Fusarium wilt of banana, caused by Fusarium oxysporum f. sp. cubense race TR4 (Foc), is one of the most destructive diseases threatening global banana production, particularly the Cavendish cultivar. Conventional control strategies, including chemical treatments and quarantine, remain largely ineffective and unsustainable, underscoring the urgent need for alternative approaches. Biological control using rhizosphere-associated microorganisms offers a promising and environmentally friendly strategy. In this study, we isolated 436 bacterial strains from the rhizosphere of healthy banana plants and screened them for antifungal activity against Foc. Out of the screened isolates, 93 exhibited significant in-vitro inhibitions, and 64 of these were subsequently evaluated in greenhouse assays. We found that 22 strains reduced Fusarium wilt severity by 45 to 85% compared to untreated controls. Among them, two isolates, DDC20 and DDC\_NEW2, consistently demonstrated strong biocontrol activity. In addition, cell-free culture media (CFCM) and crude extracts inhibited spore germination in fluorescence-based assays, indicating the involvement of secreted antifungal metabolites. Microscopy and confocal observations of GFP tagged Foc revealed hyphal abnormalities in the presence of bacterial treatments, including swelling, irregular branching, and distortion, accompanied by excessive sporulation characterized by abundant microconidia, macroconidia, and chlamydospores. Whole-genome sequencing and comparative analyses placed both isolates within the genus Bacillus. Genome mining using antiSMASH identified multiple biosynthetic gene clusters encoding known antifungal compounds such as surfactin, fengycin, bacillibactin, and difficidin, as well as putative novel clusters. LCMS confirmed the presence of surfactin and fengycin in bacterial extracts, supporting the genomic predictions. Collectively, these findings highlight the potential of DDC20 and DDC\_NEW2 (related to Bacillus spp.) from the banana rhizosphere as effective biocontrol agents against Foc TR4. This integrated approach, combining phenotypic assays, microscopy, and genome mining, provides a strong foundation for the development of sustainable strategies to manage Fusarium wilt in banana cultivation. ### Competing Interest Statement The authors have declared no competing interest.

Bacterial strains in the rhizosphere secrete volatile organic compounds (VOCs) that play critical roles in interand intra-kingdom signaling, influencing both microbe-microbe and microbe-plant interactions. In this study we evaluated the plant growth-promoting effects of VOCs emitted by Bacillus sp. NYG5 on Arabidopsis thaliana, Nicotiana tabacum, and Cucumis sativus, focusing on VOC-induced alterations in plant metabolic pathways, rhizosphere microbial communities, and soil chemical properties. NYG5 VOCs enhanced plant biomass across all tested species and induced significant shifts in rhizosphere microbial community composition, specifically increasing relative abundance of Gammaproteobacteria and reducing Deltaproteobacteria (Linear discriminant analysis Effect Size, p < 0.05). Soil analysis revealed a considerable reduction in humic substance concentrations following VOCs exposure, as detected by fluorescent spectral analysis. Using SPME-GC-MS, several novel VOCs were identified, some of which directly promoted plant growth. Transcriptomic analysis of N. tabacum exposed to NYG5 VOCs demonstrated activation of pathways related to phenylpropanoid biosynthesis, sugar metabolism, and hormone signal transduction. Within the phenylpropanoid biosynthesis pathway, a significant upregulation (p adj = 1.16e-14) of caffeic acid 3-O-methyltransferase was observed, a key enzyme leading to lignin and suberin monomer biosynthesis. These results highlight the complex mechanisms through which bacterial VOCs influence plant growth, including metabolic modulation, rhizosphere microbiome restructuring, and soil chemical changes. Collectively, this study highlights the pivotal role of bacterial VOCs in shaping plant-microbesoil interactions.

