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The pesticide active ingredient azoxystrobin is widely used in agriculture and has negative effects for the environment and contained organisms. Bacterial strains have been reported to degrade azoxystrobin, but precise methodologies for producing and storing these strains as potential biotechnological products are lacking. The study focused on creating and optimising a non-sterile, small-scale microbial fermentation protocol to produce azoxystrobin-degrading products and to test their shelf life. By testing 14 variants and sampling at three production and two storage time points, the trial demonstrated the successful production and storage of microbial products capable of pesticide degradation. Various measurement parameters such as pH value and organic acids were used to monitor the quality of the microbial products during the production and storage. Further, we developed and validated qPCR assays to rapidly and specifically assess the concentration of the two azoxystrobin degrading strains, namely Bacillus subtilis strain MK101 and Rhodococcus fascians strain MK144. To ensure good specificity, the combination of two qPCR assays targeting two different genome regions was implemented for each strain. The study highlights the significant impact of media selection and bacterial inoculum quantity on the microbial product quality.

Abstract In recent years, microbiomes and their potential applications for human, animal or plant health, food production and environmental management came into the spotlight of major national and international policies and strategies. This has been accompanied by substantial R&D investments in both public and private sectors, with an increasing number of products entering the market. Despite widespread agreement on the potential of microbiomes and their uses across disciplines, stakeholders and countries, there is no consensus on what defines a microbiome application. This often results in non‐comprehensive communication or insufficient documentation making commercialisation and acceptance of the novel products challenging. To showcase the complexity of this issue we discuss two selected, well‐established applications and propose criteria defining a microbiome application and their conditions of use for clear communication, facilitating suitable regulatory frameworks and building trust among stakeholders.

The association of the plant microbiota is a successional process that starts with the seed and its intrinsic microbiota. The recently reported relevance of seeds as carriers of microbiota has encouraged investigations of the assembly of these communities in different tissues. Here, we address the contributions of both seed and soil bacterial microbiota in the assembly of communities within endospheres of adult plants by 16S ribosomal RNA gene-based Illumina sequencing. Surface-sterilized seeds of the model plant Setaria viridis were sown in sterile conditions and seedlings were transferred onto either their native soil or a foreign soil. Soil-derived bacterial colonizers contributed to the highest portion of endophytic microbiota, with varying community composition depending on the cultivation soil. In contrast, the contribution of seed microbiota in those of adult plants was less evident. Moreover, seedlings grown in their native soils resulted in plants with consistent endophytic assemblages, whereas a dramatic increase in variability was observed for rhizosphere and endophytic root communities of plants grown in foreign soils.

Improved understanding of the complex interaction between plant metabolism, environmental conditions and the plant-associated microbiome requires an interdisciplinary approach: Our hypothesis in our multiomics study posited that several environmental and biotic factors have modulating effects on the microbiome and metabolome of the roots of wild Echium vulgare plants. Furthermore, we postulated reciprocal interactions between the root metabolome and microbiome. We investigated the metabolic content, the genetic variability, and the prokaryotic microbiome in the root systems of wild E. vulgare plants at rosette and flowering stages across six distinct locations. We incorporated the assessment of soil microbiomes and the measurement of selected soil chemical composition factors. Two distinct genetic clusters were determined based on microsatellite analysis without a consistent alignment with the geographical proximity between the locations. The microbial diversity of both the roots of E. vulgare and the surrounding bulk soil exhibited significant divergence across locations, varying soil pH characteristics, and within the identified plant genetic clusters. Notably, acidophilic bacteria were characteristic inhabitants of both soil and roots under acidic soil conditions, emphasizing the close interconnectedness between these compartments. The metabolome of E. vulgare significantly differed between root samples from different developmental stages, geographical locations, and soil pH levels. The developmental stage was the dominant driver of metabolome changes, with significantly higher concentrations of sugars, pyrrolizidine alkaloids, and some of their precursors in rosette stage plant roots. Our study featured the complex dynamics between soil pH, plant development, geographical locations, plant genetics, plant metabolome and microbiome, shedding light on existing knowledge gaps.

Abstract Endophytes isolated from extremophile plants are interesting microbes for improving the stress tolerance of agricultural plants. Here, we isolated and characterized endophytic bacteria showing plant growth-promoting (PGP) traits from plants in two extreme Chilean biomes (Atacama Desert and Chilean Patagonia). Forty-two isolates were characterized as both halotolerant auxin producers (2–51 mg L−1) and 1-aminocyclopropane-1-carboxylate (ACC)-degrading bacteria (15–28 µmol αKB mg protein−1 h−1). The most efficient isolates were tested as single strains, in dual and triple consortia, or in combination with previously reported PGP rhizobacteria (Klebsiella sp. 27IJA and 8LJA) for their impact on the germination of salt-exposed (0.15 M and 0.25 M NaCl) wheat seeds. Interestingly, strain P1R9, identified as Variovorax sp., enhanced wheat germination under salt stress conditions when applied individually or as part of bacterial consortia. Under salt stress, plants inoculated with dual consortia containing the strain Variovorax sp. P1R9 showed higher biomass (41%) and reduced lipid peroxidation (33–56%) than uninoculated plants. Although the underlying mechanisms remain elusive, our data suggest that the application of Variovorax sp. P1R9, alone or as a member of PGP consortia, may improve the salt stress tolerance of wheat plants.

