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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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2020
Topman-Rakover, S. ; Malach, E. ; Burdman, S. ; Hayouka, Z. Antibacterial lipo-random peptide mixtures exhibit high selectivity and synergistic interactions. CHEMICAL COMMUNICATIONS 2020, 56, 12053-12056.Abstract
Random peptide mixtures (RPMs) have been recently proposed as powerful antimicrobial compounds. These are unique mixtures of peptides synthesized by random combination of a cationic and a hydrophobic amino acid. Here, we introduce a new type of antimicrobial compounds, short lipo-RPMs, which result fromN-palmitoylation of RPMs. We report the characterization of 5-mer lipo-RPMs containingl-phenylalanine andd-lysine, named p-FdK5. p-FdK5 had high antibacterial activity against several bacterial strains and was able to reduce disease severity caused by a plant pathogen. We further synthesized and studied all 32 (2(5)) possible lipopeptides that compose the p-FdK5 mixture. We showed that the antibacterial activity of specific lipopeptides depends on the peptide hydrophobicity and on the location of the hydrophobic amino acids relative to the palmitic acid. Interestingly, synergism assays revealed positive interactions between different sequence-specific lipopeptides in terms of antimicrobial activity.
Covo, S. Genomic Instability in Fungal Plant Pathogens. GENES 2020, 11.Abstract
Fungi and fungal-like organisms (oomycetes) that cause diseases in plants have impacted human communities for centuries and probably from the dawn of agriculture. In modern agriculture, there is a constant race between new strategies to manage fungal plant pathogens and their ability to adapt. An important component in this race is fungal genetic diversity. Mechanisms such as sexual and parasexual recombination that contribute to the creation of novel allele combinations in fungal plant pathogens are briefly discussed in the first part of this review. Advances in genomics have enabled the investigation of chromosomal aberrations of agriculturally important fungal isolates at the nucleotide level. Some of these cases are summarized in the second part of this review; it is claimed that the effect of chromosomal aberrations on pathogenicity should be studied mechanistically. More data on the effect of gene copy number variations on phenotypes that are relevant to agriculture are especially needed. Genome rearrangements through translocations have shaped the genome of fungal plant pathogens by creating lineage-specific chromosome territories encoding for genes participating in plant diseases. Pathogenicity chromosomes are unique cases of such lineage-specific genetic elements, interestingly these chromosomes can be transferred horizontally and thus transforming a non-pathogenic strain to a pathogenic one. The third part of this review describes our attempts to reveal mutators in fungal plant pathogens by identifying fungi that lack important DNA repair genes or respond to DNA damage in an unconventional way. We found that a group of fungal plant pathogens lack conserved genes that are needed for an important Holliday junction resolution pathway. In addition, in Fusarium oxysporum, the rate-limiting step in dNTP production is not induced under DNA replication stress. This is very different from organisms from bacteria to humans. It remains to be seen if these mechanisms promote genetic instability in fungal plant pathogens.
Katan, J. Milestones and future expectations for soil disinfestation after 45 years of soil disinfestation symposia (1973-2018). In IX INTERNATIONAL SYMPOSIUM ON SOIL AND SUBSTRATE DISINFESTATION; Gamliel, A. ; Tsitsigiannis, D. ; Gkizi, D., Ed. IX INTERNATIONAL SYMPOSIUM ON SOIL AND SUBSTRATE DISINFESTATION; Int Soc Hort Sci, 2020; Vol. 1270, pp. 1-7.Abstract
The soil disinfestation (SD) symposia have contributed to the progress of SD research, and to cooperation among scientists and industry leaders. These symposia reflect the development of new or rediscovered SD measurements and their application in soilborne pathogen research, environmental awareness, regulation, and especially the impact of the methyl bromide (MB) phaseout. During these symposia, studies on microbial activities in soil were frequently discussed. The performance of old fumigants, e.g., chloropicrin, metam sodium, and MB, and the relatively new fumigants, e.g., DMDS, methyl iodide, and certain nematicides were thoroughly discussed and debated. Physical tools, e.g., soil steaming, played a minor role. In the mid-1990s, the performance of non-chemical tools and their mode of action became an important part in these symposia, including soil solarization, biofumigation, organic amendments, anaerobic SD, biocontrol, soilless culture, physical tools, and others. Related topics are integrated pest management (with an emphasis on combining methods of control), soil suppressiveness, soil health, diagnostics, decision-making tools, and others. The MB phaseout crisis, which was very severe since only a few alternatives were available at the time, had a major impact on SD research, prompting the development of chemical and non-chemical alternatives. We are now in a better situation because we depend on a larger variety of SD tools, but the long-term effects of the newly introduced technologies have to be studied, and therefore continuous monitoring of disinfested soils is necessary.
