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Department of Plant Pathology and Microbiology
The Robert H. Smith Faculty of Agriculture, Food & Environment
The Hebrew University of Jerusalem

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Rehovot 7610001 
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Publications

2021
Steinberg, S. ; Grinberg, M. ; Beitelman, M. ; Peixoto, J. ; Orevi, T. ; Kashtan, N. Two-way microscale interactions between immigrant bacteria and plant leaf microbiota as revealed by live imaging. ISME JOURNAL 2021, 15, 409-420.Abstract
The phyllosphere - the aerial parts of plants - is an important microbial habitat that is home to diverse microbial communities. The spatial organization of bacterial cells on leaf surfaces is non-random, and correlates with leaf microscopic features. Yet, the role of microscale interactions between bacterial cells therein is not well understood. Here, we ask how interactions between immigrant bacteria and resident microbiota affect the spatial organization of the combined community. By means of live imaging in a simplified in vitro system, we studied the spatial organization, at the micrometer scale, of the biocontrol agentPseudomonas fluorescensA506 and the plant pathogenP. syringaeB728a when introduced to pear and bean leaf microbiota (the corresponding native plants of these strains). We found significant co-localization of immigrant and resident microbial cells at distances of a few micrometers, for both strains. Interestingly, this co-localization was in part due to preferential attachment of microbiota cells near newly formedP. fluorescensaggregates. Our results indicate that two-way immigrant bacteria - resident microbiota interactions affect the microscale spatial organization of leaf microbiota, and possibly that of other surface-related microbial communities.
Beizman-Magen, Y. ; Grinberg, M. ; Orevi, T. ; Kashtan, N. Wet-dry cycles protect surface-colonizing bacteria from major antibiotic classes. ISME JOURNAL 2021.Abstract
Diverse antibiotic compounds are abundant in microbial habitats undergoing recurrent wet-dry cycles, such as soil, root and leaf surfaces, and the built environment. These antibiotics play a central role in microbial warfare and competition, thus affecting population dynamics and the composition of natural microbial communities. Yet, the impact of wet-dry cycles on bacterial response to antibiotics has been scarcely explored. Using the bacterium E. coli as a model organism, we show through a combination of experiments and computational modeling, that wet-dry cycles protect bacteria from beta-lactams. This is due to the combined effect of several mechanisms including tolerance induced by high salt concentrations and slow cell-growth, which are inherently associated with microscopic surface wetness-a hydration state typical to `dry' periods. Moreover, we find evidence for a cross-protection effect, where lethal doses of antibiotic considerably increase bacterial survival during the dry periods. This work focuses on beta-lactams, yet similar protection was observed for additional major antibiotic classes. Our findings shed new light on how we understand bacterial response to antibiotics, with broad implications for population dynamics, interspecies interactions, and the evolution of antibiotic resistance in vast terrestrial microbial habitats.
Tovi, N. ; Orevi, T. ; Grinberg, M. ; Kashtan, N. ; Hadar, Y. ; Minz, D. Pairwise Interactions of Three Related Pseudomonas Species in Plant Roots and Inert Surfaces. FRONTIERS IN MICROBIOLOGY 2021, 12.Abstract
Bacteria are social organisms that interact extensively within and between species while responding to external stimuli from their environments. Designing synthetic microbial communities can enable efficient and beneficial microbiome implementation in many areas. However, in order to design an efficient community, one must consider the interactions between their members. Using a reductionist approach, we examined pairwise interactions of three related Pseudomonas species in various microenvironments including plant roots and inert surfaces. Our results show that the step between monoculture and co-culture is already very complex. Monoculture root colonization patterns demonstrate that each isolate occupied a particular location on wheat roots, such as root tip, distance from the tip, or scattered along the root. However, pairwise colonization outcomes on the root did not follow the bacterial behavior in monoculture, suggesting various interaction patterns. In addition, we show that interspecies interactions on a microscale on inert surface take part in co-culture colonization and that the interactions are affected by the presence of root extracts and depend on its source. The understanding of interrelationships on the root may contribute to future attempts to manipulate and improve bacterial colonization and to intervene with root microbiomes to construct and design effective synthetic microbial consortia.
