs), and volatile organic compounds (VOCs), including hydrogen cyanide (HCN). The antifungal, antibacterial, antiviral, antitumor, and antinematicidal effects of these metabolites are well-established.

Insecticidal features displayed by the beneficial rhizobacterium Pseudomonas chlororaphis PCL1606

The biocontrol rhizobacterium Pseudomonas chlororaphis is one of the bacterial species of the P. fluorescens group where insecticide fit genes have been found. Fit toxin, supported with other antimicrobial compounds, gives the bacterial the ability to repel and to fight against eukaryotic organisms, such as nematodes and insect larvae, thus protecting the plant host and itself. Pseudomonas chlororaphis PCL1606 is an antagonistic rhizobacterium isolated from avocado roots and show efficient biocontrol against fungal soil-borne disease. The main antimicrobial compound produced by P. chlororaphis PCL606 is 2-hexyl-5-propyl resorcinol (HPR), which plays a crucial role in effective biocontrol against fungal pathogens. Further analysis of the P. chlororaphis PCL1606 genome showed the presence of hydrogen cyanide (HCN), pyrrolnitrin (PRN), and homologous fit genes. To test the insecticidal activity and to determine the bases for such activity, single and double mutants on the biosynthetic genes of these four compounds were tested in a Galleria mellonella larval model using inoculation by injection. The results revealed that Fit toxin and HPR in combination are involved in the insecticide phenotype of P. chlororaphis PCL1606, and additional compounds such as HCN and PRN could be considered supporting compounds.

Pyoluteorin Produced by the Biocontrol Agent Pseudomonas protegens Is Involved in the Inhibition of Heterobasidion Species Present in Europe.

Pseudomonas protegens (strain DSMZ 13134) is a biocontrol agent with promising antagonistic activity hinging on antibiosis against the fungal forest pathogens Heterobasidion spp. Here, by using High-Performance Liquid Chromatography coupled to Mass Spectrometry (HPLC-MS), we assessed whether monocultures of P. protegens (strain DSMZ 13134) produce the three major determinants of biocontrol activity known for the genus Pseudomonas: 2,4-diacetylphloroglucinol (2,4-DAPG), pyoluteorin (PLT), and pyrrolnitrin (PRN). At the tested culture conditions, we observed the production of PLT at concentrations ranging from 0.01 to 10.21 mg/L and 2,4-DAPG at a concentration not exceeding 0.5 mg/L. Variations of culture conditions involving culture medium, incubation temperature, and incubation period had no consistent influence on PLT production by the bacterium. Assays using culture medium amended with PLT at the same concentration of that present in cell-free filtrate of the bacterium, i.e., 3.77 mg/L, previously documented as effective against Heterobasidion spp., showed a remarkable activity of PLT against genotypes of all the four Heterobasidion species present in Europe, including the non-native invasive H. irregulare. However, such antifungal activity decreased over time, and this may be a constraint for using this molecule as a pesticide against Heterobasidion spp. When the bacterium was co-cultured in liquid medium with genotypes of the different Heterobasidion species, an increased production of PLT was observed at 4 °C, suggesting the bacterium may perform better as a PLT producer in field applications under similar environmental conditions, i.e., at low temperatures. Our results demonstrated the role of PLT in the inhibition of Heterobasidion spp., although all lines of evidence suggest that antibiosis does not rely on a single constitutively produced metabolite, but rather on a plethora of secondary metabolites. Findings presented in this study will help to optimize treatments based on Pseudomonas protegens (strain DSMZ 13134) against Heterobasidion spp.

​Control Activity and Antibiotic Gene Detection of Endophytic Bacteria in Suppressing Cocoa Black Pod Disease (Phytophthora palmivora Butl.)