Youdkes, D. ; Helman, Y. ; Burdman, S. ; Matan, O. ; Jurkevitch, E. Potential Control of Potato Soft Rot Disease by the Obligate Predators Bdellovibrio and Like Organisms. APPLIED AND ENVIRONMENTAL MICROBIOLOGY 2020, 86.Abstract
Bacterial soft rot diseases caused by Pectobacterium spp. and Dickeya spp. affect a wide range of crops, including potatoes, a major food crop. As of today, farmers mostly rely on sanitary practices, water management, and plant nutrition for control. We tested the bacterial predators Bdellovibrio and like organisms (BALOs) to control potato soft rot. BALOs are small, motile predatory bacteria found in terrestrial and aquatic environments. They prey on a wide range of Gram-negative bacteria, including animal and plant pathogens. To this end, BALO strains HD100, 1091, and a Delta merRNA derivative of HD100 were shown to efficiently prey on various rot-causing strains of Pectobacterium and Dickeya solani. BALO control of maceration caused by a highly virulent strain of Pectobacterium carotovorum subsp. brasilense was then tested in situ using a potato slice assay. All BALO strains were highly effective at reducing disease, up to complete prevention. Effectivity was concentration dependent, and BALOs applied before P. carotovorum subsp. brasilense inoculation performed significantly better than those applied after the disease-causing agent, maybe due to in situ consumption of glucose by the prey, as glucose metabolism by live prey bacteria was shown to prevent predation. Dead predators and the supernatant of BALO cultures did not significantly prevent maceration, indicating that predation was the major mechanism for the prevention of the disease. Finally, plastic resistance to predation was affected by prey and predator population parameters, suggesting that population dynamics affect prey response to predation. IMPORTANCE Bacterial soft rot diseases caused by Pectobacterium spp. and Dickeya spp. are among the most important plant diseases caused by bacteria. Among other crops, they inflict large-scale damage to potatoes. As of today, farmers have few options to control them. The bacteria Bdellovibrio and like organisms (BALOs) are obligate predators of bacteria. We tested their potential to prey on Pectobacterium spp. and Dickeya spp. and to protect potato. We show that different BALOs can prey on soft rot-causing bacteria and prevent their growth in situ, precluding tissue maceration. Dead predators and the supernatant of BALO cultures did not significantly prevent maceration, showing that the effect is due to predation. Soft rot control by the predators was concentration dependent and was higher when the predator was inoculated ahead of the prey. As residual prey remained, we investigated what determines their level and found that initial prey and predator population parameters affect prey response to predation.
Lucke, M. ; Correa, M. G. ; Levy, A. The Role of Secretion Systems, Effectors, and Secondary Metabolites of Beneficial Rhizobacteria in Interactions With Plants and Microbes. FRONTIERS IN PLANT SCIENCE 2020, 11.Abstract
Beneficial rhizobacteria dwell in plant roots and promote plant growth, development, and resistance to various stress types. In recent years there have been large-scale efforts to culture root-associated bacteria and sequence their genomes to uncover novel beneficial microbes. However, only a few strains of rhizobacteria from the large pool of soil microbes have been studied at the molecular level. This review focuses on the molecular basis underlying the phenotypes of three beneficial microbe groups; (1) plant-growth promoting rhizobacteria (PGPR), (2) root nodulating bacteria (RNB), and (3) biocontrol agents (BCAs). We focus on bacterial proteins and secondary metabolites that mediate known phenotypes within and around plants, and the mechanisms used to secrete these. We highlight the necessity for a better understanding of bacterial genes responsible for beneficial plant traits, which can be used for targeted gene-centered and molecule-centered discovery and deployment of novel beneficial rhizobacteria.