Executive summary Microbes are all pervasive in their distribution and influence on the functioning and well‐being of humans, life in general and the planet. Microbially‐based technologies contribute hugely to the supply of important goods and services we depend upon, such as the provision of food, medicines and clean water. They also offer mechanisms and strategies to mitigate and solve a wide range of problems and crises facing humanity at all levels, including those encapsulated in the sustainable development goals (SDGs) formulated by the United Nations. For example, microbial technologies can contribute in multiple ways to decarbonisation and hence confronting global warming, provide sanitation and clean water to the billions of people lacking them, improve soil fertility and hence food production and develop vaccines and other medicines to reduce and in some cases eliminate deadly infections. They are the foundation of biotechnology, an increasingly important and growing business sector and source of employment, and the centre of the bioeconomy, Green Deal, etc. But, because microbes are largely invisible, they are not familiar to most people, so opportunities they offer to effectively prevent and solve problems are often missed by decision‐makers, with the negative consequences this entrains. To correct this lack of vital knowledge, the International Microbiology Literacy Initiative–the IMiLI–is recruiting from the global microbiology community and making freely available, teaching resources for a curriculum in societally relevant microbiology that can be used at all levels of learning. Its goal is the development of a society that is literate in relevant microbiology and, as a consequence, able to take full advantage of the potential of microbes and minimise the consequences of their negative activities. In addition to teaching about microbes, almost every lesson discusses the influence they have on sustainability and the SDGs and their ability to solve pressing problems of societal inequalities. The curriculum thus teaches about sustainability, societal needs and global citizenship. The lessons also reveal the impacts microbes and their activities have on our daily lives at the personal, family, community, national and global levels and their relevance for decisions at all levels. And, because effective, evidence‐based decisions require not only relevant information but also critical and systems thinking, the resources also teach about these key generic aspects of deliberation. The IMiLI teaching resources are learner‐centric, not academic microbiology‐centric and deal with the microbiology of everyday issues. These span topics as diverse as owning and caring for a companion animal, the vast range of everyday foods that are produced via microbial processes, impressive geological formations created by microbes, childhood illnesses and how they are managed and how to reduce waste and pollution. They also leverage the exceptional excitement of exploration and discovery that typifies much progress in microbiology to capture the interest, inspire and motivate educators and learners alike. The IMiLI is establishing Regional Centres to translate the teaching resources into regional languages and adapt them to regional cultures, and to promote their use and assist educators employing them. Two of these are now operational. The Regional Centres constitute the interface between resource creators and educators–learners. As such, they will collect and analyse feedback from the end‐users and transmit this to the resource creators so that teaching materials can be improved and refined, and new resources added in response to demand: educators and learners will thereby be directly involved in evolution of the teaching resources. The interactions between educators–learners and resource creators mediated by the Regional Centres will establish dynamic and synergistic relationships–a global societally relevant microbiology education ecosystem–in which creators also become learners, teaching resources are optimised and all players/stakeholders are empowered and their motivation increased. The IMiLI concept thus embraces the principle of teaching societally relevant microbiology embedded in the wider context of societal, biosphere and planetary needs, inequalities, the range of crises that confront us and the need for improved decisioning, which should ultimately lead to better citizenship and a humanity that is more sustainable and resilient. Abstract The biosphere of planet Earth is a microbial world: a vast reactor of countless microbially driven chemical transformations and energy transfers that push and pull many planetary geochemical processes, including the cycling of the elements of life, mitigate or amplify climate change (e.g., Nature Reviews Microbiology, 2019, 17, 569) and impact the well‐being and activities of all organisms, including humans. Microbes are both our ancestors and creators of the planetary chemistry that allowed us to evolve (e.g., Life's engines: How microbes made earth habitable, 2023). To understand how the biosphere functions, how humans can influence its development and live more sustainably with the other organisms sharing it, we need to understand the microbes. In a recent editorial (Environmental Microbiology, 2019, 21, 1513), we advocated for improved microbiology literacy in society. Our concept of microbiology literacy is not based on knowledge of the academic subject of microbiology, with its multitude of component topics, plus the growing number of additional topics from other disciplines that become vitally important elements of current microbiology. Rather it is focused on microbial activities that impact us–individuals/communities/nations/the human world–and the biosphere and that are key to reaching informed decisions on a multitude of issues that regularly confront us, ranging from personal issues to crises of global importance. In other words, it is knowledge and understanding essential for adulthood and the transition to it, knowledge and understanding that must be acquired early in life in school. The 2019 Editorial marked the launch of the International Microbiology Literacy Initiative, the IMiLI. Here, we present our concept of how microbiology literacy may be achieved and the rationale underpinning it; the type of teaching resources being created to realise the concept and the framing of microbial activities treated in these resources in the context of sustainability, societal needs and responsibilities and decision‐making; and the key role of Regional Centres that will translate the teaching resources into local languages, adapt them according to local cultural needs, interface with regional educators and develop and serve as hubs of microbiology literacy education networks. The topics featuring in teaching resources are learner‐centric and have been selected for their inherent relevance, interest and ability to excite and engage. Importantly, the resources coherently integrate and emphasise the overarching issues of sustainability, stewardship and critical thinking and the pervasive interdependencies of processes. More broadly, the concept emphasises how the multifarious applications of microbial activities can be leveraged to promote human/animal, plant, environmental and planetary health, improve social equity, alleviate humanitarian deficits and causes of conflicts among peoples and increase understanding between peoples (Microbial Biotechnology, 2023, 16(6), 1091–1111). Importantly, although the primary target of the freely available (CC BY‐NC 4.0) IMiLI teaching resources is schoolchildren and their educators, they and the teaching philosophy are intended for all ages, abilities and cultural spectra of learners worldwide: in university education, lifelong learning, curiosity‐driven, web‐based knowledge acquisition and public outreach. The IMiLI teaching resources aim to promote development of a global microbiology education ecosystem that democratises microbiology knowledge.

Abstract Chile is a prominent seed exporter globally, but the seed microbiome of vegetables (46% of seeds) and its role in the early stages of plant growth have remained largely unexplored. Here, we employed DNA metabarcoding analysis to investigate the composition and putative functions of endophytic bacterial communities in ungerminated and germinated seeds of the commercial vegetables Apiaceae (parsley and carrot), Asteraceae (lettuce), Brassicaceae (cabbage and broccoli), and Solanaceae (tomato). Bacterial quantification showed 104 to 108 copies of the 16S rRNA gene per gram of ungerminated and germinated seeds. Alpha diversity analysis (e.g., Chao1, Shannon, and Simpson indices) did not indicate significant differences (Kruskal–Wallis test) between ungerminated and germinated seeds, except for Solanaceae. However, beta diversity (PCoA) analysis showed distinctions (Adonis test) between ungerminated and germinated seeds, except Apiaceae. Pseudomonadota and Bacillota were identified as the dominant and specialist taxa in both ungerminated and germinated seed samples. Chemoheterotrophy and fermentation were predicted as the main microbial functional groups in the endophytic bacterial community. Notably, a considerable number of the 143 isolated endophytic strains displayed plant growth-promoting traits (10 to 64%) and biocontrol activity (74% to 82%) against plant pathogens (Xanthomonas and Pseudomonas). This study revealed the high variability in the abundance, diversity, composition, and functionality of endophytic bacteria between ungerminated and germinated seeds in globally commercialized vegetables. Furthermore, potential beneficial endophytic bacteria contained in their seed microbiomes that may contribute to the microbiome of the early stages, development, growth and progeny of vegetables were found.