Background: Black pod rot disease of cocoa caused by (Phytophthora palmivora Butl.) is one of the major diseases on cocoa plantations worldwide. Many attempts have been made to prevent or reduce the infection of pathogens, but they have not provided optimum results. This study aims to detect antibiotic genes in endophytic bacteria that can suppress cocoa black pod disease. Methods: Eight endophytic bacteria were isolated from healthy cocoa pods and twigs that showed potentials in suppressing P. palmivora growth in vitro were used in suppressing of black pod rot disease in vivo tests. Antibiotic biosynthesis-related genes from eight endophytic bacterial isolates were confirmed by using PCR method, which includes phenazine-1-carboxylic (PCA), pyrrolnitrin (PRN), phenazine-1-carboxamide acid (PCN), pyoluteorin (PLT) and 2,4-diacetylphloroglucinol (DPAG/Phl). Result: The endophytic bacteria, 4RSI, 5BR B3 and 2RW B2 isolates showed the highest disease suppressing index to black pod rot disease in vivo, i.e., 70.27%, 70.08% and 56.64%. The isolates 5BR B3 and 2RW B2 DNA yielded PCR product by using PCA primers (1400 bp), PRN primers (700 bp) and DAPG primers (1600 bp), while the 5RSI isolate yield PCR product using PRN primers only. Endophytic bacterial isolates 5BRB3 and 2RW B2 provided partial disease suppression to pod rot disease by inhibit pathogen growth and antibiotic compounds production.

Fluorescent pseudomonads (FPs) as a potential biocontrol and plant growth promoting agent associated with tomato rhizosphere

A total of 87 FPs have been isolated from tomato (Lycopersicon esculentum) rhizosphere and evaluated by dual plate assay method for the antimicrobial activity of Ralstonia solanacearum. Of which, 30 FPs showed antagonistic activity against R. solanacearum with the zone of inhibitions (ZOI) ranging from 19 mm to 28 mm. Similarly, antagonistic FPs significantly controlled various fungal phytopathogens such as Rhizoctonia solani (6.3 mm−20 mm), Macrophomina phaseolina (8.7 mm−19.3 mm), Fusarium oxysporum (7.3 mm−30.7 mm), and Sclerotium rolfsii (5.3 mm−21 mm). The cell-free culture filtrate of Pseudomonas sp VSMKU3054 significantly suppressed the fungal pathogens and increased the root and shoot length of the tomato seedlings compared to the control. Furthermore, the antagonistic FPs effectively produced lytic enzymes and antimicrobial traits such as amylase (6), protease (30), cellulase (28), pectinase (12), chitinase (22), gelatinase (27), siderophore (28), hydrogen cyanide (25), phosphate solubilization (30), and Indole acetic acid (IAA) (23). Genetic diversity of FPs was assessed by BOX-PCR with specific primers revealed that two distinct clusters were observed, whereas, RFLP analyses were showed that 5 to 16 bands ranged from 75 bp to 1.2 bp with two major clusters using restriction enzyme HaeIII. The antibiotic encoding genes were detected from FPs, of which 10 FPs were positive for 2,4-diacetylphloroglucinol (DAPG), 9 FPs were positive for pyoleuterin (PLT), 10 FPs were positive for hydrogen cyanide (HCN) and none of them obtained pyrrolnitrin (PRN) with respective primers. Based on the superior antagonistic isolates against R. solanacearum and phytofungal pathogens and other antimicrobial traits, the isolate VSMKU3054 was selected for further studies. Based on the morphological, physiological and 98% of 16 s rDNA sequence similarity, the selected isolate VSMKU3054 was identified as Pseudomonas fluorescens. Further, the potential isolate P. fluorescens VSMKU3054 and cell free culture filtrate remarkably enhance tomato seedling growth such as root and shoot length, fresh and dry weight and vigor index compared to control. This study indicated that rhizospheric isolates of FPs have more potential for plant growth promotion and plant protection from plant pathogenic microbes.

Pseudomonas chlororaphis PA23 metabolites protect against protozoan grazing by the predator Acanthamoeba castellanii.

