Jos Raaijmakers

Prof. Jos Raaijmakers PhD




Droevendaalsesteeg 10
6708 PB Wageningen

+31 (0) 317 47 34 00

The Netherlands


My research group investigates the taxonomic and functional diversity of the plant microbiome. We developed the concept of microbiome ‘rewilding’, which posits that plant health can be improved by reinstating key members of the diverse (ancestral) microbiota that were lost through domestication. To this end, we go back to the centres of origin of the wild relatives of our crop species to identify the ‘missing microbes’ and their beneficial traits.


Jos Raaijmakers is head of the Microbial Ecology Department at the Netherlands Institute of Ecology (NIOO-KNAW) and Professor at the Institute of Biology at Leiden University. He is member of the Royal Netherlands Academy of Arts & Sciences and board member of the PhD graduate School PE&RC. The overall goal of his research program is to unravel the impact of plant domestication on the diversity and beneficial functions of microorganisms associated with plants. In this search for 'missing plant microbes', we work closely together with research institutes and universities in the centres of origin of plant species (Africa, Asia, South America). The functions of the plant microbes studied in detail are protection of plants against infections caused by fungal pathogens, parasitic weeds and insects. Jos teaches BSc and MSc courses at Leiden University, and organizes (inter)national PhD courses and conferences, including the International Plant Microbiome conference.

International visibility, activities, prizes, scholarships

Jos Raaijmakers is  member of the Royal Netherlands Academy of Arts & Sciences (KNAW), Professor of Microbial Ecology at Leiden University, board member of the graduate school Production Ecology & Resource Conservation and former board member of the Centre for Soil Ecology. He was a visiting Professor at Copenhagen University (Denmark), Oregon State University (USA) and the University of Malaga (Spain), and former Chair of the Dutch Phytobacteriology group. He is currently editor-in-chief of the scientific journal ISME Communications.

His past and present research program is conducted in an international context with projects in Asia (Vietnam, Indonesia, Korea, China), South America (Ecuador, Colombia, Brasil) and Africa (Ghana, Ethiopia). He is recipient/coordinator of several prestigious and large national and international research grants (e.g. NWO-EcoGenomics (Netherlands Genomics Initiative), NWO-Gravitation (, BE-Basic, TTW-Perspectief Back-To-Roots), EU-Horizon/Marie Curie ITN; NWO-Groot). His expertise in plant-microbiome research led to the invitation by the Bill & Melinda Gates Foundation to develop and coordinate the international program PROMISE ( In the latest international peer review, his department/institute was rated with the highest score (“excellent”) on all criteria (Quality, Relevance, Viability).

He published many articles in peer reviewed scientific journals, including papers in Nature-based journals (Nature Microbiology, Nature Chemical Biology, ISME Journal), Science, PNAS, and top Microbiology, Plant Science and Ecology journals. Over the past 5 years, he was elected in the Top 1% of the most highly cited researchers worldwide. 

He holds several international patents and works closely with industry (start-ups, medium & large enterprises), including international seed and agrochemical companies focused on developing new microbiome-based products for sustainable horti- and agriculture. He supervises multiple PhD students, postdocs and technicians, and is involved in teaching BSc and MSc courses at Leiden University as well as international PhD courses. During his time as an associate professor at Wageningen University, he was selected by the students among the best teachers. He also organized several international conferences, including the PGPR-meeting (2007), Rhizosphere 4 (2015), and the International Plant Microbiome conference (2016, 2018, 2022, 2023). Many of his former PhD and postdocs have acquired leading positions at (inter)national universities, government research institutes and R&D departments of different companies.

Professional activities

  • Promoter of PhD students: 19 graduations completed; 11 ongoing
  • Reviewer for various international scientific journals 
  • Speaker at international conferences and workshops
  • Member of (inter)national PhD committees & Scientific Advisory Boards
  • Member of professional organizations - International Society for Microbial Ecology (ISME); American Society for Microbiology (ASM); Dutch Society for Microbiology (NVvM); Dutch Phytopathological Society (KNPV); Graduate Schools - Experimental Plant Sciences (EPS); Production Ecology & Resource Conservation (PE&RC).

Teaching (past & present)

  • Microbial Ecology; Molecular Microbiology; Leiden University, Netherlands
  • Plant-Microbe Interactions, Integrated Pest Management, Living Soil, Wageningen University, Netherlands
  • Genomics, Applied Microbiology, Wageningen University & University Copenhagen

Scholarships & prizes

  • Elected member of the Royal Netherlands Academy of Arts & Sciences (KNAW) - 2023
  • Nominee Huibregtsenprize 2022 (
  • 2018 – 2022: top 1% of most Highly Cited Researchers worldwide (Clarivate Analytics)
  • Nominee of the Merck Future Insight Prize 2021
  • Academy (KNAW) fellow (2001-2006) ‘Pathogen defense against microbial antagonism’
  • Elected in 2010 & 2011 by students among best teachers of Wageningen University
  • OECD-fellowship (1998) ‘Role of antibiosis in Fusarium-wilt suppressive soils’
  • USDA-award (1997) ‘Unravelling the molecular basis of disease suppressive soils’




  • 2014–Present
    Head of Department Microbial Ecology, NIOO-KNAW
  • 2014–Present
    Professor Microbial Diversity, IBL, Leiden University, Netherlands
  • 1998–2014
    Associate Professor Plant Pathology, Wageningen University, Netherlands
  • 1995–1998
    Postdoc soil microbiology, USDA-ARS, Washington State University, USA
  • 1994–1995
    Postdoc Plant-Microbe Interactions, Utrecht University, Netherlands
  • 1989–1994
    PhD Plant-Microbe Interactions, Utrecht University, Netherlands
  • 2019
    visiting Professor at Malaga University, Spain
  • 2007
    visiting Professor Oregon State University, Corvallis, USA
  • 2006–2008
    visiting Professor, University Copenhagen, Denmark
  • 2019–2022
    Board member Graduate School PE&RC
  • 2012–2020
    Board member Centre for Soil Ecology (
  • 2009–2013
    Chair Phytobacteriology group KNPV


BSc, MSc Biology, Utrecht University, Netherlands

Editorial board memberships

Editor-in-Chief, ISME Communications



Peer-reviewed publicaties

  • Trends in Microbiology

    C4 cereal and biofuel crop microbiomes

    Xiaoyu Zai, Viviane Cordovez da Cunha, Feng Zhu, Meicheng Zhao, Xianmin Diao, Fusuo Zhang, Jos M. Raaijmakers, Chunxu Song

    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.
  • Cell Reports

    The soil microbiome modulates the sorghum root metabolome and cellular traits with a concomitant reduction of Striga infection

    Dorota Kawa, Benjamin Thiombiano, Mahdere Shimels, Tamera Taylor, Aimee Walmsley, Hannah E. Vahldick, Dominika Rybka, Marcio Fernandes Alves Leite, Zayan Musa, Alexander Bucksch, Francisco Dini-Andreote, Mario Schilder, Alexander J. Chen, Jiregna Daksa, Desalegn Etalo, Taye Tessema, Eiko Kuramae, Jos M. Raaijmakers, Harro J. Bouwmeester, Siobhan M. Brady
    Sorghum bicolor is among the most important cereals globally and a staple crop for smallholder farmers in sub-Saharan Africa. Approximately 20% of sorghum yield is lost annually in Africa due to infestation with the root parasitic weed Striga hermonthica. Existing Striga management strategies are not singularly effective and integrated approaches are needed. Here, we demonstrate the functional potential of the soil microbiome to suppress Striga infection in sorghum. We associate this suppression with microbiome-mediated induction of root endodermal suberization and aerenchyma formation and with depletion of haustorium-inducing factors, compounds required for the initial stages of Striga infection. We further identify specific bacterial taxa that trigger the observed Striga-suppressive traits. Collectively, our study describes the importance of the soil microbiome in the early stages of root infection by Striga and pinpoints mechanisms of Striga suppression. These findings open avenues to broaden the effectiveness of integrated Striga management practices.
  • Nature Communications

    bacLIFE: a user-friendly computational workflow for genome analysis and prediction of lifestyle-associated genes in bacteria

    Guillermo Guerrero-Egido, Adrian Pintado, Kevin M. Bretscher, Joseph N. Paulson, Herman P Spaink, Dennis Claessen, Cayo Ramos, Francisco M. Cazorla, Marnix H Medema, Jos M. Raaijmakers, Victor Carrion Bravo
    Bacteria have an extensive adaptive ability to live in close association with eukaryotic hosts, exhibiting detrimental, neutral or beneficial effects on host growth and health. However, the genes involved in niche adaptation are mostly unknown and their functions poorly characterized. Here, we present bacLIFE ( a streamlined computational workflow for genome annotation, large-scale comparative genomics, and prediction of lifestyle-associated genes (LAGs). As a proof of concept, we analyzed 16,846 genomes from the Burkholderia/Paraburkholderia and Pseudomonas genera, which led to the identification of hundreds of genes potentially associated with a plant pathogenic lifestyle. Site-directed mutagenesis of 14 of these predicted LAGs of unknown function, followed by plant bioassays, showed that 6 predicted LAGs are indeed involved in the phytopathogenic lifestyle of Burkholderia plantarii and Pseudomonas syringae pv. phaseolicola. These 6 LAGs encompassed a glycosyltransferase, extracellular binding proteins, homoserine dehydrogenases and hypothetical proteins. Collectively, our results highlight bacLIFE as an effective computational tool for prediction of LAGs and the generation of hypotheses for a better understanding of bacteria-host interactions.
  • FEMS Microbiology Ecology

    Impact of bacterial and fungal inoculants on the resident rhizosphere microbiome and the volatilome of tomato plants under leaf herbivory stress

    Ana Shein Lee Diaz, Zhivko Minchev Ivanov, Jos M. Raaijmakers, María José Pozo, Paolina Garbeva
    Various studies have addressed the impact of microbial inoculants on the composition of the resident microbiome. How microbial inoculants impact plant metabolism and interact with the resident rhizobiota under herbivory stress remains elusive. Here, we investigated the impact of two bacterial and two fungal inoculants, inoculated as single species and as a synthetic community, on the rhizosphere microbiome and volatilome of tomato plants (Solanum lycopersicum) comparing nonstress conditions to exposed to leaf herbivory by Spodoptera exigua. Based on amplicon sequencing analysis, rhizobacterial community composition was significantly affected by all four inoculants and the magnitude of this effect was dependent on herbivory stress. Fungal community composition was altered by the microbial inoculants but independent of herbivory stress. The rhizosphere volatilome was impacted by the microbial inoculation and differences between treatments were evened under herbivory stress. Each microbial inoculant caused unique changes in the volatilome of stressed plants but also shared similar responses, in particular the enhanced production of dimethyl disulfide and benzothiazole. In conclusion, the introduction of microbial inoculants in the tomato rhizosphere caused unique as well as common changes in the rhizosphere microbiome and volatilome, but these changes were minor compared to the microbiome changes induced by herbivory stress.
  • Phytobiomes Journal

    The Tomato’s Tale: Exploring taxonomy, biogeography, domestication and microbiome for enhanced resilience

    S.W. Sarango Flores, Viviane Cordovez da Cunha, Ben Oyserman, Nejc Stopnisek, Jos M. Raaijmakers, Pieter van 't Hof
    Plant domestication and breeding not only resulted in multiple phenotypic changes, but also impacted agricultural ecosystems where our current crops are cultivated. Most crops to date rely on the extensive use of fertilizers and pesticides to support crop growth and health. To minimize the environmental impact of these management practices, the plant microbiome got renewed attention as a large, yet untapped resource of microorganisms with beneficial effects on plant growth and health. In the past decade, it has become evident that the microbiome of plants plays a key role in nutrient acquisition, plant development and tolerance to diverse abiotic and biotic stresses. Here we review past and present knowledge of the microbiome of tomato as a model for unraveling the functional potential of plant microbiomes, the impact of domestication and the underlying genetics of microbiome assembly and activity. We also provide perspectives on how this knowledge can be adopted to enhance crop productivity and strengthen the sustainability of agricultural management practices.
  • Environmental Microbiome

    Repeated exposure of wheat to the fungal root pathogen Bipolaris sorokiniana modulates rhizosphere microbiome assembly and disease suppressiveness

    Lilian Abreu, Mírian Rabelo de Faria, Josiane B. Chiaramonte, L.W. Mendes, Edis Sepo, Mattias De Hollander, José Maurício Cunha Fernandes, Victor Carrion Bravo, Wagner Bettiol, Tim H. Mauchline, Jos M. Raaijmakers, Rodrigo Mendes

    Disease suppressiveness of soils to fungal root pathogens is typically induced in the field by repeated infections of the host plant and concomitant changes in the taxonomic composition and functional traits of the rhizosphere microbiome. Here, we studied this remarkable phenomenon for Bipolaris sorokiniana in two wheat cultivars differing in resistance to this fungal root pathogen.

    The results showed that repeated exposure of the susceptible wheat cultivar to the pathogen led to a significant reduction in disease severity after five successive growth cycles. Surprisingly, the resistant wheat cultivar, initially included as a control, showed the opposite pattern with an increase in disease severity after repeated pathogen exposure. Amplicon analyses revealed that the bacterial families Chitinophagaceae, Anaerolineaceae and Nitrosomonadaceae were associated with disease suppressiveness in the susceptible wheat cultivar; disease suppressiveness in the resistant wheat cultivar was also associated with Chitinophagaceae and a higher abundance of Comamonadaceae. Metagenome analysis led to the selection of 604 Biosynthetic Gene Clusters (BGCs), out of a total of 2,571 identified by AntiSMASH analysis, that were overrepresented when the soil entered the disease suppressive state. These BGCs are involved in the biosynthesis of terpenes, non-ribosomal peptides, polyketides, aryl polyenes and post-translationally modified peptides.

    Combining taxonomic and functional profiling we identified key changes in the rhizosphere microbiome during disease suppression. This illustrates how the host plant relies on the rhizosphere microbiome as the first line of defense to fight soil-borne pathogens. Microbial taxa and functions identified here can be used in novel strategies to control soil-borne fungal pathogens.
  • ISME Journal

    Widespread and largely unknown prophage activity, diversity, and function in two genera of wheat phyllosphere bacteria

    Peter Erdmann Dougherty, Tue Kjærgaard Nielsen, Leise Riber, Helen Helgå Lading, Laura Milena Forero-Junco, Witold Kot, Jos M. Raaijmakers, Lars Hestbjerg Hansen
    Environmental bacteria host an enormous number of prophages, but their diversity and natural functions remain largely elusive. Here, we investigate prophage activity and diversity in 63 Erwinia and Pseudomonas strains isolated from flag leaves of wheat grown in a single field. Introducing and validating Virion Induction Profiling Sequencing (VIP-Seq), we identify and quantify the activity of 120 spontaneously induced prophages, discovering that some phyllosphere bacteria produce more than 108 virions/mL in overnight cultures, with significant induction also observed in planta. Sequence analyses and plaque assays reveal E. aphidicola prophages contribute a majority of intraspecies genetic diversity and divide their bacterial hosts into antagonistic factions engaged in widespread microbial warfare, revealing the importance of prophage-mediated microdiversity. When comparing spontaneously active prophages with predicted prophages we also find insertion sequences are strongly correlated with non-active prophages. In conclusion, we discover widespread and largely unknown prophage diversity and function in phyllosphere bacteria.
  • Applied and Environmental Microbiology

    The plant stress hormone jasmonic acid evokes defensive responses in streptomycetes

    Anne Van der Meij, Somayah S. Elsayed, Chao Du, Joost Willemse, Thomas M. Wood, Nathaniel I. Martin, Jos M. Raaijmakers, Gilles van Wezel
    Actinobacteria are prevalent in the rhizosphere and phyllosphere of diverse plant species where they help to enhance tolerance of plants against biotic and abiotic stresses. Here, we show that the plant hormones jasmonic acid (JA) and methyl jasmonate (MeJA) affect the growth, development, and specialized metabolism of Streptomyces. Exposure of Streptomyces coelicolor to JA or MeJA led to enhanced production of the polyketide antibiotic actinorhodin. JA also exhibited toxicity toward Streptomyces and Streptacidiphilus at higher concentrations, whereby streptomycetes were more tolerant to JA than members of the genus Streptacidiphilus. Tolerance to JA could be linked to its conjugation by the bacteria with glutamine. Additionally, JA conjugates with valine, tyrosine, phenylalanine, and leucine/isoleucine were identified. In contrast to JA, synthetic JA conjugates failed to activate antibiotic production and showed significantly reduced toxicity. Thus, our findings provide insights into a previously unknown defense mechanism deployed by Streptomycetaceae to a plant hormone. The underlying mechanism encompasses the attachment of amino acids to JA, which in turn safeguards the bacteria against the harmful impacts of the plant hormone. This study adds to the growing body of evidence that plant hormones can have a significant impact on members of the plant microbiome by affecting their growth, development, and secondary metabolism.
    IMPORTANCEMicroorganisms that live on or inside plants can influence plant growth and health. Among the plant-associated bacteria, streptomycetes play an important role in defense against plant diseases, but the underlying mechanisms are not well understood. Here, we demonstrate that the plant hormones jasmonic acid (JA) and methyl jasmonate directly affect the life cycle of streptomycetes by modulating antibiotic synthesis and promoting faster development. Moreover, the plant hormones specifically stimulate the synthesis of the polyketide antibiotic actinorhodin in Streptomyces coelicolor. JA is then modified in the cell by amino acid conjugation, thereby quenching toxicity. Collectively, these results provide new insight into the impact of a key plant hormone on diverse phenotypic responses of streptomycetes.
  • Applied Soil Ecology

    Diversity of viruses and viroids in the rhizosphere of common bean cultivars differing in resistance to the fungal root pathogen Fusarium oxysporum

    Lucas P.P. Braga, Andrew J Tanentzap, Benjamin Lee, Siu Mui Tsai, Jos M. Raaijmakers, Rodrigo Mendes, L.W. Mendes
    The rhizosphere microbiome plays a key role in plant protection against soil-borne pathogens. Plant breeding for resistance against soil-borne pathogens can alter the rhizosphere microbiome. However, most studies have focused on bacterial and fungal communities, leaving the role of the virus and viroids unassessed. Here, we tested the influence of resistance breeding on the composition of rhizosphere viruses and viroids. By analyzing metatranscriptomes from the rhizosphere of common bean (Phaseolus vulgaris) cultivars with varying resistance to the soil-borne pathogen Fusarium oxysporum, we recovered sequences representing 78 and 23 novel populations of viruses and viroids, respectively. We compared the abundances of these infectious agents across the different cultivars and found that the Fusarium-resistant cultivar harbored >1.2 times more viroids and a more different composition of viroids and viruses than less resistant plants. Given their role in interfering with host metabolism and their potential influence on plant-fungi associations, our study suggests that changes in the rhizosphere infectome are an important consideration in breeding for resistance against soil-borne pathogens.
  • Environmental Microbiome

    Impact of the fungal pathogen Fusarium oxysporum on the taxonomic and functional diversity of the common bean root microbiome

    L.W. Mendes, Jos M. Raaijmakers, Mattias De Hollander, Edis Sepo, Ruth Gomez Exposito, Alisson Fernando Chiorato, Rodrigo Mendes, Siu Mui Tsai, Victor Carrion Bravo

    Background: Plants rely on their root microbiome as the first line of defense against soil-borne fungal pathogens. The abundance and activities of beneficial root microbial taxa at the time prior to and during fungal infection are key to their protective success. If and how invading fungal root pathogens can disrupt microbiome assembly and gene expression is still largely unknown. Here, we investigated the impact of the fungal pathogen Fusarium oxysporum (fox) on the assembly of rhizosphere and endosphere microbiomes of a fox-susceptible and fox-resistant common bean cultivar. Results: Integration of 16S-amplicon, shotgun metagenome as well as metatranscriptome sequencing with community ecology analysis showed that fox infections significantly changed the composition and gene expression of the root microbiome in a cultivar-dependent manner. More specifically, fox infection led to increased microbial diversity, network complexity, and a higher proportion of the genera Flavobacterium, Bacillus, and Dyadobacter in the rhizosphere of the fox-resistant cultivar compared to the fox-susceptible cultivar. In the endosphere, root infection also led to changes in community assembly, with a higher abundance of the genera Sinorhizobium and Ensifer in the fox-resistant cultivar. Metagenome and metatranscriptome analyses further revealed the enrichment of terpene biosynthesis genes with a potential role in pathogen suppression in the fox-resistant cultivar upon fungal pathogen invasion. Conclusion: Collectively, these results revealed a cultivar-dependent enrichment of specific bacterial genera and the activation of putative disease-suppressive functions in the rhizosphere and endosphere microbiome of common bean under siege.
  • Trends in Microbiology