Andreopoulos, W. B. ; Geller, A. M. ; Lucke, M. ; Balewski, J. ; Clum, A. ; Ivanova, N. N. ; Levy, A. Deeplasmid: deep learning accurately separates plasmids from bacterial chromosomes. Nucleic Acids Res 2021, gkab1115. Publisher's VersionAbstract
Plasmids are mobile genetic elements that play a key role in microbial ecology and evolution by mediating horizontal transfer of important genes, such as antimicrobial resistance genes. Many microbial genomes have been sequenced by short read sequencers and have resulted in a mix of contigs that derive from plasmids or chromosomes. New tools that accurately identify plasmids are needed to elucidate new plasmid-borne genes of high biological importance. We have developed Deeplasmid, a deep learning tool for distinguishing plasmids from bacterial chromosomes based on the DNA sequence and its encoded biological data. It requires as input only assembled sequences generated by any sequencing platform and assembly algorithm and its runtime scales linearly with the number of assembled sequences. Deeplasmid achieves an AUC–ROC of over 89%, and it was more accurate than five other plasmid classification methods. Finally, as a proof of concept, we used Deeplasmid to predict new plasmids in the fish pathogen Yersinia ruckeri ATCC 29473 that has no annotated plasmids. Deeplasmid predicted with high reliability that a long assembled contig is part of a plasmid. Using long read sequencing we indeed validated the existence of a 102 kb long plasmid, demonstrating Deeplasmid's ability to detect novel plasmids.
Geller, A. M. ; Pollin, I. ; Zlotkin, D. ; Danov, A. ; Nachmias, N. ; Andreopoulos, W. B. ; Shemesh, K. ; Levy, A. The extracellular contractile injection system is enriched in environmental microbes and associates with numerous toxins. 2021, 12, 3743. Publisher's VersionAbstract
The extracellular Contractile Injection System (eCIS) is a toxin-delivery particle that evolved from a bacteriophage tail. Four eCISs have previously been shown to mediate interactions between bacteria and their invertebrate hosts. Here, we identify eCIS loci in 1,249 bacterial and archaeal genomes and reveal an enrichment of these loci in environmental microbes and their apparent absence from mammalian pathogens. We show that 13 eCIS-associated toxin genes from diverse microbes can inhibit the growth of bacteria and/or yeast. We identify immunity genes that protect bacteria from self-intoxication, further supporting an antibacterial role for some eCISs. We also identify previously undescribed eCIS core genes, including a conserved eCIS transcriptional regulator. Finally, we present our data through an extensive eCIS repository, termed eCIStem. Our findings support eCIS as a toxin-delivery system that is widespread among environmental prokaryotes and likely mediates antagonistic interactions with eukaryotes and other prokaryotes.
Meroz, N. ; Tovi, N. ; Sorokin, Y. ; Friedman, J. Community composition of microbial microcosms follows simple assembly rules at evolutionary timescales. 2021, 12, 2891. Publisher's VersionAbstract
Managing and engineering microbial communities relies on the ability to predict their composition. While progress has been made on predicting compositions on short, ecological timescales, there is still little work aimed at predicting compositions on evolutionary timescales. Therefore, it is still unknown for how long communities typically remain stable after reaching ecological equilibrium, and how repeatable and predictable are changes when they occur. Here, we address this knowledge gap by tracking the composition of 87 two- and three-species bacterial communities, with 3–18 replicates each, for ~400 generations. We find that community composition typically changed during evolution, but that the composition of replicate communities remained similar. Furthermore, these changes were predictable in a bottom-up approach—changes in the composition of trios were consistent with those that occurred in pairs during coevolution. Our results demonstrate that simple assembly rules can hold even on evolutionary timescales, suggesting it may be possible to forecast the evolution of microbial communities.
