Bacillus cabrialesii BH5 Protects Tomato Plants Against Botrytis cinerea by Production of Specific Antifungal Compounds

The gray mold caused by the phytopathogen Botrytis cinerea presents a threat to global food security. For the biological regulation of several plant diseases, Bacillus species have been extensively studied. In this work, we explore the ability of a bacterial strain, Bacillus cabrialesii BH5, that was isolated from tomato rhizosphere soil, to control the fungal pathogen B. cinerea. Strain B. cabrialesii BH5 showed a strong antifungal activity against B. cinerea. A compound was isolated and identified as a cyclic lipopeptide of the fengycin family by high-performance liquid chromatography and tandem mass spectrometry (ESI-MS/MS) that we named fengycin H. The fengycin H-treated hyphae of B. cinerea displayed stronger red fluorescence than the control, which is clearly indicating that fengycin H triggered the hyphal cell membrane defects. Moreover, root inoculation of tomato seedlings with BH5 effectively promoted the growth of tomato plants. Transcription analysis revealed that both BH5 and fengycin H stimulate induced systemic resistance of tomato plants via the jasmonic acid signaling pathway and provide a strong biocontrol effect in vivo. Therefore, the strain BH5 and fengycin H are very promising candidates for biological control of B. cinerea and the associated gray mold

Characterization of plant growth-promoting rhizobacteria from perennial ryegrass and genome mining of novel antimicrobial gene clusters

Background Plant growth-promoting rhizobacteria (PGPR) are good alternatives for chemical fertilizers and pesticides, which cause severe environmental problems worldwide. Even though many studies focus on PGPR, most of them are limited in plant-microbe interaction studies and neglect the pathogens affecting ruminants that consume plants. In this study, we expand the view to the food chain of grass-ruminant-human. We aimed to find biocontrol strains that can antagonize grass pathogens and mammalian pathogens originated from grass, thus protecting this food chain. Furthermore, we deeply mined into bacterial genomes for novel biosynthetic gene clusters (BGCs) that can contribute to biocontrol. Results We screened 90 bacterial strains from the rhizosphere of healthy Dutch perennial ryegrass and characterized seven strains (B. subtilis subsp. subtilis MG27, B. velezensis MG33 and MG43, B. pumilus MG52 and MG84, B. altitudinis MG75, and B. laterosporus MG64) that showed a stimulatory effect on grass growth and pathogen antagonism on both phytopathogens and mammalian pathogens. Genome-mining of the seven strains discovered abundant BGCs, with some known, but also several potential novel ones. Further analysis revealed potential intact and novel BGCs, including two NRPSs, four NRPS-PKS hybrids, and five bacteriocins. Conclusion Abundant potential novel BGCs were discovered in functional protective isolates, especially in B. pumilus, B. altitudinis and Brevibacillus strains, indicating their great potential for the production of novel secondary metabolites. Our report serves as a basis to further identify and characterize these compounds and study their antagonistic effects against plant and mammalian pathogens

l-2,3-Diaminopropionate: One of the building blocks for the biosynthesis of Zwittermicin A in Bacillus thuringiensis subsp. kurstaki strain YBT-1520

AbstractZwittermicin A (ZwA) is a hybrid polyketide–non-ribosomal peptide that is thought to be biosynthesized from five proposed building blocks, including the 2,3-diaminopropionate. Candidate genes for de novo biosynthesis of 2,3-diaminopropionate, zwa5A and zwa5B, have been identified in a previous study. In this research, zwa5A was interrupted and chemically synthesized 2,3-diaminopropionate was used to feed the zwa5A− mutant. Results showed that feeding with 2,3-diaminopropionate restored the ability of the zwa5A− mutant to produce ZwA. Another non-ribosomal peptide synthase gene, designated orf3, was identified. Amino acid dependent PPi release assay showed that the adenylation domain ZWAA2 of ORF3 acyl-adenylated l-2,3-diaminopropionate effectively. Taken together, it can be concluded that l-2,3-diaminopropionate is indeed one of the building blocks for the biosynthesis of Zwittermicin A

The Ecological Role of Volatile and Soluble Secondary Metabolites Produced by Soil Bacteria

The rich diversity of secondary metabolites produced by soil bacteria has been appreciated for over a century, and advances in chemical analysis and genome sequencing continue to greatly advance our understanding of this biochemical complexity. However, we are just at the beginning of understanding the physicochemical properties of bacterial metabolites, the factors that govern their production and ecological roles. Interspecific interactions and competitor sensing are among the main biotic factors affecting the production of bacterial secondary metabolites. Many soil bacteria produce both volatile and soluble compounds. In contrast to soluble compounds, volatile organic compounds can diffuse easily through air- and gas-filled pores in the soil and likely play an important role in long-distance microbial interactions. In this review we provide an overview of the most important soluble and volatile classes of secondary metabolites produced by soil bacteria, their ecological roles, and their possible synergistic effects

Exploring plant-microbe interactions of the rhizobacteria Bacillus subtilis and Bacillus mycoides by use of the CRISPR-Cas9 system

