Aer receptors influence the Pseudomonas chlororaphis PCL1606 lifestyle.

Pseudomonas chlororaphis PCL1606 (PcPCL1606) is a rhizobacterium isolated from avocado roots, which is a favorable niche for its development. This strain extensively interacts with plant roots and surrounding microbes and is considered a biocontrol rhizobacterium. Genome sequencing has shown the presence of thirty-one potential methyl-accepting chemotaxis proteins (MCPs). Among these MCPs, two candidates are putative functional aerotaxis receptors, encoded at locus PCL1606_41090 (aer1- 1) and locus PLC1606_20530 (aer1-2), that are homologous to the Aer receptor of Pseudomonas aeruginosa strain PaO1. Single- and double-deletion mutants in one or both genes have led to motility deficiencies in oxygen-rich areas, particularly reduced swimming motility compared with that of wildtype PcPCL1606. No differences in swarming tests were detected, and less adhesion by the aer double mutant was observed. However, the single and double mutants on avocado plant roots showed delayed biocontrol ability. During the first days of the biocontrol experiment, the aer-defective mutants also showed delayed root colonization. The current research characterizes the presence of aer transductors on P. chlororaphis. Thus, the functions of the PCL1606_41090 and PCL1606_20530 loci, corresponding to genes aer1-1 and aer1-2, respectively, are elucidated.This research was supported by the Spanish Plan Nacional ICDCI. Grant AGL2017-83368-C2-1-R and partially supported by the European Union (FEDER)

La rizobacteria Pseudomonas chlororaphis PCL1606, no presenta actividad promotora del crecimiento como mecanismo adicional de biocontrol

Pseudomonas chlororaphis PCL1606 es una rizobacteria, que muestra capacidad antagonista y actividad de biocontrol frente a diferentes hongos fitopatógenos de suelo, entre ellos Rosellinia necatrix, que produce la enfermedad denominada podredumbre blanca radicular en la planta de aguacate. Se ha demostrado que PCL1606 produce el antifúngico 2 hexil, 5 propil resorcinol (HPR), entre otros compuestos antifúngicos. HPR es clave para el antagonismo y la actividad biocontrol contra R. necatrix, así como en la colonización de la rizosfera de aguacate. En este trabajo se analiza la posible actividad PGPR (actividad promotora del crecimiento de la planta mediada por rizobacterias) de P. chlororaphis PCL1606, como mecanismo adicional implicado en el control biológico. Para ello, se realizaron ensayos in vitro con semillas de tomate, así como ensayos en plantas comerciales de aguacate de 6 meses. También se estudiaron actividades relacionadas con el PGPR, como la actividad 1-aminociclopropano-1-carboxylato (ACC) deaminasa, la producción de ácido indol acético (IAA), la solubilización de fosfatos o la producción de sideróforos. Los resultados obtenidos de los distintos ensayos, muestran que la cepa P. chlororaphis PCL1606 no tiene actividad promotora del crecimiento vegetal.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Construcción de una colección de cepas de Pseudomonas syringae pv. syringae asociadas a mango para su uso en estudios comparativos

La necrosis apical es una enfermedad que afecta a las principales áreas de cultivo de mango de clima mediterráneo, y cuyo agente causal es Pseudomonas syringae pv. syringae (Pss). Se han descrito diferentes genes implicados en el desarrollo de los síntomas de la enfermedad, así como en aumentar el fitness epifítico de la bacteria; como la producción de mangotoxina, o la resistencia al cobre respectivamente. En estudios previos mediante análisis filogenéticos, se han agrupado todas las cepas de Pss aisladas de mango y productoras de mangotoxina en el filotipo I. El objetivo de este trabajo es aislar cepas de Pss de árboles de mango de las distintas zonas de estudio (España, Portugal, Italia, Israel y Australia), para así poder abordar un análisis comparativo de las cepas de Pss aisladas antes del año 2000 y disponibles en nuestro laboratorio, con los nuevos aislamientos (2016-2017). Tras proceder a la identificación de las cepas, se caracterizarán las mismas mediante diferentes pruebas fenotípicas y genotípicas (producción de mangotoxina, resistencia al cobre, detección de genes específicos por PCR, etc).Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Papel del HPR como molécula señal en las interacciones multitróficas en la bacteria de control biológico pseudomonas chlororaphis PCL1606.