2020
Tian, L. ; Wang, X. - W. ; Wu, A. - K. ; Fan, Y. ; Friedman, J. ; Dahlin, A. ; Waldor, M. K. ; Weinstock, G. M. ; Weiss, S. T. ; Liu, Y. - Y. Deciphering functional redundancy in the human microbiome. NATURE COMMUNICATIONS 2020, 11.Abstract
Although the taxonomic composition of the human microbiome varies tremendously across individuals, its gene composition or functional capacity is highly conserved - implying an ecological property known as functional redundancy. Such functional redundancy has been hypothesized to underlie the stability and resilience of the human microbiome, but this hypothesis has never been quantitatively tested. The origin of functional redundancy is still elusive. Here, we investigate the basis for functional redundancy in the human microbiome by analyzing its genomic content network - a bipartite graph that links microbes to the genes in their genomes. We find that this network exhibits several topological features that favor high functional redundancy. Furthermore, we develop a simple genome evolution model to generate genomic content network, finding that moderate selection pressure and high horizontal gene transfer rate are necessary to generate genomic content networks with key topological features that favor high functional redundancy. Finally, we analyze data from two published studies of fecal microbiota transplantation (FMT), finding that high functional redundancy of the recipient's pre-FMT microbiota raises barriers to donor microbiota engraftment. This work elucidates the potential ecological and evolutionary processes that create and maintain functional redundancy in the human microbiome and contribute to its resilience. Here, the authors develop a genome evolution model to investigate the origin of functional redundancy in the human microbiome by analyzing its genomic content network and illustrate potential ecological and evolutionary processes that may contribute to its resilience.
Marano, R. B. M. ; Fernandes, T. ; Manaia, C. M. ; Nunes, O. ; Morrison, D. ; Berendonk, T. U. ; Kreuzinger, N. ; Telson, T. ; Corno, G. ; Fatta-Kassinos, D. ; et al. A global multinational survey of cefotaxime-resistant coliforms in urban wastewater treatment plants. ENVIRONMENT INTERNATIONAL 2020, 144.Abstract
The World Health Organization Global Action Plan recommends integrated surveillance programs as crucial strategies for monitoring antibiotic resistance. Although several national surveillance programs are in place for clinical and veterinary settings, no such schemes exist for monitoring antibiotic-resistant bacteria in the environment. In this transnational study, we developed, validated, and tested a low-cost surveillance and easy to implement approach to evaluate antibiotic resistance in wastewater treatment plants (WWTPs) by targeting cefotaxime-resistant (CTX-R) coliforms as indicators. The rationale for this approach was: i) coliform quantification methods are internationally accepted as indicators of fecal contamination in recreational waters and are therefore routinely applied in analytical labs; ii) CTX-R coliforms are clinically relevant, associated with extended-spectrum beta-lactamases (ESBLs), and are rare in pristine environments. We analyzed 57 WWTPs in 22 countries across Europe, Asia, Africa, Australia, and North America. CTX-R coliforms were ubiquitous in raw sewage and their relative abundance varied significantly (< 0.1% to 38.3%), being positively correlated (p < 0.001) with regional atmospheric temperatures. Although most WWTPs removed large proportions of CTX-R coliforms, loads over 10(3) colony-forming units per mL were occasionally observed in final effluents. We demonstrate that CTX-R coliform monitoring is a feasible and affordable approach to assess wastewater antibiotic resistance status.
