Improving crop salt tolerance through soil legacy effects.

Soil salinization poses a critical problem, adversely affecting plant development and sustainable agriculture. Plants can produce soil legacy effects through interactions with the soil environments. Salt tolerance of plants in saline soils is not only determined by their own stress tolerance but is also closely related to soil legacy effects. Creating positive soil legacy effects for crops, thereby alleviating crop salt stress, presents a new perspective for improving soil conditions and increasing productivity in saline farmlands. Firstly, the formation and role of soil legacy effects in natural ecosystems are summarized. Then, the processes by which plants and soil microbial assistance respond to salt stress are outlined, as well as the potential soil legacy effects they may produce. Using this as a foundation, proposed the application of salt tolerance mechanisms related to soil legacy effects in natural ecosystems to saline farmlands production. One aspect involves leveraging the soil legacy effects created by plants to cope with salt stress, including the direct use of halophytes and salt-tolerant crops and the design of cropping patterns with the specific crop functional groups. Another aspect focuses on the utilization of soil legacy effects created synergistically by soil microorganisms. This includes the inoculation of specific strains, functional microbiota, entire soil which legacy with beneficial microorganisms and tolerant substances, as well as the application of novel technologies such as direct use of rhizosphere secretions or microbial transmission mechanisms. These approaches capitalize on the characteristics of beneficial microorganisms to help crops against salinity. Consequently, we concluded that by the screening suitable salt-tolerant crops, the development rational cropping patterns, and the inoculation of safe functional soils, positive soil legacy effects could be created to enhance crop salt tolerance. It could also improve the practical significance of soil legacy effects in the application of saline farmlands.

C4 cereal and biofuel crop microbiomes.

Microbiomes provide multiple life-support functions for plants, including nutrient acquisition and tolerance to abiotic and biotic stresses. Considering the importance of C4 cereal and biofuel crops for food security under climate change conditions, more attention has been given recently to C4 plant microbiome assembly and functions. Here, we review the current status of C4 cereal and biofuel crop microbiome research with a focus on beneficial microbial traits for crop growth and health. We highlight the importance of environmental factors and plant genetics in C4 crop microbiome assembly and pinpoint current knowledge gaps. Finally, we discuss the potential of foxtail millet as a C4 model species and outline future perspectives of C4 plant microbiome research.

Lipid transfer protein StLTPa enhances potato disease resistance against different pathogens by binding and disturbing the integrity of pathogens plasma membrane.

Potato is the third most important food crop worldwide. Potato production suffers from severe diseases caused by multiple detrimental plant pathogens, and broad-spectrum disease resistance genes are rarely identified in potato. Here we identified the potato non-specific lipid transfer protein StLTPa, which enhances species none-specific disease resistance against various pathogens, such as the oomycete pathogen Phytophthora infestans, the fungal pathogens Botrytis cinerea and Verticillium dahliae, and the bacterial pathogens Pectobacterium carotovorum and Ralstonia solanacearum. The StLTPa overexpression potato lines do not show growth penalty. Furthermore, we provide evidence that StLTPa binds to lipids present in the plasma membrane (PM) of the hyphal cells of P. infestans, leading to an increased permeability of the PM. Adding of PI(3,5)P2 and PI(3)P could compete the binding of StLTPa to pathogen PM and reduce the inhibition effect of StLTPa. The lipid-binding activity of StLTPa is essential for its role in pathogen inhibition and promotion of potato disease resistance. We propose that StLTPa enhances potato broad-spectrum disease resistance by binding to, and thereby promoting the permeability of the PM of the cells of various pathogens. Overall, our discovery illustrates that increasing the expression of a single gene in potato enhances potato disease resistance against different pathogens without growth penalty.

Metagenomic insights into genetic factors driving bacterial niche differentiation between bulk and rhizosphere soils.