Feldman, D. ; Yarden, O. ; Hadar, Y. Seeking the Roles for Fungal Small-Secreted Proteins in Affecting Saprophytic Lifestyles. FRONTIERS IN MICROBIOLOGY 2020, 11.Abstract
Small secreted proteins (SSPs) comprise 40-60% of the total fungal secretome and are present in fungi of all phylogenetic groups, representing the entire spectrum of lifestyles. They are characteristically shorter than 300 amino acids in length and have a signal peptide. The majority of SSPs are coded by orphan genes, which lack known domains or similarities to known protein sequences. Effectors are a group of SSPs that have been investigated extensively in fungi that interact with living hosts, either pathogens or mutualistic systems. They are involved in suppressing the host defense response and altering its physiology. Here, we aim to delineate some of the potential roles of SSPs in saprotrophic fungi, that have been bioinformatically predicted as effectors, and termed in this mini-review as ``effector-like'' proteins. The effector-like Ssp1 from the white-rot fungus Pleurotus ostreatus is presented as a case study, and its potential role in regulating the ligninolytic system, secondary metabolism, development, and fruiting body initiation are discussed. We propose that deciphering the nature of effector-like SSPs will contribute to our understanding of development and communication in saprophytic fungi, as well as help, to elucidate the origin, regulation, and mechanisms of fungal-host, fungal-fungal, and fungal-bacterial interactions.
Fedorenko, A. ; Grinberg, M. ; Orevi, T. ; Kashtan, N. Survival of the enveloped bacteriophage Phi6 (a surrogate for SARS-CoV-2) in evaporated saliva microdroplets deposited on glass surfaces. SCIENTIFIC REPORTS 2020, 10.Abstract
Survival of respiratory viral pathogens in expelled saliva microdroplets is central to their transmission, yet the factors that determine survival in such microdroplets are not well understood. Here we combine microscopy imaging with virus viability assays to study survival of three bacteriophages suggested as good models for respiratory pathogens: the enveloped Phi6 (a surrogate for SARS-CoV-2), and the non-enveloped PhiX174 and MS2. We measured virus viability in human saliva microdroplets, SM buffer, and water following deposition on glass surfaces at various relative humidities (RH). Saliva and water microdroplets dried out rapidly, within minutes, at all tested RH levels (23%, 43%, 57%, and 78%), while SM microdroplets remained hydrated at RH >= 57%. Generally, the survival of all three viruses in dry saliva microdroplets was significantly greater than those in SM buffer and water under all RH (except PhiX174 in water under 57% RH survived the best among 3 media). Thus, atmosphere RH and microdroplet hydration state are not sufficient to explain virus survival, indicating that the virus-suspended medium, and association with saliva components in particular, likely play a role in virus survival. Uncovering the exact properties and components that make saliva a favorable environment for the survival of viruses, in particular enveloped ones like Phi6, is thus of great importance for reducing transmission of viral respiratory pathogens including SARS-CoV-2.
Lifshitz, N. ; Hazanov, L. ; Fine, M. ; Yarden, O. Seasonal Variations in the Culturable Mycobiome of Acropora loripes along a Depth Gradient. Microorganisms 2020, 8. Publisher's VersionAbstract
Coral associated fungi are widespread, highly diverse and are part and parcel of the coral holobiont. To study how environmental conditions prevailing near the coral-host may affect fungal diversity, the culturable (isolated on potato dextrose agar) mycobiome associated with Acropora loripes colonies was seasonally sampled along a depth gradient in the Gulf of Aqaba. Fragments were sampled from both apparently healthy coral colonies as well as those exhibiting observable lesions. Based on phylogenetic analysis of 197 fungal sequences, Ascomycota were the most prevalent (91.9%). The abundance of fungi increased with increasing water depth, where corals sampled at 25 m yielded up to 70% more fungal colony forming units (CFUs) than those isolated at 6 m. Fungal diversity at 25 m was also markedly higher, with over 2-fold more fungal families represented. Diversity was also higher in lesioned coral samples, when compared to apparently healthy colonies. In winter, concurrent with water column mixing and increased levels of available nutrients, at the shallow depths, Saccharomytacea and Sporidiobolacea were more prevalent, while in spring and fall Trichocomacea (overall, the most prevalent family isolated throughout this study) were the most abundant taxa isolated at these depths as well as at deeper sampling sites. Our results highlight the dynamic nature of the culturable coral mycobiome and its sensitivity to environmental conditions and coral health.
