When genome-based approach meets the “Old but Good”: revealing genes involved in the antibacterial activity of Pseudomonas sp. P482 against soft rot pathogens

Dickeya solani and Pectobacterium carotovorum subsp. brasiliense are recently established species of bacterial plant pathogens causing black leg and soft rot of many vegetables and ornamental plants. Pseudomonas sp. strain P482 inhibits the growth of these pathogens, a desired trait considering the limited measures to combat these diseases. In this study, we determined the genetic background of the antibacterial activity of P482, and established the phylogenetic position of this strain. Pseudomonas sp. P482 was classified as Pseudomonas donghuensis. Genome mining revealed that the P482 genome does not contain genes determining the synthesis of known antimicrobials. However, the ClusterFinder algorithm, designed to detect atypical or novel classes of secondary metabolite gene clusters, predicted 18 such clusters in the genome. Screening of a Tn5 mutant library yielded an antimicrobial negative transposon mutant. The transposon insertion was located in a gene encoding an HpcH/HpaI aldolase/citrate lyase family protein. This gene is located in a hypothetical cluster predicted by the ClusterFinder, together with the downstream homologs of four nfs genes, that confer production of a non-fluorescent siderophore by P. donghuensis HYS(T). Site-directed inactivation of the HpcH/HpaI aldolase gene, the adjacent short chain dehydrogenase gene, as well as a homolog of an essential nfs cluster gene, all abolished the antimicrobial activity of the P482, suggesting their involvement in a common biosynthesis pathway. However, none of the mutants showed a decreased siderophore yield, neither was the antimicrobial activity of the wild type P482 compromised by high iron bioavailability. A genomic region comprising the nfs cluster and three upstream genes is involved in the antibacterial activity of P. donghuensis P482 against D. solani and P. carotovorum subsp. brasiliense. The genes studied are unique to the two known P. donghuensis strains. This study illustrates that mining of microbial genomes is a powerful approach for predictingthe presence of novel secondary-metabolite encoding genes especially when coupled with transposon mutagenesis

Understanding disease suppressive soils: molecular and chemical identification of microorganisms and mechanisms involved in soil suppressiveness to Fusarium culmorum of wheat

Soil is a home for an unbelievable diversity and abundance of microbial life that is essential for supporting life on our planet. Microorganisms living in soil take part in cleaning our water, degrading toxic compounds and recycling nutrients, and last but not least, they are essential partners to plants. Through their roots, plants release a mix of secretions to attract microorganisms, creating a remarkable environment called the rhizosphere. The rhizosphere is populated by microbes who often provide beneficial services to plants, like nutrient acquisition, growth promotion and protection against diseases. Modern agriculture suffers from losses caused by crop diseases, and a common way of controlling diseases is using pesticides. Pesticides often have a negative impact on the environment, and disease-causing agents (pathogens), can become resistant with time. One of the possible solutions to this problem is based on soil microbial communities. Due to the activity of their microbiome, some soils possess a natural capacity to protect plants against diseases. These soils are called disease suppressive soils, and the investigation of the microbial mechanisms leading to the natural protection of crops is the topic of this thesis. In our work we used a common pathogen of cereals, fungus Fusarium culmorum, and wheat, to first, identify suppressive soils able to protect this plant from the pathogen, and, investigate the mechanisms of protection using e.g. sequencing and mass spectrometry. During our project we identified potential microbes, genes and metabolites involved in soil disease suppressiveness. Moreover, we evaluated the impact of microplastic on the soil disease suppressiveness.Microbial Biotechnolog

Disentangling soil microbiome functions by perturbation

Soil biota contribute to diverse soil ecosystem services such as greenhouse gas mitigation, carbon sequestration, pollutant degradation, plant disease suppression and nutrient acquisition for plant growth. Here, we provide detailed insight into different perturbation approaches to disentangle soil microbiome functions and to reveal the underlying mechanisms. By applying perturbation, one can generate compositional and functional shifts of complex microbial communities in a controlled way. Perturbations can reduce microbial diversity, diminish the abundance of specific microbial taxa and thereby disturb the interactions within the microbial consortia and with their eukaryotic hosts. Four different microbiome perturbation approaches, namely selective heat, specific biocides, dilution-to-extinction and genome editing are the focus of this mini-review. We also discuss the potential of perturbation approaches to reveal the tipping point at which specific soil functions are lost and to link this change to key microbial taxa involved in specific microbiome-associated phenotypes.Microbial Biotechnolog

Dissecting Disease-Suppressive Rhizosphere Microbiomes by Functional Amplicon Sequencing and 10x Metagenomics

Disease-suppressive soils protect plants against soilborne fungal pathogens that would otherwise cause root infections. Soil suppressiveness is, in most cases, mediated by the antagonistic activity of the microbial community associated with the plant roots. Considering the enormous taxonomic and functional diversity of the root-associated microbiome, identification of the microbial genera and mechanisms underlying this phenotype is challenging. One approach to unravel the underlying mechanisms is to identify metabolic pathways enriched in the disease-suppressive microbial community, in particular, pathways that harbor natural products with antifungal properties. An important class of these natural products includes peptides produced by nonribosomal peptide synthetases (NRPSs). Here, we applied functional amplicon sequencing of NRPS-associated adenylation domains (A domains) to a collection of eight soils that are suppressive or nonsuppressive (i.e., conducive) to Fusarium culmorum, a fungal root pathogen of wheat. To identify functional elements in the root-associated bacterial community, we developed an open-source pipeline, referred to as dom2BGC, for amplicon annotation and putative gene cluster reconstruction through analyzing A domain co-occurrence across samples. We applied this pipeline to rhizosphere communities from four disease-suppressive and four conducive soils and found significant similarities in NRPS repertoires between suppressive soils. Specifically, several siderophore biosynthetic gene clusters were consistently associated with suppressive soils, hinting at competition for iron as a potential mechanism of suppression. Finally, to validate dom2BGC and to allow more unbiased functional metagenomics, we performed 10× metagenomic sequencing of one suppressive soil, leading to the identification of multiple gene clusters potentially associated with the disease-suppressive phenotyp