BackgroundPseudomonas chlororaphis strain PA23 is a biocontrol agent that is able to protect canola against the pathogenic fungus Sclerotinia sclerotiorum. This bacterium secretes a number of metabolites that contribute to fungal antagonism, including pyrrolnitrin (PRN), phenazine (PHZ), hydrogen cyanide (HCN) and degradative enzymes. In order to be successful, a biocontrol agent must be able to persist in the environment and avoid the threat of grazing predators. The focus of the current study was to investigate whether PA23 is able to resist grazing by the protozoan predator Acanthamoeba castellanii (Ac) and to define the role of bacterial metabolites in the PA23-Ac interaction.MethodsAc was co-cultured with PA23 WT and a panel of derivative strains for a period of 15 days, and bacteria and amoebae were enumerated on days 1, 5, 10 and 15. Ac was subsequently incubated in the presence of purified PRN, PHZ, and KCN and viability was assessed at 24, 48 and 72 h. Chemotactic assays were conducted to assess whether PA23 compounds exhibit repellent or attractant properties towards Ac. Finally, PA23 grown in the presence and absence of amoebae was subject to phenotypic characterization and gene expression analyses.ResultsPRN, PHZ and HCN were found to contribute to PA23 toxicity towards Ac trophozoites, either by killing or inducing cyst formation. This is the first report of PHZ-mediated toxicity towards amoebae. In chemotaxis assays, amoebae preferentially migrated towards regulatory mutants devoid of extracellular metabolite production as well as a PRN mutant, indicating this antibiotic has repellent properties. Co-culturing of bacteria with amoebae led to elevated expression of the PA23 phzI/phzR quorum-sensing (QS) genes and phzA and prnA, which are under QS control. PHZ and PRN levels were similarly increased in Ac co-cultures, suggesting that PA23 can respond to predator cues and upregulate expression of toxins accordingly.ConclusionsPA23 compounds including PRN, PHZ and HCN exhibited both toxic and repellent effects on Ac. Co-culturing of bacteria and amoebae lead to changes in bacterial gene expression and secondary metabolite production, suggesting that PA23 can sense the presence of these would-be predators and adjust its physiology in response.

Antibiotics of Pseudomonas protegens FD6 are essential for biocontrol activity

Pseudomonas protegens (formerly Pseudomonas fluorescens) FD6, which was isolated from the canola rhizosphere, is a biocontrol agent that protects against the fungal pathogens Botrytis cinerea and Monilinia fructicola. The complete sequence of the 6.7-Mb Pseudomonas protegens FD6 genome was determined. Genomic analysis of strain FD6 led to the identification of twelve putative gene clusters of secondary metabolites, including the antibiotics 2,4-diacetylphloroglucinol (2,4-DAPG), pyoluteorin (PLT) and pyrrolnitrin (PRN). To assess the role of 2,4-DAPG and PLT in the biocontrol activity of P. protegens FD6, two mutants of P. protegens FD6, including strains ΔphlC and ΔpltD, were constructed using homologous recombination technology. The results showed that no significant differences were observed in the antagonistic action of the ΔphlC mutant compared with wild-type FD6. However, the ΔpltD mutant no longer exhibited antifungal activity. HPLC analysis indicated that ΔphlC mutant could not biosynthesize 2,4-DAPG, whereas PLT production in the ΔphlC mutant was significantly increased by 67-fold compared with wild-type FD6. Biosynthesis of PLT in the ΔpltD mutant was not detected, whereas the level of 2,4-DAPG was vastly decreased over sixteenfold compared with strain FD6. The major antagonistic activity produced by P. protegens FD6 corresponded to 2,4-diacetylphloroglucinol and pyoluteorin. There is an inverse interaction between 2,4-DAPG and PLT.

Diversity of Antibiotic Biosynthesis Gene-possessing Rhizospheric Fluorescent Pseudomonads in Japan and Their Biocontrol Efficacy.