Hou, L. W. ; Groenewald, J. Z. ; Pfenning, L. H. ; Yarden, O. ; Crous, P. W. ; Cai, L. The phoma-like dilemma. 2020. Publisher's VersionAbstract
Species of Didymellaceae have a cosmopolitan distribution and are geographically widespread, occurring in diverse ecosystems. The family includes several important plant pathogenic fungi associated with fruit, leaf, stem and root diseases on a wide variety of hosts, as well as endophytic, saprobic and clinically relevant species. The Didymellaceae was recently revised based on morphological and phylogenetic analyses of ex-type strains subjected to DNA sequencing of partial gene data of the LSU, ITS, rpb2 and tub2 loci. Several poly- and paraphyletic genera, including Ascochyta, Didymella and Phoma were redefined, along with the introduction of new genera. In the present study, a global collection of 1 124 Didymellaceae strains from 92 countries, 121 plant families and 55 other substrates, including air, coral, human tissues, house dust, fungi, insects, soil, and water were examined via multi-locus phylogenetic analyses and detailed morphological comparisons, representing the broadest sampling of Didymellaceae to date. Among these, 97 isolates representing seven new genera, 40 new species and 21 new combinations were newly introduced in Didymellaceae. In addition, six epitypes and six neotypes were designated to stabilise the taxonomy and use of older names. A robust, multi-locus reference phylogenetic tree of Didymellaceae was generated. In addition, rpb2 was revealed as the most effective locus for the identification of Didymellaceae at species level, and is proposed as a secondary DNA marker for the family.
Meyer, V. ; Basenko, E. Y. ; Benz, J. P. ; Braus, G. H. ; Caddick, M. X. ; Csukai, M. ; de Vries, R. P. ; Endy, D. ; Frisvad, J. C. ; Gunde-Cimerman, N. ; et al. Growing a circular economy with fungal biotechnology: a white paper. 2020, 7 5. Publisher's VersionAbstract
Fungi have the ability to transform organic materials into a rich and diverse set of useful products and provide distinct opportunities for tackling the urgent challenges before all humans. Fungal biotechnology can advance the transition from our petroleum-based economy into a bio-based circular economy and has the ability to sustainably produce resilient sources of food, feed, chemicals, fuels, textiles, and materials for construction, automotive and transportation industries, for furniture and beyond. Fungal biotechnology offers solutions for securing, stabilizing and enhancing the food supply for a growing human population, while simultaneously lowering greenhouse gas emissions. Fungal biotechnology has, thus, the potential to make a significant contribution to climate change mitigation and meeting the United Nation’s sustainable development goals through the rational improvement of new and established fungal cell factories. The White Paper presented here is the result of the 2nd Think Tank meeting held by the EUROFUNG consortium in Berlin in October 2019. This paper highlights discussions on current opportunities and research challenges in fungal biotechnology and aims to inform scientists, educators, the general public, industrial stakeholders and policymakers about the current fungal biotech revolution.
Kan, Y. ; Lyu, Q. ; Jiang, N. ; Han, S. ; Li, J. ; Burdman, S. ; Luo, L. iTRAQ-based proteomic analyses of the plant-pathogenic bacterium Acidovorax citrulli during entrance into and resuscitation from the viable but nonculturable state. Journal of Proteomics 2020, 211. Publisher's VersionAbstract
Acidovorax citrulli, the causal agent of bacterial fruit blotch (BFB) disease, infects cucurbit crops including watermelon and melon. This bacterium can enter the viable but nonculturable (VBNC) state following exposure to copper sulfate. Moreover, copper-induced VBNC A. citrulli cells can be resuscitated by EDTA. In this study, isobaric tag for relative and absolute quantification (iTRAQ) was used to compare protein profiles of VBNC cells, resuscitated cells at different stages and log-phase cells of the A. citrulli model strain AAC00-1. A total of 2672 proteins were identified, with 60 being differentially abundant in VBNC cells compared with log-phase cells, and 469 being differentially abundant in resuscitated cells compared with VBNC cells. Proteins involved in the arginine and proline metabolism pathway and degradation of aromatic compounds could be important for the VBNC cells. In the early resuscitation process, proteins associated with carbon metabolism, and degradation of naphthalene and aromatic compounds were significantly enriched, while proteins involved in oxidative phosphorylation, bacterial chemotaxis, ABC transporters and quorum sensing were significantly enriched at the late resuscitation stages. This is the first study reporting thorough protein profile analyses of VBNC and resuscitating cells of a plant-pathogenic bacterium. Biological significance: The VBNC state is a dormant-like condition that was reported to occur in many bacterial species, upon facing a variety of environmental stresses. Acidovorax citrulli is a seed borne pathogenic bacterium that threatens cucurbit production worldwide. Moreover, A. citrulli can enter into the VBNC state after treatment of copper sulfate, thus increasing its survival and dissemination probabilities. This study enriches our understanding of the mechanisms of entrance into and resuscitation from the VBNC state of this important plant-pathogenic bacterium. This knowledge could be exploited in the future to develop novel approaches to interfere with these processes, thus contributing to a more efficient management of this pathogen. In a broader perspective, the knowledge emerging from this study has implications to the general understanding of the VBNC state in bacteria. © 2019 Elsevier B.V.