Screening the olive tree phyllosphere: Search and find potential antagonists against Pseudomonas savastanoi pv. savastanoi

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2020.02051/full#supplementary-materialOlive knot (OK) is a widespread bacterial disease, caused byPseudomonas savastanoipv.savastanoi(Pss), which currently has not effective control methods. The use of naturally occurring microbial antagonists, such as bacteria, as biocontrol agents could be a strategy to manage this disease. The objective of this work was to select bacteria from olive tree phyllosphere able to antagonizePssusingin vitroandin plantaexperiments. The elucidation of their modes of action and the potential relationship between antagonism and bacteria origin has been investigated, as well. To this end, 60 bacterial isolates obtained from the surface and inner tissues of different organs (leaves, twigs, and knots), from two olive cultivars of varying susceptibilities to OK, were screened for theirin vitroantagonistic effect againstPss. A total of 27 bacterial strains were able to significantly inhibitPssgrowth, being this effect linked to bacteria origin. Strains from OK-susceptible cultivar and colonizing the surface of plant tissues showed the strongest antagonistic potential. The antagonistic activity was potentially due to the production of volatile compounds, siderophores and lytic enzymes.Bacillus amyloliquefaciensP41 was the most effective antagonistic strain and their capacity to control OK disease was subsequently assayed usingin plantaexperiments. This strain significantly reduces OK disease severity (43.7%), knots weight (55.4%) and population size ofPss(26.8%), while increasing the shoot dry weight (55.0%) and root water content (39.6%) ofPss-infected olive plantlets. Bacterial isolates characterized in this study, in particularB. amyloliquefaciensP41, may be considered as promising biocontrol candidates for controlling OK disease.This work was funded by FEDER funds through COMPETE (Programa Operacional Factores de Competitividade), national funds through FCT (Fundacao para a Ciencia e a Tecnologia) and by Horizon 2020, the European Union's Framework Programme for Research and Innovation, within the project PRIMA/0002/2018 (INTOMED - Innovative tools to combat crop pests in the Mediterranean), and the Mountain Research Center - CIMO (UIDB/00690/2020 and UIDB/04046/2020)

Dissecting disease-suppressive rhizosphere microbiomes by functional amplicon sequencing and 10× metagenomics

Microbial Biotechnolog

MIBiG 4.0: advancing biosynthetic gene cluster curation through global collaboration

Specialized or secondary metabolites are small molecules of biological origin, often showing potent biological activities with applications in agriculture, engineering and medicine. Usually, the biosynthesis of these natural products is governed by sets of co-regulated and physically clustered genes known as biosynthetic gene clusters (BGCs). To share information about BGCs in a standardized and machine-readable way, the Minimum Information about a Biosynthetic Gene cluster (MIBiG) data standard and repository was initiated in 2015. Since its conception, MIBiG has been regularly updated to expand data coverage and remain up to date with innovations in natural product research. Here, we describe MIBiG version 4.0, an extensive update to the data repository and the underlying data standard. In a massive community annotation effort, 267 contributors performed 8304 edits, creating 557 new entries and modifying 590 existing entries, resulting in a new total of 3059 curated entries in MIBiG. Particular attention was paid to ensuring high data quality, with automated data validation using a newly developed custom submission portal prototype, paired with a novel peer-reviewing model. MIBiG 4.0 also takes steps towards a rolling release model and a broaderinvolvement of the scientific community. MIBiG 4.0 is accessible online at https://mibig.secondarymetabolites.org/

MIBiG 4.0 : advancing biosynthetic gene cluster curation through global collaboration

Specialized or secondary metabolites are small molecules of biological origin, often showing potent biological activities with applications in agriculture, engineering and medicine. Usually, the biosynthesis of these natural products is governed by sets of co-regulated and physically clustered genes known as biosynthetic gene clusters (BGCs). To share information about BGCs in a standardized and machine-readable way, the Minimum Information about a Biosynthetic Gene cluster (MIBiG) data standard and repository was initiated in 2015. Since its conception, MIBiG has been regularly updated to expand data coverage and remain up to date with innovations in natural product research. Here, we describe MIBiG version 4.0, an extensive update to the data repository and the underlying data standard. In a massive community annotation effort, 267 contributors performed 8304 edits, creating 557 new entries and modifying 590 existing entries, resulting in a new total of 3059 curated entries in MIBiG. Particular attention was paid to ensuring high data quality, with automated data validation using a newly developed custom submission portal prototype, paired with a novel peer-reviewing model. MIBiG 4.0 also takes steps towards a rolling release model and a broader involvement of the scientific community. MIBiG 4.0 is accessible online at https://mibig.secondarymetabolites.org/