Alder-Rangel, A. ; Idnurm, A. ; Brand, A. C. ; Brown, A. J. P. ; Gorbushina, A. ; Kelliher, C. M. ; Campos, C. B. ; Levin, D. E. ; Bell-Pedersen, D. ; Dadachova, E. ; et al. The Third International Symposium on Fungal Stress - ISFUS. FUNGAL BIOLOGY 2020, 124, 235-252.Abstract
Stress is a normal part of life for fungi, which can survive in environments considered inhospitable or hostile for other organisms. Due to the ability of fungi to respond to, survive in, and transform the environment, even under severe stresses, many researchers are exploring the mechanisms that enable fungi to adapt to stress. The International Symposium on Fungal Stress (ISFUS) brings together leading scientists from around the world who research fungal stress. This article discusses presentations given at the third ISFUS, held in Sao Jose dos Campos, Sao Paulo, Brazil in 2019, thereby summarizing the state-of-the-art knowledge on fungal stress, a field that includes microbiology, agriculture, ecology, biotechnology, medicine, and astrobiology. (C) 2020 British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Levi-Zada, A. ; Levy, A. ; Rempoulakis, P. ; Fefer, D. ; Steiner, S. ; Gazit, Y. ; Nestel, D. ; Yuval, B. ; Byers, J. A. Diel rhythm of volatile emissions of males and females of the peach fruit fly Bactrocera zonata. JOURNAL OF INSECT PHYSIOLOGY 2020, 120.Abstract
Fruit flies in the genus Bactrocera are among the most destructive insect pests of fruits and vegetables throughout the world. A number of studies have identified volatiles from fruit flies, but few reports have demonstrated behavioral effects or sensitivities of fly antennae to these compounds. We applied a recently developed method of automated headspace analysis using SPME (Solid Phase Microextraction) fibers and GC MS (gas chromatography mass spectrometry), termed SSGA, to reveal volatiles specific to each sex of B. zonata that are emitted in a diel periodicity. The volatiles released primarily at dusk were identified by GC MS and chemical syntheses as several spiroacetals, pyrazines, and ethyl esters. Solvent extraction of male rectal glands or airborne collections from each sex, followed by GC MS, showed that certain of the volatiles increase or decrease in quantity sex-specifically with age of the flies. Electroantennographic (EAG) analysis of dose-response indicates differences in sensitivities of male and female antenna to the various volatiles. Our study provides a comprehensive analysis of the volatile chemicals produced and released by B. zonata and their antennal responses. The possible pheromone and semiochemical roles of the various volatiles released by each sex and the difficulties of establishing behavioral functions are discussed.
Joshi, J. R. ; Khazanov, N. ; Khadka, N. ; Charkowski, A. O. ; Burdman, S. ; Carmi, N. ; Yedidia, I. ; Senderowitz, H. Direct Binding of Salicylic Acid to Pectobacterium N-Acyl-Homoserine Lactone Synthase. ACS CHEMICAL BIOLOGY 2020, 15, 1883-1891.Abstract
Salicylic acid (SA) is a hormone that mediates systemic acquired resistance in plants. We demonstrated that SA can interfere with group behavior and virulence of the soft-rot plant pathogen Pectobacterium spp. through quorum sensing (QS) inhibition. QS is a population density-dependent communication system that relies on the signal molecule acyl-homoserine lactone (AHL) to synchronize infection. P. parmentieri mutants, lacking the QS AHL synthase (expI(-)) or the response regulator (expR(-)), were used to determine how SA inhibits QS. ExpI was expressed in DHS alpha, the QS negative strain of Escherichia coli, revealing direct interference of SA with AHL synthesis. Docking simulations showed SA is a potential ExpI ligand. This hypothesis was further confirmed by direct binding of SA to purified ExpI, shown by isothermal titration calorimetry and microscale thermophoresis. Computational alanine scanning was employed to design a mutant ExpI with predicted weaker binding affinity to SA. The mutant was constructed and displayed lower affinity to the ligand in the binding assay, and its physiological inhibition by SA was reduced. Taken together, these data support a likely mode of action and a role for SA as potent inhibitor of AHL synthase and QS.