En este trabajo, se analizará a nivel transcriptómico, la expresión/represión de los genes cuando se compara un mutante defectivo en la producción de HPR (mutante en el gen darB) frente a la cepa silvestre, identificando los fenotipos dependientes de la producción de HPR durante el proceso de interacción multitrófica, que se produce durante el control biológico de la podredumbre blanca radicular.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech . Este trabajo se ha financiado gracias a el Plan Nacional de l+D+I del Ministerio de Economía (AGL2014-52518-C2-l-R y AGL2017-83368-C2-l-R; MINECO, España) y cofinanciado con fondos PEDER (EU). S. Tienda está siendo financiada con una ayuda del programa FPI del MINECO

Insecticidal features displayed by the beneficial rhizobacterium Pseudomonas chlororaphis PCL1606

The biocontrol rhizobacterium Pseudomonas chlororaphis is one of the bacterial species of the P. fuorescens group where insecticide ft genes have been found. Fit toxin, supported with other antimicrobial compounds, gives the bacterial the ability to repel and to fght 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 efcient 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 efective biocontrol against fungal pathogens. Further analysis of the P. chlororaphis PCL1606 genome showed the presence of hydrogen cyanide (HCN), pyrrolnitrin (PRN), and homologous ft 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 compoundsOpen Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. Funding for open access charge: Universidad de Málaga / CBUA. This work was supported by the research project AGL2017-83368-CO2-1-R of Ministerio de Ciencias y Tecnología, and the project UMA-FEDERJA-046 of Junta de Andalucía

The Iturin and Fengycin Families of Lipopeptides Are Key Factors in Antagonism of Bacillus subtilis Toward Podosphaera fusca

Podosphaera fusca is the main causal agent of cucurbit powdery mildew in Spain. Four Bacillus subtilis strains, UMAF6614, UMAF6619, UMAF6639, and UMAF8561, with proven ability to suppress the disease on melon in detached leaf and seedling assays, were subjected to further analyses to elucidate the mode of action involved in their biocontrol performance. Cell-free supernatants showed antifungal activities very close to those previously reported for vegetative cells. Identification of three lipopeptide antibiotics, surfactin, fengycin, and iturin A or bacillomycin, in butanolic extracts from cell-free culture filtrates of these B. subtilis strains pointed out that antibiosis could be a major factor involved in their biocontrol ability. The strong inhibitory effect of purified lipopeptide fractions corresponding to bacillomycin, fengycin, and iturin A on P. fusca conidia germination, as well as the in situ detection of these lipopeptides in bacterial-treated melon leaves, provided interesting evidence of their putative involvement in the antagonistic activity. Those results were definitively supported by site-directed mutagenesis analysis, targeted to suppress the biosynthesis of the different lipopeptides. Taken together, our data have allowed us to conclude that the iturin and fengycin families of lipopeptides have a major role in the antagonism of B. subtilis toward P. fusca.

The Iturin and Fengycin Families of Lipopeptides Are Key Factors in Antagonism of Bacillus subtilis Toward Podosphaera fusca

Podosphaera fusca is the main causal agent of cucurbit powdery mildew in Spain. Four Bacillus subtilis strains, UMAF6614, UMAF6619, UMAF6639, and UMAF8561, with proven ability to suppress the disease on melon in detached leaf and seedling assays, were subjected to further analyses to elucidate the mode of action involved in their biocontrol performance. Cell-free supernatants showed antifungal activities very close to those previously reported for vegetative cells. Identification of three lipopeptide antibiotics, surfactin, fengycin, and iturin A or bacillomycin, in butanolic extracts from cell-free culture filtrates of these B. subtilis strains pointed out that antibiosis could be a major factor involved in their biocontrol ability. The strong inhibitory effect of purified lipopeptide fractions corresponding to bacillomycin, fengycin, and iturin A on P. fusca conidia germination, as well as the in situ detection of these lipopeptides in bacterial-treated melon leaves, provided interesting evidence of their putative involvement in the antagonistic activity. Those results were definitively supported by site-directed mutagenesis analysis, targeted to suppress the biosynthesis of the different lipopeptides. Taken together, our data have allowed us to conclude that the iturin and fengycin families of lipopeptides have a major role in the antagonism of B. subtilis toward P. fusca.