2019
Feldman, D. ; Amedi, N. ; Carmeli, S. ; Yarden, O. ; Hadar, Y. Manipulating the Expression of Small Secreted Protein 1 (Ssp1) Alters Patterns of Development and Metabolism in the White-Rot Fungus Pleurotus ostreatus. Applied and Environmental Microbiology 2019, 85. Publisher's VersionAbstract
The function of small secreted proteins (SSPs) in saprotrophic fungi is, for the most part, unknown. The white-rot mushroom Pleurotus ostreatus produces considerable amounts of SSPs at the onset of secondary metabolism, during colony development, and in response to chemical compounds such as 5-hydroxymethylfurfural and aryl alcohols. Genetic manipulation of Ssp1, by knockdown (KDssp1) or overexpression (OEssp1), indicated that they are, in fact, involved in the regulation of the ligninolytic system. To elucidate their potential involvement in fungal development, quantitative secretome analysis was performed during the trophophase and the idiophase and at a transition point between the two growth phases. The mutations conferred a time shift in the secretion and expression patterns: OEssp1 preceded the entrance to idiophase and secondary metabolism, while KDssp1 was delayed. This was also correlated with expression patterns of selected genes. The KDssp1 colony aged at a slower pace, accompanied by a slower decline in biomass over time. In contrast, the OEssp1 strain exhibited severe lysis and aging of the colony at the same time point. These phenomena were accompanied by variations in yellow pigment production, characteristic of entrance of the wild type into idiophase. The pigment was produced earlier and in a larger amount in the OEssp1 strain and was absent from the KDssp1 strain. Furthermore, the dikaryon harboring OEssp1 exhibited a delay in the initiation of fruiting body formation as well as earlier aging. We propose that Ssp1 might function as a part of the fungal communication network and regulate the pattern of fungal development and metabolism in P. ostreatus.IMPORTANCE Small secreted proteins (SSPs) are common in fungal saprotrophs, but their roles remain elusive. As such, they comprise part of a gene pool which may be involved in governing fungal lifestyles not limited to symbiosis and pathogenicity, in which they are commonly referred to as “effectors.” We propose that Ssp1 in the white-rot fungus Pleurotus ostreatus regulates the transition from primary to secondary metabolism, development, aging, and fruiting body initiation. Our observations uncover a novel regulatory role of effector-like SSPs in a saprotroph, suggesting that they may act in fungal communication as well as in response to environmental cues. The presence of Ssp1 homologues in other fungal species supports a common potential role in environmental sensing and fungal development.