Dror, B. ; Wang, Z. ; Brady, S. F. ; Jurkevitch, E. ; Cytryn, E. Elucidating the Diversity and Potential Function of Nonribosomal Peptide and Polyketide Biosynthetic Gene Clusters in the Root Microbiome. MSYSTEMS 2020, 5.Abstract
Polyketides (PKs) and nonribosomal peptides (NRPs) are two microbial secondary metabolite (SM) families known for their variety of functions, including antimicrobials, siderophores, and others. Despite their involvement in bacterium-bacterium and bacterium-plant interactions, root-associated SMs are largely unexplored due to the limited cultivability of bacteria. Here, we analyzed the diversity and expression of SM-encoding biosynthetic gene clusters (BGCs) in root microbiomes by culture-independent amplicon sequencing, shotgun metagenomics, and metatranscriptomics. Roots (tomato and lettuce) harbored distinct compositions of nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) relative to the adjacent bulk soil, and specific BGC markers were both enriched and highly expressed in the root microbiomes. While several of the highly abundant and expressed sequences were remotely associated with known BGCs, the low similarity to characterized genes suggests their potential novelty. Low-similarity genes were screened against a large set of soil-derived cosmid libraries, from which five whole BGCs of unknown function were retrieved. Three clusters were taxonomically affiliated with Actinobacteria, while the remaining were not associated with known bacteria. One Streptomyces-derived BGC was predicted to encode a polyene with potential antifungal activity, while the others were too novel to predict chemical structure. Screening against a suite of metagenomic data sets revealed higher abundances of retrieved clusters in roots and soil samples. In contrast, they were almost completely absent in aquatic and gut environments, supporting the notion that they might play an important role in root ecosystems. Overall, our results indicate that root microbiomes harbor a specific assemblage of undiscovered SMs. IMPORTANCE We identified distinct secondary-metabolite-encoding genes that are enriched (relative to adjacent bulk soil) and expressed in root ecosystems yet almost completely absent in human gut and aquatic environments. Several of the genes were distantly related to genes encoding antimicrobials and siderophores, and their high sequence variability relative to known sequences suggests that they may encode novel metabolites and may have unique ecological functions. This study demonstrates that plant roots harbor a diverse array of unique secondary-metabolite-encoding genes that are highly enriched and expressed in the root ecosystem. The secondary metabolites encoded by these genes might assist the bacteria that produce them in colonization and persistence in the root environment. To explore this hypothesis, future investigations should assess their potential role in interbacterial and bacterium-plant interactions.
Abreu, C. I. ; Woltz, V. L. A. ; Friedman, J. ; Gore, J. Microbial communities display alternative stable states in a fluctuating environment. PLOS COMPUTATIONAL BIOLOGY 2020, 16.Abstract
Author summary The effect of environmental fluctuations on community structure and function is a fundamental question in ecology. A significant body of work suggests that fluctuations increase diversity due to a variety of proposed mechanisms. In this study, we compare the effects of constant and fluctuating dilution regimes on simple microbial communities with two or three species. We find that in all cases, the outcome in a fluctuating environment is the same as that in a constant environment in which the fluctuations are time-averaged. This surprising result highlights that in some communities, ecological stable states may be predicted by averaging environmental parameters, rather than by the variation itself. The effect of environmental fluctuations is a major question in ecology. While it is widely accepted that fluctuations and other types of disturbances can increase biodiversity, there are fewer examples of other types of outcomes in a fluctuating environment. Here we explore this question with laboratory microcosms, using cocultures of two bacterial species, P. putida and P. veronii. At low dilution rates we observe competitive exclusion of P. veronii, whereas at high dilution rates we observe competitive exclusion of P. putida. When the dilution rate alternates between high and low, we do not observe coexistence between the species, but rather alternative stable states, in which only one species survives and initial species' fractions determine the identity of the surviving species. The Lotka-Volterra model with a fluctuating mortality rate predicts that this outcome is independent of the timing of the fluctuations, and that the time-averaged mortality would also lead to alternative stable states, a prediction that we confirm experimentally. Other pairs of species can coexist in a fluctuating environment, and again consistent with the model we observe coexistence in the time-averaged dilution rate. We find a similar time-averaging result holds in a three-species community, highlighting that simple linear models can in some cases provide powerful insight into how communities will respond to environmental fluctuations.