Cellular motility is crucial for effective colonization of the rhizosphere, but it is not yet clear whether bacterial motility is particularly linked to other genetic traits. Here, we applied genome-resolved metagenomics and phylogenomics to investigate the ecological significance of cellular motility for niche differentiation and the links between the genetic makeup of motile bacteria and rhizosphere colonization within a four-decade maize field experiment. Indeed, highly diverse sets of genes encoding cellular motility, including chemotaxis, flagellar assembly and motility proteins, and utilization of polymeric carbon were the important predictors of bacterial niche differentiation between bulk and rhizosphere soils. This is well exemplified by metagenome-assembled genomes encoding high motility capacity (hmc_MAGs). Their collective abundance was, on average, sixfold higher in rhizosphere soil than in bulk soil. All bulk-soil-derived MAGs showed low motility capacities (lmc). The hmc_MAGs were highly enriched in beneficial traits involved in carbohydrate utilization, assimilatory (nasA) and dissimilatory (nirBD) nitrate reduction, inorganic phosphate solubilization (gcd), and organic phosphate mineralization (phoD). Belonging to the families Sphingomonadaceae, Burkholderiaceae and Steroidobacteraceae, the hmc_MAGs showed a ninefold greater enrichment in these traits than proteobacterial lmc_MAGs and a twofold greater enrichment than 264 genomes publicly available for the above three families, thereby substantiating that a specific rhizosphere effect acted on the microbes represented by the hmc_MAGs. The particular link between the genetic capacities for high cellular motility and increased carbohydrate depolymerization as the key determinant for plant-selected rhizosphere colonization was further substantiated by the analysis of public bulk-rhizosphere soil metagenomes retrieved from wheat and cucumber field sites.

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.

Designing a home for beneficial plant microbiomes.

The plant microbiome comprises a highly diverse community of saprotrophic, mutualistic, and pathogenic microbes that can affect plant growth and plant health. There is substantial interest to exploit beneficial members of plant microbiomes for new sustainable management strategies in crop production. However, poor survival and colonization of plant tissues by introduced microbial isolates as well as lack of expression of the plant growth-promoting or disease-suppressive traits at the right time and place are still major limitations for successful implementation of microbiomes in future agricultural practices and plant breeding programs. Similar to building a home for humans, we discuss different strategies of building a home for beneficial plant microbiomes, here referred to as the 'MicrobiHome'.

Antimicrobial activity screening of rhizosphere soil bacteria from tomato and genome-based analysis of their antimicrobial biosynthetic potential.

BACKGROUND: Tomato plant growth is frequently hampered by a high susceptibility to pests and diseases. Traditional chemical control causes a serious impact on both the environment and human health. Therefore, seeking environment-friendly and cost-effective green methods in agricultural production becomes crucial nowadays. Plant Growth Promoting Rhizobacteria (PGPR) can promote plant growth through biological activity. Their use is considered to be a promising sustainable approach for crop growth. Moreover, a vast number of biosynthetic gene clusters (BGCs) for secondary metabolite production are being revealed in PGPR, which helps to find potential anti-microbial activities for tomato disease control. RESULTS: We isolated 181 Bacillus-like strains from healthy tomato, rhizosphere soil, and tomato tissues. In vitro antagonistic assays revealed that 34 Bacillus strains have antimicrobial activity against Erwinia carotovora, Pseudomonas syringae; Rhizoctonia solani; Botrytis cinerea; Verticillium dahliae and Phytophthora infestans. The genomes of 10 Bacillus and Paenibacillus strains with good antagonistic activity were sequenced. Via genome mining approaches, we identified 120 BGCs encoding NRPs, PKs-NRPs, PKs, terpenes and bacteriocins, including known compounds such as fengycin, surfactin, bacillibactin, subtilin, etc. In addition, several novel BGCs were identified. We discovered that the NRPs and PKs-NRPs BGCs in Bacillus species are encoding highly conserved known compounds as well as various novel variants. CONCLUSIONS: This study highlights the great number of varieties of BGCs in Bacillus strains. These findings pave the road for future usage of Bacillus strains as biocontrol agents for tomato disease control and are a resource arsenal for novel antimicrobial discovery.

Novel Modifications of Nonribosomal Peptides from Brevibacillus laterosporus MG64 and Investigation of Their Mode of Action.