    Importance of Bacteroidetes in host–microbe interactions and ecosystem functioning

    Xinya Pan, Jos M. Raaijmakers, Victor Carrion Bravo
    Bacteroidetes are prevalent in soil ecosystems and are associated with various eukaryotic hosts, including plants, animals, and humans. The ubiquity and diversity of Bacteroidetes exemplify their impressive versatility in niche adaptation and genomic plasticity. Over the past decade, a wealth of knowledge has been obtained on the metabolic functions of clinically relevant Bacteroidetes, but much less attention has been given to Bacteroidetes living in close association with plants. To improve our understanding of the functional roles of Bacteroidetes for plants and other hosts, we review the current knowledge of their taxonomy and ecology, in particular their roles in nutrient cycling and host fitness. We highlight their environmental distribution, stress resilience, genomic diversity, and functional importance in diverse ecosystems, including, but not limited to, plant-associated microbiomes.
  • Bioinformatics Advances

    baseLess: lightweight detection of sequences in raw MinION data

    Ben Noordijk, Reindert Nijland, Victor Carrion Bravo, Jos M. Raaijmakers, Dick de Ridder, Carlos de Lannoy
    With its candybar form factor and low initial investment cost, the MinION brought affordable portable nucleic acid analysis within reach. However, translating the electrical signal it outputs into a sequence of bases still requires mid-tier computer hardware, which remains a caveat when aiming for deployment of many devices at once or usage in remote areas. For applications focusing on detection of a target sequence, such as infectious disease monitoring or species identification, the computational cost of analysis may be reduced by directly detecting the target sequence in the electrical signal instead. Here, we present baseLess, a computational tool that enables such target-detection-only analysis. BaseLess makes use of an array of small neural networks, each of which efficiently detects a fixed-size subsequence of the target sequence directly from the electrical signal. We show that baseLess can accurately determine the identity of reads between three closely related fish species and can classify sequences in mixtures of 20 bacterial species, on an inexpensive single-board computer.
  • Nature Communications

    Disentangling the genetic basis of rhizosphere microbiome assembly in tomato

    Ben Oyserman, S.W. Sarango Flores, Thom Griffioen, Xinya Pan, Elmar van der Wijk, Wouter Lokhorst, Azkia Nurfikari, Joseph N. Paulson, Mercedeh Movassagh, Nejc Stopnisek, Anne Kupczok, Viviane Cordovez da Cunha, Victor Carrion Bravo, Wilco Ligterink, Basten Snoek, Marnix H Medema, Jos M. Raaijmakers
    Microbiomes play a pivotal role in plant growth and health, but the genetic factors involved in microbiome assembly remain largely elusive. Here, we map the molecular features of the rhizosphere microbiome as quantitative traits of a diverse hybrid population of wild and domesticated tomato. Gene content analysis of prioritized tomato quantitative trait loci suggests a genetic basis for differential recruitment of various rhizobacterial lineages, including a Streptomyces-associated 6.31 Mbp region harboring tomato domestication sweeps and encoding, among others, the iron regulator FIT and the water channel aquaporin SlTIP2.3. Within metagenome-assembled genomes of root-associated Streptomyces and Cellvibrio, we identify bacterial genes involved in metabolism of plant polysaccharides, iron, sulfur, trehalose, and vitamins, whose genetic variation associates with specific tomato QTLs. By integrating ‘microbiomics’ and quantitative plant genetics, we pinpoint putative plant and reciprocal rhizobacterial traits underlying microbiome assembly, thereby providing a first step towards plant-microbiome breeding programs.
  • Science

    Rewilding plant microbiomes

    Jos M. Raaijmakers, E. Toby Kiers
    Microbiota of crop ancestors may offer a way to enhance sustainable food production Over the past decade, research has shown that microorganisms living on and inside eukaryotes—the microbiota—are drivers of host health. For plants, microbiota can greatly expand their genomic capabilities by enhancing immunity, nutrient acquisition, and tolerance to environmental stresses ( 1 ). More than ever, plant microbiota are being considered as a lever to increase the sustainability of food production under a changing climate. Emerging from this global interest to harness the largely unexplored functional potential of microbiota, the microbiome rewilding hypothesis posits that plant and animal health can be improved by reinstating key members of the diverse (ancestral) microbiota that were lost through domestication and industrialization processes, including changes in diet, plant and animal breeding, and the (over)use of antibiotics, pesticides, and fertilizers ( 2 – 4 ).
  • Weed Research

    Molecular detection and quantification of the Striga seedbank in agricultural soils

    Getahun Mitiku Benti, Dominika Rybka, P.J.A. Klein Gunnewiek, Taye Tessema, Jos M. Raaijmakers, Desalegn Etalo
    Striga hermonthica (Del.) Benth is a devastating parasitic weed in Sub-Saharan Africa (SSA) and its soil seedbank is the major factor contributing to its prevalence and persistence. To date, there is a little information on the Striga seedbank density in agricultural fields in SSA due to the lack of reliable detection and quantification methods. We developed a high-throughput method that combines density- and size-based separation techniques with quantitative polymerase chain reaction (qPCR)-based detection of Striga seeds in soil. The method was optimised and validated by introducing increasing numbers of Striga seeds in two physicochemically different Striga-free agricultural soils. The results showed that as little as one seed of S. hermonthica per 150 g of soil could be detected. This technique was subsequently tested on soil samples of 48 sorghum fields from different agro-ecological zones in Ethiopia to map the geospatial distribution of the Striga seedbank along a trajectory of more than 1500 km. Considerable variation in Striga seed densities was observed. Striga seeds were detectable in 75% of the field soils with densities up to 86 seeds per 150 g of soil. The Striga seed density in soil and the number of emerged Striga plants in the field showed a non-linear relationship. In conclusion, the method developed allows for accurate mapping of the Striga seedbank in physicochemically diverse SSA field soils and can be used to assess the impact of management strategies on Striga seedbank dynamics.
  • ISME Communications


    Elizabeth A. McDaniel, J.J.M. van Steenbrugge, Daniel R. Noguera, Katherine D. McMahon, Jos M. Raaijmakers, Marnix H Medema, Ben Oyserman
    A grand challenge in microbial ecology is disentangling the traits of individual populations within complex communities. Various cultivation-independent approaches have been used to infer traits based on the presence of marker genes. However, marker genes are not linked to traits with complete fidelity, nor do they capture important attributes, such as the timing of gene expression or coordination among traits. To address this, we present an approach for assessing the trait landscape of microbial communities by statistically defining a trait attribute as a shared transcriptional pattern across multiple organisms. Leveraging the KEGG pathway database as a trait library and the Enhanced Biological Phosphorus Removal (EBPR) model microbial ecosystem, we demonstrate that a majority (65%) of traits present in 10 or more genomes have niche-differentiating expression attributes. For example, while many genomes containing high-affinity phosphorus transporter pstABCS display a canonical attribute (e.g. up-regulation under phosphorus starvation), we identified another attribute shared by many genomes where transcription was highest under high phosphorus conditions. Taken together, we provide a novel framework for unravelling the functional dynamics of uncultivated microorganisms by assigning trait-attributes through genome-resolved time-series metatranscriptomics.
  • Pathogens

    Plant- and bacteria-derived compounds with anti-Philasterides dicentrarchi activity

    Rosa Ana Sueiro, José Manuel Leiro, Verónica Blanco-Abad, Jos M. Raaijmakers, Irene de Bruijn, Ron P.H. Dirks, Jesús Lamas
    Philasterides dicentrarchi is a scuticociliate that causes high mortalities in farmed fish. Although vaccination is an effective method to prevent scuticociliatosis caused by the homologous serotype, a universal vaccine has not been developed yet. Many compounds have been shown to be toxic to this ciliate species; moreover, most of them are toxic to aquatic life and cannot be used to prevent the disease. We have evaluated the toxicity to P. dicentrarchi of several compounds of natural origin to be used to reduce parasite levels in the seawater. Ciliates were exposed to several compound concentrations, and the mortality was determined at several incubation times. Tomatine, plumbagin and 2′,4′-dihydroxychalcone displayed the highest anticiliate activity, with a dose-dependent response. The effects of these compounds on the EPC cell line were also evaluated, finding that 2′,4′-dihydroxychalcone displayed the lowest toxicity to fish cells. At 7.54 μM, 2′,4′-dihydroxychalcone inhibited 50% parasite growth but only killed about 10% of EPC cells after 24 h incubation. Finally, we evaluated the toxicity of Pseudomonas H6 surfactant (PS) to P. dicentrarchi, finding that PS was toxic to the ciliate but showed lower toxicity to EPC cells. At a concentration of 7.8 μg/mL (LC50 for the ciliate after 3 h incubation), PS killed 14.9% of EPC cells. We conclude that 2′,4′-dihydroxychalcone, and PS could be used to reduce parasite levels in seawater, thus decreasing the risk of scuticociliatosis infection in cultured fish.
  • Plant, Cell and Environment

    Metabolic signatures of rhizobacteria-induced plant growth promotion

    Je Seung Jeon, Dominika Rybka, N. Carreno Quintero, Ric C.H De Vos, Jos M. Raaijmakers, Desalegn Etalo
    Various root-colonizing bacterial species can promote plant growth and trigger systemic resistance against aboveground leaf pathogens and herbivore insects. To date, the underlying metabolic signatures of these rhizobacteria-induced plant phenotypes are poorly understood. To identify core metabolic pathways that are targeted by growth-promoting rhizobacteria, we used combinations of three plant species and three rhizobacterial species and interrogated plant shoot chemistry by untargeted metabolomics. A substantial part (50-64%) of the metabolites detected in plant shoot tissue was differentially affected by the rhizobacteria. Among others, the phenylpropanoid pathway was targeted by the rhizobacteria in each of the three plant species. Differential regulation of the various branches of the phenylpropanoid pathways showed an association with either plant growth promotion or growth reduction. Overall, suppression of flavonoid biosynthesis was associated with growth promotion, while growth reduction showed elevated levels of flavonoids. Subsequent assays with twelve Arabidopsis flavonoid biosynthetic mutants revealed that the proanthocyanidin branch plays an essential role in rhizobacteria-mediated growth promotion. Our study also showed that a number of pharmaceutically and nutritionally relevant metabolites in the plant shoot were significantly increased by rhizobacterial treatment, providing new avenues to use rhizobacteria to tilt plant metabolism towards the biosynthesis of valuable natural plant products.
  • Trends in Plant Science

    Ecology and functional potential of phyllosphere yeasts

    Linda Gouka, Jos M. Raaijmakers, Viviane Cordovez da Cunha
    The phyllosphere (i.e., the aerial parts of plants) harbors a rich microbial life, including bacteria, fungi, viruses, and yeasts. Current knowledge of yeasts stems primarily from industrial and medical research on Saccharomyces cerevisiae and Candida albicans, both of which can be found on plant tissues. For most other yeasts found in the phyllosphere, little is known about their ecology and functions. Here, we explore the diversity, dynamics, interactions, and genomics of yeasts associated with plant leaves and how tools and approaches developed for model yeasts can be adopted to disentangle the ecology and natural functions of phyllosphere yeasts. A first genomic survey exemplifies that we have only scratched the surface of the largely unexplored functional potential of phyllosphere yeasts.
  • Fungal Systematics and Evolution

    Fusarium diversity associated with the Sorghum-Striga interaction in Ethiopia

    Lorenzo Lombard, R. Van Doorn, Ewald Groenewald, Taye Tessema, Eiko Kuramae, D.W. Etolo, Jos M. Raaijmakers, Pedro W. Crous
    Sorghum production is seriously threatened by the root parasitic weeds (RPWs) Striga hermonthica and Striga asiatica in sub-Saharan Africa. Research has shown that Striga control depends on eliminating its seed reserves in soil. Several species of the genus Fusarium (Nectriaceae, Hypocreales), which have been isolated from diseased Striga plants have proven to be highly pathogenic to all developmental stages of these RPWs. In the present study 439 isolates of Fusarium spp. were found associated with soils from Sorghum growing fields, Sorghum rhizosphere, or as endophytes with Sorghum roots and seeds, or as endophytes of Striga stems and seeds. Based on multi-locus phylogenies of combinations of CaM, tef1, rpb1 and rpb2 alignments, and morphological characteristics, 42 species were identified, including three species that are newly described, namely F. extenuatum and F. tangerinum from Sorghum soils, and F. pentaseptatum from seed of Striga hermonthica. Using a previously published AFLP-derived marker that is specific to detect isolates of F. oxysporum f.sp.strigae, an effective soil-borne biocontrol agent against Striga, we also detected the gene in several other Fusarium species. As these isolates were all associated with the Striga/Sorghum pathosystem, the possibility of horizontal gene transfer among these fusaria will be of interest to further investigate in future.
  • FEMS Microbiology Ecology

    Back to the Roots

    Sewunet Abera Dinke, Mahdere Shimels, Taye Tessema, Jos M. Raaijmakers, Fernando Dini Andreote
    Sorghum is a major staple crop in sub-Saharan Africa with yields severely impacted by biotic and abiotic factors. Here, we analysed the taxonomic diversity and biogeographical distribution of bacterial taxa of 48 agricultural fields along a transect of approximately 2000 km across the Ethiopian sorghum belt, the centre of origin of sorghum. The ultimate goal is to identify—yet-unexplored—beneficial plant–microbe associations. Based on bulk soil bacterial communities and DArT-SNP analyses of 59 sorghum accessions, we selected three microbiologically distinct field soils and 12 sorghum genotypes, including commercial varieties, wild relatives, and farmer-preferred landraces. The results showed a core rhizosphere microbiome of 2125 amplicon sequence variants (ASVs), belonging to eight bacterial families consistently found across the three soil types and the 12 sorghum genotypes. Integration of the rhizosphere bacterial community analysis with DArT-SNP sorghum genotyping revealed the association of differentially abundant ASVs with sorghum genotypic traits, including the distinct recruitment of Pseudomonadaceae by the stay-green, drought-tolerant, and wild sorghum genotypes. Collectively, these results provide new insights into the core and accessory bacterial taxa in the sorghum rhizosphere in the centre of origin, setting a baseline for targeted isolation and functional characterization of putative beneficial rhizobacteria.
  • Frontiers in Plant Science

    Genetic, phenotypic and metabolic diversity of yeasts from wheat flag leaves

    Linda Gouka, Caroline Vogels, Lars Hestbjerg Hansen, Jos M. Raaijmakers, Viviane Cordovez da Cunha
    The phyllosphere, the aboveground part of a plant, is a harsh environment with diverse abiotic and biotic stresses, including oscillating nutrient availability and temperature as well as exposure to UV radiation. Microbial colonization of this dynamic environment requires specific adaptive traits, including tolerance to fluctuating temperatures, the production of secondary metabolites and pigments to successfully compete with other microorganisms and to withstand abiotic stresses. Here, we isolated 175 yeasts, comprising 15 different genera, from the wheat flag leaf and characterized a selection of these for various adaptive traits such as substrate utilization, tolerance to different temperatures, biofilm formation, and antagonism towards the fungal leaf pathogen Fusarium graminearum. Collectively our results revealed that the wheat flag leaf is a rich resource of taxonomically and phenotypically diverse yeast genera that exhibit various traits that can contribute to survival in the harsh phyllosphere environment.
  • Molecules

    Exploring the Volatiles Released from Roots of Wild and Domesticated Tomato Plants under Insect Attack

    Ana Shein Lee Diaz, Muhammad Syamsu Rizaludin, Hans Zweers, Jos M. Raaijmakers, Paolina Garbeva

    Plants produce volatile organic compounds that are important in communication and defense. While studies have largely focused on volatiles emitted from aboveground plant parts upon exposure to biotic or abiotic stresses, volatile emissions from roots upon aboveground stress are less studied. Here, we investigated if tomato plants under insect herbivore attack exhibited a different root volatilome than non-stressed plants, and whether this was influenced by the plant’s genetic background. To this end, we analyzed one domesticated and one wild tomato species, i.e., Solanum lycopersicum cv Moneymaker and Solanum pimpinellifolium, respectively, exposed to leaf herbivory by the insect Spodoptera exigua. Root volatiles were trapped with two sorbent materials, HiSorb and PDMS, at 24 h after exposure to insect stress. Our results revealed that differences in root volatilome were species-, stress-, and material-dependent. Upon leaf herbivory, the domesticated and wild tomato species showed different root volatile profiles. The wild species presented the largest change in root volatile compounds with an overall reduction in monoterpene emission under stress. Similarly, the domesticated species presented a slight reduction in monoterpene emission and an increased production of fatty-acid-derived volatiles under stress. Volatile profiles differed between the two sorbent materials, and both were required to obtain a more comprehensive characterization of the root volatilome. Collectively, these results provide a strong basis to further unravel the impact of herbivory stress on systemic volatile emissions.
  • New Phytologist

    Plant neighbours can make or break the disease transmission chain of a fungal root pathogen

    Eline Ampt, Jasper van Ruijven, Mark Zwart, Jos M. Raaijmakers, Aad J Termorshuizen, Liesje Mommer
    Biodiversity can reduce or increase disease transmission. These divergent effects suggest that community composition rather than diversity per se determines disease transmission. In natural plant communities, little is known about the functional roles of neighbouring plant species in belowground disease transmission.
    Here, we experimentally investigated disease transmission of a fungal root pathogen (Rhizoctonia solani) in two focal plant species in combinations with four neighbour species of two ages. We developed stochastic models to test the relative importance of two transmission-modifying mechanisms: (1) infected hosts serve as nutrient supply to increase hyphal growth, so that successful disease transmission is self-reinforcing; and (2) plant resistance increases during plant development.
    Neighbouring plants either reduced or increased disease transmission in the focal plants. These effects depended on neighbour age, but could not be explained by a simple dichotomy between hosts and nonhost neighbours. Model selection revealed that both transmission-modifying mechanisms are relevant and that focal host–neighbour interactions changed which mechanisms steered disease transmission rate.
    Our work shows that neighbour-induced shifts in the importance of these mechanisms across root networks either make or break disease transmission chains. Understanding how diversity affects disease transmission thus requires integrating interactions between focal and neighbour species and their pathogens.
  • Current Opinion in Plant Biology

    Designing a home for beneficial plant microbiomes

    Chunxu Song, Kemo Jin, Jos M. Raaijmakers
    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’.
  • Scientific Reports

    Impact of root-associated strains of three Paraburkholderia species on primary and secondary metabolism of Brassica oleracea

    Je Seung Jeon, N. Carreno Quintero, Henriëtte D L M van Eekelen, Ric C.H De Vos, Jos M. Raaijmakers, Desalegn Etalo

    Several root-colonizing bacterial species can simultaneously promote plant growth and induce systemic resistance. How these rhizobacteria modulate plant metabolism to accommodate the carbon and energy demand from these two competing processes is largely unknown. Here, we show that strains of three Paraburkholderia species, P. graminis PHS1 (Pbg), P. hospita mHSR1 (Pbh), and P. terricola mHS1 (Pbt), upon colonization of the roots of two Broccoli cultivars led to cultivar-dependent increases in biomass, changes in primary and secondary metabolism and induced resistance against the bacterial leaf pathogen Xanthomonas campestris. Strains that promoted growth led to greater accumulation of soluble sugars in the shoot and particularly fructose levels showed an increase of up to 280-fold relative to the non-treated control plants. Similarly, a number of secondary metabolites constituting chemical and structural defense, including flavonoids, hydroxycinnamates, stilbenoids, coumarins and lignins, showed greater accumulation while other resource-competing metabolite pathways were depleted. High soluble sugar generation, efficient sugar utilization, and suppression or remobilization of resource-competing metabolites potentially contributed to curb the tradeoff between the carbon and energy demanding processes induced by Paraburkholderia-Broccoli interaction. Collectively, our results provide a comprehensive and integrated view of the temporal changes in plant metabolome associated with rhizobacteria-mediated plant growth promotion and induced resistance.
  • Science of the Total Environment