Prusky, D. ; de Assis, L. J. ; Baroncelli, R. ; Benito, E. P. ; Casado del Castillo, V. ; Chaya, T. ; Covo, S. ; Maria Diaz-Minguez, J. ; Donofrio, N. M. ; Espeso, E. ; et al. Nutritional factors modulating plant and fruit susceptibility to pathogens: BARD workshop, Haifa, Israel, February 25-26, 2018. PHYTOPARASITICA 2020, 48, 317-333.Abstract
The molecular dialog between fungal pathogens and their plant hosts is governed by signals from the plant, secreted pathogen effectors and enzymes, and the plant immune system. There is an increasing awareness that nutritional factors are also central to fungal-plant interactions. Nutritional factors include carbon and nitrogen metabolism, local pH and redox state, and manipulation of host metabolism by secreted pathogen effectors. A diverse combination of approaches from genetics, biochemistry and fungal and plant cell biology addresses these questions, and a workshop whose abstracts accompany this note was held in 2018 to bring these together. Questions were asked about how the lifestyles and nutritional strategies of eukaryotic filamentous phytopathogens are related to the metabolic architectures and pathogenic processes affecting both plant hosts and their pathogens. The aim for future work will be to provide metabolism-based strategies for pathogen control.
Zhao, M. ; Dutta, B. ; Luo, X. ; Burdman, S. ; Walcott, R. Genetically Distinct Acidovorax citrulli Strains Display Cucurbit Fruit Preference Under Field Conditions. PHYTOPATHOLOGY 2020, 110, 973-980.Abstract
Strains of Acidovorax citrulli, the causal agent of bacterial fruit blotch (BFB) of cucurbits, can be assigned to two groups, I and II. The natural association of group I and II strains with different cucurbit species suggests host preference; however, there are no direct data to support this hypothesis under field conditions. Hence, the objective of this study was to assess differences in the prevalence of group I and II A. citrulli strains on cucurbit species in the field. From 2017 to 2019, we used group I and II strains to initiate BFB outbreaks in field plots planted with four cucurbit species. At different times, we collected symptomatic tissues and assayed them for group I and II strains using a group-specific PCR assay. Binary distribution data analysis revealed that the odds of melon, pumpkin, and squash foliage infection by group I strains were 21.7, 11.5, and 22.1 times greater, respectively, than the odds of watermelon foliage infection by the group I strain (P < 0.0001). More strikingly, the odds of melon fruit infection by the group I strain were 97.5 times greater than watermelon fruit infection by the same strain (P < 0.0001). Unexpectedly, some of the group II isolates recovered from the 2017 and 2019 studies were different from the group II strains used as inocula. Overall, data from these experiments confirm that A. citrulli strains exhibit a preference for watermelon and melon, which is more pronounced in fruit tissues.