Soil fatigue, well documented in various crops, presents a significant challenge to banana production by causing fast and then gradual declines in plant growth and yield over years of cultivation. Despite its impact on profitability, the underlying mechanisms driving soil fatigue remain poorly understood; however, a strong link to shifts in the soil microbiome has been suggested. We investigated the dynamics of microbial communities in relation to soil fatigue, using a novel semi-controlled outdoor experimental system. Soil at different stages of fatigue (0 to 42 months of banana cultivation) was generated in large containers filled with initially healthy soil. Banana plants grown in these soils were replaced with new plants which showed soil age-dependent growth. Three months postplanting, soil and root samples were collected for analyses of soil parameters and microbial community composition using bacterial (16S) and fungal (ITS) amplicon sequencing. We identified minor age-related shifts in mainly pH, potassium, and organic matter in the soil. While alpha diversity remained unchanged, significant shifts in bacterial and fungal community composition were observed in fatigued soils. Notably, the relative abundance of bacterial families such as Flavobacteriaceae, Pseudomonaceae, and Acidibacter increased, as did some fungal taxa (many from groups with known pathogens)—Ceratobasidiaceae (including Rhizoctonia), Dothideomycetes, and Stachybotryaceae. Simultaneously, the relative abundance of bacterial families with known beneficial members, including Gemmatimonadaceae, Moraxellaceae, Sphingomonadaceae, and Azospirillaceae, as well as symbiotic fungal taxa such as Glomeraceae and Lasiosphaeriaceae, declined. Thus, soil fatigue may be correlated to the proliferation of pathogenic populations and a loss of beneficial microorganisms.

Peanuts (Arachis hypogaea) are of significant agricultural importance due to their versatile uses, providing a valuable source of edible oil and protein-rich food products and serving in industrial applications. Peanuts are susceptible to various diseases, one of them being net syndrome, a poorly understood threat triggered by an undefined causal agent, with symptoms including black coloring and lesions of the peanut pod that affect product quality. In this study, amplicon sequencing was harnessed to explore the fungal and bacterial populations associated with the syndrome. Using this approach, we identified a specific Fusarium sp. amplicon sequence variant that demonstrated a significant correlation with the presence of net syndrome symptoms in both young and mature peanuts. This finding was further validated by Koch's postulates testing, including isolation, cultivation, and application of this strain to corroborate its involvement in net syndrome in peanuts. Our findings contribute to a better understanding of the interactions between peanuts and their microbial communities and to identification of the previously undetermined agent involved in net syndrome and the etiology of the disease.

Background Bacterial strains in the rhizosphere may secrete an array of volatile organic compounds (VOCs), many of which are vital for inter- and intra-kingdom signaling, modulating microbe-microbe and microbe-plant interactions. In this study, we tested the plant growth promoting effect of VOCs emitted by Bacillus sp. NYG5 on several plants, focusing on VOC-induced changes in plant metabolic pathways, soil chemical properties and rhizo-microbiome. Results NYG5 emitted VOCs were found to promote growth of Arabidopsis thaliana, Nicotiana tabacum and Cucumis sativus, while also altering the community composition of the rhizosphere microbiome. Furthermore, fluorescent spectral analysis of the soil showed a significant decrease in concentration of humic substances following exposure to NYG5-emitted VOCs. Utilizing SPME-GC-MS analysis, several novel VOCs were identified, some of which were shown to promote plant growth. Transcriptomic analysis of N. tabacum plants exposed to NYG5-emitted VOCs revealed the induction of several plant growth related pathways such as phenylpropanoid biosynthesis, metabolism of sugars, and signal transduction of plant hormones. Conclusions Exposure to NYG5-emitted VOCs significantly impacted several plant growth related pathways, increasing plant biomass. Additionally, these VOCs altered the community composition of the rhizosphere microbiome, and decreased the concentration of humic substances in the soil. These findings may suggest that bacterial VOCs significantly impact plant growth via various mechanisms.