For a long time, microbial applications in crop production focused primarily on the symbiosis between mycorrhizal fungi and plants and the relationship between N2-fixing rhizobia and legumes. However, plant-associated microorganisms and microbiomes in general have lately gained immense attention, and the available information on the ecology and functions of microbiomes in the soil and plant environment exploded in the last decade. With this understanding, researchers as well as the industry have realized the vast potential of microorganisms to be used in agriculture and most importantly, to make farming more sustainable. Microorganisms have been shown to be applicable for plant protection and to improve plant nutrition. As a consequence, microbiome management can help to replace agrochemicals and increase environmental and human health. Furthermore, agriculture is facing various challenges related to climate change such as drought or emerging pathogens. Also, for these scenarios, microorganisms may find application to alleviate various abiotic and biotic stresses. [Graphic: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Abstract Within the European‐funded Coordination and Support Action MicrobiomeSupport (https://www.microbiomesupport.eu/), the Workshop ‘Education in Food Systems Microbiome Related Sciences: Needs for Universities, Industry and Public Health Systems’ brought together over 70 researchers, public health and industry partners from all over the world to work on elaborating microbiome‐related educational needs in food systems. This publication provides a summary of discussions held during and after the workshop and the resulting recommendations.

Abstract Background Due to climate change and reduced use of fertilizers combined stress scenarios are becoming increasingly frequent in crop production. In a field experiment we tested the effect of combined water and phosphorus limitation on the growth performance and plant traits of eight tetraploid and two diploid potato varieties as well as on root-associated microbiome diversity and functional potential. Microbiome and metagenome analysis targeted the diversity and potential functions of prokaryotes, fungi, plasmids, and bacteriophages and was linked to plant traits like tuber yield or timing of canopy closure. Results The different potato genotypes responded differently to the combined stress and hosted distinct microbiota in the rhizosphere and the root endosphere. Proximity to the root, stress and potato genotype had significant effects on bacteria, whereas fungi were only mildly affected. To address the involvement of microbial functions, we investigated well and poorly performing potato genotypes (Stirling and Desirée, respectively) under stress conditions and executed a metagenome analysis of rhizosphere microbiomes subjected to stress and no stress conditions. Functions like ROS detoxification, aromatic amino acid and terpene metabolism were enriched and in synchrony with the metabolism of stressed plants. In Desirée, Pseudonocardiales had the genetic potential to take up assimilates produced in the fast-growing canopy and to reduce plant stress-sensing by degrading ethylene, but overall yield losses were high. In Stirling, Xanthomonadales had the genetic potential to reduce oxidative stress and to produce biofilms, potentially around roots. Biofilm formation could be involved in drought resilience and nutrient accessibility of Stirling and explain the recorded low yield losses. In the rhizosphere exposed to combined stress, the relative abundance of plasmids was reduced, and the diversity of phages was enriched. Moreover, mobile elements like plasmids and phages were affected by combined stresses in a genotype-specific manner. Conclusion Our study gives new insights into the interconnectedness of root-associated microbiota and plant stress responses in the field. Functional genes in the metagenome, phylogenetic composition and mobile elements play a role in potato stress adaption. In a poor and a well performing potato genotype grown under stress conditions, distinct functional genes pinpoint to a distinct stress sensing, water availability and compounds in the rhizospheres.

Abstract The overarching biological impact of microbiomes on their hosts, and more generally their environment, reflects the co-evolution of a mutualistic symbiosis, generating fitness for both. Knowledge of microbiomes, their systemic role, interactions, and impact grows exponentially. When a research field of importance for planetary health evolves so rapidly, it is essential to consider it from an ethical holistic perspective. However, to date, the topic of microbiome ethics has received relatively little attention considering its importance. Here, ethical analysis of microbiome research, innovation, use, and potential impact is structured around the four cornerstone principles of ethics: Do Good; Don’t Harm; Respect; Act Justly. This simple, but not simplistic approach allows ethical issues to be communicative and operational. The essence of the paper is captured in a set of eleven microbiome ethics recommendations, e.g., proposing gut microbiome status as common global heritage, similar to the internationally agreed status of major food crops.

Winter wheat is an important cereal consumed worldwide. However, current management practices involving chemical fertilizers, irrigation, and intensive tillage may have negative impacts on the environment. Conservation agriculture is often presented as a sustainable alternative to maintain wheat production, favoring the beneficial microbiome. Here, we evaluated the impact of different water regimes (rainfed and irrigated), fertilization levels (half and full fertilization), and tillage practices (occasional tillage and no-tillage) on wheat performance, microbial activity, and rhizosphere- and root-associated microbial communities of four winter wheat genotypes (Antequera, Allez-y, Apache, and Cellule) grown in a field experiment. Wheat performance (i.e., yield, plant nitrogen concentrations, and total nitrogen uptake) was mainly affected by irrigation, fertilization, and genotype, whereas microbial activity (i.e., protease and alkaline phosphatase activities) was affected by irrigation. Amplicon sequencing data revealed that habitat (rhizosphere vs. root) was the main factor shaping microbial communities and confirmed that the selection of endophytic microbial communities takes place thanks to specific plant–microbiome interactions. Among the experimental factors applied, the interaction of irrigation and tillage influenced rhizosphere- and root-associated microbiomes. The findings presented in this work make it possible to link agricultural practices to microbial communities, paving the way for better monitoring of these microorganisms in the context of agroecosystem sustainability.

Abstract Background Tremendous amounts of data generated from microbiome research studies during the last decades require not only standards for sampling and preparation of omics data but also clear concepts of how the metadata is prepared to ensure re-use for integrative and interdisciplinary microbiome analysis. Results In this Commentary, we present our views on the key issues related to the current system for metadata submission in omics research, and propose the development of a global metadata system. Such a system should be easy to use, clearly structured in a hierarchical way, and should be compatible with all existing microbiome data repositories, following common standards for minimal required information and common ontology. Although minimum metadata requirements are essential for microbiome datasets, the immense technological progress requires a flexible system, which will have to be constantly improved and re-thought. While FAIR principles (Findable, Accessible, Interoperable, and Reusable) are already considered, international legal issues on genetic resource and sequence sharing provided by the Convention on Biological Diversity need more awareness and engagement of the scientific community. Conclusions The suggested approach for metadata entries would strongly improve retrieving and re-using data as demonstrated in several representative use cases. These integrative analyses, in turn, would further advance the potential of microbiome research for novel scientific discoveries and the development of microbiome-derived products.