Mosquito, S. ; Meng, X. ; Devescovi, G. ; Bertani, I. ; Geller, A. M. ; Levy, A. ; Myers, M. P. ; Bez, C. ; Covaceuszach, S. ; Venturi, V. LuxR Solos in the Plant Endophyte Kosakonia sp. Strain KO348. APPLIED AND ENVIRONMENTAL MICROBIOLOGY 2020, 86.Abstract
Endophytes are microorganisms that live inside plants and are often beneficial for the host. Kosakonia is a novel bacterial genus that includes several species that are diazotrophic and plant associated. This study revealed two quorum sensing-related LuxR solos, designated LoxR and PsrR, in the plant endophyte Kosakonia sp. strain KO348. LoxR modeling and biochemical studies demonstrated that LoxR binds N-acyl homoserine lactones (AHLs) in a promiscuous way. PsrR, on the other hand, belongs to the subfamily of plant-associated-bacterium (PAB) LuxR solos that respond to plant compounds. Target promoter studies as well as modeling and phylogenetic comparisons suggest that PAB LuxR solos are likely to respond to different plant compounds. Finally, LoxR is involved in the regulation of T6SS and PsrR plays a role in root endosphere colonization. IMPORTANCE Cell-cell signaling in bacteria allows a synchronized and coordinated behavior of a microbial community. LuxR solos represent a subfamily of proteins in proteobacteria which most commonly detect and respond to signals produced exogenously by other microbes or eukaryotic hosts. Here, we report that a plant-beneficial bacterial endophyte belonging to the novel genus of Kosakonia possesses two LuxR solos; one is involved in the detection of exogenous N-acyl homoserine lactone quorum sensing signals and the other in detecting a compound(s) produced by the host plant. These two Kosakonia LuxR solos are therefore most likely involved in interspecies and interkingdom signaling.
Zolti, A. ; Green, S. J. ; Sela, N. ; Hadar, Y. ; Minz, D. The microbiome as a biosensor: functional profiles elucidate hidden stress in hosts. MICROBIOME 2020, 8.Abstract

Background

Microbial communities are highly responsive to environmental cues, and both their structure and activity can be altered in response to changing conditions. We hypothesized that host-associated microbial communities, particularly those colonizing host surfaces, can serve as in situ sensors to reveal environmental conditions experienced by both microorganisms and the host. For a proof-of-concept, we studied a model plant-soil system and employed a non-deterministic gene-centric approach. A holistic analysis was performed using plants of two species and irrigation with water of low quality to induce host stress. Our analyses examined the genetic potential (DNA) and gene expression patterns (RNA) of plant-associated microbial communities, as well as transcriptional profiling of host plants.

Results

Transcriptional analysis of plants irrigated with treated wastewater revealed significant enrichment of general stress-associated root transcripts relative to plants irrigated with fresh water. Metagenomic analysis of root-associated microbial communities in treated wastewater-irrigated plants, however, revealed enrichment of more specific stress-associated genes relating to high levels of salt, high pH and lower levels of oxygen. Meta-analysis of these differentially abundant genes obtained from other metagenome studies, provided evidence of the link between environmental factors such as pH and oxygen and these genes. Analysis of microbial transcriptional response demonstrated that enriched gene content was actively expressed, which implies contemporary response to elevated levels of pH and salt.

Conclusions

We demonstrate here that microbial profiling can elucidate stress signals that cannot be observed even through interrogation of host transcriptome, leading to an alternate mechanism for evaluating in situ conditions experienced by host organisms. This study is a proof-of-concept for the use of microbial communities as microsensors, with great potential for interrogation of a wide range of host systems.

Dror, B. ; Jurkevitch, E. ; Cytryn, E. State-of-the-art methodologies to identify antimicrobial secondary metabolites in soil bacterial communities-A review. SOIL BIOLOGY & BIOCHEMISTRY 2020, 147.Abstract
Non-ribosomal peptides (NRPs) and polyketides (PKs) are among the most profuse families of secondary metabolites (SM) produced by bacteria. These compounds are believed to play an important ecological role in microbe-microbe and microbe-plant interactions in soil and roots microbiomes. Over the years, screening of NRPs and PKs in soil bacteria has resulted in high rates of rediscovery, mainly due to challenges associated with bacterial isolation. The quest to expose compounds in the hidden ``unculturable'' fraction of the soil microbiome, and to activate existing and novel SM gene clusters in cultivated bacteria, has facilitated a paradigm shift from traditional isolation-based natural product identification platforms to novel `ecologically inspired' culturing techniques and cutting-edge culture-independent methods. This review provides a comprehensive overview of platforms and applications for studying secondary metabolites in soil and root environments, deliberating the benefits and limitations of the various approaches. Initially, it highlights innovative methodologies to ``culture the unculturable'' to uncover novel soil bacterial SM. Next, it explores ``culture-independent'' platforms for the identification of SM-synthesizing gene clusters through next generation sequencing and bioinformatics. It then evaluates innovative approaches for heterologous expression of metabolites from complex soil environments. Finally, it presents a conceptual integrated pipeline for evaluating the potential function and role of root-associated bacterial SM in suppressive soils that inhibit plant pathogens. This pipeline can be modified to address the ecological role of SM in other soil and root ecosystems, which can ultimately enhance our understanding of microbe-microbe and bacterial-plant interactions.