More than 3,000 isolates of fluorescent pseudomonads have been collected from plant roots in Japan and screened for the presence of antibiotic-synthesizing genes. In total, 927 hydrogen cyanide (HCN)-, 47 2,4-diacetylphloroglucinol (PHL)-, 6 pyoluteorin (PLT)-, 14 pyrrolnitrin (PRN)-, and 8 phenazine (PHZ)-producing isolates have been detected. A cluster analysis (≥99% identity) identified 10 operational taxonomic units (OTUs) in antibiotic biosynthesis gene-possessing pseudomonads. OTU HLR (PHL, PLT, and PRN) contained four antibiotics: HCN, PHL, PLT, and PRN, while OTU RZ (PRN and PHZ) contained three: HCN, PRN, and PHZ. OTU H1, H2, H3, H4, H5, H6, and H7 (PHL1-7) contained two antibiotics: HCN and PHL, while OTU H8 (PHL8) contained one: PHL. Isolates belonging to OTU HLR and RZ suppressed damping-off disease in cabbage seedlings caused by Rhizoctonia solani. Effective strains belonging to OTU HLR and RZ were related to Pseudomonas protegens and Pseudomonas chlororaphis, respectively. Antibiotic biosynthesis gene-possessing fluorescent pseudomonads are distributed among different geographical sites in Japan and plant species.

Pyrrolnitrin from Rhizospheric Serratia marcescens NCIM 5696: Optimization of Process Parameters Using Statistical Tools and Seed-Applied Bioprotectants for Vigna radiata (L.) Against Fusarium oxysporum MTCC 9913.

The extensive use of chemical fungicide in the health and agriculture sectors has increased environmental concerns and promoted an extensive search for alternative bioactives from the microbial system. In the present study, two rhizospheric strains of Serratia spp. (TO-2 and TW-3) have been shown to secrete pyrrolnitrin (PRN) in the range of 11.35 to 35.97 μg ml-1 using MSG and MSD medium after 72 h under static and shake conditions, respectively, but thereafter marginally declined in 96 to 240 h. Alternative one variable assortment at a time (OVAT) for PRN secretion by TW-3 yielded 59.27 μg ml-1 using (gl-1) glycerol (20), monosodium glutamate (14), KH2PO4 (14), NH4Cl (3), Na2HPO4 (4), and MgSO4 (0.3) at pH 7, 120 rpm within 72 h. Further, the Placket-Burman Design (PBD) identified KH2PO4, glycerol, pH, and monosodium glutamate as significant variables and optimized by centered composite design. Accordingly, 3% glycerol, 1.72% KH2PO4, 1.1% monosodium glutamate, 0.4% Na2HPO4, 0.03% MgSO4, 0.05% FeSO4, and 0.01% ZnSO4 were found to enhance the yield of PRN to 96.54 μg ml-1 by TW-3 in 72 h, 120 rpm. Thus, the statistical tool employed in the present study showed a threefold hike in PRN secretion over the OVAT approach, thereby indicating the scope for more PRN production from rhizobacteria. Further, seed application of low PRN (30 μg ml-1) concentration in treatments I and II showed > 90% germination in the initial seed germination and pot assay with the Fusarium oxysporum challenge compared to the control. Also, various growth parameters calculated during 11 days of experiment were significantly increased compared to the negative control (seed + fungus) in both treatments. Thus, the application of PRN at a low concentration to seeds of Vigna radiata (L.) offered protection against the phytopathogenic F. oxysporum MTCC 9913 challenge, suggesting biocontrol activity potential for use in agriculture soils particularly salt-affected soil.

Microbial Pyrrolnitrin: Natural Metabolite with Immense Practical Utility.