In addition to challenges like climate change and biodiversity loss, the sustainability and resilience of agrifood systems worldwide are currently challenged by new threats, such as the COVID-19 pandemic and the Ukraine war. Furthermore, the resilience and sustainability of our agrifood systems need to be enhanced in ways that simultaneously increase agricultural production, decrease post-harvest food losses and food waste, protect the climate, environment and health, and preserve biodiversity. The precarious situation of agrifood systems is also illustrated by the fact that overall, around 3 billion people worldwide still do not have regular access to a healthy diet. This results in various forms of malnutrition, as well as increasing number of people suffering from overweight and obesity, and diet-related, non-communicable diseases (NCDs) around the world. Findings from microbiome research have shown that the human gut microbiome plays a key role in nutrition and diet-related diseases and thus human health. Furthermore, the microbiome of soils, plants, and animals play an equally important role in environmental health and agricultural production. Upcoming, microbiome-based solutions hold great potential for more resilient, sustainable, and productive agrifood systems and open avenues toward preventive health management. Microbiome-based solutions will also be key to make better use of natural resources and increase the resilience of agrifood systems to future emerging and already-known crises. To realize the promises of microbiome science and innovation, there is a need to invest in enhancing the role of microbiomes in agrifood systems in a holistic One Health approach and to accelerate knowledge translation and implementation.

ABSTRACT Plants are naturally associated with diverse microbial communities, which play significant roles in plant performance, such as growth promotion or fending off pathogens. The roots of Alkanna tinctoria L. are rich in naphthoquinones, particularly the medicinally used enantiomers alkannin and shikonin and their derivatives. Former studies already have shown that microorganisms may modulate plant metabolism. To further investigate the potential interaction between A. tinctoria and associated microorganisms, we performed a greenhouse experiment in which A. tinctoria plants were grown in the presence of three distinct soil microbiomes. At four defined plant developmental stages, we made an in-depth assessment of bacterial and fungal root-associated microbiomes as well as all extracted primary and secondary metabolite content of root material. Our results showed that the plant developmental stage was the most important driver influencing the plant metabolite content, revealing peak contents of alkannin/shikonin derivatives at the fruiting stage. Plant root microbial diversity was influenced both by bulk soil origin and to a small extent by the developmental stage. The performed correlation analyses and cooccurrence networks on the measured metabolite content and the abundance of individual bacterial and fungal taxa suggested a dynamic and at times positive or negative relationship between root-associated microorganisms and root metabolism. In particular, the bacterial genera Labrys and Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium as well as four species of the fungal genus Penicillium were found to be positively correlated with higher content of alkannins. IMPORTANCE Previous studies have shown that individual, isolated microorganisms may influence secondary metabolism of plants and induce or stimulate the production of medicinally relevant secondary metabolism. Here, we analyzed the microbiome-metabolome linkage of the medicinal plant Alkanna tinctoria, which is known to produce valuable compounds, particularly the naphthoquinones alkannin and shikonin and their derivatives. A detailed bacterial and fungal microbiome and metabolome analysis of A. tinctoria roots revealed that the plant developmental stage influenced root metabolite production, whereas soil inoculants from three different geographical origins in which plants were grown shaped root-associated microbiota. Metabolomes of plant roots of the same developmental stage across different soils were highly similar, pinpointing to plant maturity as the primary driver of secondary metabolite production. Correlation and network analyses identified bacterial and fungal taxa showing a positive relationship between root-associated microorganisms and root metabolism. In particular, the bacterial genera Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium and Labrys as well as the fungal species of genus Penicillium were found to be positively correlated with higher content of alkannins.

Increasing knowledge of the microbiome has led to significant advancements in the agrifood system. Case studies based on microbiome applications have been reported worldwide and, in this review, we have selected 14 success stories that showcase the importance of microbiome research in advancing the agrifood system. The selected case studies describe products, methodologies, applications, tools, and processes that created an economic and societal impact. Additionally, they cover a broad range of fields within the agrifood chain: the management of diseases and putative pathogens; the use of microorganism as soil fertilizers and plant strengtheners; the investigation of the microbial dynamics occurring during food fermentation; the presence of microorganisms and/or genes associated with hazards for animal and human health (e.g., mycotoxins, spoilage agents, or pathogens) in feeds, foods, and their processing environments; applications to improve HACCP systems; and the identification of novel probiotics and prebiotics to improve the animal gut microbiome or to prevent chronic non-communicable diseases in humans (e.g., obesity complications). The microbiomes of soil, plants, and animals are pivotal for ensuring human and environmental health and this review highlights the impact that microbiome applications have with this regard.

Abstract In the past, the potato plant microbiota and rhizosphere have been studied in detail to improve plant growth and fitness. However, less is known about the postharvest potato tuber microbiome and its role in storage stability. The storage stability of potatoes depends on genotype and storage conditions, but the soil in which tubers were grown could also play a role. To understand the ecology and functional role of the postharvest potato microbiota, we planted four potato varieties in five soil types and monitored them until the tubers started sprouting. During storage, the bacterial community of tubers was analysed by next-generation sequencing of the 16S rRNA gene amplicons. The potato tubers exhibited soil-dependent differences in sprouting behaviour. The statistical analysis revealed a strong shift of the tuber-associated bacterial community from harvest to dormancy break. By combining indicator species analysis and a correlation matrix, we predicted associations between members of the bacterial community and tuber sprouting behaviour. Based on this, we identified Flavobacterium sp. isolates, which were able to influence sprouting behaviour by inhibiting potato bud outgrowth.