Bez, C. ; Javvadi, S. G. ; Bertani, I. ; Devescovi, G. ; Guarnaccia, C. ; Studholme, D. J. ; Geller, A. M. ; Levy, A. ; Venturi, V. AzeR, a transcriptional regulator that responds to azelaic acid in Pseudomonas nitroreducens. 2019. Publisher's VersionAbstract
Azelaic acid is a dicarboxylic acid that has recently been shown to play a role in plant-bacteria signalling and also occurs naturally in several cereals. Several bacteria have been reported to be able to utilize azelaic acid as a unique source of carbon and energy, including . In this study, we utilize as a model organism to study bacterial degradation of and response to azelaic acid. We report genetic evidence of azelaic acid degradation and the identification of a transcriptional regulator that responds to azelaic acid in DSM 9128. Three mutants possessing transposons in genes of an acyl-CoA ligase, an acyl-CoA dehydrogenase and an isocitrate lyase display a deficient ability in growing in azelaic acid. Studies on transcriptional regulation of these genes resulted in the identification of an IclR family repressor that we designated as AzeR, which specifically responds to azelaic acid. A bioinformatics survey reveals that AzeR is confined to a few proteobacterial genera that are likely to be able to degrade and utilize azelaic acid as the sole source of carbon and energy.
Petrenko, M. ; Friedman, S. P. ; Fluss, R. ; Pasternak, Z. ; Huppert, A. ; Jurkevitch, E. Spatial heterogeneity stabilizes predator–prey interactions at the microscale while patch connectivity controls their outcome. Environmental Microbiology 2019, n/a. Publisher's VersionAbstract
Summary Natural landscapes are both fragmented and heterogeneous, affecting the distribution of organisms, and their interactions. While predation in homogeneous environments increases the probability of population extinction, fragmentation/heterogeneity promotes coexistence and enhances community stability as shown by experimentation with animals and microorganisms, and supported by theory. Patch connectivity can modulate such effects but how microbial predatory interactions are affected by water-driven connectivity is unknown. In soil, patch habitability by microorganisms, and their connectivity depend upon the water saturation degree (SD). Here, using the obligate bacterial predator Bdellovibrio bacteriovorus, and a Burkholderia prey, we show that soil spatial heterogeneity profoundly affects predatory dynamics, enhancing long-term co-existence of predator and prey in a SD-threshold dependent-manner. However, as patches and connectors cannot be distinguished in these soil matrices, metapopulations cannot be invoked to explain the dynamics of increased persistence. Using a set of experiments combined with statistical and physical models we demonstrate and quantify how under full connectivity, predation is independent of water content but depends on soil microstructure characteristics. In contrast, the SD below which predation is largely impaired corresponds to a threshold below which the water network collapses and water connectivity breaks down, preventing the bacteria to move within the soil matrix.
Mosquito, S. ; Bertani, I. ; Licastro, D. ; Compant, S. ; Myers, M. P. ; Hinarejos, E. ; Levy, A. ; Venturi, V. In Planta Colonization and Role of T6SS in Two Rice Kosakonia Endophytes. Molecular Plant-Microbe Interactions® 2019, MPMI-09-19-0256-R. Publisher's VersionAbstract
Endophytes live inside plants and are often beneficial. Kosakonia is a novel bacterial genus that includes many diazotrophic plant-associated isolates. Plant–bacteria studies on two rice endophytic Kosakonia beneficial strains were performed, including comparative genomics, secretome profiling, in planta tests, and a field release trial. The strains are efficient rhizoplane and root endosphere colonizers and localized in the root cortex. Secretomics revealed 144 putative secreted proteins, including type VI secretory system (T6SS) proteins. A Kosakonia T6SS genomic knock-out mutant showed a significant decrease in rhizoplane and endosphere colonization ability. A field trial using rice seed inoculated with Kosakonia spp. showed no effect on plant growth promotion upon nitrogen stress and microbiome studies revealed that Kosakonia spp. were significantly more present in the inoculated rice. Comparative genomics indicated that several protein domains were enriched in plant-associated Kosakonia spp. This study highlights that Kosakonia is an important, recently classified genus involved in plant–bacteria interaction.