    Successive plant growth amplifies genotype-specific assembly of the tomato rhizosphere microbiome

    Viviane Cordovez da Cunha, Cristina Rotoni, Francisco Dini-Andreote, Ben Oyserman, Victor Carrion Bravo, Jos M. Raaijmakers
    Plant microbiome assembly is a spatial and dynamic process driven by root exudates and influenced by soil type, plant developmental stage and genotype. Genotype-dependent microbiome assembly has been reported for different crop plant species. Despite the effect of plant genetics on microbiome assembly, the magnitude of host control over its root microbiome is relatively small or, for many plant species, still largely unknown. Here we cultivated modern and wild tomato genotypes for four successive cycles and showed that divergence in microbiome assembly between the two genotypes was significantly amplified over time. Also, we show that the composition of the rhizosphere microbiome of modern and wild plants became more dissimilar from the initial bulk soil and from each other. Co-occurrence analyses further identified amplicon sequence variants (ASVs) associated with early and late successions of the tomato rhizosphere microbiome. Among the members of the Late Successional Rhizosphere microbiome, we observed an enrichment of ASVs belonging to the genera Acidovorax, Massilia and Rhizobium in the wild tomato rhizosphere, whereas the modern tomato rhizosphere was enriched for an ASV belonging to the genus Pseudomonas. Collectively, our approach allowed us to study the dynamics of rhizosphere microbiome over successional cultivation as well as to categorize rhizobacterial taxa for their ability to form transient or long-term associations with their host plants.
  • Environmental Microbiology

    Towards meaningful scales in ecosystem microbiome research

    Francisco Dini-Andreote, George Kowalchuk, James I. Prosser, Jos M. Raaijmakers
    Summary Studies of microbial communities in natural ecosystems have been generally focused on mapping patterns of species and gene distributions. Although highly instrumental in expanding our understanding of microbial diversity and distribution patterns, such census studies often lack a meaningful and explicit definition of scale. Here, we discuss the importance of scale in environmental microbiology assessments and consider how patterning ecology can be redirected towards advancing concept and theory formation in ecosystem microbiome research. This article is protected by copyright. All rights reserved.
  • Metabolites

    The Chemistry of Stress: Understanding the ‘Cry for Help’ of Plant Roots

    Muhammad Syamsu Rizaludin, Nejc Stopnisek, Jos M. Raaijmakers, Paolina Garbeva
    Plants are faced with various biotic and abiotic stresses during their life cycle. To withstand these stresses, plants have evolved adaptive strategies including the production of a wide array of primary and secondary metabolites. Some of these metabolites can have direct defensive effects, while others act as chemical cues attracting beneficial (micro)organisms for protection. Similar to aboveground plant tissues, plant roots also appear to have evolved “a cry for help” response upon exposure to stress, leading to the recruitment of beneficial microorganisms to help minimize the damage caused by the stress. Furthermore, emerging evidence indicates that microbial recruitment to the plant roots is, at least in part, mediated by quantitative and/or qualitative changes in root exudate composition. Both volatile and water-soluble compounds have been implicated as important signals for the recruitment and activation of beneficial root-associated microbes. Here we provide an overview of our current understanding of belowground chemical communication, particularly how stressed plants shape its protective root microbiome.
  • Frontiers in Microbiology

    Extracting the GEMs: Genotype, Environment and Microbiome interactions shaping host phenotypes

    Ben Oyserman, Viviane Cordovez da Cunha, Stalin W. Sarango Flores, Harm Nijveen, Marnix H Medema, Jos M. Raaijmakers
    One of the fundamental tenets of biology is that the phenotype of an organism (Y) is determined by its genotype (G), the environment (E) and their interaction (GE). Quantitative phenotypes can then be modeled as Y=G+E+GE+e, where e is the biological variance. This simple and tractable model has long served as the basis for studies investigating the heritability of traits and decomposing the variability in fitness. Increasingly, the importance of microbe interactions on organismal phenotypes is being recognized, but it is currently unclear what the relative contribution of microbiomes to a given host phenotype is and how this translates into the traditional GE model. Here we address this fundamental question and propose an expansion of the original model, referred to as GEM, which explicitly incorporates the contribution of the microbiome (M) to the host phenotype, while maintaining the simplicity and tractability of the original GE model. We show that by keeping host, environment and microbiome as separate but interacting variables, the GEM model can capture the nuanced ecological interactions between these variables. Finally, we demonstrate with an in vitro experiment how the GEM model can be used to statistically disentangle the relative contributions of each component on specific host phenotypes.
  • mSystems

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

    Vittorio Tracanna, Adam Ossowicki, M.L.C. Petrus, S. Overduin, B. Terlouw, G. Lund, S. Robinson, Sven Warris, Elio G.W.M. Schijlen, Gilles van Wezel, Jos M. Raaijmakers, Paolina Garbeva, Marnix H Medema
    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 10x metagenomic sequencing of one suppressive soil, leading to the identification of multiple gene clusters potentially associated with the disease-suppressive phenotype.
  • Environmental Microbiology Reports

    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.
  • Ecological Entomology

    Volatiles from the fungus Fusarium oxysporum affect interactions of Brassica rapa plants with root herbivores

    Kay Moisan, Marcel Dicke, Jos M. Raaijmakers, Elvira Rachmawati, Viviane Cordovez da Cunha
    1. Soil is a diverse and heterogeneous environment where chemicals mediate numerous interactions between soil organisms and plants. To date, studies have extensively addressed volatile-mediated interactions between soil microorganisms and the effects of microbial volatiles on plant growth. Yet, to our knowledge, it remains to be explored whether volatiles from soil-borne fungi can influence plant interactions with root herbivores, facilitating or hampering performance of competitors that share the same host plant.
    2. In the present study, we investigated the effects of volatiles emitted by the soil-borne fungus Fusarium oxysporum on the performance of two root herbivores: the plant parasitic cyst nematode, Heterodera schachtii, and the insect root herbivore, Delia radicum, upon infestation of Brassica rapa roots.
    3. Fungal volatiles slowed down the development of the root nematode cysts but increased their size, suggesting an enhanced egg load. In contrast, the performance of the insect root herbivore was unaffected by the exposure of roots to fungal volatiles. Additionally, fungal volatiles promoted the growth of plants infested with the root nematode, but not of those infested with the insect root herbivore.
    4. Together, our data show that volatiles from a soil-borne fungus can affect root interactions with root herbivores. Increased production of nematode eggs and plant growth promotion suggest a specific modulation of root-herbivore interactions by fungal volatiles.
  • Biomolecules

    Effects of Sulfur Assimilation in Pseudomonas fluorescens SS101 on Growth, Defense, and Metabolome of Different Brassicaceae

    Je Seung Jeon, Desalegn Etalo, Natalia Carreno-Quintero, Ric C.H De Vos, Jos M. Raaijmakers
    Genome-wide analysis of plant-growth-promoting Pseudomonas fluorescens strain SS101 (PfSS101) followed by site-directed mutagenesis previously suggested that sulfur assimilation may play an important role in growth promotion and induced systemic resistance in Arabidopsis. Here, we investigated the effects of sulfur metabolism in PfSS101 on growth, defense, and shoot metabolomes of Arabidopsis and the Brassica crop, Broccoli. Root tips of seedlings of Arabidopsis and two Broccoli cultivars were treated with PfSS101 or with a mutant disrupted in the adenylsulfate reductase cysH, a key gene in cysteine and methionine biosynthesis. Phenotyping of plants treated with wild-type PfSS101 or its cysH mutant revealed that sulfur assimilation in PfSS101 was associated with enhanced growth of Arabidopsis but with a reduction in shoot biomass of two Broccoli cultivars. Untargeted metabolomics revealed that cysH-mediated sulfur assimilation in PfSS101 had significant effects on shoot chemistry of Arabidopsis, in particular on chain elongation of aliphatic glucosinolates (GLSs) and on indole metabolites, including camalexin and the growth hormone indole-3-acetic acid. In Broccoli, PfSS101 sulfur assimilation significantly upregulated the relative abundance of several shoot metabolites, in particular, indolic GLSs and phenylpropanoids. These metabolome changes in Broccoli plants coincided with PfSS101-mediated suppression of leaf infections by Xanthomonas campestris. Our study showed the metabolic interconnectedness of plants and their root-associated microbiota.
  • Journal of Natural Products

    Discovery of Thanafactin A, a Linear Proline-containing Octa-Lipopeptide from Pseudomonas sp. SH-C52, Motivated by Genome Mining

    Norbert Kirchner, Carolina Cano-Prieto, Anna Schulz-Finke, M. Gütschow, Nico Ortlieb, Julia Moschny, Timo Niedermeyer, Jeannie Horak, Michael Lämmerhofer, Menno van der Voort, Jos M. Raaijmakers, Harald Gross
    Genome mining of the bacterial strains Pseudomonas sp. SH-C52 and Pseudomonas fluorescens DSM 11579 showed that both strains contained a highly similar gene cluster encoding an octamodular nonribosomal peptide synthetase (NRPS) system which was not associated with a known secondary metabolite. Insertional mutagenesis of an NRPS component followed by comparative profiling led to the discovery of the corresponding novel linear octalipopeptide thanafactin A, which was subsequently isolated and its structure determined by two-dimensional NMR and further spectroscopic and chromatographic methods. In bioassays, thanafactin A exhibited weak protease inhibitory activity and was found to modulate swarming motility in a strain-specific manner.
  • Agronomy Journal

    Optimizing biocontrol activity of Paenibacillus xylanexedens for management of hairy root disease in tomato grown in hydroponic greenhouses

    Pablo Vargas, Lien Bosmans, Stef Van Kerckhove, Bart Van Calenberge, Jos M. Raaijmakers, Bart Lievens, Hans Rediers
    Hairy root disease (HRD) caused by rhizogenic Agrobacterium biovar 1 strains affect tomato, cucumber, eggplant, and bell pepper grown in hydroponic greenhouses and can cause considerable yield losses worldwide. Recently, Paenibacillus xylanexedens strains (ST15.15/027 and AD117) with antagonistic activity against rhizogenic agrobacteria were identified. In this study, we present results of greenhouse trials of two consecutive growing seasons (2019 and 2020) to examine the potential of these two biocontrol organisms (BCOs) under practical conditions. BCO-treatment at a 107 colony forming units (CFU)/mL density resulted in a considerable reduction of the HRD infestation rate, confirming the biocontrol potential of the two P. xylanexedens strains. Results revealed that a single BCO strain (ST15.15/027) performed equally well as the mixed inoculum of both strains. The same level of biocontrol activity was even achieved when the BCO inoculum density was reduced to 105 CFU/mL. qPCR analysis further showed that Paenibacillus was still present in rockwool substrate near the end of both trials, indicating that they persist well in a rockwool environment and that application at the start of the trial is sufficient to protect tomato plants until the end of the trial. Altogether, these results are highly valuable for further optimization and exploitation of P. xylanexedens as a biocontrol product for the control of HRD in hydroponic greenhouses.
  • Annual Review of Plant Biology

    Extension of Plant Phenotypes by the Foliar Microbiome

    Christine V. Hawkes, Rasmus Kjøller, Jos M. Raaijmakers, Leise Riber, Svend Christensen, Anders Bjorholm Dahl, Jesper Cairo Westergaard, Mads Nielsen, Gina Brown-Guedira, Lars Hestbjerg Hansen
    The foliar microbiome can extend the host plant phenotype by expanding its genomic and metabolic capabilities. Despite increasing recognition of the importance of the foliar microbiome for plant fitness, stress physiology, and yield, the diversity, function, and contribution of foliar microbiomes to plant phenotypic traits remain largely elusive. The recent adoption of high-throughput technologies is helping to unravel the diversityand spatiotemporal dynamics of foliar microbiomes, but we have yet to resolve their functional importance for plant growth, development, and ecology. Here, we focus on the processes that govern the assembly of the foliar microbiome and the potential mechanisms involved in extended plant phenotypes. We highlight knowledge gaps and provide suggestions for new research directions that can propel the field forward. These efforts will be instrumental in maximizing the functional potential of the foliar microbiome for sustainable crop production.
  • Plant and Soil

    Plant functional group drives the community structure of saprophytic fungi in a grassland biodiversity experiment

    Davide Francioli, Sophie van Mastrigt-van Rijssel, Jasper van Ruijven, Aad J Termorshuizen, T. E. Anne Cotton, Alex J. Dumbrell, Jos M. Raaijmakers, Alexandra Weigelt, Liesje Mommer
    Saprophytic fungi are important agents of soil mineralization and carbon cycling. Their community structure is known to be affected by soil conditions such as organic matter and pH. However, the effect of plant species, whose roots provide the litter input into the soil, on the saprophytic fungal community is largely unknown.

    We examined the saprophytic fungi in a grassland biodiversity experiment with eight plant species belonging to two functional groups (grasses and forbs), combining DNA extraction from plant roots, next-generation sequencing and literature research.

    We found that saprophyte richness increased with plant species richness, but plant functional group richness was the best predictor. Plant functional group was also the main factor driving fungal saprophytic community structure. This effect was correlated with differences in root lignin content and C:N ratio between grasses and forbs. In monocultures, root traits and plant functional group type explained 16% of the variation in community structure. The saprophyte taxa detected in mixed plant communities were to a large extent subsets of those found in monocultures.

    Our work shows that the richness and community structure of the root-associated saprophytic fungi can largely be predicted by plant functional groups and their associated root traits. This means that the effects of plant diversity on ecosystem functions such as litter decomposition may also be predictable using information on plant functional groups in grasslands.
  • Ecological Entomology

    No evidence of modulation of indirect plant resistance of Brassica rapa plants by volatiles from soil-borne fungi

    Kay Moisan, Dani Lucas-Barbosa, Alexandre Villela, Liana O. Greenberg, Viviane Cordovez da Cunha, Jos M. Raaijmakers, Marcel Dicke
    Upon herbivory, plants emit specific herbivore‐induced plant volatiles (HIPVs) that can attract natural enemies of the herbivore thus serving as indirect plant resistance. Not only insect herbivores, but microorganisms may also affect HIPV emission before or after plant colonisation, which in turn can affect behaviour of natural enemies of the herbivore. Yet, it remains elusive whether volatiles from microorganisms influence HIPV emission and indirect plant resistance.
    In this study, we investigated whether exposure of Brassica rapa roots to volatiles from soil‐borne fungi influence HIPV emission and the recruitment of natural enemies of Pieris brassicae larvae.
    Using a two‐compartment pot system, we performed greenhouse and common‐garden experiments, and we profiled plant HIPV emission.
    We found that exposure of plant roots to fungal volatiles did not affect the number of P. brassicae larvae recollected from the plants, suggesting a neutral effect of the fungal volatiles on natural predation. Likewise, in a greenhouse, similar numbers of larvae were parasitised by Cotesia glomerata wasps on control plants as on fungal volatile‐exposed plants. Additionally, chemical analysis of HIPV profiles revealed no qualitative and quantitative differences between control plants and fungal volatile‐exposed plants that were both infested with P. brassicae larvae.
    Together, our data indicate that root exposure to fungal volatiles did not affect indirect plant resistance to an insect herbivore. These findings provide new insight into the influence of indirect plant resistance by fungal volatiles that are discussed together with the effects of fungal volatiles on direct plant resistance.
  • Proceedings of the Royal Society B-Biological Sciences

    Microbial and volatile profiling of soils suppressive to Fusarium culmorum of wheat

    Adam Ossowicki, Vittorio Tracanna, M.L.C. Petrus, Gilles van Wezel, Jos M. Raaijmakers, Marnix H Medema, Paolina Garbeva

    In disease-suppressive soils, microbiota protect plants from root infections. Bacterial members of this microbiota have been shown to produce specific molecules that mediate this phenotype. To date, however, studies have focused on individual suppressive soils and the degree of natural variability of soil suppressiveness remains unclear. Here, we screened a large collection of field soils for suppressiveness to Fusarium culmorum using wheat ( Triticum aestivum) as a model host plant. A high variation of disease suppressiveness was observed, with 14% showing a clear suppressive phenotype. The microbiological basis of suppressiveness to F. culmorum was confirmed by gamma sterilization and soil transplantation. Amplicon sequencing revealed diverse bacterial taxonomic compositions and no specific taxa were found exclusively enriched in all suppressive soils. Nonetheless, co-occurrence network analysis revealed that two suppressive soils shared an overrepresented bacterial guild dominated by various Acidobacteria. In addition, our study revealed that volatile emission may contribute to suppression, but not for all suppressive soils. Our study raises new questions regarding the possible mechanistic variability of disease-suppressive phenotypes across physico-chemically different soils. Accordingly, we anticipate that larger-scale soil profiling, along with functional studies, will enable a deeper understanding of disease-suppressive microbiomes.
  • FEMS Microbiology Ecology

    Multitrophic interactions in the rhizosphere microbiome of wheat: from bacteria and fungi to protists

    Maike Rossmann, Juan Esteban Perez Jaramillo, Vanessa N. Kavamura, Josiane B. Chiaramonte, Kenneth Dumack, Anna Maria Fiore-Donno, L.W. Mendes, Márcia M. C. Ferreira, Michael Bonkowski, Jos M. Raaijmakers, Tim H. Mauchline, Rodrigo Mendes
    Plants modulate the soil microbiota by root exudation assembling a complex rhizosphere microbiome with organisms spanning different trophic levels. Here, we assessed the diversity of bacterial, fungal and cercozoan communities in landraces and modern varieties of wheat. The dominant taxa within each group were the bacterial phyla Proteobacteria, Actinobacteria and Acidobacteria; the fungi phyla Ascomycota, Chytridiomycota and Basidiomycota; and the Cercozoa classes Sarcomonadea, Thecofilosea and Imbricatea. We showed that microbial networks of the wheat landraces formed a more intricate network topology than that of modern wheat cultivars, suggesting that breeding selection resulted in a reduced ability to recruit specific microbes in the rhizosphere. The high connectedness of certain cercozoan taxa to bacteria and fungi indicated trophic network hierarchies where certain predators gain predominance over others. Positive correlations between protists and bacteria in landraces were preserved as a subset in cultivars as was the case for the Sarcomonadea class with Actinobacteria. The correlations between the microbiome structure and plant genotype observed in our results suggest the importance of top-down control by organisms of higher trophic levels as a key factor for understanding the drivers of microbiome community assembly in the rhizosphere.
  • Microbiology Resource Announcements

    Draft Genome Sequence of Lipopeptide-Producing Strain Pseudomonas fluorescens DSM 11579 and Comparative Genomics with Pseudomonas sp. Strain SH-C52, a Closely Related Lipopeptide-Producing Strain

    Norbert Kirchner, Carolina Cano-Prieto, Menno van der Voort, Jos M. Raaijmakers, Harald Gross
    Pseudomonas fluorescens DSM 11579 is known to be a producer of the lipopeptides brabantamide and thanamycin. Its draft genome gives insight into the complete secondary metabolite production capacity of the strain and builds the basis for a comparative study with Pseudomonas sp. strain SH-C52, a lipopeptide-producing strain involved in natural disease-suppressive soils.
  • ISME Journal

    Production of glycine-derived ammonia as a low-cost and long-distance antibiotic strategy by Streptomyces

    Mariana Avalos, Paolina Garbeva, Jos M. Raaijmakers, Gilles van Wezel
    Soil-inhabiting streptomycetes are Nature’s medicine makers, producing over half of all known antibiotics and many other bioactive natural products. However, these bacteria also produce many volatile compounds, and research into these molecules and their role in soil ecology is rapidly gaining momentum. Here we show that streptomycetes have the ability to kill bacteria over long distances via air-borne antibiosis. Our research shows that streptomycetes do so by producing surprisingly high amounts of the low-cost volatile antimicrobial ammonia, which travels over long distances and antagonises both Gram-positive and Gram-negative bacteria. Glycine is required as precursor to produce ammonia, and inactivation of the glycine cleavage system annihilated air-borne antibiosis. As a resistance strategy, E. coli cells acquired mutations resulting in reduced expression of the porin master regulator OmpR and its cognate kinase EnvZ, which was just enough to allow them to survive. We further show that ammonia enhances the activity of the more costly canonical antibiotics, suggesting that streptomycetes adopt a low-cost strategy to sensitize competitors for antibiosis over longer distances.
  • Plant, Cell and Environment