Pyrrolnitrin (PRN) is a microbial pyrrole halometabolite of immense antimicrobial significance for agricultural, pharmaceutical and industrial implications. The compound and its derivatives have been isolated from rhizospheric fluorescent or non-fluorescent pseudomonads, Serratia and Burkholderia. They are known to confer biological control against a wide range of phytopathogenic fungi, and thus offer strong plant protection prospects against soil and seed-borne phytopathogenic diseases. Although chemical synthesis of PRN has been obtained using different steps, microbial production is still the most useful option for producing this metabolite. In many of the plant-associated isolates of Serratia and Burkholderia, production of PRN is dependent on the quorum-sensing regulation that usually involves N-acylhomoserine lactone (AHL) autoinducer signals. When applied on the organisms as antimicrobial agent, the molecule impedes synthesis of key biomolecules (DNA, RNA and protein), uncouples with oxidative phosphorylation, inhibits mitotic division and hampers several biological mechanisms. With its potential broad-spectrum activities, low phototoxicity, non-toxic nature and specificity for impacts on non-target organisms, the metabolite has emerged as a lead molecule of industrial importance, which has led to developing cost-effective methods for the biosynthesis of PRN using microbial fermentation. Quantum of work narrating focused research efforts in the emergence of this potential microbial metabolite is summarized here to present a consolidated, sequential and updated insight into the chemistry, biology and applicability of this natural molecule.

Polyhydroxyalkanoate (PHA) Polymer Accumulation and pha Gene Expression in Phenazine (phz⁻) and Pyrrolnitrin (prn⁻) Defective Mutants of Pseudomonas chlororaphis PA23.

Pseudomonas chlororaphis PA23 was isolated from the rhizosphere of soybeans and identified as a biocontrol bacterium against Sclerotinia sclerotiorum, a fungal plant pathogen. This bacterium produces a number of secondary metabolites, including phenazine-1-carboxylic acid, 2-hydroxyphenazine, pyrrolnitrin (PRN), hydrogen cyanide, proteases, lipases and siderophores. It also synthesizes and accumulates polyhydroxyalkanoate (PHA) polymers as carbon and energy storage compounds under nutrient-limited conditions. Pseudomonads like P. chlororaphis metabolize glucose via the Entner-Doudoroff and Pentose Phosphate pathways, which provide precursors for phenazine production. Mutants defective in phenazine (PHZ; PA23-63), PRN (PA23-8), or both (PA23-63-1) accumulated higher concentrations of PHAs than the wild-type strain (PA23) when cultured in Ramsay’s Minimal Medium with glucose or octanoic acid as the carbon source. Expression levels of six pha genes, phaC1, phaZ, phaC2, phaD, phaF, and phaI, were compared with wild type PA23 by quantitative real time polymerase chain reaction (qPCR). The qPCR studies indicated that there was no change in levels of transcription of the PHA synthase genes phaC1 and phaC2 in the phz- (PA23-63) and phz- prn- (PA23-63-1) mutants in glucose medium. There was a significant increase in expression of phaC2 in octanoate medium. Transcription of phaD, phaF and phaI increased significantly in the phz- prn- (PA23-63-1) mutant. Mutations in regulatory genes like gacS, rpoS, and relA/spoT, which affect PHZ and PRN production, also resulted in altered gene expression. The expression of phaC1, phaC2, phaF, and phaI genes was down-regulated significantly in gacS and rpoS mutants. Thus, it appears that PHZ, PRN, and PHA production is regulated by common mechanisms. Higher PHA production in the phz- (PA23-63), prn- (PA23-8), and phz- prn- (PA23-63-1) mutants in octanoic medium could be correlated with higher expression of phaC2. Further, the greater PHA production observed in the phz- and prn- mutants was not due to increased transcription of PHA synthase genes in glucose medium, but due to more accessibility of carbon substrates and reducing power, which were otherwise used for the synthesis of PHZ and PRN.

Functional identification of the prnABCD operon and its regulation in Serratia plymuthica.