Chalupowicz, L. ; Reuven, M. ; Dror, O. ; Sela, N. ; Burdman, S. ; Manulis-Sasson, S. Characterization of Acidovorax citrulli strains isolated from solanaceous plants. PLANT PATHOLOGY 2020, 69, 1787-1797.Abstract
Acidovorax citrulli is the causal agent of bacterial fruit blotch disease of cucurbits. Strains of this pathogen are distributed into two major groups: Group I strains have been mainly isolated from melon and other non-watermelon cucurbits, while Group II strains have been mainly recovered from watermelon. Here we report the characterization of strains T1 and EP isolated from diseased tomato and eggplant plants, respectively, and further confirmed to belong to A. citrulli species. Based on PCR, PFGE, and rep-PCR, these strains showed high similarity to the Group II strain 7a1. Sequencing and comparative analyses revealed that the genomes of T1 and EP aligned with that of the Group II model strain AAC00-1, over 97.88% and 99.22%, respectively. The virulence of T1, EP, and 7a1 determined on tomato, eggplant, and watermelon was similar and significantly higher than that of Group I strain M6. In contrast, M6 was more virulent on melon. Expression levels of seven virulence genes measured 24 hr after inoculation of tomato, eggplant, watermelon, and melon showed that the expression pattern was generally similar in strains 7a1, T1, and EP, whereas for M6 the expression was high only on melon. Overall, our results indicate that the solanaceous strains belong to Group II. To the best of our knowledge, this is the first study that reports characterization of A. citrulli strains isolated from solanaceous species. The fact that A. citrulli is able to naturally colonize and cause disease in non-cucurbit crops poses additional challenges for management of this important pathogen.
Usyskin-Tonne, A. ; Hadar, Y. ; Yermiyahu, U. ; Minz, D. Elevated CO2 has a significant impact on denitrifying bacterial community in wheat roots. SOIL BIOLOGY & BIOCHEMISTRY 2020, 142.Abstract
Elevated CO2 (eCO(2)) stimulates plant growth and photosynthesis, which affect root deposition, leading to altered structure and function of the root microbiome. We studied the effect of eCO(2) on wheat-root microbiome composition and plant development, with an emphasis on denitrifying communities. Wheat plants were grown in a greenhouse with continuous fertigation for 6 weeks under ambient CO2 (400 ppm) or eCO(2) (850 ppm). The total bacterial community was quantified using qPCR with universal 16S rRNA gene primers, and denitrifying genes (nirK, nirS, nosZ) were measured. In addition, total (16S-based) and N2O-reducing (nosZ-based) bacterial community compositions in the soil and roots were analyzed by amplicon sequencing during plant growth. eCO(2) had a significant impact on abundance of the studied denitrifying genes, particularly during the late stages of wheat growth before spike formation. Moreover, eCO(2) had a significant impact on N2O-reducing community structure in roots. This effect was more pronounced on Burkholderiales and Rhizobiales, with a minor effect on Pseudomonadales. In addition, as expected, bacterial community structure (total and N2O-reducing bacteria), and denitrifying gene abundance, were primary influenced by habitat (soil vs. roots), and secondarily by plant developmental stage. In summary, it is suggested that eCO(2) may change root microbiome, enhance wheat development and N demand without an increase in N2O emission.