    Volatiles from soil-borne fungi affect directional growth of roots

    Kay Moisan, Jos M. Raaijmakers, Marcel Dicke, Dani Lucas-Barbosa, Viviane Cordovez da Cunha
    Volatiles play major roles in mediating ecological interactions between soil (micro)organisms and plants. It is well-established that microbial volatiles can increase root biomass and lateral root formation. To date, however, it is unknown whether microbial volatiles can affect directional root growth. Here, we present a novel method to study belowground volatile-mediated interactions. As proof-of-concept, we designed a root Y-tube olfactometer, and tested the effects of volatiles from four different soil-borne fungi on directional growth of Brassica rapa roots in soil. Subsequently, we compared the fungal volatile organic compounds (VOCs) previously profiled with Gas Chromatography?Mass Spectrometry (GC?MS). Using our newly designed setup, we show that directional root growth in soil is differentially affected by fungal volatiles. Roots grew more frequently toward volatiles from the root pathogen Rhizoctonia solani, whereas volatiles from the other three saprophytic fungi did not impact directional root growth. GC?MS profiling showed that six VOCs were exclusively emitted by R. solani. These findings verify that this novel method is suitable to unravel the intriguing chemical cross-talk between roots and soil-borne fungi and its impact on root growth.
  • FEMS Microbiology Ecology

    Restoring degraded microbiome function with self-assembled communities

    Carlos Gutierrez, J. Gómez, Jos M. Raaijmakers, Ben Oyserman
    The natural microbial functions of many soils are severely degraded. Current state-of-the-art technology to restore these functions is through the isolation, screening, formulation and application of microbial inoculants and synthetic consortia. These approaches have inconsistent success, in part due to the incompatibility between the biofertilizer, crop, climate, existing soil microbiome and physicochemical characteristics of the soils. Here, we review the current state of the art in biofertilization and identify two key deficiencies in current strategies: the difficulty in designing complex multispecies biofertilizers and the bottleneck in scaling the production of complex multispecies biofertilizers. To address the challenge of producing scalable, multispecies biofertilizers, we propose to merge ecological theory with bioprocess engineering to produce ‘self-assembled communities’ enriched for particular functional guilds and adapted to a target soil and host plant. Using the nitrogen problem as an anchor, we review relevant ecology (microbial, plant and environmental), as well as reactor design strategies and operational parameters for the production of functionally enriched self-assembled communities. The use of self-assembled communities for biofertilization addresses two major hurdles in microbiome engineering: the importance of enriching microbes indigenous to (and targeted for) a specific environment and the recognized potential benefits of microbial consortia over isolates (e.g. functional redundancy). The proposed community enrichment model could also be instrumental for other microbial functions such as phosphorus solubilization, plant growth promotion or disease suppression.
  • Functional Ecology

    Fungal volatiles influence plant defence against aboveground and belowground herbivory

    Kay Moisan, Marcela Aragón, Gerrit Gort, Marcel Dicke, Viviane Cordovez da Cunha, Jos M. Raaijmakers, Dani Lucas-Barbosa
    Plants have evolved resistance traits that negatively affect attackers, and tolerance traits that sustain plant growth despite herbivore damage. These mechanisms often co‐occur in a mixed‐defence strategy, balancing resistance and tolerance. These plant defences can be enhanced upon interaction with soil micro‐organisms.
    Here we investigated the effects of volatiles emitted by soil‐borne fungi on plant defence to insect herbivory, and on plant phenology.
    We exposed roots of Brassica rapa plants to volatiles emitted by four soil‐borne fungi. As a proxy of plant resistance, we assessed the performance of Pieris brassicae, a caterpillar feeding on leaves and inflorescences, and of Delia radicum, an insect root herbivore. As a proxy of plant tolerance, we compared growth of volatile‐exposed plants challenged with or without insects. Additionally, we assessed the effects on plant phenology by recording bolting time and by counting the number of buds and flowers.
    Plant exposure to fungal volatiles differentially affected plant resistance to above‐ and below‐ground herbivory. Performance of P. brassicae caterpillars differed between the fungal volatile‐exposed plants but was variable between experimental batches. In contrast, the effects of fungal volatiles on D. radicum performance were predominantly negative, indicating an increased plant resistance. Despite root consumption by D. radicum, root dry weight remained unchanged in infested plants compared with uninfested ones, irrespectively of the volatile exposure, suggesting compensation for the tissue loss, sometimes at the cost of undamaged above‐ground tissues. When B. rapa plants were attacked by P. brassicae caterpillars, only exposure to volatiles of some fungi led to compensation for the loss of above‐ground tissues consumed by the caterpillars, which differed between leaves and inflorescences. Furthermore, bolting was accelerated in response to volatiles of some fungi, resulting in more buds and flowers, which suggests a potential enhancement of plant fitness.
    Our data show that fungal volatiles can modulate the mixed‐defence strategies of B. rapa plants, balancing plant resistance and tolerance to above‐ and below‐ground herbivory. These effects may be variable and were fungus specific. Ultimately, plant fitness may be enhanced upon root exposure to fungal volatiles.
  • Oecologia

    Volatiles of pathogenic and non-pathogenic soil-borne fungi affect plant development and resistance to insects

    Kay Moisan, Viviane Cordovez da Cunha, Els M. van de Zande, Jos M. Raaijmakers, Marcel Dicke, Dani Lucas-Barbosa
    Plants are ubiquitously exposed to a wide diversity of (micro)organisms, including mutualists and antagonists. Prior to direct contact, plants can perceive microbial organic and inorganic volatile compounds (hereafter: volatiles) from a distance that, in turn, may affect plant development and resistance. To date, however, the specificity of plant responses to volatiles emitted by pathogenic and non-pathogenic fungi and the ecological consequences of such responses remain largely elusive. We investigated whether Arabidopsis thaliana plants can differentiate between volatiles of pathogenic and non-pathogenic soil-borne fungi. We profiled volatile organic compounds (VOCs) and measured CO2 emission of 11 fungi. We assessed the main effects of fungal volatiles on plant development and insect resistance. Despite distinct differences in VOC profiles between the pathogenic and non-pathogenic fungi, plants did not discriminate, based on plant phenotypic responses, between pathogenic and non-pathogenic fungi. Overall, plant growth was promoted and flowering was accelerated upon exposure to fungal volatiles, irrespectively of fungal CO2 emission levels. In addition, plants became significantly more susceptible to a generalist insect leaf-chewing herbivore upon exposure to the volatiles of some of the fungi, demonstrating that a prior fungal volatile exposure can negatively affect plant resistance. These data indicate that plant development and resistance can be modulated in response to exposure to fungal volatiles.
  • Current Opinion in Microbiology

    Harnessing the microbiome to control plant parasitic weeds

    Raul Masteling, Lorenzo Lombard, Wietse de Boer, Jos M. Raaijmakers, Francisco Dini-Andreote
    Microbiomes can significantly expand the genomic potential of plants, contributing to nutrient acquisition, plant growth promotion and tolerance to (a)biotic stresses. Among biotic stressors, root parasitic weeds (RPWs), mainly of the genera Orobanche, Phelipanche and Striga, are major yield-limiting factors of a wide range of staple crops, particularly in developing countries. Here, we provide a conceptual synthesis of putative mechanisms by which soil and plant microbiomes could be harnessed to control RPWs. These mechanisms are partitioned in direct and indirect modes of action and discussed in the context of past and present studies on microbe-mediated suppression of RPWs. Specific emphasis is given to the large but yet unexplored potential of root-associated microorganisms to interfere with the chemical signalling cascade between the host plant and the RPWs. We further provide concepts and ideas for future research directions and prospective designs of novel control strategies.
  • Soil Biology & Biochemistry

    Impacts of long-term plant residue management on soil organic matter quality, Pseudomonas community structure and disease suppressiveness

    Bryony E.A. Dignam, Maureen O'Callaghan, Leo M. Condron, Jos M. Raaijmakers, George Kowalchuk, Steve A. Wakelin
    The microbiome of grassland soils provides ecosystem services essential to plant health and productivity, including nutrient cycling and suppression of soil-borne diseases. Understanding how soil management practices affect soil microbial communities will provide opportunities by which indigenous soil microbes and their functions can be managed to sustain or promote plant growth and enhance disease suppressiveness. Here, we investigated the impact of 20 years of plant residue management in a long-term grassland field trial on soil chemical and (micro)biological properties, in particular the suppression of damping-off disease of kale caused by the fungal root pathogen Rhizoctonia solani AG 2–1. Plant residue management led to significant variation in the community structure of the bacterial genus Pseudomonas between treatments. Soil organic matter quality (inferred carbon recalcitrance) was responsible for 80% of the observed variation in Pseudomonas community structure. Furthermore, increased Pseudomonas species diversity (Shannon's index), microbial activity, soil organic matter content, and carbon availability distinguished suppressive (low disease) soils from conducive (high disease) soils. More specifically, Pseudomonas species diversity and richness (Margalef's) were identified as the primary parameters explaining the greatest proportion (>30%) of variation in the disease suppressive capacity of soils across treatments. Collectively, our results suggest that management-induced shifts in Pseudomonas community composition, notably species diversity and richness, provide a better indicator of disease conduciveness for a broad-host range fungal pathogen than soil chemical parameters. In conclusion, our study indicates that frequent addition of organic residues to agricultural grassland soils enhances the diversity and activity of plant-beneficial bacterial taxa.
  • European Journal of Plant Pathology

    Linking ecology and plant pathology to unravel the importance of soil-borne fungal pathogens in species-rich grasslands

    Eline A. Ampt, Jasper van Ruijven, Jos M. Raaijmakers, Aad J Termorshuizen, Liesje Mommer
    Soil-borne fungal diseases are a major problem in agriculture. A century ago, the Dutch plant pathologist Johanna Westerdijk recognized the importance of linking fungal biology with ecology to understand plant disease dynamics. To explore new ways to manage soil-borne fungal disease in agriculture by ‘learning from nature’, we follow in her footsteps: we link below ground plant-fungal pathogen interactions to ecological settings, i.e. natural grasslands. Ecological research hypothesised that the build-up of ‘enemies’ is reduced in species-rich vegetation compared to monocultures. To understand how plant diversity can suppress soil-borne fungal pathogens, we first need to identify fungal actors in species-rich grasslands. Next-generation sequencing revealed a first glimpse of the potential fungal actors, but their ecological functions often remain elusive. Databases are becoming available to predict the ecological fungal guild, but classic phytopathology studies that isolate and characterize – taxonomically and functionally -, remain essential. Secondly, we need to set-up experiments that reveal ecological mechanisms underlying the complex below ground interactions between plant diversity and fungal pathogens. Several studies suggested that disease incidence of (host-specific) pathogens is related to abundance of the host plant species. However, recent studies suggest that next to host species density, presence of heterospecific species additionally affects disease dynamics. We explore the direct and indirect ways of these neighboring plants diluting pathogen pressure. We argue that combining the expertise of plant pathologists and ecologists will improve our understanding of belowground plant-fungal pathogen interactions in natural grasslands and contribute to the design of sustainable and productive intercropping strategies in agriculture.
  • Science

    Pathogen-induced activation of disease-suppressive functions in the endophytic root microbiome

    Victor Carrion Bravo, Juan Esteban Perez Jaramillo, Viviane Cordovez da Cunha, Vittorio Tracanna, Mattias De Hollander, Daniel Ruiz Buck, L.W. Mendes, Wilfried F.J. van IJcken, Ruth Gomez Exposito, Somayah S. Elsayed, Prarthana Mohanraju, Adini Arifah, John van der Oost, Joseph N. Paulson, Rodrigo Mendes, Gilles van Wezel, Marnix H Medema, Jos M. Raaijmakers
    Microorganisms living inside plants can promote plant growth and health, but their genomic and functional diversity remain largely elusive. Here, metagenomics and network inference show that fungal infection of plant roots enriched for Chitinophagaceae and Flavobacteriaceae in the root endosphere and for chitinase genes and various unknown biosynthetic gene clusters encoding the production of nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs). After strain-level genome reconstruction, a consortium of Chitinophaga and Flavobacterium was designed that consistently suppressed fungal root disease. Site-directed mutagenesis then revealed that a previously unidentified NRPS-PKS gene cluster from Flavobacterium was essential for disease suppression by the endophytic consortium. Our results highlight that endophytic root microbiomes harbor a wealth of as yet unknown functional traits that, in concert, can protect the plant inside out.
  • Annual Review of Microbiology

    Ecology and Evolution of Plant Microbiomes

    Viviane Cordovez da Cunha, Francisco Dini-Andreote, Victor Carrion Bravo, Jos M. Raaijmakers
    Microorganisms colonizing plant surfaces and internal tissues provide a number of life-support functions for their host. Despite increasing recognition of the vast functional capabilities of the plant microbiome, our understanding of the ecology and evolution of the taxonomically hyperdiverse microbial communities is limited. Here, we review current knowledge of plant genotypic and phenotypic traits as well as allogenic and autogenic factors that shape microbiome composition and functions. We give specific emphasis to the impact of plant domestication on microbiome assembly and how insights into microbiomes of wild plant relatives and native habitats can contribute to reinstate or enrich for microorganisms with beneficial effects on plant growth, development, and health. Finally, we introduce new concepts and perspectives in plant microbiome research, in particular how community ecology theory can provide a mechanistic framework to unravel the interplay of distinct ecological processes—i.e., selection, dispersal, drift, diversification—that structure the plant microbiome.
  • Frontiers in Microbiology

    Resistance breeding of common bean shapes the physiology of the rhizosphere microbiome

    L.W. Mendes, Miriam Gonçalves de Chaves, Mariley de Cássia da Fonseca, Rodrigo Mendes, Jos M. Raaijmakers, Siu Mui Tsai
    The taxonomically diverse rhizosphere microbiome contributes to plant nutrition, growth and health, including protection against soil-borne pathogens. We previously showed that breeding for Fusarium-resistance in common bean changed the rhizosphere microbiome composition and functioning. Here, we assessed the impact of Fusarium-resistance breeding in common bean on microbiome physiology. Combined with metatranscriptome data, community-level physiological profiling by Biolog EcoPlate analyses revealed that the rhizosphere microbiome of the Fusarium-resistant accession was distinctly different from that of the Fusarium-susceptible accession, with higher consumption of amino acids and amines, higher metabolism of xylanase and sialidase, and higher expression of genes associated with nitrogen, phosphorus and iron metabolism. The resistome analysis indicates higher expression of soxR, which is involved in protecting bacteria against oxidative stress induced by a pathogen invasion. These results further support our hypothesis that breeding for resistance has unintentionally shaped the assembly and activity of the rhizobacterial community towards a higher abundance of specific rhizosphere competent bacterial taxa that can provide complementary protection against fungal root infections.
  • Microbiome

    The wild side of plant microbiomes

    Juan Esteban Perez Jaramillo, Victor Carrion Bravo, Mattias De Hollander, Jos M. Raaijmakers
  • Annual Report of the Bean Improvement Cooperative

    The rhizosphere microbiome as an auxiliary breeding component in common bean against Fusarium oxysporum

    L.W. Mendes, Jos M. Raaijmakers, Rodrigo Mendes
  • Trends in Plant Science

    Embracing community ecology in plant microbiome research

    Francisco Dini-Andreote, Jos M. Raaijmakers
    Community assembly is mediated by selection, dispersal, drift, and speciation. Environmental selection is mostly used to date to explain patterns in plant microbiome assembly, whereas the influence of the other processes remains largely elusive. Recent studies highlight that adopting community ecology concepts provides a mechanistic framework for plant microbiome research.
  • Biological Control

    Isolation, characterization and comparative analysis of plant-associated bacteria for suppression of soil-borne diseases of field-grown groundnut in Vietnam

    C.N. Le, T.K. Hoang, T.H. Thai, T.L. Tran, T.P.N. Phan, Jos M. Raaijmakers
    Abstract Groundnut (Arachis hypogaea L.) is an important oil seed crop worldwide and used extensively for feed and food. In Vietnam, groundnut cultivation is hampered by several soil-borne fungal pathogens, in particular Sclerotium rolfsii. To develop sustainable measures to control stem rot disease caused by S. rolfsii, plant-associated bacteria were isolated from the stem base and roots of groundnut plants grown in farmer fields in central Vietnam and tested for activity against S. rolfsii. Among a total of 3,360 randomly selected bacterial isolates, only thirteen (0.4%) inhibited hyphal growth of S. rolfsii. BOX-PCR and 16S-rDNA sequence analyses revealed that these bacterial isolates were genetically diverse and belonged to three bacterial Phyla, i.e. the γ-Proteobacteria (Pseudomonas), Firmicutes (Bacillus) and Bacteroidetes (Chryseobacterium). Nethouse and field experiments conducted in central Vietnam showed that treatment of groundnut seeds or field soil with strains of each of these three bacterial genera significantly reduced the incidence of stem rot disease, led to significant yield increases of up to 21% and did not have adverse effects on nodulation. The level of disease protection provided by the bacterial strains was similar to that achieved by the fungicide tebuconazole. Comparative analysis of the biocontrol efficacy of the indigenous Pseudomonas strain R4D2 with that of two exogenous, antagonistic Pseudomonas strains from the Netherlands showed that in field trials the indigenous strain R4D2 better colonized the roots of groundnut, reduced stem rot (S. rolfsii), black collar rot (Aspergillus niger), and bacterial wilt (Ralstonia solanacearum), and more consistently enhanced groundnut yield.
  • mBio

    Secondary metabolism and interspecific competition affect accumulation of spontaneous mutants in the gacS/gacA regulatory system in Pseudomonas protegens

    Qing Yan, Lucas D. Lopes, Brenda T. Schaffer, Teresa Kidarsa, Oliver Vining, Benjamin Philmus, Chunxu Song, Virginia O. Stockwell, Jos M. Raaijmakers, Kerry L. McPhail, Fernando Dini Andreote, Jeff H. Chang, Joyce E. Loper
    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.
  • Current Opinion in Microbiology

    Healthy scents: microbial volatiles as new frontier in antibiotic research?