Abstract The field of microbiome research has evolved rapidly over the past few decades and has become a topic of great scientific and public interest. As a result of this rapid growth in interest covering different fields, we are lacking a clear commonly agreed definition of the term “microbiome.” Moreover, a consensus on best practices in microbiome research is missing. Recently, a panel of international experts discussed the current gaps in the frame of the European-funded MicrobiomeSupport project. The meeting brought together about 40 leaders from diverse microbiome areas, while more than a hundred experts from all over the world took part in an online survey accompanying the workshop. This article excerpts the outcomes of the workshop and the corresponding online survey embedded in a short historical introduction and future outlook. We propose a definition of microbiome based on the compact, clear, and comprehensive description of the term provided by Whipps et al. in 1988, amended with a set of novel recommendations considering the latest technological developments and research findings. We clearly separate the terms microbiome and microbiota and provide a comprehensive discussion considering the composition of microbiota, the heterogeneity and dynamics of microbiomes in time and space, the stability and resilience of microbial networks, the definition of core microbiomes, and functionally relevant keystone species as well as co-evolutionary principles of microbe-host and inter-species interactions within the microbiome. These broad definitions together with the suggested unifying concepts will help to improve standardization of microbiome studies in the future, and could be the starting point for an integrated assessment of data resulting in a more rapid transfer of knowledge from basic science into practice. Furthermore, microbiome standards are important for solving new challenges associated with anthropogenic-driven changes in the field of planetary health, for which the understanding of microbiomes might play a key role. Video Abstract

Seed-associated bacteria represent an important reservoir of microorganisms passed onto progeny plants and have been postulated to be important for early plant development and early plant vigor. According to a few reports, some bacterial taxa seem to be transferred from seed to seed and some seed-associated microorganisms may derive from insect visits during flowering; however, the origin of seed endophytes is poorly understood. To better understand the origin, ecology, and functional role of seed bacterial endophytes, we planted Setaria viridis seeds over several generations in a sterile growth substrate. Seed microbiota of each generation were analyzed by next generation sequencing of 16S rRNA genes and seeds were characterized regarding to their germination and plant growth. Growing plants in a sterile (or highly depleted) substrate resulted in seed microbiota, which were largely less diverse and which had altered community composition, particularly at later generations, indicating that soil is an important reservoir of seed microbiota. Some taxa were inherited to the next generations seeds; however, different subsets of taxa were inherited in different seeds/seed batches and across different generations. This suggests that other factors than the host control the establishment of most seed endophytes and only few, e.g., obligate endophytes, might be consistently inherited. Furthermore, we observed a drastic decline in seed vigor and later generations were particularly affected. Overall, our results demonstrated that the supply of endophytes from external sources such as the soil/rhizosphere environment is highly important for the build-up of a healthy seed microbiome warranting early plant establishment and vigor of next generation plants.

Plants have evolved with a plethora of microorganisms having important roles for plant growth and health. A considerable amount of information is now available on the structure and dynamics of plant microbiota as well as on the functional capacities of isolated community members. Due to the interesting functional potential of plant microbiota as well as due to current challenges in crop production there is an urgent need to bring microbial innovations into practice. Different approaches for microbiome improvement exist. On the one hand microbial strains or strain combinations can be applied, however, field success is often variable and improvement is urgently required. Smart, knowledge-driven selection of microorganisms is needed as well as the use of suitable delivery approaches and formulations. On the other hand, farming practices or the plant genotype can influence plant microbiota and thus functioning. Therefore, selection of appropriate farming practices and plant breeding leading to improved plant-microbiome interactions are avenues to increase the benefit of plant microbiota. In conclusion, different avenues making use of a new generation of inoculants as well as the application of microbiome-based agro-management practices and improved plant lines could lead to a better use of the plant microbiome. This paper reviews the importance and functionalities of the bacterial plant microbiome and discusses challenges and concepts in regard to the application of plant-associated bacteria. Keywords: Plant microbiome, Composition, Functionalities, Inoculation, Farming practices, Plant microbiome modulation

Plant growth-promoting bacteria (PGPB) are promising alternatives in the reduction of the use of chemical fertilizers. Likewise, humic acid (HA) can improve plant growth and/or the establishment of endophytic PGPB. Although the effects of PGPB colonization or HA treatment have been studied separately, little information is available on plant response to the combined applications of PGPB and HA. Thus, the aim of this work was to understand the physiological effects, bacterial colonization and transcriptional responses activated by endophytic bacterial strains in tomato roots and shoots in the absence (control condition) and presence of HA (HA condition). Tomato shoot length was promoted by seed inoculation with Paraburkholderia phytofirmans PsJN, Pantoea agglomerans D7G, or Enterobacter sp. 32A in the presence of HA, indicating a possible complementation of PGPB and HA effects. Tomato colonization by endophytic bacterial strains was comparable in the control and HA condition. The main transcriptional regulations occurred in tomato roots and the majority of differentially expressed genes (DEGs) was upregulated by endophytic bacterial strains in the HA condition. Half of the DEGs was modulated by two or three strains as possible common reactions to endophytic bacterial strains, involving protein metabolism, transcription, transport, signal transduction, and defense. Moreover, strain-specific tomato responses included the upregulation of signal transduction, transcription, hormone metabolism, protein metabolism, secondary metabolism, and defense processes, highlighting specific traits of the endophyte-tomato interaction. The presence of HA enhanced the upregulation of genes related to signal transduction, hormone metabolism, transcription, protein metabolism, transport, defense, and growth-related processes in terms of number of involved genes and fold change values. This study provides detailed information on HA-dependent enhancement of growth-related processes stimulated by endophytic bacterial strains in tomato plants and reports the optimized dosages, complementation properties and gene markers for the further development of efficient PGPB- and HA-based biostimulants.

Microbacterium species have been isolated from a wide range of hosts and environments, including heavy metal-contaminated sites. Here, we present a comprehensive analysis on the phylogenetic distribution and the genetic potential of 70 Microbacterium belonging to 20 different species isolated from heavy metal-contaminated and non-contaminated sites with particular attention to secondary metabolites gene clusters. The analyzed Microbacterium species are divided in three main functional clades. They share a small core genome (331 gene families covering basic functions) pointing to high genetic diversity. The most common secondary metabolite gene clusters encode pathways for the production of terpenoids, type III polyketide synthases and non-ribosomal peptide synthetases, potentially responsible of the synthesis of siderophore-like compounds. In vitro tests showed that many Microbacterium strains produce siderophores, ACC deaminase, auxins (IAA) and are able to solubilize phosphate. Microbacterium isolates from heavy metal contaminated sites are on average more resistant to heavy metals and harbor more genes related to metal homeostasis (e.g., metalloregulators). On the other hand, the ability to increase the metal mobility in a contaminated soil through the secretion of specific molecules seems to be widespread among all. Despite the widespread capacity of strains to mobilize several metals, plants inoculated with selected Microbacterium isolates showed only slightly increased iron concentrations, whereas concentrations of zinc, cadmium and lead were decreased.