BACKGROUND:Fungal phytopathogens are a significant threat to crops and food security, and there is a constant need to develop safe and effective compounds that antagonize them. In-planta assays are complex and tedious and are thus not suitable for initial high-throughput screening of new candidate antifungal compounds. We propose an in vitro screening pipeline that integrates five rapid quantitative and qualitative methods to estimate the efficacy and mode of action of prospective antifungal compounds. RESULTS:The pipeline was evaluated using five documented antifungal compounds (benomyl, catechol, cycloheximide, 2,4-diacetylphloroglucinol, and phenylacetic acid) that have different modes of action and efficacy, against the model soilborne fungal pathogen Fusarium oxysporum f. sp. radicis cucumerinum. We initially evaluated the five compounds' ability to inhibit fungal growth and metabolic activity using green fluorescent protein (GFP)-labeled F. oxysporum and PrestoBlue staining, respectively, in multiwell plate assays. We tested the compounds' inhibition of both conidial germination and hyphal elongation. We then employed FUN-1 and SYTO9/propidium iodide staining, coupled to confocal microscopy, to differentiate between fungal growth inhibition and death at the cellular level. Finally, using a reactive oxygen species (ROS)-detection assay, we were able to quantify ROS production in response to compound application. CONCLUSIONS:Collectively, the proposed pipeline provides a wide array of quantitative and qualitative data on the tested compounds that can help pinpoint promising novel compounds; these can then be evaluated more vigorously using in planta screening assays. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Volatile organic compounds (VOCs) produced by bacteria play an important, yet relatively unexplored role in interactions between plants and phytopathogens. In this study, the soil bacterium Bacillus halotolerans NYG5 was identified as a potent biocontrol agent against several phytopathogenic fungi (Macrophomina phaseolina, Rhizoctonia solani, Pythium aphanidermatum, and Sclerotinia sclerotiorum) through the production of VOCs. NYG5emitted VOCs also inhibited the growth of bacterial pathogens (Agrobacterium tumefaciens, Xanthomonas campestris, Clavibacter michiganensis, and Pseudomonas syringae). When cultured in various growth media, NYG5 produced a variety of VOCs. Five distinct VOCs (2-methylbutanoic acid, 5-methyl-2-hexanone, 2,3-hexanedione, 2-ethyl-1-hexanol, and 6-methyl-2-heptanone) were identified using headspace GC-MS. 2,3-Hexanedione exhibited potent lethal effects on the tested phytopathogens and nematicidal activity against Meloidogyne javanica at a concentration of 50 ppm. In addition, 0.05 ppm 2,3-hexanedione stimulated the expression of pathogenesis-related genes 1 and 2 in Arabidopsis thaliana. Interestingly, 2,3-hexanedione is used as a food additive at higher concentrations than those tested in this study. Hence, 2,3-hexanedione is a promising biologically active compound that might serve as a sustainable alternative to common chemical pesticides and an elicitor of plant defense.

Organic amendment, and especially the use of composts, is a well-accepted sustainable agricultural practice. Compost increases soil carbon and microbial biomass, changes enzymatic activity, and enriches soil carbon and nitrogen stocks. However, relatively little is known about the immediate and long-term temporal dynamics of agricultural soil microbial communities following repeated compost applications. Our study was conducted at two field sites: Newe Ya’ar (NY, Mediterranean climate) and Gilat (G, semi-arid climate), both managed organically over 4 years under either conventional fertilization (0, zero compost) or three levels of compost amendment (20, 40 and 60 m 3 /ha or 2, 4, 6 L/m 2 ). Microbial community dynamics in the soils was examined by high-and low-time-resolution analyses. Annual community composition in compost-amended soils was significantly affected by compost amendment levels in G (first, second and third years) and in NY (third year). Repeated sampling at high resolution (9–10 times over 1 year) showed that at both sites, compost application initially induced a strong shift in microbial communities, lasting for up to 1 month, followed by a milder response. Compost application significantly elevated alpha diversity at both sites, but differed in the compost–dose correlation effect. We demonstrate higher abundance of taxa putatively involved in organic decomposition and characterized compost-related indicator taxa and a compost-derived core microbiome at both sites. Overall, this study describes temporal changes in the ecology of soil microbiomes in response to compost vs. conventional fertilization. Highlights Dose-dependent changes in soil microbiome structure by manure compost application Dynamic short-and long-term changes in soil microbiomes by compost amendment Climate, soil properties and management influence compost-amendment effects Immediate and temporal cumulative effects of compost on soil α and β diversity

Bacillus cereus sensu lato (Bcsl) strains are widely explored due to their capacity to antagonize a broad range of plant pathogens. These include B. cereus sp. UW85, whose antagonistic capacity is attributed to the secondary metabolite Zwittermicin A (ZwA). We recently isolated four soil and root-associated Bcsl strains (MO2, S−10, S-25, LSTW-24) that displayed different growth profiles and in-vitro antagonistic effects against three soilborne plant pathogens models: Pythium aphanidermatum (oomycete) Rhizoctonia solani (basidiomycete), and Fusarium oxysporum (ascomycete). To identify genetic mechanisms potentially responsible for the differences in growth and antagonistic phenotypes of these Bcsl strains, we sequenced and compared their genomes, and that of strain UW85 using a hybrid sequencing pipeline. Despite similarities, specific Bcsl strains had unique secondary metabolite and chitinase-encoding genes that could potentially explain observed differences in in-vitro chitinolytic potential and anti-fungal activity. Strains UW85, S-10 and S-25 contained a (~500 Kbp) mega-plasmid that harbored the ZwA biosynthetic gene cluster. The UW85 mega-plasmid contained more ABC transporters than the other two strains, whereas the S-25 mega-plasmid carried a unique cluster containing cellulose and chitin degrading genes. Collectively, comparative genomics revealed several mechanisms that can potentially explain differences in in-vitro antagonism of Bcsl strains toward fungal plant pathogens.