    Mariana Avalos Garcia, Gilles van Wezel, Jos M. Raaijmakers, Paolina Garbeva
    Microorganisms represent a large and still resourceful pool for the discovery of novel compounds to combat antibiotic resistance in human and animal pathogens. The ability of microorganisms to produce structurally diverse volatile compounds has been known for decades, yet their biological functions and antimicrobial activities have only recently attracted attention. Various studies revealed that microbial volatiles can act as infochemicals in long-distance cross-kingdom communication as well as antimicrobials in competition and predation. Here, we review recent insights into the natural functions and modes of action of microbial volatiles and discuss their potential as a new class of antimicrobials and modulators of antibiotic resistance.
  • Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology

    Inter- and intracellular colonization of Arabidopsis roots by endophytic actinobacteria and the impact of plant hormones on their antimicrobial activity

    Anne Van der Meij, Joost Willemse, Martinus A. Schneijderberg, Rene Geurts, Jos M. Raaijmakers, Gilles van Wezel
    Many actinobacteria live in close association with eukaryotes like fungi, insects, animals and plants. Plant-associated actinobacteria display (endo)symbiotic, saprophytic or pathogenic life styles, and can make up a substantial part of the endophytic community. Here, we characterised endophytic actinobacteria isolated from root tissue of Arabidopsis thaliana (Arabidopsis) plants grown in soil from a natural ecosystem. Many of these actinobacteria belong to the family of Streptomycetaceae with Streptomyces olivochromogenes and Streptomyces clavifer as well represented species. When seeds of Arabidopsis were inoculated with spores of Streptomyces strain coa1, which shows high similarity to S. olivochromogenes, roots were colonised intercellularly and, unexpectedly, also intracellularly. Subsequent exposure of endophytic isolates to plant hormones typically found in root and shoot tissues of Arabidopsis led to altered antibiotic production against Escherichia coli and Bacillus subtilis. Taken together, our work reveals remarkable colonization patterns of endophytic streptomycetes with specific traits that may allow a competitive advantage inside root tissue.
  • New Phytologist

    Lost in diversity: the interactions between soil-borne fungi, biodiversity and plant productivity

    Liesje Mommer, T. E. Anne Cotton, Jos M. Raaijmakers, Aad J Termorshuizen, Jasper van Ruijven, Marloes Hendriks, Sophie van Mastrigt-van Rijssel, Judith E. van de Mortel, Jan Willem van der Paauw, Elio G.W.M. Schijlen, Annemiek E. Smit-Tiekstra, Frank Berendse, Hans de Kroon, Alex J. Dumbrell
    There is consensus that plant species richness enhances plant productivity within natural grasslands, but the underlying drivers remain debated. Recently, differential accumulation of soil-borne fungal pathogens across the plant diversity gradient has been proposed as a cause of this pattern. However, the below-ground environment has generally been treated as a ‘black box’ in biodiversity experiments, leaving these fungi unidentified.
    Using next generation sequencing and pathogenicity assays, we analysed the community composition of root-associated fungi from a biodiversity experiment to examine if evidence exists for host specificity and negative density dependence in the interplay between soil-borne fungi, plant diversity and productivity.
    Plant species were colonised by distinct (pathogenic) fungal communities and isolated fungal species showed negative, species-specific effects on plant growth. Moreover, 57% of the pathogenic fungal operational taxonomic units (OTUs) recorded in plant monocultures were not detected in eight plant species plots, suggesting a loss of pathogenic OTUs with plant diversity.
    Our work provides strong evidence for host specificity and negative density-dependent effects of root-associated fungi on plant species in grasslands. Our work substantiates the hypothesis that fungal root pathogens are an important driver of biodiversity-ecosystem functioning relationships.
  • ISME Journal

    Involvement of Burkholderiaceae and sulfurous volatiles in disease suppressive soils

    Victor Carrion Bravo, Viviane Cordovez da Cunha, Olaf Tyc, Desalegn Etalo, Irene de Bruijn, Victor de Jager, Marnix H Medema, Leo Eberl, Jos M. Raaijmakers
    Disease-suppressive soils are ecosystems in which plants suffer less from root infections due to the activities of specific microbial consortia. The characteristics of soils suppressive to specific fungal root pathogens are comparable to those of adaptive immunity in animals, as reported by Raaijmakers and Mazzola (Science 352:1392–3, 2016), but the mechanisms and microbial species involved in the soil suppressiveness are largely unknown. Previous taxonomic and metatranscriptome analyses of a soil suppressive to the fungal root pathogen Rhizoctonia solani revealed that members of the Burkholderiaceae family were more abundant and more active in suppressive than in non-suppressive soils. Here, isolation, phylogeny, and soil bioassays revealed a significant disease-suppressive activity for representative isolates of Burkholderia pyrrocinia, Paraburkholderia caledonica, P. graminis, P. hospita, and P. terricola. In vitro antifungal activity was only observed for P. graminis. Comparative genomics and metabolite profiling further showed that the antifungal activity of P. graminis PHS1 was associated with the production of sulfurous volatile compounds encoded by genes not found in the other four genera. Site-directed mutagenesis of two of these genes, encoding a dimethyl sulfoxide reductase and a cysteine desulfurase, resulted in a loss of antifungal activity both in vitro and in situ. These results indicate that specific members of the Burkholderiaceae family contribute to soil suppressiveness via the production of sulfurous volatile compounds.
  • Applied and Environmental Microbiology

    Priming of plant growth promotion by volatile compounds of root-associated Microbacterium

    Viviane Cordovez da Cunha, S. Schop, Cees Hordijk, H. Dupré de Boulois, F. Coppens, I. Hanssen, Jos M. Raaijmakers, Victor Carrion Bravo
    Volatile compounds produced by plant-associated microorganisms represent a diverse resource to promote plant growth and health. Here we investigated the effect of volatiles from root-associated Microbacterium species on plant growth and development. Volatiles of eight strains induced significant increases in shoot and root biomass of Arabidopsis, but differed in their effects on root architecture. Microbacterium strain EC8 also enhanced root and shoot biomass of lettuce and tomato. Biomass increases were also observed for plants exposed only shortly to volatiles from EC8 prior to transplantation of the seedlings to soil. These results indicate that volatiles from EC8 can prime plants for growth promotion without direct and prolonged contact. We further showed that the induction of plant growth promotion is tissue specific: exposure of roots to volatiles from EC8 led to an increase in plant biomass whereas shoot exposure resulted in no or less growth promotion. GC-QTOF analysis revealed that EC8 produces a wide array of sulfur containing compounds as well as ketones. Bioassays with synthetic sulfur volatile compounds revealed that the plant growth response to dimethyl trisulfide was concentration-dependent with a significant increase in shoot weight at 1 μM and negative effects on plant biomass at concentrations higher than 1 mM. Genome-wide transcriptome analysis of volatile-exposed Arabidopsis seedlings showed up-regulation of genes involved in assimilation and transport of sulfate and nitrate. Collectively, these results show that root-associated Microbacterium primes plants, via the roots, for growth promotion most likely via modulation of sulfur and nitrogen metabolism.

    Importance In the past decade, various studies have described the effects of microbial volatiles on other (micro)organisms in vitro, but their broad-spectrum activity in vivo and the mechanisms underlying volatile-mediated plant growth promotion have not been addressed in detail. Here, we revealed that volatiles from root-associated bacteria of the genus Microbacterium can enhance growth of different plant species and can prime plants for growth promotion without direct and prolonged contact between the bacterium and the plant. Collectively, these results provide new opportunities for sustainable agriculture and horticulture by exposing roots of plants only briefly to a specific blend of microbial volatile compounds prior to transplantation of the seedlings to the greenhouse or field. This strategy has no need for large-scale introduction, root colonization and survival of the microbial inoculant.
  • FEMS Microbiology Ecology

    Exploring fish microbial communities to mitigate emerging diseases in aquaculture

    Irene de Bruijn, Yiying Liu, Geert F. Wiegertjes, Jos M. Raaijmakers
    Aquaculture is the fastest growing animal food sector worldwide and expected to further increase to feed the growing human population. However, existing and (re-)emerging diseases are hampering fish and shellfish cultivation and yield. For many diseases, vaccination protocols are not in place and the excessive use of antibiotics and other chemicals is of substantial concern. A more sustainable disease control strategy to protect fish and shellfish from (re-)emerging diseases could be achieved by introduction or augmentation of beneficial microbes. To establish and maintain a ‘healthy’ fish microbiome, a fundamental understanding of the diversity and temporal-spatial dynamics of fish-associated microbial communities and their impact on growth and health of their aquatic hosts is required. This review describes insights in the diversity and functions of the fish bacterial communities elucidated with next-generation sequencing and discusses the potential of the microbes to mitigate (re-)emerging diseases in aquaculture.
  • ISME Journal

    Breeding for soil-borne pathogen resistance impacts active rhizosphere microbiome of common bean

    L.W. Mendes, Rodrigo Mendes, Jos M. Raaijmakers, Siu Mui Tsai
    Over the past century, plant breeding programs have substantially improved plant growth and health, but have not yet
    considered the potential effects on the plant microbiome. Here, we conducted metatranscriptome analysis to determine if and
    how breeding for resistance of common bean against the root pathogen Fusarium oxysporum (Fox) affected gene expression
    in the rhizobacterial community. Our data revealed that the microbiome of the Fox-resistant cultivar presented a significantly
    higher expression of genes associated with nutrient metabolism, motility, chemotaxis, and the biosynthesis of the antifungal
    compounds phenazine and colicin V. Network analysis further revealed a more complex community for Fox-resistant
    cultivar and indicated Paenibacillus as a keystone genus in the rhizosphere microbiome. We suggest that resistance breeding
    in common bean has unintentionally co-selected for plant traits that strengthen the rhizosphere microbiome network structure
    and enrich for specific beneficial bacterial genera that express antifungal traits involved in plant protection against infections
    by root pathogens.
  • ISME Journal

    Saving Seed Microbiomes

    Gabriele Berg, Jos M. Raaijmakers
    Plant seeds are home to diverse microbial communities whose composition is determined by plant genotype, environment, and management practices. Plant domestication is now recognized as an important driver of plant-associated microbial diversity. To what extent and how domestication affects seed microbiomes is less well studied. Here we propose a ‘back-to-the-future’ approach to harness seed microbiomes of wild relatives of crop cultivars to save and re-instate missing beneficial seed microbes for improved plant tolerance to biotic and abiotic stress.
  • Frontiers in Microbiology

    Microbial extracellular polymeric substances – ecological functions and impact on soil aggregation

    A wide range of microorganisms produce extracellular polymeric substances (EPS), highly hydrated polymers that are mainly composed of polysaccharides, proteins and DNA. EPS are fundamental for microbial life and provide an ideal environment for chemical reactions, nutrient entrapment and protection against environmental stresses such as salinity and drought. Microbial EPS can enhance the aggregation of soil particles and benefit plants by maintaining the moisture of the environment and trapping nutrients. In addition, EPS have unique characteristics, such as biocompatibility, gelling and thickening capabilities, with industrial applications. However, despite decades of research on the industrial potential of EPS, only a few polymers are widely used in different areas, especially in agriculture. This review provides an overview of current knowledge on the ecological functions of microbial extracellular polymeric substances (EPS) and their application in agricultural soils to improve soil particle aggregation, an important factor for soil structure, health and fertility.
  • Frontiers in Microbiology

    Comparative microbiome analysis of a Fusarium wilt suppressive soil and a Fusarium wilt conducive soil from the Châteaurenard region

    Katarzyna Siegel-Hertz, Véronique Edel-Hermann, E. Chapelle, Sébastien Terrat, Jos M. Raaijmakers, Christian Steinberg
    Disease-suppressive soils are soils in which specific soil-borne plant pathogens cause only limited disease although the pathogen and susceptible host plants are both present. Suppressiveness is in most cases of microbial origin. We conducted a comparative metabarcoding analysis of the taxonomic diversity of fungal and bacterial communities from suppressive or non-suppressive (conducive) soils as regards Fusarium wilts sampled from the Châteaurenard region (France). Bioassays confirmed that disease incidence was significantly lower in the suppressive soil than in the conducive soil. Furthermore, we succeeded in partly transferring Fusarium wilt-suppressiveness to the conducive soil by mixing 10% (w/w) of the suppressive soil into the conducive soil. Fungal diversity differed significantly between the suppressive and conducive soils. Among dominant fungal operational taxonomic units (OTUs) affiliated to known genera, seventeen OTUs were detected exclusively in the suppressive soil. These OTUs were assigned to the Acremonium, Chaetomium, Cladosporium, Clonostachys, Fusarium, Ceratobasidium, Mortierella, Penicillium, Scytalidium, and Verticillium genera. Additionally, the relative abundance of specific members of the bacterial community was significantly higher in the suppressive and mixed soils than in the conducive soil. OTUs found more abundant in Fusarium wilt-suppressive soils were affiliated to the bacterial genera Adhaeribacter, Massilia, Microvirga, Rhizobium, Rhizobacter, Arthrobacter, Amycolatopsis, Rubrobacter, Paenibacillus, Stenotrophomonas, and Geobacter. Several of the fungal and bacterial genera detected exclusively or more abundantly in the Fusarium wilt-suppressive soil included genera known for their activity against F. oxysporum. Overall, this study supports the potential role of known fungal and bacterial genera in Fusarium wilt suppressive soils from Châteaurenard and pinpoints new bacterial and fungal genera for their putative role in Fusarium wilt suppressiveness.
  • Journal of Fish Diseases

    Impact of Pseudomonas H6 surfactant on all external life cycle stages of the fish parasitic ciliate Ichthyophthirius multifiliis

    A. Al-Jubury, Cao lu, P Walter Kania, L. von Gersdorff Jørgensen, Yiying Liu, Irene de Bruijn, Jos M. Raaijmakers, Kurt Buchman
    A bacterial biosurfactant isolated from Pseudomonas (strain H6) has previously been shown to have a lethal effect on the oomycete Saprolegnia diclina infecting fish eggs. The present work demonstrates that the same biosurfactant has a strong in vitro antiparasitic effect on the fish pathogenic ciliate Ichthyophthirius multifiliis. Three life cycle stages (the infective theront stage, the tomont and the tomocyst containing tomites) were all susceptible to the surfactant. Theronts were the most sensitive showing 100% mortality in as low concentrations as 10 and 13 μg/ml within 30 min. Tomonts were the most resistant but were killed in concentrations of 100 μg/ml. Tomocysts, which generally are considered resistant to chemical and medical treatment, due to the surrounding protective cyst wall, were also sensitive. The surfactant, in concentrations of 10 and 13 μg/ml, penetrated the cyst wall and killed the enclosed tomites within 60 min. Rainbow trout fingerlings exposed to the biosurfactant showed no adverse immediate or late signs following several hours incubation in concentrations effective for killing the parasite. This bacterial surfactant may be further developed for application as an antiparasitic control agent in aquaculture.
  • ISME Journal

    Influence of resistance breeding in common bean on rhizosphere microbiome composition and function

    L.W. Mendes, Jos M. Raaijmakers, Mattias De Hollander, Rodrigo Mendes, S.M. Tsai
    The rhizosphere microbiome has a key role in plant growth and health, providing a first line of defense against root infections by soil-borne pathogens. Here, we investigated the composition and metabolic potential of the rhizobacterial community of different common bean (Phaseolus vulgaris) cultivars with variable levels of resistance to the fungal root pathogen Fusarium oxysporum (Fox). For the different bean cultivars grown in two soils with contrasting physicochemical properties and microbial diversity, rhizobacterial abundance was positively correlated with Fox resistance. Pseudomonadaceae, bacillaceae, solibacteraceae and cytophagaceae were more abundant in the rhizosphere of the Fox-resistant cultivar. Network analyses showed a modular topology of the rhizosphere microbiome of the Fox-resistant cultivar, suggesting a more complex and highly connected bacterial community than in the rhizosphere of the Fox-susceptible cultivar. Metagenome analyses further revealed that specific functional traits such as protein secretion systems and biosynthesis genes of antifungal phenazines and rhamnolipids were more abundant in the rhizobacterial community of the Fox-resistant cultivar. Our findings suggest that breeding for Fox resistance in common bean may have co-selected for other unknown plant traits that support a higher abundance of specific beneficial bacterial families in the rhizosphere with functional traits that reinforce the first line of defense.
  • Current Opinion in Microbiology

    Road MAPs to engineer host microbiomes

    Ben Oyserman, Marnix H Medema, Jos M. Raaijmakers
    Microbiomes contribute directly or indirectly to host health and fitness. Thus far, investigations into these emergent traits, referred to here as microbiome-associated phenotypes (MAPs), have been primarily qualitative and taxonomy-driven rather than quantitative and trait-based. We present the MAPs-first approach, a theoretical and experimental roadmap that involves quantitative profiling of MAPs across genetically variable hosts and subsequent identification of the underlying mechanisms. We outline strategies for developing ‘modular microbiomes’ — synthetic microbial consortia that are engineered in concert with the host genotype to confer different but mutually compatible MAPs to a single host or host population. By integrating host and microbial traits, these strategies will facilitate targeted engineering of microbiomes to the benefit of agriculture, human/animal health and biotechnology.
  • Natural Product Reports

    Modulation of plant chemistry by beneficial root microbiota

    Desalegn Etalo, Je Seung Jeon, Jos M. Raaijmakers
    Covering: 1981-2017Plants are colonized by an astounding number of microorganisms that can reach cell densities much greater than the number of plant cells. Various plant-associated microorganisms can have profound beneficial effects on plant growth, development, physiology and tolerance to (a)biotic stress. In return, plants release metabolites into their direct surroundings, thereby feeding the microbial community and influencing their composition, gene expression and the production of secondary metabolites. Similarly, microbes living on and in plant tissue may induce known and yet unknown biosynthetic pathways in plants leading to diverse alterations in the plant metabolome. Here, we provide an overview of the impact of beneficial microbiota on plant chemistry, with an emphasis on bacteria living on or inside root tissues. We will also provide new perspectives on deciphering the yet untapped potential of microbe-mediated alteration of plant chemistry as an alternative platform to discover new pathways, genes and enzymes involved the biosynthesis of high value natural plant products.
  • Frontiers in Microbiology

    Current insights into the role of rhizosphere bacteria in disease suppressive soils

    Ruth Gomez Exposito, Irene de Bruijn, Joeke Postma, Jos M. Raaijmakers
    Disease suppressive soils offer effective protection to plants against infection by soil-borne pathogens, including fungi, oomycetes, bacteria, and nematodes. The specific disease suppression that operates in these soils is, in most cases, microbial in origin. Therefore, suppressive soils are considered as a rich resource for the discovery of beneficial microorganisms with novel antimicrobial and other plant protective traits. To date, several microbial genera have been proposed as key players in disease suppressiveness of soils, but the complexity of the microbial interactions as well as the underlying mechanisms and microbial traits remain elusive for most disease suppressive soils. Recent developments in next generation sequencing and other ‘omics’ technologies have provided new insights into the microbial ecology of disease suppressive soils and the identification of microbial consortia and traits involved in disease suppressiveness. Here, we review the results of recent ‘omics’-based studies on the microbial basis of disease suppressive soils, with specific emphasis on the role of rhizosphere bacteria in this intriguing microbiological phenomenon.
  • Frontiers in Plant Science

    Plant phenotypic and transcriptional changes induced by volatiles from the fungal root pathogen Rhizoctonia solani

    Viviane Cordovez da Cunha, Liesje Mommer, Kay Moisan, D. Lucas-Barbosa, R. Pierik, Roland Mumm, Victor Carrion Bravo, Jos M. Raaijmakers
    Beneficial soil microorganisms can affect plant growth and resistance by the production of volatile organic compounds (VOCs). Yet, little is known on how VOCs from soil-borne plant pathogens affect plant growth and resistance. Here we show that VOCs released from mycelium and sclerotia of the fungal root pathogen Rhizoctonia solani enhance growth and accelerate development of Arabidopsis thaliana. Seedlings briefly exposed to the fungal VOCs showed similar phenotypes, suggesting that enhanced biomass and accelerated development are primed already at early developmental stages. Fungal VOCs did not affect plant resistance to infection by the VOC-producing pathogen itself but reduced aboveground resistance to the herbivore Mamestra brassicae. Transcriptomics of A. thaliana revealed that genes involved in auxin signaling were up-regulated, whereas ethylene and jasmonic acid signaling pathways were down-regulated by fungal VOCs. Mutants disrupted in these pathways showed similar VOC-mediated growth responses as the wild-type A. thaliana, suggesting that other yet unknown pathways play a more prominent role. We postulate that R. solani uses VOCs to predispose plants for infection from a distance by altering root architecture and enhancing root biomass. Alternatively, plants may use enhanced root growth upon fungal VOC perception to sacrifice part of the root biomass and accelerate development and reproduction to survive infection.
  • Biochimica et Biophysica Acta (BBA) - Biomembranes

    Membrane Interactions of Natural Cyclic Lipodepsipeptides of the Viscosin Group

    Niels Geudens, Mehmet Nail Nasir, Jean-Marc Crowet, Jos M. Raaijmakers, Krisztina Fehér, Tom Coenye, José C. Martins, Laurence Lins, Davy Sinnaeve, Magali Deleu
    Abstract Many Pseudomonas spp. produce cyclic lipodepsipeptides (CLPs), which, besides their role in biological functions such as motility, biofilm formation and interspecies interactions, are antimicrobial. It has been established that interaction with the cellular membrane is central to the mode of action of CLPs. In this work, we focus on the CLPs of the so-called viscosin group, aiming to assess the impact of the main structural variations observed within this group on both the antimicrobial activity and the interaction with model membranes. The antimicrobial activity of viscosin, viscosinamide A, WLIP and pseudodesmin A were all tested on a broad panel of mainly Gram-positive bacteria. Their capacity to permeabilize or fuse PG/PE/cardiolipin model membrane vesicles is assessed using fluorescent probes. We find that the Glu2/Gln2 structural variation within the viscosin group is the main factor that influences both the membrane permeabilization properties and the minimum inhibitory concentration of bacterial growth, while the configuration of the Leu5 residue has no apparent effect. The CLP-membrane interactions were further evaluated using CD and FT-IR spectroscopy on model membranes consisting of PG/PE/cardiolipin or POPC with or without cholesterol. In contrast to previous studies, we observe no conformational change upon membrane insertion. The CLPs interact both with the polar heads and aliphatic tails of model membrane systems, altering bilayer fluidity, while cholesterol reduces CLP insertion depth.
  • ISME Journal