Characterisation of the mgo operon in Pseudomonas syringae pv. syringae UMAF0158 that is required for mangotoxin production

<p>Abstract</p> <p>Background</p> <p>Mangotoxin is an antimetabolite toxin that is produced by strains of <it>Pseudomonas syringae </it>pv. <it>syringae</it>; mangotoxin-producing strains are primarily isolated from mango tissues with symptoms of bacterial apical necrosis. The toxin is an oligopeptide that inhibits ornithine N-acetyl transferase (OAT), a key enzyme in the biosynthetic pathway of the essential amino acids ornithine and arginine. The involvement of a putative nonribosomal peptide synthetase gene (<it>mgo</it>A) in mangotoxin production and virulence has been reported.</p> <p>Results</p> <p>In the present study, we performed a RT-PCR analysis, insertional inactivation mutagenesis, a promoter expression analysis and terminator localisation to study the gene cluster containing the <it>mgo</it>A gene. Additionally, we evaluated the importance of <it>mgo</it>C, <it>mgo</it>A and <it>mgo</it>D in mangotoxin production. A sequence analysis revealed an operon-like organisation. A promoter sequence was located upstream of the <it>mgo</it>B gene and was found to drive <it>lac</it>Z transcription. Two terminators were located downstream of the <it>mgo</it>D gene. RT-PCR experiments indicated that the four genes (<it>mgo</it>BCAD) constitute a transcriptional unit. This operon is similar in genetic organisation to those in the three other <it>P. syringae </it>pathovars for which complete genomes are available (<it>P. syringae </it>pv. <it>syringae </it>B728a, <it>P. syringae </it>pv. <it>tomato </it>DC3000 and <it>P. syringae </it>pv. <it>phaseolicola </it>1448A). Interestingly, none of these three reference strains is capable of producing mangotoxin. Additionally, extract complementation resulted in a recovery of mangotoxin production when the defective mutant was complemented with wild-type extracts.</p> <p>Conclusions</p> <p>The results of this study confirm that <it>mgo</it>B, <it>mgo</it>C, <it>mgo</it>A and <it>mgo</it>D function as a transcriptional unit and operon. While this operon is composed of four genes, only the last three are directly involved in mangotoxin production.</p

Contribution of mangotoxin to the virulence and epiphytic fitness of Pseudomonas syringae pv. syringae

Mangotoxin is an antimetabolite toxin that inhibits ornithine acetyl transferase, a key enzyme in the biosynthetic pathway of ornithine and arginine and recently reported in strains of Pseudomonas syringae pv. syringae (Pss) isolated from mango. Since symptoms on mango tissues are very difficult to reproduce, in this study the role of mangotoxin in Pss virulence was addressed by analyzing the in planta growth and development of disease symptoms on tomato leaflets. Inoculation experiments were carried out following several procedures using the wild-type strain Pss UMAF0158, two Tn5-mutant derivative strains defective in mangotoxin production, and their complemented derivative strains in which mangotoxin production is restored. The ability of the mangotoxin-defective mutants to grow in planta was similar, and their epiphytic survival on the tomato leaf surface identical to the wild-type and complemented strains. However, both the disease index data of incidence and the severity of necrotic symptoms indicated that mangotoxin-defective mutants were less virulent, indicating that mangotoxin is a virulence factor. Furthermore, competition experiments showed that the survival values of the wild-type strain were slightly but significantly higher than those of the mangotoxin-defective mutants, suggesting that mangotoxin production would improve the epiphytic fitness of Pss. [Int Microbiol 2009; 12(2):87-95

Fitness Features Involved in the Biocontrol Interaction of Pseudomonas chlororaphis With Host Plants: The Case Study of PcPCL1606

The goal of this mini review is to summarize the relevant contribution of some beneficial traits to the behavior of the species Pseudomonas chlororaphis, and using that information, to give a practical point of view using the model biocontrol strain P. chlororaphis PCL1606 (PcPCL1606). Among the group of plant-beneficial rhizobacteria, P. chlororaphis has emerged as a plant- and soil-related bacterium that is mainly known because of its biological control of phytopathogenic fungi. Many traits have been reported to be crucial during the multitrophic interaction involving the plant, the fungal pathogen and the soil environment. To explore the different biocontrol-related traits, the biocontrol rhizobacterium PcPCL1606 has been used as a model in recent studies. This bacterium is antagonistic to many phytopathogenic fungi and displays effective biocontrol against fungal phytopathogens. Antagonistic and biocontrol activities are directly related to the production of the compound 2-hexyl, 5-propyl resorcinol (HPR), despite the production of other antifungal compounds. Furthermore, PcPCL1606 has displayed additional traits regarding its fitness in soil and plant root environments such as soil survival, efficient plant root colonization, cell-to-cell interaction or promotion of plant growth.This research was supported by the Spanish Plan Nacional I C D C I. Grant AGL2017-83368-C2-1-R and partially supported by the European Union (FEDER). CV and ST were supported by a grant from FPI, Ministerio de Ciencia e Innovación, Spain