Bacteria emit a large number of volatile organic compounds (VOCs) into the environment. VOCs are species-specific and their emission depends on environmental conditions, such as growth medium, pH, temperature, incubation time and interaction with other microorganisms. These VOCs can enhance plant growth, suppress pathogens and act as signaling molecules during plant-microorganism interactions. Some bacterial VOCs have been reported to show strong antimicrobial, nematicidal, pesticidal, plant defense, induced tolerance and plant-growth-promoting activities under controlled conditions. Commonly produced antifungal VOCs include dimethyl trisulfide, dimethyl disulfide, benzothiazole, nonane, decanone and 1-butanol. Species of Bacillus, Pseudomonas, Arthrobacter, Enterobacter and Burkholderia produce plant growth promoting VOCs, such as acetoin and 2,3-bute-nediol. These VOCs affect expression of genes involved in defense and development in plant species (i.e., Ara-bidopsis, tobacco, tomato, potato, millet and maize). VOCs are also implicated in altering pathogenesis-related genes, inducing systemic resistance, modulating plant metabolic pathways and acquiring nutrients. However, detailed mechanisms of action of VOCs need to be further explored. This review summarizes the bioactive VOCs produced by diverse bacterial species as an alternative to agrochemicals, their mechanism of action and chal-lenges for employment of bacterial VOCs for sustainable agricultural practices. Future studies on technological improvements for bacterial VOCs application under greenhouse and open field conditions are warranted.

Freshwater bodies are critical components of terrestrial ecosystems. The microbial communities of freshwater ecosystems are intimately linked water quality. These microbes interact with, utilize and recycle inorganic elements and organic matter. Here, we present three metagenomic sequence datasets (total of 182.9 Gbp) from different freshwater environments in Israel. The first dataset is from diverse freshwater bodies intended for different usages - a nature reserve, irrigation and aquaculture facilities, a tertiary wastewater treatment plant and a desert rainfall reservoir. The second represents a two-year time-series, collected during 2013-2014 at roughly monthly intervals, from a water reservoir connected to an aquaculture facility. The third is from several time-points during the winter and spring of 2015 in Lake Kinneret, including a bloom of the cyanobacterium Microcystis sp. These datasets are accompanied by physical, chemical, and biological measurements at each sampling point. We expect that these metagenomes will facilitate a wide range of comparative studies that seek to illuminate new aspects of freshwater microbial ecosystems and inform future water quality management approaches.

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.

Root selection of their associated microbiome composition and activities is determined by the plant’s developmental stage and distance from the root. Total gene abundance, structure and functions of root-associated and rhizospheric microbiomes were studied throughout wheat growth season under field conditions. On the root surface, abundance of the well-known wheat colonizers Proteobacteria and Actinobacteria decreased and increased, respectively, during spike formation, whereas abundance of Bacteroidetes was independent of spike formation. Metagenomic analysis combined with functional co-occurrence networks revealed a significant impact of plant developmental stage on its microbiome during the transition from vegetative growth to spike formation. For example, gene functions related to biofilm and sensorial movement, antibiotic production and resistance and carbons and amino acids and their transporters. Genes associated with these functions were also in higher abundance in root vs. the rhizosphere microbiome. We propose that abundance of transporter-encoding genes related to carbon and amino acid, may mirror the availability and utilization of root exudates. Genes related to antibiotic resistance mechanisms were abundant during vegetative growth, while after spike formation, genes related to the biosynthesis of various antibiotics were enriched. This observation suggests that during root colonization and biofilm formation, bacteria cope with competitor’s antibiotics, whereas in the mature biofilm stage, they invest in inhibiting new colonizers. Additionally, there is higher abundance of genes related to denitrification in rhizosphere compared to root-associated microbiome during wheat growth, possibly due to competition with the plant over nitrogen in the root vicinity. We demonstrated functional and phylogenetic division in wheat root zone microbiome in both time and space: pre- and post-spike formation, and root-associated vs. rhizospheric niches. These findings shed light on the dynamics of plant–microbe and microbe–microbe interactions in the developing root zone.