Cohen, O. ; Bar (Kutiel), P. ; Gamliel, A. ; Katan, J. ; Kurzbaum, E. ; Weber, G. ; Schubert, I. ; Riov, J. Rain-based soil solarization for reducing the persistent seed banks of invasive plants in natural ecosystems – Acacia saligna as a model. Pest Management Science 2019, 75, 1933-1941. Publisher's VersionAbstract
BACKGROUND: A large persistent seed bank of invasive plants is a significant obstacle to restoration programs. Soil solarization was demonstrated to be an effective method for reducing the seed bank of Australian acacias. However, use of this method in natural habitats might be limited due to the requirement to moisten the soil by irrigation. This study examined the possibility of replacing irrigation by trapping the soil moisture caused by the most recent rainfall, i.e. rain-based soil solarization (RBS). RESULTS: Exposure of Acacia saligna seeds to 57 °C at 20% soil moisture for 68 h resulted in almost complete loss of seed viability. Similarly, RBS treatment significantly reduced the viability of A. saligna seeds buried at a soil depth of 1–19 cm as well as seed density in the natural seed bank, and almost completely eliminated seedling emergence from natural seed banks of A. saligna and other environmental weeds. CONCLUSION: Our results indicate that RBS is an effective method for reducing the seed bank of invasive plants in natural habitats located in various climate regions characterized by different soil types. This is the first demonstration of a successful application of RBS for soil disinfestation. © 2018 Society of Chemical Industry. © 2018 Society of Chemical Industry
Yoav, S. ; Stern, J. ; Salama-Alber, O. ; Frolow, F. ; Anbar, M. ; Karpol, A. ; Hadar, Y. ; Morag, E. ; Bayer, E. A. Directed evolution of clostridium thermocellum β-glucosidase a towards enhanced thermostability. International Journal of Molecular Sciences 2019, 20. Publisher's VersionAbstract
β-Glucosidases are key enzymes in the process of cellulose utilization. It is the last enzyme in the cellulose hydrolysis chain, which converts cellobiose to glucose. Since cellobiose is known to have a feedback inhibitory effect on a variety of cellulases, β-glucosidase can prevent this inhibition by hydrolyzing cellobiose to non-inhibitory glucose. While the optimal temperature of the Clostridium thermocellum cellulosome is 70 °C, C. thermocellum β-glucosidase A is almost inactive at such high temperatures. Thus, in the current study, a random mutagenesis directed evolutionary approach was conducted to produce a thermostable mutant with Kcat and Km, similar to those of the wild-type enzyme. The resultant mutant contained two mutations, A17S and K268N, but only the former was found to affect thermostability, whereby the inflection temperature (Ti) was increased by 6.4 °C. A17 is located near the central cavity of the native enzyme. Interestingly, multiple alignments revealed that position 17 is relatively conserved, whereby alanine is replaced only by serine. Upon the addition of the thermostable mutant to the C. thermocellum secretome for subsequent hydrolysis of microcrystalline cellulose at 70 °C, a higher soluble glucose yield (243%) was obtained compared to the activity of the secretome supplemented with the wild-type enzyme. © 2019 by the authors. Licensee MDPI, Basel, Switzerland.
Tovi, N. ; Frenk, S. ; Hadar, Y. ; Minz, D. Host specificity and spatial distribution preference of three Pseudomonas isolates. Frontiers in Microbiology 2019, 10. Publisher's VersionAbstract
Plant hosts recruit and maintain a distinct root-associated microbiota based on host and bacterium traits. However, past studies disregarded microbial strain-host specificity and spatial micro-heterogeneity of the root compartment. Using genetic manipulation, confocal laser scanning microscopy, real-time quantitative PCR, and genome sequencing we characterized the colonization patterns of three Pseudomonas spp. isolates native to wheat roots, on the micro-scale. Namely, isolates P. fluorescens NT0133, P. stutzeri NT124, and P. stutzeri NT128. All three isolates preferentially colonized wheat over cucumber roots that served as control for host specificity. Furthermore, not only had the isolates strong host specificity but each isolate had a distinct spatial distribution on the root, all within a few millimeters. Isolate P. stutzeri-NT0124 preferentially colonized root tips, whereas P. fluorescens-NT0133 showed a preference for zones distant from the tip. In contrast, isolate P. stutzeri-NT0128 had no preference for a specific niche on the root. While all isolates maintained genetic potential for motility and biofilm formation their phenotype varied significantly and corresponded to their niche preference. These results demonstrate the importance of spatial colonization patterns, governed by both niche and bacterial characteristics which will have great importance in future attempts to manipulate the plant microbiome by constructing synthetic microbial consortia. © 2019 Frontiers Media S.A. All Rights Reserved.