    Linking rhizosphere microbiome composition of wild and domesticated Phaseolus vulgaris to genotypic and root phenotypic traits

    Juan Esteban Perez Jaramillo, Victor Carrion Bravo, Mirte Bosse, Luiz F. V. Ferrão, Mattias De Hollander, Antonio A. F. Garcia, Camilo A. Ramirez, Rodrigo Mendez, Jos M. Raaijmakers
    Plant domestication was a pivotal accomplishment in human history, but also led to a reduction in genetic diversity of crop species compared to their wild ancestors. How this reduced genetic diversity affected plant–microbe interactions belowground is largely unknown. Here, we investigated the genetic relatedness, root phenotypic traits and rhizobacterial community composition of modern and wild accessions of common bean (Phaseolus vulgaris) grown in agricultural soil from the highlands of Colombia, one of the centers of common bean diversification. Diversity Array Technology-based genotyping and phenotyping of local common bean accessions showed significant genetic and root architectural differences between wild and modern accessions, with a higher specific root length for the wild accessions. Canonical Correspondence Analysis indicated that the divergence in rhizobacterial community composition between wild and modern bean accessions is associated with differences in specific root length. Along the bean genotypic trajectory, going from wild to modern, we observed a gradual decrease in relative abundance of Bacteroidetes, mainly Chitinophagaceae and Cytophagaceae, and an increase in relative abundance of Actinobacteria and Proteobacteria, in particular Nocardioidaceae and Rhizobiaceae, respectively. Collectively, these results establish a link between common bean domestication, specific root morphological traits and rhizobacterial community assembly.
  • Environmental Microbiology

    Genome-wide analysis of bacterial determinants of plant growth promotion and induced systemic resistance by Pseudomonas fluorescens

    Xu Cheng, Desalegn Etalo, Judith E. van de Mortel, Ester Dekkers, Linh Nguyen, Marnix H Medema, Jos M. Raaijmakers
    Pseudomonas fluorescens strain SS101 (Pf.SS101) promotes growth of Arabidopsis thaliana, enhances greening and lateral root formation, and induces systemic resistance (ISR) against the bacterial pathogen Pseudomonas syringae pv. tomato (Pst). Here, targeted and untargeted approaches were adopted to identify bacterial determinants and underlying mechanisms involved in plant growth promotion and ISR by Pf.SS101. Based on targeted analyses, no evidence was found for volatiles, lipopeptides and siderophores in plant growth promotion by Pf.SS101. Untargeted, genome-wide analyses of 7,488 random transposon mutants of Pf.SS101 led to the identification of 21 mutants defective in both plant growth promotion and ISR. Many of these mutants, however, were auxotrophic and impaired in root colonization. Genetic analysis of three mutants followed by site-directed mutagenesis, genetic complementation and plant bioassays revealed the involvement of the phosphogluconate dehydratase gene edd, the response regulator gene colR and the adenylsulfate reductase gene cysH in both plant growth promotion and ISR. Subsequent comparative plant transcriptomics analyses strongly suggest that modulation of sulfur assimilation, auxin biosynthesis and transport, steroid biosynthesis and carbohydrate metabolism in Arabidopsis are key mechanisms linked to growth promotion and ISR by Pf.SS101. This article is protected by copyright. All rights reserved.
  • Frontiers in Microbiology

    Potential for biocontrol of hairy root disease by a Paenibacillus clade

    Lien Bosmans, Irene de Bruijn, Saskia Gerards, Rob Moerkens, Lore Van Looveren, Lieve Wittemans, Bart Van Calenberge, Anneleen Paeleman, Stef Van Kerckhove, Jef Rozenski, Rene de Mot, Hans Rediers, Jos M. Raaijmakers, Bart Lievens
    Rhizogenic Agrobacterium biovar 1 is the causative agent of hairy root disease (HRD) in the hydroponic cultivation of tomato and cucumber causing significant losses in marketable yield. In order to prevent and control the disease chemical disinfectants such as hydrogen peroxide or hypochlorite are generally applied to sanitize the hydroponic system and/or hydroponic solution. However, effective control of HRD sometimes requires high disinfectant doses that may have phytotoxic effects. Moreover, several of these chemicals may be converted to unwanted by-products with human health hazards. Here we explored the potential of beneficial bacteria as a sustainable means to control HRD. A large collection of diverse bacterial genera was screened for antagonistic activity against rhizogenic Agrobacterium biovar 1 using the agar overlay assay. Out of more than 130 strains tested only Paenibacillus strains showed antagonistic activity. Strikingly, phylogenetic analysis showed that antagonistic activity was restricted to a particular Paenibacillus clade, representing the species P. illinoisensis, P. pabuli, P. taichungensis, P. tundrae, P. tylopili, P. xylanexedens and P. xylanilyticus. Assessment of the spectrum of activity revealed that some strains were able to inhibit the growth of all 35 rhizogenic agrobacteria strains tested, while others were only active against part of the collection, suggesting a different mode of action. Preliminary characterization of the compounds involved in the antagonistic activity of two closely related Paenibacillus strains, tentatively identified as P. xylanexedens, revealed that they are water-soluble and have low molecular weight. Application of a combination of these strains in greenhouse conditions resulted in a significant reduction of HRD, indicating the great potential of these strains to control HRD.
  • Frontiers in Plant Science

    Role of the GacS Sensor Kinase in the Regulation of Volatile Production by Plant Growth-Promoting Pseudomonas fluorescens SBW25

    Xu Cheng, Viviane Cordovez da Cunha, Desalegn Etalo, Menno van der Voort, Jos M. Raaijmakers
    In plant-associated Pseudomonas species, the production of several secondary metabolites and exoenzymes is regulated by the GacS/GacA two-component regulatory system (the Gac-system). Here, we investigated if a mutation in the GacS sensor kinase affects the production of volatile organic compounds (VOCs) in P. fluorescens SBW25 (Pf.SBW25) and how this impacts on VOCs-mediated growth promotion and induced systemic resistance of Arabidopsis and tobacco. A total of 205 VOCs were detected by Gas Chromatography Mass Spectrometry (GC-MS) for Pf. SBW25 and the gacS-mutant grown on 2 different media for 3 and 6 days. Discriminant function analysis followed by hierarchical clustering revealed 24 VOCs that were significantly different in their abundance between Pf.SBW25 and the gacS-mutant, which included three acyclic alkenes (3-nonene, 4-undecyne, 1-undecene). These alkenes were significantly reduced by the gacS mutation independently of the growth media and of the incubation time. For Arabidopsis, both Pf.SBW25 and the gacS-mutant enhanced, via VOCs, root and shoot biomass, induced systemic resistance against leaf infections by P. syringae and rhizosphere acidification to the same extent. For tobacco, however, VOCs-mediated effects on shoot and root growth were significantly different between Pf.SBW25 and the gacS-mutant. While Pf.SBW25 inhibited tobacco root growth, the gacS-mutant enhanced root biomass and lateral root formation relative to the non-treated control plants. Collectively these results indicate that the sensor kinase GacS is involved in the regulation of VOCs production in Pf.SBW25, affecting plant growth in a plant species-dependent manner.
  • Nature Microbiology

    Indexing the Pseudomonas specialized metabolome enabled the discovery of poaeamide B and the bananamides

    D. Nguyen, Alexey V Melnik, N. Koyama, X. Lu, M. Schorn, Jinyu Fang, K. Aguinaldo, T. Lincecum Jr., M. Ghequire, Victor Carrion Bravo, T. Cheng, J. Malone, Tim H. Mauchline, Laura M. Sanchez, A. Marm Kilpatrick, Jos M. Raaijmakers, Rene de Mot, B. Moore, Marnix H Medema, Pieter C. Dorrestein
    Pseudomonads are cosmopolitan microorganisms able to produce a wide array of specialized metabolites. These molecules allow Pseudomonas to scavenge nutrients, sense population density and enhance or inhibit growth of competing microorganisms. However, these valuable metabolites are typically characterized one-molecule–one-microbe at a time, instead of being inventoried in large numbers. To index and map the diversity of molecules detected from these organisms, 260 strains of ecologically diverse origins were subjected to mass-spectrometry-based molecular networking. Molecular networking not only enables dereplication of molecules, but also sheds light on their structural relationships. Moreover, it accelerates the discovery of new molecules. Here, by indexing the Pseudomonas specialized metabolome, we report the molecular-networking-based discovery of four molecules and their evolutionary relationships: a poaeamide analogue and a molecular subfamily of cyclic lipopeptides, bananamides 1, 2 and 3. Analysis of their biosynthetic gene cluster shows that it constitutes a distinct evolutionary branch of the Pseudomonas cyclic lipopeptides. Through analysis of an additional 370 extracts of wheat-associated Pseudomonas, we demonstrate how the detailed knowledge from our reference index can be efficiently propagated to annotate complex metabolomic data from other studies, akin to the way in which newly generated genomic information can be compared to data from public databases.
  • Science

    Soil immune responses

    Jos M. Raaijmakers, Mark Mazzola
    Soil microorganisms are central to the provision of food, feed, fiber, and medicine. Engineering of soil microbiomes may promote plant growth and plant health, thus contributing to food security and agricultural sustainability (1, 2). However, little is known about most soil microorganisms and their impact on plant health. Disease-suppressive soils offer microbiome-mediated protection of crop plants against infections by soil-borne pathogens. Understanding of the microbial consortia and mechanisms involved in disease suppression may help to better manage plants while reducing fertilizer and pesticide inputs.
  • Frontiers in Microbiology

    Editorial: Smelly fumes: volatile-mediated communication between bacteria and other organisms

    Laure Weisskopf, Paolina Garbeva, Choo-Min Ryu, Jos M. Raaijmakers
    Volatiles are small (<300 Da), smelly molecules emitted by all organisms. They have very diverse roles for the producing organism (e.g., as infochemicals or antimicrobial compounds) and fulfill important ecosystem functions. While the importance of plant volatiles has been recognized for more than 30 years, research on microbial volatiles attracted attention only in the last decades. This special issue focuses on several new findings and recent developments in the field of microbial (fungal and bacterial) volatiles, their biological functions and chemical identification, which are highlighted in this editorial.
  • International Journal of Molecular Sciences

    Elucidating the diversity of aquatic Microdochium and Trichoderma species and their activity against the fish pathogen Saprolegnia diclina

    Yiying Liu, Christin Zachow, Jos M. Raaijmakers, Irene de Bruijn
    Animals and plants are increasingly threatened by emerging fungal and oomycete diseases. Amongst oomycetes, Saprolegnia species cause population declines in aquatic animals, especially fish and amphibians, resulting in significant perturbation in biodiversity, ecological balance and food security. Due to the prohibition of several chemical control agents, novel sustainable measures are required to control Saprolegnia infections in aquaculture. Previously, fungal community analysis by terminal restriction fragment length polymorphism (T-RFLP) revealed that the Ascomycota, specifically the genus Microdochium, was an abundant fungal phylum associated with salmon eggs from a commercial fish farm. Here, phylogenetic analyses showed that most fungal isolates obtained from salmon eggs were closely related to Microdochium lycopodinum/Microdochium phragmitis and Trichoderma viride species. Phylogenetic and quantitative PCR analyses showed both a quantitative and qualitative difference in Trichoderma population between diseased and healthy salmon eggs, which was not the case for the Microdochium population. In vitro antagonistic activity of the fungi against Saprolegnia diclina was isolate-dependent; for most Trichoderma isolates, the typical mycoparasitic coiling around and/or formation of papilla-like structures on S. diclina hyphae were observed. These results suggest that among the fungal community associated with salmon eggs, Trichoderma species may play a role in Saprolegnia suppression in aquaculture.
  • ISME Journal

    Insect pathogenicity in plant-beneficial pseudomonads: phylogenetic distribution and comparative genomics

    Pascale Flury, Nora Aellen, Beat Ruffner, Maria Pechy-Tarr, Shakira Fataar, Zane Metla, Ana Dominguez-Ferreras, Guido Bloemberg, Joachim Frey, Alexander Goesmann, Jos M. Raaijmakers, Brion Duffy, Monica Hofte, Jochen Blom, Theo H M Smits, Christoph Keel, Monika Maurhofer
    Bacteria of the genus Pseudomonas occupy diverse environments. The Pseudomonas fluorescens group is particularly well-known for its plant-beneficial properties including pathogen suppression. Recent observations that some strains of this group also cause lethal infections in insect larvae, however, point to a more versatile ecology of these bacteria. We show that 26 P. fluorescens group strains, isolated from three continents and covering three phylogenetically distinct sub-clades, exhibited different activities toward lepidopteran larvae, ranging from lethal to avirulent. All strains of sub-clade 1, which includes Pseudomonas chlororaphis and Pseudomonas protegens, were highly insecticidal regardless of their origin (animals, plants). Comparative genomics revealed that strains in this sub-clade possess specific traits allowing a switch between plant- and insect-associated lifestyles. We identified 90 genes unique to all highly insecticidal strains (sub-clade 1) and 117 genes common to all strains of sub-clade 1 and present in some moderately insecticidal strains of sub-clade 3. Mutational analysis of selected genes revealed the importance of chitinase C and phospholipase C in insect pathogenicity. The study provides insight into the genetic basis and phylogenetic distribution of traits defining insecticidal activity in plant-beneficial pseudomonads. Strains with potent dual activity against plant pathogens and herbivorous insects have great potential for use in integrated pest management for crops.
  • ISME Journal

    Fungal invasion of the rhizosphere microbiome

    E. Chapelle, R. Mendes, P.A.H.M. Bakker, Jos M. Raaijmakers
    The rhizosphere is the infection court where soil-borne pathogens establish a parasitic relationship with the plant. To infect root tissue, pathogens have to compete with members of the rhizosphere microbiome for available nutrients and microsites. In disease-suppressive soils, pathogens are strongly restricted in growth by the activities of specific rhizosphere microorganisms. Here, we sequenced metagenomic DNA and RNA of the rhizosphere microbiome of sugar beet seedlings grown in a soil suppressive to the fungal pathogen Rhizoctonia solani. rRNA-based analyses showed that Oxalobacteraceae, Burkholderiaceae, Sphingobacteriaceae and Sphingomonadaceae were significantly more abundant in the rhizosphere upon fungal invasion. Metatranscriptomics revealed that stress-related genes (ppGpp metabolism and oxidative stress) were upregulated in these bacterial families. We postulate that the invading pathogenic fungus induces, directly or via the plant, stress responses in the rhizobacterial community that lead to shifts in microbiome composition and to activation of antagonistic traits that restrict pathogen infection.
  • Environmental Microbiology

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

    Chunxu Song, Teresa Kidarsa, J. van de Mortel, J.E. Loper, Jos M. Raaijmakers
    Swarming motility is a flagella-driven multicellular behavior 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. This article is protected by copyright. All rights reserved.
  • Ecology Letters

    Impact of soil heat on reassembly of bacterial communities in the rhizosphere microbiome and plant disease suppression

    Menno van der Voort, M. Kempenaar, M. van Driel, Jos M. Raaijmakers, R. Mendes
    The rhizosphere microbiome offers a range of ecosystem services to the plant, including nutrient acquisition and tolerance to (a)biotic stress. Here, analysing the data by Mendes et al. (2011), we show that short heat disturbances (50 or 80 °C, 1 h) of a soil suppressive to the root pathogenic fungus Rhizoctonia solani caused significant increase in alpha diversity of the rhizobacterial community and led to partial or complete loss of disease protection. A reassembly model is proposed where bacterial families that are heat tolerant and have high growth rates significantly increase in relative abundance after heat disturbance, while temperature-sensitive and slow-growing bacteria have a disadvantage. The results also pointed to a potential role of slow-growing, heat-tolerant bacterial families from Actinobacteria and Acidobacteria phyla in plant disease protection. In conclusion, short heat disturbance of soil results in rearrangement of rhizobacterial communities and this is correlated with changes in the ecosystem service disease suppression.
  • Soil Biology & Biochemistry

    Challenges and opportunities in harnessing soil disease suppressiveness for sustainable pasture production

    B.E.A. Dignam, Maureen O'Callaghan, Leo M. Condron, Jos M. Raaijmakers, George Kowalchuk, Steve A. Wakelin
    Grasslands are an important source of biodiversity, providing a range of essential ecosystem services such as ensuring water quality and soil carbon storage. An increasing proportion of grasslands are used for pastoral agriculture, supporting production of domestic livestock. Pasture productivity is significantly affected by soil-borne microbial pathogens. Reducing the impact of soil-borne diseases in pastures is challenging given the complexity of interactions within the soil/rhizosphere microbiome and the diverse impacts of vegetation, land management, soil conditions and climate. Furthermore, there are fewer opportunities to control plant pathogens in pastures compared to arable cropping systems. The greater diversity of vegetation leads to the development of more diverse and less well characterized pathogen complexes, and the application of agrochemicals for control of soil-borne diseases is economically prohibitive and ecologically undesirable. Soil-borne plant pathogens can be suppressed through the general activity of the total soil microbiota acting in competition with the pathogenic microbiota, or by increases in the abundance and activity of specific microbes or microbial consortia that are antagonistic against selected pathogens. The development of strategies that enhance disease suppressiveness in pastures will depend not only on phylogenetic assessment of microbial communities, but also on a mechanistic understanding of the functional potential and properties (i.e. disease suppressive traits) of the soil microbiome. Collectively, this fundamental knowledge will be essential to identify the factors driving the emergence of desired disease suppressive microorganisms and traits. To understand and predict disease suppressive functionality, the spatial and temporal variability of the soil and plant-associated microbial populations and their activities must be taken into account. A systems-based approach is therefore required to identify the obstacles and opportunities related to controlling plant pathogens in pasture systems. Such an integrated approach should incorporate a “microbial” perspective to examine traits, drivers and activities of soil-borne microbes, while utilizing emerging tools in ecological genomics, as well as computational, statistical and modelling approaches that also accommodate the chemical and physical complexity of soil ecosystems.
  • Plant Molecular Biology

    Impact of plant domestication on rhizosphere microbiome assembly and functions

    Juan Esteban Perez Jaramillo, Rodrigo Mendes, Jos M. Raaijmakers
    The rhizosphere microbiome is pivotal for plant health and growth, providing defence against pests and diseases, facilitating nutrient acquisition and helping plants to withstand abiotic stresses. Plants can actively recruit members of the soil microbial community for positive feedbacks, but the underlying mechanisms and plant traits that drive microbiome assembly and functions are largely unknown. Domestication of plant species has substantially contributed to human civilization, but also caused a strong decrease in the genetic diversity of modern crop cultivars that may have affected the ability of plants to establish beneficial associations with rhizosphere microbes. Here, we review how plants shape the rhizosphere microbiome and how domestication may have impacted rhizosphere microbiome assembly and functions via habitat expansion and via changes in crop management practices, root exudation, root architecture, and plant litter quality. We also propose a “back to the roots” framework that comprises the exploration of the microbiome of indigenous plants and their native habitats for the identification of plant and microbial traits with the ultimate goal to reinstate beneficial associations that may have been undermined during plant domestication.
  • Nature Chemical Biology