Serendipita (=Piriformospora) indica is a fungal endophytic symbiont with the capabilities to enhance plant growth and confer resistance to different stresses. However, the application of this fungus in the field has led to inconsistent results, perhaps due to antagonism with other microbes. Here, we studied the impact of individual bacterial isolates from the endophytic bacterial community on the in vitro growth of S. indica. We further analyzed how combinations of bacteria and S. indica influence plant growth and protection against the phytopathogens Fusarium oxysporum and Rhizoctonia solani. Bacterial strains of the genera Bacillus, Enterobacter and Burkholderia negatively affected S. indica growth on plates, whereas Mycolicibacterium, Rhizobium, Paenibacillus strains and several other bacteria from different taxa stimulated fungal growth. To further explore the potential of bacteria positively interacting with S. indica, four of the most promising strains belonging to the genus Mycolicibacterium were selected for further experiments. Some dual inoculations of S. indica and Mycolicibacterium strains boosted the beneficial effects triggered by S. indica, further enhancing the growth of tomato plants, and alleviating the symptoms caused by the phytopathogens F. oxysporum and R. solani. However, some combinations of S. indica and bacteria were less effective than individual inoculations. By analyzing the genomes of the Mycolicibacterium strains, we revealed that these bacteria encode several genes predicted to be involved in the stimulation of S. indica growth, plant development and tolerance to abiotic and biotic stresses. Particularly, a high number of genes related to vitamin and nitrogen metabolism were detected. Taking into consideration multiple interactions on and inside plants, we showed in this study that some bacterial strains may induce beneficial effects on S. indica and could have an outstanding influence on the plant-fungus symbiosis.

Beneficial insects and mites, including generalist predators of the family Miridae, are widely used in biocontrol programs against many crop pests, such as whiteflies, aphids, lepidopterans and mites. Mirid predators frequently complement their carnivore diet by feeding plant sap with their piercing–sucking mouthparts. This implies that mirids may act as vectors of phytopathogenic and beneficial microorganisms, such as plant growth-promoting bacterial endophytes. This work aimed at understanding the role of two beneficial mirids (Macrolophus pygmaeus and Nesidiocoris tenuis) in the acquisition and transmission of two plant growth-promoting bacteria, Paraburkholderia phytofirmans strain PsJN (PsJN) and Enterobacter sp. strain 32A (32A). Both bacterial strains were detected on the epicuticle and internal body of both mirids at the end of the mirid-mediated transmission. Moreover, both mirids were able to transmit PsJN and 32A between tomato plants and these bacterial strains could be re-isolated from tomato shoots after mirid-mediated transmission. In particular, PsJN and 32A endophytically colonised tomato plants and moved from the shoots to roots after mirid-mediated transmission. In conclusion, this study provided novel evidence for the acquisition and transmission of plant growth-promoting bacterial endophytes by beneficial mirids.

Abstract Potato (Solanum tuberosum) is an important staple crop worldwide, it has been cultivated in the Andean Altiplano under low-input farming practices at high altitudes and under harsh environment for centuries. We analyzed secondary metabolite (SM) gene diversity encoded in the potato rhizosphere microbiome during plant growth at three distinct sites located in the Andes at high altitudes by 454-pyrosequencing of non-ribosomal peptide and polyketide biosynthetic genes. Phylogenetic analysis indicated that the majority of rhizosphere SM-encoding sequences differed from previously known sequences and may have distinct ancestors. In particular, actinobacterial methyl-malonyl-CoA transferase and acyl carrier protein from Firmicutes, both involved in the synthesis of SMs, showed widespread distribution of clades which were clearly distinct from sequences deposited in public databases, and only 11% of these sequences could be linked to the production of specific classes of SMs. Although the same cultivar was analyzed, SM gene composition radically differed among plant growth stages and across sites, suggesting a distinct repertoire of SM genes that likely encode diverse SM structures. Also, great diversity of non-ribosomal peptide and polyketide biosynthetic pathways in potato-associated microbiomes in the Andean highlands may represent a rich source of novel natural products.

Strong efforts have been made to understand the bacterial communities in potato plants and the rhizosphere. Research has focused on the effect of the environment and plant genotype on bacterial community structures and dynamics, while little is known about the origin and assembly of the bacterial community, especially in potato tubers. The tuber microbiota, however, may be of special interest as it could play an important role in crop quality, such as storage stability. Here, we used 16S rRNA gene amplicon sequencing to study the bacterial communities that colonize tubers of different potato cultivars commonly used in Austrian potato production over three generations and grown in different soils. Statistical analysis of sequencing data showed that the bacterial community of potato tubers has changed over generations and has become more similar to the soil bacterial community, while the impact of the potato cultivar on the bacterial assemblage has lost significance over time. The communities in different tuber parts did not differ significantly, while the soil bacterial community showed significant differences to the tuber microbiota composition. Additionally, the presence of OTUs in subsequent tuber generation points to vertical transmission of a subset of the tuber microbiota. Four OTUs were common to all tuber generations and all potato varieties. In summary, we conclude that the microbiota of potato tubers is recruited from the soil largely independent from the plant variety. Furthermore, the bacterial assemblage in potato tubers consists of bacteria transmitted from one tuber generation to the next and bacteria recruited from the soil.

An amendment to this paper has been published and can be accessed via the original article.

Virtually all studied plant tissues are internally inhabited by endophytes. Due to their relevance for plant growth and health, bacterial microbiota of crop plants have been broadly studied. In plant microbiome research the root is the most frequently addressed environment, whereas the ecology of microbiota associated with reproductive organs still demands investigation. In this work, we chose the model grasses Setaria viridis and Setaria pumila to better understand the drivers shaping bacterial communities associated with panicles (representing a reproductive organ) as compared to those associated with roots. We collected wild individuals of both grass species from 20 different locations across Austria and investigated the bacterial assemblages within roots and ripe grain-harboring panicles by 16S rRNA gene-based Illumina sequencing. Furthermore, plant samples were subjected to genotyping by genetic diversity-focused Genotyping by Sequencing. Overall, roots hosted more diverse microbiota than panicles. Both the plant organ and sampling site significantly shaped the root and panicle-associated microbiota, whereas the host genotype only affected root communities. In terms of community structure, root-specific assemblages were highly diverse and consisted of conserved bacterial taxa. In contrast, panicle-specific communities were governed by Gammaproteobacteria, were less diverse and highly origin-dependent. Among OTUs found in both plant tissues, relative abundances of Gammaproteobacteria were higher in panicles, whereas Rhizobiales dominated root communities. We further identified core and non-core taxa within samples of both Setaria species. Non-core taxa included members of the Saccharibacteria and Legionelalles, while core communities encompassed eleven OTUs of seven bacterial orders, together with a set of ten panicle-enriched OTUs. These communities were widespread across root and panicle samples from all locations, hinting toward an evolved form of mutualism through potential vertical transmission of these taxa within Setaria species.