The antibiotic pyrrolnitrin (PRN) is a tryptophan-derived secondary metabolite that plays an important role in the biocontrol of plant diseases due to its broad-spectrum of antimicrobial activities. The PRN biosynthetic gene cluster remains to be characterised in Serratia plymuthica, though it is highly conserved in PRN-producing bacteria. To better understand PRN biosynthesis and its regulation in Serratia, the prnABCD operon from S. plymuthica G3 was cloned, sequenced and expressed in Escherichia coli DH5α. Furthermore, an engineered strain prnind which is a conditional mutant of G3 prnABCD under the control of the Ptac promoter was constructed. This mutant was able to overproduce PRN with isopropylthiogalactoside (IPTG) induction by overexpressing prnABCD, whilst behaving as a conditional mutant of G3 prnABCD in the absence of IPTG. These results confirmed that prnABCD is responsible for PRN biosynthesis in strain G3. Further experiments involving lux-/dsRed-based promoter fusions, combined with site-directed mutagenesis of the putative σS extended -10 region in the prnA promoter, and liquid chromatography-mass spectrometry (LC-MS) analysis extended our previous knowledge about G3, revealing that quorum sensing (QS) regulates PRN biosynthesis through cross talk with RpoS, which may directly activated prnABCD transcription. These findings suggest that PRN in S. plymuthica G3 is produced in a tightly controlled manner, and has diverse functions, such as modulation of cell motility, in addition to antimicrobial activities. Meanwhile, the construction of inducible mutants could be a powerful tool to improve PRN production, beyond its potential use for the investigation of the biological function of PRN.

Anti-fungal activity of bacterial endophytes associated with legumes against Fusarium solani: Assessment of fungi soil suppressiveness and plant protection induction

Legumes (Fabacea) plants are mainly known for their symbiotic relationship with soil nitrogen-fixing bacteria (rhizobia). This symbiosis requires the formation of new root structures called nodules. Besides rhizobia, nodules host several microbial species that may serve to enhance plant growth and disease resistance. In this study, we demonstrate that several endophytic bacteria isolated from nodules harbour plant growth promotion and biocontrol traits. A collection of 120 bacterial strains isolated from Faba bean (Vicia faba) and chickpea (Cicer arietinum) nodules were screened for their ability to inhibit phytopathogenic Fusarium solani on “In vitro” antibiosis tests. Sixteen best effective isolates were selected, identified and sequences were deposited in Genbank. These strains were all isolated from Faba bean nodules. These have the characteristics to produce siderophores and auxin as well as expression of some genes coding the production of the antibiotic compounds as Pyrrolnitrin (PRN), Phenazine (PHZ)… Based on the former PGPR and biocontrol characteristics, three strains; Rahnella aquatilis B16C, Pseudomonas yamanorum B12 and Pseudomonas fluorescens B8P were analyzed for their “In vivo” biocontrol potential in suppressing F. solani root rot of three cultivars of Faba bean under greenhouse conditions. The three strains significantly reduced the pathogen symptom severity. R. aquatilis B16C showed the best protecting potentiality with the three Faba bean cultivars and it is consequently, suggested as biocontrol agent for field application. Then again, our study confirms previous suggestion of legume nodules as untapped suitable source of beneficial microorganisms that can be used to control pathogens in a sustainable way.

Hydrogen cyanide, which contributes to Pseudomonas chlororaphis strain PA23 biocontrol, is upregulated in the presence of glycine