Vetvicka, V. ; Gover, G. ; Hayby, H. ; Danay, O. ; Ezov, N. ; Hadar, Y. ; Schwartz, B. Immunomodulating effects exerted by glucans extracted from the king oyster culinary-medicinal mushroom pleurotus eryngii (agaricomycetes) grown in substrates containing various concentrations of olive mill waste. International Journal of Medicinal Mushrooms 2019, 21, 765-781. Publisher's VersionAbstract
We have recently demonstrated that we could enhance glucan content in Pleurotus eryngii following cultivation of the mushrooms on a substrate containing different concentrations of olive mill solid waste (OMSW). These changes are directly related to the content of OMSW in the growing substrate. Using dextran sulfate sodium (DSS)-inflammatory bowel disease (IBD) mice model, we measured the colonic inflammatory response to the different glucan preparations. We found that the histology damaging score (HDS) resulting from DSS treatment reach a value of 11.8 ± 2.3 were efficiently downregulated by treatment with the fungal extracted glucans. Glucans extracted from stalks cultivated at 20% OMSW downregulated to a HDS value of 6.4 ± 0.5 whereas those cultivated at 80% OMSW showed the strongest effects (5.5 ± 0.6). Similar downregulatory effects were obtained for expression of various intestinal cytokines. All tested glucans were equally effective in regulating the number of CD14/CD16 monocytes from 18.2 ± 2.7% for DSS to 6.4 ± 2.0 for DSS + glucans extracted from stalks cultivated at 50% OMSW. We tested the effect of glucans on lipopolysaccharide- induced production of TNF-α, which demonstrated that stalk-derived glucans were more effective than caps-derived glucans. Isolated glucans competed with anti-Dectin-1 and anti-CR3 antibodies, indicating that they contain β-glucans recognized by these receptors. In conclusion, the most effective glucans in ameliorating IBD-associated symptoms induced by DSS treatment in mice were glucan extracts prepared from the stalk of P. eryngii grown at higher concentrations of OMSW. We conclude that these stress-induced growing conditions may be helpful in selecting more effective glucans derived from edible mushrooms. © 2019 by Begell House, Inc.
Feldman, D. ; Kowbel, D. J. ; Cohen, A. ; Glass, N. L. ; Hadar, Y. ; Yarden, O. Identification and manipulation of Neurospora crassa genes involved in sensitivity to furfural. Biotechnology for Biofuels 2019, 12. Publisher's VersionAbstract
Background: Biofuels derived from lignocellulosic biomass are a viable alternative to fossil fuels required for transportation. Following plant biomass pretreatment, the furan derivative furfural is present at concentrations which are inhibitory to yeasts. Detoxification of furfural is thus important for efficient fermentation. Here, we searched for new genetic attributes in the fungus Neurospora crassa that may be linked to furfural tolerance. The fact that furfural is involved in the natural process of sexual spore germination of N. crassa and that this fungus is highly amenable to genetic manipulations makes it a rational candidate for this study. Results: Both hypothesis-based and unbiased (random promotor mutagenesis) approaches were performed to identify N. crassa genes associated with the response to furfural. Changes in the transcriptional profile following exposure to furfural revealed that the affected processes were, overall, similar to those observed in Saccharomyces cerevisiae. N. crassa was more tolerant (by ∼ 30%) to furfural when carboxymethyl cellulose was the main carbon source as opposed to sucrose, indicative of a link between carbohydrate metabolism and furfural tolerance. We also observed increased tolerance in a Δcre-1 mutant (CRE-1 is a key transcription factor that regulates the ability of fungi to utilize non-preferred carbon sources). In addition, analysis of aldehyde dehydrogenase mutants showed that ahd-2 (NCU00378) was involved in tolerance to furfural as well as the predicted membrane transporter NCU05580 (flr-1), a homolog of FLR1 in S. cerevisiae. Further to the rational screening, an unbiased approach revealed additional genes whose inactivation conferred increased tolerance to furfural: (i) NCU02488, which affected the abundance of the non-anchored cell wall protein NCW-1 (NCU05137), and (ii) the zinc finger protein NCU01407. Conclusions: We identified attributes in N. crassa associated with tolerance or degradation of furfural, using complementary research approaches. The manipulation of the genes involved in furan sensitivity can provide a means for improving the production of biofuel producing strains. Similar research approaches can be utilized in N. crassa and other filamentous fungi to identify additional attributes relevant to other furans or toxic chemicals. © 2019 The Author(s).