[This corrects the article DOI: 10.1371/journal.pone.0198107.].

Bacillus spp. produce a broad spectrum of lipopeptide biosurfactants, among which surfactin, iturin and fengycin are widely studied families. The goals of this study were to characterize the biosurfactant activity of Bacillus spp. and to investigate their motility and biofilm formation capabilities. In addition, we extracted lipopeptides from these bacteria to assess their antifungal activities and analyzed these products by mass spectrometry (MS). B. amyloliquefaciens FZB42, Bacillus sp. NH 217 and B. subtilis NH-100 exhibited excellent biosurfactant and surface spreading activities, whereas B. atrophaeus 176s and Paenibacillus polymyxa C1225 showed moderate activity, and B. subtilis 168 showed no activity. Strains FZB42, NH-100, NH-217, 176s and CC125 exhibited excellent biofilm formation capabilities. Lipopeptide extracts displayed good antifungal activity against various phytopathogens and their associated diseases, such as Fusarium moniliforme (rice bakanae disease), Fusarium oxysporum (root rot), Fusarium solani (root rot) and Trichoderma atroviride (ear rot and root rot). Lipopeptide extracts of these strains also showed hemolytic activity, demonstrating their strong potential to produce surfactants. LCMS-ESI analyses identified the presence of surfactin, iturin and fengycin in the extracts of Bacillus strains. Thus, the strains assayed in this study show potential as biocontrol agents against various Fusarium and Trichoderma species.

Bacillus and related genera in the Bacillales within the Firmicutes harbor a variety of secondary metabolite gene clusters encoding polyketide synthases and non-ribosomal peptide synthetases responsible for remarkable diverse number of polyketides (PKs) and lipopeptides (LPs). These compounds may be utilized for medical and agricultural applications. Here, we summarize the knowledge on structural diversity and underlying gene clusters of LPs and PKs in the Bacillales. Moreover, we evaluate by using published prediction tools the potential metabolic capacity of these bacteria to produce type I PKs or LPs. The huge sequence repository of bacterial genomes and metagenomes provides the basis for such genome-mining to reveal the potential for novel structurally diverse secondary metabolites. The otherwise cumbersome task to isolate often unstable PKs and deduce their structure can be streamlined. Using web based prediction tools, we identified here several novel clusters of PKs and LPs from genomes deposited in the database. Our analysis suggests that a substantial fraction of predicted LPs and type I PKs are uncharacterized, and their functions remain to be studied. Known and predicted LPs and PKs occurred in the majority of the plant associated genera, predominantly in Bacillus and Paenibacillus. Surprisingly, many genera from other environments contain no or few of such compounds indicating the role of these secondary metabolites in plant-associated niches.

The African pepper bark tree, Warburgia ugandensis, accumulates antimicrobial drimane sesquiterpenes in all of its organs. One hypothesis states that plant defense compounds determine endophyte community structure. Another hypothesis suggests that they just facilitate the endophytic lifestyle by exerting a balanced antagonism. To explore this, a representative selection of endophytic bacterial and fungal isolates from this tree species was assayed together with six non-endophytic strains to determine their tolerance and susceptibility to the root and leaf extract fraction containing high and low drimane sesquiterpene amounts respectively. Inhibitory effects were explored by assessing both growth and growth efficiency, the latter of which relates respiratory activity to growth. The susceptibility of the tested strains showed considerable variation and the obtained patterns did not allow a clear distinction between root and leaf endophytes as well as endophytes and non-endophytes. In addition, all strains were also assayed against juglone, an antimicrobial and redox-active aromatic naphthoquinone. A comparison of differential pulse voltammograms and efficacy in variants of the deoxyribose degradation assay revealed that drimane sesquiterpenes possess anti- and pro-oxidant activities that compare to those of juglone. Leaf endophytes showed higher resistance to oxidative stress than root endophytes, quite contrary to the actual exposure. The obtained results support the notion that structural diverse plant defense compounds can contribute to a balanced antagonism against but not to structuring of endophyte communities. Oxidative stress seems to be involved in generating this effect albeit it cannot explain it alone.

ABSTRACT It is widely accepted that bacterial endophytes actively colonize plants, interact with their host, and frequently show beneficial effects on plant growth and health. However, the mechanisms of plant-endophyte communication and bacterial adaption to the plant environment are still poorly understood. Here, whole-transcriptome sequencing of B. phytofirmans PsJN colonizing potato (Solanum tuberosum L.) plants was used to analyze in planta gene activity and the response of strain PsJN to plant stress. The transcriptome of PsJN colonizing in vitro potato plants showed a broad array of functionalities encoded in the genome of strain PsJN. Transcripts upregulated in response to plant drought stress were mainly involved in transcriptional regulation, cellular homeostasis, and the detoxification of reactive oxygen species, indicating an oxidative stress response in PsJN. Genes with modulated expression included genes for extracytoplasmatic function (ECF) group IV sigma factors. These cell surface signaling elements allow bacteria to sense changing environmental conditions and to adjust their metabolism accordingly. TaqMan quantitative PCR (TaqMan-qPCR) was performed to identify ECF sigma factors in PsJN that were activated in response to plant stress. Six ECF sigma factor genes were expressed in PsJN colonizing potato plants. The expression of one ECF sigma factor was upregulated whereas that of another one was downregulated in a plant genotype-specific manner when the plants were stressed. Collectively, our study results indicate that endophytic B. phytofirmans PsJN cells are active inside plants. Moreover, the activity of strain PsJN is affected by plant drought stress; it senses plant stress signals and adjusts its gene expression accordingly. IMPORTANCE In recent years, plant growth-promoting endophytes have received steadily growing interest as an inexpensive alternative to resource-consuming agrochemicals in sustainable agriculture. Even though promising effects are recurrently observed under controlled conditions, these are rarely reproducible in the field or show undesirably strong variations. Obviously, a better understanding of endophyte activities in plants and the influence of plant physiology on these activities is needed to develop more-successful application strategies. So far, research has focused mainly on analyzing the plant response to bacterial inoculants. This prompted us to study the gene expression of the endophyte Burkholderia phytofirmans PsJN in potato plants. We found that endophytic PsJN cells express a wide array of genes and pathways, pointing to high metabolic activity inside plants. Moreover, the strain senses changes in the plant physiology due to plant stress and adjusts its gene expression pattern to cope with and adapt to the altered conditions.