Nonribosomal peptides (NRPs) are a class of secondary metabolites usually produced by microorganisms. They are of paramount importance in different applications, including biocontrol and pharmacy. Brevibacillus spp. are a rich source of NRPs yet have received little attention. In this study, we characterize four novel bogorol variants (bogorols I to L, cationic linear lipopeptides) and four succilins (succilins I to L, containing a succinyl group that is attached to the Orn3/Lys3 in bogorols I to L) from the biocontrol strain Brevibacillus laterosporus MG64. Further investigation revealed that the bogorol family of peptides employs an adenylation pathway for lipoinitiation, different from the usual pattern, which is based on an external ligase and coenzyme A. Moreover, the formation of valinol was proven to be mediated by a terminal reductase domain and a reductase encoded by the bogI gene. Furthermore, succinylation, which is a novel type of modification in the family of bogorols, was discovered. Its occurrence requires a high concentration of the substrate (bogorols), but its responsible enzyme remains unknown. Bogorols display potent activity against both Gram-positive and Gram-negative bacteria. Investigation of their mode of action reveals that bogorols form pores in the cell membrane of both Gram-positive and Gram-negative bacteria. The combination of bogorols and relacidines, another class of NRPs produced by B. laterosporus MG64, displays a synergistic effect on different pathogens, suggesting the great potential of both peptides as well as their producer B. laterosporus MG64 for broad applications. Our study provides a further understanding of the bogorol family of peptides as well as their applications.IMPORTANCE NRPs form a class of secondary metabolites with biocontrol and pharmaceutical potential. This work describes the identification of novel bogorol variants and succinylated bogorols (namely, succilins) and further investigates their biosynthetic pathway and mode of action. Adenylation domain-mediated lipoinitiation of bogorols represents a novel pathway by which NRPs incorporate fatty acid tails. This pathway provides the possibility to engineer the lipid tail of NRPs without identifying a fatty acid coenzyme ligase, which is usually not present in the biosynthetic gene cluster. The terminal reductase domain (TD) and BogI-mediated valinol formation and their effect on the biological activity of bogorols are revealed. Succinylation, which is rarely reported in NRPs, was discovered in the bogorol family of peptides. We demonstrate that bogorols combat bacterial pathogens by forming pores in the cell membrane. We also report the synergistic effect of two natural products (relacidine B and bogorol K) produced by the same strain, which is relevant for competition for a niche.

Beyond Plant Microbiome Composition: Exploiting Microbial Functions and Plant Traits via Integrated Approaches.

Plants recruit specific microorganisms to live inside and outside their roots that provide essential functions for plant growth and health. The study of the microbial communities living in close association with plants helps in understanding the mechanisms involved in these beneficial interactions. Currently, most of the research in this field has been focusing on the description of the taxonomic composition of the microbiome. Therefore, a focus on the plant-associated microbiome functions is pivotal for the development of novel agricultural practices which, in turn, will increase plant fitness. Recent advances in microbiome research using model plant species started to shed light on the functions of specific microorganisms and the underlying mechanisms of plant-microbial interaction. Here, we review (1) microbiome-mediated functions associated with plant growth and protection, (2) insights from native and agricultural habitats that can be used to improve soil health and crop productivity, (3) current -omics and new approaches for studying the plant microbiome, and (4) challenges and future perspectives for exploiting the plant microbiome for beneficial outcomes. We posit that integrated approaches will help in translating fundamental knowledge into agricultural practices.

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.

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.

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.

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.

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.

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 ΔasbA (petropectin-deficient) mutant, but not the ΔdhbB (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. © 2018 The Authors.

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

Interest in secondary metabolites such as RiPPs (ribosomally 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 ORF-calling. 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 web-interface which simplifies data evaluation. BAGEL4 is freely accessible at http://bagel4.molgenrug.nl.

Secondary Metabolism and Interspecific Competition Affect Accumulation of Spontaneous Mutants in the GacS-GacA Regulatory System in Pseudomonas protegens.