    Minimum Information about a Biosynthetic Gene cluster

    Marnix H Medema, Renzo Kottmann, Pelin Yilmaz, Matthew Cummings, John B Biggins, Kai Blin, Irene de Bruijn, Yit Heng Chooi, Jan Claesen, R Cameron Coates, Pablo Cruz-Morales, Srikanth Duddela, Stephanie Dusterhus, Daniel J Edwards, David P Fewer, Neha Garg, Christoph Geiger, Juan Pablo Gomez-Escribano, Anja Greule, Michalis Hadjithomas, Anthony S Haines, Eric J N Helfrich, Matthew L Hillwig, Keishi Ishida, Adam C Jones, Carla S Jones, Katrin Jungmann, Carsten Kegler, Hyun Uk Kim, Peter Kotter, Daniel Krug, Joleen Masschelein, Alexey V Melnik, Simone M Mantovani, Emily A Monroe, Marcus Moore, Nathan Moss, Hans-Wilhelm Nutzmann, Guohui Pan, Amrita Pati, Daniel Petras, F Jerry Reen, Federico Rosconi, Zhe Rui, Zhenhua Tian, Nicholas J Tobias, Yuta Tsunematsu, Philipp Wiemann, Elizabeth Wyckoff, Xiaohui Yan, Grace Yim, Fengan Yu, Yunchang Xie, Bertrand Aigle, Alexander K Apel, Carl J Balibar, Emily P Balskus, Francisco Barona-Gómez, Andreas Bechthold, Helge B Bode, Rainer Borriss, Sean F Brady, Axel A Brakhage, Patrick Caffrey, Yi-Qiang Cheng, Jon Clardy, Russell J Cox, Rene de Mot, Stefano Donadio, Mohamed S Donia, Wilfred A van der Donk, Pieter C. Dorrestein, Sean Doyle, Arnold J M Driessen, Monika Ehling-Schulz, Karl-Dieter Entian, Michael A Fischbach, Lena Gerwick, William H Gerwick, Harald Gross, Bertolt Gust, Christian Hertweck, Monica Hofte, Susan E Jensen, Jianhua Ju, Leonard Katz, Leonard Kaysser, Jonathan L Klassen, Nancy P Keller, Jan Kormanec, Oscar P Kuipers, Tomohisa Kuzuyama, Nikos C Kyrpides, Hyung-Jin Kwon, Sylvie Lautru, Rob Lavigne, Chia Y Lee, Bai Linquan, Xinyu Liu, Wen Liu, Andriy Luzhetskyy, Taifo Mahmud, Yvonne Mast, Carmen Mendez, Mikko Metsä-Ketelä, Jason Micklefield, Douglas A Mitchell, Bradley S Moore, Leonilde M Moreira, Rolf Müller, Brett A Neilan, Markus Nett, Jens Nielsen, Fergal O'Gara, Hideaki Oikawa, Anne Osbourn, Marcia S Osburne, Bohdan Ostash, Shelley M Payne, Jean-Luc Pernodet, Miroslav Petricek, Jörn Piel, Olivier Ploux, Jos M. Raaijmakers, Jose A Salas, Esther K Schmitt, Barry Scott, Ryan F Seipke, Ben Shen, David H Sherman, Kaarina Sivonen, Michael J Smanski, Margherita Sosio, Evi Stegmann, Roderich D. Süssmuth, Kapil Tahlan, Christopher M Thomas, Yi Tang, Andrew W Truman, Muriel Viaud, Jonathan D Walton, Christopher T Walsh, Tilmann Weber, Gilles van Wezel, Barrie Wilkinson, Joanne M Willey, Wolfgang Wohlleben, Gerard D Wright, Nadine Ziemert, Changsheng Zhang, Sergey B Zotchev, Rainer Breitling, Eriko Takano, Frank Oliver Glockner
    A wide variety of enzymatic pathways that produce specialized metabolites in bacteria, fungi and plants
    are known to be encoded in biosynthetic gene clusters. Information about these clusters, pathways and
    metabolites is currently dispersed throughout the literature, making it difficult to exploit. To facilitate
    consistent and systematic deposition and retrieval of data on biosynthetic gene clusters, we propose the
    Minimum Information about a Biosynthetic Gene cluster (MIBiG) data standard.
  • Scientific Reports

    Molecular and chemical dialogues in bacteria-protozoa interactions

    Chunxu Song, M. Mazzola, Xu Cheng, Janina Oetjen, Theodore Alexandrov, Pieter C. Dorrestein, Jeramie Watrous, Menno van der Voort, Jos M. Raaijmakers
    Protozoan predation of bacteria can significantly affect soil microbial community composition and ecosystem functioning. Bacteria possess diverse defense strategies to resist or evade protozoan predation. For soil-dwelling Pseudomonas species, several secondary metabolites were proposed to provide protection against different protozoan genera. By combining whole-genome transcriptome analyses with (live) imaging mass spectrometry (IMS), we observed multiple changes in the molecular and chemical dialogues between Pseudomonas fluorescens and the protist Naegleria americana. Lipopeptide (LP) biosynthesis was induced in Pseudomonas upon protozoan grazing and LP accumulation transitioned from homogeneous distributions across bacterial colonies to site-specific accumulation at the bacteria-protist interface. Also putrescine biosynthesis was upregulated in P. fluorescens upon predation. We demonstrated that putrescine induces protozoan trophozoite encystment and adversely affects cyst viability. This multifaceted study provides new insights in common and strain-specific responses in bacteria-protozoa interactions, including responses that contribute to bacterial survival in highly competitive soil and rhizosphere environments.
  • Microbial Biotechnology

    The Rsm regulon of plant growth-promoting Pseudomonas fluorescens SS101: role of small RNAs in regulation of lipopeptide biosynthesis

    Chunxu Song, Menno van der Voort, J. van de Mortel, Karl Hassan, L.D.H. Elbourne, Ian T. Paulsen, J.E. Loper, Jos M. Raaijmakers
    Interpretive Summary: Biological control provides a promising strategy for managing plant diseases, but has not yet been utilized widely in agriculture due, in part, to unexplained variation in its success in managing disease. Our research goals are to identify sources of variation in biological control, and devise ways to make it more reliable. We focus on Pseudomonas fluorescens, which is a species of bacteria that occurs naturally on plant surfaces such as leaves and roots. In this study, we evaluated the roles of two small RNAs (called rsmY and rsmZ) of gene expression in the plant-associated bacterium P. fluorescens SBW25. We show that these small RNAs influenced the transcription of many genes that have diverse functions in this bacterium. We conclude that the rsmY and rsmZ of P. fluorescens SS101 plays critical role in the regulation of lipopeptide biosynthesis and control the expression of other genes involved in motility, competition and survival in the plant rhizosphere.
    Technical Abstract: The rhizobacterium Pseudomonas fluorescens SS101 inhibits growth of oomycete and fungal pathogens, and induces resistance in plants against pathogens and insects. To unravel regulatory pathways of secondary metabolite production in SS101, we conducted a genome-wide search for sRNAs and performed transcriptomic analyses to identify genes associated with the Rsm (repressor of secondary metabolites) regulon. In silico analysis led to the identification of sixteen putative sRNAs in the SS101 genome. In frame deletion of the sRNAs rsmY and rsmZ showed that the Rsm system regulates the biosynthesis of the lipopeptide massetolide A and involves the two repressor proteins RsmA and RsmE, with the LuxR-type transcriptional regulator MassAR as their most likely target. Transcriptome analyses of the rsmYZ mutant further revealed that genes associated with iron acquisition, motility and chemotaxis were significantly upregulated, whereas genes of the type VI secretion system were downregulated. Comparative transcriptomic analyses showed that most, but not all, of the genes controlled by RsmY/RsmZ are also controlled by the GacS/GacA two-component system. We conclude that the Rsm regulon of P. fluorescens SS101 plays a critical role in the regulation of lipopeptide biosynthesis and controls the expression of other genes involved in motility, competition and survival in the plant rhizosphere.
  • New Zealand Plant Protection

    A bioassay to compare the disease suppressive capacity of pasture soils

    B.E.A. Dignam, Maureen O'Callaghan, Leo M. Condron, Jos M. Raaijmakers, George Kowalchuk, Steve A. Wakelin
    Dynamic pathogen complexes can develop under pastures, thereby substantially reducing potential productivity. Suppression of such pathogen complexes is therefore of great importance, and bioassays can quantify disease suppression in soils. This study describes the development of a pasture-relevant system: Rhizoctonia solani AG 2-1 induced damping-off (wirestem) of kale (Brassica oleracea). As kale is not a component of traditional ryegrass clover pasture swards, the assay allows assessment of general disease suppression, considered more enduring in multiple-host-multiple-pathogen systems. A pathogenic Rhizoctonia solani isolate was obtained from New Zealand pastoral soil. Inoculation of soils with this isolate resulted in a level of damping-off disease comparable to that induced by reference Rhizoctonia solani isolate Rs043-2. Significantly different levels of inoculum-induced disease incidence and progression were found in four distinct pastoral soils. In combination with soil physicochemical data and environmental DNA approaches, this bioassay can be used to further advance understanding of the influence of farm management practices on disease suppression in pasture soils.
  • ISME Journal

    Cross-kingdom similarities in microbiome functions

    R. Mendes, Jos M. Raaijmakers
    Recent advances in medical research have revealed how humans rely on their microbiome for diverse traits and functions. Similarly, microbiomes of other higher organisms play key roles in disease, health, growth and development of their host. Exploring microbiome functions across kingdoms holds enormous potential to understand common mechanisms and concepts underlying microbiome assembly and microbial processes that sustain life of Eukaryotes. The gut and plant rhizosphere are both open systems with large surface areas overpopulated with microbes. Despite distinct differences in microbiome composition, these two ecosystems share striking similarities in microbiome functions related to nutrient acquisition, immune system modulation and protection against infections. We also discuss how humans and plants exchange microbes, for better or for worse. We propose that adopting ecological theory, combined with modeling and synthetic microbial ecosystems, provides a promising strategy to identify host traits and cues involved in microbiome assembly on and in Eukaryotes
  • PLoS One

    Diversity of aquatic Pseudomonas species and their activity against the fish pathogenic oomycete Saprolegnia

    Yiying Liu, E. Rzeszutek, Menno van der Voort, C.H. Wu, E. Thoen, I. Skaar, Vincent Bulone, Pieter C. Dorrestein, Jos M. Raaijmakers, Irene de Bruijn
    Emerging fungal and oomycete pathogens are increasingly threatening animals and plants globally. Amongst oomycetes, Saprolegnia species adversely affect wild and cultivated populations of amphibians and fish, leading to substantial reductions in biodiversity and food productivity. With the ban of several chemical control measures, new sustainable methods are needed to mitigate Saprolegnia infections in aquaculture. Here, PhyloChip-based community analyses showed that the Pseudomonadales, particularly Pseudomonas species, represent one of the largest bacterial orders associated with salmon eggs from a commercial hatchery. Among the Pseudomonas species isolated from salmon eggs, significantly more biosurfactant producers were retrieved from healthy salmon eggs than from Saprolegnia-infected eggs. Subsequent in vivo activity bioassays showed that Pseudomonas isolate H6 significantly reduced salmon egg mortality caused by Saprolegnia diclina. Live colony mass spectrometry showed that strain H6 produces a viscosin-like lipopeptide surfactant. This biosurfactant inhibited growth of Saprolegnia in vitro, but no significant protection of salmon eggs against Saprolegniosis was observed. These results indicate that live inocula of aquatic Pseudomonas strains, instead of their bioactive compound, can provide new (micro)biological and sustainable means to mitigate oomycete diseases in aquaculture.
  • BMC Genomics

    Comparative genomics and metabolic profiling of the genus Lysobacter

    Irene de Bruijn, Xu Cheng, Victor de Jager, Ruth Gomez Exposito, Jeramie Watrous, N. Patel, Joeke Postma, Pieter C. Dorrestein, D. Kobayashi, Jos M. Raaijmakers
    Lysobacter species are Gram-negative bacteria widely distributed in soil, plant and freshwater habitats. Lysobacter owes its name to the lytic effects on other microorganisms. To better understand their ecology and interactions with other (micro)organisms, five Lysobacter strains representing the four species L. enzymogenes, L. capsici, L. gummosus and L. antibioticus were subjected to genomics and metabolomics analyses.

    Comparative genomics revealed a diverse genome content among the Lysobacter species with a core genome of 2,891 and a pangenome of 10,028 coding sequences. Genes encoding type I, II, III, IV, V secretion systems and type IV pili were highly conserved in all five genomes, whereas type VI secretion systems were only found in L. enzymogenes and L. gummosus. Genes encoding components of the flagellar apparatus were absent in the two sequenced L. antibioticus strains. The genomes contained a large number of genes encoding extracellular enzymes including chitinases, glucanases and peptidases. Various nonribosomal peptide synthase (NRPS) and polyketide synthase (PKS) gene clusters encoding putative bioactive metabolites were identified but only few of these clusters were shared between the different species. Metabolic profiling by imaging mass spectrometry complemented, in part, the in silico genome analyses and allowed visualisation of the spatial distribution patterns of several secondary metabolites produced by or induced in Lysobacter species during interactions with the soil-borne fungus Rhizoctonia solani.

    Our work shows that mining the genomes of Lysobacter species in combination with metabolic profiling provides novel insights into the genomic and metabolic potential of this widely distributed but understudied and versatile bacterial genus.
  • Microbiology

    The lipopeptide biosurfactant viscosin enhances dispersal of Pseudomonas fluorescens SBW25 biofilms

    Lise Bonnichsen, Nanna Bygvraa Svenningsen, Morten Rybtke, Irene de Bruijn, Jos M. Raaijmakers, Tim Tolker-Nielsen, Ole Nybroe
    Pseudomonads produce several lipopeptide biosurfactants that have antimicrobial properties, but also facilitate surface motility and influence biofilm formation. Detailed studies addressing the significance of lipopeptides for biofilm formation and architecture are rare. Hence the current study sets out to determine the specific role of the lipopeptide viscosin for Pseudomonas fluorescens SBW25 biofilm formation, architecture and dispersal, and to relate viscA gene expression with viscosin production and effect. Initially, we compared biofilm formation of SBW25 and the viscosin-deficient mutant strain SBW25ΔviscA in static microtiter assays. These experiments demonstrated that viscosin had little influence on the amount of biofilm formed by SBW25 during the early stages of biofilm development. Later, however, SBW25 formed significantly less biofilm than SBW25ΔviscA. The indication that viscosin is involved in biofilm dispersal was confirmed by chemical complementation of the mutant biofilm. Further, a fluorescent bioreporter showed that viscA expression was induced in biofilms 4 hours prior to dispersal. Subsequent detailed studies of biofilms formed in flow-cells for up to 5 days revealed that SBW25 and SBW25ΔviscA developed comparable biofilms dominated by well-defined mushroom-shaped structures. Carbon-starvation was required to obtain biofilm dispersal in this system. Dispersal of SBW25 biofilms was significantly larger than of SBW25ΔviscA biofilms after 3 hours, and importantly, carbon-starvation strongly induced viscA expression, in particular for cells that were apparently leaving the biofilm. Hence the current study points towards a role for viscosin-facilitated motility in dispersal of SBW25 biofilms.
  • Environmental Microbiology Reports

    Gac-mediated changes in pyrroloquinoline quinone biosynthesis enhance the antimicrobial activity of Pseudomonas fluorescens SBW25

    Xu Cheng, Menno van der Voort, Jos M. Raaijmakers
    In Pseudomonas species, production of secondary metabolites and exoenzymes is regulated by the GacS/GacA two-component regulatory system. In P. fluorescens SBW25, mutations in the Gac-system cause major transcriptional changes and abolished production of the lipopeptide viscosin and of an exoprotease. In contrast to many other Pseudomonas species and strains, inactivation of the Gac-system in strain SBW25 significantly enhanced its antimicrobial activities against oomycete, fungal and bacterial pathogens. Here, random plasposon mutagenesis of the gacS mutant led to the identification of seven mutants with reduced or loss of antimicrobial activity. In four mutants, the plasposon insertion was located in genes of the pyrroloquinoline quinone (PQQ) biosynthesis pathway. Genetic complementation, ectopic expression, activity bioassays and RP-HPLC analyses revealed that a gacS mutation in SBW25 leads to enhanced expression of pqq genes, resulting in an increase in gluconic and 2-ketogluconic acid production, which in turn acidified the extracellular medium to levels that inhibit growth of other microorganisms. We also showed that PQQ-mediated acidification comes with a growth penalty for the gacS mutant in the stationary phase. In conclusion, PQQ-mediated acidification compensates for the loss of several antimicrobial traits in P. fluorescens SBW25 and may help gac mutants to withstand competitors.
  • Frontiers in Microbiology

    Diversity and functions of volatile organic compounds produced by Streptomyces from a disease-suppressive soil

    Viviane Cordovez da Cunha, Victor Carrion Bravo, Desalegn Etalo, Roland Mumm, Hua Zhu, Gilles van Wezel, Jos M. Raaijmakers
    BACKGROUND: In disease-suppressive soils, plants are protected from infections by specific root pathogens due to the antagonistic activities of soil and rhizosphere microorganisms. For most disease-suppressive soils, however, the microorganisms and mechanisms involved in pathogen control are largely unknown. Our recent studies identified Actinobacteria as the most dynamic phylum in a soil suppressive to the fungal root pathogen Rhizoctonia solani. Here we isolated and characterized 300 isolates of rhizospheric Actinobacteria from the Rhizoctonia-suppressive soil. Streptomyces species were the most abundant, representing approximately 70% of the isolates. Streptomyces are renowned for the production of an exceptionally large number of secondary metabolites, including volatile organic compounds (VOCs). VOC profiling of 12 representative Streptomyces isolates by SPME-GC-MS allowed a more refined phylogenetic delineation of the Streptomyces isolates than the sequencing of 16S rRNA and the house-keeping genes atpD and recA only. VOCs of several Streptomyces isolates inhibited hyphal growth of R. solani and significantly enhanced plant shoot and root biomass. Coupling of Streptomyces VOC profiles with their effects on fungal growth, pointed to VOCs potentially involved in antifungal activity. Subsequent assays with five synthetic analogs of the identified VOCs showed that methyl 2-methylpentanoate, 1,3,5-trichloro-2-methoxy benzene and the VOCs mixture have antifungal activity. In conclusion, our results point to a potential role of VOC-producing Streptomyces in disease suppressive soils and show that VOC profiling of rhizospheric Streptomyces can be used as a complementary identification tool to construct strain-specific metabolic signatures.
  • ChemBioChem

    Investigations into the biosynthesis, regulation and self-resistance of toxoflavin in Pseudomonas protegens Pf-5.