Bacillus subtilis HS3 and Bacillus mycoides EC18 are two rhizosphere-associated bacteria with plant growth-promoting activity. The CRISPR-Cas9 system was implemented to study various aspects of plant-microbe interaction mechanisms of these two environmental isolates. The results show that fengycin and surfactin are involved in the antifungal activity of B. subtilis HS3. Moreover, this strain emits several other volatile organic compounds than 2,3-butanediol, contributing to plant growth promotion. Confocal laser scanning microscopy observations of the GFP-labelled strain showed that HS3 selectively colonizes root hairs of grass (Lolium perenne) in a hydroponic system. For B. mycoides EC18, we found that the wild-type EC18 strain and a Delta asbA (petropectin-deficient) mutant, but not the Delta dhbB (bacillibactin-deficient) and ADKO (asbA and dhbB double knockout) mutants, can increase the plant biomass and total chlorophyll. All the mutant strains have a reduced colonization capability on Chinese cabbage (Brassica rapa) roots, at the root tip and root hair region compared with the wild-type strain. These results indicate that the siderophore, bacillibactin, is involved in the plant growth promoting activity and could affect the root colonization of B. mycoides. Collectively, the CRISPR-Cas9 system we developed for environmental isolates is broadly applicable and will facilitate deciphering the mechanisms of Bacillus-plant interactions. (c) 2018 The Authors

Draft Genome Sequences of Six Bacillus Strains and One Brevibacillus Strain Isolated from the Rhizosphere of Perennial Ryegrass (Lolium perenne)

Seven strains of endospore-forming bacteria with antagonistic activity against a series of plant pathogens were sequenced in order to investigate their antimicrobial gene clusters and antimicrobial modes of action. The selected strains include six Bacillus strains and one Brevibacillus strain

Draft Genome Sequences of 10 Paenibacillus and Bacillus sp. Strains Isolated from Healthy Tomato Plants and Rhizosphere Soil

In order to investigate the underlying interaction mechanisms between plants and Gram-positive bacteria, 10 Paenibacillus and Bacillus strains were isolated from healthy tomato rhizosphere and plant tissues

The symbiotic bacteria Alcaligenes faecalis of the entomopathogenic nematodes Oscheius spp. exhibit potential biocontrol of plant- and entomopathogenic fungi

Soil-dwelling entomopathogenic nematodes (EPNs) kill arthropod hosts by injecting their symbiotic bacteria into the host hemolymph and feed on the bacteria and the tissue of the dying host for several generations cycles until the arthropod cadaver is completely depleted. The EPN-bacteria-arthropod cadaver complex represents a rich energy source for the surrounding opportunistic soil fungal biota and other competitors. We hypothesized that EPNs need to protect their food source until depletion and that the EPN symbiotic bacteria produce volatile and non-volatile exudations that deter different soil fungal groups in the soil. We isolated the symbiotic bacteria species (Alcaligenes faecalis) from the EPN Oscheius spp. and ran infectivity bioassays against entomopathogenic fungi (EPF) as well as against plant pathogenic fungi (PPF). We found that both volatile and non-volatile symbiotic bacterial exudations had negative effects on both EPF and PPF. Such deterrent function on functionally different fungal strains suggests a common mode of action of A.faecalis bacterial exudates, which has the potential to influence the structure of soil microbial communities, and could be integrated into pest management programs for increasing crop protection against fungal pathogens

Characterization of two relacidines belonging to a novel class of circular lipopeptides that act against Gram-negative bacterial pathogens

The development of sustainable agriculture and the increasing antibiotic resistance of human pathogens call for novel antimicrobial compounds. Here, we describe the extraction and characterization of a class of cationic circular lipopeptides, for which we propose the name relacidines, from the soil bacterium Brevibacillus laterosporus MG64. Relacidines are composed of a fatty acid side chain (4-methylhexanoic acid) and 13 amino acid residues. A lactone ring is formed by the last five amino acid residues and three positively charged ornithines are located in the linear fragment. Relacidines selectively combat Gram-negative pathogens, including phytopathogens and human pathogens. Further investigation of the mode of action revealed that relacidine B binds to the lipopolysaccharides but does not form pores in the cell membrane. We also provide proof to show that relacidine B does not affect the biosynthesis of the cell wall and RNA. Instead, it affects the oxidative phosphorylation process of cells and diminishes the biosynthesis of ATP. Transcription of relacidines is induced by plant pathogens, which strengthens the potential of B. laterosporus MG64 to be used as a biocontrol agent. Thus, we identified a new group of potent antibiotic compounds for combating Gram-negative pathogens of plants or animals

BAGEL4:a user-friendly web server to thoroughly mine RiPPs and bacteriocins

Interest in secondary metabolites such as RiPPs (ri-bosomally synthesized and posttranslationally modified peptides) is increasing worldwide. To facilitate the research in this field we have updated our mining web server. BAGEL4 is faster than its predecessor and is now fully independent from ORFcalling. Gene clusters of interest are discovered using the core-peptide database and/or through HMM motifs that are present in associated context genes. The databases used for mining have been updated and extended with literature references and links to UniProt and NCBI. Additionally, we have included automated promoter and terminator prediction and the option to upload RNA expression data, which can be displayed along with the identified clusters. Further improvements include the annotation of the context genes, which is now based on a fast blast against the prokaryote part of the UniRef90 database, and the improved web-BLAST feature that dynamically loads structural data such as internal cross-linking from UniProt. Overall BAGEL4 provides the user with more information through a user-friendly webinterface which simplifies data evaluation. BAGEL4 is freely accessible at http://bagel4.molgenrug.nl