Secondary metabolites are synthesized by many microorganisms and provide a fitness benefit in the presence of competitors and predators. Secondary metabolism also can be costly, as it shunts energy and intermediates from primary metabolism. In Pseudomonas spp., secondary metabolism is controlled by the GacS-GacA global regulatory system. Intriguingly, spontaneous mutations in gacS or gacA (Gac- mutants) are commonly observed in laboratory cultures. Here we investigated the role of secondary metabolism in the accumulation of Gac- mutants in Pseudomonas protegens strain Pf-5. Our results showed that secondary metabolism, specifically biosynthesis of the antimicrobial compound pyoluteorin, contributes significantly to the accumulation of Gac- mutants. Pyoluteorin biosynthesis, which poses a metabolic burden on the producer cells, but not pyoluteorin itself, leads to the accumulation of the spontaneous mutants. Interspecific competition also influenced the accumulation of the Gac- mutants: a reduced proportion of Gac- mutants accumulated when P. protegens Pf-5 was cocultured with Bacillus subtilis than in pure cultures of strain Pf-5. Overall, our study associated a fitness trade-off with secondary metabolism, with metabolic costs versus competitive benefits of production influencing the evolution of P. protegens, assessed by the accumulation of Gac- mutants.IMPORTANCE Many microorganisms produce antibiotics, which contribute to ecologic fitness in natural environments where microbes constantly compete for resources with other organisms. However, biosynthesis of antibiotics is costly due to the metabolic burdens of the antibiotic-producing microorganism. Our results provide an example of the fitness trade-off associated with antibiotic production. Under noncompetitive conditions, antibiotic biosynthesis led to accumulation of spontaneous mutants lacking a master regulator of antibiotic production. However, relatively few of these spontaneous mutants accumulated when a competitor was present. Results from this work provide information on the evolution of antibiotic biosynthesis and provide a framework for their discovery and regulation.

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.

The prey's scent - Volatile organic compound mediated interactions between soil bacteria and their protist predators.

Protists are major predators of bacteria in soils. However, it remains unknown how protists sense their prey in this highly complex environment. Here, we investigated whether volatile organic compounds (VOCs) of six phylogenetic distinct soil bacteria affect the performance of three different soil protists and how that relates to direct feeding interactions. We observed that most bacteria affected protist activity by VOCs. However, the response of protists to the VOCs was strongly dependent on both the bacterial and protist interacting partner. Stimulation of protist activity by volatiles and in direct trophic interaction assays often coincided, suggesting that VOCs serve as signals for protists to sense suitable prey. Furthermore, bacterial terpene synthase mutants lost the ability to affect protists, indicating that terpenes represent key components of VOC-mediated communication. Overall, we demonstrate that volatiles are directly involved in protist-bacterial predator-prey interactions.

Living on the edge: emergence of spontaneous gac mutations in Pseudomonas protegens during swarming motility.

Swarming motility is a flagella-driven multicellular behaviour that allows bacteria to colonize new niches and escape competition. Here, we investigated the evolution of specific mutations in the GacS/GacA two-component regulatory system in swarming colonies of Pseudomonas protegens Pf-5. Experimental evolution assays showed that repeated rounds of swarming by wildtype Pf-5 drives the accumulation of gacS/gacA spontaneous mutants on the swarming edge. These mutants cannot swarm on their own because they lack production of the biosurfactant orfamide A, but they do co-swarm with orfamide-producing wildtype Pf-5. These co-swarming assays further demonstrated that ΔgacA mutant cells indeed predominate on the edge and that initial ΔgacA:wildtype Pf-5 ratios of at least 2:1 lead to a collapse of the swarming colony. Subsequent whole-genome transcriptome analyses revealed that genes associated with motility, resource acquisition, chemotaxis and efflux were significantly upregulated in ΔgacA mutant on swarming medium. Moreover, transmission electron microscopy showed that ΔgacA mutant cells were longer and more flagellated than wildtype cells, which may explain their predominance on the swarming edge. We postulate that adaptive evolution through point mutations is a common feature of range-expanding microbial populations and that the putative fitness benefits of these mutations during dispersal of bacteria into new territories are frequency-dependent.