Pseudomonas chlororaphis strain PA23 is a biocontrol agent capable of suppressing the pathogenic fungus Sclerotinia sclerotiorum. This bacterium secretes the antibiotics pyrrolnitrin (PRN) and phenazine (PHZ), together with degradative enzymes and siderophores. Strain PA23 also produces hydrogen cyanide (HCN); however, the role of this compound in PA23 antifungal (AF) activity remains unknown. The aim of the current study was to characterize an hcn mutant and determine whether HCN contributes to biocontrol. Analysis of an hcn mutant revealed decreased AF activity both in vitro and in a newly established model of S. sclerotiorum root-rot infection in lettuce. When glycine (20mM) and ferric chloride (100µM) were included as media amendments, elevated AF activity was observed. Moreover glycine, but not FeCl3, increased HCN production and hcnA-lacZ transcriptional activity in the wild type. As the metabolic precursor for HCN biosynthesis, glycine is expected to enhance HCN production at the post-transcriptional level. We postulated that glycine might be indirectly affecting HCN transcription through the PhzRI quorum-sensing (QS) system. Analysis of phzR-lacZ and phzI-lacZ activity together with autoinducer levels revealed upregulation by glycine. This compound also increased expression of the QS-controlled prnA and phzA and their endproducts but, for reasons unknown, FeCl3 exerted a repressive effect. In a QS-deficient background the opposite occurred; FeCl3 increased hcnA-lacZ expression whereas glycine had no effect. HCN is under control of QS and the global regulator ANR. We present a model depicting how these regulators, in combination with media amendments, impact PA23 hcn expression.

The global regulator ANR is essential for Pseudomonas chlororaphis strain PA23 biocontrol.

Pseudomonas chlororaphis PA23 is a biocontrol agent capable of protecting canola from stem rot disease caused by the fungus Sclerotinia sclerotiorum. The focus of the current study was to elucidate the role of the transcriptional regulator ANR in the biocontrol capabilities of this bacterium. An anr mutant was created, PA23anr, that was devoid antifungal activity. In other pseudomonads, ANR is essential for regulating HCN production. Characterization of PA23anr revealed that, in addition to HCN, ANR controls phenazine (PHZ), pyrrolnitrin (PRN), protease and autoinducer (AHL) signal molecule production. In gene expression studies, hcnA, phzA, prnA and phzI were found to be downregulated, consistent with our endproduct analysis. Because the phenotype of PA23anr closely resembles that of quorum sensing (QS)-deficient strains, we explored whether there is a connection between ANR and the PhzRI QS system. Both phzI and phzR are positively regulated by ANR, whereas PhzR represses anr transcription. Complementation of PA23anr with pUCP-phzR, C6-HSL or both yielded no change in phenotype. Conversely, PA23phzR harbouring pUCP23-anr exhibited partial-to-full restoration of antifungal activity, HCN, PRN and AHL production together with hcnA, prnA, phzI and rpoS expression. PHZ and protease production remained unchanged indicating that ANR can complement the QS-deficient phenotype with respect to some but not all traits. Our experiments were conducted at atmospheric O2 levels underscoring the fact that ANR has a profound effect on PA23 physiology under aerobic conditions.

Pyrrolnitrin and Hydrogen Cyanide Production by Pseudomonas chlororaphis Strain PA23 Exhibits Nematicidal and Repellent Activity against Caenorhabditis elegans.

Pseudomonas chlororaphis strain PA23 is a biocontrol agent able to suppress growth of the fungal pathogen Sclerotinia sclerotiorum. This bacterium produces an arsenal of exometabolites including pyrrolnitrin (PRN), phenazine (PHZ), hydrogen cyanide (HCN), and degradative enzymes. Production of these compounds is controlled at both the transcriptional and posttranscriptional levels by the Gac-Rsm system, RpoS, PsrA, and the Phz quorum-sensing system. Beyond pathogen-suppression, the success of a biocontrol agent is dependent upon its ability to establish itself in the environment where predation by bacterivorous organisms, including nematodes, may threaten persistence. The focus of this study was to investigate whether PA23 is able to resist grazing by Caenorhabditis elegans and to define the role played by exoproducts in the bacterial-nematode interaction. We discovered that both PRN and HCN contribute to fast- and slow-killing of C. elegans. HCN is well-established as having lethal effects on C. elegans; however, PRN has not been reported to be nematicidal. Exposure of L4 stage nematodes to purified PRN reduced nematode viability in a dose-dependent fashion and led to reduced hatching of eggs laid by gravid adults. Because bacterial metabolites can act as chemoattractants or repellents, we analyzed whether PA23 exhibited attractant or repulsive properties towards C. elegans. Both PRN and HCN were found to be potent repellents. Next we investigated whether the presence of C. elegans would elicit changes in PA23 gene activity. Co-culturing the two organisms increased expression of a number of genes associated with biocontrol, including phzA, hcnA, phzR, phzI, rpoS and gacS. Exoproduct analysis showed that PHZ and autoinducer signals were upregulated, consistent with the gene expression profiles. Collectively, these findings indicate that PA23 is able to sense the presence of C. elegans and it is able to both repel and kill the nematodes, which should facilitate environmental persistence and ultimately biocontrol.