Background Roots select their associated microbiome and affect its composition and activities through exudates that provide a nutrient-rich environment based on distance from the root. Root-exudation patterns depend on the plant's developmental stage. We followed field-grown wheat from emergence to spike maturation and compared the structure and functions of the microbiomes in two niches: adjacent, root-associated bacteria and the rhizosphere. The effects of growth stage on root-associated and rhizospheric communities structure and functions were investigated to enhance our understanding of plant–microbe interactions. Results A significant impact of wheat developmental stage during the transition from vegetative growth to spike formation was observed on structure and functions of both root-associated and rhizosphere microbiomes. On the root surface, abundance of the well-known wheat colonizers Proteobacteria and Actinobacteria decreased and increased, respectively, during spike formation, whereas abundance of Bacteroidetes was independent of spike formation. Three microbiome functional clusters were specifically associated with root proximity: (1) biofilm and sensorial movement; (2) antibiotic production and resistance; and (3) carbon biosynthesis, degradation and transporters, and amino acid biosynthesis and transporters. Interestingly, in the root-associated microbiome, genes related to all of these functions were influenced by spike formation. Abundance of genes related to nine other functions (lipopolysaccharide transporter and biosynthesis, beta-lactam resistance, metabolism of methane, alanine-aspartate-glutamate and arginine-proline, and biosynthesis of peptidoglycan, lysine and enediyne antibiotics) was significantly influenced by spike formation in both roots and rhizosphere. All of these genes were abundant in the rhizosphere, more so before spike formation when root exudation is high, supporting the notion that some of the root exudates are not fully utilized by the root-associated bacteria and subsequently diffuse into the surrounding soil, creating the rhizosphere. Conclusions We demonstrated functional division in the microbiome of the wheat root zone in both time and space: pre- and post-spike formation and root-associated vs . rhizospheric niches. The responses of the root microbiome were driven by both the plant and the microbial physiology and activities, both of which respond to environmental cues. These findings shed light on the dynamics of plant–microbe and microbe–microbe interactions in the root zone.

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

No-tillage (NT) is a common soil-conservation management practice with known agricultural advantages and drawbacks. However, its short- and long-term effects on the soil microbiome have not been well established. Here, we compared conventional (CT), minimal (MT) and NT practices in two agricultural fields in the north of Israel over a period of 3 years. Edaphic properties, plant-associated pests, weed species abundance and soil microbial community structure were assessed to examine the effects of tillage. Tillage significantly altered physical and chemical soil properties, and a significant increase in hydrolytic and redox microbial activities was observed in NT soils from both sites. Consistent with this, the microbial community structure of NT samples diverged significantly over time from those of CT samples. Repetitive tillage and even a single tillage event caused significant changes in the relative abundance of microorganisms at taxonomic levels ranging from phylum to OTU. However, no significant difference between treatments was found in microbial community alpha-diversity or crop yield. Conversely, higher levels of weed diversity and some pests number were found in NT samples. Overall, we demonstrate that tillage plays a major role in shaping microbial community structure, and in influencing additional environmental, ecological and agricultural soil parameters.

Excessive use of antimicrobials in aquaculture is concerning, given possible environmental ramifications and the potential contribution to the spread of antimicrobial resistance (AR). In this study, we explored seasonal abundance of antimicrobial resistance genes and bacterial community composition in the water column of an intensive aquaculture pond stocked with Silver Carp (Hypophthalmichthys molitrix) prophylactically treated with sulfamethoprim (25% sulfadiazine; 5% trimethoprim), relative to an adjacent unstocked reservoir. Bacterial community composition was monitored using high-throughput sequencing of 16S rRNA gene amplicons in eight sampling profiles to determine seasonal dynamics, representing principal stages in the fish fattening cycle. In tandem, qPCR was applied to assess relative abundance of selected antimicrobial resistance genes (sul1, sul2, dfrA1, tetA and blaTEM) and class-1 integrons (int1). Concomitantly, resistomes were extrapolated from shotgun metagenomes in representative profiles. Analyses revealed increased relative abundance of sulfonamide and tetracycline resistance genes in fishpond-03, relative to pre-stocking and reservoir levels, whereas no significant differences were observed for genes encoding resistance to antimicrobials that were not used in the fishpond-03. Seasons strongly dictated bacterial community composition, with high abundance of cyanobacteria in summer and increased relative abundance of Flavobacterium in the winter. Our results indicate that prophylactic use of sulfonamides in intensive aquaculture ponds facilitates resistance suggesting that prophylactic use of these antimicrobials in aquaculture should be restricted.