Heterotrophic plate counts (HPC) are routinely determined within the scope of water quality assessment. However, variable HPC methods with different cultivation parameters (i.e., temperature and media type) are applied, which could lead to significant effects in the outcome of the analysis. Therefore the effect of different HPC methods, according to DIN EN ISO 6222 and EPA, on the culturable microbial community composition was investigated by 16S rRNA gene sequence analysis and statistical evaluation was performed. The culturable community composition revealed significant effects assigned to temperature (p < 0.01), while for media type no statistical significance was observed. However, the abundance of certain detected bacteria was affected. Lower temperature (22 °C) showed the abundance of naturally occurring Pseudomonadaceae and Aeromonadaceae, whereas at high temperature (37 °C) numerous Enterobacteriaceae, Citrobacter spp. and Bacilli were identified. The highest biodiversity was detected at lower temperature, especially on R2A medium. These results indicate that different temperatures (low and high) should be included into HPC measurement and selection of media should, ideally, be adjusted to the monitored water source. Accordingly, it can be inferred that the HPC method is more suitable for continuous monitoring of the same water source than for single assessments of a water sample.

INTRODUCTION:Esca disease has become a major threat for viticulture. Phaeoacremonium aleophilum is considered a pioneer of the esca complex pathosystem, but its colonisation behaviour inside plants remains poorly investigated. MATERIAL AND METHODS:In this study, P. aleophilum::gfp7 colonisation was assessed six and twelve weeks post-inoculation in two different types of tissues: in the node and the internode of one year-old rooted cuttings of Cabernet Sauvignon. These processes of colonisation were compared with the colonisation by the wild-type strain using a non-specific lectin probe Alexa Fluor 488-WGA. RESULTS:Data showed that six weeks post-inoculation of the internode, the fungus had colonised the inoculation point, the bark and xylem fibres. Bark, pith and xylem fibres were strongly colonised by the fungus twelve weeks post-inoculation and it can progress up to 8 mm from the point of inoculation using pith, bark and fibres. P. aleophilum was additionally detected in the lumen of xylem vessels in which tyloses blocked its progression. Different plant responses in specific tissues were additionally visualised. Inoculation of nodes led to restricted colonisation of P. aleophilum and this colonisation was associated with a plant response six weeks post-inoculation. The fungus was however detected in xylem vessels, bark and inside the pith twelve weeks post-inoculation. CONCLUSIONS:These results demonstrate that P. aleophilum colonisation can vary according to the type of tissues and the type of spread using pith, bark and fibres. Woody tissues can respond to the injury and to the presence of this fungus, and xylem fibres play a key role in the early colonisation of the internode by P. aleophilum before the fungus can colonise xylem vessels.

Different regions of the bacterial 16S rRNA gene evolve at different evolutionary rates. The scientific outcome of short read sequencing studies therefore alters with the gene region sequenced. We wanted to gain insight in the impact of primer choice on the outcome of short read sequencing efforts. All the unknowns associated with sequencing data, i.e. primer coverage rate, phylogeny, OTU-richness and taxonomic assignment, were therefore implemented in one study for ten well established universal primers (338f/r, 518f/r, 799f/r, 926f/r and 1062f/r) targeting dispersed regions of the bacterial 16S rRNA gene. All analyses were performed on nearly full length and in silico generated short read sequence libraries containing 1175 sequences that were carefully chosen as to present a representative substitute of the SILVA SSU database. The 518f and 799r primers, targeting the V4 region of the 16S rRNA gene, were found to be particularly suited for short read sequencing studies, while the primer 1062r, targeting V6, seemed to be least reliable. Our results will assist scientists in considering whether the best option for their study is to select the most informative primer, or the primer that excludes interferences by host-organelle DNA. The methodology followed can be extrapolated to other primers, allowing their evaluation prior to the experiment.

Plant inoculation with endophytic bacteria that normally live inside the plant without harming the host is a highly promising approach for biological disease control. The mechanism of resistance induction by beneficial bacteria is poorly understood, because pathways are only partly known and systemic responses are typically not seen. The innate endophytic community structures change in response to external factors such as inoculation, and bacterial endophytes can exhibit direct or indirect antagonism towards pathogens. Earlier we showed that resistance induction by an endophytic Methylobacterium sp. in potato towards Pectobacterium atrosepticum was dependent on the density of the inoculum, whereas the bacterium itself had no antagonistic activity. To elucidate the role of innate endophyte communities in plant responses, we studied community changes in both in vitro and greenhouse experiments using various combinations of plants, endophyte inoculants, and pathogens. Induction of resistance was studied in several potato (Solanum tuberosum L.) cultivars by Methylobacterium sp. IMBG290 against the pathogens P. atrosepticum, Phytophthora infestans and Pseudomonas syringae pv. tomato DC3000, and in pine (Pinus sylvestris L.) by M. extorquens DSM13060 against Gremmeniella abietina. The capacities of the inoculated endophytic Methylobacterium spp. strains to induce resistance were dependent on the plant cultivar, pathogen, and on the density of Methylobacterium spp. inoculum. Composition of the endophyte community changed in response to inoculation in shoot tissues and correlated with resistance or susceptibility to the disease. Our results demonstrate that endophytic Methylobacterium spp. strains have varying effects on plant disease resistance, which can be modulated through the endophyte community of the host.

There are indications that the more frequent use of untreated organic residues for fertilization results in increased risk of contamination with human pathogens. Here, we evaluate the ability of two different strains of Campylobacter jejuni to persist in manure and soil as well as spread to spinach plants. It was revealed that different strategies for inoculation of C. jejuni contribute to the persistence of the bacterium in soil, roots, and shoots. Upon inoculation of the bacteria into manure prior to soil application, the amount of C. jejuni subsequently recovered in soil was higher than that from treatments involving the addition of C. jejuni cells to the soil after plant emergence. Irrespective of the bacterial inoculation dose and strategy employed, the C. jejuni content in soil remained relatively constant, whereas the majority of C. jejuni cells applied to spinach leaves could be recovered during the whole evaluation period of 21 days.