Protozoa Drive the Dynamics of Culturable Biocontrol Bacterial Communities

Some soil bacteria protect plants against soil-borne diseases by producing toxic secondary metabolites. Such beneficial biocontrol bacteria can be used in agricultural systems as alternative to agrochemicals. The broad spectrum toxins responsible for plant protection also inhibit predation by protozoa and nematodes, the main consumers of bacteria in soil. Therefore, predation pressure may favour biocontrol bacteria and contribute to plant health. We analyzed the effect of Acanthamoeba castellanii on semi-natural soil bacterial communities in a microcosm experiment. We determined the frequency of culturable bacteria carrying genes responsible for the production of the antifungal compounds 2,4-diacetylphloroglucinol (DAPG), pyrrolnitrin (PRN) and hydrogen cyanide (HCN) in presence and absence of A. castellanii. We then measured if amoebae affected soil suppressiveness in a bioassay with sugar beet seedlings confronted to the fungal pathogen Rhizoctonia solani. Amoebae increased the frequency of both DAPG and HCN positive bacteria in later plant growth phases (2 and 3 weeks), as well as the average number of biocontrol genes per bacterium. The abundance of DAPG positive bacteria correlated with disease suppression, suggesting that their promotion by amoebae may enhance soil health. However, the net effect of amoebae on soil suppressiveness was neutral to slightly negative, possibly because amoebae slow down the establishment of biocontrol bacteria on the recently emerged seedlings used in the assay. The results indicate that microfaunal predators foster biocontrol bacterial communities. Understanding interactions between biocontrol bacteria and their predators may thus help developing environmentally friendly management practices of agricultural systems.

The PhzI/PhzR quorum-sensing system is required for pyrrolnitrin and phenazine production, and exhibits cross-regulation with RpoS in Pseudomonas chlororaphis PA23

The aim of the current study was to determine how quorum sensing (QS) affects the production of secondary metabolites in Pseudomonas chlororaphis strain PA23. A phzR mutant (PA23phzR) and an N-acylhomoserine lactone (AHL)-deficient strain (PA23-6863) were generated that no longer inhibited the fungal pathogen Sclerotinia sclerotiorum in vitro. Both strains exhibited reduced pyrrolnitrin (PRN), phenazine (PHZ) and protease production. Moreover, phzA-lacZ and prnA-lacZ transcription was significantly reduced in PA23phzR and PA23-6863. As the majority of secondary metabolites are produced at the onset of stationary phase, we investigated whether cross-regulation occurs between QS and RpoS. Analysis of transcriptional fusions revealed that RpoS has a positive and negative effect on phzI and phzR, respectively. In a reciprocal manner, RpoS is positively regulated by QS. Characterization of a phzRrpoS double mutant showed reduced antifungal activity as well as PRN and PHZ production, similar to the QS-deficient strains. Furthermore, phzR but not rpoS was able to complement the phzRrpoS double mutant for the aforementioned traits, indicating that the Phz QS system is a central regulator of PA23-mediated antagonism. Finally, we discovered that QS and RpoS have opposing effects on PA23 biofilm formation. While both QS-deficient strains produced little biofilm, the rpoS mutant showed enhanced biofilm production compared with PA23. Collectively, our findings indicate that QS controls diverse aspects of PA23 physiology, including secondary metabolism, RpoS and biofilm formation. As such, QS is expected to play a crucial role in PA23 biocontrol and persistence in the environment.