    Benjamin Philmus, Brenda Shaffer, Teresa Kidarsa, Qing Yan, Jos M. Raaijmakers, Tadhg Begley, Joyce E. Loper
    Pseudomonas spp. are prolific producers of natural products from many structural classes. Here we show that the soil bacterium Pseudomonas protegens Pf-5 is capable of producing trace levels of the triazine natural product toxoflavin (1) under microaerobic conditions. We evaluated toxoflavin production by derivatives of Pf-5 having deletions in specific biosynthesis genes, which led us to propose a new biosynthetic pathway for toxoflavin that shares the first two steps with riboflavin biosynthesis. We also report that toxM, which is not present in the well-characterized cluster of Burkholderia glumae, encodes a monooxygenase that degrades toxoflavin. The toxoflavin degradation product of ToxM is identical to that of TflA, the toxoflavin lyase from Paenibacillus polymyxa. Toxoflavin production by P. protegens causes inhibition of several plant-pathogenic bacteria, and introduction of toxM into the toxoflavin-sensitive strain P. syringae DC3000 results in resistance to toxoflavin.
  • BMC Microbiology

    Lipopeptide biosynthesis in Pseudomonas fluorescens is regulated by the protease complex ClpAP

    Chunxu Song, Gustav Sundqvist, Erik Malm, Irene de Bruijn, Aundy Kumar, J. van de Mortel, Vincent Bulone, Jos M. Raaijmakers
  • ISME Journal

    Volatile affairs in microbial interactions

    Ruth Schmidt, Viviane Cordovez da Cunha, Wietse de Boer, Jos M. Raaijmakers, Paolina Garbeva
    Microorganisms are important factors in shaping our environment. One key characteristic that has been neglected for a long time is the ability of microorganisms to release chemically diverse volatile compounds. At present, it is clear that the blend of volatiles released by microorganisms can be very complex and often includes many unknown compounds for which the chemical structures remain to be elucidated. The biggest challenge now is to unravel the biological and ecological functions of these microbial volatiles. There is increasing evidence that microbial volatiles can act as infochemicals in interactions among microbes and between microbes and their eukaryotic hosts. Here, we review and discuss recent advances in understanding the natural roles of volatiles in microbe–microbe interactions. Specific emphasis will be given to the antimicrobial activities of microbial volatiles and their effects on bacterial quorum sensing, motility, gene expression and antibiotic resistance.
  • Frontiers in Microbiology

    Diversity and activity of Lysobacter species from disease suppressive soils

    Ruth Gomez Exposito, Joeke Postma, Jos M. Raaijmakers, Irene de Bruijn
    BACKGROUND: The genus Lysobacter includes several species that produce a range of extracellular enzymes and other metabolites with activity against bacteria, fungi, oomycetes and nematodes. Lysobacter species were found to be more abundant in soil suppressive against the fungal root pathogen Rhizoctonia solani, but their actual role in disease suppression is still unclear. Here, the antifungal and plant growth-promoting activities of 18 Lysobacter strains, including 11 strains from Rhizoctonia-suppressive soils, were studied both in vitro and in vivo. Based on 16S rRNA sequencing, the Lysobacter strains from the Rhizoctonia-suppressive soil belonged to the four species L. antibioticus, L. capsici, L. enzymogenes and L. gummosus. Most strains showed strong in vitro activity against R. solani and several other pathogens, including Pythium ultimum, Aspergillus niger, Fusarium oxysporum and Xanthomonas campestris. When the Lysobacter strains were introduced into soil, however, no significant and consistent suppression of R. solani damping-off disease of sugar beet and cauliflower was observed. Subsequent bioassays further revealed that none of the Lysobacter strains was able to promote growth of sugar beet, cauliflower, onion and Arabidopsis thaliana, either directly or via volatile compounds. The lack of in vivo activity is most likely attributed to poor colonization of the rhizosphere by the introduced Lysobacter strains. In conclusion, our results demonstrated that Lysobacter species have strong antagonistic activities against a range of pathogens, making them an important source for putative new enzymes and antimicrobial compounds. However, their potential role in R. solani disease suppressive soil could not be confirmed. In-depth omics’- based analyses will be needed to shed more light on the potential contribution of Lysobacter species to the collective activities of microbial consortia in disease suppressive soils.
  • Frontiers in Microbiology

    Genome mining and metabolic profiling of the rhizosphere bacterium Pseudomonas sp. SH-C52 for antimicrobial compounds

    Menno van der Voort, Harold J G Meijer, Yvonne Schmidt, Jeramie Watrous, Ester Dekkers, Rodrigo Mendes, Pieter C. Dorrestein, Harald Gross, Jos M. Raaijmakers
    BACKGROUND: The plant microbiome represents an enormous untapped resource for discovering novel genes and bioactive compounds. Previously, we isolated Pseudomonas sp. SH-C52 from the rhizosphere of sugar beet plants grown in a soil suppressive to the fungal pathogen Rhizoctonia solani and showed that its antifungal activity is, in part, attributed to the production of the chlorinated 9-amino-acid lipopeptide thanamycin (Mendes et al. 2011. Science). To get more insight into its biosynthetic repertoire, the genome of Pseudomonas sp. SH-C52 was sequenced and subjected to in silico, mutational and functional analyses. The sequencing revealed a genome size of 6.3 Mb and 5,579 predicted ORFs. Phylogenetic analysis placed strain SH-C52 within the Pseudomonas corrugata clade. In silico analysis for secondary metabolites revealed a total of six nonribosomal peptide synthetase (NRPS) gene clusters, including the two previously described NRPS clusters for thanamycin and the 2-amino acid antibacterial lipopeptide brabantamide. Here we show that thanamycin also has activity against an array of other fungi and that brabantamide A exhibits anti-oomycete activity and affects phospholipases of the late blight pathogen Phytophthora infestans. Most notably, mass spectrometry led to the discovery of a third LP, designated thanapeptin, with a 22-amino-acid peptide moiety. Seven structural variants of thanapeptin were found with varying degrees of activity against P. infestans. Of the remaining four NRPS clusters, one was predicted to encode for yet another and unknown lipopeptide with a predicted peptide moiety of 8-amino acids. Collectively, these results show an enormous metabolic potential for Pseudomonas sp. SH-C52, with at least three structurally diverse lipopeptides, each with a different antimicrobial activity spectrum.
  • Molecular Plant-Microbe Interactions

    The novel lipopeptide Poaeamide of the endophyte Pseudomonas poae RE*1-1-14 is involved in pathogen suppression and root colonization

    Christin Zachow, Jahanshah Ghazaleh, Irene de Bruijn, Chunxu Song, Federica Ianni, Zoltán Pataj, Heike Gerhardt, Isabelle Pianet, Michael Lämmerhofer, Gabriele Berg, Harald Gross, Jos M. Raaijmakers
    Endophytic Pseudomonas poae strain RE*1-1-14 was originally isolated from internal root tissue of sugar beet plants and shown to suppress growth of the fungal pathogen Rhizoctonia solani both in vitro and in the field. To identify genes involved in its biocontrol activity, RE*1-1-14 random mutagenesis and sequencing led to the identification of a nonribosomal peptide synthetase (NRPS) gene cluster predicted to encode a lipopeptide (LP) with a 10-amino acid peptide moiety. The two unlinked gene clusters consisted of three NRPS genes, designated poaA (cluster 1), and poaB and poaC (cluster 2), spanning approximately 33.7 kb. In silico analysis followed by chemical analyses revealed that the encoded LP designated poaeamide, is a structurally new member of the orfamide family. Poaeamide inhibited mycelial growth of R. solani and different oomycetes including Phytophthora capsici, Ph. infestans, and Pythium ultimum. The novel LP was shown to be essential for swarming motility of strain RE*1-1-14 and had an impact on root colonization of sugar beet seedlings The poaeamide-deficient mutant colonized the rhizosphere and upper plant cortex at higher densities and with more scattered colonization patterns than the wildtype. Collectively these results indicate that P. poae RE*1-1-14 produces a structurally new LP that is relevant for its antagonistic activity against soil-borne plant pathogens and for colonization of sugar beet roots.
  • 2015

    The minimal rhizosphere microbiome

    The rhizosphere provides a home to numerous (micro)organisms that in turn may affect plant growth, development, and tolerance to abiotic and biotic stresses. How plants shape the rhizosphere microbiome has been subject of many past and present studies with the ultimate goal to identify plant genetic traits that select and support beneficial microorganisms. Novel ‘omics technologies have provided more in-depth knowledge of the diversity and functioning of the rhizosphere microbiome and significant advances are being made to uncover mechanisms, genes and metabolites involved in the multitrophic interactions in the rhizosphere. To better understand this intriguing complexity, both reductionists’ and systems ecology approaches are needed to identify the biotic and abiotic factors involved in microbiome assembly. Here, different strategies are discussed to re-shape the rhizosphere microbiome in favour of microbial consortia that promote root development and plant growth, and that prevent the proliferation of pests and diseases.
  • Biochemical and Biophysical Research Communications

    Promotion of plant growth by Pseudomonas fluorescens strain SS101 via novel volatile organic compounds

    Yong-Soon Park, Swarnalee Dutta, Mina Ann, Jos M. Raaijmakers, Kyungseok Park
    Abstract Volatile organic compounds (VOCs) from plant growth-promoting rhizobacteria (PGPR) play key roles in modulating plant growth and induced systemic resistance (ISR) to pathogens. Despite their significance, the physiological functions of the specific VOCs produced by Pseudomonas fluorescens SS101 (Pf.SS101) have not been precisely elucidated. The effects of Pf.SS101 and its VOCs on augmentation of plant growth promotion were investigated in vitro and in planta. Significant growth promotion was observed in plants exposed Pf.SS101 under both conditions, suggesting its VOCs play a key role in promoting plant growth. Solid-phase micro-extraction (SPME) and a gas chromatography-mass spectrophotometer (GC–MS) system were used to characterize the VOCs emitted by Pf.SS101 and 11 different compounds were detected in samples inoculated this bacterium, including 13-Tetradecadien-1-ol, 2-butanone and 2-Methyl-n-1-tridecene. Application of these compounds resulted in enhanced plant growth. This study suggests that Pf.SS101 promotes the growth of plants via the release of VOCs including 13-Tetradecadien-1-ol, 2-butanone and 2-Methyl-n-1-tridecene, thus increasing understanding of the role of VOCs in plant-bacterial inter-communication.
  • Environmental Microbiology

    Biosynthetic genes and activity spectrum of antifungal polyynes from Collimonas fungivorans Ter331

    K. Fritsche, M. Van den Berg, Wietse de Boer, T.A. Van Beek, Jos M. Raaijmakers, Hans van Veen, Johan Leveau
    The antifungal activity of bacteria from the genus Collimonas has been well documented, but the chemistry and gene functions that underlie this phenotype are still poorly understood. Screening of a random plasposon insertion library of Collimonas fungivorans Ter331 for loss-of-function mutants revealed the importance of gene cluster K, which is annotated to code for the biosynthesis of a secondary metabolite and which features genes for fatty acid desaturases and polyketide synthases. Mutants in gene cluster K had lost the ability to inhibit hyphal growth of the fungus Aspergillus niger and were no longer able to produce and secrete several metabolites that after extraction and partial purification from wildtype strain Ter331 were shown to share a putative ene-triyne moiety. Some but not all of these metabolites were able to inhibit growth of A. niger, indicating functional variation within this group of Collimonas-produced polyyne-like ‘collimomycins’. Polymerase chain reaction analysis of isolates representing different Collimonas species indicated that the possession of cluster K genes correlated positively with antifungal ability, further strengthening the notion that this cluster is involved in collimomycin production. We discuss our findings in the context of other bacterially produced polyynes and the potential use of collimomycins for the control of harmful fungi.
  • ChemBioChem

    Biosynthetic Origin of the Antibiotic Cyclocarbamate Brabantamide A (SB-253514) in Plant-Associated Pseudomonas

    Y. Schmidt, Menno van der Voort, M. Crüsemann, J. Piel, M. Josten, H.-G. Sahl, H. Miess, Jos M. Raaijmakers, H. Gross
    Within the framework of our genome-based program to discover new antibiotic lipopeptides from Pseudomonads, brabantamides A–C were isolated from plant-associated Pseudomonas sp. SH-C52. Brabantamides A–C displayed moderate to high in vitro activities against Gram-positive bacterial pathogens. Their shared structure is unique in that they contain a 5,5-bicyclic carbamate scaffold. Here, the biosynthesis of brabantamide A (SB-253514) was studied by a combination of bioinformatics, feeding experiments with isotopically labelled precursors and in vivo and in vitro functional analysis of enzymes encoded in the biosynthetic pathway. The studies resulted in the deduction of all biosynthetic building blocks of brabantamide A and revealed an unusual feature of this metabolite: its biosynthesis occurs via an initially formed linear di-lipopeptide that is subsequently rearranged by a novel FAD-dependent Baeyer–Villiger monooxygenase. [KEYWORDS: biosynthesis carbamates monooxygenases peptides Pseudomonas SB-253514]
  • PLoS One

    Unravelling the Microbiome of Eggs of the Endangered Sea Turtle Eretmochelys imbricata Identifies Bacteria with Activity against the Emerging Pathogen Fusarium falciforme

    J.M. Sarmiento-Ramirez, Menno van der Voort, Jos M. Raaijmakers, J. Dieguez-Uribeondo
    Habitat bioaugmentation and introduction of protective microbiota have been proposed as potential conservation strategies to rescue endangered mammals and amphibians from emerging diseases. For both strategies, insight into the microbiomes of the endangered species and their habitats is essential. Here, we sampled nests of the endangered sea turtle species Eretmochelys imbricata that were infected with the fungal pathogen Fusarium falciforme. Metagenomic analysis of the bacterial communities associated with the shells of the sea turtle eggs revealed approximately 16,664 operational taxonomic units, with Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes as the most dominant phyla. Subsequent isolation of Actinobacteria from the eggshells led to the identification of several genera ( Streptomyces, Amycolaptosis, Micromomospora Plantactinospora and Solwaraspora) that inhibit hyphal growth of the pathogen F. falciforme. These bacterial genera constitute a first set of microbial indicators to evaluate the potential role of microbiota in conservation of endangered sea turtle species.
  • ISME Journal

    Deciphering microbial landscapes of fish eggs to mitigate emerging diseases

    Yiying Liu, Irene de Bruijn, A. Jack, K. Drynan, A.H. van den Berg, E. Thoen, V. Sandoval-Sierra, I. Skaar, P. van West, J. Diéguez-Uribeondo, Menno van der Voort, R. Mendes, M. Mazzola, Jos M. Raaijmakers
    Animals and plants are increasingly suffering from diseases caused by fungi and oomycetes. These emerging pathogens are now recognized as a global threat to biodiversity and food security. Among oomycetes, Saprolegnia species cause significant declines in fish and amphibian populations. Fish eggs have an immature adaptive immune system and depend on nonspecific innate defences to ward off pathogens. Here, meta-taxonomic analyses revealed that Atlantic salmon eggs are home to diverse fungal, oomycete and bacterial communities. Although virulent Saprolegnia isolates were found in all salmon egg samples, a low incidence of Saprolegniosis was strongly correlated with a high richness and abundance of specific commensal Actinobacteria, with the genus Frondihabitans (Microbacteriaceae) effectively inhibiting attachment of Saprolegniato salmon eggs. These results highlight that fundamental insights into microbial landscapes of fish eggs may provide new sustainable means to mitigate emerging diseases. The ISME Journal advance online publication, 27 March 2014; doi:10.1038/ismej.2014.44 Subject Category: Microbe-microbe and microbe-host interactions Keywords: salmon; Saprolegniosis; Actinobacteria; microbiome; emerging pathogens
  • BMC Microbiology

    Mangotoxin production of Pseudomonas syringae pv. syringae is regulated by MgoA

    Victor Carrion Bravo, Menno van der Voort, E. Arrebola, J.A. Gurierrez-Barranquero, A. de Vicente, Jos M. Raaijmakers, Francisco M. Cazorla
  • FEMS Microbiology Letters

    Discovery of new regulatory genes of lipopeptide biosynthesis in Pseudomonas fluorescens

    Chunxu Song, K. Aundy, J. van de Mortel, Jos M. Raaijmakers
    Pseudomonas fluorescens SS101 produces the cyclic lipopeptide massetolide with diverse functions in antimicrobial activity, motility, and biofilm formation. To understand how massetolide biosynthesis is genetically regulated in SS101, c. 8000 random plasposon mutants were screened for reduced or loss of massetolide production. Of a total of 58 putative mutants, 45 had a mutation in one of the three massetolide biosynthesis genes massA, massB, or massC. For five mutants, the insertions were located in the known regulatory genes gacS, gacA, and clpP. For the remaining eight mutants, insertions were located in clpA, encoding the ClpP chaperone, in phgdh, encoding D-3-phosphoglycerate dehydrogenase, in the heat shock protein-encoding dnaK, or in the transmembrane regulatory gene prtR. Genetic, chemical, and phenotypic analyses showed that phgdh, dnaK, and prtR are indeed involved in the regulation of massetolide biosynthesis, most likely by transcriptional repression of the LuxR-type regulator genes massAR and massBCR. In addition to their role in massetolide biosynthesis, dnaK and prtR were found to affect siderophore and extracellular protease(s) production, respectively. The identification of new regulatory genes substantially extended insights into the signal transduction pathways of lipopeptide biosynthesis in P. fluorescens and into regulation of other traits that may contribute to its life-style in the rhizosphere.
  • Environmental Microbiology Reports

    The Gac regulon of Pseudomonas fluorescensSBW25

    Xu Cheng, Irene de Bruijn, Menno van der Voort, Joyce E. Loper, Jos M. Raaijmakers

    Transcriptome analysis of Pseudomonas fluorescensSBW25 showed that 702 genes were differentially regulated in a gacS::Tn5 mutant, with 300 and 402 genes up- and downregulated respectively. Similar to the Gac regulon of other Pseudomonas species, genes involved in motility, biofilm formation, siderophore biosynthesis and oxidative stress were differentially regulated in the gacS mutant of SBW25. Our analysis also revealed, for the first time, that transcription of 19 rhizosphere-induced genes and of genes involved in type II secretion, (exo)polysaccharide and pectate lyase biosynthesis, twitching motility and an orphan non-ribosomal peptide synthetase (NRPS) were significantly affected in the gacS mutant. Furthermore, the gacS mutant inhibited growth of oomycete, fungal and bacterial pathogens significantly more than wild type SBW25. Since RP-HPLC analysis did not reveal any potential candidate metabolites, we focused on the Gac-regulated orphan NRPS gene cluster that was predicted to encode an eight-amino-acid ornicorrugatin-like peptide. Site-directed mutagenesis indicated that the encoded peptide is not involved in the enhanced antimicrobial activity of the gacS mutant but may function as a siderophore. Collectively, this genome-wide analysis revealed that a mutation in the GacS/A two-component regulatory system causes major transcriptional changes in SBW25 and significantly enhances its antimicrobial activities by yet unknown mechanisms.
  • Nature Reviews Microbiology

    Going back to the roots: the microbial ecology of the rhizosphere

    L. Philippot, Jos M. Raaijmakers, P. Lemanceau, Wim H. van der Putten
    The rhizosphere is the interface between plant roots and soil where interactions among a myriad of microorganisms and invertebrates affect biogeochemical cycling, plant growth and tolerance to biotic and abiotic stress. The rhizosphere is intriguingly complex and dynamic, and understanding its ecology and evolution is key to enhancing plant productivity and ecosystem functioning. Novel insights into key factors and evolutionary processes shaping the rhizosphere microbiome will greatly benefit from integrating reductionist and systems-based approaches in both agricultural and natural ecosystems. Here, we discuss recent developments in rhizosphere research in relation to assessing the contribution of the micro- and macroflora to sustainable agriculture, nature conservation, the development of bio-energy crops and the mitigation of climate change.
  • FEMS Microbiology Reviews

    The rhizosphere microbiome: significance of plant beneficial, plant pathogenic, and human pathogenic microorganisms

    Microbial communities play a pivotal role in the functioning of plants by influencing their physiology and development. While many members of the rhizosphere microbiome are beneficial to plant growth, also plant pathogenic microorganisms colonize the rhizosphere striving to break through the protective microbial shield and to overcome the innate plant defense mechanisms in order to cause disease. A third group of microorganisms that can be found in the rhizosphere are the true and opportunistic human pathogenic bacteria, which can be carried on or in plant tissue and may cause disease when introduced into debilitated humans. Although the importance of the rhizosphere microbiome for plant growth has been widely recognized, for the vast majority of rhizosphere microorganisms no knowledge exists. To enhance plant growth and health, it is essential to know which microorganism is present in the rhizosphere microbiome and what they are doing. Here, we review the main functions of rhizosphere microorganisms and how they impact on health and disease. We discuss the mechanisms involved in the multitrophic interactions and chemical dialogues that occur in the rhizosphere. Finally, we highlight several strategies to redirect or reshape the rhizosphere microbiome in favor of microorganisms that are beneficial to plant growth and health.
  • Science

    Deciphering the rhizosphere microbiome for disease-suppressive bacteria

    R. Mendes, M. Kruijt, Irene de Bruijn, E. Dekkers, Menno van der Voort, J.H.M. Schneider, Y.M. Piceno, Todd Z. DeSantis, Gary L. Andersen, Peter A.H.M. Bakker, Jos M. Raaijmakers
  • ISME Journal

    Transcriptional and antagonistic responses of Pseudomonas fluorescens Pf0-1 to phylogenetically different bacterial competitors

    The ability of soil bacteria to successfully compete with a range of other microbial species is crucial for their growth and survival in the nutrient-limited soil environment. In the present work, we studied the behavior and transcriptional responses of soil-inhabiting Pseudomonas fluorescens strain Pf0-1 on nutrient-poor agar to confrontation with strains of three phylogenetically different bacterial genera, that is, Bacillus, Brevundimonas and Pedobacter. Competition for nutrients was apparent as all three bacterial genera had a negative effect on the density of P. fluorescens Pf0-1; this effect was most strong during the interaction with Bacillus. Microarray-based analyses indicated strong differences in the transcriptional responses of Pf0-1 to the different competitors. There was higher similarity in the gene expression response of P. fluorescens Pf0-1 to the Gram-negative bacteria as compared with the Gram-positive strain. The Gram-negative strains did also trigger the production of an unknown broad-spectrum antibiotic in Pf0-1. More detailed analysis indicated that expression of specific Pf0-1 genes involved in signal transduction and secondary metabolite production was strongly affected by the competitors’ identity, suggesting that Pf0-1 can distinguish among different competitors and fine-tune its competitive strategies. The results presented here demonstrate that P. fluorescens Pf0-1 shows a species-specific transcriptional and metabolic response to bacterial competitors and provide new leads in the identification of specific cues in bacteria–bacteria interactions and of novel competitive strategies, antimicrobial traits and genes.
  • Journal of Bacteriology

    Massetolide A biosynthesis in Pseudomonas fluorescens

    Irene de Bruijn, M.J.D. De Kock, P.T. de Waard, T.A. Van Beek, Jos M. Raaijmakers

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